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COORDINATION OF SULFUR AND NITROGEN IN THE PROTECTION OF PHOTOSYNTHESIS, GROWTH AND YIELD OF CADMIUM-TREATED MUSTARD (Brassicajuncea)

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ABSTRACT

THESIS / / '

SUBMITTED FOR THE AWARD OF THE DEGREE OF

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Bottor of $litlo!£opi)p

BOTANY

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SARVAJEET SINGH

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DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2008

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Coordination of sulfur and nitrogen in ttie protection of

photosynthesis, growth and yield of cadmium-treated

mustard (Brassicajuncea)

Abstract

Sarvajeet Singh

Abstract of the thesis submitted to the Aligarh Muslim University, Aligarh, India for

the degree of Doctor of Philosophy in Botany.

The present thesis comprises of six chapters.

The importance of the problem and justifications for the present work undertaken

were emphasized in Chapter 1.

Chapter 2 is the review of literature. It includes literature available on the

aspects of the physiological analysis of various growth-, photosynthetic- and,

biochemical-characteristics, antioxidant studies and yield of various crop plants under

cadmium stress. The importance of sulfur and nitrogen nutrients in the alleviation of

stress and in the regulation of plant growth and development under stress condition

were also reviewed. The chapter has been divided in to sections and sub-sections for

better understanding of the work of other research reports in this field of study. In

addition, the critical appraisal of the review of literature has also been included to

identify the gap in the field of study.

The details of the material used in the study and methodology adopted to

determine various characteristics recorded in the three experiments have been

described in the Chapter 3. In addition the Chapter 3 also mentions the relevant

information on the location of the study and the environmental conditions during the

data sampling times.

Chapter 4 includes the results on the response of mustard to cadmium, sulfur

and nitrogen treatments in three Experiments. The data obtained were statistically

analyzed and the significance was determined at/7<0.05.

The results obtained in the Experiments have been discussed in Chapter 5 in

the light of observations recorded and supported with the earlier findings, if available.

This chapter also presents the possible explanations of the data obtained to reach a

conclusion and possible future prospects.

Chapter 6 present the summary of the work reported in this thesis.

In the following pages a brief account of the importance of the study

undertaken, results obtained and conclusion are given.

Importance of the study undertaken

Abiotic stresses such as salinity, heavy metals, cold, drought and heat are the primary

cause of crop loss worldwide and the relative decreases in potential maximum yields

vary between 50-80% (Bray et al. 2000). Among abiotic stresses, heavy metal

contamination is a serious environmental problem that limits plant productivity and

threatens human health (Wagner 1993, Sanita di Toppi and Gabbrielli 1999, Khan and

Singh 2008). Cadmium (Cd) is of special concern due to its wide availability and

potential toxicity to biota even at low concentrations (Das et al. 1997, Sanita di Toppi

and Gabbrielli 1999). It is released into the environment by power stations, heating

systems, metal working industries, waste incinerators, urban traffic, cement factories

and as a byproduct of sewage sludge and phosphatic fertilizers (Sanita di Toppi and

Gabbrielli 1999, Lombi et al 2000, Nolan et al. 2003, McLaughlin et al. 2006,

Sharma and Dudey 2006, Xie et al. 2006, Verma et al. 2007, Singh et al. 2008).

For sustainable crop production, it is important to develop methods or

techniques for alleviating the Cd-induced growth inhibition and reducing its

accumulation in plants. Increasing evidence suggests that mineral-nutrient status of

plants plays a critical role in increasing plant resistance to environmental stresses

(Marschner 1995, Vassilev et al. 2005, Anjum et al. 2008a,b, Hassan et al. 2008,

Khan et al. 2008). The use of sulfur (S) and nitrogen (N) fertilizers opens potential

option which can be used to alleviate Cd toxicity. Sulfiir is a structural constituent of

several coenzymes and prosthetic groups, such as ferredoxin, which are also

important for N assimilation. Thus, S plays an important role in plant growth and in

the regulation of plant development (Ernst et al. 2008). It has been found that S

nutrition is a critical factor for the alleviation of Cd toxicity (Popovic et al. 1996,

Chen and Huerta 1997, Hassan et al. 2005a, Vassilev et al. 2005, Anjum et al. 2008a,

b). Further, N is an important component of many structural, genetic and metabolic

compounds in plant cells. Plant N status is highly dependent on N fertilization

(Hernandez et al. 1997). Nitrogen is also a major component of chlorophyll and

amino acids, the building blocks of proteins. Increase in N supply can stimulate plant

growth and productivity (Joel et al. 1997) as well as photosynthetic activity (Makino

et al. 1992) through increased amounts of stromal and thylakoid proteins in leaves

f U 31?

(Bungard et al. 1997). Pankovic et al. (2000) have shown that optimal N supply

decreased the inhibitory effects of Cd on photosynthesis of Helianthus annuus. A

proper N supply has been shown to have a positive effect in overcoming the adverse

effects caused by Cd toxicity in Oryza sativa (Hassan et al. 2005b). The assimilation

pathways of S and N are well coordinated (Brunold 1993, Takahashi and Saito 1996,

Abdin et al. 2003, Hawkesford et al. 2006). Therefore, coordination of S and N could

play a prominent role in alleviating the Cd-induced toxicity in crop plants.

The oleiferous Brassica is the third most important source of vegetable oil in

the world after palm and soybean oil and grown as an edible or an industrial oil crop

which is used as a source of edible protein, in much the same way as soybean protein

(Ahmad and Abdin 2000, Zhang et al. 2003). The major mustard oil-producing

countries include Canada, China, France, Germany, India and UK. According to a

report of United States Department of Agriculture (USDA), the world oilseed

production is 397metric tons in 2006-07. This total production is an increase of

9metric tons, or 2% of the last season. Indian agriculture contributes about 15% and

8% to the world total acreage under oilseed cultivation and production, respectively.

However, the productivity in India is only 791kg ha"' as compared to the world

average of 1718kg ha'' (Damodaran and Hedge 2002). About 90% of the total land

under oilseed cultivation in India is occupied by Brassica juncea (Khan et al. 2007).

Despite a large area under cultivation of mustard, the productivity of the crop has

dropped because of anthropogenic activities emitting heavy metals in the

environment.

Discussed the importance of sulftir and nitrogen in plant metabolism and their

role in stress tolerance it was assumed that mustard (Brassica juncea L. Czem &

Coss.) may serve as a model crop in the study of the influence of S and N in plant

metabolism under Cd stress. Further, the fertilization of crop with balanced S and N

may prove fruitftil in alleviating Cd stress and improving crop productivity. Studies

on crops with a view of alleviating Cd stress with balanced S and N application have

not been carried out. Therefore, the reported research was undertaken with the

following objectives:

• To screen and select mustard cultivars as Cd tolerant and Cd non-tolerant

grown under Cd treatments.

• To study the influence of S application in the alleviation of Cd-induced effects

in Cd tolerant and Cd non-tolerant cultivars of mustard.

• To study the combined influence of S and N in the alleviation of Cd-induced

effects in Cd tolerant and Cd non-tolerant cultivars of mustard.

Experimental Results

The results of the experiments are summarized below: Experiment 1

The Experiment was conducted to study the effect of five concentrations of Cd viz., 0,

25, 50, 100 and I50mg kg"' soil on Cd accumulation in root and leaf, growth,

photosynthetic and yield characteristics of five mustard cultivars {Brassica juncea L.)

namely, Alankar, Varuna, Pusa Bold, Sakha and RH30. The treatments in this

Experiment were arranged in a factorial randomized block design. Cadmium

accumulation, growth and photosynthetic characteristics were studied at pre-flowering

(30DAS), flowering (60DAS) and post-flowering (90DAS) growth stages, while yield

characteristics were noted at harvest (120DAS). Tolerance index of the five cultivars

of mustard was calculated and the cultivars were designated as Cd tolerant and Cd

non-tolerant on the basis of their performance under Cd stress. The effect of Cd on

growth, photosynthetic and yield characteristics was found significant at all sampling

times. The interaction of Cd treatments and cultivars was also found significant. For

growth characteristics, the interaction effect was found non-significant at 30 DAS.

The increase in Cd levels decreased the growth, photosynthetic and yield

characteristics of all the five cultivars at all sampling stages. The observations

recorded at the three sampling stages showed similar pattern of cultivar response to

Cd treatments. Maximum reduction in growth, photosynthesis and yield

characterisfics was noted with 150mg Cd kg'' soil followed by 100, 50 and 25mg Cd

kg"' soil.

Among cultivars, Alankar showed lesser decrease in growth, photosynthetic

and yield characteristics followed by Varuna and Pusa Bold, whereas, RH30 and

Sakha exhibited greater reduction in growth characteristics under Cd stress. The

tolerance index of cultivars was: Alankar >Varuna >Pusa Bold >Sakha >RH30.

Experiment 2

Experiment 2 was conducted on the basis of findings of Experiments 1. As observed

in Experiment 1, Alankar emerged as Cd-tolerant and R_H30 as Cd non-tolerant

cuhivar. Among Cd levels, 150mg Cd kg"' soil was found most toxic and caused

4 ' ti.'.*" *'

maximum reductions in the characteristics studied. In the present Experiment, the aim

was to study the alleviation potential of elemental S levels (50 and lOOmg S kg' soil)

on the effects of 50 and 150mg Cd kg"' soil. The treatments in this Experiment were

arranged in a factorial randomized block design. The assessment was done by

studying the changes in Cd accvmiulation in root and leaf, growth and photosynthetic

characteristics, S and N assimilation, components of antioxidant defense system and

yield characteristics of tolerant (Alankar) and non-tolerant (RH30) cultivars. The

sampling stages for Cd accumulation, growth and photosynthetic characteristics, S

and N assimilation, components of antioxidant defense system were pre-flowering

(30DAS), flowering (60DAS) and post-flowering (90DAS). The yield characteristics

were noted at harvest (120DAS).

The growth and photosynthetic characteristics and N assimilation decreased

maximally in both the cultivars treated with 150mg Cd kg"' soil. Contrarily, there was

significant increase in the Cd accumulation, S assimilation, H2O2 and TBARS content

and components of enzymatic antioxidant system and glutathione content was found.

Non-tolerant cultivar RH30 exhibited greater Cd effects than tolerant cultivar

Alankar. In comparison to lOOmg S kg"' soil, the application of 50mg S kg"' soil

maximally alleviated the toxic effect of 50mg Cd kg"' soil and improved growth,

photosynthesis, S and N assimilation and components of enzymatic and non-

enzymatic antioxidant system thus yield characteristics of both the cultivars. Further,

no ameliorative effect of lOOmg S kg"' soil was observed in plants treated with 150mg

Cd kg"' soil. Alleviation effects of S were higher in Alankar than RH30.

Experiment 3

Experiment 3 was conducted on the basis of the findings of Experiments 2. In

Experiment 2, it was observed that the application of 50mg S kg"' soil to plants treated

with 50mg Cd kg"' soil alleviated Cd-induced toxicity in both the culdvars.

Supplementafion of 50mg S kg"' soil maximally overcome the toxic effects of 50mg

Cd kg"' soil in Alankar (tolerant cultivar), while, the same combination lowered the

Cd-induced toxic effects in RH30 to some extent. The present experiment was aimed

to study the effect of supplementation of different N levels (40, 80 and 120mg N kg"'

soil) to 50mg S kg"' soil for alleviation of adverse effects of 50mg Cd kg"' soil. The

treatments in this Experiment were arranged in a factorial randomized block design.

The assessment was done by analyzing the changes in Cd accumulation in root and

leaf, growth and photosynthetic characteristics, S and N assimilation, components of

antioxidant defense system at 30, 60 and 90DAS and yield characteristics at harvest.

The alterations in growth, photosynthesis, S and N assimilation, components

of antioxidant system and yield characteristics caused by 50mg Cd kg" soil were

alleviated by 50mg S kg"' soil in tolerant (Alankar) and non-tolerant (RH30) cultivars,

but the alleviation potential of S varied between the cultivars. Application of different

levels of N (40, 80 and 120mg N kg"' soil) further enhanced the alleviation effect of S

(50mg S kg"' soil) in both the cultivars. Among the N levels used, the application of

80mg N kg'' soil proved superior than the other N levels. A combined application of S

and N (50mg S kg"' soil plus SOmg N kg"' soil) not only ameliorated the Cd-induced

(50mg Cd kg"' soil) effects but also increased the growth, photosynthesis, S and N

assimilation and components of enzymatic and non-enzymatic antioxidant system

thus yield characteristics in Alankar. In RH30, this combination (SOmg S kg"' soil

plus SOmg N kg"' soil) only lowered the adverse effects of Cd.

Conclusion

Conclusively, nutrients are known to decipher essential role in plant metabolism and

augmenting growth and productivity of crops. However, a nutrient package is

required for sustainable agriculture under varied environmental conditions. Crop

cultivars display their inherent potential. The cultivar Alankar surpassed other

cultivars tested and tolerated Cd stress to a significant degree. The cultivar RH30 was

weak in performance and least tolerant to Cd among the cultivars tested. Therefore,

Alankar exhibited lesser decreases in growth, photosynthetic and yield characteristics

under Cd stress. Correspondingly, Alankar also showed lesser oxidative stress and

increased antioxidant system than RH30 to protect photosynthetic machinery and

consequent effects on other attributes. Sulfur proved significant potential in the

alleviation of Cd stress in both the cultivars. The decreases in the characteristics

observed due to Cd stress were lowered by S application and SOmg S kg"' soil proved

effective in alleviation of Cd stress than the high doses of S. The coordination of S

and N proved most effective in nullifying the Cd stress effects. The potential of SOmg

S kg'' soil in the alleviation of Cd stress was substantially enhanced by the

simultaneous application of SOmg N kg"' soil. The combined application of SOmg S

kg" soil plus SOmg N kg"' soil not only resulted in restricting the decrease caused by

Cd but also nullified the Cd effects and even enhanced the characteristics values over

the control. A package of SOmg S kg"' soil plus SOmg N kg"' soil appears to be most

6

effective in the cultivation of mustard under Cd stress. The effect of this combination

was due to coordination of S and N in maintaining plant metabolism and alleviating

Cd stress.

New Report in the Thesis and Future Prospects

Mineral nutrient status of plants plays a critical role in increasing plant resistance to

environmental stress factors. Of the mineral nutrients, S and N are major

macronutrients necessary for the plant life cycle. The uptake and assimilation of S and

N in higher plants are the crucial factors determining plant growth and vigor and crop

yield. The present study showed that a balanced S and N supply can mitigate the

inhibitory effects of Cd on mustard metabolism. The individual effects of S and N and

other nutrients have been studied. However, no effort has been made to study

combined application of S and N in tolerance of plants, although coordinated

functions of these nutrients are well known. For sustainable agriculture a nutrient

protocol with balanced S and N is necessary in the growth and development of crops

under Cd stress. The results reported in this thesis have confirmed this. The effect of

combined application of S and N on oxidative stress and antioxidants under Cd stress

has been reported for the first time. Efforts have been made for the first time to

develop S and N protocol for alleviation of Cd stress in mustard.

Further, the efforts should be focused to dissect the mechanism(s) of

regulation of sulfate and nitrate assimilation at the molecular level in crop plants

under varied environmental conditions. Strategies should also aim to manipulate steps

of pathways leading to the production of thiols and their products in plants through

overexpressing Ser acetyl transferase (SAT), y-ECS and GSHS enzymes of S and N

nutrition under Cd stress. More detailed studies are required to understand the Cd-

induced stress response modulated by S and N metabolism at molecular and

mechanistic levels. This would help to develop an effective strategy to raise

transgenic species for stress resistance.

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with other nutrients. In: Abrol YP, Ahmad A (Eds) Sulphur in plants. Kluwer

Academic Publishers, The Netherlands, pp359-374.

Ahmad A, Abdin MZ (2000) Effect of sulphur application on lipid, RNA, protein

content and fatty acid composition in developing seeds of rapeseed {Brassica

campesths L). Plant Sci 150:71-75.

7

Anjum NA, Umar S, Ahmad A, Iqbal M, Khan NA (2008a) Sulphur protects mustard

(Brassica campestris L.) from cadmium toxicity by improving leaf ascorbate and

glutathione. Plant Growth Regul 54:271-279.

Anjum NA, Umar S, Singh S, Nazar R, Khan NA (2008b) Sulfur assimilation and

cadmium tolerance in plants. In: Khan NA, Singh S, Umar S (Eds) Sulfur

assimilation and abiotic stresses in plants. Springer-Verlag, The Netherlands,

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^ ^

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10 , . . - . .

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COORDINATION OF SULFUR AND NITROGEN IN THE PROTECTION OF PHOTOSYNTHESIS, GROWTH AND YIELD OF CADMIUM-TREATED MUSTARD (Brassi'ca juiicea)

i • \

/ / THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

II Bottor of $t)ilosiop[)p IN / /

\ \ A < BOTANY

SARVAJEET SINGH

iS^ DEPARTMENT OF BOTANY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2008

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f e d U. CoraiJUi C<8it:x

2 7 JAN d

T6547

Dedicated to my father.

< ^ s\5

<Dr. fNafeesA- Xfian ^Pmfessof of (plant Hysiotogy

Department of Botany Aligarh Muslim University Aligarli-202 002, INDIA Off: +91-571-2702016 Mobile:+91-9411488881 E-mail: naf9(^lycos.com

Dated: ^t)- Z> ^OO^

CERTIFICATE

This is to certify that the thesis entitled, '"Coordination of sulfur and

nitrogen in the protection of photosynthesis, growth and yield of

cadmium-treated mustard (Brassica juncea)" submitted for the degree

of Doctor of Philosophy in Botany is a faithful record of bonafide

research work carried out at the Department of Botany, Aligarh

Muslim University, Aligarh by Mr. Sarvajeet Singh under my

guidance and supervision and that no part of it has been submitted for any

other degree or diploma.

i ^ : . \ ^

(P fafees A. Khan)

ACKNOWLEDGEMENTS

I bow in reverence to GOD the Almighty, Who blessed me with an innumerable

favour of academic work.

Like all creative works, completing a thesis in any discipline is really a hard

work to accomplish. I have no words to express my heartfelt gratitude to my

supervisor Professor Nafees A. Khan, Department of Botany, Aligarh Muslim

University, Aligarh for his excellent guidance, valuable and timely suggestions,

stimulating discussions and academic alertness, sense of perfection and precision

which enabled me to complete this work. His affectionate instinct and constant

encouragement were always been boon to me. Without his unending support and

encouragement this thesis would have never been completed.

I am extremely obliged to Professor Ainul H. Khan, Chaimian, Department of

Botany for providing necessary research facilities. Thanks in this regard are also due

to Ex-Chairman Professor SamiuUah.

I have always been inspired by my respected teachers. I duly acknowledge

their moral support. I am also thankful to my friend Ms. Priya and lab colleagues

Ms. Rabat Nazar and Ms. Noushina Iqbal for the help they extended whenever

required.

I also place on record the assistance rendered by Dr. Naser A. Anjum,

Dr. Moinudddin Khan, Dr. Asgar Ali Shah, Dr. Lamabam Peter Singh, Dr. Sandeep

Sharma, Dr. Naeem, Dr. Jarrar Ahmad, Dr. Shuaib Khan, Dr. Riyasat Ali and

Mr. Rais A. Khan during the gruelling task.

I am also thankful to the Council of Scientific and Industrial Research,

Government of India, New Delhi for providing financial assistance as Senior

Research Fellowship to me.

Finally, I would like to express my greatest gratitude to people who gave me

the greatest encouragement and motivation to carry out my research work; my

parents, paternal aunt Surjit, brothers Gurvinder, Dev and Ranjeet and sister Jasbeer.

(Sarvajeet Singh)

ABBREVIATIONS

%

^g Ag ALA AsA As ATP ATP-S CA Car CAT Cd CdCb CdBP Chi a Chlb Chi cm Co CO2 Cr Cu/Zn-SOD cv. Cys d DAS DHAR DMSO DW EDTA FAD Fe Fe-SOD Fv/Fm FW g GDH Gly Glu GPOX GR GS GSH GSSG GST h H2O2

Percent Gravity Silver 5-aminolaevulinic acid Ascorbic acid Arsenic Adenosine triphosphate ATP-sulfiirylase Carbonic anhydrase Carotenoids Catalase Cadmium Cadmium chloride Cd-binding peptides Chlorophyll a Chlorophyll b Chlorophyll Centimeter Cobalt Carbon dioxide Cromium Copper/zinc superoxide dismutases Cultivar Cysteine Days Days after sowing Dehydroascorbate reductase Dimethyl sulphoxide Dry weight Ethylenediamine tetraacetic acid Flavin adenin dinucleotide Iron Iron superoxide dismutase Photosynthetic quantum yield Fresh weight Gram Glutamate dehydrogenase Glycine Glutamate Guaiacol peroxidase Glutathione reductase Glutamine synthetase Reduced glutathione Oxidized glutathione Glutathione S-transferase Hour Hydrogen peroxide

HEPES Hg. HMW hPC LMW M MDA MDHAR Mg mg Met min mM Mn-SOD Mo mt MT N NaOCl NADH-GDH NADPH NBT Ni NiR ng NPT NR O2 'O2 O2-°C OH-Pb PC PDM pH ppm PSI PSII ROS rpm Rubisco Sb -SH sec SOD TBARS Va liM

^g

Hydroxyethylenepiperazine ethanesulfonic acid Mercury High molecular weight Homophytochelatin Low molecular weight Molar Malondialdehyde Monodehydroascorbate reductase Magnecium Miligram Methionine Minute Millimolar Manganese superoxide dismutase Molybdenum Metric ton Metallothionein Nitrogen Sodium hypochlorite Glutamate dehydrogenase Nicotinamide adenine dinucleotide phosphate Nitroblue tetrazolium Nickel Nitrite reductase Nano gram Non-protein thiol Nitrate reductase Molecular oxygen Singlet oxygen Superoxide anion Degree celcius Hydroxyl radical Lead Phytochelatin Plant dry mass Hydrogen ion potential Parts per million Photosystem I Photosystem II Reactive oxygen species Revolutions per minute Ribulose 1,5 bisphosphate carboxylase Antimony Sulfhydryl group Second Superoxide dismutase Thiobarbituric acid reactive substances Vanadium Micro mol Micro gram

Chapters

1. Introduction

2. Review of Literature

3. Material and Methods

4. Experimental Results

5. Discussion

6. Summary

Bibliography

CONTENTS

Page No.

1

7

48

65

85

117

121

INTRODUCTION

INTRODUCTION Chapter 1

Environmental stress is referred to the extreme environmental conditions that lead to

alterations in plant metabolism resulting in decreased rate of plants growth processes,

loss in productivity or inducing damaging effect in any of plant's organ/part,

alteration in anatomical, biochemical or molecular regulation.

Abiotic stress such as salinity, heavy metal, cold, drought or heat is the

primary cause of crop loss worldwide. The relative decrease in potential maximum

yields caused by abiotic stress varies between 50-80% (Bray et al. 2000, Mahajan and

Tuteja 2005, Khan et al. 2006). Among the abiotic factors, heavy metals are

ubiquitous in the enviroimient, depending on both geological origin and

anthropogenic activities. Metal contamination to soil is considered as major threat

because of their acute toxicity, non-biodegradable nature and gradual build up of high

concentrations (Prasad 1995, Prasad and Hagemeyer 1999, Khan and Singh 2008).

Although, heavy metals at minute quantities are natural components of soil, but the

anthropogenic activities such as mining, industry, and localized agriculture have

contributed to undesirable accumulations of toxic metals in the environment (Alloway

1995, Kabata-Pendias and Pendias 2001). Heavy metal concentration in soil ranges

from traces to as high as 100,000mg kg"' soil (Blaylock and Huang 2000), depending

on the location and the type of metal.

Among heavy metals, cadmium (Cd) is of special concern due to its wide

availability and potential toxicity to biota at low concentrations (Das et al. 1997,

Sanita di Toppi and Gabbrielli 1999). It is released into the enviroimient by power

stations, heating systems, metal working industries, waste incinerators, urban traffic,

cement factories, and as a byproduct of sewage sludge and phosphatic fertilizers

(Sanita di Toppi and Gabbrielli 1999, Lombi et al. 2000, McLaughlin et al. 2000,

Nolan et al. 2003, Sharma and Dubey 2006, Xie et al. 2006, Verma et al. 2007, Singh

et al. 2008b). Cadmium in unpolluted soil is mainly originated from soil parent ores.

Globally, Cd content in soil is about 0.01~2mg kg'' soil, with an average of 0.35mg

kg'' soil (Xu and Yang 1995). The global production of Cd has increased steadily

since 1910 when Cd electroplating was developed commercially. The average armual

global production of Cd was 12000tons in 1960-1969 which increased to 15000-

20000tons in 1980-1985 (Sandra 1986).

t^^

Cadmium contamination may pose a serious hazard to plants and human

health through its uptake in food chain (Jackson and Alloway 1992, Wagner 1993,

Hail 2002). The Food and Agriculture OrganizationAVorld Health Organization

recommended 400-500^g Cd per week as a maximum tolerable intake (Fassett 1980).

Average dietary intakes of Cd around the world range between 25 and 75^g per day

(Page et al. 1981). Although Cd is not essential for plant growth, but it is readily

taken up by roots and translocated into the leaves in m ^ y plant species (Prasad

1995). Root uptake of Cd from soil depends on the Cd concentration in soil, soil pH,

level of organic matter and Zn concentration in soil (Eriksson et al. 1996, Ciecko et

al. 2001, Yu et al. 2005, Singh et al. 2008b). Soil factors affect chemical availability

of soil Cd to plant roots (Oliver et al. 1995). Most of the Cd absorbed is accumulated

in the roots and very little amount of Cd is transported from roots to shoots. Retention

of Cd in roots is advantageous as the entry of Cd into human food chain is reduced.

According to Grant et al. (1998) Cd concentration in grains reduced when Cd

translocation from roots to shoots is restricted. It has been shown that Cd toxicity is a

major factor limiting plant growth in many soils (Sandalio et al. 2001, Artetxe et al.

2002, Fediuc and Erdei 2002). The most characteristic symptoms of Cd toxicity

are brown and short roots, chlorosis, fewer tillers, senescence and reduced plant

growth and biomass (Greger e/^/. 1991, Arduini et al. 1994, Boussama et al. 1999,

Wu and Zhang 2002b, Wu et al. 2003, Drazkiewicz and Baszynski 2005, Khan et

al. 2006, 2007a, Gianazza et al. 2007, Anjum et al. 2008a,c,d, Singh et al. 2008a) and

plant death in extreme cases (Sanita di Toppi and Gabbrielli 1999). It causes

inhibition of shoot and root growth (Schutzendubel et al. 2001), disorganization of the

grana structures and reduction in the biosynthesis of chlorophyll (Padmaja et al. 1990,

Somashekaraiah et al. 1992, Siedlecka and Krupa 1995^. Cadmiimi can also interfere

with photosynthesis (Baszynski 1986, Prasad 1995, Sandalio et al. 2001, Fargasova

2004, Ben Ammar et al. 2005, Khan et al. 2006, 2007a, 2008b, Samiullah et al. 2007,

Mobin and Khan 2007, Singh et al. 2008a), respiration, water relations and

reproduction (Barcelo and Poschenreider 1990, Prasad 1997). It causes changes in

organelles by disruption of membrane structure and fiinction (Sandalio et al. 2001,

Perfiis-Barbeoch et al. 2002, Gratao et al. 2006). Moreover, Cd can also inhibit the

activity of several groups of enzymes such as those of the Calvin cycle (Sandalio et

al. 2001), nitrogen metabolism (Boussama et al. 1999, Gouia et al. 2000, 2003,

Chaffei et al. 2003, Ghnaya et al. 2005, Anjana et al. 2006, Hasan et al. 2008),

2

carbohydrate metabolism (Sanita di Toppi and Gabbrielli 1999, Verma and Dubey

2001) and phosphorus metabolism (Shah and Dubey 1998, Sharma and Dubey 2006).

It also limits the uptake of both cations (K"", Ca "", Mg "", Mn " , Zn "", Fe "") and anions

like NO3". The toxicity symptoms observed in plants in the presence of excessive

amounts of Cd are due to a range of interactions at the cellular level (Hall 2002). It

enhances lipid peroxidation and alters functionality of membranes, and causes

changes in enzymatic and non-enzymatic antioxidant systems (De Knecht et al. 1995,

Piqueras et al. 1999, Chien and Kao 2000, Dixit et al. 2001, Olmos et al. 2003, Kuo

and Kao 2004, Ben Youssef er al. 2005, Cho and Seo 2005, Gratao et al. 2006, Mobin

and Khan 2007, Khan et al. 2007a, Anjum et al. 2008a,c,d, Singh et al. 2008a,b).

It has also been reported that excess of Cd stimulates the formation of free

radicals and reactive oxygen species (ROS) (Hendry et al. 1992, Groppa et al. 2001,

Sandalio et al. 2001, Fomazier et al. 2002a,b, lannelli et al. 2002, Milone et al. 2003,

Romero-Puertas et al. 2004, Ben Youssefet al. 2005, Gratao et al. 2006, Mobin and

Khan 2007, Singh et al. 2008a,b), that triggers activation of plant antioxidative

defense system which involves several enzymes and low molecular weight quenchers,

present in plant cells. Among antioxidant enzymes, superoxide dismutase (SOD; EC

1.15.1.1) constitutes the primary step of cellular defense and dismutates superoxide

radicals (O2") to H2O2 and O2. Accumulation of H2O2, a strong oxidant, is prevented

in the cell either by catalase (CAT; EC 1.11.1.6) or by the Ascorbate-Glutathione

cycle, where ascorbate peroxidase (APX; EC 1.1.11.1) converts it to H2O. Finally,

glutathione reductase (GR; EC 1.6.4.2) catalyzes the NADPH-dependent reduction of

oxidized glutathione (GSSG) to the reduced glutathione (GSH) (Noctor et al. 2002).

Ascorbate (AsA) and glutathione (GSH) are important components of Ascorbate-

Glutathione cycle responsible for the removal of H2O2 in different cellular

compartments (Foyer et al. 2002). Ascorbate is the major, probably the only,

antioxidant buffer in the apoplast and is an essential metabolite involved with vital

cell functions. It is a key primary antioxidant that reacts directly with OH- radicals,

O2", and O2 (Chen and Gallic 2004). Glutathione, present in plant cell is a major

non-enzymatic scavenger of ROS (Mishra et al. 2006). In particular, GSH play a very

important role in plants exposed to Cd and sequester it into the vacuoles by the

contribution of a protein with properties similar to those of glutathione-S-transferase

(GST; EC 2.5.1.18) (Marrs and Walbot 1997). Glutathione in the reduced form

protects the thiol (-SH) groups of many enzymes from oxidative conditions. For this

3

reason, its concentration is controlled by a complex homeostatic mechanism where

the availability of sulfiir (S) seems to be required (May et al. 1998a,b). Both AsA and

GSH can also react directly and scavenge certain ROS. However, a general

conclusion on the regulation of the antioxidant system by S assimilation has not been

drawn. Cadmium has a high affinity to metabolic processes of S assimilation by its

effect on the activity of ATP-sulfurylase (ATP-S; EC 2.7.7.4) (Sanita di Toppi and

Gabbrielli 1999, Khan et al. 2007a), and the enzymes involved in the activation of

sulfur (Dominguez-Solis et al. 2001, Harada et al. 2002, Hassan et al. 2006). The

entry of Cd into the cytosol promptly activates S metabolism for the accumulation of

reduced S-containing compound, GSH, which serves as substrates for the biosynthesis

of phytochelatins (PCs) (Rauser 2000, Cobbett 2000b, Leustek et al. 2000, Cobbett

and Goldsbrough 2002), and cysteine (Cys) is a precursor for the biosynthesis of GSH

and its derivatives (Rauser 1990, Inouhe 2005). The rate-limiting step in PC

biosynthesis is the provision of Cys. Thus, the availability of reduced S is considered

as of prime importance (Lee and Leustek 1999, Goldsbrough 2000). Both Cys and.

GSH hence are said to be involved in heavy metal detoxification (Inouhe 2005).

As GSH contains three moles of nitrogen (N) per mole of S, it may be

assumed that GSH biosynthesis is also dependent on the availability of N precursors

and thus N nutrition of plants. However, the sink strength of GSH biosynthesis for N

may be low compared with the other major N sinks such as the synthesis of proteins

or nucleotides (Kopriva and Rennenberg 2004). The GSH biosynthesis is, therefore,

regulated not only by the Cys availability (S, N and carbon metabolism) but also on

the availability of Glutamate (Glu) and Glycine (Gly). Glutamate the primary product

of the GS-GOGAT pathway of N assimilation is produced in photoautotrophic cells

(Gebler et al. 1998). Glycine the C-terminal amino acid of GSH, is an intermediate of

photorespiration in photoautotrophic cells. The availability of Gly hence is also

dependent on N assimilation and the Citric Acid cycle in mitochondria. Thus, GSH

biosynthesis has a tighter relationship with S and N, and also to carbon metabolism

(Kopriva and Rennenberg 2004).

Increasing evidence suggests that mineral-nutrient status of plants plays a

critical role in increasing plant resistance to environmental stress (Marschner 1995,

Vassilev et al. 2005, Hassan et al. 2006, 2008a,b, Anjum et al. 2008a,b, Khan et al.

2008a). The use of S and N fertilizers is a potential option which can be used to

alleviate Cd toxicity. The plant nutrients, S and N are of great importance. Sulfur is an

4

essential macronutrient of plants that plays a vital role in the regulation of plant

growth and development (Ernst et al. 2008). In addition, it is a structural constituent

of several coenzymes and prosthetic groups, such as ferredoxine, which is also

important for N assimilation. Nitrogen is an important component of several

important structural, genetic and metabolic compounds in plant cells. Plant N status is

highly dependent on N fertilization (Hernandez et al. 1997). N is also a major

component of chlorophyll and amino acids, the building blocks of proteins. Increase

in N supply can stimulate plant growth and productivity (Joel et al. 1997), as well as

photosynthetic activity (Makino et al. 1992) through increased amounts of stromal

and thylakoid proteins in leaves (Bungard et al. 1997).

Several studies have established regulatory interactions between assimilatory

sulfate and nitrate reduction in plants (Rennenberg et al. 1979, Reuveny et al. 1980,

Brunold and Suter 1984, Haller et al. 1986, Takahashi and Saito 1996, Abdin et al.

2003, Hawkesford et al. 2006). Most of the reduced S and N in a plant are utilized in

protein synthesis. As the nitrate reductase (NR; EC 1.6.6.1) (Abrol et al. 1976,

Ahmad et al. 1999, Abdin et al. 2003, Abrol 2007) and ATP-sulfiirylase (Brunold

1990, Kopriva et al. 2002, Saito 2004, Anjum et al. 2008b) enzymes catalyze the first

steps of the nitrate and sulfate assimilation pathways, respectively, they can be used

as indicators of the states of regulation of these pathways (Barney and Bush 1985).

Also, the assimilatory pathways of these elements have been considered convergent

and well coordinated (Brunold 1993, Abdin et al. 2003, Hawkesford et al. 2006). A

positive role of sulfate in regulating nitrate reductase was found by Pal et al. (1976)

and Friedrich and Schrader (1978), and role of N in the regulation of sulfate

assimilation at the ATP-sulfurylase step was observed by Smith (1975). Absence of

readily available external N source decreases ATP-sulfurylase activity (Brunold and

Suter 1984). The interrelationship of the regulation of nitrate and sulfate assimilation

has been found to be an effective mechanism to coordinate and meet the requirement

of net protein synthesis (Reuveny et al. 1980). It has also been established through

genetic studies that sulfate reduction is regulated by N nutrition at the transcriptional

level (Koprivova et al. 2000).

The oleiferous Brassica is the third most important source of vegetable oil in

the world after palm and soybean oil and grown as an edible or an industrial oil crop

which is used as a source of edible protein, in much the same way as soybean protein

(Zhang et al. 2003). Brassica is the Latin name of a genus that is taxonomically

5

placed within the Brassicaceae (Cruciferae), which is one of the tenth most

economically important plant families in the world. The major mustard oil-producing

countries include Canada, China, France, Germany, India and UK. According to a

report of United States Department of Agriculture (USDA), the world oilseed

production was 397metric tons in 2006-07. Indian agriculture contributes about 15%

and 8% to the world total acreage under oilseed cultivation and production,

respectively. However, the productivity in India is only 791kg ha"' as compared to the

world average of 1718kg ha'' (Damodaran and Hedge 2002). About 90% of the total

land under oilseed cultivation in India is occupied by Brassica juncea (Khan et al.

2007b). Despite a large area under cultivation of mustard, the productivity of the crop

has dropped because plant growth and development are affected by heavy metals

introduced into soil by anthropogenic activities.

Discussed the importance of S and N in plant metabolism and their role in

stress tolerance it was assumed that mustard {Brassica juncea L. Czem & Coss.) may

serve as a model crop in the study of the influence of S and N on plant metabolism

under Cd stress. Further, the fertilization of crop with balanced S and N may prove

fruitful in alleviating Cd stress and improving crop productivity. Studies on crops

with a view of alleviating Cd stress with balanced S and N application have not been

carried out. Therefore, the reported research was undertaken with the following

objectives:

• To screen and select mustard cuhivars as Cd tolerant and Cd non-tolerant

grown vmder Cd treatments.

• To study the influence of S application in the alleviation of Cd-induced effects

in Cd tolerant and Cd non-tolerant cultivars of mustard.

• To study the influence of combined application of S and N in the alleviation of

Cd-induced effects in Cd tolerant and Cd non-tolerant cultivars of mustard.

REVIEW OF LITERATURE

Contents

REVIEW OF LITERATURE

2.1 2.2 2.2.1 2.2.2 2.3 2.4 2.4.1 2.5 2.5.1 2.5.1.1 2.5.1.2 2.5.2 2.6 2.7 2.8 2.9 2.9.1 2.9.2 2.10 2.10.1 2.10.1.1 2.10.1.2 2.10.1.3 2.10.1.4 2.10.1.5 2.10.1.6 2.10.1.7 2.10.1.8 2.10.2 2.10.2.1 2.10.2.2 2.11

2.12 2.13 2.14

Physical and Chemical Characteristics of Cd Sources of Cd Contamination in Soil Natural sources Anthropogenic sources Uptake, Accumulation, Transport and Localization of Cd Morphological Alterations under Cd Stress Plant growth Physiochemical Alterations under Cd Stress Photosynthetic pigments Chlorophyll Carotenoids Photosynthesis Sulfur Metabolism imder Cd Stress Nitrogen Metabolism under Cd Stress Yield Characteristics under Cd Stress Cadmium-induced Oxidative Stress Lipid peroxidation Hydrogen peroxide content Cadmium-tolerance Mechanism in Plants Enzymatic antioxidant system Superoxide dismutases Catalase Ascorbate peroxidase Glutathione reductase Monodehydroascorbate reductase Glutathione S-transferase Glutathione peroxidase Guaiacol peroxidase Non-enzymatic antioxidant system Ascorbic acid Glutathione Coordination of Sulfur and Nitrogen in Plant Growth and Productivity Coordination of Sulfur and Nitrogen under Environmental Stress Research Work Reported from Our Laboratory Critical Appraisal of Review of Literature

7 8 8 9 9

12 12 14 14 14 15 16 18 20 21 23 25 26 27 28 28 30 31 32 33 34 34 34 35 35 36

38 39 42 46

REVIEW OF LITERATURE Chapter 2

Heavy metal contamination is a major environmental problem worldwide.

Environmental pollution by metals increased with the increase in mining and

industrial activities in the late 19* and early 20'*' century. The current worldwide mine

production of Cu, Cd, Pb and Hg is considerably large (Pinto et al. 2004). They are

released into the environment through various means including natural and

anthropogenic sources. The metal pollutants, derived from a growing number of

diverse anthropogenic sources, have had enormous impact on different ecosystems

(Macfarlane and Burchett 2001). In fact, heavy metals enlist a relatively large series

of elements with specific density over 5g cm" and relative atomic mass above 40.

Many of these metals manifest high affinity for sulfur-containing ligands and strongly

bind to the latter.

Fifty-three of the ninety naturally occurring elements are heavy metals (Weast

1984). Of these, Fe, Mo and Mn are important as micronutrients, while Zn, Ni, Cu,

Co, Cr and Va are toxic element, but have importance as trace elements. On the other

hand, Ag, As, Cd, Hg, Pb and Sb have no known function as nutrients, and seem to be

toxic to plants and microorganisms (Nies 1999). The presence of heavy metals in the

atmosphere, soil and water in excess amount, can cause serious problems to all

organisms. Among heavy metals, Cd is considered as an extremely potent pollutant

affecting all life forms due to its high toxicity, long half life and greater solubility in

water (Pinto et al. 2004). It is readily taken up by plants from soil via the root system

(Sharma and Dubey 2006). The level of Cd in soil is continuously increasing over

time (Jones et al. 1992). Soils contaminated with Cd show a sharp decline in crop

productivity and therefore, Cd serves as a serious problem for agriculture (Shah and

Dubey 1997).

2.1 Physical and Chemical Characteristics of Cd

Pure cadmium is a soft, silver-white, malleable, ductile and flexible metal. It is not

usually found in the environment as a metal, but occurs normally as a mineral

combined with other elements such as oxygen (cadmium oxide), chlorine (cadmium

chloride) or sulfur (cadmium sulfate, cadmium sulfide).

Atomic number

Relative atomic mass ('^C=12.000)

Melting point/°C

Boiling point/°C

Density/g cm'

Electron affinity (M-M-)/kJ mol"'

48

112.41

321

765

8.64

+26

Source: Chatterjee and Dube (2006)

Cadmium is the second member of Class II Group b of the Periodic Table. The

stable state of Cd in the natural environment is Cd" . It has a medium Class b

character compared to Zn and Hg. This imparts moderate covalency in bonds and high

affinity for sulfhydryl groups, leading to increased lipid solubility, bioaccumulation

and toxicity.

2.2 Sources of Cd Contamination in Soil

Some of the heavy metals are abundant in serpentine soils (Ni, Cr, Co etc.), some

others in calamine soils (Zn, Pb, Cd etc.). As they cannot be degraded or destroyed,

they are persistent in all parts of the environment. Humans also affect the natural,

geological and biological redistribution of heavy metals by altering the chemical form

of heavy metals released into the environment. Among heavy metals, Cd inputs have

also natural as well as anthropogenic sources. Cadmium is naturally occurring

metallic element, one of the components of earth's crust and present everywhere in

the environment (Chatterjee and Dube 2006). Different types of sources are given

below.

2.2.1 Natural sources

The natural occurrence of Cd in the environment results mainly from gradual

phenomena, such as rock erosion/abrasion, that estimates for 15,000mt Cd per annum

(OECD 1994, WHO 1992). Natural calamities such as volcanic eruptions, add 820mt

Cd per annum to the environment (Nriagu 1980, 1989, OECD 1994, WHO 1992). The

naturally existing concentration of Cd in the atmosphere is 0.1-0.5ng m"''. In the

earth's crust, its concentration varies from 0.1 to 0.5|ag g"', but there are reports of a

much higher accumulation in the sedimentary rocks and marine phosphates.

Phosphorites have been reported to contain Cd levels as high as a SOOppm (Cook and

Morrow 1995). Forest fires are also regarded as a natural source of Cd emissions in

the air that range from l-70mt per annum (Nriagu 1990).

8

Atmosphere Earth's crust Marine sediment Sea-water

0.1 toSngm"^* O.ltoO.S^gg"'

- l^gg' ' ~o.l^g^'

* no — ng - nanograms = 10" g

Source: McLaughlin et al. (2000), Chaney and Homick (1978)

2.2.2 Anthropogenic sources

Cadmium contamination of soils may be categorized as i) inputs to agricultural soils,

ii) inputs to non-agricultural soils, and iii) depositions in controlled landfills. In the

first case, the main chunk of Cd comes from atmospheric deposition, sewage sludge

application, and phosphate fertilizers application (Jensen and Bro-Rasmussen 1992,

Van Assche and Ciarletta 1993, Chen et al. 2007). In the second case, inputs arise

mainly from the iron and steel industry, non-ferrous metals production, fossil fuel

combustion, and cement manufacture (ERL 1990, Jackson and MacGillivray 1993,

OECD 1994). In fact, Cd is discharged into the soil to the tune of 22,000ton per year

globally (Nriagu and Pacyna 1988). In the case of controlled landfills, presence of Cd

is mainly due to the disposal of products, which may contain Cd impurities, and

naturally-occurring wastes such as grass, food and soil which inherently contain trace

levels of Cd (Chandler 1996).

2.3 Uptake, Accumulation, Transport and Localization of Cd

Cadmium is taken up by plants from soil \ia the root system and to a lesser extent

through leaves. As soon as Cd enters the roots, it can reach the xylem through an

apoplastic and/or symplastic pathway. Plants show a differing metal distribution and

accumulation pattern among different parts. Most of Cd that enters the plant system

accumulates in the roots and only a small portion is translocated to the above ground

parts (Schutzendubel et al. 2001, Vitoria et al. 2001, Pereira et al. 2002, Ramos et al.

2002, Patel et al. 2005, Kovacik et al. 2006, Sharma and Agrawal 2006, Mobin and

Khan 2007, Ekmekci et al. 2008, Liu et al. 2007, Singh et al. 2008a,b). The actual

accumulation of Cd in plant parts depends on the plant species and soil properties

(Fediuc and Erdei 2002, Arao et al. 2003). Substantial variability among 99 Pisum

sativum genotypes in tolerance to Cd and uptake of different heavy metals was

reported by Belimov et al. (2003). Zhang et al. (2002) reported significant differences

among Triticum aestivum genotypes in shoot Cd concentration. Significant

differences in Cd accumulation and tolerance were found in Sedum alfredii

populations (Deng et al. 2007). Ten times higtier Cd accumulation in the roots than

above ground parts was reported in Hordeum vulgare plants by Vassilev et al. (1998).

Cadmium was accumulated 1826 times more in the roots of Allium sativum than the

control with the application of lO' M Cd and very low amount was transported to the

bulbs and shoots (Jiang et al. 2001). Kovacik et al. (2006) reported that Cd

accumulation was seven- (60^M Cd) to eleven- (120^M Cd) fold higher in the roots

than in the leaves of Matricaria chamomilla, whereas, only 6% of Cd was

accumulated in the leaves of Crotalaria juncea compared to roots with 2mM CdCb

(Pereira et al. 2002). The Milyang 23 rice accumulated 10-15% of the total soil Cd in

its shoot (Murakami et al. 2007). Djebali et al. (2008) reported that the roots of Cd

treated Solarium lycopersicum plants accumulated four to five fold Cd as much as

mature leaves. Liu et al. (2007) suggested that the root tissue was a barrier to Cd

uptake and translocation within rice plants. Retention of Cd in roots might be due to

its cross linking with carboxyl groups of cell wall proteins (Barcelo and Poschenreider

1990) and/or an interaction with the thiol groups of soluble proteins and non-protein

thiols operating as a tolerance mechanism in root cells (Chaoui et al. 1997a). Low

temperature inhibits the uptake of Cd as well as its transport across plasma membrane

(Hart era/. 1998).

The uptake of Cd by plants varies not only among plant species but also

among cultivars (Salt et al. 1995, Arao et al. 2003, Metwally et al. 2005, Grant et al.

2008). Li et al. (1997) reported significant variation in the grain Cd level of

Helianthus annuus, Triticum aestivum and Linum usitatissimum. Arao et al. (2003)

found that lower level of Cd in the seeds of certain soybean varieties was due to the

lower initial uptake. Studies of Cd uptake by plants have indicated that at lower Cd

concentrations (2.5-90nM), its transport across membrane is an active, energy

requiring H" -ATPase mediated process, whereas at high concentrations of Cd the

uptake is a non metabolic (passive) process, involving diffusion coupled with

sequestration (Grant et al. 1998). Although the mechanism of transport of Cd across

the plasmalemma of root cells is not well understood, the electrochemical potential

across the membrane appears to be an important factor (Grant et al. 1998). It is

suggested that the uptake of cationic solutes is likely to be driven largely by the

negative membrane potential across the plasma membrane, which is generated in part

by metabolically dependent processes such as proton extrusion via the plasma

membrane H''-ATPase (Kochian 1991). Following uptake by plant roots, Cd gets

10

accumulated in cytosol, cytosolic organelles and vacuoles. At low level of exposure,

Cd forms complexes in the cytosol with glutathione whereas, at higher Cd exposure

levels, it is transported into the vacuoles, where it forms complexes with organic acids

and phytochelatins (Grant et al. 1998). hi most of the plant species much of the Cd

taken up by plants is retained in root and its translocation to aerial portion is low. In

Glycine max plants, about 98% of the accumulated Cd is strongly retained by roots

and only 2% is transported to the shoot system (Cataldo et al. 1983). Rice plants

absorb Cd from the rooting medium against the concentration gradient and the

localization of absorbed Cd in rice is greater in roots than in shoots. When rice

seedlings were raised in sand cultures for 20d in nutrient medium containing 500|iM

Cd, localization of absorbed Cd was about 3 times in roots compared to its level in

shoots (Shah and Dubey 1998). Movement of Cd from root to shoot is also likely to

occur via the xylem and is driven by the transpiration stream from the leaves that

include metal uploading into root xylem cells, long distance transport from root to

shoot within xylem and reabsorption of metal from the xylem stream by leaf

mesophyll cells (Raskin and Ensley 2000). Evidence for this was provided by Salt et

al. (1995) who showed that ABA-induced stomatal closure reduced Cd accumulation

in shoots of Brassicajuncea. Reduced movement of Cd from roots to shoots in plants

is believed to result from barrier fimction of root endodermis and mechanism

involving sequestration and decreased xylem loading of Cd (Hart et al. 1998). The

casparian strips of root endodermis retard the entrance of Cd into the central cylinder

(Seregin et al. 2004), vascular compartmentation of Cd tends to limit symplastic

movement of Cd. Movement of Cd across the tonoplast occurs via Cd^VH' -antiport

system (Salt and Wagner 1993) as well as by phytochelatin-Cd transporter (Vogeli-

Lange and Wagner 1990) that appears to be Mg-ATP dependent (Salt and Rauser

1995). The absorption of Cd by green microalgae, Chlorella vulgaris, Ankistrodesmus

braunii and Eremosphera viridis revealed that Cd is mainly absorbed in the cell wall.

The binding sites seem to be an acidic group in the cell wall structure (Geisweid and

Urbach 1983). In Eichhornia crassipes, Cd was found to accumulate throughout the

roots (Hosayama et al. 1994). Ammar et al. (2007) reported that Cd was found to be

mainly accumulated in roots, but a severe inhibition of biomass production occurred

in Lycopersicon esculentum leaves, even at its low concentration (l|uM). In Solidago

altissima, Cd was found in most parts of the plant, but located mainly in the cambium,

cortex and phloem tissues (Hosayama et al. 1994).

11

Most of the Cd accumulates in the vacuole within a cell. The fact that Cd is

found in golgi apparatus and endoplasmic reticulum is apparently related to metal

secretion through the cell surface and into the vacuole. A small quantity of Cd reaches

nuclei, chloroplast, and mitochondria and exerts toxic effects on these organelles

(Miller et al. 1973, Malik et al. 1992a). Localization of Cd in different parts of a plant

appears to be maximum in roots (Hart et al. 1998).

2.4 Morphological Alterations under Cd Stress

2.4.1 Plant growth

The growth of whole plant or of plant parts is frequently used as an easily

measurable parameter to monitor the effects of various stressors. The root system

of plants acts usually as the first barrier or acceptor to heavy metals in soil.

Reduction in growth and biomass yield with increased levels of Cd in growth

media arises because of altered physiological phenomena (Demirevska-Kepova

et al. 2006). The most characteristic symptoms of Cd stress are brown and short

roots, chlorosis, fewer tillers, senescence and reduced plant growth and biomass

(Arduini et al. 1994, Wu and Zhang 2002b, Wu et al. 2003, Cosio et al. 2006). In

Elodea canadensis, a thiimer stem, less expanded leaves with partial bleaching of

green tissues and 40% internode shortening were observed in response Cd

treatments when compared with control plants (Vecchia et al. 2005). Bachir et

al. (2004) found decrease in root length, plant height and fruiting branch number

in cotton with increasing Cd concentration in a pot experiment. Cadmium

treatments inhibited the shoot and root growth and dry weight of two wheat

cultivars, C-1252 and Balcali-85. The decrease was more distinct in C-1252

(Ozturk et al. 2003). Sandalio et al. (2001) reported significant reduction in root

grov^h in Pisum sativum plants. Roots of Pisum sativum plants were more

sensitive to Cd toxicity than shoot (Metwally et al. 2005). Increasing

concentration of Cd in liquid culture and pot experiment decreased the

germination and root growth of carrot and radish plants (Chen et al. 2003). Rai et

al. (2005) observed significant decrease in root and shoot length and fresh and

dry weight of Phyllanthus amarus under Cd stress. Cadmium inhibited root dry

mass and induced changes in biomass allocation pattern without any effect on

biomass accumulation at the whole plant level in Hordeum vulgare (Vassilev et

al. 2004). Leaf expansion and root growth were inhibited significantly at high Cd

concentrations in Sedum alfredii, and Cd was suggested to suppress cell

12

expansion and induced senescence (Zhou and Qiu 2005). Exposure to increasing

Cd concentrations reduced the fresh weight of the upper part

(hypocotyis+cotyledons) of the seedlings of Brassica napus more strongly than

that of the root system, which was accompanied by higher Cd accumulation in

these tissues (Filek et al. 2007). Ekmekci et al. (2008) reported that increasing Cd

concentration significantly reduced the leaf and root dry weight of Zea mays cultivars.

Prince et al. (2002) reported significant decrease in plant height, number of

leaves and leaf yield of Cd exposed Morus alba plants. Root and shoot growth of

Crotalaria juncea seedlings was strongly inhibited by Cd concentrations above

0.2mM CdCb (Pereira et al. 2002). Di Cagno et al. (1999) observed Cd-induced

inhibition of growth parameters in Halianthus annuus seedlings in both mature

and young leaves after 7d of exposure. The presence of Cd decreased the

seedling growth of Zea mays, Triticum aestivum, Cucumis sativus and Sorghum

bicolor in terms of root and shoot growth (An 2004). Ghnaya et al. (2005)

reported that Cd severely inhibited Mesembryanthemum crystallinum growth

even at low concentration. Cadmium caused gradual decrease in plant growth of

Lemna minor (Razinger et al. 2007). Patel et al. (2005) observed Cd-induced

decrease in dry matter production of Colocassia esculentum plants. Increasing Cd

concentration in the solution significantly decreased the shoot and root dry

weight of two selected Brassica napus and Brassica juncea plants (Su and Wong

2004). Wahid et al. (2007) observed increased shoot Cd accumulation and leaf

chlorosis with a concomitant reduction in shoot dry weight, leaf area, relative

growth rate, net assimilation rate and relative leaf expansion rate in Vigna

radiata seedlings under Cd stress. Krantev et al. (2007) reported that exposure of

Zea mays plants to Cd caused a gradual decrease in the shoot and root dry weight

accumulation. Ahsan et al. (2007) reported that increasing Cd concentration in

the medium significantly decreased the germination rate, shoot elongation and

biomass of Oryza sativa. Kuriakose and Prasad (2008) reported that at

concentrations above 3mM Cd, seed germination of Sorghum bicolor was

adversely affected with a complete cessation of seedling growth.

The over all reduction in growth has been attributed to repression of the

elongation and the growth rates of cells because of an irreversible inhibition

exerted by Cd on proton pump (Aidid and Okamoto 1993). However, slight

increase in the growth was noticed when the plants were exposed to very low Cd

13

concentrations. Stimulatory effect of lower doses of Cd on growth has also been

observed in Lycopersicon esculentum and Solanum melongena (Khan and Khan

1983), Beta vulgaris (Greger and Lindberg 1986), Medicago saliva seedlings

(Peralta et al. 2000), Hordeum vulgare (Wu et al. 2003), Miscanthus sinensis

(Arduini et al. 2004) and Lycopersicon esculentum seedlings (Dong et al. 2005).

2.5 Physiochemical Alterations under Cd Stress

2.5.1 Photosynthetic pigments

Chlorophyll (Chi) and carotenoids (Car) are the most abundant biological pigments in

plants (Hall and Rao 1999). The presence of these pigments in plants reflects the

nutritional status, growth as well as crop productivity (Seyyedi et al. 1999). Chlorosis

and retardation of plant growth that is frequently observed in metal polluted

enviroimient indicate that an impairment of photosynthetic pigment biosynthetic

pathway is among the earlier targets of heavy metal influence on plant metabolism.

Lower content of photosynthetic pigments induces changes in plastid development,

photosynthetic efficiency as well as in general metabolism. Better is the status of

these pigments, better will be the light harvesting capability and the capability of

plants to fix CO2.

2.5.1.1 Chlorophyll

Chlorophylls play a fundamental role in the process of photosynthesis because of its

ability to absorb light. Baszynski et al. (1980) reported about the negative effect of Cd

on Lycopersicon esculentum plants. Lately, Cd-induced decrease in Chi content was

observed in Triticum aestivum and Cucumis sativus (Buszek 1984, Malik et al.

1992a,b), Zea mays (El-Enany 1995, Stiborova et al. 1986, Krantev et al. 2007,

Ekmekci et al. 2008), Phaeolus vulgaris (Barcelo et al. 1988a,b, Siedlecka and Krupa

1996), Hordeum vulgare genotypes (Wu et al. 2003), Oryza sativa (Kuo and Kao

2004), Glycine max seedlings (Drazic et al. 2004), Matricaria chamomilla (Kovacik

et al. 2006), Brassica juncea (Mobin and Khan 2007), Bechmeria nivea (Liu et al.

IQQl), Brassica campestris (Anjum et al. 2008a) and Vigna mungo (Singh et al.

2008a). Following Cd treatment concentration of chlorophyll a (Chi a) is reduced

more than chlorophyll b (Chi b) in Phyllanthus amarus (Rai et al. 2005), Brassica

juncea plants (Mobin and Khan 2007) and Vigna mungo (Singh et al. 2008a). The

increased Chi a:b ratio was linked with the change in pigment composition of

photosynthetic apparatus which posses lower level of light harvesting Chi proteins

(Loggini et al. 1999). Skorzynska-Polit and Baszynski (1997) reported that Cd-

14

induced necrosis of leaf tissues might be the reason of Cd mobilization and its

transport to above ground plant parts.

Most researchers connect the reduction of Chi in Cd-treated plants with

inhibition of its biosynthesis. Cadmium-induced inhibition of Chi biosynthesis was

even suggested to be a primary event as compared to the inhibition of photosynthesis

(Baszynski et al. 1980). Stobart et al. (1985) established that Cd inhibited Chi

biosynthesis at two levels: in the synthesis of 5-aminolaevulinic acid (ALA) and in

the formation of photoactive protochlorophyllide reductase complex. Horvath et al.

(1996) reported that the photoconversion of protochlorophyllide was not inhibited, but

Cd disturbed Chi molecules integration in stable complexes. On the other hand,

Greger and Lindberg (1986) suggested that the lower Chi concentrations in plants

were a result of the deficiency of Mg and Fe in the leaves of Cd-treated Beta vulgaris

plants. The decrease in Mg and Fe content in leaves as a response to Cd treatment has

been established in other plant species also (Breckle and Kahle 1992, Rubio et al.

1994). More convincing data about the interactions between Fe deficiency (this

element is a co-factor of an enzyme taking a part in Chi biosynthesis) and Chi

concentrations were reported by Krupa and Baszynski (1995). They showed that

50^M Cd induced Fe deficiency and decreased Chi by 55% in bean plants. Lang et al.

(1995) confirmed that translocation of the labeled Fe to the over-ground organs of Cd-

treated Cucumis sativus plants was inhibited and decreased Chi. Somashekaraiah et

al. (1992) showed that treatment of 100|aM Cd for 6d increased the lipoxygenase

activity and decreased Chi concentration in Phaseolus vulgaris plants.

2.5.1.2 Carotenoids

Carotenoids carry out three major functions in plants. First, they absorb light at

wavelength between 400 and 550nm and transfer it to the Chi (an accessory light-

harvesting role) (Siefermann-Harms 1987). Second, they protect the photosynthetic

apparatus by quenching a triplet sensitizer (ChP), singlet oxygen and other harmful

free radicals which are naturally formed during photosynthesis (an antioxidant

ftmction) (Oelmuller 1989, Thiele et al. 1996, Havaux et al. 2000, Collins 2001).

Third, they are important for the photosystem (PS) 1 assembly and the stability of

light harvesting complex proteins as well as thylakoid membrane stabilization (a

structural role) (Mayfield and Taylor 1984, Siefermann-Harms 1987, Niyogi et al.

2001).

15

The influence of Cd on Car has been investigated in different plants.

Cadmium-induced decrease in Car content in the seedlings of Raphanus sativus, Ulva

lactuca, Triticum aestivum and Oryza sativa was reported by Naguib et al. (1982).

Baszynski et al. (1980) observed Cd induced decrease in Car content in Lycopersicon

esculentum. The toxic effect of Cd was partially reversed by the addition of Mn;

however, the Car content never reached the value of control plants. Chi and Car

contents were increased initially and decreased thereafter in Zea mays leaves under

Cd stress (Prochazkova et al. 2001). Baryla et al. (2001) reported reduced Car content

in Brassica napus plants imder Cd stress. Rai et al. (2005) and Singh et al. (2008a)

reported decreased Car and Chi contents in Phyllanthus amarus and Vigna mungo

plants with increasing Cd concentration, respectively. In Brassica napus, Cd reduced

the total Chi and Car contents and increased the non-photochemical quenching

(Larsson et al. 1998). Ekmekci et al. (2008) reported that the increase in Cd

concentration caused loss of Car in Zea mays cultivars. Collin et al. (2008) also

reported decreased concentration of Car in Arabidopsis plants. Car content of

Hordeum vulgare seedlings decreased under Cd-stress (Demirevska-Kepova et al.

2006) or remained less affected (Clijsters and Van Assche 1985). An increase in Car

content was also reported following Cd stress (Foyer and Harbinson 1994).

2.5.2 Photosynthesis

Cadmium has been shown to affect the photosynthetic functions through interacting

with photosynthetic apparatus at various levels of organization and architecture viz.,

accumulation of metal in leaf (main photosynthetic organ), partitioning in leaf tissues

like stomata, mesophyll and bundle sheath cells, interaction with cytosolic enzymes

and alteration of the functions of chloroplast membranes. Cadmium has been shown

to be the most effective inhibitor of photosynthetic activity (Bazzaz et al. 1974,

Huang et al. 1974, Hampp et al. 1976), particularly the oxygen evolving reactions of

photosystem II (Bazzaz and Govindjee 1974, Baszynski et al. 1980, Atal et al. 1991).

With only small amount of Cd in chloroplasts, many direct and indirect effects are

observed, resulting in strong inhibition of photosynthesis. Earlier investigations have

demonstrated a marked reduction in the rate of photosynthesis by Cd in different plant

species (Baszynski et al. 1980, Sawhney et al. 1990, Sheoran et al. 1990a,b, Chugh

and Sawhney 1999, Arduini et al. 2004, Wojcik and Tukiendorf 2005, Jing et al.

2005, Mobin and Khan 2007, Singh et al. 2008a, Khan et al. 2006, 2007a, Anjum et

al. 2008a). Deleterious effects of Cd on various facets of photosynthesis, such as Chi

16

metabolism (Padmaja et al. 1990, Stobart et al. 1985, Sandalio et al. 2001, Chaffei et

al. 2004, Hsu and Kao 2004, Vassilev et al. 2005), functioning of photochemical

reactions (Li and Miles 1975, Skorzynska-Polit and Baszynski 1995) and the activities

of the Calvin cycle enzymes (Ascencio and Cedeno-Maldonado 1979, Weigel 1985,

Krupa 1999) have also been reported. Baszynski et al. (1980) observed that the

decline in chlorophyll content preceded that of CO2 fixation and proposed this to be

the primary cause of diminished photosynthetic activity. Cadmium-induced reduction

in the activity of ribulose 1,5 bisphosphate carboxylase (Rubisco) has been reported in

Hordeum vulgare (Stiborova et al. 1986, Vassilev et al. 2004), Cajanus cajan

(Sheoran et al. 1990b), Triticum aestivum (Malik et al. 1992b), Pisum sativum

seedlings (Chugh and Sawhney 1999), Zea mays (Krantev et al. 2007) and Brassica

juncea (Mobin and Khan 2007). Wahid et al. (2007) reported Cd-induced reduction in

transpiration rate, stomatal conductance and net photosynthesis due to reduced CO2

fixation by Rubisco in Vigna radiata plants. Di Cagno et al. (2001) also observed

significant reduction in CO2 fixation rate and Rubisco activity while stomatal

conductance and Fv:Fm ratio remain unchanged in Cd treated sunflower plants.

Carbonic anhydrase (CA), a zinc metalloenzyme related to photosynthesis in higher

plants (Khan 1994), was significantly reduced by Cd in Brassica juncea (Mobin and

Khan 2007) and Vigna mungo (Singh et al. 2008a). The loss of water from crop plants

is controlled mainly by the stomatal movement on leaves that has been shown to be

sensitive to various environmental factors including Cd stress (Barcelo et al. 1986a,b).

Stomatal closure to minimize water loss has been identified as an early event in plant

response to Cd-induced water deficiency leading to limitations in carbon uptake by

leaves (Barcelo et al. 1986a,b, Chaves 1991, Poschenreider et al. 1989). In addition,

conductance and index of stomata, transpiration and net CO2 uptake are greatly

reduced with elevated Cd levels in the growth media (Bindhu and Bera 2001,

Balakhnina et al. 2005). Siedlecka and Krupa (1996) found that leaf chlorophyll

content and Hill activity decreased with increased chlorophyllase activity under Cd

stress. Cadmium interacts with the water balance (Costa and Morel 1994) and

damages the photosystem apparatus, in particularly the PSI and PSII (Siedlecka and

Krupa 1996). In Medicago sativa, Becerril et al. (1989) found that Cd inhibited

transpiration and CO2 assimilation in a drastic manner, whereas, in Picea abies

seedlings decreased CO2 assimilation was mainly due to stomatal closure (Schlegel et

al. 1987). Cd-induced reduction in photosynthesis and transpiration was attributed to

17

stomatal closure in Beta vulgaris (Greger and Johansson 1992). Mobin and Khan

(2007) found that the Cd-induced decrease in net photosynthetic rate in non-tolerant

cultivar (RH30) of Brassica juncea was accompanied by an increased transpiration

rate and stomatal conductance but in tolerant cultivar (Varuna), it remained unaltered.

2.6 Sulfur Metabolism under Cd Stress

Plants can manage in a wide range of environmental stress conditions using

appropriate physiological responses. In the case of heavy metals stress, the processes

consist of modulation of the activity of plasma membrane and/or vacuolar transporters

and biosynthesis of intracellular metal-chelators (Clemens 2006). In this context,

sulfur plays a pivotal role because sulfate transporters can mediate the entry of

sulfate-analogues into the cells (Maruyama-Nakashita et al. 2007), and S-containing

compounds like GSH, PCs and metallothioneifis (MTs) often improve the tolerance of

plants to several metals and metalloids through complexation and/or further

sequestration of toxic forms inside cellular vacuoles (Xiang and Oliver 1998, Cobbett

and Goldsbrough 2002, Hall 2002).

Sulfur is an essential macronutrient that plays a vital role in the regulation of

plant growth and development (Ernst 1998, Singh 2004, Anjum et al. 2008b,

Bimbraw 2008, Ernst et al. 2008, Kumaran et al. 2008, Ratti and Giordano 2008). It is

constituent of amino acids, Cys and Met, GSH and of varieties of other plant

metabolites involved in stress tolerance (Saito 2000, Rausch and Wachter 2005,

Bouranis et al. 2008, Burritt 2008). A significant induction in S assimilation has been

reported in heavy metal-exposed higher plants (Tukiendorf and Rauser 1990).

Exposure of plants to Cd induces enzymes involved in the sulfate assimilation

pathway (Herbette et al. 2006, Khan et al. 2007a). It has been noted that genes

involved in S assimilation pathway are rapidly up-regulated such as Sultrl;l and

Sultr2;l encoding two sulfate transporters which are up-regulated after 2 or 6h of Cd-

treatment and 12-24h after sulfate-depletion (Takahashi et al. 2000, Herbette et al.

2006, Sarry et al. 2006). Nussbaum et al. (1988) reported that Cd accumulation

increased ATP-sulftorylase (ATP-S) and adenosine 5-phosphosulfate reductase (AR)

activities in Zea mays seedlings. Ruegsegger et al. (1990) showed that AR activity is

induced coordinately with glutathione synthetase in Cd-treated Pisum sativum plants.

In fact the capacity of plants to survive in a polluted environment is partially linked to

the efficiency of their reductive sulfate assimilation pathway. Upon heavy metal

stress, some genes involved in the S assimilation pathway are known to be

18

transcriptionally activated, resulting in an elevation of enzymatic activity (Xiang and

Oliver 1988, Schafer et al. 1998, Heiss et al. 1999, Lee and Leustek 1999, Barroso et

al. 2006). The involvement of S in Cd-tolerance mechanism was reported in

Arahidopsis thaliana (Dominguez-Solis et al. 2001, Harada et al. 2002), Brassica

juncea (Zhu et al. 1999a,b), Nicotiana tabacum (Harada et al. 2001), Triticum

aestivum (Khan et al. 2007a) and Brassica campestris (Anjum et al. 2008a).

A good part of S incorporated into organic molecules in plants is located in

thiol (-SH) groups in proteins (cys-residues) or non-protein thiols, GSH (Noji and

Saito 2003, Tausz et al. 2003, De Kok et al. 2005, Anjum et al. 2008b). Sulfur being a

component of PC may play an important role in their synthesis and ultimately in

detoxification of Cd through the formation of Cd-binding peptides (CdBP) (Cobbett

2000a,b, Harada et al. 2002, Cobbett and Goldsbrough 2002). In many plants, PC

synthesis is reported to be activated by heavy metal treatment and a supply of

precursors, including Cys, y-glutamylcysteine and GSH (Zenk 1996, Chen et al. 1997,

Goldsbrough 2000, Xiang et al. 2001, Nocito et al. 2002, 2006). Studies have

demonstrated the critical role of PCs in Cd detoxification and tolerance of plants to

Cd (Kuboi et al. 1987, Howden et al. 1995a,b, Inouhe et al. 2000, Harada et al. 2001,

2002, Ebbs et al. 2002, Srivastava et al. 2004, Wojcik et al. 2005, Zhang et al. 2008).

It has been showed that PC formation was induced in the leaf, stem and root tissues of

Sedum alfredii upon exposure to 400|aM Cd (Zhang et al. 2008). PCs rapidly form a

'low molecular weight' (LMW) complex with Cd (Sanita di Toppi and Gabbrielli

1999). These complexes acquire acid labile S at the tonoplast and form a 'high

molecular weight' (HMW) complex with a higher affinity for Cd ions (Hu et al.

2001). Gupta et al. (2002) reported an increase in Cys and GSH in the shoots of

Arabidopsis and suggested that these changes can be expected to have a positive

effect via transport on the PC/hPC synthesis and/or the other stress responses in roots

in a Cd-contaminated environment.

Moreover, evidence suggests that CdS solids may be formed and coated by

CdBP-Cd complexes after exposure of cultured suspension cells to high levels of Cd

(Rauser 1990) and the co-occurrence of CdS and CdBP-Cd has been thought to play a

role in stabilizing CdBP-Cd complexes (Rauser 1990, Wang and Evangelou 1995). As

CdBP synthesis and CdS solids formation are closely dependent on S metabolism and

increase the need for thiol compounds by cells, there is a need for maintaining high S

19

ions in the roots to sustain a high S-assimilation rate during phytochelatin

biosynthesis to detoxify Cd.

2.7 Nitrogen Metabolism under Cd Stress

The presence of Cd in plants results in many physiological alterations affecting both

nitrogen and carbohydrate metabolism (Greger and Bertell 1992, Hernandez et al.

1997, Chaffei et al. 2003). The nitrate assimilation process consumes about 25% of

energy produced by photosynthesis (Solomonson and Barber 1990). In most of the

plants nitrate reduction takes place in leaves where major part of the reducing power

arises directly from light via ferredoxin. A high rate of CO2 assimilation favours an

efficient N assimilation and vice versa (Ferrario et al. 1998). Cadmium as a potential

inhibitor of photosynthetic process considerably inhibits nitrate assimilation. It has

been shown that enzymes of N metabolism are differentially affected by Cd stress

(Petrovic et al. 1990, Chugh et al. 1992, Singh et al. 1994, Syntichaki et al. 1996,

Boussama et al. 1999). Among various enzymes nitrate reductase (NR) is a key

enzyme in the conversion of nitrate to nitrite, and its sustained activity is crucial to N

assimilation (Srivastava 1992, Gouia et al. 2000, Ghnaya et al. 2005, 2007). Nitrate

reductase and nitrite reductase (NiR) activities are significantly decreased by Cd,

leading to reduced nitrate assimilation by plants (Ferretti et al. 1993, Chaffei et al.

2004, Rai et al. 1998, Khudsar et al. 2001, Balestrasse et al. 2004, Anjana et al. 2006,

Wahid et al. 2007, Wang et al. 2008). Cadmium reduces absorption of nitrate and its

transport from root to shoot by inhibiting the NR activity in shoots (Hernandez et al.

1996). Cadmium treatment can also result in endogenous ammonium increase through

deamination of some free amino acids and other N forms. Chaffei et al. (2003)

demonstrated that Cd treatment produced ammonium accumulation through an

increase in protease activity. Glutamine synthetase (GS) is one of the key enzymes in

the main pathway of ammonium assimilation in higher plants (Lea and Miflin 2004),

and deleterious effects of Cd on its activity have been observed in several species

(Astolfi et al. 2004, Balestrasse et al. 2006a, Chaffei et al. 2004). In contrast,

glutamate dehydrogenase (GDH), an important enzyme in the "shunt" of N

metabolism, was induced under Cd stress, but the physiological role of GDH and the

increase in its activity are still controversial (Hernandez et al. 1997, Astolfi et al.

2004, Chaffei et al. 2004, Lea and Miflin 2004, Balestrasse et al. 2006a, Hsu et al.

2006). Nitrate assimilation also requires carbon skeletons, especially in the form of

2-oxoglutarate, which is produced via the anaplerotic pathway in the cytosol. 2-

20

Table 1. Effect of Cd on activities of enzymes of different metabolic processes. '+' and '- ' signs denote stimulatory and inhibitory effects of Cd on enzymes, respectively.

Chlorophyll Synthesis

Photosynthesis

ALA synthase

ALA dehydratase

NADPH:protochlorophyll -ide oxidoreductase

RUB? Carboxylase

PEP Carboxylase

Carbonic anhydrase

Phaseolus vulgaris Hordeum vulgare Phaseolus vulgaris Hordeum vulgare Triticum aestivum Phaseolus vulgaris Brassica juncea Triticum aestivum Cajanus cajan Zea mays

Vigna radiata Phaseolus vulgaris Cajanus cajan Zea mays

Phaseolus vulgaris Ceratophyllu m demersum Brassica juncea Vigna mungo Triticum aestivum Brassica juncea Cicer arietinum Cicer arietinum

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

Padmaja et al. 1990 Stobart et al. 1985 Padmaja et al. 1990 Stobart et al. 1985 Boddi et al. 1995 Siedlecka et al. 1997 Mobin and Khan 2007 Malik et al. 1992b Sheoran et al. 1990b Krantev et al. 2007 Anjum et al. 2008c Siedlecka et al. 1997 Sheoran et al. 1990b Krantev et al. 2007 Gouia et al. 2003 Aravind and Prasad 2004 Mobin and Khan 2007 Singh et al. 2008a Khan et al. 2007a Hayat et al. 2007 Hasan et al. 2007 Hasan et al. 2008

N metabolism

S metabolism

Nitrate reductase

Glutamine synthatase

Glutamate dehydrogenase ATP-sulfurylase

ATP-sulfurylase, APS reductase, Sulfite reductase ATP-sulfurylase, APS reductase OASTL

Glutamate dehydrogenase, Serine acetyltransferase and 0-acetyl-L-serine (thiol)lyase iS-nitrosoglutathione reductase

Sesamum indicum Vigna radiata Brassica juncea Cicer arietinum Phaseolus vulgaris Pisum sativum Phaseolus vulgaris Triticum aestivum Arabidopsis thaliana

Brassica juncea

Chlamydom o-nas reinhardtii

Pisum sativum

—

—

—

—

—

—

+ +

+ +

+ +

+ +

+ +

+ +

—

Singh et a/. 1994 Wahid et al. 2007 Hayat et al. 2007 Hasan et al. 2008 Gouia et al. 2000 Chugh et al. 1992 Gouia et al. 2000,2003 Khan et al. 2007a Harada et al. 2002

Lee and Leustekl999 Barroso et al. 1999 Dominguez et al. 2003

Barroso et al. 2006

Oxoglutarate is imported into the chloroplasts (Lancien et al. 2000) where it serves as

the NH4 acceptor by the coupled reaction of giutamine synthase and glutamate

synthase under physiological conditions (Oaks 1994, Ouariti et al. 1997, Gouia et al.

2000). Wahid et al. (2007) reported that Cd-induced inhibition of growth was mainly

due to the damaged photosynthetic apparatus and disruption of the coordination

between C and N metabolism in Vigna radiata. A high rate of CO2 assimilation

favours an efficient N assimilation and vice-versa (Ferrario et al. 1998). Nitrate

reductase and giutamine synthetase enzymes activities are sensitive to Cd stress

whereas, NAD-dependent glutamate dehydrogenase (GDH) shows a substantial rise

under the influence of Cd with dramatic build-up of ammonium pool (Gouia et al.

2003, Boussama et al. 1999). The induction of GDH activity by Cd results from de

novo synthesis and/or activation of specific isoenzymes that removes excess

ammonium (Syntichaki et al. 1996). GDH isoenzymes appear to remove in part the

excess of ammonium under Cd toxicity conditions. Under physiological conditions

the incorporation of ammonium into organic compounds occurs mainly via the

GS/GOGAT cycle (Gouia et al. 2000). The most striking change in Cd treated plants

appears to be a rapid decay of GS/Fd-GOGAT and NADH-GOGAT activities and the

accumulation of ammonia (Boussama et al. 1999, Balestrasse et al. 2006a). This

implies a reduced capacity of GS/GOGAT cycle and induced activity of the

alternative mode of ammonium assimilation by NADH-GDH pathway under Cd

treatment.

Upon exposure to Cd, plants often synthesize a set of N-containing

metabolites through N metabolism, such as proline, glutathione and phytochelatins,

which play a significant role in Cd tolerance of plants (Sharma and Dietz 2006).

Accordingly, plants might exhibit a higher Cd tolerance by the maintenance of normal

N metabolism levels under Cd stress (Gussarsson et al. 1996).

2.8 Yield Characteristics under Cd Stress

Stress factors prevent the plants from reaching their fiill genetic potential and limit the

crop productivity worldwide (Mahajan and Tuteja 2005). A comparison of record

yields and average yields for various crop plants indicates that crops mainly attain

only 20% of their genetic potential for yield due to various biotic and abiotic stress

factors. Abiotic stress is in fact the principal cause of crop failure worldwide, dipping

the average yield for most major crops by more than 50% (Boyer 1982, Bray et al.

2000, Mahajan and Tuteja 2005, Khan et al. 2006).

2 ^ l ^ ^ l f ^

Cadmium affects growth and yield through disturbances in several morpho-

physiological processes and nutrient uptake. Reduction in growth and yield with

increased levels of Cd in growth media arises because of increased leaf rolling and

chlorosis of leaf and stem (Ghani and Wahid 2007) and reduced photosynthetic rate

(Chugh and Sawhney 1999). Wahid and Ghani (2007) reported significant reduction

in number of pods per plant and seeds per pod, 100 seed weight, seed yield and

harvest index of Vigna radiata genotypes as a result of Cd toxicity. It has been

suggested that although varietal difference exists, the accumulated Cd is mainly toxic

to the mesophyll tissue, most probably by interfering with the uptake of essential

nutrients, thereby reducing growth and yield at various stages. Cadmium significantly

reduced the number of ear, ear weight, ear length, spikelet number, grains per ear,

1000-grain weight and grain yield of Triticum aestivum cultivars and the decrease was

correlated with the reduced photosynthetic capacity of the cultivars (Khan et al. 2006,

2007a).

It has been shown that Cd stress significantly reduced grain yield, panicle

number per plant, spikelets per panicle, filled spikelet rate and grain weight of Oryza

sativa genotypes and reduction in yield components of rice genotypes was

proportional to grain Cd content (Cheng et al. 2006). Genotypic differences on the

basis of biomass production, yield and yield components were observed in Triticum

aestivum and it was noted that Cd significantly reduced the root and stem biomass and

spikes per plant but the grains per ear and grain weight were not significantly reduced

(Zhang et al. 2002). They found that the inconsistent decrease in number of grains per

ear and single grain weight among wheat genotypes were because of the

compensation among yield components.

Wu et al. (2004) reported reduction in yield of three Gossypium hirsutum

genotypes under Cd stress and found that the reduction in yield was proportional to

Cd accumulation. The Gossypium hirsutum genotype Simian 3 showed higher Cd

concentration and greater decrease in yield than the other two genotypes (Zhongmian

16 and Zhonmian 16-2). Liu et al. (2007) noted great variation among Oryza sativa

cultivars in their tolerance to soil Cd stress with respect to tillering, plant height, leaf

area, dry matter accumulation and grain yield. The relative change in the number of

grains per panicle showed a strong positive correlation with relative change in grain

yield. Of the four grain yield components measured (panicles per pot, grains per

22

panicle, filled grain percentage, weight per grain), the reduction of grains per panicle

was the main cause of grain yield loss under Cd stress.

2.9 Cadmium-induced Oxidative Stress

Plant cells are continuously exposed to various environmental and biotic stresses

which lead to the increased production of reactive oxygen species (ROS). The

responses of plants to these excess ROS have recently been analyzed extensively at

biochemical and molecular levels (Kreps et al. 2002, Gachomo et al. 2003, Kouril et

al. 2003, Rizhsky et al. 2004, Kotchoni and Gachomo 2006). Under normal

physiological conditions, ROS are continuously produced in the chloroplasts,

mitochondria, peroxisomes as byproducts of aerobic metabolic processes like

photosynthesis, respiration and photorespiration. These ROS are scavenged by both

enzymatic and non-enzymatic antioxidant pathways for the maintenance of normal

plant growth (Jimenez et al. 1998, del Rio et al. 2002, Mittler et al. 2004, Davletova

et al. 2005, Asada 2006, Kotchoni and Gachomo 2006, Nathawat et al. IQQl, Mobin

and Khan 2007, Chalapathi Rao and Reddy 2008). The production of ROS through

Cd-exposed oxidative stress is common in a variety of plants species (Sandalio et al.

2001, Ali et al. 2002, Ranieri et al. 2005, Smeets et al. 2005, Singh et al. 2008a,b).

Oxidative stress occurs when there is a serious imbalance in any cell compartment

between the production of ROS and antioxidant defense, leading to significant

physiological challenges (Foyer and Noctor 2000). These excess ROS cause damage

to proteins, lipids, carbohydrates, DNA and ultimately may result in cell death

(Fadzilla et al. 1997, Gueta-Dahan et al. 1997, Fahmy et al. 1998, Mittler et al. 2004,

Foyer and Noctor 2005, Sairam et al. 2005, Shulaev and Oliver 2006, Djebali et al.

2008).

Photosynthesizing plants are naturally prone to oxidative stress because they

have an array of photosensitizing pigments. These pigments produce and consume

oxygen which can easily donate electrons to form ROS. The chlorophyll pigments

associated with the electron transport system are the primary source of singlet oxygen

('O2). It may also arise as a by product of lipoxygenase activity and is highly

destructive, reacting with most biological molecules at near diffusion-controlled rates.

Superoxides, produced by the transport of electron to oxygen, are not compatible with

metabolism and are required to be eliminated by the antioxidative defense system

while recycling of phosphoglycolate to phosphoglycerate (re-enter the Bassam-Calvin

cycle) results in a considerable loss of assimilated carbon.

23

Cadmium has been found to induce oxidative stress in plants (Hendry et al.

1992, Somashekaraiah et al. 1992, Piqueras et al. 1999, Chien et al. 2001, Dixit et al.

2001, Sandalio et al. 2001, Okamoto et al. 2001, Pereira et al. 2002, Milone et al.

2003, Wu et al. 2003, Kuo and Kao 2004, Skorzynska-Polit et al. 2003/04, Liu et al.

2007, Djebali et al. 2008, Singh et al. 2008a,b). But in contrast with other heavy

metals, such as Cu, it does not seem to act directly on the production of ROS through

Fenton type reactions (Salin 1988). Evidence that Cd causes the production of ROS in

plants (Foyer et al. 1997) arised from the observations that new isozymes of

peroxidases were detectable in both root and leaves of Phaseolus vulgaris (Van

Assche and Clijsters 1990). Further evidence of the Cd-induced oxidative stress arised

from the detection of lipid peroxidation, increased lipoxygenase activity, chlorophyll

degradation and inhibition or stimulation of the activity of several antioxidant

enzymes (Padmaja et al. 1990, Stochs and Bagchi 1995, Shaw 1995a,b, Dixit et al.

2001, lannelli et al. 2002, Leon et al. 2002, Skorzynska-Polit et al. 2003/04, Mobin

and Khan 2007, Agrawal and Mishra 2008, Anjum et al. 2008a). Cadmium provokes

significant disturbances in the structural organization and fiinctional activity of

photosynthetic apparatus (Baszynski 1986, Krupa and Baszynski 1995, Vassilev et al.

1995, Dahlin et al. 2000). The main targets of toxic Cd effects are the pigment

apparatus and photosynthetic gas exchange system (Clijsters and Van Assche 1985,

Tukiendrof and Baszynski 1991, Lang etal. 1995).

Heavy metal exposed plants adopt the process of avoidance of the production

of ROS as the first line of defense against oxidative stress. Once formed, ROS must

be detoxified as efficiently as possible to minimize eventual damage. Thus, the

detoxification mechanisms constitute the second line of defense against the

detrimental effects of ROS (Moller 2001). In fact, compounds having the property of

quenching the ROS without undergoing conversion to a destructive radical can be

described as 'antioxidant'. Antioxidant enzymes are considered as those that either

catalyses such reactions, or are involved in the direct processing of ROS (Medici et al.

2004). Hence, antioxidants (enzymatic and non-enzymatic) function to interrupt the

cascades of imcontrolled oxidation (Noctor and Foyer 1998). Though, the expression

for antioxidant enzymes is altered under stress conditions, their up regulation has a

key role in combating the abiotic stress-induced oxidative stress. However, the level

of up regulation is subject to type and magnitude of the stress. Superoxide dismutase

(SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR),

24

monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR),

guaiacol peroxidase (GOPX) and glutathione S-transferase (GST) showed great

variations in their activities depending on the Cd concentration and the plant species

used.

2.9.1 Lipid peroxidation

Photosynthesizing plants are especially at the risk of oxidative damage, because of

their oxygenic conditions and the abundance of the photosensitizers and

polyunsaturated fatty acids in the chloroplast envelope. It is reported that 1% of the

oxygen consumed by plants is diverted to produce activated oxygen species like

hydroxyl radical (0H-), singlet oxygen ('O2) and superoxide radicals (O2") (Asada

and Takahashi 1987). Free radicals and other derivatives of oxygen are inevitable by

products of biological redox reactions. Their production is considered to be a

universal and common feature of living world under natural conditions as a by

product of respiration and photosynthesis during electron transport systems of

mitochondria and chloroplast. Their concentration increases under unfavorable

conditions. Litracellular structures like membranes and biomolecules like proteins,

enzymes, lipids and DNA have a high degree of organization that is at the risk of

being destructed by these oxidative radicals.

Lipids are more prone to oxidative damage. The peroxidation of lipids is

considered as the most damaging process known to occur in every living organism

(Zhang and Kirkham 1996, Hung and Kao 1998). Membrane damage is sometimes

taken as a single parameter to determine the level of lipid destruction (i.e., lipid

peroxidation). Now, it has been recognized that during the lipid peroxidation,

products are formed from polyunsaturated precursors that include small hydrocarbon

fragments such as ketones, malondialdehyde (MDA), etc. and compounds related to

them (de Vos et al. 1993, Weckx and Clijsters 1996). Some of these compounds react

with thiobarbituric acid (TBA) to form coloured products called thiobarbituric acid

reactive substances (TBARS) that can be measured by monitoring their absorption at

around 530nm (Gray 1978).

Plants exposed to heavy metal stress exhibited an increase in lipid

peroxidation due to the generation of free radicals (Van Assche and Clijsters 1990,

Shaw 1995b, Chaoui et al. 1997b, Lozano-Rodriguez et al. 1997, Vanaja et al. 2000).

Increase in TBARS with increasing Cd concentration has been reported in

germinating Phaseolus vulgaris seedlings (Somashekaraiah et al. 1992). This was

25

related to blockage of electron flow in PSII by metal ions that lead to the formation of

excited Chi which in turn causes the production of free radicals (Kato and Shimizu

1985). Treatment with Cd notably increased the accumulation of lipid peroxides in

Phaseolus vulgaris (Chaoui et al. 1997b), Pisum sativum (Lozano-Rodriguez et al.

1997, Dixit et al. 2001, Metwally et al. 2005), Oryza sativa (Chien et al. 2001, Guo et

al. 2007, Ahsan et al. 2007), different Hordeum vulgare genotypes (Wu et al. 2003),

Helianthus annuus (Gallego et al. 1996a,b, Groppa et al. 2001), Arabidopsis seedlings

(Cho and Seo 2005), Lemna minor (Razinger et al. 2007), Brassica juncea (Mobin

and Khan 2007), Glycine max (Balestrasse et al. 2004, Noriega et al. 10()1),

Bechmeria nivea (Liu et al. 2007), Lycopersicon esculentum (Ammar et al. 2007),

Brassica napus (Filek et al. 2007) and Vigna mungo (Singh et al. 2008a). Contrarily,

decreased rate of lipid peroxidation in peroxisomes of Pisum sativum plants has been

reported due to Cd (Romero-Puertas et al. 1999). However, no lipid peroxidation was

observed in the hairy roots of Daucus carota under Cd stress (Sanita di Toppi et al.

1998). Piqueras et al. (1999) reported an increased lipid peroxidation in BY2 cell

cultures of Lycopersicon esculentum plants exposed to 5mM Cd. The increase was

related to a rapid generation of H2O2 followed by an alteration of the antioxidant

system. Dixit et al. (2001) reported an increase in lipid peroxidation in roots and

leaves of Pisum sativum plants exposed to varying range of Cd in hydroponic system.

Okamoto et al. (2001) observed oxidative damage to lipids in isolated chloroplasts of

unicellular alga, Ganyaulax polyendra under Cd stress.

2.9.2 Hydrogen peroxide content

H2O2 is produced at high flux rates by two processes associated with photosynthesis,

the Mehler reaction and the Glycolate oxidase reaction of photorespiration (Foyer and

Noctor 2000). In addition, there are a number of other enzymes in leaves that are

capable of producing significant amounts of H2O2, including peroxidases, NADPH

oxidases and oxalate oxidase (Bema and Bemier 1999, Bolwell et al. 1999, Sagi and

Fluhr2001).

The accumulation of H2O2 after Cd exposure has been detected in the leaf of

different plant species such as Pisum sativum (Dixit et al. 2001, Romero-Puertas et al.

2004), Arabidopsis thaliana (Cho and Seo 2005), Brassica juncea (Mobin and Khan

2007), Vigna mungo (Singh et al. 2008a). The formation of H2O2 after Cd exposure

has also been detected in Solanum tuberosum tuber discs (Stroinski and Zielezinska

1997), suspension cultures of Nicotiana tabacum cells (Piqueras et al. 1999), and

26

Pinus sylvesths and Pisum sativum roots (Schutzendubel et al. 2001, Romero-Puertas

et al. 2003). Balestrasse et al. (2006b) also reported that Cd increased concentrations

and in situ accumulation of H2O2 and O2" in soybean leaves. Exposure to increasing

Cd concentration increased the H2O2 content in the upper part

(hypocotyls+cotyledons) and roots of Brassica napus (Filek et al. 2007). Guo et al.

(2007) reported that exposure to 50mM Cd significantly increased the H2O2 content in

the roots of Oryza sativa. Zawoznik et al. (2007) reported that leaves of wild type

Arabidopsis thaliana plants exposed to Cd showed accumulation of H2O2. It has also

been reported that Cd increased the accumulation of H2O2 in soybean root tips (Yang

era/. 2007).

2.10 Cadmium-tolerance Mechanism in Plants

Plants adopt several strategies such as physically avoiding the metal-contaminated

environment. These include making exudates of complexing agents into rhizosphere

region, binding metal ions in the cell wall, effluxing the metal ions from the

symplasm, preventing the upward transport of metal ions to the above ground parts,

transporting metal-peptide/ligand complexes into the vacuole, storing metal ions in

the vacuoles by complexation with vacuolar peptides/ligands and or forming metal-

resistant enzymes or metabolites to minimize metal-induced severe internal metabolic

injuries (Hall 2002, McGrath and Zhao 2003, Gratao et al. 2005, Zhang et al. 2006).

Plants have evolved a complex array of mechanisms to maintain optimal metal

levels and avoid the detrimental effects of excessively high concentrations (Clemens

2001). When these homeostatic mechanisms are overwhelmed, plants suffer metal-

induced damage and pro-oxidant conditions within cells. However, higher plants are

very well equipped with antioxidant mechanisms (Mittler et al. 2004, Gratao et al.

2006, Singh et al. 2008b). Plant cells display an antioxidant network including

numerous soluble and membrane compounds, particularly in mitochondria and in

chloroplasts where respiratory and photosynthetic electron transfer chains take place.

Antioxidant enzymes are considered as those that either catalyze such reactions, or are

involved in the direct processing of ROS (Medici et al. 2004). Plants possess very

efficient enzymatic (SOD, CAT, APX, GR, MDHAR, DHAR, GPX, GOPX and GST)

and non-enzymatic (AsA, GSH, phenolic compounds, alkaloids, non-protein amino

acids and a-tocopherols) antioxidant defense systems. Components of antioxidant

defense systems control the cascades of uncontrolled oxidation (Noctor and Foyer

1998) and protect plant cells from oxidative damage by scavenging of ROS.

27 ^^^iV-::-^-^

2.10.1 Enzymatic antioxidant system

2.10.1.1 Superoxide dismutase

Superoxide dismutase (SOD; EC 1.15.1.1) was first isolated by Mann and Keilin

(1938) and thought to be a copper-storage protein. Subsequently, it was identified by

different names, erythrocuprein, indophenol oxidase, and tetrazolium oxidase until its

catalytic function was discovered by McCord and Fridovich (1969). SOD catalyzes

the disproportionation of superoxide (02~) to hydrogen peroxide and molecular

oxygen. It removes Or' and hence decreases the risk of hydroxyl radical formation

fi-om 02~ via the metal catalyzed Haber-Weiss-type reaction.

Sensitive to SOD isozymes

Fe-SOD

Mn-SOD

Cu/Zn-SOD

Location

Chloroplast

Mitochondria and

Peroxisomes

Resistant to

KCN

KCN and H2O2

Chloroplast and Cytosol

H202

H2O2 and KCN

There are three distinct types of SOD classified on the basis of the metal

cofactor: the copper/zinc (Cu/Zn-SOD), the manganese (Mn-SOD) and the iron (Fe-

SOD) isozymes have been reported in various plant species (Bannister et al. 1987,

Alscher et al. 2002). These isozymes can be separated by native polyacrylamide gel

electrophoresis. Their activity is detected by negative staining and identified on the

basis of their sensitivity to KCN and H2O2. The Mn-SOD is resistant to both

inhibitors; Cu/Zn-SOD is sensitive to both inhibitors whereas; Fe-SOD is resistant to

KCN and sensitive to H2O2. The subcellular distribution of these isozymes is also

distinctive. The Mn-SOD is found in the mitochondria of eukaryotic cells and in

peroxisomes (del Rio et al. 2003); some Cu/Zn-SOD isozymes are found in the

cytosolic fractions, and also in chloroplasts of higher plants (del Rio et al. 2002). The

Fe-SOD isozymes, often not detected in plants (Ferreira et al. 2002) are usually

associated with the chloroplast compartment when present (Bowler et al. 1992,

Alscher et al. 2002). The prokaryotic Mn-SOD and Fe-SOD, and the eukaryotic

Cu/Zn-SOD enzymes are dimers, whereas Mn-SOD of mitochondria are tetramers

(Scandalios 1993). Peroxisomes and glyoxysomes of Citrillus vulgaris have been

shown to contain both Cu/Zn- and Mn-SOD activity (Sandalio and del Rio 1988), but

there are no reports of extracellular SOD enzymes in plants. All forms of SOD are

28

nuclear-encoded and targeted to their respective subcellular compartments by an

amino terminal targeting sequence. Several forms of SOD have been cloned from a

variety of plants (Scandalios 1990, Bowler et al. 1992). The response of SOD to

heavy metal stress varies considerably depending upon plant species, stage of the

plant development, metal in the experiment and the exposure time. SOD activity in

leaves exhibited increase in response to Cd, whereas, in roots there was no significant

variation in its activity was noted (Vitoria et al. 2001). Activity staining for SOD in

Glycine max revealed seven isozymes in leaves and eight in roots, corresponding to

Mn-SOD and Cu/Zn SOD isozymes. Although a clear effect of Cd on plant growth

was observed, the activities of the SOD isozymes were unaltered (Ferreira et al.

2002). In Saccharum officinarum seedlings several isozymes have been observed, but

growth in the presence of Cd did not result in any significant alteration in SOD

activity (Fomazier et al. 2002a). In Pisum sativum plants, a strong reduction in

chloroplastic and cytosolic Cu/Zn SODs by Cd was reported and to a lesser extent for

Fe-SOD, while Mn-SOD was only affected by the highest Cd concentration tested.

This showed that Mn-SOD was the isozyme more resistant to Cd (Sandalio et al.

2001). In Pisum sativum leaf peroxisomes, the Mn-SOD activity did not change in

response to Cd treatment (Romero-Puertas et al. 1999). Contrarily, increases in total

SOD activity were detected following the application of Cd in Pisum sativum

(Dalurzo et al. 1997), Solarium tuberosum (Stroinski and Kozlowska 1997), Hordeum

vulgare (Guo et al. 2004), Arabidopsis thaliana (Skorzynska-Polit et al. 2003/04),

Oryza sativa (Hsu and Kao 2004), Triticum aestivum (Khan et al. 2007a), Zea mays

(Krantev et al. 2007), Brassica juncea (Mobin and Khan 2007), Vigna mungo (Singh

et al. 2008a), Cicer arietinum (Hasan et al. 2008), Vigna radiata (Anjum et al.

2008c), Brassica campestris (Anjum et al. 2008d) and hyperaccumulator plants of the

genus Alyssum (Schickler and Caspi 1999). SOD activity remained unaltered in

Helianthus annuus (Gallego et al. 1996b, 1999) and declined in Amaranthus lividus

(Bhattacharjee 1998), Phragmites australis (lannelli et al. 2002), Capsicum annuum

plants (Leon et al. 2002), Glycine max (Noriega et al. 2007) under Cd stress. In

clonal, hydroponically grown poplar plants {Populus x canescens, a hybrid of

Populus termula x Populus alba) (Schutzendubel et al. 2002) and Arabidopsis lividus

(Bhattachagee 1998) exposure to Cd resulted in inhibition of SOD activity. Romero-

Puertas et al. (2004) studied the involvement of H2O2 and O2" in the signaling events

29

that lead to the variation of the transcript levels of Cu/Zn-SOD in Pisum sativum

plants under Cd stress.

2.10.1.2 Catalase

Catalase (CAT; EC 1.11.1.6) is a heme-containing enzyme that catalyzes the

dismutation of hydrogen peroxide into water and oxygen (Frugoli et al. 1996). Present

in all aerobic eukaryotes, this is important in the removal of hydrogen peroxide

generated in peroxisomes (microbodies) by oxidases involved in P-oxidation of fatty

acids, the glyoxylate cycle (photorespiration) and purine catabolism. CAT is one of

the first enzymes isolated in a highly purified state. The isozymes of catalase have

been studied extensively in higher plants (Polidoros and Scandalios 1999). Scandalios

et al. (2000) characterized three genetically distinct CAT isozymes in maize plants.

All forms of the enzyme are tetramers in excess of 220,000 molecular weight.

Multiple forms of catalase have been described in many plants. Zea mays has 3

isoforms termed as CAT 1, CAT 2 and CAT 3, and are found on separate

chromosomes and are differentially expressed and independently regulated

(Scandalios 1990). CAT 1 and CAT 2 aie localized in peroxisomes and the cytosol,

whereas CAT 3 is mitochondrial. Plants contain multiple CAT isozymes e.g., 2 in

Hordeum vulgare (Azevedo et al. 1998), 4 in Helianthus annuus cotyledons

(Azpilicueta et al. 2007) and as many as 12 isozymes in Brassica (Frugoli et al.

1996). Catalase isozymes have been shown to be regulated temporally and spatially

and may respond differentially to light (Willekens et al. 1994, Skadsen et al. 1995).

The variable response of CAT activity has been observed under Cd stress.

CAT activity declined in Helianthus annuus leaves (Gallego et al. 1996b), Phaseolus

vulgaris (Chaoui et al. 1997b), Phaseolus aureus (Shaw 1995b), Pisum sativum

(Daliu^o et al. 1997), Lemna minor (Mohan and Hossetti 1997), Amaranthus lividus

(Bhattacharjee 1998), Glycine max roots (Balestrasse et al. 2001), Phragmites

australis (lannelli et al. 2002), Capsicum annuum (Leon et al. 2002) and Arabidopsis

thaliana (Cho and Seo 2005) under Cd stress conditions. A significant decline in CAT

activity was reported after 50|aM Cd applications for 48h in the roots and shoots of

Bacopa monnieri (Singh et al. 2006). However, CAT activity increased in Agropyron

repens (Brej 1998), Helianthus annuus (Gallego et al. 1999), Glycine max nodules

(Balestrasse et al. 2001), Oryza sativa leaves (Hsu and Kao 2004), in tolerant varieties

oiSolanum tuberosum (Stroinski and Kozlowska 1997), in roots of Raphanus sativus

seedlings (Vitoria et al. 2001), Brassica juncea (Mobin and Khan 2007), Triticum

30

aestivum (Khan et al. 2007a), Vigna mungo roots (Singh et al. 2008a), Vigna radiata

(Anjum et al. 2008c), Brassica campestris (Anjum et al. 2008d) and Cicer arietinum

(Hasan et al. 2008). Azpihcueta et al. (2007) reported that incubation of Helianthus

annuus leaf discs with 300 and 500|iM CdCh under light conditions increased CATA3

transcript level but this transcript was not induced by Cd in etiolated plants.

Moreover, in roots of the transgenic CAT-deficient tobacco lines {CAT IAS), the

DNA damage induced by Cd was higher than in wild type tobacco (SR 1) roots

(Gichner et al. 2004). Furthermore, CAT activity remained unaltered under Cd stress

in Glycine max leaves (Ferreira et al. 2002).

2.10.1.3 Ascorbate peroxidase

Ascorbate peroxidase (APX; EC 1.11.1.11) is a heme protein, and its primary function

is the rapid removal of H2O2 at the site of generation (Asada 1992). APX isozymes

are distributed in at least four distinct cell compartments, the stroma (sAPX),

thylakoid membrane (tAPX), the mitochondria (mAPX), and the cytosol (cAPX)

(Asada 1992, Miyake and Asada 1992, Ishikawa et al. 1998). The various isoforms of

APX respond differentially to metabolic and environmental signals (Kubo et al.

1995). Thylakoid membrane-bound APX is limiting factor of antioxidant systems

under photoxidative stress in chloroplasts and the enhanced tAPX activity maintain

the redox status of ascorbate under stress conditions (Yabuta et al. 2002).

Chloroplasts contain APX in two isoforms, thylakoid-bound and soluble stromal

enzymes. At least one half of the chloroplastic APX is tAPX, but the ratio of

tAPX/sAPX varies according to the plant species, possibly, leaf age, but the

biosynthetic ratio of the two APXs is controlled by alternative splicing (Asada 1999).

tAPX bind to the stroma thylakoids where the PSI complex is located, while sAPX is

thought to be localized in the stroma (Asada 1999). Plants also contain the cytosolic

isoforms of APX (cAPX), which has a different amino acid sequence in comparison

to chloroplastic APXs, but participate in the scavenging of H2O2 in compartments

other than chloroplasts. cAPX is a homodimer and its electron donor is not so specific

for ascorbate, unlike tAPX and sAPX (Asada 1999).

APX has an important role in the scavenging of H2O2 under stressed

conditions but its activity depends on the Cd concentration applied. Increased leaf

APX activity under Cd stress has been reported in Ceratophyllum demersum (Aravind

and Prasad 2003), Brassica juncea (Mobin and Khan 2007), Pisum sativum (Romero-

Puertas et al. 1999), Phaseolus aureus (Shaw 1995b), Phaseolus vulgaris (Chaoui et

31

al. 1997b), Zea mays (Krantev et al. 2007), Triticum aestivum (Khan et al. 2007a),

Vigna radiata (Anjum et al. 2008c), Brassica campestris (Anjum et al. 2008d) and

Vigna mungo (Singh et al. 2008a), however, in Hordeum vulgare roots, the APX

activity was reduced at high concentration of Cd (Hegedus et al. 2001). Balestrasse et

al. (2001) reported that low Cd levels led to an increased APX activity in Glycine max

roots and nodules, but the activity decreased with high Cd concentration. The lower

APX activity was also noted in Cucumis sativus chloroplasts with increasing Cd

concentration (Zhang et al. 2003). Whereas, Cd induced inhibition of APX activity

was also observed in clonal, hydroponically grown Populus x canescens.

(Schutzendubel et al. 2002) and Helianthus annuus plants (Gallego et al. 1996b).

APX activity in Ceratophyllum demersum showed a very high increase in its activity

in Cd+Zn treated plants as compared to Cd or Zn alone, indicating efficient

antioxidant and ROS scavenging activities by Zn against Cd induced free radicals and

oxidative stress (Aravind and Prasad 2003). Khan et al. (2008b) also reported

increased APX activity in Triticum aestivum plants treated with Cd under low Zn

levels.

2.10.1.4 Glutathione reductase

Glutathione reductase (GR; EC 1.6.4.2) is a flavo-protein oxidoreductase, found in

both prokaryotes and eukaryotes (Mullineaux and Creissen 1997, Romero-Puertas et

al. 2006). This enzyme was first reported in eukaryotes and yeast (Meldrum and Tarr

1935) as well as in plants (Corm and Vennesland 1951, Mapson and Goddord 1951).

GR maintains the balance between reduced glutathione (GSH) and ascorbate pools,

which in turn maintain cellular redox state (Lascano et al. 1999, 2001, Ansel et al.

2006, Reddy and Raghavendra 2006, Romero-Puertas et al. 2006, Chalapathi Rao and

Reddy 2008). The enzyme protein, although synthesized in the cytoplasm, can be

targeted to both chloroplast and mitochondria (Mullineaux and Creissen 1997). In

higher plants, GR is involved in defense against oxidative stress, whereas, GSH plays

an important role within the cell system, which includes participation in the

Ascorbate-GIutathione cycle, maintenance of the sulfhydryl (-SH) group and a

substrate for glutathione-S-transferases (Noctor et al. 2002, Reddy and Raghavendra

2006). Glutathione reductase and GSH play a crucial role in determining the tolerance

of a plant during environmental stresses (Chalapathi Rao and Reddy 2008). In almost

all the biological functions, GSH is oxidized to GSSG which should be converted

back to GSH in plant cell to perform normal physiological functions. Hence, rapid

32

recycling of GSH is more essential rather than synthesis of GSH, which is a highly

regulated and ATP requiring process. GR activity increases as part of the defense

against Cd-exposure, which is dose-dependent and variable over time (Fomazier et al.

2002a). GR activity increased in the presence of Cd in Phaseolus vulgaris (Chaoui et

al. 1997b), Solarium tuberosum (Stroinski and Kozlowska 1997), Raphanus sativus

(Vitoria et al. 2001), Crotolaria juncea (Pereira et al. 2002), Glycine max (Ferreira et

al. 2002), Saccharum ojficinarum (Fomazier et al. 2002b), Capsicum annuum (Leon

et al. 2002), Arabidopsis thaliana (Skorzynska-Polit et al. 2003/4), Triticum aestivum

(Khan et al. 2007a), Brassica juncea (Mobin and Khan 2007), Brassica campestris

(Anjum et al. 2008d) and Vigna mungo (Singh et al. 2008a). In Raphanus sativus, it

exhibited very little variation in the roots and leaves of control plants, indicating a

direct correlation with Cd accumulation (Vitoria et al. 2001). In Pisum sativum, GR

activity was enhanced more with 40nM than with 4)4,M Cd (Dixit et al. 2001).

However, a decrease in GR activity after application of Cd has also been reported for

a few plant species such as Helianthus annuus (Gallego et al. 1996a,b), Pisum

sativum (Dalurzo et al. 1997) and Solanum tuberosum (Stroinski and Kozlowska

1997).

2.10.1.5 Monodehydroascorbate reductase

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a flavin adenin

dinucleotide (FAD) enzyme that is present as chloroplastic and cytosolic isozymes

which share similar properties. Monodehydroascorbate reductase exhibits a high

specificity for monodehydro asorbate (MDHA) as the electron acceptor, preferring

NADH rather than NADPH as the electron donor. Asada (1999) studied the multi-step

reduction of FAD in detail. The first step is the reduction of the enzyme-FAD to form

a charge transfer complex. The reduced enzyme donates electrons successively to

MDHA, producing two molecules of ascorbate via a semiquinone form [E-FAD-

NADP(P)' ]. The disproportionation by photoreduced ferredoxin (redFd) in the

thylakoids is of great importance. Since redFd (reduced ferredoxin) can reduce

MDHA more effectively than NADP" , MDHAR caimot participate in the reduction of

MDHA in the thylakoidal scavenging system. Therefore, MDHAR would function at

a site where NAD(P)H is available, but redFd is not (Asada 1999).

MDHAR is also located in peroxisomes and mitochondria accompanying

APX. MDHAR could remove and scavenge H2O2 in these organelles similar to that in

chloroplasts, which has escaped from break down by peroxisomal CAT (del Rio et al.

33

2002). Schutzendubel et al. (2001) have noted enhanced MDHAR activity in Cd-

exposed Pinus sylvestris and a declined MDHAR activity in Cd-exposed poplar

hybrids {Populus x canescens).

2.10.1.6 Glutathione S-transferase

Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the conjugation of tripeptide

GSH into a variety of hydrophobic, electrophylic and cytotoxic substrates (Marrs

1996). Noctor et al. (2002) observed that GSTs can remove genotoxic or cytotoxic

compounds that have potential to damage or react with genetic material (DNAs and

RNAs) and protein. In fact, glutathione-S-transferases can reduce peroxides with the

help of GSH and produce scavengers of cytotoxic and genotoxic compounds. An

increased GST activity was found in leaves and roots of Cd-exposed Pisum sativum

plants by Dixit et al. (2001) and in roots of Oryza sativa and Phragmites australis

plants (lannelli et al. 2002, Moons 2003).

2.10.1.7 Glutathione peroxidase

H2O2 can be metabolized by another plant peroxidase-scavenging enzyme called

glutathione peroxidase (GPX; EC 1.11.1.9) (Noctor et al. 2002). Millar et al. (2003)

identified a family of seven related proteins in cytosol, chloroplast, mitochondria and

endoplasmic reticulum, named AtGPXl-AtGPX7 in Arabidopsis. Stress increases

GPX activity in cultivars of Capsicum annuum plants (Leon et al. 2002) but decreases

in roots and causes no significant change in the leaves of Cd-exposed Pisum sativum

plants (Dixit e/a/. 2001).

2.10.1.8 Guaiacol peroxidase

Ascorbate peroxidase (APX) can be distinguished fi-om plant-isolated guaiacol

peroxidase (GPOX; EC 1.11.1.7) in terms of differences in sequences and

physiological fiinctions (Chen et al. 1992). GPOX decomposes indole acetic acids

(lAAs) and has a role in the biosynthesis of lignin and defense against pathogens by

consuming H2O2. GPOX prefers aromatic electron donors such as guaiacol and

pyragallol usually oxidizing ascorbate at the rate of around 1% that of guaiacol

(Asada 1999).

The activity of GPOX varies considerably depending upon plant species and

the concentration of Cd used. It increased in Cd-exposed plants of Triticum aestivum

(Milone et al. 2003), Arabidopsis thaliana (Cho and Seo 2005) and Ceratophyllum

demersum (Aravind and Prasad 2003) and decreased in Cd-exposed Pisum sativum

plants (Sandalio et al. 2001). Radotic et al. (2000) noted an initial increase in GPOX

34

activity in spruce needles subjected to Cd stress and subsequent Cd-treatments caused

a decline in the activity.

2.10.2 Non-enzymatic antioxidant system

2.10.2.1 Ascorbic acid

All plants can synthesize ascorbate, which can accumulate to millimolar

concentrations in both photosynthetic and non-photosynthetic tissues (Foyer et al.

1983). Ascorbate is one of the most powerful antioxidants (Noctor and Foyer 1998,

Smirnoff et al. 2001), reacts directly with hydroxyl radicals, superoxide and singlet

oxygen and reduces H2O2 to water via ascorbate peroxide reaction (Noctor and Foyer

1998). Ascorbate also acts as an electron donor in the regeneration of a-tocopherol.

Under physiological conditions it exists mostly in reduced form in leaves and

chloroplast and its intracellular concentration can build up to millimolar range (viz.,

20mM in the cytosol and 20-300niM in the chloroplast stroma) (Foyer and Lelandais

1996). The ability to donate electrons in a wide range of enzymatic and non-

enzymatic reactions makes ascorbate the main ROS-detoxifying compound in the

aqueous phase. In addition to the importance of ascorbate in the Ascorbate-

Glutathione cycle, it plays a role in preserving the activities of enzymes that contain

prosthetic transition metal ions (Noctor and Foyer 1998). The ascorbate redox system

consists of L-ascorbic acid, MDHA and DHA. Both oxidized forms of ascorbate are

relatively unstable in aqueous environments while DHA can be chemically reduced

by GSH to ascorbate (Foyer and Halliwell 1976). Evidence to support the actual role

of DHAR, GSH and GR in maintaining the foliar ascorbate pool has been observed in

transformed plants overexpressing GR (Foyer et al. 1995). Nicotiana tabacum and

Populus X canescens plants have higher foliar ascorbate contents and improved

tolerance to oxidative stress (Aono et al. 1993, Foyer et al. 1995). Demirevska-

Kepova et al. (2006) reported that the content of oxidized ascorbate increased during

Cd exposure in Hordeum vulgare plants. A decrease in the ascorbate content in the

roots and nodules of Glycine max under Cd stress has been observed (Balestrasse et

al. 2001). Cadmium also decreases ascorbate content in Cucumis sativus chloroplast

and in the leaves of Arabidopsis thaliana, Pisum sativum and Brassica campestris

(Zhang et al. 2003, Skorzynska-Polit et al. 2003/04, Romero-Puertas et al. 2007,

Anjum et al. 2008a,d), respectively, whereas, it remained unaffected in Populus x

canescens roots (Schutzendubel et al. 2002).

35

2.10.2.2 Glutathione

The tripeptide (y-GluCysGly) glutathione (GSH) plays a central role in several

physiological processes, including regulation of sulfate transport, signal transduction,

conjugation of metabolites, detoxification of xenobiotics (Xiang et al. 2001) and the

expression of stress-responsive genes (Muliineaux and Rausch 2005). The reduced

form of glutathione, GSH, is an abundant compound in plant tissue that exists

interchangeably with the oxidized form, GSSG. GSH is abundant (3-lOmM) in

cytoplasm, nuclei and mitochondria and is the major soluble antioxidant in these cell

compartments. GSH has been associated with several growth and development related

events in plants, including cell differentiation, cell death and senescence, pathogen

resistance and enzymatic regulation (Ogawa 2005, Rausch and Wachter 2005) and its

content is affected by S nutrition (Blake-Kalff e/ al. 2000). GSH is the major reservoir

of non-protein sulfur. It is the major redox buffer in most aerobic cells, and plays an

important role in physiological functions, including redox regulation, conjugation of

metabolites, detoxification of xenobiotics and homeostasis and cellular signaling that

triggers adaptive responses (Noctor et al. 2002, Kopriva and Koprivova 2005). It also

plays an indirect role in protecting membranes by maintaining a-tocopherol and

zeaxanthin in the reduced state. It can also function directly as a free radical

scavenger by reacting with superoxide, singlet oxygen and hydroxyl radicals. GSH

protects proteins against denaturation caused by the oxidation of protein thiol groups

under stress. In addition, GSH is a substrate for glutathione peroxidase (GPX) and

glutathione-S-transferases (GST), which are also involved in the removal of ROS

(Noctor et al. 2002). GSH is a precursor of PCs, which are crucial in controlling

cellular heavy metal concentrations. GSH and its oxidized form, GSSG maintains a

redox balance in the cellular compartments. This property of glutathione is of great

biological importance since it allows fine-tuning of the cellular redox environment

under normal conditions and upon onset of stress, and provides the basis for GSH

stress signaling. A central nucleophilic Cys residue is responsible for higher reductive

potential of GSH. It scavenges cytotoxic H2O2, and reacts non-enzymatically with

other ROS i.e., O2", OH- and 'O2 (Larson 1988). The central role of GSH in the

antioxidative defense system is due to its ability to regenerate another water soluble

antioxidant, ascorbate, in Ascorbate-Glutathione cycle (Foyer and Halliwell 1976,

Noctor and Foyer 1998). The role of GSH in the antioxidant defense system provides

a strong basis for its use as a stress marker. However, the concentration of cellular

36

GSH has a major effect on its antioxidant function and it varies considerably under Cd

stress. Furthermore, strong evidence has indicated that an elevated GSH concentration

is correlated with the ability of plants to withstand metal-induced oxidative stress

(Freeman et al. 2004). Xiang et al. (2001) observed that plants with low levels of

glutathione were highly sensitive to even low levels of Cd due to limited PC

synthesis. The increased demand for GSH can be met by the activation of pathways

involved in S assimilation and Cys biosynthesis. Its concentration is controlled by a

complex homeostatic mechanism where the availability of S seems to be required

(May et al. 1998a). Manipulation of GSH biosynthesis increases resistance to

oxidative stress (Youssefian et al. 2001, Sirko et al. 2004). It has been observed that

upon Cd exposure, one of the mam responses observed was the induction of genes

involved in S assimilation-reduction and GSH metabolism in the roots of Arabidopsis

(Herbette et al. 2006).

Feed back inhibition of y-glutamylcysteine synthase (y-ECS) by GSH has been

considered as a fundamental central point for GSH synthesis. In vitro studies with the

enzymes from tobacco and parsley cells showed that the plant y-ECS was inhibited by

GSH (Noctor and Foyer 1998). Oxidation of GSH to GSSG decreases GSH levels and

allows increased y-ECS activity under stressed conditions (Noctor and Foyer 1998).

Environmental stresses trigger an increase in ROS levels in plants and the

response of GSH can be crucial for adaptive responses. Antioxidant activity in the

leaves and chloroplast of Phragmites australis Trin. (cav.) ex Steudel was associated

with a large pool of GSH, protecting the activity of many photosynthetic enzymes

against the thiophilic bursting of Cd exerting a direct important protective role in the

presence of Cd (Pietrini et al. 2003). Increased concentration of GSH has been

observed wdth the increasing Cd concentration in Pisum sativum (Gupta et al. 2002),

Lactuca sativa (Maier et al. 2003), Phragmites australis (Pietrini et al. 2003),

Brassica juncea (Qadir et al. 2004), Pisum sativum (Metwally et al. 2005), Sedum

alfredii (Sun et al. 2007), Oryza sativa (Hassan et al. 2008). However, decay in GSH

content in Glycine max roots (Balestrasse et al. 2001), Helianthus annuus leaves

(Gallego et al. 1996b), Zea mays seedlings (Rauser 1990), Pisum sativum

(Ruegsegger et al. 1990), Pinus sylvestris roots (Schutzendubel et al. 2001), Cucumis

sativus chloroplast (Zhang et al. 2003), Populus x canescens roots (Schutzendubel et

al. 2002) and Oryza sativa leaves (Hsu and Kao 2004) has been reported under Cd

stress. Furthermore, unaltered GSH content was observed in the nodules of

37

Table 2. Antioxidative enzymes in economically important crop plants grown under cadmium stress. Symbols used: - - Decrease; + + Increase/overexpressed; x x Unaltered

Antioxidants

Superoxide dismutase (SOD)

Ascorbate peroxidase (APX)

Plant species/ part studied

Raphanus sativus leaf

Raphanus sativus root Pisum sativum leaf Solarium tuberosum

Hordeum vulgare Arabidopsis thaliana

Oryza sativa leaves Alyssum spp

Helianthus annuus Amaranthus lividus Phragmites australis Helianthus annuus leaves Capsicum annuum plant Bacopa monnieri Vigna mungo roots Vigna mungo leaves Triticum aestivum leaves Brassicajuncea leaves

Brassica campestris leaves Zea mays Cicer arietinum Pisum sativum

Oryza sativa Arabidopsis thaliana Oryza sativa shoots and roots Oryza sativa roots Zea mays Glycine max Brassica napus Ceratophyllum demersum Pisum sativum leaves

Phaseolus aureus

Response

+ +

X X

+ + + +

+ + + +

+ + + +

X X

— —

—

+ +

+ + + + + +

+ +

+ + + + + +

+ + + + + +

+ + — —

+ +

+ +

+ +

Reference (s)

VitoriaeM/. 2001

Vitoriae/a/. 2001 DalurzoeM/. 1997 Stroinski and Kozlowska 1997 Guo et al. 2004 Skorzynska-Polit et al. 2003/04 Hsu and Kao 2004 Schickler and Caspi 1999 Gallegoera/. 1999 Bhattacharjee 1998 lannelli et al. 2002 Gallego et al. 1996b Leon et al. 2002 Mishra^/a/. 2006 Singh et al. 2008a Singh et al. 2008a Khan et al. 2007a Mobin and Khan 2007 Anjum et al. 2008d

Ekmekci et al. 2008 Hasan et al. 2008 Agrawal and Mishra 2008 Shah e/a/. 2001 Cho and Seo 2005 Wu et al. 2006

Guo et al. 2007 Krantev et al. 2007 Noriega et al. 2007 Filek et al. 2007 Aravind and Prasad 2003 Romero-Puertas et al. 1999 Shaw 1995b

Catalase (CAT)

Hordeum vulgare roots Ceratophyllum demersum Glycine max roots and nodules Cucumis chloroplast Populus plant

Vigna mungo leaves and roots Triticum aestivum leaves Zea mays

Helianthus annuus leaves Brassicajuncea leaves

Brassica campestris leaves Arabidopsis thaliana Bacopa monnieri Brassica napus Helianthus annuus leaves Phaseolus vulgaris Phaseolus aureus Pisum sativum Lemna minor

Amaranthus lividus Glycine max leaves

Phargmitis australis Capsicum annuum Agropyron repens Glycine max nodules

Helianthus annuus

Oryza sativa leaves Solanum tuberosum

Raphanus sativus roots Saccharum officinarum In vitro callus Glycine max leaves Phaseolus vulgaris roots and leaves Bacopa monnieri

—

+ +

+ +

—

—

+ +

+ + + +

—

+ +

+ +

+ + + + + + —

—

—

—

—

—

—

—

—

+ + + +

+ +

+ + + +

+ + + +

X X

—

—

Hegeduse/a/. 2001 Aravind and Prasad 2003 Balestrasse et al. 2001 Zhang et al. 2003 Schutzendubel et al. 2002 Singh et al. 2008a

Khan et al. 2007a Krantev et al. 2007, Ekmekci et al. 2008 Gallego et al. 1996b, 2002 Mobin and Khan 2007 Anjum et al. 2008d

Cho and Seo 2005 Mishra et al. 2006 Filek et al. 2007 Gallego et al. 1996b Chaouie/a/. 1997b Shaw 1995b Dalurzoe/a/. 1997 Mohan and Hossetti 1997 Bhattacharjee 1998 Balestrasse et al. 2001 lannelli et al. 2002 Leon et al. 2002 Brej 1998 Balestrasse et al. 2001 Gallego e/a/. 1999, 2002 Hsu and Kao 2004 Stroinski and Kozlowska 1997 Vitoriae^a/. 2001 Fomazier et al. 2002b Ferreira et al. 2002 Chaouie/a/. 1997b

Mishra et al. 2006

Guaiacol peroxidase (GPOX)

Dehydroascorbate reductase (DHAR)

Glutathione peroxidase (GPX)

Glutathione reductase (GR)

•

Phaseolus vulgaris stem Vigna mungo roots Vigna mungo leaves Triticum aestivum leaves Brassicajuncea leaves

Brassica campestris leaves Pisum sativum

Oryza sativa Cicer arietinum Miscanthuss sinensis leaves and roots Brassica napus Pisum sativum plants

Phaseolus aureus Phaseolus vulgaris Glycine max roots and nodules Arabidopsis thaliana Triticum aestivum Capsicum annuum plants Helianthus annuus Glycine max Arabidopsis thaliana

Bacopa monnieri Brassica napus Helianthus annuus leaves Pinus sylvestyris

Glycine max roots and nodules Capsicum annuum plants

Pisum sativum roots Pisum sativum leaves Triticum aestivum leaves Raphanus sativus

Crotolaria juncea Capsicum annuum plants Glycine max Saccharum officinarum leaves Phaeodactylum

X X

—

+ + + + + +

+ +

—

+ + + + + +

—

—

+ + + + —

+ + + + + + — —

+ +

+ + —

—

X X

+ +

+ +

X X

+ + + +

+ + + + + + + +

+ +

Chaouiera/. 1997b Singh et al. 2008a Singh et al. 2008a Khan e/a/. 2007a Mobin and Khan 2007 Anjumera/. 2008d

Agrawal and Mishra 2008 Hassan et al. 2008 Hasan et al. 2008 Scebba et al. 2006

Filek et al. 2001 Sandalio^/flf/. 2001

Shaw 1995b Chaouie/a/. 1997b Balestrasse et al. 2001 Cho and Seo 2005 Milone et al. 2003 Leon et al. 2002 Gallegoe/a/. 2002 Noriega et al. 2007 Skorzynska-Polit et al. 2003/04 Mishra et al. 2006 Filek et al. 2007 Gallego et al. 1996b Schutzendubel et al. 2001 Balestrasse et al. 2001 Leon et al. 2002

Dixit era/. 2001 Dixit era/. 2001 Khan et al. 2007a Vitoriaera/. 2001

Pereira et al. 2002 Leon et al. 2002 Ferreira et al. 2002 Fomazier et al. 2002a Morelli and

Glutathione S-transferase (GST)

Monodehydroascorbate reductase (MDHAR)

Ascorbic acid (AsA)

Glutathione (GSH)

tricomutum Pisum sativum roots and leaves Alyssum sp.

Cucumis chloroplast Pisum sativum Solarium tuberosum

Helanthus annuus

Viciafaba shoots Bacopa monnieri Viciafaba roots Vigna mungo leaves and roots Triticum aestivum leaves Brassicajuncea leaves

Brassica campestris leaves Zea mays Arabidopsis thaliana Pisum sativum plants

Oryza sativa roots Phragmites australis Pinus sylvestris

Populus canescens (poplar hybrid roots) Glycine max roots and nodules Arabidopsis thaliana leaves Cucumis chloroplast Populus canescens roots

Brassica juncea leaves Pisum sativum

Brassica campestris leaves Oryza sativa roots Sedum alfredii

Pisum sativum

+ +

—

—

— —

—

+ + + + —

+ +

+ + + +

+ +

+ + + + + +

+ + + + + +

—

—

—

— X X

—

—

+ + + +

+ +

Scarano 2004 Dixit e/a/. 2001

Schickler and Caspi 1999 Zhang et al. 2003 DaluTzo etal. 1997 Stroinski and Kozlowska 1997 Gallego et al. 1999b Rosa et al. 2003 Mishra et al. 2006 Rosa et al. 2003 Singh et al. 2008a

Khan et al. 2007a Mobin and Khan 2007 Anjum et al. 2008d

Ekmekci et al. 2008 Cho and Seo 2005 Dixit etal. 2001

Moons 2003 lannelli et al. 2002 Schutzendubel et al. 2001 Schutzendubel et al. 2001 Balestrasse et al. 2001 Skorzynska-Polit et al. 2003/04 Zhang et al. 2003 Schutzendubel et al. 2002 Qadir et al. 2004 Romero-Puertas et al. 2007, Agrawal and Mishra 2008 Anjum et al. 2008a,d Guo et al. 2007 Sun et al. 2007

Metwally et al. 2005

Brassica juncea leaves Phragmites australis Lactuca sativa Oryza sativa Oryia sativa roots Glycine max roots

Helianthus annuus leaves Zea mays seedlings Pisum sativum

Pinus sylvestris roots

Cucumis chloroplast Populus canescens roots

Oryza sativa leaves Glycine max Brassica campestris

Glycine max nodules

Pisum sativum

+ + + + + + + + + + —

—

—

—

—

—

—

—

—

—

X X

+ +

Qadir eM/. 2004 Pietrini et al. 2003 Maier era/. 2003 Hassan et al. 2008 Guo et al. 2007 Balestrasse et al. 2001 Gallego et al. 1996b Rauser 1990 Ruegsegger et al. 1990 Schutzendubel et al. 2001 Zhang et al. 2003 Schutzendubel et al. 2002 Hsu and Kao 2004 Noriega et al. 2007 Anjum et al. 2008a,d Balestrasse et al. 2001 Gupta et al. 2002

Glycine max (Balestrasse et al. 2001). Cadmium-induced depletion of GSH has been

mainly attributed to phytochelatin synthesis (Grill et al. 1985). Phytochelatin-heavy

metal complexes have been reported to be accumulated in the vacuole of Nicotiana

tabacum leaves (Vogeli-Lange and Wagner 1990) and in Avena sativa. These

complexes have been shown to be transported across the tonoplast (Salt and Rauser

1995). The decline in the levels of GSH might also be attributed to an increased

utilization for ascorbate synthesis or for a direct interaction with Cd (Pietrini et al.

2003). The variety of response to Cd-induced oxidative stress is probably related not

only to the levels of Cd supplied, but also to the plant species, the age of the plant and

duration of the treatment.

2.11 Coordination of Sulfur and Nitrogen in Plant Growth and Productivity

The improvement in crop production and quality depend upon a number of plant- soil-

and nutrients-associated factors i.e. balanced fertilization, optimization of nutrient

replenishment, minunization of nutrient losses to the enviromnent and the concept of

co-ordination in action among plant nutrients. Among the plant nutrients, S and N are

of great importance and are required by plants for maintaining normal growth and

development (Scherer 2008, Bimbraw 2008). Several studies have established

regulatory interactions between assimilatory sulfate and nitrate reduction in plants

(Rennenberg et al. 1979, Reuveny et al. 1980, Brunold and Suter 1984, Haller et al.

1986, Takahashi and Saito 1996). The two pathways are very well coordinated

(Brunold 1993, Hawkesford et al. 2006). A deprivation of one leads to a reduction of

the metabolism of the other (Reuveny et al. 1980, Prosser et al. 2001, Hesse et al.

2004, Scherer 2008). It has also been established through genetic studies that sulfate

reduction is regulated by N nutrition at the transcriptional level (Koprivova et al.

2000).

The S and N affect enzyme activity in their respective assimilatory pathways

(Reuveny et al. 1980, Brunold and Suter 1984, Barney and Bush 1985, Bell et al.

1995, Ahmad et al. 1999). While N directly affects the photosynthesis efficiency of

the plant, S affects the photosynthesis efficiency indirectly by improving the N

utilization efficiency of the plants as evident from the relationship between N content

and photosynthetic rate in the leaves of S-treated and S-untreated plants (Ahmad and

Abdin 2000a). It has also been shown in Hordeum vulgare that high levels of nitrate

and ammonium can induce a high affinity sulfate transporter gene and hence sulfate

38

uptake in N-fed plants suggesting that a N metabolite may affect sulfate transporter

gene expression (Vidmar et al. 1999).

Positive interaction between S and N results in increased crop productivity

(Podlesna and Cacak-Pietrzak 2008). Brassica genotypes require higher amounts of S

in addition to N for optimum growth and yield (Aulakh et al. 1980, Lakkineni and

Abrol 1992, 1994, Abdin et al. 2003, Aulakh 2003). Application of S along with N

lead to enhanced biomass production and increased leaf area as both nutrients are

involved in biosynthesis of proteins and several other molecules. Nitrogen is a basic

constituent of proteins and with the increase in the rate of N application, the N

availability increases. Similarly, increased S supply increases seed yield with higher

protein content. Combined application of S and N promotes the uptake of S and N,

which leads to significant enhancement in seed protein and oil content in Brassica

juncea and Brassica campestris (McGrath and Zhao 1996, Ahmad and Abdin 2000b,

Abdin et al. 2003). Sulfur and nitrogen relationship in terms of crop yield and quality

has been established in many studies (Singh and Bairathi 1980, Sachdev and Deb

1990, Lakkineni and Abrol 1992, McGrath and Zhao 1996, Ahmad et al. 1998,

Fismes et al. 2000).

2.12 Coordination of Sulfur and Nitrogen under Environmental Stress

Plants have developed strategies resulting in optimal adaptation to environmental

resources needed for growth, respiration, and propagation (Rennenberg and Brunold

1994, Brunold et al. 1996). Among these adaptations, strategic co-ordination of

macronutrient assimilation, i.e., carbon, nitrogen, phosphorous, water, and sulfur

['CHONSP'] is especially important, as these pathways are dependent on the varying

availability of mineral nutrients and CO2 and changes in light energy (Hesse et al.

2004). The assimilatory pathways interact in the formation of products and are

regulated in a coordinated maimer to balance macro- and micronutrients in possible

synergistic or antagonistic effects (Brunold 1993, Leustek et al. 2000, Saito 2000).

As heavy metals can induce essential nutrient deficiency and even decrease

the concentration of several macronutrients in plants (Siedlecka 1995), it may be

possible to reverse or reduce (at least partly) some of the metal induced negative

effects on the plants by optimization of mineral nutrition. Chen and Huerta (1997)

showed that S is a critical nutritional factor for reduction of Cd toxicity. These authors

observed that negative effects of Cd on growth and photosynthesis are stronger in

Hordeum vulgare plants supplied with 0.1 mM S than in plants receiving ImM S. A

39

positive effect of S nutrition on Cd detoxification in Beta vulgaris plants has also

been established (Popovic et al. 1996). It has been found that at sub-optimal S

nutrition Cd exposed plants preferably allocate S to PCs synthesis, which may

provoke S deficiency (McMahon and Anderson 1998). Probably, the improved S

nutrition allows a more adequate plant defense response to Cd and also prevents S

deficiency. Good S nutrition diminishes the toxicity of Cd by restoring a new steady

state of the GSH level earlier than in plants grown at low S supply (McMahon and

Anderson 1998). Glutathione represents the major pool of non-protein reduced S in

plants (Kunert and Foyer 1993) and it accounts only for 2% of the total organic S

content in plants ranging from 120 to 380mmol S kg"' dry matter (McMahon and

Anderson 1998, Hawkesford and De Kok 2006, Sun et al. 2007, Ernst 2008).

Glutathione has critical functions aside from its role as a redox buffer. It serves as the

first line of defense against the products of oxygen metabolism, ROS, and heavy

metals (May et al. 1998a,b). Recently, El-Shintinawy (1999) showed that exogenous

GSH counteracted all retardation effects in Glycine max seedlings induced by Cd. He

provided data to conclude that the dual fimction of GSH; first, as being an antioxidant

and oxy-radicals scavenger, it protects chloroplast from oxidative damage by trapping

the hydroxyl radicals, secondly as a substrate for PCs synthesis that mainly sequesters

and detoxifies excess Cd ions. When both functions are realized, repairing membrane

damage and sequestration of Cd are achieved by GSH. The electron transport rate as

well as photosynthetic parameters is not disturbed and this results in a photosynthetic

performance similar to that in the control plants. The crucial role of plant PCs in Cd

detoxification was strongly supported by the investigations on two mutants from

Arabidopsis, cadi and cad2, which were deficient in PC and GSH biosynthesis,

respectively, and are consequently more sensitive to Cd (Howden et al. 1995a,b,

Cobbett et al. 1998, Cobbett and Goldsbrough 2002). Inouhe et al. (2000) found a

close relationship between high Cd sensitivity and lack of phytochelatin synthase in

the cultured cells of Vigna angularis. In a recent study, Ranieri et al. (2005) reported

that the leaves of Triticum aestivum exposed to Cd stress were coimteracted by PC

biosynthesis. Furthermore, Anjum et al. (2008a) reported that S supplementation

increased the production of GSH content under low level of Cd which protects the

Brassica campestris plants by improving the growth and photosynthesis.

Pankovic et al. (2000) have shown that optimal N supply decreased the

inhibitory effects of Cd on photosynthesis of Helianthus annuus plants. The authors

40

studied the effects of Cd on Helianthus annuus plants grown at optimal, sub-optimal

and supra-optimal N supplies. They found the lowest inhibition of photosynthetic

activity by Cd at optimal N supply, when an investment in soluble proteins and

Rubisco were at their maximum. Higher N supplies did not alleviate the toxic Cd

effects; therefore the authors concluded that N nutrition can be manipulated as a

means of decreasing Cd phytotoxicity. A proper N supply has positive effects in

overcoming the adverse effects caused by Cd toxicity in Oryza sativa (Hassan et al.

2005a). An exhaustive research by Hassan et al. (2005a) has revealed that different

forms of N show differential response in the alleviation of Cd toxicity in Oryza sativa.

They concluded that among the N forms that unproved growth and photosynthetic

traits to a large extent was Ca(N03)2 followed by NH4NO3 and (NH4)2S04. Beside, a

substantial difference was noted among N forms in their effect on Cd and N uptake,

(NH4)2S04-fed plants had less Cd and more N uptake than both Ca(N03)2- and

NHtNOs-fed plants, suggesting potential antagonist effect between Cd and

ammonium-N, and synergist effect between Cd and nitrate-N.

Phytochelatin biosynthesis is closely dependent on S metabolism (Cobbett

2000a,b, Leustek et al. 2000), synthesized from different isoforms of GSH (Grill et al.

1985). It is important to note here that GSH with the help of Cys takes part in the

biosynthesis of PCs. In plants PCs play a crucial role in Cd detoxification (Howden et

al. 1995b). It complexes cations (Cd " ), and makes them less harmful to plant cells

(Weigel and Jager 1980). Expression of genes involved in reductive sulphate

assimilation pathway and enzyme activities are stimulated by Cd (Herbette et al.

2006, Ernst et al. 2008). Cadmium exposure induces the activity of enzymes (y-

glutamyl-Cys synthatase, yECS and glutathione synthatase, GS) involved in the

biosynthesis of GSH. Herbette et al. (2006) suggested that plants activate the S

assimilation pathway by increasing transcription of related genes to provide an

enhanced supply of GSH for PC biosynthesis to cope with Cd toxicity. In addition,

when plants encounter reactive oxygen species, GSH is a direct source of electrons for

stress mitigation by the enzyme glutathione peroxidase (GPX) or an indirect means to

maintain a reduced pool of ascorbate, another antioxidant. In fact, the biosynthesis of

y-glutamylcysteinylglycine (GSH) depends on the linking and the availability of its

constituent amino acids Cys, Glu and Gly. The biosynthesis of GSH in plants is well

established: two sequential ATP-dependent reactions allow the synthesis of y-

glutamylcysteine (y-EC) from L-Glu and L-Cys, followed by the formation of Gly to

41

the C-terminal end of y-EC (Meister 1988). These reactions are catalyzed by y-ECS

and GS (May et al. 1998a, Noctor et al. 1998b). Glutathione contains three moles of

N per mole of S and its GSH biosynthesis may depend on the availability of N

precursors and thus N nutrition of plants. However, the sink strength of GSH

biosynthesis for N may be low compared with the other major N sinks such as the

synthesis of proteins or nucleotides (Kopriva and Rennenberg 2004). The GSH

biosynthesis is, therefore, regulated not only by the dependency of Cys availability on

S, N and C metabolism, but also on the availability of Glu and Gly. In this way, the

coordinative functions of S and N may strengthen the stress tolerance ability of plants

growing under abiotic stresses.

2.13 Research Work Reported from Our Laboratory

We have conducted research to study the effects of Cd on growth, photosynthesis and

response of the components of antioxidant defense system in different plants such as

Triticum aestivum, Vigna mungo, Brassica juncea, Brassica campestris and Lepidium

sativum. Following is the summary of the published work.

Wheat (Triticum aestivum L.)

Khan et aL (2006) World J Agric Sci 2: 223-226

The variation in growth, photosynthesis and yield characteristics of five Triticum

aestivum cultivars, namely PBW343, HT2329, PBW373, UP2338 and WH542 at

three plant growth stages, treated with 0, 25, 50 and lOOmg Cd kg'' soil was studied.

Maximal significant reductions in the growth characteristics were observed with

lOOmg Cd kg"' soil in all the cultivars at all the sampling times. Among cultivars,

PBW343 proved tolerant and showed lesser decrease in the characteristics, whereas,

WH542 emerged as non-tolerant and suffered maximum decrease.

Khan et aL (2007a) J Agron Crop Sci 193:435-444

The activities of antioxidative enzymes and S assimilation were studied in Cd-treated

Triticum aestivum cultivars to assess their involvement in determining yield potential.

The cultivar WH542 (low yielding type) accumulated Cd to a greater amount in both

root and leaf, and also exhibited higher contents of H2O2 and TEARS and the activity

of SOD than cultivar PBW343 (high yielding type). The activities of other

antioxidative enzymes, CAT, APX, GR and GPOX, activity of ATP-sulfurylase, S

content, photosynthetic, growth and yield characteristics were higher in PBW343 than

in WH542 in Cd treatment compared to the control. The results suggest that the

efficient fimctioning of enzymes of the antioxidative system and S assimilation helped

42

in alleviating the effects of Cd in PBW343, protected photosynthetic ability and

maintained high yielding potential of the cultivar.

Khan et aL (2008b) J Plant Interactions 3:31-37.

Leaf carbonic anhydrase (CA) activity, net photosynthetic rate (PN), stomatal

conductance (gs) and plant dry mass (DM) exhibited a dose-dependent response to Cd

in Triticum aestivum grown under low and sufficient Zn levels at 30 d after seedling

emergence in sand culture. Cadmium at lOmM concentration enhanced CA, PN and

dry mass at low Zn level but these remain unaltered at sufficient Zn. Stomatal

conductance decreased with increasing CdCb concentration at both the Zn levels.

Cadmium higher than lOmM inhibited the characteristics irrespective of Zn level. The

activity of SOD was not saturated with the highest Cd concentration (50mM) at both

the Zn levels. However, the activities of APX and GR increased up to 25mM Cd and

decreased at 50mM Cd at both the Zn levels. It is suggested that CA activity was

induced at low Cd (lOmM) under low Zn level mcreasing the photosynthesis. The

synergies among the activities of antioxidative enzymes helped to maintain CA and

thus photosynthesis and plant dry mass at low Cd concentration under low Zn level.

Blackgram (Vigna mungo L.)

Singh et aL (2008a) Amer J Plant Physiol 3:25-32

Vigna mungo plants were treated with 0, 25, 50 and lOOmg Cd kg"' soil as CdCb for

30d. The changes in total chlorophyll (Chi), Chi a^, net photosynthetic rate (PN),

stomatal conductance (gs), water use efficiency (WUE) and carbonic anhydrase (CA)

activity were noted. The activities of antioxidative enzymes in root and leaf were also

assayed together with the content of TBARS and H2O2. The concentration of Cd in

root and leaf increased with the increasing Cd concentration. Greatest decrease in

photosynthetic traits was observed with lOOmg Cd kg"' soil. The activity of SOD

increased in leaf but decreased in root, whereas, the activity of CAT decreased in both

root and leaf By contrast to CAT, the activity of APX increased in root and leaf

However, GR activity increased in root and decreased in leaf The results suggest that

the antioxidative enzymes showed differential pattern in root and leaf and the

decrease in photosynthesis with lOOmg Cd kg"' soil was associated with the

accumulation of TBARS and H2O2 content and reduction in Chi content, stomatal

conductance and CA activity.

43

Mustard {Brassica juncea L.)

Mobin and Khan (2007) J Plant Physiol 164:601-610

Photosynthetic performance, contents of chlorophyll and associated pigments, cellular

damage and activities of antioxidative enzymes were investigated in two Brassica

juncea cultivars differing in photosynthetic capacity subjected to Cd stress. Exposure

to Cd severely restricted the PN of RH30 (low photosynthetic potential) compared to

Varuna (high photosynthetic potential). This corresponded to the reductions in the

activities of CA and ribulose-l,5-bisphosphate carboxylase (Rubisco) in both the

cultivars. Decline in Chi (a+b) and Chi a content was observed but decrease in Chi b

was more conspicuous in Varuna under Cd treatments, which was responsible for

higher Chi a:b ratio. Additionally, the relative amount of anthocyanin remained higher

in Varuna compared to RH-30 even in the presence of high Cd concentration, while

percent pheophytin content increased in RH-30 at low Cd concentration. A higher

concentration of Cd (lOOmg Cd kg'' soil) resulted in elevated H2O2 content in both

the cultivars. However, Varuna exhibited lower content of H2O2 in comparison to

RH30. This was reflected in the increased cellular damage in RH30, expressed by

greater TBARS content and electrolyte leakage. The enhanced activities of

antioxidative enzymes, APX, CAT and GR and also lower activity of SOD in Varuna

alleviated Cd stress and protected the photosynthetic activity.

Anjum et aL (2007) XXXth Indian Botanical Conference pp. 148

The differential response of two Brassica juncea cultivars, Varuna and RH30

differing in photosynthetic potential to 0, 50, 100, 150 and 200mg Cd kg"' soil were

investigated. Varuna (high photosynthetic potential) showed higher rate of

photosynthesis, greater production of Cys, non-protein thiol and PC and the activity of

ATP-sulfurylase than the cultivar RH30 (low photosynthetic potential). In both the

cultivars the response was greatest at lOOmg Cd kg"' soil and decreased at higher Cd

concentration. The activities of antioxidative enzymes, SOD, GR, CAT and APX

showed a dose-dependent response to Cd in both the cultivars. But the cultivar Varuna

showed greater increase in the activities of enzymes such as APX, CAT and GR with

respect to control than the cultivar RH30 at lOOmg Cd kg'' soil. However, the

repression in the activities of enzymes at 150 and 200mg Cd kg"' soil was greater in

RH30 than Varuna compared to control. The cultivar RH30 exhibited a higher degree

of oxidative stress than Varuna and therefore, the activity of SOD, the contents of

TBARS, H2O2 and electrolyte leakage were found greater in RH30 cultivar than

44

Varuna with increasing Cd concentrations. The results show that high photosynthetic

mustard cultivar Varuna protected photosynthesis through enhanced production of

cysteine, non-protein thiol and phytochelatins and simultaneously by reducing

oxidative stress through efficient antioxidative enzyme system.

Rapeseed {Brassica campestris L.)

Anjum et aL (2008a) Plant Growth Regul 54:271-279.

The ameliorative effect of S (0 or 40mg S kg"' soil) on Cd (0, 25, 50 and lOOmg Cd

kg' soil)-induced growth inhibition and oxidative stress in Brassica campestris

cultivar Pusa Gold was studied. Cadmium at lOOmg kg'' soil caused maximum

increase in the contents of Cd and TBARS in leaves. Maximum reductions in growth

(plant dry mass, leaf area), Chi content, PN and the contents of ascorbate and

glutathione were observed with lOOmg Cd kg"' soil compared to control. The

application of S helped in reducing Cd toxicity, which was greater for 25 and 50mg

Cd kg"' soil compared tolOOmg Cd kg"' soil. Addition of S to Cd-treated plants

showed decrease in Cd and TBARS content in leaves and restoration of growth and

photosynthesis through increase in the contents of ascorbate and glutathione. Net

photosynthetic rate and plant dry mass were strongly and positively correlated with

the contents of ascorbate and glutathione. It is suggested that S may ameliorate Cd

toxicity and protects growth and photosynthesis of mustard involving ascorbate and

glutathione.

Anjum etaL (2008b) J Plant Interactions DOI 10.1080/17429140701823164

Brassica campestris cultivar Pusa Gold plants, exposed to different Cd levels (0, 25,

50 and lOOmg kg"' soil), were analyzed for the distribution of metal, biomass and the

degree of growth stage to Cd-sensitivity. A significant maximum decrease in plant

biomass was observed at flowering stage followed by pre-flowering and post-

flowering stages. Activities of enzymatic antioxidants such as SOD, CAT, APX and

GR differentially increased; while, the concentrations of non-enzymatic antioxidants

such as ascorbate and glutathione drastically decreased in plants exposed to Cd at

various growth stages. However, the concentrations of glutathione and ascorbate

decreased maximally in plant groups exposed to Cd at flowering stage. Maximum Cd-

accumulation occurred in roots followed by leaves and stem. Various Cd levels

inhibited also the contents of plant nutrients such as N, P, K and S in leaves. The

study concludes the existence of close relationships among growth parameters, Cd-

45

sensitivity of phenological stages of the crop and the components of antioxidant

system in rapeseed plants.

Garden cress (Lepidium sativum L.)

Singh etaL (2007) XXXth Indian Botanical Conference pp. 161

Lepidium sativum is a medicinally and economically important plant which contains

significant amounts of iron, calcium and folic acid, in addition to vitamins A and C.

An experiment was conducted to examine the effect of different concentrations of

cadmium (0, 25, 50 and lOOmg kg'' soil) on the performance of garden cress plants.

Cadmium accumulation in roots and leaves increased with the increasing Cd

concentration in soil, and roots accumulated more Cd than leaves. Cadmium

treatments inhibited leaf area and plant dry mass. Dose dependent increase in TEARS,

H2O2 content and the activity of antioxidant enzymes (viz. SOD, CAT, APX and GR)

was observed under Cd stress. A progressive decline in relative water content,

photosynthetic rate, stomatal conductance, intercellular CO2, Chi content (Chi a, Chi

b, total Chi), CA activity, Rubisco, NR activity and N content was also observed with

the increasing Cd concentration. However, ATP-sulfiirylase activity, S content and

activities of antioxidant enzymes were increased. The decrease in NR activity and N

content showed that Cd affects nitrogen metabolism; whereas, the increase in ATP-

sulfurylase activity and S content suggests the up-regulation of S assimilation

pathway for possible Cd tolerance.

2.14 Critical Appraisal of Review of Literature

The literature reviewed above includes studies on the physiological analysis of

various growth-, photosynthetic- and biochemical-traits, antioxidant studies and yield

of various crop plants xmder Cd stress. It appears that there are few reports concerning

the effect of fertilization (especially S and N) under abiotic stress on various

physiological processes, including productivity of mustard, an important oilseed crop.

In fact, the work on various crop plants is invariably aimed at establishing

mechanisms of antioxidant machinery of plants exposed to various abiotic stresses.

However, in-depth understanding of the response of various mineral nutrients in

enhancing and/or strengthening the antioxidant machinery to coimteract the effects of

abiotic stress-induced ROS production has not been done.

Only few reports are available on the individual effects of S and N in the

amelioration of Cd toxicity, and that no work has been taken to study the interactive

effects of S and N in crop plants grown under Cd stress. The reported study was

46

undertaken on Brassica juncea (S requiring crop) to explore the possibility of

ameliorating the adverse effects of Cd through the balanced application of S and N.

Following the application of S and N to Cd-treated Brassica juncea plants, concurrent

changes in growth, physiological and biochemical traits, components of antioxidant

defense system and the productivity oi Brassica juncea were studied.

47

MATERIAL AND METHODS

Contents

MATERIAL AND METHODS

3.1 3.1.1 3.1.1.1 3.1.1.2 3.1.1.3 3.2 3.3 3.3.1 3.3.2 3.3.3 3.4 3.4.1 3.4.2 3.4.3 3.5 3.6 3.7 3.8 3.8.1 3.8.2 3.8.2.1 3.8.2.2 3.8.2.2.1 3.8.2.2.2 3.8.2.2.3 3.8.2.3 3.8.2.3.1 3.8.2.4 3.8.2.4.1 3.8.2.5 3.8.2.5.1 3.8.2.5.2 3.8.2.6 3.8.2.6.1 3.8.2.6.2 3.8.2.7 3.8.2.7.1 3.8.2.7.2 3.8.2.8 3.8.2.9 3.8.2.10 3.8.2.11 3.8.2.11.1 3.8.2.11.2 3.8.2.11.2.1 3.8.2.11.2.1.:

Plant Material Botanical description of mustard Nomenclature Botanical description Genomic relationships of Brassica juncea Agro-Climatic Conditions of Aligarh Experimentation Preparation and analysis of soil Pot preparation and treatments Sowing of seeds Experimental Set-up Experiment 1 Experiment 2 Experiment 3 Sampling Technique Chemicals Parameters Studied Methodology Growth characteristics Physiological and biochemical characteristics Net photosynthetic rate and stomatal conductance Chlorophyll and carotenoid contents Extraction Estimation Calculation for chlorophyll and carotenoids content Assay of carbonic anhydrase activity Estimation Assay of nitrate reductase activity Estimation Assay of ATP-sulfurylase activity Extraction Estimation Determination of nitrogen Digestion of leaf powder Estimation of nitrogen Determination of sulfur Digestion of leaf powder Estimation of sulfur Determination of cadmium Determination of thiobarbituric acid reactive substances Determination of hydrogen peroxide content Components of en2ymatic and non-enzymatic antioxidant system Activities of antioxidant enzymes En2yme extraction Superoxide dismutase activity

1 Enzyme assay

48 48 48 48 49 50 50 50 51 51 52 52 52 53 53 54 54 55 55 55 55 55 55 55 55 56 56 56 57 57 57 57 58 58 58 59 59 59 59 60 60 60 60 60 60 61

3.8.2.11.2.2 Catalase activity 61 3.8.2.11.2.2.1 Enzyme assay 61 3.8.2.11.2.3 Ascorbate peroxidase activity 61 3.8.2.11.2.3.1 Enzyme assay 61 3.8.2.11.2.4 Glutathione reductase activity 62 3.8.2.11.2.4.1 Enzyme assay 62 3.8.2.11.2.5 Soluble protein content 62 3.8.2.11.2 Non-enzymatic antioxidants 62 3.8.2.11.2.1 Ascorbic acid content 62 3.8.2.11.2.1.1 Procedure 63 3.8.2.11.2.2 Glutathione content 63 3.8.2.11.2.2.1 Procedure 63 3.8.3 Yield characteristics 63 3.8.4 Statistical analysis 64

MATERIAL AND METHODS Chapter 3

The chapter deals with the description of material used for the study and methods

adopted for the experimentation and determination of various traits during the course

of the investigation. The details of agro-climatic conditions, soil analyses,

experimentation and the techniques and procedures employed in this regard are given

below.

3.1 Plant Material

The Experimental material selected for the present study was mustard {Brassica

juncea L. Czem & Coss.) cultivars. Healthy and authentic seeds of mustard cultivars

namely, Alankar, Varuna, Pusa Bold, Sakha and RH30, were obtained from National

Research Centre on Plant Biotechnology (NRCPB) of the Indian Agricultural

Research Institute (lARI), New Delhi.

3.1.1 Botanical description of mustard

3.1.1.1 Nomenclature

The oleiferous Brassica grown in India are divided into four groups:

1. Brown mustard: Commonly known as rai, raya or laha {Brassica juncea L.

Czem & Coss.)

2. Sarson

a. Yellow sarson: Brassica campestris L. var. Sarson Prain

b. Brown sarson: Brassica campestris L. var. Dichotoma Watt

3. Toria: lahi or maghi lahi Brassica campestris L. var. Toria Duth

4. Taramira or Tara {Eruca sativa Mill.)

In addition, there are two other species, namely Brassica nigra Koch. (Banarasi

rai) and Brassica juncea var. Rugosa (Pahadi rai). These two species do not fall under

any of the four groups. These are, moreover, grown to a limited extent. Mustard

{Brassica juncea L. Czem & Coss.) is the dominant species grown in India (Prakash

1980).

3.1.1.2 Botanical description

Rape and mustard include annual herbs. Roots, in general, are long and tapering.

Toria is more or less a surface feeder but Brown sarson bears long roots with limited

lateral spread enabling its successful cultivation under drier conditions. The height of

the stem varies from 45cm (in some varieties of Toria) to 190cm (in Yellow sarson).

In Toria and Brown sarson, the branches arise at an angle of 30° to 40°. In Yellow

48

sarson, the branches arise laterally at an angle of about 10° to 20° and give the plant a

narrow and pyramidal shape. The inflorescence is a corymbose raceme. In the case of

Yellow sarson, the four petals are spread apart, whereas, in Brown sarson and Toria,

the petals overlap or may be placed apart, depending upon the cultivar. The flowers

bear a hypogynous ovary. In Brown sarson and Toria, the ovary is bicarpellary,

whereas, in Yellow sarson, it may also be tri-or tetra-carpellary. The fruit is sliliqua.

The sliliqua are two-valved, three-valved or four-valved, depending upon the number

of carpels in the ovary. The flowers begin to open from 8:00h and continue up to

12:00hnoon.

3.1.1.3 Genomic relationships of Brassica juncea

There are six species of Br assica that merit attention for their economic importance.

Among the six species, three are diploid: B. campestris, B. nigra and B. oleraceae,

and the other three are amphidiploids: B. juncea, B. napus and B. carinata. The

botanical and genomic relationships between these six species may be represented in

the form of atriangle usually knovm as triangle of U (U 1935) (Figure 1).

Fig 1. The "Triangle of U" representing the genomic relationships among Brassica

species.

Brassica juncea is generally thought to have originated in the Middle East,

whereas, B. rapa and B. nigra species overlapped in the wild, but central Asia and

China are suggested as the sites of primary origin (Prakash 1980). Hemingway

49

(1976), however, considers that B. juncea L. may also have arisen by independent

hybridization at secondary centers in India, China and Caucasus, as B. nigra was

widely used as the commercial spice from early times.

3.2 Agro-Climatic Conditions of Aiigarh

Aligarh has an area of about 5,024sq kms and is situated at 27°52'N latitude, 78°5rE

longitude, and 187.45m altitude above sea level. It has semi-arid and subtropical

climate, with hot dry summers and cold winters. The winter extends from the middle

of October to the end of March. The mean temperatures for December and January,

the coldest months, are about 15°C and 13°C and minimum recorded for any single

day is 2°C and 0.5°C, respectively. The simimer season extends from April to June

and the average temperature for May is 34.5°C and 45.5°C, respectively. The

monsoon extends from the end of June to middle of October. The mean annual

rainfall is about 847.3mm.

More than 85% of the total down pour is delivered during a short span of four

months from Jime to September. The remaining showers are received during winter.

Winter rainfall is especially usefiil for 'Rabi' (winter) crops. The district of Aligarh

has the same soil composition and appearances as those found generally in the plains

of Uttar Pradesh. Different types of soils, such as sandy, loamy, sandy-loam and clay-

loam are found in the district.

3.3 Experimentation

Experiments were conducted in the earthen pots during the winter season of 2003,

2004 and 2005 on mustard (Brassica juncea L. Czem & Coss.) in the green house of

the Department of Botany, Aligarh Muslim University, Aligarh, India under natural

day/night conditions. The Experimental period varied from October to March.

3.3.1 Preparation and analysis of soil

To ensure maximum soil-aeration, thoroughly ploughed soil was collected from

University Agriculture Farm, Aligarh Muslim University, Aligarh for the filling of

earthen pots. Before sowing, soil samples were collected randomly from different pots

for the analysis of the selected soil-characteristics. The physicochemical properties of

soil are given in Table 3.

50

Sandy loam 7.80 90.54 8.89 112.37 15.52 0.310

Sandy loam 8.10 100.16 8.75 110.55 16.00 0.300

Sandy loam 8.00 98.72 8.58 108.65 16.31 0.323

Table 3. Some physicochemical properties of the soil used in the present study.

Experiment 1 Experiment 2 Experiment 3 Soil characteristics (2003-2004) (2004-2005) (2005-2006) Texture pH Available N (mg kg"' soil) Available P (mg kg"' soil) Available K (mg kg"' soil) Available S (mg kg"' soil) Total Cd (mg kg"'soil)

3.3.2 Pot preparation and treatments

Before the start of each Experiment, earthen pots of 23-cm diameter were filled with

soil thoroughly mixed with farmyard manure in 3:1 ratio making a total of 4.0kg soil

pot"'. Appropriate amount i.e., 0, 163.20, 326.16, 652.28, 978.44mg CdCb was

mixed thoroughly with 4.0kg soil to achieve 0, 25, 50, 100 and 150mg Cd

kg"'soil, respectively. In Experiments where application of sulfur was done at the

rate of 0, 50 and lOOmg S kg"'soiI, elemental S was applied to the soil 15 days

before sowing for complete mobilization by microbial activity. For nitrogen

application, it was given at the rate of 0, 40, 80 and 120mg N kg"'soil before

sowing as urea. In Experiment 1 different mustard cultivars were treated with Cd

alone, whereas, in Experiment 2 tolerant and non-tolerant mustard cultivars were

treated with Cd alone or Cd in combination with S. Further, in Experiment 3,

combined doses of S and N were given to Cd-treated tolerant and non-tolerant

mustard cultivars. Plants grown without any treatment (no Cd, S or N) served as

control.

3.3.3 Sowing of seeds

Healthy seeds of mustard cultivars were surface sterilized with 0.5% (v/v) sodium

hypochlorite for 15min and then rinsed three times with deionized water. Before

sowing light application of deionized water was given in each pot to provide

necessary moisture for germination of the seeds. Ten seeds per pot were sown to

avoid germination failure and after the establishment of seedlings, thiiming was done

to retain only two healthy plants of nearly equal size in each pot. In Experiment 1,

twelve pots for each treatment were maintained. Four sets of pots with three pots

(each pot with two plants) in each set were maintained. Each set of pots served as

sampling material at pre-flowering (30days after sowing, DAS), flowering (60DAS),

51

post-flowering (90DAS) stages and harvest (120DAS). Thus, total number of pots

was sixty for each cultivar in Experiment 1. One plant in a pot of each set was

considered as one replicate. Thus, there were three replicates. Diagrammatic

representation of the arrangement of pots for Experiment 1 is shown in Scheme 1. In

Experiment 2, similar distribution of pots was made, but due to seven treatments, the

total number of pots was eighty four for each cultivar. The distribution of pots for

Experiment 2 for determining various characteristics at 30, 60, 90 and 120DAS is

described in Scheme 2. Distribution of pots for Experiment 3 was same as in

Experiments 2 but due to six treatments, total number of pots was sixty for each

cultivar. The distribution of pots for Experiment 3 for determining various

characteristics at 30, 60, 90 and 120DAS is described in Scheme 3.

Crop was irrigated with tap water as and when required. In order to check the

aphid contagion, if any, insecticidal spray of Dimecron-100 was done.

3.4 Experimental Set-up

The Experiments were carried out according to the standard agricultural practices.

Calendar of various operations in each Experiment is given in Table 4, which shows

time schedule for experimentation, treatment and the data collection.

3.4.1 Experiment 1

Experiment 1 was conducted in the winter season of 2003-2004. Surface sterilized

seeds of five mustard cultivars namely, Alankar, Varuna, Pusa Bold, Sakha and RH30

were sovm on 15* October 2003 and the crop was harvested on 13* February 2004.

The treatments in this Experiment were arranged in a factorial randomized block

design. The aim of the Experiment 1 was to study the effects of five levels of Cd (0,

25, 50, 100 and 150mg Cd kg'' soil) on growth, photosynthetic and yield

characteristics and select tolerant and non-tolerant cultivars. Samplings for growth

and photosynthetic characteristics were done at pre-flowering (30DAS), flowering

(60DAS), post-flowering stage (90DAS) and yield characteristics were recorded at

harvest (120DAS). The scheme of treatments for Experiment 1 is given in Table 5.

3.4.2 Experiment 2

Experiment 2 was conducted on the basis of the findings of Experiment 1 in the

winter season of 2004-2005. The sovdng of the seeds of Alankar (tolerant) and RH30

(non-tolerant) cultivars of mustard was done on 20* October 2004 and the crop was

harvested on 18* February 2005. The treatments in this Experiment were arranged in

a factorial randomized block design. The aim of the Experiment 2 was to assess the

52

effect of S application in the alleviation of Cd stress in Cd-tolerant and non-tolerant

mustard cultivars. Sulfur was given at the rate of 0, 50 and lOOmg S kg'' soil prior to

15 days of sowing to plants treated with 0, 50 and 150mg Cd kg'' soil. The response

of Cd tolerant and Cd non-tolerant cultivars to S application under Cd stress was

assessed in terms of the growth, photosynthetic and S and N assimilation, components

of antioxidant system and the yield characteristics. The timing of sampling was same

as for Experiment 1. The scheme of treatments for Experiment 2 is given in Table 6.

3.4.3 Experiment 3

Experiment 3 was conducted on the basis of the findings of Experiments 2 in the

winter season of 2005-2006. The sowing of mustard seeds (tolerant and non-tolerant

mustard cultivars as in Experiment 2) was done on 17* October 2005 and the crop

was harvested on 15* February 2006. The treatments in this Experiment were

arranged in a factorial randomized block design. The aim of the Experiment 3 was to

assess the effect of N application on the effectiveness of S in the alleviation of Cd

stress in Cd-tolerant and non-tolerant mustard cultivars. Sulfur was given at the rate of

0 and 50mg kg"' soil along with 0, 40, 80 and 120mg N kg"' soil to plants treated with

0 and 50mg Cd kg'' soil. The timing of sampling was same as for Experiment 1 and 2.

The scheme of treatments for Experiment 3 is given in Table 7.

3.5 Sampling Technique

To study the growth and photosynthetic characteristics, Cd accumulation in root and

leaf, S and N assimilation and components of antioxidant system, samplings were

done at 30, 60 and 90DAS corresponding to pre-flowering, flowering and post-

flowering stages. For the determination of growth one plant from each pot of a set

corresponding to the sampling time in each cultivar was taken. In a set of pots, there

were three pots for each sampling time. Therefore, three plants served as three

replicates. The remaining one plant in each pot at various sampling stages was used

for photosynthetic measurements and other analyses. There was a separate set of pots

for the determination of yield characteristics. For growth, destructive sampling was

done and plzints were taken to the laboratory. Photosynthetic measurements were

made on intact plants and the plants on which photosynthetic measurements were

made also taken to laboratory for further analyses. For recording yield, plants were

cut at the base, siliqua were plucked and thrashed manually. Seeds were cleaned and

collected separately from each treatment for the measurement of seed yield and its

attributes.

53

3 plants were 3 plants were 3 plants were Yield used for growth used for growth used for growth characteristics & 3 for & 3 for & 3 for were recorded photosynthesis at photosynthesis at photosynthesis at at 120DAS 30DAS 60DAS 90DAS

,.....»„„W,W>W nffM MffM MffM Treatments

.,-"^T^/v/ \^nU \7UU V/V/U

25Cd

50Cd

lOOCd

150Cd

WWW WWW www www UUU V/UV/ UUV UUU

www www www www UUU UUU UUU UUU

www www www www UUU UUU UUU UUU

www www www www ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

Scheme 1. Diagrammatic representation of the arrangement of pots for Experiment 1. Scheme shows the arrangement of pots for one cultivar. The other cuhivars also had similar arrangement of pots.

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 30DAS

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 60DAS

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 90DAS

Yield characteristics were recorded at 120DAS

Treatments f l f t f (mgkg-'soil)^y/ ^ 1 / ^

0

50Cd

l50Cd

50Cd +50S

50Cd

150Cd +50S

150Cd

ft ft ft Y y Y y Y y

ft ft ft

M/ Mf M,

ft ft ft

wv YY WW

ft ft ft

ft ft ft M/ M/ %

ft ft ft Y y TV Yy ft ft ft Y y Y y Y y ft ft ft Y y T y Y y ft ft ft Y y Y y Y y

ft ft ft Y y Y y Y y ft ft ft Y y Y y Y y ft ft ft Y y Y y Y y ft ft ft Y y Y y \ y ft ft ft Y y Y y \ y

ft ft ft Y y Y y T 7 ft ft ft Y y Y y Y y ft ft ft Y y Y y T 7 ft ft ft Y y Y y Y y ft ft ft Y y Y y \ y

f t f t ft ft ft ft ft ft ft ft ft ft

m/ M, M, M, M/ M/ M/ Mi w% M, M, M/

ft ft ft ft ft ft ft

V t Tt Tt Tt ft Tt Tt Tt ft ft ft ml f^^xy ^ ^ ^ ^ ^ ^ \ [ j / ^ ^

Scheme 2. Digramatic representation of the arrangement of pots for Experiment 2. Scheme shows the arrangement of pots for one cultivar. The other cultivar also had similar arrangement of pots.

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 30DAS

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 60DAS

3 plants were used for growth & 3 for photosynthesis, S, N & antioxidants analysis at 90DAS

Yield characteristics were recorded at 120DAS

Treatments ( m g k g - ' s o i l ) ^ ^ ^ ^ ^ ^

50Cd

50Cd +50S

fl ft ft ft ft ft ft ft ft vvYy Y y \ 7 \ y \ y \v \ y \ y

ft ft ft ft ft ft ft ft ft M, M, M, M, M, M, M, M, M,

ft ft ft Y y Y y \ y

ft ft ft ft ft ft Y y Y y Yy Yy \v \v

ft ft ft Y y Y y Y t ft ft ft Y y Y y Y /

ft ft ft Yy YV Yy

50Cd +50S +40N

50Cd +50S +80N

ft ft ft ft ft ft ft ft ft ft ft ft Y y Y y Yy \ y Yy Yy Yy \v \ t \ y \ t Yy

ft ft ft ft ft ft ft ft ft ft ft ft Y y Y y \ y \ y Y y \ y \ y \ y \ y T y Y y Y y

50Cd +50S +120N

ft ft ft ft ft ft ft ft ft ft ft ft Y y Y y Y y Y y Y y \ y Y y Y y Y t Y y Y t Y /

Scheme 3. Digramatic representation of the arrangement of pots for Experiment 3. Scheme shows the arrangement of pots for one cultivar. The other cultivar also had similar arrangement of pots.

Table 4. Experimental calendar.

Preparation of pots Sowing Treatments Cadmium

Sulfur

Nitrogen

Replications

Cultivars

Samplings

Pre-flowering (SODAS) Flowering (60DAS) Post-flowering (90DAS) Harvest (120D AS)

Experiment 1 (2003-2004)

11.10.2003 15.10.2003

19.11.2003 (0,25,50, 100 or 150mgCdkg"'soil)

Three

Alankar, Vanma, Pusa Bold, Sakha, RH30

14.11.2003 14.12.2003 13.01.2004 13.02.2004

Experiment 2 (2004-2005)

04.10.2004 20.10.2004

17.11.2004 (0, 50or lOOmg Cd kg-' soil)

05.10.2004 (0,50orl00mg S kg ' soil)

Three

Alankar (Tolerant) RH30 (Non-tolerant)

19.11.2004 19.12.2004 19.01.2005 18.02.2005

Experiment 3 (2005-2006)

02.10.2005 17.10.2005

18.11.2005 (0 or 50mg Cd kg-' soil)

03 10 2005 (0 or 50mg S kg-' soil)

15.10.2005 (0, 40, 80 or 120mgkg-' soil)

Three

Alankar (Tolerant) RH30 (Non-tolerant)

16.11.2005 16.12.2004 16.01.2005 15.02.2006

Table 5. Scheme of treatments for Experiment 1.

v_.uiiivurs

Alankar

Vanma

Pusa Bold

Sakha

RH30

3Cd

+

+

+

+

+

Treatments (mg kg''

25Cd

+

+

+

+

+

SOCd

Table 6. Scheme of treatments for Experiment 2.

Treatments (mg kg'' soil) OCd + OS

50Cd

I50Cd

50Cd + SOS

50Cd+100S

150Cd + 50S

150Cd+100S

+

+

+

+

+

soil)

lOOCd

Cultivars Alankar (Cd tolerant)

+

+

+

+

+

+

+

Table 7. Scheme of treatments for Experiment 3.

Treatments (mg kg'' soil) OCd + OS + ON

50Cd

50Cd + SOS

SOCd + SOS + 40N

SOCd + SOS + SON

SOCd + SOS+ 120N

+

+

+

+

+

RH30 (Cd

Cultivars Alankar (Cd tolerant)

+

+

+

+

+

+

RH30 (Cd

ISOCd

+

+

+

+

+

non-tolerant)

+

+

+

+

+

+

+

non-tolerant)

+

+

+

+

+

+

3.6 Chemicals

For the analysis of various enzymes activities, chemicals were obtained from Sigma-

Aldrich (St. Louis Mo, USA). Other major and minor salts, buffer components were

procured from MERK and/or SRL. All chemicals were obtained in highest purity

available commercially.

3.7 Parameters Studied

Following parameters were studied at different sampling times.

> Growth characteristics

Root length per plant

Shoot length per plant

Leaf area per plant

Plant dry mass per plant

Physiological and biochemical characteristics

Net photosynthetic rate

Stomatal conductance

Total chlorophyll content

Carotenoids content

Carbonic anhydrase activity

Nitrate reductase activity

ATP-sulfurylase activity

N, S and Cd content

Superoxide dismutase activity

Catalase activity

Ascorbate peroxidase activity

Glutathione reductase activity

Soluble protein content

Glutathione content

Ascorbic acid content

Yield characteristics

Nimiber of siliqua per plant

Number of seeds per siliqua

1000 seeds weight

Seed yield per plant

54

' MlS ' *''^'^ t.y*

3.8 Methodology

3.8.1 Growth characteristics \

Plants were uprooted and length of the main tap root and shoot length wa§,ga3^sured

on a meter scale. For dry mass accumulation, plants were dried in a hot air oven at

80°C till constant weight. Weights of the samples were recorded with the help of an

electronic balance (CY204, Scalteo Ins., Germany).

After separating all the leaves at petiole and stem junction, leaf area per plant

was recorded vnih the help of a leaf area meter (LA21, Systronics, Ahmedabad,

India).

3.8.2 Physiological and biochemical characteristics

3.8.2.1 Net photosynthetic rate and stomatal conductance

Net photosynthetic rate (PN) and stomatal conductance (gs) were measured on fully

expanded uppermost leaves of plants using close chamber infra-red gas analyzer

(IRGA, LiCOR, 6200, Lincoln, NE, USA) at light saturating intensity between 11:00-

12:00h. The capacity of leaf chamber was 1 litre. During the measurements, the air

relative humidity, photosynthetically active radiation, ambient temperature and CO2

concentration were 68±4%, 785±22|amol photons m^s"', 24±3°C and 350±5^mol

mol"', respectively.

3.8.2.2 Chlorophyll and carotenoids content

Total chlorophyll (Chi) and carotenoids (Car) were extracted using the method of

Hiscox and Israelstam (1979) by using dimethyl sulphoxide (DMSO) as an extraction

medium, and estimated and calculated by the method of Amon (1949).

3.8.2.2.1 Extraction

Fresh leaves (lOOmg) were cut into small pieces and collected in test tubes containing

7.0ml of DMSO. The test tubes were covered with black paper and incubated at 45°C

for 40min for the extraction. The content was transferred to a graduated tube and the

final volume was made to 10.0ml with DMSO.

3.8.2.2.2 Estimation

Extract measuring 3.0ml was transferred to a cuvette and the absorbance was read at

645 and 663nm for Chi content and at 480 and 510nm for Car content on UV-VIS

spectrophotometer (SL164, Elico, Hyderabad, India).

3.8.2.2.3 Calculation for chlorophyll and carotenoids content

Total Chi and Car content were calculated according to the following equations.

55

Total Chi content (mg g ' FW) = [(20.2 x OD645) + (8.02 x OD663)] x

Car content (mg g"' FW) = [(7.6 x OD480) - (1.49 x OD510)] x

V dxlOOOxW

dxlOOOxW

Where, V = volume of the extract

W = mass of the leaf tissue taken

d = distance travelled by the light path

OD = optical density at the given wave lengths viz. 645, 663,480 and 5 lOnm

3.8.2.3 Assay of carbonic anhydrase activity

Carbonic anhydrase (CA) facilitates the supply of CO2 to the carboxylation sites. It

catalyzes the reversible hydration of CO2 (Raven 1995, Khan et al. 2004).

H2O + CO2 ^ ^ ' ^ H^ + HCOs"

3.8.2.3.1 Estimation

Carbonic anhydrase activity was measured by adopting the method of Dwivedi and

Randhava (1974). Leaves selected for photosynthetic measurements were used for the

enzyme assay. Leaves were cut into small pieces (2-3mm) in 10.0ml of 0.2M cysteine

in petri dish at 0-4°C. The solution adhering to the leaf surface was removed with the

help of blotting paper followed by the immediate transfer of leaves to test tube having

4.0ml of 0.2M Na-phosphate buffer (pH 6.8). To this, 4.0ml of 0.2M sodium

bicarbonate in 0.02M sodium hydroxide solution and 0.2ml 0.002% bromothymol

blue indicator were added to the tubes. The tubes were kept at 4°C for 20min.

CO2 librated during catalytic action of the enzyme on sodium bicarbonate was

estimated by titration of the reaction mixture against 0.05N hydrochloric acid, using

methyl red as an indicator. The control reaction mixture was also titrated against

0.05N hydrochloric acid. The difference of the sample and the control readings was

noted for the calculation of the enzyme activity.

3.8.2.4 Assay of nitrate reductase activity

The nitrate reductase (NR) activity was estimated by the method of Jaworski (1971),

which is based on the reduction of nitrate to nitrite.

56

NOs' + NADH^ H^ ^ ^ > NO2' + NAD^ + H2O

3.8.2.4.1 Estimation

Fresh leaves (200mg) were cut into small pieces and transferred to plastic vial. To the

vial, 2.5ml of O.IM K-phosphate buffer (pH 7.5) and 0.5ml of 0.2M potassium nitrate

solution were added, followed by the addition of 2.5ml of 5% isopropanol. Later, two

drops of chloramphenicol solution were added to avoid bacterial growth in the

medium. Each vial was incubated for 2h in the dark at 30°C.

From the incubated mixture, 0.4ml was taken in a test tube and 0.3ml each of

1% sulphanilamide and 0.02% N-(l-Naphthyl) ethylenediamine dihydrochloride

(NED-HCL) was added. The test tube was kept for 20min at room temperature for

colour development. The mixture was diluted to 5.0ml by adding sufficient amount of

deionized water. The optical density was read at 540rmi. A blank consisting of 4.4ml

of deionized water and 0.3ml each of sulphanilamide and NED-HCl was also run side

by side.

Standard curve was plotted by taking known graded dilutions of sodium nitrite

from a standard aqueous solution of this salt. The optical density of the samples was

compared with the calibrated curve and nitrate reductase activity was calculated.

3.8.2.5 Assay of ATP-sulfurylase activity

The method of Wilson and Bandurski (1958) was followed for the assay of ATP-

sulfurylase (ATP-S) activity. The details of the procedure are given below.

3.8.2.5.1 Extraction

Extract was prepared by grinding 500mg of fresh leaves in 5.0ml of extraction buffer

[O.IM Tris-HCl (pH 8.0) containing 2mM MgCh, lOOmM KCl and lOmM DTE] in a

chilled glass homogenizer and centrifuged at 5,000x rpm for 15min at 4°C.

3.8.2.5.2 Estimation

The assay was started by adding 0.1ml of extract and 0.4ml of reaction mixture

[consists of 8.0ml deionized water (phosphate and sulphate free), 4.0ml 40mM

MgCl2.6H20, 3.0ml 0.4mM Tris buffer (pH 8.0), 40mg Na2Mo04.2H20, 45mg

Na2ATP and 20^L of inorganic pyrophosphate]. The resultant solution was incubated

at 30°C for lOmin. The reaction was stopped by adding 1.0ml of stop solution

[consists of 18.0ml 0.5M sodium acetate and 100ml 0.5M acetic acid]. Control

was also prepared by substituting NaCl in place of sodium molybdate.

57

ATP + SO4 ^TP-sulfiuylase ^ ^ ^ ^ pp.

pp. Inorganic Pyrophosphatase. .

The inorganic phosphate was estimated by the procedure of Fiske and

SubbaRow (1925). In this procedure, 1.0ml of 5N H2SO4, 0.5ml of 2.5% ammonium

molybdate and 0.1ml of reducing agent [Ig of l-amino-2-naphthol-4-sulphonic acid,

3mg of Na2S03.7H20 and 6mg of Na2S205 in a mortar and pestle and stored in dark at

4°C. 0.25g of this mixture was dissolved in 10.0ml of deionized water] were added to

the aliquot. The volume was made up to 10.0ml by adding deionized water. After a

lapse of 20min, the absorbance was measured at 660nm. The calibration curve was

dravm by using KH2PO4 solution as standard.

3.8.2.6 Determination of nitrogen

The leaves were dried in hot air oven at 80°C for 24h. Oven-dried leaves were

powdered with mortar and pestle and meshed through a 72mm mesh sieve.

3.8.2.6.1 Digestion of leaf powder

Oven-dried leaf powder (lOOmg) was carefully transferred to a digestion tube and

2.0ml of concentrated H2SO4 was added to it. It was heated on a temperature

controlled assembly for about 2h to allow complete reduction of nitrates present in the

plant material. After heating, the contents of the tube turned black. It was cooled for

about 15min at room temperature and then 0.5ml 30% H2O2 was added drop by drop.

The solution was heated again till the colour of the solution changed from black to

light yellow. Again after cooling for about BOmin, additional 3 to 4 drops of 30%

H2O2 were added, followed by reheating for another 15min. The addition of H2O2

followed by careful heating was repeated until the contents of the tube turned

colourless. The peroxide-digested-material was transferred from the tube to a 100ml

volumetric flask with three washings of deionized water. The volume of the

volumetric flask was then made up to the mark (100ml) with deionized water. This

aliquot was used to estimate nitrogen content.

3.8.2.6.2 Estimation of nitrogen

Nitrogen was estimated according to the method of Lindner (1944). A lO.OmI aliquot

of the digested material was taken in a 50.0ml volumetric flask. To this, 2.0ml of 2.5N

sodium hydroxide and 1.0ml of 10% sodium silicate solutions were added to

neutralize the excess of acid and to prevent turbidity, respectively. The volume was

58

made up to the mark with deionized water. In a 10.0ml graduated test tube, 5.0ml

aliquot of this solution was taken and 0.5ml Nessler's reagent was added. The

contents of the test tubes were allowed to stand for 5min for maximum colour

development. The optical density of the solution was read at 525nm.

A standard curve was plotted using different concentrations of ammonium

sulphate solution versus optical density of the solution. The reading of each sample

was compared with the standard calibration curve to estimate the nitrogen content.

3.8.2.7 Determination of sulfur

Oven-dried leaves were powdered with mortar and pestle and meshed through a

72mm mesh sieve.

3.8.2.7.1 Digestion of leaf powder

Oven-dried leaf powder (lOOmg) was taken in digestion tube of 75ml capacity. In

digestion tube, 4.0ml acid mixture (consists of concentrated nitric acid and perchloric

acid in the ratio of 1:1) and 0.0075g of selenium dioxide as catalyst was added. The

digestion was carried out till the digested solution become colourless. Subsequent to

digestion, the volume was made up to 75.0ml with deionized water. The interference

of silica was checked by filtering the contents of the tube.

3.8.2.7.2 Estimation of sulfur

Total sulfur in plant samples was estimated according to turbidimetric method of

Chesnin and Yien (1951). A 5.0ml aliquot was pippeted out from the digested

solution for turbidity development in 25.0ml volumetric flasks. Turbidity was

developed by adding 2.5ml gum acacia (0.25%) solution, l.Og barium chloride

(BaCh, sieved through 40-60mm mesh) and the volume was made up to the mark,

with deionized water. Contents of 25.0ml volumetric flask were thoroughly shaken till

BaCl2 completely dissolved. Turbidity was allowed to develop for 2min. The values

were recorded at 415imi with in lOmin after the turbidity development. A blank was

also run simultaneously after each set of determination.

The amount of sulfate was calculated with the help of a calibration curve

drawn afresh using a series of K2SO4 solutions, prepared following the procedure

similar to aliquot, described as above.

3.8.2.8 Determination of cadmium

Cadmium content was determined in root and leaf samples. Leaves were washed with

deionized water, and the roots were washed with an ice-cold 5mM CaCb solution for

1 Omin to displace extracellular Cd (Rauser 1987). The root/leaf samples were dried

59

for 48h at 80°C, weighed and ground to fine powder, and digested with concentrated

HNO3-HCIO4 (3:1). Cadmium concentration was determined by atomic absorption

spectrophotometer (GBG, 932 plus, Australia).

3.8.2.9 Determination of thiobarbituric acid reactive substances

The level of lipid peroxidation products in the leaves was determined by

thiobarbituric acid reactive substances (TBARS) as described by Dhindsa et al.

(1981). Fresh leaf tissue (200mg) was ground in 0.25% 2-thiobarbituric acid (TBA) in

10% trichloroacetic acid (TCA) using mortar and pestle. After heating at 95°C for

30min, the mixture was quickly cooled on ice bath and centrifuged at 10,000 xg for

lOmin. The absorbance of the supematant was read at 532nm and corrected for non­

specific turbidity by subtracting the absorbance of the same at 600nm. The blank was

0.25% TBA in 10% TCA. The TBARS content was calculated using the extinction

coefficient (155 mM"' cm'').

3.8.2.10 Determination of hydrogen peroxide content

The content of H2O2 in leaves was measured as described by Jana and Chaudhuri

(1981). Fresh leaves (lOOmg) were homogenized with 6.0ml of K-phosphate buffer

(50mM, pH 6.5). The homogenate was centrifiiged at 6,000xg for 25min. To 3.0ml of

the extracted solution, 1.0ml of 0.1% titanium sulfate in 20% H2SO4 was added, and

then centrifiiged at 6,000xg for 15min. The colour intensity of the supematant was

measured at 410nm. The H2O2 content was calculated using the extinction coefficient

(0.28^imor'cm'').

3.8.2.11 Components of enzymatic and non-enzymatic antioxidant system

3.8.2.11.1 Activities of antioxidant enzymes

3.8.2.11.2 Enzyme extraction

Fresh leaf tissue (200mg) was homogenized with an extraction buffer containing

lOOmM K-phosphate buffer (pH 7.0), 0.5% Triton X-100 and 1%

polyvinylpyrrolidone (PVP) using chilled mortar and pestle. The homogenate

was centrifuged at 15,000xg for 20min at 4°C. The supernatant obtained was

used for the enzymatic assays. For ascorbate peroxidase, extraction buffer was

supplemented with 2mM ascorbate. Enzyme extract was used for the assay of

superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase.

3.8.2.11.2.1 Superoxide dismutase activity

The method described by Dhindsa et a/. (1981) was followed with slight modification

for the estimation of superoxide dismutase activity.

60

3.8.2.11.2.1.1 Enzyme assay

Superoxide dismutase activity in the supernatant was assayed by its ability to inhibit

the photochemical reduction. The assay mixture, consisting of 1.5ml O.IM K-

phosphate buffer (pH 7.8), 0.2ml of methionine, 0.1ml enzyme extract with equal

amount of IM NaCOs, 2.25mM NBT solution, 3mM EDTA, 60^M riboflavin and

1.0ml of deionized water, was taken in test tubes which were incubated under light of

15W inflorescent lamp for lOmin at 25°C. Blank 'A' containing all the above

substances of the reaction mixture, along with the enzyme extract, was placed in the

dark. Blank 'B' containing all the above substances of reaction mixture except the

enzyme was placed in light along with the sample. The reaction was terminated by

switching off the light, and the tubes were covered with black cloth. The non-

irradiated reaction mixture containing enzyme extract did not develop light blue

colour. Absorbance of the samples along with Blank 'B' was read at 560nm against

the Blank 'A'. The difference of percent reduction in color between Blank 'B' and the

sample was then calculated.

A reduction of 50% in the colour was considered as one Unit of enzyme

activity.

3.8.2.11.2.2 Catalase activity

Catalase activity in leaves was determined by the method of Aebi (1984) with slight

modification.

3.8.2.11.2.2.1 Enzyme assay

Catalase activity was determined by monitoring the disappearance of H2O2 by

measuring the absorbance at 240run. Reaction was carried in a final volume of 2.0ml

of reaction mixture containing 0.5M K-phosphate buffer (pH 7.2) with 0.1ml 3niM

EDTA, 0.1ml of enzyme extract and 0.1ml of 3niM H2O2. The reaction was allowed

to run for 5min. Activity was calculated by using extinction coefficient 0.036mM"'

cm" . One Unit of enzyme activity is defined as the amount necessary to decompose

1 mol of H2O2 per min at 25°C.

3.8.2.11.2.3 Ascorbate peroxidase activity

Ascorbate peroxidase activity was estimated by the method used by Nakano and

Asada(1981).

3.8.2.11.2.3.1 Enzyme assay

Ascorbate peroxidase activity was determined by the decrease in absorbance of

ascorbate at 290nm, due to its enzymatic breakdown. 1.0ml of 50mM K-phosphate

61

buffer (pH 7.2) contained 0.5mM ascorbate, 0.1 mM H2O2, 0.1 mM EDTA and 0.1ml

enzyme extract. The reaction was allowed to run for 5min at 25°C. Ascorbate

peroxidase activity was calculated by using extinction coefficient 2.8 mM" cm" . One

Unit of enzyme activity is defined as the amount necessary to decompose 1 fimol of

substrate consumed per min at 25°C.

3.8.2.11.2.4 Glutathione reductase activity

Glutathione reductase activity was determined by the method of Foyer and Halliwell

(1976) as modified by Rao (1992).

3.8.2.11.2.4.1 Enzyme assay

Glutathione reductase activity was determined by monitoring the glutathione-

dependent oxidation of NADPH at its absorption maxima of wavelength 340nm.

Reaction mixture (1.0ml) contained 0.2mM NADPH, 0.5mM GSSG and 50|al of the

enzyme extract. The reaction was allowed to run for 5min at 25°C. Corrections were

made for any GSSG oxidation in the absence of NADPH. The activity was calculated

by using extinction coefficient (6.2 mNT cm"'). One Unit of enzyme activity is

defined as the amount necessary to decompose 1 famol of NADPH per min at 25 °C.

3.8.2.11.2.5 Soluble protein content

Soluble protein content of leaves was estimated following the method of Bradford

(1976).

Fresh leaf tissue (200mg) was homogenized in 2.0ml of extraction buffer with

the help of pre-chilled mortar and pestle at 4°C. The homogenate was transferred to

2.0ml tube and centrifiiged at 5000x rpm at 4°C for lOmin. An equal volume of 10%

trichloroacetic acid (TCA) was added to 1.0ml of the crude extract, and allowed to

centrifuge at 3300x rpm for lOmin. The precipitate was then discarded and the pellet

was dissolved completely in O.IN NaOH. To 0.1ml of aliquot, 5.0ml Bradford's

reagent was added and mixed vigorously. The intensity of blue colour developed

within 2min was read at 595nm. Calibration curve was drawn using different

concentrations of Bovine Serum Albumin (BSA) processed similarly as that of

aliquots to calculate protein content.

3.8.2.11.2 Non-enzymatic antioxidants

3.8.2.11.2.1 Ascorbic acid content

Estimation of ascorbic acid (AsA) content was done by the method of Law et al.

(1983) with slight modifications.

62

3.8.2.11.2.1.1 Procedure

Fresh leaf tissue (200mg) was ground in 2.0ml of lOOmM K-phosphate buffer (pH

7.0) with ImM EDTA and centrifuged at 10,000xg for lOmin. The supernatant was

collected and 200nl of 10% TCA was added. After mixing the solution by vortex, it

was allowed to stand for 5min. Following mixing, 10|al of 5M NaOH was added and

centriftjged for 2min. To 200^1 of supernatant, 200(J1 of 150mM K-phosphate buffer

(pH 7.4), 100^1 of lOmM 5',5'-dithiobis-2-nitrobenzoic acid (DTT) were added,

mixed thoroughly and left at room temperature for 15min. After mixing 100^1 of

0.5% N'N-ethylemaleimide (NEM) was added. Samples were mixed by vortex and

incubated at 24°C for 30sec. To each sample 400^1 of 10% TCA, 400^1 H3PO4,400^1

of 4% bipyridyl dye (N'N-dimethyl bipyridyl) and 200^1 of 3% FeCb were added.

After vortex-mixing, samples were incubated at 37°C for Ih and the absorbance was

recorded at 525imi. A standard curve in the range of 10-lOOnmol of ascorbic acid was

used for calibration. Values in both cases were corrected for the absorbance

eliminating the supernatant in the blank prepared separately for AsA and AsA+DHAs.

3.8.2.11.2.2 Glutathione content

Reduced (GSH) content was determined by the glutathione recycling method of

Anderson (1985). The details are as follows.

3.8.2.11.2.2.1 Procedure

Fresh leaf tissue (200mg) was homogenized in 2.0ml of 5%) sulphosalicylic acid under

cold conditions. The homogenate was centriftaged at 10,000xg for lOmin. To 0.5ml of

supernatant, 0.6ml of lOOmM K-phosphate buffer (pH 7.0) and 40|al of 5',5'-dithiobis-

2-nitrobenzoic acid (DTNB) were added. After 2min the absorbance was read at

412mn. Standard curve for calculations was prepared from GSH, covering a range of

10-lOOnmol.

3.8.3 Yield characteristics

Yield is the final manifestation of morphological, physiological and

biochemical traits of a crop.

At harvest (120DAS) following parameters were recorded.

1. Number of siliqua per plant

2. Number of seeds per siliqua

3. 1000 seed weight

4. Seed yield per plant

63

At harvest, siliqua were collected and counted. The number of seeds from each

siliqua was counted. From the produce of the pot, a sample of thousand seeds was

randomly drawn and the weight was recorded using electronic balance. The total

seeds from a plant in each treatment were cleared, sun-dried and weighed to compute

seed yield per plant.

3.8.4 Statistical analysis

The data were analyzed statistically using two way analysis of variance (ANOVA) in

each Experiment. F-value was calculated and the level of significance was determined

at p<0.05 using the SPSS statistical program (ver. 12.0 Inc., Chicago, USA). Least

significant difference (LSD) was calculated for the significant data at/7<0.05. Model

of ANOVA for the Experiments is given in Tables 8, 9 and 10.

64

Table 8. Model of analysis of variance (ANOVA) of Experiment 1.

Source of Variation df SS MSS F. value Sig.

Replication 2

Cultivar 4

Treatment 4

Interaction 16

Error 48

Total 74

Table 9. Model of analysis of variance (ANOVA) of Experiment 2.

Source of Variation df SS MSS F. value Sig

Replication

Cultivar

Treatment

Interaction

Error

Total

2

1

6

6

26

41

Table 10. Model of analysis of variance (ANOVA) of Experiment 3.

Source of Variation df SS MSS F. value SigT

Replication

Cultivar

Treatment

Interaction

Error

Total

2

1

5

5

22

35

EXPERIMENTAL RESULTS

Contents

EXPERIMENTAL RESULTS

4.1 Experiment 1: Screening of Mustard Cultivars Treated with Different Levels of Cadmium 65

4.1.1 Growth characteristics 65 4.1.2 Photosynthetic characteristics 66 4.1.3 Cadmium accumulation 68 4.1.4 Yield characteristics 68 4.2 Summary of Experiment 1 69 4.3 Experiment 2: Study of Alleviation of Cd-induced Effects With

Sulfur Application Cd-tolerant and non-tolerant Mustard Cultivars 69 4.3.1 Growth characteristics 69 4.3.2 Photosynthetic characteristics 70 4.3.3 Sulfur and nitrogen assimilation 71 4.3.4 Cadmivmi accumulation 72 4.3.5 Thiobarbituric acid reactive substances and hydrogen peroxide

content 73 4.3.6 Components of antioxidant system 74 4.3.6.1 Enzymatic antioxidants 74 4.3.6.2 Non-en2ymatic antioxidants 75 4.3.7 Yield characteristics 76 4.4 Summary of Experiment 2 77 4.5 Experiment 3: Coordination of Sulfur and Nitrogen Application in

the Alleviation of Cd-toxicity in Cd tolerant and non-tolerant Mustard Cultivars 78

4.5.1 Growth characteristics 78 4.5.2 Photosynthetic characteristics 78 4.5.3 Sulfur and nitrogen assimilation 79 4.5.4 Cadmium accumulation 80 4.5.5 Thiobarbitxiric acid reactive substances and hydrogen peroxide

content 80 4.5.6 Components of antioxidant system 81 4.5.6.1 Enzymatic antioxidants 81 4.5.6.2 Non-enzymatic antioxidants 82 4.5.7 Yield characteristics 83 4.6 Summary of Experiment 3 83

EXPERIMENTAL RESULTS Chapter 4

Experimental results are presented herein mainly as changes in growth and

photosynthetic characteristics, sulfur and nitrogen assimilation, components of

antioxidant system and yield characteristics of mustard cultivars as influenced by

different concentrations of cadmium, sulfur and nitrogen.

4.1 Experiment 1: Screening of Mustard Cultivars Treated with Different Levels

of Cadmium

The Experiment was conducted to study the effect of five concentrations of Cd viz., 0,

25, 50, 100 and 150mg kg'' soil on Cd accumulation, growth, photosynthetic and

yield characteristics of five mustard cultivars, namely Alankar, Varuna, Pusa Bold,

Sakha and RH30. Tolerance index of the five cultivars of mustard was calculated and

cultivars were designated as Cd tolerant and Cd non-tolerant on the basis of their

performance under Cd stress. Results are described below in detail.

4.1.1 Growth characteristics

The effect of Cd on growth characteristics was found significant at the three sampling

stages. The interaction of Cd treatments and cultivars was also found significant at 60

and 90DAS but was non-significant at SODAS for all the growth characteristics

studied (Figures 2-5). The increase in Cd concentration decreased the growth

characteristics of all the five cultivars at all sampling stages. The observations

recorded at all the sampling stages showed similar pattern of cultivar response to Cd

treatments. The pattern of decrease in growth characteristics for the cultivars was

RH30 >Sakha >Pusa Bold >Varuna >Alankar. Maximum reduction in growth

characteristics was noted with 150mg Cd kg'' soil followed by 100, 50 and 25mg Cd

kg'' soil at all growth stages.

Among cultivars, Alankar showed lesser decrease in growth characteristics

followed by Varuna and Pusa Bold, whereas, RH30 and Sakha exhibited greater

reduction in growth characteristics under Cd stress.

In Alankar, root length was decreased by 9.1, 15.1, 24.2, 37.1% at 30DAS;

7.1, 11.9, 20.2, 32.1% at 60DAS and 5.2, 10.4, 18.0, 27.7% at 90DAS due to 25, 50,

100, and 150mg Cd kg'' soil, respectively, over their respective control. Contrarily, a

higher decrease of 17.0, 27.0, 40.0, 58.0% at 30DAS; 16.4, 30.3, 45.0, 60.6% at

60DAS and 13.0, 23.7, 33.6, 42.6% at 90DAS in root length of RH30 was noted with

65

1

I I

30DAS LSDoo5(CxT) = NS

60DAS ,0.05 (CxT) = 0.89

18

16 ^

14

12 -I

10

8

6 18

16 •

14 •

12

10

8

6 18

16

14

12 H

10

8

6 16

14

12

10

8

6

4 14 1

12

10

8

6 ^

4

2

90DAS LSDo„5(CxT) = NS

Varuna

Pusa Bold

Sakha

RH30

25 50 100 150

-1 Treatments (mg kg" soil)

Fig. 2. Effect of Cd treatments (T) on root length (cm plant'') of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

SODAS

100 90 80 70 60 50 40 30 20

100 90 80 70 60 50 40 30

y-N

•4-'

c SJ O.

F u x: tt, c _a) ^-i

o o C/1

20 90 80

70

60

50 40

30

20 80

70

60

50

40

30

20

10 70

60

50

40

30

20

10

60DAS LSDoo5(CxT) = 2.32

Aiankar

90DAS LSD„o3 (CxT) = 2.08

Varuna

Pusa

Sakha

RH30

25 50 100 150

Treatments (mg kg soil)

Fig. 3. Effect of Cd treatments (T) on shoot length (cm plant"') of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

- • - 30DAS LSDoo5(CxT) = NS

1000

800

600

400 -

200 •

0 1000

800

600 ^

400

200

0 800 1

§ 600

1 400 i to

200 •

0 800

600

400

200

0 700

600

500

400

300

200

100

0

60DAS LSDoo5(CxT)= 10.78

Alankar

90DAS LSDoo5(CxT) = 7.80

Varuna

Pusa Bold

Sakha

RH30

25 50 100

Treatments (mg kg soil)

150

Fig. 4. Effect of Cd treatments (T) on leaf area (cm^ plant"') of five cultivars (C) of mustard (Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

SODAS LSDoo5(CxT) = NS

60DAS LSDo<,5(CxT) = 0.28

Alankar

90DAS LSD„o3(CxT) = 0.57

Treatments (mg kg" soil)

Fig. 5. Effect of Cd treatments (T) on plant dry mass (g plant'') of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days afler sowing (DAS).

25, 50, 100 and 150mg Cd kg"' soil, respectively, over their respective control

(Figures 2,6).

The decrease in shoot length followed the same pattern as observed in root

length. In comparison to control, shoot length of Alankar decreased by 5.4, 10.2, 17.4,

26.2% at 30DAS; 4.6, 8.9, 14.7, 24.8% at 60DAS and 3.4, 6.5, 12.5, 20.4% at 90DAS

with 25, 50, 100 and 150mg Cd kg'' soil, respectively. In RH30, reductions of 10.3,

22.7, 29.6, 47.0% at 30DAS; 14.3, 26.8, 30.6, 50.2% at 60DAS and 8.4, 20.3, 25.6,

42.2% at 90DAS were noted with 25, 50, 100 and 150mg Cd kg'' soil, respectively,

over their respective control (Figures 3,6).

When compared to control, leaf area of Alankar decreased by 8.6, 15.5, 29.3,

37.9% at 30DAS; 9.5, 15.5, 30.5, 40.0% at 60DAS and 5.0, 7.7, 16.9, 26.5% at

90DAS with 25, 50, 100 and 150mg Cd kg'' soil, respectively. Whereas, RH30

showed reductions of 16.7, 26.2, 38.1, 54.7% at 30DAS; 21.9, 30.9, 40.8, 58.1% at

60DAS and 15.9, 21.6, 32.7, 42.5% at 90DAS with 25, 50, 100 and 150mg Cd kg"'

soil, respectively, over their respective control (Figures 4,7).

The trend of plant response to Cd treatments in terms of plant dry mass was

almost similar to those observed for the individual growth characteristics. Plant dry

mass of Alankar decreased by 6.0, 11.3, 25.3, 33.3% at 30DAS; 5.4, 9.5, 21.2, 29.8%

at 60DAS and 3.4, 7.1, 20.3, 26.9% at 90DAS due to 25, 50, 100 and 150mg Cd kg"'

soil, respectively, in comparison to their respective control. While, in RH30, plant dry

mass was decreased by 13.2, 21.5, 39.6, 54.5% at 30DAS; 15.5, 25.3, 41.4, 60.3% at

60DAS and 9.6, 18.1, 31.6, 43.7% at 90DAS with 25, 50, 100 and 150mg Cd kg"'

soil, respectively, over their respective control (Figures 5, 7). The trend of sensitivity

of mustard cultivars to Cd toxicity in terms of tolerance index (calculated on the basis

of plant dry mass) was: RH30 >Sakha >Pusa Bold >Varuna >Alankar (Figure 8).

4.1.2 Photosynthetic characteristics

Photosynthetic characteristics (total chlorophyll content, carotenoids content, carbonic

anhydrase activity, net photosynthetic rate and stomatal conductance) of the five

mustard cultivars decreased significantly with the increasing Cd concentration at all

growth stages (Figures 9-13). The interaction of Cd treatments and cultivars was

significant at all the sampling stages except for carotenoids content which remained

non-significant at 30DAS. The greatest significant reduction in photosynthetic

characteristics was recorded with highest Cd level (150mg Cd kg"' soil) in all the

66

Cultivars

Fig. 6. Per cent change in root and shoot length of five cultivars of mustard {Brassica juncea L.) due to 150mg Cd kg"' soil over control at 30, 60 and 90days after sowing (DAS).

40 -a

N \ - ^ ' ^ 0 ° ' ' S ' " ^ N X - ^ ' % 0 ° ' ' S * " ^ Cultivars

Fig. 7. Per cent change in leaf area and plant dry mass of five cultivars of mustard (Brassicajuncea L.) due to 150mg Cd kg"' soil over control at 30, 60 and 90days after sowing (DAS).

X V

u o B (8

O

v\j -

80 -

60 -

40 -

20 -

0 -

T ,

I I ^ d I 1 I I ^^^9H^^^I ^^^SM^^H ^^^M^^^I ^^BSJ^^B

^^^^H ^ H ^ H ^H^^H ^ l ^ ^ l ^ ^ ^ ^ 1 ^ ^ ^ ^ 1 ^ ^ ^ ^ 1 ^ I' S I

• ^ 30DAS g ^ l bODAb

• • 90DAS

1 1 m

1 ^H *(l l 1 I ^^H

1 19 Alankar Varuna Pusa Bold

Cultivars

Sakha RH30

Fig. 8. Tolerance index of five cultivars of mustard (Brassica juncea L.) treated with 150mg Cd kg"' soil. Tolerance index was calculated as percentage of the plant dry mass obtained in 150mg Cd kg"' soil and control at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates ± S.E.

cultivars. The pattern of decrease in photosynthetic characteristics was: RH30 >Sakha

>Pusa Bold >Varuna >Alankar.

Maximum reduction in total chlorophyll and carotenoids content was observed

in RH30 and least in Alankar under Cd stress at all sampling stages. In Alankar, total

chlorophyll content was decreased by 13.7, 21.6, 32.7, 44.4% at 30DAS; 10.9, 17.4,

22.3, 35.3% at 60DAS; 13.0, 20.0, 28.0, 41.0% at 90DAS and in carotenoids content

by 5.3, 17.4, 21.1, 32.6% at 30DAS; 4.3, 17.1, 25.2, 34.2% at 60DAS; 4.8, 18.6,25.7,

38.3% at 90DAS with 25, 50, 100 and 150mg Cd kg"' soil, respectively, over their

respective control. RH30 showed greater reduction in total chlorophyll content, which

was 20.3, 31.6, 43.6, 60.1% at 30DAS; 26.3, 41.3, 52.6, 67.7% at 60DAS; 26.9, 46.1,

53.8, 69.2% at 90DAS and carotenoids content decreased by 16.0, 33.3, 40.7, 49.3%

at 30DAS; 19.7, 34.3, 44.5, 54.0% at 60DAS; 18.7, 34.9, 45.5, 55.3% at 90DAS with

25, 50, 100 and 150mg Cd kg'' soil, respectively, over their respective control

(Figures 9,10,14).

The decrease in carbonic anhydrase activity in Alankar was 8.6, 14.5, 32.7,

40.7% at 30DAS; 9.1, 16.4, 36.8, 44.7% at 60DAS and 9.3, 20.3, 39.4, 45.2% at

90DAS with 25, 50, 100 and 150mg Cd mg"' soil, respectively, over their respective

control. Carbonic anhydrase activity in RH30 was decreased by 22.1, 35.0, 55.4,

67.5% at 30DAS; 26.6, 46.9, 58.3, 70.6% at 60DAS and 29.4, 48.8, 63.5, 78.9% at

90DAS with 25, 50, 100 and 150mg Cd mg'' soil, respectively, over their respective

control (Figures 11,15).

In comparison to control, net photosynthetic rate of Alankar was decreased by

7.9, 14.3, 32.5, 37.7% at 30DAS; 7.1,13.2, 30.8, 33.8% at 60DAS and 5.9, 12.5, 27.6,

29.9% at 90DAS with 25, 50, 100 and 150mg Cd kg'' soil, respectively, over their

respective control. Whereas, in RH30, it was decreased by 12.4, 27.9, 46.1, 53.5% at

30DAS; 14.3, 32.0,47.4, 54.7% at 60DAS and 14.1, 33.3,47.8, 60.7% at 90DAS with

25, 50, 100 and 150mg Cd kg'' soil, respectively, over their respective control

(Figures 12,16).

The decrease in stomatal conductance followed the same pattern as observed

in net photosynthetic rate. When compared with the respective control, reduction in

stomatal conductance in Alankar was 9.7, 16.1, 25.2, 40.3% at 30DAS; 11.3, 17.7,

30.0, 42.3% at 60DAS and 9.8, 20.2, 30.4, 45.5% at 90DAS with 25, 50, 100 and

150mg Cd kg"' soil, respectively. RH30 showed decrease in stomatal conductance by

16.4, 26.5, 49.8, 57.8% at 30DAS; 18.9, 30.4, 52.6, 60.3% at 60DAS and 21.0, 34.9,

67

30DAS LSD„o5(CxT) = 0.04

2.0

1.5 ^

1.0

0.5 ^

0.0 2.0

1.5 -

1.0 -

0.5

u.

au 00

e N w /

c (1>

e o u • > .

J: a. o t^

o JS u 4-<

0.0 ?,.o

1.6

1.2

0.8

0.4 1.6

1.2

0.8

0.4 •

0.0 1.6 1

1.2

0.8 -

0.4

0.0

60DAS LSDooj (CxT) = 0.06

Alankar

Varuna

Pusa Bold

Sakha

RH30

25 50 100

Treatments (mg kg soil)

90DAS LSDo.05 (CxT) = 0.04

150

Fig. 9. Effect of Cd treatments (T) on total chlorophyll content (mg g'' FW) of five cultivars (C) of mustard (Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

30DAS LSD,o5(C'<T) = NS

0.20

0.15

0.10

0.05 •

to

o u CO

-a '5 c <u o u ca

U

0.00 0.25

0.20

0.15

0.10

0.05

0.00 0.20

0.18

0.16

0.14

0.12

0.10

0.08 0.20

0.15

0.10

0.05

0.00 0.16

0.12 -

0.08

0.04

0.00

60DAS LSDoo5 (CxT) = 0.007

Alankar

90DAS LSDQQ5 (CXT) = 0.007

Varuna

Pusa Bold

Sakha

RH30

25 50 100 150

Treatments (mg kg' soil)

Fig. 10. Effect of Cd treatments (T) on carotenoids content (mg g"' FW) of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

O U

>

< u

SODAS LSDoo5(CxT) = 0.21

10

8^

6

4

2

0 10

8

6

4

2

0 10

8

6

4

2

0

4 ^

2

60DAS LSDg jj (CxT) = 0.22

Aiankar

90DAS LSDooj (CxT) = 0.26

Varuna

Pusa Bold

Sakha

RH30

25 50 100

Treatments (mg kg' ' soil)

150

Fig. 11. Effect of Cd treatments (T) on carbonic anhydrase activity (mmol CO2 g"' FW) of five cultivars (C) of mustard (Brassica Juncea L.) at 30, 60 and 90days after sowing (DAS).

o u "o e

c

o o.

Z

30DAS LSDo„5(CxT) = 0.40

25

20

15

10

5

0 20

15 -

10 -

5 -

0 20

18

16

14

12

10

8

6 18 16 14 12 10 8 6 4 2 0

16 14 12 10 8 6 •

4 •

2 0

60DAS LSDoo5(CxT) = 0.51

Alankar

90DAS LSDo<,3(CxT) = 0.33

Pusa Bold

Sakha

RH3G

25 50 100 150

-1 Treatments (mg kg" soil)

Fig. 12. Effect of Cd treatments (T) on net photosynthetic rate ( trnol CO2 m" s'') of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

30DAS LSD(,o5(CxT)= 11.02

25

20 -i

15

10 -

5 •

0 20

15

10

5 •

l/l

's "o

g ^•m^

0)

u s CQ

u 3

o u BJ

o on

0 20

18

16

14

12 10

X 6

18

14 12 10 8 6 4 2 0

16 14 12 10 8

6 4 -I 2 0

60DAS LSDo,„(CxT)= 12.21

Alankar

90DAS LSDoo5 (CxT) = 48.40

Varuna

Pusa Bold

Sakha

RH30

25 50 100 150

Treatments (mg kg soil)

Fig. 13. Effect of Cd treatments (T) on stomatal conductance (mmol m' s'') of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

N\-^' '^<vv^^°' ' S-^" ^ ' ^ ' N X - ^ ' ^ < ^ . . ^ ° ' ^ ^-^ ' ^ ' '

Cultivars

Fig. 14. Per cent change in total chlorophyll content and carotenoids content of five cultivars of mustard {Brassica juncea L.) due to 150mg Cd kg"' soil over control at 30, 60 and 90days after sowing (DAS).

Alankar Varuna PusaBold Sakha RH30

Cultivars

Fig. 15. Per cent change in carbonic anhydrase activity of five cultivars of mustard {Brassica juncea L.) due to 150mg Cd kg'' soil over control at 30, 60 and 90days after sowing (DAS).

^X.^''\^^,M' S- ' ^ ' ^ ' N X ^ ^ ' % < ^ S . ^ ° ' ' ^-^ ' ^ ' '

Cultivars

Fig. 16. Per cent change in net photosynthetic rate and stomatal conductance of five cultivars of mustard {Brassica juncea L.) due to 150mg Cd kg"' soil over control at 30, 60 and 90days after sowing (DAS).

54.2, 68.1% at 90DAS with 25, 50, 100 and 150mg Cd mg'' soil, respectively, in

comparison to their respective control (Figures 13,16).

4.1.3 Cadmium accumulation

Root and leaf Cd accumulation increased with the increase in soil Cd concentration

and was significant at 30, 60 and 90DAS (Figures 17-18). The effect of Cd treatment

and cultivar interaction was also found significant at all the growth stages. For every

concentration used in the study, Cd concentration was more pronounced in the roots

than the leaves in all the five cultivars. The trend of Cd accumulation in root and leaf

of five cultivars was: RH30 >Sakha >Pusa Bold >Varuna >Alankar at all growth

stages.

Among cultivars, RH30 accumulated highest amount of Cd in root and leaf,

whereas, Alankar accumulated least. Maximum accumulation of Cd in the root of

RH30 was 354.2, 403.7, 452.8^g Cd g"' DW and in the leaf the accumulation was

137.1, 205.6, 130.2^g Cd g"' DW at 30, 60 and 90DAS, respectively of plants treated

with 150mg Cd kg"' soil. Root and leaf of Alankar accumulated 273.1, 330.8,

381.2^g Cd g-' DW and 70.7, 95.3, 51.2^g Cd g- DW at 30, 60 and 90DAS,

respectively, when treated with 150mg Cd kg'' soil (Figures 17-19).

4.1.4 Yield characteristics

The increasing concentration of Cd significantly decreased the yield characteristics of

all the five cultivars (Figure 20). The interaction of Cd treatments and cultivars was

also significant. Maximum decrease in yield characteristics was noted with 150mg Cd

kg'' soil. The cultivars RH30 and Sakha exhibited similar and greatest decrease in

yield characteristics, whereas, Alankar showed lowest decrease followed by Varuna

and Pusa Bold. The order of performance for yield characteristics of cultivars was

Alankar >Varuna >Pusa Bold >Sakha >RH30.

In Alankar, the decrease in number of siliqua per plant was 11.5, 22.7, 30.4,

35.7%; number of seeds per siliqua was 10.6, 22.7, 32.2, 33.8%; 1000 seed weight

was 13.7, 25.0, 35.8, 41.4% and seed yield was 13.7, 21.6, 28.1, 32.5% due to 25, 50,

100 and 150mg Cd kg" soil, respectively, over their respective control. In contrast,

RH30 showed greater reduction in number of siliqua per plant which was 24.5, 35.5,

43.5, 53.8%, number of seeds per siliqua was decreased by 25.2, 39.3, 51.1, 55.9%,

1000 seed weight by 25.6, 37.4, 53.2, 60.0% and seed yield by 22.4, 35.3, 45.4,

52.7% with to 25, 50, 100 and 150mg Cd kg"' soil, respectively (Figures 20-21).

68

SODAS LSD„o3(CxT) = 6.21

Q

'oo 60

B

c o o

U 100 i o o

60DAS LSDo_o3(CxT)= 13.79

Alankar

90DAS LSDoo5(CxT) = 6.33

25 50 100 150

Treatments (mg kg" soil)

Fig. 17. Effect of Cd treatments (T) on root Cd content (i^g g"' DW) of five cultivars (C) of mustard (Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

SODAS LSDoo5(CxT) = 2.92

60DAS LSDo,o5(CxT) = 2.37

Alankar

90DAS LSDoos (CxT) = 2.42

25 50 100 150

Treatments (mg kg soil)

Fig. 18. Effect of Cd treatments (T) on leaf Cd content (^g g"' DW) of five cultivars (C) of mustard {Brassica juncea L.) at 30, 60 and 90days after sowing (DAS).

N \ - ^ ^ 0 ° ' ' S ' ^^' N\- '%<,s. °' ^-^' ^ ' ' ^j-o>'

Cultivars

Fig. 19. Per cent change in root Cd content and leaf Cd content of five cultivars of mustard {Brassica juncea L.) due to 150mg Cd kg"' soil over control at 30, 60 and 90days after sowing (DAS).

100

80

60 ^

40

20

0 100

80

60

40 -

20 -

Number of siliqua per plant LSDoo5(CxT)=1.83

Number of seeds per siliqua LSDoo5(CxT) = 0.31

Atankar

1000 seed weight (g) LSD„o3(CxT) = 0.12

Seed yield (g plant'') LSDo„5(CxT) = 0,14

-8 S- ^

0 80

60

40

20

Varuna

60

50

40

30

20

10

0

Pusa Bold

s ^ ^

25 50 100 150

-1 Treatments (mg kg soil)

Fig. 20. Effect of Cd treatments (T) on number of siliqua per plant, number of seeds per siliqua, 1000 seed weight (g) and seed yield (g plant'') of five cultivars (C) of mustard {Brassica juncea L.) at harvest i.e., 120days after sowing (DAS).

Alankar Vanina PusaBold Sakha RH30 Alankar Vanina PusaBold Saidia RH30

Cultivars

Fig. 21. Per cent change in number of siliqua per plant, number of seeds per siliqua, 1000 seed weight and seed yield of five cultivars of mustard (Brassica juncea L.) due to 150mg Cd kg"' soil over control at harvest i.e., 120days after sowing (DAS).

4.2 Summary of Experiment 1

• The accumulation of Cd in root and leaf was found Cd dose dependent. The

accumulation was maximum in RH30 followed by Sakha, Pusa Bold, Varuna

and Alankar.

• Cadmium treatments resulted in an overall reduction in growth, photosynthetic

and yield characteristics in mustard cultivars with more pronounced decrease

with the 150mg Cd kg'' soil treatment.

• Among cultivars, RH30 showed greater reduction in growth, photosynthetic

and yield characteristics, whereas, least decrease in these characteristics was

found in Alankar.

• The trend of sensitivity of mustard cultivars to Cd toxicity was: RH30 >Sakha

>Pusa Bold >Varuna >Alankar.

• On the basis of overall performance of the cultivars under Cd stress, Alankar

emerged as Cd tolerant and RH30 as Cd non-tolerant.

4.3 Experiment 2: Study of Alleviation of Cd-induced Effects with Sulfur

Application in Cd-tolerant and non-tolerant Mustard Cultivars

Experiment 2 was conducted on the basis of the findings of Experiments 1. As

observed in Experiment 1, Alankar emerged as Cd-tolerant and RH30 as Cd non-

tolerant cultivar. The treatment 150mg Cd kg"' soil caused maximum decrease in the

observed characteristics and 50mg Cd kg"' soil was moderate toxic in effects.

Therefore, the present Experiment was aimed to study the alleviation potential of

elemental S (0, 50 and lOOmg S kg'' soil) under the moderate (50mg Cd kg'' soil) and

extreme (150mg Cd kg'' soil) Cd levels by studying the changes in Cd accumulation,

growth and photosynthetic characteristics, sulfur and nitrogen assimilation,

components of antioxidant system and yield characteristics of tolerant (Alankar) and

non-tolerant (RH30) mustard cultivars.

Detailed results of this Experiment are presented in the following section.

4.3.1 Growth characteristics

Growth characteristics (root length, shoot length, leaf area and plant dry mass)

decreased in both the cultivars treated with Cd and the decrease was proportional to

the concentration of Cd (50 and 150mg Cd kg'' soil). Sulfur application nullified the

effects of 50mg Cd kg"', but could not ameliorate the effects of 150mg Cd kg' soil.

Ironically, the application of 50mg S kg"' soil proved most effective in improving

growth characteristics of both the cultivars treated with 50mg Cd kg'' soil. Whereas,

69

no such effect was observed for the supplementation of lOOmg S kg"' soil (Figures 22-

23).

Both the levels of Cd (50 and 150mg Cd kg'' soil) significantly reduced the

growth characteristics of both the cultivars. The extent of decrease was greater in non-

tolerant cultivar RH30 than the tolerant cultivar Alankar. In Alankar, the variation in

the reduction of plant dry mass was 10.5 and 35.7% at 30DAS; 7.9 and 27.9% at

60DAS and 7.1 and 27.9% at 90DAS due to 50 and 150mg Cd kg"' soil, respectively,

in comparison to their respective control. In RH30, the decrease in plant dry mass due

to 50mg Cd kg"' soil was about 25% and 50% due to 150mg Cd kg"' soil at all

sampling times (Figures 24-25).

The application of 50mg S kg"' soil completely overcome the effects of 50mg

Cd kg"' soil and also increased the growth characteristics. This increasing effect of S

was seen in Alankar. Root length, shoot length, leaf area and plant dry mass of

Alankar treated with 50mg Cd kg"' soil were increased by 1.5, 3.9, 1.6, 1.4% at

30DAS; 3.3, 4.7, 4.3, 2.6% at 60DAS and 2.6, 3.6, 3.9, 1.9% at 90DAS, respectively,

over their respective control due to 50mg S kg"' soil (Figures 22-25).

In RH30, supplementation of 50mg S kg"' soil reduced the decrease in growth

characteristics caused by 50mg Cd kg"' soil (Figures 22-25). The effect of

supplementation of lOOmg S kg"' soil in the alleviation of the effects of 50mg Cd kg"'

soil was lesser than 50mg S kg"' soil. Furthermore, no ameliorative effect of lOOmg S

kg'' soil was observed in plants treated with 150mg Cd kg"' soil (Figures 22-25).

4.3.2 Photosynthetic characteristics

Significant reductions in photosynthetic characteristics (total chlorophyll content,

carotenoids content, carbonic anhydrase activity, net photosynthetic rate and stomatal

conductance) were noted with 50 and 150mg Cd kg'' soil in both the cultivars.

However, the extent of decrease was higher in non-tolerant cultivar RH30 than

Alankar (tolerant). The application of 50mg S kg"' soil alleviated and improved the

photosynthetic characteristics of both the cultivars treated with 50mg Cd kg" soil.

The application of lOOmg S kg"' soil proved ineffective in the alleviation of 150mg

Cd kg"' soil-induced toxicity (Figures 26-28, 29-31).

Lesser decrease in photosynthetic characteristics was noted when plants were

treated with 50mg S kg"' soil plus 50mg Cd kg"' soil in comparison to SOmg Cd kg"'

soil alone. In Alankar, supplementation of 50mg Cd kg"' soil treated plants with 50mg

S kg"' soil limited the decrease in total chlorophyll and carotenoids content and

70

— n.

OS ^ - '

ri II

16

14

12

10

8

6

4

2

0

25

20

15 •

10

LSD„o5(CxT) = 0.50

oc

OS > - ^

0

25

20 •

15 -

10

5 ]

•— Alankar RH30

LSD„„3(CxT) = 0.32

30DAS I • • I — - — T

LSDoo5 (CxT) = 0.40 LSD„„(CxT) = 0.48

60DAS T ' I-" ' T . f • — - . -^ -

LSD„„, (CxT) = 0.39 LSD<,03 (CxT) =1.61

90DAS

r 50

• 40

30 f,-r

20

10

0

100

80

60 t C w a .

h40 ^ 1

- 20

0

120

100

80

0 0 ' * j

c c 60 -^ "=

40

20

Control 50Cd 150Cd 50Cd 50Cd 150Cd ISOCd Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S + 1 0 0 S + 5 0 S + lOOS + SOS + lOOS + 50S + lOOS

Treatments (mg kg'' soil)

Fig. 22. Effect of sulfur (S) application on root and shoot length (cm plant'') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

80

60

<*- a. 40

20

0

1000

800

o -^ 600 u c

-I i 400-1

200 -

0 800

600 -

« c ;2 -E. 400

200

LSD„„3(CxT) = 2.99 Alankar RH30 LSDoo5 (CxT) = 0.04

30DAS

LSDooj (CxT) = 20.41 LSDoo, (CxT) = 0.10

60DAS

LSD„„3 (CxT) =19.40 LSD„„, (CxT) = 0.79

90DAS

r 2.0

1.6

1 2 =3 -

0.8 I ^

0.4

0.0

r 14

12

I- 10

6

• 4

• 2

0

40

30

20

10

Control 50Cd l50Cd 50Cd 50Cd 150Cd 150Cd

+ SOS + lOOS + SOS + IOCS

Control SOCd ISOCd SOCd SOCd ISOCd ISOCd

+ 50S + IOCS + 5 0 S + IOCS

•^ a . c oc a '—-

to •—'

Treatments (mg kg" soil)

Fig. 23. Effect of sulfur (S) application on leaf area (cm^ plant"') and plant dry mass (g plant'') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

50Cd ISOCd 50Cd

+ 50S

50Cd

+ lOOS

150Cd

+ 50S

ISOCd

+ 100S

SOCd ISOCd SOCd

+ 50S

SOCd

+ IOCS

ISOCd

+ 50S

ISOCd

+ lOOS

Treatments (mg kg" soil)

Fig. 24. Per cent change in root and shoot length of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

50Cd 150Cd 50Cd

+ 50S

50Cd

+ lOOS

ISOCd

+ 50S

150Cd

+ lOOS

50Cd ISOCd 50Cd

+ 50S

50Cd

+ lOOS

ISOCd

+ 50S

ISOCd

+ lOOS

Treatments (mg kg' soil)

Fig. 25. Per cent change in leaf area and plant dry mass of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) imder Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

2.0

1,5 •

1.0 •

0.5

0.0

2.5

2.0 -

1.5 •

1.0

0.5

0.0

1.2

1.0 -

0.8 -

0.6

0.4 -

0.2

0.0

LSDoo5(CxT) = 0.05 Alankar RH30 LSDoo5(CxT) = 0.002

30DAS r r

LSDoo5 (CxT) = 0.05 LSD„o5(CxT) = 0.003

60DAS

LSDo.o3(CxT) = 0.03 LSDoo5 (CxT) = 0.003

90DAS

Control 50Cd 150Cd 50Cd 50Cd I50Cd l50Cd Conlroi 50Cd l50Cd 50Cd 50Cd l50Cd 150Cd

+ SOS + lOOS + SOS + lOOS + SOS + lOOS + SOS + lOOS

0.25

0.20

0.15

0 10

o o V5

•o o c (L>

o

u. ~p

0/J OJJ E "•—'

0.05

0.00

0.25

0.20

c

0.15 I i~ T3 T •5 M

\ 0.10 B. E o --'

0.05

0.00

r 0.20

0.15

0.10 Y'^ C 00

£ E

• 0.05

0.00

Treatments (mg kg" soil)

Fig. 26. Effect of sulfur (S) application on total chlorophyll content (mg g"' FW) and carotenoids content (mg g'' FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

10 2 -

0 I

^1

• > ofi

0

12

10

8 -I

3 -

1 -

LSDQ 05 (CxT) = 0.03 Alankar RH30

30DAS

LSD„„ (CxT) = 0.09

60DAS

LSDo„5 (CxT) = 0.04

90DAS

Control 50Cd 150Cd 50Cd + 50S

50Cd + lOOS

!50Cd + 50S

150Cd + 100S

Treatments (mg kg" soil)

Fig. 27. Effect of sulfur (S) application on carbonic anhydrase activity (nmol CO2 g"' FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

20 1

16

12

4 -

0

24

20 -

16 -

12 -

0

16

12

8 -

LSD„„3(CxT) = 0.45 -•— Alankar - O - RH30 LSDoo5(CxT) = 8.03

400

• 300

- 200

100

30DAS

LSD<,<,3(CxT) = 0.92 LSD„o3 (CxT) = 8.99

0

500

400

- 300

- 200

100

60DAS

LSDoo3(CxT) = 0.37 LSDoo5 (CxT) = 8.29

0

400

- 300

• 200

100

90DAS

Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 505 + lOOS + 505 + lOOS

Control 50Cd 150Cd 50Cd 50Cd l50Cd 150Cd

+ 505 + lOOS + 505 + lOOS

Treatments (mg kg" soil)

Fig. 28. Effect of sulfur (S) application on net photosynthetic rate (fjmol CO2 m" s"') and stomatal conductance (mmol m" s"') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

Alankar ^ O RH30

50Cd ISOCd 50Cd

+ 50S

50Cd

+ lOOS

150Cd

+ 50S

150Cd

+ lOOS

50Cd ISOCd 50Cd

+ 50S

50Cd

+ lOOS

150Cd

+ 50S

ISOCd

+ lOOS

Treatments (mg kg"' soil)

Fig. 29. Per cent change in total chlorophyll content and carotenoids content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

-80

10

0

-10 ^

-20

.^ -30 -> 1 -40 <

U -50

-60

-70

-80 50Cd 150Cd 50Cd + 50S

50Cd + lOOS

150Cd + 50S

150Cd + lOOS

90DAS

Treatments (mg kg'' soil)

Fig. 30. Per cent change in carbonic anhydrase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulflir (S) doses at 30, 60 and 90days after sowing (DAS).

10 1

« -30

S -40

Alankar ^ ^ RH30 30DAS

-70

10

0

-10 •

-20

-50 -

-60

-70

1 r-

60DAS

-10

-20

-30

• -40

-50

50Cd rSOCd 50Cd

+ 50S

50Cd l50Cd

+ lOOS + SOS

ISOCd

+ lOOS

50Cd ISOCd SOCd

+ 50S

50Cd ISOCd ISOCd

+ lOOS + SOS + lOOS

-60

10

0

-10

-20

-30

-40

-50

a C8

E o

-60

-70

-70

Treatments (mg kg' soil)

Fig. 31. Per cent change in net photosynthetic rate and stomatal conductance of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

stomatal conductance caused by 50mg Cd kg"' soil alone. Finally, the decrease noted

in total chlorophyll and carotenoids content and stomatal conductance was 7.9, 2.7

and 8.0% at 30DAS; 4.6, 2.7 and 7.1% at 60DAS and 3.3, 0.7 and 5.3% at 90DAS,

respectively. Similar effect was noted for carbonic anhydrase activity and net

photosynthetic rate, which was decreased by 1.7 and 0.6% at 30DAS and 1.5 and

0.4% at 90DAS, respectively. At 60DAS, the application of 50mg S kg'' soil not only

reduced the adverse effects of Cd (50mg Cd kg'' soil) but overcome the effect and

increased the carbonic anhydrase activity and net photosynthetic rate. The increase

was found to be 0.3 and 1.5% at 60DAS (Figures 29-31).

In RH30, the application of 50mg S kg'' soil only reduced the adverse effects

of 50mg Cd kg'' soil at all the growth stages. The decrease in total chlorophyll

content, carotenoids content, carbonic anhydrase activity, net photosynthetic rate and

stomatal conductance was 19.4, 21.8, 20.0, 14.6, 15.0% at 30DAS; 22.6, 18.8, 21.5,

13.6, 16.2% at 60DAS and 25.0, 25.4, 24.6, 15.9, 20.7% at 90DAS, respectively, over

their respective control (Figures 26-31). Whereas, no ameliorative effect of lOOmg S

kg'' soil was observed in plants treated with 150mg Cd kg'' soil in both the cultivars

(Figures 29-31).

4.3.3 Sulfur and nitrogen assimilation

Sulfur and nitrogen assimilation varied differentially under Cd stress. Cadmium stress

increased the ATP-sulfurylase activity and S content, whereas, nitrate reductase

activity and N content were decreased in both cultivars at all growth stages (Figures

32-33). ATP-sulfurylase activity and S content increased in Cd treated plants and the

increases were higher in Alankar than RH30. In Alankar, the increase in ATP-

sulfurylase activity and S content due to 50 and 150mg Cd kg'' soil was 39.2, 44.5%

and 21.9, 25.5% at 30DAS; 35.4, 45.5% and 21.3, 24.7% at 60DAS and 27.3, 36.1%

and 15.0, 20.0% at 90DAS, respectively, in comparison to their respective control.

This increase in RH30 was 22.6, 30.3% and 7.4, 12.0% at 30DAS; 23.4, 33.8% and

10.4, 13.3% at 60DAS and 20.0, 25.5% and 5.0, 10.1% at 90DAS, respectively, in

comparison to their respective control (Figure 34).

The application of 50mg S kg'' soil further increased the ATP-sulfurylase

activity and S content of Cd treated plants of both the cultivars. Maximum significant

increase was noted with 50mg S kg'' soil and 50mg Cd kg"' soil treated plants. The

effect of lOOmg S kg'' soil plus 50mg Cd kg'' soil was almost similar to 50mg S kg''

71

50 1

40

B. 30

20

10 -

0

60

50

40 -

30 -

20

10

0

40

Z^'.S 30

20 •

10 -

LSDoo5(CxT)=1.68 -•— Alankar

- O - RH30 LSD„o5(CxT) = 0.08

30DAS T " 1 I

LSDooj (CxT) = 2.02 LSDoo5(CxT) = 0.09

60DAS

•7 ' I -

LSD„,o5 (CxT) =1.28 LSDoo5 (CxT) = 0.05

90DAS

Control 50Cd l50Cd 50Cd 50Cd 150Cd l50Cd

+ SOS + lOOS + SOS + lOOS

Control SOCd ISOCd SOCd SOCd ISOCd ISOCd

+ SOS + lOOS +503 - lOOS

2.5

2.0

- 1.5 S

c o

1.0 " (/2

• 0.5

0.0

r 3,0

2.5

20 _

• 1.5 S c o

• 1.0

0.5

0.0

r 2.0

1.5

1.0

0.5

0.0

Treatments (mg kg" soil)

Fig. 32. Effect of sulfur (S) application on ATP-suifurylase activity (|imol Pi mg"' protein min'') and sulfur content (%) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

500

400

300

Z 200 -

100 •

600 1

500

400

300 -

200 -

100 -

0

400

300

200

100

LSDoo3 (CxT) = 7.25 -•— Alankar

-O- RH30 LSDoo5 (CxT) = 0.09 r 5

30DAS ' I - T

LSDooj (CxT) =10.55 LSDo,o5 (CxT) = 0.07

60DAS • • f ^ ~ - ' 1 • • , • • •—• . . , . 11

LSD„„5 (CxT) = 8.16 LSDooj (CxT) = 0.08

9GDAS

3 e c 1) B O

2 o

0

r 6

3 I c o o

[2 ^

• 1

0

5

• 4

• 3 c o c o o

Control 50Cd 150Cd 50Cd 50Cd ISOCd 150Cd

+ SOS + lOOS + 50S + lOOS Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S + lOOS +50S + lOOS

Treatments (mg kg"' soil)

Fig. 33. Effect of sulfur (S) application on nitrate reductase activity (nmol NO2 g"' FW h'') and nitrogen content (%) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are the mean of three replicates.

Alankar P ? ^ RH30

U 50Cd 150Cd 50Cd 50Cd

+ 50S

150Cd 150Cd

lOOS + SOS + lOOS 50Cd 150Cd 50Cd 50Cd ISOCd ISOCd

+ SOS + lOOS + SOS + IOCS

Treatments(mgkg' soil)

Fig. 34. Per cent change in ATP-sulfuryiase activity and sulfur content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

soil plus 50mg Cd kg'' soil. Further, no ameliorative effect of lOOmg S kg'' soil was

observed in plants treated with 150mg Cd kg'' soil.

Cadmium treatments significantly decreased NR activity and N content of

both the cultivars but the decrease was greater in RH30 than Alankar. The activity of

NR and N content of Alankar was decreased by 14.6, 11.0% and 27.2, 25.4% at

30DAS; 15.5, 10.6% and 29.9, 25.6% at 60DAS and 21.0, 17.2% and 34.4, 30.4% at

90DAS with 50 and 150mg Cd kg''soil, respectively, in comparison to their respective

control. Whereas, in RH30 the decrease in NR activity and N content was 26.4,19.4%

and 48.1, 38.6% at 30DAS; 34.7, 22.5% and 50.0, 39.4% at 60DAS and 37.7, 27.7%

and 58.6, 45.7% at 90DAS with 50 and 150mg Cd kg''soil, respectively, in

comparison to their respective control (Figure 35).

The reductions in NR activity and N content due to Cd were lowered by S

supplementation. Lesser decrease in NR activity and N content was observed when

50mg Cd kg"' soil treated plants were given 50mg S kg'' soil in comparison to 50mg

Cd kg'' soil alone, hi Alankar, the decrease in NR activity and N content due to 50mg

Cd kg'' soil was limited to 1.8 and 1.7% at 30DAS; 1.1 and 1.4% at 60DAS and 8.8

and 10.1% at 90DAS with 50mg S kg"' soil. In RH30, these decreases were restricted

to 15.1 and 7.9% at 30DAS, 18.6 and 13.2% at 60DAS and 20.9 and 16.5% at 90DAS

with 50mg S kg"' soil (Figure 35).

4.3.4 Cadmium accumulation

Cadmivmi content in the root and leaf of both the cultivars increased with the

increasing Cd concentration in soil. Non-tolerant cultivar (RH30) accumulated more

Cd in root and leaf than tolerant cultivar Alankar (Figure 36). In comparison to root,

the content of Cd was less in the leaf of both the cultivars due to S supplementation.

The application of 50mg S kg"' soil maximally lowered the Cd content in the root and

leaf of both the cultivars treated with 50mg Cd kg"' soil. The application of lOOmg S

kg"' soil also lowered the Cd content in root and leaf of 50mg Cd kg"' soil treated

plants but the values obtained were almost similar to that of 50mg S kg"' soil plus

50mg Cd kg"' soil. Application of lOOmg S kg"' soil did not decrease the Cd content

in root and leaf of 150mg Cd kg"' soil treated plants.

Cadmium content in the root and leaf of Alankar was 110.8, 27.7 and 274.3,

70.0^g g"' DW at 30DAS; 141.3, 40.7 and 331.5, 95.8^g g"' DW at 60DAS and

184.4, 24.5 and 380.5, SOA^g g'' DW at 90DAS due to 50 and 150mg Cd kg'' soil,

respectively. The addition of 50mg S kg'' soil maximally lowered the Cd content in

72

Alankar felMM RH30 30DAS

a: 2

-20

-40

-60

10

0

-10

^ -20

:> - 30 -» o

oi -40

-50

-60

-70

T T -

-1 1 f • -•} r -

60DAS

- 1 p— I • I

90DAS

10

0

-10

F -20 r c u

-30 § Z

-40

-50

-60

10

• - 20

-10

-20 r c u

-30 § Z

-40

-50

-60

0

C

c o o

-40 2

-60

50Cd 150Cd 50Cd 50Cd 150Cd ISOCd

+ SOS + lOOS + SOS + lOOS

SOCd ISOCd 50Cd SOCd ISOCd ISOCd

+ SOS + lOOS +50S ^ IOCS

Treatments (mg kg" soil)

Fig. 35. Per cent change in nitrate reductase activity and nitrogen content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

400

300

200 •

100 •

LSD„„,(CxT)= 10.66

Alankar RH-30

LSD„„5(CxT) = 3.68

0

600

500

400

300

200

100

0

700

600

500

400 -I

Z. op 300 o =1.

200

100

LSDoo5(CxT)= 14.03 LSD„„5(CxT) = 5.40

60DAS

LSDo 05 (CxT)= 16.95 LSDo_o5(CxT) = 3.48

200

150

100

50

0

250

200

50

00

c o u

U

^ Q

'ofi

a. -

50

0

200

150

c a ^ 8 Q

50

Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ SOS + lOOS + SOS + lOOS + SOS + IOCS + SOS + lOOS

Treatments (mg kg' soil)

Fig. 36. Effect of sulfur (S) application on root Cd content and leaf Cd content (|ig g"' DW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

root and leaf of plants treated with 50mg Cd kg'' soil and the values were 85.5 and

18.8^g g"' DW at SODAS; 106.7 and 25.4^g g"' DW at 60DAS and 154.6 and 18.3^g

g"' DW at 90DAS, respectively (Figure 37).

In RH30, Cd content in the root and leaf was 135.8, 63.3 and 357.5, 138.1^g

g"' DW at 30DAS; 190.7, 92.6 and 488.3, 206.2^g g"' DW at 60DAS and 286.1, 62.4

and 556.4, 132.3^g g'' DW at 90DAS due to 50 and 150mg Cd kg"' soil, respectively.

The combined application of 50mg S kg'' soil and 50mg Cd kg'' soil lowered the Cd

content in root and leaf and the values were 115.3 and 49.8|ig g'' DW at 30DAS;

161.6 and 72.1^g g'' DW at 60DAS and 260.2 and 53.3|ag g"' DW at 90DAS,

respectively (Figure 37).

4.3.5 Thiobarbituric acid reactive substances and hydrogen peroxide content

Significant Cd-dose dependent increase in TBARS and H2O2 content was observed in

both cultivars and the extent of increase was found greater in RH30 than Alankar.

Maximum increase in TBARS and H2O2 content was noted with 150mg Cd kg'' soil

in both the cultivars at all growth stages. Application of 50mg S kg'' soil maximally

lowered the TBARS and H2O2 content of 50mg Cd kg"' soil treated plants at all

growth stages. The values obtained for TBARS and H2O2 content with lOOmg S kg''

soil plus 50mg Cd kg'' soil were almost similar to the values obtained in plants treated

with 50mg S kg'' soil plus 50mg Cd kg'' soil. Application of lOOmg S kg'' soil did

not lower the TBARS and H2O2 content of 150mg Cd kg'' soil treated plants (Figure

38).

In Alankar, the increase in TBARS and H2O2 content was 146.3, 317.6% and

74.8, 142.6% at 30DAS; 168.1, 324.4% and 85.1, 145.2% at 60DAS and 173.3,

363.7% and 86.4, 150.5% at 90DAS due to 50 and 150mg Cd kg'' soil, respectively,

over their respective control. Supplementation of 50mg S kg'' soil to plants treated

with 50mg Cd kg"' soil significantly lowered the TBARS and H2O2 content by 41.4

and 28.4% at 30DAS; 34.9 and 25.9% at 60DAS and 62.4 and 33.1% at 90DAS,

respectively, in Alankar (Figure 39).

The content of TBARS and H2O2 in RH30 was increased by 224.8, 412.4%

and 144.2, 197.7% at 30DAS; 339.4, 427.8% and 156.0, 206.5% at 60DAS and 403.5,

445.1% and 161.5, 228.0% at 90DAS due to 50 and 150mg Cd kg"' soil, respectively,

over their respective control. Whereas, supplementation with 50mg S kg' soil

lowered the TBARS and H2O2 content by 162.9 and 99.7% at 30DAS; 180.2 and

73

7000 Alankar ^ ^ RH30 30DAS r 5000

4->

c o u •a U o o

5000

4000

3000

2000

0 8000

«3^

o u T3 U o o ai

c <D * - » B O

^ 4000 o o

50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S + lOOS +50S +100S

50Cd l50Cd 50Cd 50Cd ISOCd ISOCd

+ 50S + lOOS +50S + lOOS

Treatments (mg kg' soil)

Fig. 37. Per cent change in root Cd content and leaf Cd content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

100 1

80 •

60 -

40 -

20 -

LSDo„5(CxT)=1.85 Alankar RH30

LSDoo5(CxT) = 7.97

0

100

80

60 ^

40

20

0

100

80 •

60

40 -

20

30DAS

LSDQOS (CXT) = 2.08 LSD„„,(CxT) = 9.56

60DAS I I

LSD„„, (CxT) = 3.49 LSDoo5 (CxT) =10.23

90DAS

r 300

250

200 „ ^

150 2 '60

100

50

0

400

300

c _ 200 S 'ej)

100

0

400

300

200 o _M

- 100

Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd Control 50Cd I50Cd 50Cd 50Cd 150Cd ISOCd

+ 50S +100S+50S + lOOS + SOS + lOOS + SOS + lOOS

Treatments (mg kg' soil)

Fig. 38. Effect of sulfur (S) application on thiobarbituric acid reactive substances (nmol g"' FW) and hydrogen peroxide content (^mol g"' FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

50Cd 150Cd 50Cd

+ 50S

50Cd 150Cd 150Cd

+ lOOS + SOS + lOOS 50Cd ISOCd 50Cd

+ 50S

50Cd ISOCd ISOCd

+ lOOS +50S + lOOS

Treatments (mg kg" soil)

Fig. 39. Per cent change in thiobarbituric acid reactive substances and hydrogen peroxide content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica j'uncea L.) under Cd stress with sulftir (S) doses at 30, 60 and 90days after sowing (DAS).

100.9% at 60DAS and 250.3 and 112.5% at 90DAS, respectively, in plants treated

with 50mg Cd kg"' soil (Figure 39).

4.3.6 Components of antioxidant system

4.3.6.1 Enzymatic antioxidants

The response of antioxidant enzymes was differential to Cd stress in both the

cultivars. In general, S application (50mg S kg'' soil) maximally increased the

activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase

(GR) under Cd stress (50mg Cd kg'' soil). Whereas, the application of 50mg S kg"'

soil lowered the increase in SOD activity caused by 50mg Cd kg'' soil alone (Figures

40-41).

The activity of SOD increased with increasing Cd concentration in soil and

extent of increase was greater in RH30 than Alankar. Sulfur supplementation lowered

the Cd induced increase in SOD activity of both the cultivars. In Alankar, significant

increase in SOD activity was 51.3, 71.6% at 30DAS; 43.4, 57.2% at 60DAS and 42.7,

33.9% at 90DAS due to 50 and 150mg Cd kg'' soil, respectively, over the control.

However, the activity of SOD was reduced to 24.5, 27.5 and 26.8% at 30, 60 and

90DAS, respectively, when the plants were given 50mg S kg'' soil in the presence of

50mg Cd kg"' soil (Figure 42).

The activity of SOD was increased by 71.4, 116.1% at 30DAS; 76.4, 100.0%

at 60DAS and 50.3, 69.2% at 90DAS in RH30 due to 50 and 150mg Cd kg"' soil,

respectively, over the control. Its activity was restricted to 40.3, 33.8 and 28.5% at 30,

60 and 90DAS, respectively, with the addition of 50mg S kg"' soil to 50mg Cd kg"'

soil-treated RH30 plants (Figure 42).

The activity of CAT was increased with the increasing Cd concentration in the

soil at 30, 60 and 90DAS in Alankar. Whereas, in RH30, the increase in CAT activity

was noted only at 30DAS. At later stages (60 and 90DAS), Cd significantly decreased

the CAT activity. Sulfur supplementation further increased the Cd induced increase in

CAT activity at all growth stages in tolerant cultivar Alankar. In RH30, addition of S

lowered the decrease in CAT activity only at 60 and 90DAS, whereas, slight increase

in its activity was noted at 30DAS.

Significant increase in CAT activity of Alankar due to 50 and 150mg Cd kg"'

soil was 33.3, 56.4% at 30DAS; 24.1, 42.1% at 60DAS and 23.6, 28.3% at 90DAS,

respectively, in comparison to their respective control. Addition of 50mg S kg"' soil

74

significantly increased the CAT activity by 40.2, 48.3 and 30.2% at 30, 60 and

90DAS, respectively; in Alankar treated with 50mg Cd kg"' soil (Figure 42).

In RH30, the increase in CAT activity was 10.7 and 13.9% at 30DAS,

whereas, its activity was decreased by 20.4, 36.7% at 60DAS and 18.1, 35.3% at

90DAS due to 50 and 150mg Cd kg'' soil, respectively. S supplementation (50mg S

kg'' soil) increased the CAT activity by 16.1% at 30DAS, while, at 60 and 90DAS its

application only lowered the decrease in CAT activity by 9.2 and 10.6%, respectively,

in plants treated with 50mg Cd kg'' soil (Figure 42).

Supplementation of 50mg S kg'' soil significantly and maximally increased

the APX activity of both the cultivars treated with 50mg Cd kg'" soil at all growth

stages.

The increase in APX activity of Alankar due to 50 and 150mg Cd kg' soil was

70.4, 128.4% at 30DAS; 158.4, 193.1% at 60DAS and 109.7, 144.7% at 90DAS,

respectively, over control. Further, the addition of 50mg S kg'' soil caused maximal

significant increase of 200.7, 231.8 and 175.0% in APX activity at 30, 60 and 90DAS,

respectively, of plants treated with 50mg Cd kg'' soil (Figure 43). However, in RH30

lesser increase was observed in plants treated with 50mg Cd kg"' soil or these plants

supplemented with 50mg S kg'' soil (Figure 43).

Glutathione reductase activity of both the cultivars was increased under Cd

stress. Maximal significant increase in GR activity was noted in both the cultivars

treated with 50mg Cd kg"' soil. Treatment of 150mg Cd kg"' soil significantly

decreased the GR activity in non-tolerant cultivar RH30. Addition of S (50mg S kg"'

soil) further increased the GR activity of both the cultivars at all growth stages and

extent of increase was higher in Alankar than RH30.

The increase in GR activity of Alankar due to 50 and 150mg Cd kg"' soil was

61.4, 40.6% at 30DAS; 55.3, 38.3% at 60DAS and 39.5, 26.3% at 90DAS,

respectively, over the control. Treating these plants with 50mg S kg"' soil further

increased the activity, which was maximum compared to other S treatments. Similar

response of RH30 was noted with respect to Cd and S treatments (Figure 43).

4.3.6.2 Non-enzymatic antioxidants

Cadmium treatments significantly decreased the ascorbic acid content at all growth

stages and greater decrease was found in RH30 than Alankar. Supplementation of Cd-

treated plants with S lowered the Cd-induced reduction in ascorbic acid content of

both the cultivars at all growth stages and 50mg S kg"' soil maximally lowered the

75

18 1

15

•S 12-

6 -

3 •

0

21

18 •

15 -

12

9 -

3 •

0

15

12

9 -

6 -

LSDooj (CxT) = 0.24 Alankar RH30

LSDoos (CxT) =1.44

30DAS

LSDoo5 (CxT) = 0.15 LSDoo5 (CxT) =1.32

60DAS

LSD005 (CxT) = 0.03 LSD005 (CxT) =1.09

90DAS

250

• 200

150

100

• 50

0

300

I- 250

200

1- 150

100

(- 50

0

200

150

100

50

Control 50Cd 150Cd 50Cd 50Cd 150Cd I50Cd Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S +100S+50S + lOOS + SOS + lOOS + SOS + lOOS

Treatments (mg kg" soil)

Fig. 40. Effect of sulfur (S) application on superoxide dismutase activity (Units mg"' protein) and catalase activity (Units mg"' protein) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

2.5

2.0 -

o -

0- a 1.0

0.5

/—V

.s .^^ > t.

^ - » * - * •

w so X E O N ^

< •? D N — ^

-—N

c

> t . * - » *- o — « 'OO X E tt^ « < c

3

0.0

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0 4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

LSDooj (CxT) = 0.008 -•— Alankar - O - RH30

LSD„o5 (CxT) = 0.008

30DAS

LSD„„5 (CxT) = 0.092 LSD„„, (CxT) = 0.004

60DAS

" I I I

LSD, LSD„„5 (CxT) = 0.004

90DAS

0.5

- 0.4

0.3 2:> £

E 0.2 Q S

c

0.1

0.0

0.6

0.5

• 0.4

0.3

• 0.2

£-> t j

a: a

'33 o D.

'nn f t /1

-t—t

c

0.1

0.0 r 0.5

0.4

0.3 ^ e

0-2 o .-s

T •" I

- 0.1

0.0 Control 50Cd 150Cd 50Cd 50Cd ISOCd ISOCd Control 50Cd 150Cd 50Cd 50Cd l50Cd ISOCd

+ 50S +100S+50S + lOOS + SOS +100S+50S + lOOS

Treatments (mg kg"' soil)

Fig. 41. Effect of sulfur (S) application on ascorbate peroxidase activity (Units mg"' protein) and glutathione reductase activity (Units mg'' protein) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

Alankar C2E3 RH30 30DAS

m

- 70

' 60

50

40

• 30

• 20

10

. n

> t> ta (-< U

50Cd 150Cd 50Cd

+ 50S

50Cd

+ IOOS

!50Cd

+ 50S

I50Cd

+ lOOS

50Cd l50Cd 50Cd

+ 50S

50Cd

+ lOOS

ISOCd

+ 50S

150Cd

+ lOOS

Treatments (mg kg"' soil)

Fig. 42. Per cent change in superoxide dismutases activity and catalase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica j'uncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S + lOOS +50S +100S

50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 50S + lOOS +50S + lOOS

Treatments (mg kg" soil)

Fig. 43. Per cent change in ascorbate peroxidase activity and glutathione reductase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

ascorbic acid content. Treatment of lOOmg S kg"' soil to 50mg Cd kg'' soil-treated

plants also lowered the reductions but was less effective compared to 50mg S kg"

soil. Application of lOOmg S kg"' soil did not improve the ascorbic acid content of

150mg Cd kg"' soil treated plants (Figure 44)

Ascorbic acid content of Alankar was decreased by 27.9, 44.2% at 30DAS;

24.2, 49.1% at 60DAS and 27.2, 46.0% at 90DAS with 50 and 150mg Cd kg"'soil,

respectively, over the control. Addition of S (50mg S kg"' soil) maximally lowered the

reduction in ascorbic acid content of 50mg Cd kg"' soil treated plants by 11.9, 5.3 and

10.6% at 30, 60 and 90DAS, respectively, over the control (Figure 45).

In RH30, the decrease in ascorbic acid content was 44.6, 61.2% at 30DAS;

50.0, 64.0% at 60DAS and 57.5, 68.7% at 90DAS with 50 and 150mg Cd kg"'soil,

respectively, over the control. Supplementation of 50mg S kg"' soil maximally

lowered the reduction in ascorbic acid content of 50mg Cd kg"' soil treated plants by

27.3,25.8 and 30.3% at 30, 60 and 90DAS, respectively, over the control (Figure 45).

Glutathione content increased in Cd-treated both the cultivars. The increase

was higher in Alankar than RH30. Sulfur supplementation further increased the

glutathione content of both the cultivars and maximum significant increase in its

content was noted in plants treated with 50mg S kg"' soil plus 50mg Cd kg"' soil at all

growth stages.

The increase in glutathione content of Alankar due to 50 and 150mg Cd kg"'

soil was 19.7, 24.4% at 30DAS; 25.3, 36.0% at 60DAS and 25.3, 31.9% at 90DAS,

respectively, over the control. Further maximal significant increase in its content

occurred with the addition of 50mg S kg"' soil to 50mg Cd kg"' soil treated plants.

RH30 also responded similarly to Cd and S treatments (Figure 45).

4.3.7 Yield characteristics

Cadmium treatments significantly decreased the yield characteristics (expressed as

number of siliqua per plant, number of seeds per siliqua, 1000 seed weight and seed

yield per plant) at harvest and the extent of decrease was greater in RH30 than

Alankar. Sulfiir supplementation (50mg S kg"' soil) lowered the Cd (50mg Cd kg"'

soil)-induced reduction in yield characteristics of both the cultivars (Figure 46). The

application of lOOmg S kg"' soil also lowered the reductions in yield characteristics

caused by 50mg Cd kg"' soil but was less effective than 50mg S kg"' soil. Further, the

application of lOOmg S kg"' soil did not alleviate the reduction in yield characteristics

caused by 150mg Cd kg"' soil.

76

400 1

300 •

200 •

100

0

500

400 •

300 •

200

100

0

500

400

300

200

100

LSDo,o5(CxT) = 2.81 Alankar

RH30 LSDoo, (CxT) = 5.76

30DAS I I

LSDoo5 (CxT) = 2.25 LSDQOS ( C X T ) = 2.60

1000

800

600

400 g I

200

0

800

600

• 400

• 200

60DAS

LSDoo3 (CxT) = 2.18 LSDoo, (CxT) =1.15

90DAS

0

600

400

• 200

Control 50Cd 150Cd 50Cd 50Cd 150Cd 150Cd

+ 505 + lOOS + 505 + 1005

Control 50Cd 150Cd 50Cd 50Cd 150Cd l50Cd

+ 505 + 1005 +505 +1005

Treatments (mg kg" soil)

Fig. 44. Effect of sulfur (S) application on ascorbic acid content (nmol g"' FW) and glutathione content (nmol g"' FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

-70

10

0

-10

-20

-30

-40

-50

-60

It l l lMII 1 -

in i i 1 ii ! i

w

60DAS

50Cd 150Cd 50Cd

+ 50S

50Cd

+ lOOS

150Cd

+ 50S

150Cd

+ lOOS

50Cd ISOCd 50Cd

+ 50S

50Cd

+ lOOS

130Cd

+ 50S

ISOCd

+ lOOS

Treatments (mg kg' soil)

Fig. 45. Per cent change in ascorbic acid content and glutathione content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulfur (S) doses at 30, 60 and 90days after sowing (DAS).

100

c (8 O. u u O.

3 O"

crt t«-o k-

<u £l

B 3

z

8U

60

40

20

0 8

OD

.SP '5 ? <u

o o o

LSDoo3(CxT)=1.29

Alankar RH30

LSDQOS (CXT) = 6.69

I I I " " I

LSDoo5 (CxT) = 0.45 LSDoo5 (CxT) = 0.52

r 20

15§-

o.

i o |

5 3 Z

• 6

• 4 -^

Control 50Cd 150Cd 50Cd 50Cd 150Cd ISOCd

+ SOS + lOOS + SOS + IOCS

Control SOCd ISOCd SOCd SOCd ISOCd ISOCd

+ 50S + lOOS +SOS + lOOS

Treatments (mg kg"' soil)

2 u

(U aj

C/D

Fig. 46. Effect of sulfur (S) application on number of siliqua per plant, number of seeds per siliqua, 1000 seed weight (g) and seed yield (g plant'') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at harvest i.e., 120days after sowing (DAS). Data are mean of three replicates.

c as

a. (8 3

X)

E 3

eo

u

u

o o o

Control 50Cd ISOCd 50Cd 50Cd 150Cd I50Cd Control 50Cd 150Cd 50Cd 50Cd ISOCd 150Cd

+ 50S +1005+505 +1005 +505 +1005+505 +1005

Treatments (mg kg' soil)

Fig. 47. Per cent change in number of siliqua per plant, number of seeds per siliqua, 1000 seed weight and seed yield of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulfur (S) doses at harvest i.e., 120days after sowing (DAS).

Number of siliqua per plant, number of seeds per siliqua, \1

and seed yield per plant of Alankar due to 50 and 150mg Cd M'*5pil was decreased

by 22.7, 22.8, 26.2, 21.6% and 35.7, 33.8, 42.4, 32.5%, respecti>^^ver the control.

Application of 50mg S kg' soil maximally lowered the 50mg Cd kg'^oil-induced

reductions in these characteristics, which were 8.3, 13.3, 12.7 and 10.8%,

respectively, over the control. RH30 also responded similarly to Alankar for Cd and S

treatments but the cultivar was less responsive (Figure 47).

4.4 Summary of Experiment 2

• Application of 50mg S kg'' soil proved most effective in alleviating the effects

of 50mg Cd kg'' soil on growth, photosynthetic, biochemical and yield

characteristics of Alankar and RH30 cultivars.

• Cd treatments up-regulated the S assimilation pathway and increased ATP-

sulfurylase activity and S content. Whereas, N assimilation pathway was

down-regulated and decrease in nitrate reductase activity and N content were

observed. Sulfur supplementation (50mg S kg'' soil) to plants treated with

50mg Cd kg'' soil further increased the ATP-sulflirylase activity and S content

and also lowered the decrease in nitrate reductase activity and N content.

• Application of 50mg S kg'' soil decreased the Cd content maximally in root

and leaf of both the cultivars treated with 50mg Cd kg'' soil.

• Cd-induced accimiulation of TBARS and H2O2 was lowered by the application

of50mgSkg''soil.

• Responses of antioxidant enzyme activities were found differential in both the

cultivars under Cd stress. S application (50mg S kg'' soil) increased the

activities of CAT, APX and GR of 50mg Cd kg'' soil treated plants.

• Application of 50mg S kg'' soil maximally lowered the 50mg Cd kg'' soil-

induced decrease in ascorbic acid content and increased the glutathione

content in both the cultivars.

• Application of 50mg S kg'' soil also lowered the Cd-induced decrease in yield

characteristics of both the cultivars.

• The application of 50mg S kg'' soil was found most effective in mitigating the

50mg Cd kg'' soil induced reductions in growth, photosynthetic, biochemical

and yield characteristics in Alankar, whereas, reductions in these

characteristics were only lowered in RH30.

77 ^V^v

4.5 Experiment 3: Coordination of Sulfur and Nitrogen Application in the

Alleviation of Cd-induced Effects on Cd tolerant and non-tolerant Mustard

Cultivars

Experiment 3 was conducted on the basis of the findings of Experiments 2. The

present Experiment was aimed to study the effectiveness of addition of N levels (40,

80 and 120mg N kg"' soil) to the alleviation potential of 50mg S kg'' soil on 50mg Cd

kg'' soil-induced reductions in Alankar (Cd tolerant) and RH30 (Cd non-tolerant)

cultivars.

Results of this Experiment are presented in the following section.

4.5.1 Growth characteristics

Application of different N levels (40, 80 and 120mg N kg'' soil) further enhanced the

alleviation effect of 50mg S kg'' soil in Alankar and RH30 cultivars treated with

50mg Cd kg'' soil. Among the N levels used, the addition of 80mg N kg'' soil to

50mg S kg"' soil nulhfied the adverse effects of 50mg Cd kg'' soil and also maximally

increased the growth characteristics of Alankar. Other N levels proved less effective

(Figures 48-49).

The treatment 80mg N kg'' soil plus 50mg S kg"' soil resulted in maximal

increase in shoot length, root length, leaf area and plant dry mass by 6.5, 5.7, 5.6,

4.1% at 30DAS; 7.3, 6.9, 5.9, 5.3% at 60DAS and 5.2, 5.6, 4.8, 4.8% at 90DAS of

50mg Cd kg"' soil treated Alankar plants, respectively, over their respective control

(Figures 50-51).

Similar effect of the treatment (80mg N kg"' soil plus 50mg S kg'' soil) was

observed in RH30. Cd-induced reductions in growth characteristics were lowered by

all N levels applied in combination with 50mg S kg'' soil. However, application of

80mg N kg'' soil with 50mg S kg'' soil maximally lowered the reductions in shoot

length, root length, leaf area and plant dry mass and these were limited to 8.4, 8.4, 8.2,

8.5% at 30DAS; 6.6, 6.4, 8.6, 6.4% at 60DAS and 7.0, 7.7, 9.7, 6.1% at 90DAS of

50mg Cd kg"' soil treated plants, respectively, over their respective control (Figures

50-51).

4.5.2 Photosynthetic characteristics

The reductions caused by 50mg Cd kg'' soil in photosynthetic characteristics (total

chlorophyll content, carotenoids content, carbonic anhydrase activity, net

photosynthetic rate and stomatal conductance) were reversed by the application of

50mg S kg' soil at all the growth stages in both the cultivars. Ameliorative effect of

78

20

1 5 •

5 7 60 g

l l 10

LSD„„5(CxT) = 0.08

0

25

20 -

15 -

10

0

25

20

15

10

5 ]

Alankar RH30

LSD„o,(CxT) = 0.57

30DAS

LSDo(,5(CxT) = 0.12 LSDo,o5(CxT) = 0.72

60DAS

LSDo,„3(CxT) = 0.17 LSDoo5(CxT) = 0.44

« • •

90DAS

60

50

40

30 - -5.

20

10

0

120

100

80

60

40

20

0

140

120

100

I- 80

60

40

20

Control 50Cd OCd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+120N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ I20N

Treatments (mg kg' soil)

Fig. 48. Effect of sulfur (S) and nitrogen (N) application on root and shoot length (cm plant'') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

(0 ' •«

CO <N

-J o

100 1

80 •

60

40 ^

20

0

1000

800

CO -^ 600 u c c3 i2

( i i a ,

S " -• i , 400

200 ^

0

800

LSDoo5(CxT)=1.12 -•— Alankar - O - RH30

r I • T I r

LSD„„5(CxT) = 8.29

600

<2 -Q. 400

"1

200 -

LSDo„5(CxT) = 2.30

- • •

LSD„„,(CxT) = 0.02

30DAS

LSD„„(CxT) = 0.13

60DAS

LSDoo5(CxT) = 0.39

90DAS

2.0

- 1.5

1.0

« .-^ i=-„

C 6 0 CO ^ - -

• 0.5

0.0

16

12

s | l c 00

r 50

40

30

20

i £

T3

1 § Q.

3

10

Control 50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

Control 50Cd 50Cd 50Cd 50Cd 50Cd + 50S +50S +50S +50S

+40N +80N -^nON

Treatments (mg kg"' soil)

Fig. 49. Effect of sulfur (S) and nitrogen (N) application on leaf area (cm^ plant'') and plant dry mass (g plant"') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

10 1

•10

-20

-30

1 • • • Alankar \":

H ^1 ^1 Hi ^1

111 1

^ ^ RH30

1 '

L

S ''

30DAS

r" 1 1

•

•

50Cd 50Cd +50S +50S +80N +120N

-10

-20

-30

Treatments (mg kg' soil)

Fig. 50. Per cent chaise in root and shoot length of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress with sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +I20N

Treatments (mg kg'' soil)

Fig. 51. Per cent change in leaf area and plant dry mass of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

different levels of N along with 50mg S kg"' soil was almost similar as observed for

growth characteristics. Among the N levels used, 80mg N kg'' soil along with 50mg S

kg"' soil appeared as the most effective combination for reversing the Cd-induced

(50mg Cd kg'' soil) reductions in photosynthetic characteristics (Figures 52-54).

Application of 50mg S kg"' soil together with 80mg N kg'' soil increased the

total chlorophyll and carotenoids content in Alankar by 3.2 and 3.1% at 30DAS and

5.3 and 4.8% at 60DAS but at 90DAS, this combination only lowered the reductions,

which limited to 3.9 and 2.4% in plants treated with 50mg Cd kg'' soil, respectively,

over their respective control. The activity of carbonic anhydrase, net photosynthetic

rate and stomatal conductance in Alankar were increased by 4.6, 6.2, 4.0% at 30DAS;

5.7, 6.6, 5.7% at 60DAS and 3.5, 4.2, 2.3% at 90DAS, respectively, over their

respective control due to 80mg N kg'' soil plus 50mg S kg'' soil of 50mg Cd kg'' soil-

treated plants (Figures 55-57).

Cadmium-caused reductions in photosynthetic characteristics in RH30 were

only lowered by all N levels in combination with 50mg S kg'' soil. The application of

80mg N kg"' soil along with 50mg S kg'' soil maximally lowered the reductions in

photosynthetic characteristics of RH30 plants treated with 50mg Cd kg'' soil (Figures

55-57).

4.5.3 Sulfur and nitrogen assimilation

Application of 50mg S kg'' soil together with 80mg N kg"' soil substantially increased

the ATP-sulfurylase activity and S content in both the cultivars treated with 50mg Cd

kg"' soil at all growth stages. Among the combinations, 80mg N kg"' soil and 50mg S

kg"' soil given to 50mg Cd kg"' soil treated plants resulted in maximum ATP-

sulfurylase activity and S content at all growth stages. Whereas, the same combination

maximally lowered the reductions in nitrate reductase activity and N content in both

the cultivars at all growth stages. Application of other N levels with 50mg S kg"' soil

proved less effective (Figures 58-59).

In Alankar, application of 80mg N kg"' soil and 50mg S kg ' soil maximally

increased ATP-sulfurylase activity and S content by 82.1, 54.4% at 30DAS; 85.5,

55.5% at 60DAS and 72.6, 42.1% at 90DAS of plants treated with 50mg Cd kg"' soil.

In RH30, ATP-sulfurylase activity and S content were increased by 60.0, 29.9% at

30DAS; 61.6% at 60DAS and 47.5, 21.4% at 90DAS over their respective control

(Figure 60).

79

2.0

1.5

1.0 •

0.5

0.0

2.5

2.0 •

1.5 -

1.0

0.5

0.0

1.6

1.2

0.8

0.4 -

0.0

LSDoo5(CxT) = 0.04 -•— Alankar - O - RH30

LSDoo5(CxT) = 0.002

30DAS T I

LSD„„5(CxT) = 0.02 LSDoos (CxT) = 0.002

— •

60DAS r I

LSD(,o5 (CxT) = 0.02 LSD„„, (CxT) = 0.004

90DAS

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ I20N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ I20N

0.25

0.20

0.15

0.10

o c/l

•V

O c u o

^ u. , OJJ 01)

E N — i ^

• 0.05

0.00

0.25

0.20

c 1)

0.15 I ^

C 00

0.10 B E o ~-

0.05

0.00

0.20

0.15

• 0.10

- 0.05

0.00

Treatments (mg kg" soil)

Fig. 52. Effect of sulfur (S) and nitrogen (N) application on total chlorophyll content (mg g"' FW) and carotenoids content (mg g FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

•5 60 ••3 A"

u 1

3 •

•> SO

CO JJ

61

1 •

0

12

10

LSDo„(CxT) = 0.02

8

2 ^

0

5

3 -

-•— Alankar • O - RH30

30DAS

LSDoo5(CxT) = 0.14

•

^ \

m m

0

60DAS

LSD005 (CxT) = 0.03

90DAS

Control 50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N Treatments (mg kg"' soil)

Fig. 53. Effect of sulfur (S) and nitrogen (N) application on carbonic anhydrase activity (mmol CO2 g"* FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica Juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

25

20 •

15

10 -

5 -

0

25

20

15

10 ]

LSDoo3(CxT) = 0.17 -#— Alankar - C ^ RH30 LSDoo3(CxT) = 3.56

30DAS

0 16

12

LSDoo5(CxT) = 0.41

r T I

LSDoo3 (CxT) = 7.26

60DAS

8

4 -

LSD„o5 (CxT) = 0.18 LSD„„, (CxT) = 4.27

500

400 u u c

300 i T3 ' C o u

200 1 E o

uri

100

0

600

500

400 5

300

200 I

100

0 500

400 1) <j c

300 i ' • a ' c o u

^ 2 0 0 1 E o

100

90DAS

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ I20N

Control 50Cd 50Cd 50Cd 50Cd 50Cd + 50S +50S +50S +50S

+40N +80N +I20N

Treatments (mg kg" soil)

Fig. 54. Effect of sulfur (S) and nitrogen (N) application on net photosynthetic rate (^mol CO2 m' s"') and stomatal conductance (mmol m" s"') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica Juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

-25

-30

-35 -

-40

10

Alankar ^ ^ RH30

-30

-40 -

-50 10

I -10 s o u

-20

-30

-40

-50

30DAS

I

^

90DAS

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S +120N

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S + 120N

5

0

-5

10 ^

-15

-20 ^

u -25 o

-30

-35

-40

10

•10 s

-20

-30

-40

-50

10

•10 ^

-20

-30

-40

-50

Treatments (mg kg' soil)

Fig. 55. Per cent change in total chlorophyll content and carotenoids content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

Alankar f^PTl RH30

lU -

0 -

^ -10 • 5? ^ > -20 • U

< U -30 •

-40 •

<n .

^ ^ • n k

:|!%

1 p ^

'-<•

•fC

>^.

* • ? ! '

l..» 1

_

w u V

b'iH •

I »>;;.

:.f

60DAS • -i

. 1 ! •

? ^

Ik,'*.

'r * J "

90DAS

50Cd 50Cd 50Cd +50S +50S

+40N

50Cd 50Cd +50S +50S +80N +120N

-1 Treatments (mg kg' soil)

Fig. 56. Per cent change in carbonic anhydrase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

10

-40 10

-40

.a -10 -

2 -20

-30

90DAS

I % ^

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

• - 5

-10

-15 B

-20 I • -25

-30

10

-10 5

-20 -=

• -30

-40 10

-10 B

-20

-30

-40

Treatments (mg kg" soil)

Fig. 57. Per cent change in net photosynthetic rate and stomatal conductance of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cuitivars of mustard {Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

60

50 -

40

30

20

10

0

50

40

B 30

20

10

0

70

60 •

50 -

40 -

30

20 ^

10

LSDo„5(CxT) = 2.99 -•— Alankar - O - RH30

LSDoo5(CxT) = 0.04

30DAS

LSDoo,(CxT)=1.44 LSDoo5(CxT) = 0.04

60DAS r t

LSDo,o5(CxT) = 0.22 LSDoo5 (CxT) = 0.03

90DAS

2.5

• 2.0

1.5 ^ .

c I- 1.0 S

0.5

0.0

3.0

2.5

I- 2.0

- 1.5 I c o o

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ 120N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ 120N

1.0

0.5

0.0

r 2.5

• 2.0

1.5

• 1.0

• 0.5

0.0

Treatments (mg kg' soil)

Fig. 58. Effect of sulfur (S) and nitrogen (N) application on ATP-sulfurylase activity (nmol Pi mg"' protein min"*) and sulfur content (%) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

600 1

500

400

300 •

200

100 -

LSDpoj (CxT) = 0.95 -•— Alankar - O - RH30

0

600

500 ^

400

• | ' « ^ 300

200

100

0

400

300

200

100

I I

LSDoo5(CxT) = 2.58

I • ' " • I

LSDoo5(CxT) = 2.43

LSDo„(CxT) = 0.03

30DAS

LSD„.,(CxT) = 0.04

60DAS

LSD„„(CxT) = 0.02

90DAS

r 6

• 5

4 ^

3 I c o u

• 1

c 3 B

c o u

• 4

• 3

c o

2 " Z

Control 50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

Treatments (mg kg" soil)

Control 50Cd 50Cd 50Cd 50Cd 50Cd + 50S +503 +50S +50S

+40N +80N +120N -1

Fig. 59. Effect of sulfur (S) and nitrogen (N) application on nitrate reductase activity (nmol NO2 g' FW h'') and nitrogen content (%) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

SOCd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

SOCd SOCd SOCd +50S +SOS

+40N

SOCd +S0S +80N

SOCd ••-SOS +120N

Treatments (mg kg" soil)

Fig. 60. Per cent change in ATP-sulfiirylase activity and sulfiir content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

The reductions in nitrate reductase activity and N content were reversed by S

supplementation in Cd stressed plants. Lesser decrease in nitrate reductase activity

and N content was observed when plants were given 50mg S kg'' soil plus 50mg Cd

kg"' soil in comparison to 50mg Cd kg"' soil alone. In Alankar, application of 80mg N

kg"' soil and 50mg S kg"' soil maximally increased the nitrate reductase activity and N

content by 5.2, 3.8% at SODAS; 5.2, 4.2% at 60DAS but at 90DAS, this combination

only lowered the reductions by 2.8, 5.4% in plants treated with 50mg Cd kg" soil,

respectively, over their respective control. In RH30, 50mg S kg"' soil alone and in

combination with 80mg N kg"' soil lowered the reductions in nitrate reductase activity

and N content of 50mg Cd kg"' soil treated plants (Figure 61).

4.5.4 Cadmium accumulation

In both the cultivars, increased accumulation of Cd in root and leaf due to 50mg Cd

kg"' soil was lowered by the application of 50mg S kg"' soil alone and in combination

with 40, 80 and 120mg N kg"' soil at all growth stages. Application of 40mg N kg"'

soil plus 50mg S kg"' soil also lowered the Cd accumulation in root and leaf in both

the cultivars but to a lesser extent than 80mg N kg"' soil. The effect of 120mg N kg"'

soil was equal to 80mg N kg"' soil on Cd accumulation. In Alankar, combined

application of 50mg S kg"' soil and 80mg N kg"' soil to 50mg Cd kg"' soil treated

plants maximally lowered the Cd content in root and leaf, which was 73.1, 14.6|ig g"'

DW at 30DAS; 88.5, 20.6^g g"' DW at 60DAS and 145.6, 15.4^g g"' DW at 90DAS,

respectively. Cadmium content in the root and leaf of RH30 with the same

combination was 104.3, 42.9^g g"' DW at 30DAS; 145.0, 61.9^g g"' DW at 60DAS

and 244.3, 50.3^g g"' DW at 90DAS, respectively (Figures 62-63).

4.5.5 Thiobarbituric acid reactive substances and hydrogen peroxide content

Application of 50mg S kg"' soil lowered the TEARS and H2O2 content of 50mg Cd

kg"' soil treated plants at all growth stages. Supplementation of 50mg S kg"' soil with

different levels of N (40, 80 and 120mg N kg"' soil) again lowered the contents of

TEARS and H2O2 of both the cultivars treated with 50mg Cd kg"' soil at all growth

stages. Combined application of 40mg N kg"' soil plus 50mg S kg"' soil also lowered

the TEARS and H2O2 content in both the cultivars but to a lesser extent than 80mg N

kg"' soil. Whereas, the effect of 120mg N kg"' soil plus 50mg S kg"' soil was equal to

80mg N kg"' soil plus 50mg S kg"' soil on TEARS and H2O2 content. In particular,

combined application of 80mg N kg"' soil plus 50mg S kg"' soil maximally lowered

80

10

5

0

? -5

1 -10 <J

(8

oi -15 Z

-20

-25

-30

10

Alankar RH30 30DAS

-10

-20

-30

-40

5

0

-5 ^

-10

-15

-20

-25

-30

-35

-40

—1 1—

60DAS

• T • 1 r~

90DAS

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S +120N

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S + 120N

10

5

0

-10 I c o

•15 z

-20

-25

-30

10

•10

c o

-20 " Z

-30

-40

5

0

-5

-10

-15

-20

-25

-30

-35

-40

Treatments (mg kg' soil)

Fig. 61. Per cent change in nitrate reductase activity and nitrogen content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

160

140

120

100 -

80 •

60 -

40

20

LSDoo3(CxT)= 11.19

Alankar RH-30 LSD„o5(CxT) = 4,14

0

250

200

150

100 -

50 •

0 350

300

250

200

150

100

50

0

LSDoo5(CxT)= 15.02 LSDoo5(CxT) = 6.08

LSD„„5(CxT) = 23.24 LSD„,o5(CxT) = 4.49

80

60

40

20

0

100

80

(- 60

40

20

0

80

60

40

20

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ 120N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ I20N

Treatments (mg kg'' soil)

Fig. 62. Effect of sulfur (S) and nitrogen (N) application on root Cd content and leaf Cd content (ng g'' DW) of Alankar (Cd tolerant) and RH30 (Cd non-toierant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

2500 1 Alankar F T ^ RH30

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

1000

Treatments (mg kg' soil)

Fig. 63. Per cent change in root Cd content and leaf Cd content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

the TBARS and H2O2 contents by 27.1, 15.2% at 30DAS; 21.8, 11.6% at 60DAS and

45.2, 26.5% at 90DAS, respectively, over their respective control, in 50mg Cd kg'

soil treated Alankar plants (Figures 64-65).

In RH30, combined application of 80mg N kg'' soil plus 50mg S kg'' soil

lowered the TBARS and H2O2 contents by 97.1, 80.2% at 30DAS; 90.5, 77.6% at

60DAS and 149.2, 97.6% at 90DAS, respectively, over their respective control, in

plants treated with 50mg Cd kg"' soil (Figure 65).

4.5.6 Components of antioxidant system

4.5.6.1 Enzymatic antioxidants

The increase in SOD activity in plants treated with 50mg Cd kg"' soil was lowered by

the application ofSOmg S kg"' soil alone and in combination with 40, SO and 120mg

N kg"' soil in both the cultivars at all growth stages. Application of 40mg N kg"' soil

plus 50mg S kg"' soil also lowered the SOD activity in both the cultivars but to a

lesser extent than 80mg N kg"' soil. Whereas, the effect of 120mg N kg"' soil plus

50mg S kg"' soil was equal to 80mg N kg'' soil plus 50mg S kg"' soil on SOD activity

when treated with 50mg Cd kg'' soil. In Alankar, combination of 50mg S kg"' soil and

80mg N kg"' soil to 50mg Cd kg"' soil fed plants maximally lowered the SOD activity

by 20.6, 19.5 and 25.3% at 30, 60 and 90DAS, respectively, over their respective

control. Same combination lowered the SOD activity of RH30 plants treated with

50mg Cd kg"' soil by 36.7, 30.8 and 28.3% at 30, 60 and 90DAS, respectively, over

their respective control (Figure 66,68).

In Alankar, the increase in CAT activity due to 50mg Cd kg ' soil was further

increased by the application of 50mg S kg"' soil alone and in combination with 40, 80

and 120mg N kg'' soil at all growth stages. Combined application of 50mg S kg'' soil

and 80mg N kg"' soil to 50mg Cd kg"' soil treated plants maximally increased the

CAT activity by 52.2, 58.9, 34.3% at 30, 60 and 90DAS, respectively, over their

respective control. In RH30, the application of 50mg S kg"' soil and 80mg N kg"' soil

increased the CAT activity by 23.9% at 30DAS, while, at 60 and 90DAS its

application only limited the decrease in CAT activity to 1.02 and 2.30%, respectively,

in plants treated with 50mg Cd kg"' soil (Figure 66,68).

The increase in APX activity due to 50mg Cd kg"' soil was further increased

by the application of 50mg S kg"' soil alone and in combination with 40, 80 and

120mg N kg"' soil in both the cultivars at all growth stages. In Alankar, combined

application of 50mg S kg'' soil and SOmg N kg"' soil to 50mg Cd kg"' soil treated

81

50

40 •

30

20

10

LSD„„5(CxT) = 0.90

0

100

80

60

40

20

0

100

80

60

40

20 -I

-•— Alankar

- O - RH30 LSDo„5(CxT)= 14.60 r 250

200

• 150

- 100

- 50

30DAS

LSDo.o3(CxT) = 0.90 LSDoo5(CxT)= 16.86

60DAS

LSD„„,(CxT) = 0.J LSDoo5(CxT)= 18.63

90DAS

0

300

• 250

200

150

100

50

0

r 300

250

200

150

100

50

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd 50Cd +50S +50S

+80N +120N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ 120N

Treatments (mg kg" soil)

Fig. 64. Effect of sulfur (S) and nitrogen (N) application on thiobarbituric acid reactive substances (nmol g"' FW) and hydrogen peroxide content (|imol g'' FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

SOCd SOCd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

SOCd SOCd +50S

SOCd SOCd +SOS +SOS +40N ^80N

SOCd +50S + I20N

Treatments (mg kg" soil)

Fig. 65. Per cent change in thiobarbituric acid reactive substances and hydrogen peroxide content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

15

12

D C OS 6

D

3 •

0

15

12

3 •

0

15

12 •

LSDoo5(CxT) = 0.95 Alankar RH30 LSDoo3(CxT) = 2.75

30DAS -T 1 r— t —I ~r—

LSDo„(CxT)=1.00 LSDo„5(CxT) = 3.12

60DAS

LSD„„,(CxT) = 0.95 LSDoo5 (CxT) = 0.63

-O O-

90DAS

250

200

150 .?

I- 100

50

0

350

300

250

- 200

150

- 100

- 50

0

250

- 200

- 150 .e

100

Control 50Cd 50Cd SOCd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

Control SOCd SOCd + SOS

SOCd +50S

+40N

SOCd +SOS

+80N

SOCd +50S

+ 120N

Treatments (mg kg"' soil)

50

Fig. 66. Effect of sulfur (S) and nitrogen (N) application on superoxide dismutases activity (Units mg'' protein) and catalase activity (Units mg'' protein) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

3.5

3.0

I §. 2.0

X ^ 15 a, =2 < c

B 1.0 0.5 -

LSDQOJ (CXT) = 0.03

0.0

5.0

4.0 -

.S •f S 3.0 o -

X 6 0- a 2.0

1.0

0.0 4.0

3.0

2.0 -

1.0

0.0

Alankar RH30 LSDoo5 (CxT) = 0.006

30DAS

LSDooj (CxT) = 0.28 LSD(,o5 (CxT) = 0.005

60DAS

LSD„„5 (CxT) = 0.03 LSDooj (CxT) = 0.003

90DAS

r 0.6

0.5

• 0.4

Control 50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+ 120N

.E

0.3 o 'oo

a .- 0.2 5

0.1

0.0

0.8

0.6 ^ _c '53

0.4 5 'oo

0.2

0.0

0.5

0.4

0.3

0.2

0.1

0.0

Treatments (mg kg" soil)

Fig. 67. Effect of sulfur (S) and nitrogen (N) application on ascorbate peroxidase activity (Units mg"' protein) and glutathione reductase activity (Units mg'' protein) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

50Cd 50Cd 50Cd SOCd 50Cd +50S +50S +50S +50S

+40N +80N +120N

SOCd SOCd SOCd SOCd SOCd +50S +50S +50S +50S

+40N +80N +120N

Treatments (mg kg" soil)

Fig. 68. Per cent change in superoxide dismutases activity and catalase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

-•""s

^

^ > 4 ^

u ea X O. < •

350

300

250

200

150

100

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S + 120N

Treatments (mg kg" soil)

Fig. 69. Per cent change in ascorbate peroxidase activity and glutathione reductase activity of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30,60 and 90days after sowing (DAS).

plants maximally increased the APX activity by 297.3, 301.2, 209.1% at 30, 60 and

90DAS, respectively, over their respective control. Whereas, the increase in APX

activity of RH30 by this combination was 135.0, 175.5 and 120.4% at 30, 60 and

90DAS, respectively, over their respective control (Figure 67,69).

The increase in GR activity was same as observed in the case of APX activity.

In Alankar, combined application of 50mg S kg"' soil and 80mg N kg'' soil to 50mg

Cd kg'' soil treated plants maximally increased the GR activity by 150.8, 157.3,

92.1% at 30, 60 and 90DAS, respectively, over their respective control. Whereas, the

increase in APX activity of RH30 by this combination was 85.5,94.0,47.8% at 30, 60

and 90DAS, respectively, over their respective control (Figure 67,69).

4.5.6.2 Non-enzymatic antioxidants

The decrease in ascorbic acid content due to 50mg Cd kg"' soil was lowered by the

application of 50mg S kg'' soil alone and in combination with 40, 80 and 120mg N

kg'' soil in both the cultivars at all growth stages. Combined application of 40mg N

kg"' soil plus 50mg S kg'' soil also lowered the decrease in ascorbic acid content of

both the cultivars but to a lesser extent than 80mg N kg"' soil. The effect of 120mg N

kg"' soil plus 50mg S kg'' soil was equal to 80mg N kg'' soil plus 50mg S kg"' soil on

ascorbic acid content when treated with 50mg Cd kg"' soil. In Alankar, combined

application of 50mg S kg"' soil and 80mg N kg'' soil to 50mg Cd kg'' soil treated

plants lowered the decrease in ascorbic acid content by 5.4, 4.3, 7.5%) at 30, 60 and

90DAS, respectively, over their respective control. Whereas, in RH30, this

combination lowered the decrease in ascorbate content by 15.2, 10.1, 18.1%) at 30, 60

and 90DAS, respectively, over their respective control (Figures 70-71).

The increase in glutathione content due to 50mg Cd kg'' soil was further

increased by the application of 50mg S kg'' soil alone and in combination with 40, 80

and 120mg N kg'' soil in both the cultivars at all growth stages. Combination of 40mg

N kg'' soil plus 50mg S kg"' soil also increased the glutathione content of both the

cultivars but to a lesser extent than 80mg N kg'' soil plus 50mg S kg'' soil. Whereas,

the effect of combined application of 120mg N kg'' soil plus 50mg S kg'' soil was

almost equal to 80mg N kg'' soil plus 50mg S kg"' soil on glutathione content when

treated with 50mg Cd kg"' soil. In Alankar, combined application of 50mg S kg"' soil

and 80mg N kg"' soil to 50mg Cd kg"' soil fed plants maximally increased the

glutathione content by 58.8, 60.7, 47.3% at 30, 60 and 90DAS, respectively, over

their respective control. While, the same combination increased the glutathione

82

400

300 •

200 -

100 -

0

500

400

300

200 ^

100

400

300

200 -

100 -

LSD„„,(CxT) = 0.63 Alankar RH30 LSDoo5 (CxT) = 2.03

30DAS I I

LSDo„ (CxT) = 0.74 LSD„„ (CxT) = 4.49

60DAS

LSD„o5 (CxT) = 0.85 LSD„„ (CxT) = 2.17

90DAS

800

700

600

500 c > "J rT

S 'wi • 400

- 300

200

100

0

r 1200

• 1000

• 800

• 600

• 400

- 200

0

700

600

500

400 ^ ta-

300 ^ I

200

100

Control 50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

Treatments (mg kg' soil)

Control 50Cd 50Cd 50Cd 50Cd 50Cd + 50S +50S +50S +50S

+40N +80N +120N

1

Fig. 70. Effect of sulfur (S) and nitrogen (N) application on ascorbic acid content (nmol g' FW) and glutathione content (nmol g FW) of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard {Brassica juncea L.) under Cd stress (T) at 30, 60 and 90days after sowing (DAS). Data are mean of three replicates.

50Cd 50Cd +50S

50Cd +50S +40N

50Cd +50S +80N

50Cd +50S + 120N

50Cd 50Cd 50Cd +50S +50S

+40N

50Cd +50S +80N

50Cd +50S + 120N

Treatments(mgkg' soil)

Fig. 71. Per cent change in ascorbic acid content and glutathione content of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard {Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at 30, 60 and 90days after sowing (DAS).

M

.2P ' S

-a u <u o o o

100

e « o. u u a. a 3 CT-

'« O Urn

<u £ 3

:^

80

60

40

20

0

8

LSDoo3(CxT) = 3.32

Alankar RH30

LSD„„,(CxT)=1.21

r I

LSD„.o5(CxT) = 0.28 LSD„„5(CxT) = 0.14

-O

16

• 14

12 §•

• 10

• 6

• 4

Control 50Cd 50Cd +50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+I20N

Control 50Cd 50Cd + 50S

50Cd +50S

+40N

50Cd +50S

+80N

50Cd +50S

+120N

Treatments (mg kg' soil)

a. • a

u Xi

3

u c/1

Fig. 72. Effect of sulfur (S) and nitrogen (N) application on number of siliqua per plant, number of seeds per siliqua, 1000 seed weight (g) and seed yield (g plant"') of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars (C) of mustard (Brassica j'uncea L.) under Cd stress (T) at harvest i.e., 120days after sowing (DAS). Data are mean of three replicates.

Alankar

3

o.

u s> B 3

2

00

.2P ' S

• o <u (U

o o o

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

50Cd 50Cd 50Cd 50Cd 50Cd +50S +50S +50S +50S

+40N +80N +120N

C

D. a 3 a-

x> E 3

z

2 u

'?> T3

on

Treatments (mg kg' soil)

Fig. 73. Per cent change in number of siliqua per plant, number of seeds per siliqua, 1000 seed weight and seed yield of Alankar (Cd tolerant) and RH30 (Cd non-tolerant) cultivars of mustard (Brassica juncea L.) under Cd stress due to sulfur (S) and nitrogen (N) doses at harvest i.e., 120days after sowing (DAS).

content in RH30 by 37.1, 40.5, 33.8% at 30, 60 and 90DAS, respectively, over their

respective control (Figures 70-71).

4.5.7 Yield characteristics

The reductions in yield characteristics (number of siliqua per plant, number of seeds

per siliqua, 1000 seed weight and seed yield per plant) due to 50mg Cd kg'' soil were

lowered by the application of 50mg S kg'' soil alone and in combination with 40, 80

and 120mg N kg'' soil in both the tolerant (Alankar) and non-tolerant (RH30)

cultivars. A combination of 50mg S kg'' soil and 80mg N kg'' soil maximally lowered

the reductions in yield characteristics. In Alankar, lesser decrease in number of siliqua

per plant, number of seeds per siliqua, 1000 seed weight and seed yield per plant due

to 50mg S kg'' soil and 80mg N kg"' soil was 1.9, 5.0, 4.8 and 1.9%, respectively,

over their respective control. Whereas, in RH30, the decrease remained to 19.2, 23.0,

21.0 and 20.4% due to 80mg N kg'' soil plus 50mg S kg'' soil (Figures 72-73).

4.6 Summary of Experiment 3

• Coordination of sulfiir and nitrogen proved effective in mustard cultivation. A

package of these nutrients not only helped in increasing crop productivity but

also exhibited its potential in the alleviation of Cd effects.

• Among the N levels applied, 80mg N kg'' soil strengthened the effectiveness

of 50mg S kg'' soil in the alleviation of 50mg Cd kg'' soil induced reductions

in both the cultivars at all growth stages. The effectiveness of the combined

application of 50mg S kg'' soil plus 80mg N kg'' soil was different in tolerant

and non-tolerant cultivars. In Alankar (tolerant cultivar), the Cd-induced

reductions in growth, photosynthetic, biochemical and yield characteristics

were completely overcome, whereas, the reductions in these characteristics

were only lowered by this combination in RH30 (non-tolerant cultivar).

• Application of 50mg S kg'' soil and 80mg N kg'' soil to 50mg Cd kg'' soil

treated plants increased the ATP-sulfurylase activity and S content and also

improved the NR activity and N content to a greater extent.

• Cadmium-induced increase in root and leaf Cd content was lowered by the

combined application of 50mg S kg'' soil and 80mg N kg'' soil to 50mg Cd

kg'' soil treated plants in both the cultivars at all growth stages. The extent of

decrease in Cd content was more in Alankar than RH30.

• The accumulation of TBARS and H2O2 due to 50mg Cd kg'' soil was lowered

by the combined application of 50mg S kg'' soil and 80mg N kg'' soil in both

83

the cultivars at all growth stages. The extent of decrease in TBARS and H2O2

content was greater in Alankar than RH30.

The activities of antioxidant enzymes (catalase, ascorbate peroxidase and

glutathione reductase) were maximally increased by the combined application

of 50mg S kg'' soil and 80mg N kg'' soil in 50mg Cd kg"' soil treated plants.

Whereas, activity of SOD was lowered by this combination of sulfur and

nitrogen in both the cultivars at all growth stages.

Combined application of 50mg S kg'' soil and 80mg N kg'' soil lowered the

Cd-induced decrease in ascorbic acid content. This combination, however,

increased the glutathione content in both the cultivars at all sampling stages.

Application of 50mg S kg'' soil plus 80mg N kg'' soil reversed the reduction

in yield characteristics of Alankar and values reached on par to control.

Whereas, this combination only lowered the reduction in yield characteristics

ofRH30.

84

DISCUSSION

Contents

DISCUSSION

5.1 Screening of Mustard Cultivars Treated with Different Levels of Cadmium 86 Growth characteristics 86 Photosynthetic characteristics 88 Cadmium accumulation 92 Yield characteristics 93 Alleviation of Cadmium-toxicity by Sulfur and Nitrogen in Alankar (Cd tolerant) and RH30 (Cd non-tolerant) Cultivars of Mustard 95 Growth characteristics 96 Photosynthetic characteristics 97 Sulfur and nitrogen metabolism 100 Cadmium accumulation 103 Cadmium-induced oxidative stress 104 Components of antioxidant system 105 Enzymatic antioxidants 105 Non-enzymatic antioxidants 110 Yield characteristics 113 New Report in the Thesis and Future Prospects 115

5.1.1 5.1.2 5.1.3 5.1.4 5.2

5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.6.1 5.2.6.2 5.2.7 5.3

DISCUSSION Chapter 5

Every year India loses hundreds of millions of rupees from reductions in crop

productivity caused by abiotic stresses (Mahajan and Tuteja 2005). According to Bray

et al. (2000), the relative decreases in potential maximum yields associated with

abiotic stress factors vary between 50-80%. Among abiotic stresses, heavy metal

contamination is a serious envirormiental problem that limits plant productivity and

threatens human health (Wagner 1993, Sanita di Toppi and Gabbrielli 1999). The

agricultural soil may have toxic levels of heavy metals due to agricultural and

industrial practices such as application of pesticides and chemical fertilizers, waste

water irrigation, precipitation from heavy coal combustion, and smelter wastes and

residues from metalliferous mining (Lombi et al. 2000, Vassilev et al. 2006, Xie et al.

2006, Verma et al. 2007).

For sustainable crop production, it is important to develop methods or

techniques for alleviating the Cd-induced growth inhibition and reducing its

accumulation in plants. One option is the use of balanced S and N fertilizers for

alleviating Cd stress and maintaining productivity of cultivated soils. As Cd induces

essential nutrient deficiency and even decreased concentration of several

macronutrients in plants (Jiang et al. 2004), it seems possible to reverse or at least

partly reduce the Cd-induced growth inhibition by optimization of S and N.

Increasing evidence suggests that mineral-nutrient status of plants plays a critical role

in increasing plant resistance to environmental stresses (Marschner 1995, Vassilev et

al. 2005, Hassan et al. 2005a,b, 2008a,b, Anjum et al. 2008a,b, Khan et al. 2008a).

Sulfur and N are essential nutrients for normal growth and development of plants.

Their assimilation pathways are well coordinated (Brunold 1993, Takahashi and Saito

1996, Abdin et al. 2003, Hawkesford et al. 2006). Deficiency of one element was

shown to repress the other pathway (Neuenschwander et al. 1991, Kim et al. 1999,

Koprivova et al. 2000, Migge et al. 2000, Prosser et al. 2001, Hesse et al. 2004,

Scherer 2008). S is a structural constituent of several coenzymes and prosthetic

groups, such as ferredoxin, which are also important for N assimilation. Thus, S plays

an important role in plant growth and in the regulation of plant development. It has

also been found that S nutrition is a critical factor for the alleviation of Cd toxicity

(Popovic et al. 1996, Chen and Huerta 1997, Hassan et al. 2005b, 2006, Vassilev et

al. 2005, Anjum et al. 2008a,b). A positive effect of S nutrition on Cd detoxification

85

in Beta vulgaris plants has also been established (Popovic et al. 1996). It has been

found that at suboptimal S nutrition Cd exposed plants preferably allocate S to

phytochelatin synthesis (McMahon and Anderson 1998). Anjum et al. (2008a) also

reported that S supplementation increased the production of glutathione content under

low level of Cd which protects the Brassica campestris plants by improving the

growth and photosynthesis. Pankovic et al. (2000) have shown that optimal N supply

decreased the inhibitory effects of Cd on photosynthesis of Helianthus annuus plants.

A proper N supply has been shown to have a positive effect in overcoming the

adverse effects caused by Cd toxicity in various crop species (Hassan et al. 2005a).

Therefore, coordination of S and N may alleviate the Cd-induced toxicity in crop

plants.

The present study was undertaken to understand the response of Brassica

juncea cultivars to Cd stress and the use of S nutrition alone and in combination with

N in the amelioration of Cd toxicity in Brassica juncea cultivars.

5.1 Screening of Mustard Cultivars Treated with Different Levels of Cadmium

The order of decrease in growth, photosynthetic and yield characteristics for all the

cultivars was RH30 >Sakha >Pusa Bold >Varuna >Alankar. RH30 accumulated

greatest amount of Cd and Alankar accumulated lowest Cd in root and leaf (Figures 2-

21).

5.1.1 Growth characteristics

Plant growth and development are an outcome of coordination of the main biological

processes in plants (Vassilev et al. 1998). Plant growth and development are

susceptible to stresses of all kinds including those of heavy metals. Cd adversely

affects plant growth and metabolism. The most common effect of Cd toxicity in plants

is stunted growth, leaf chlorosis and alteration in the activity of many key enzymes of

various metabolic pathways (Godbold and Hutterman 1985, Arduini et al. 1994, Wu

and Zhang 2002b, Chen et al. 2003, Wu et al. 2003, Rai et al. 2005). Bachir et

al. (2004) found decrease in root length, plant height and fruiting branch number

in cotton plants with increasing Cd concentration in a pot experiment. Growth

reduction in Cd-treated plants has been described (Ouzounidou et al. 1997, Wu and

Zhang 2002b, Wu et al. 2003, Demirevska-Kepova et al. 2006), due to the higher

accumulation of Cd and reductions in the availability of other nutrients resulting in

disturbed metabolism (Jalil et al. 1994, Wu et al. 2006). In my studies shoot length,

root length, leaf area and plant dry mass of all the Brassica Juncea cultivars were

86

decreased with different concentrations of Cd (0, 25, 50, 100 and 150mg Cd kg"' soil).

Root growth was found more susceptible to Cd toxicity compared to the shoot.

Metwally et al. (2005) also reported that the roots of Pisum sativum plants were

more sensitive to Cd toxicity than shoot. Increasing concentration of Cd in liquid

culture and pot experiment decreased the germination and root growth of

Raphanus sativus and Daucus carota plants (Chen et al. 2003). In the present

study, greater impact of Cd observed on root growth as compared to shoot in all

Brassica juncea cultivars, could be due to higher accumulation of Cd in root, leading

to impaired growth metabolism. A greater reduction in the root length could also be

an adaptation of the cultivars studied in response to high external Cd concentration, so

as to reduce the uptake of Cd along with water and their subsequent transport to the

shoot. Parameters such as root length, shoot length, leaf area as well as plant dry mass

have been used as indicators of metal toxicity in plants (Ouzounidou et al. 1997). In

my study, Alankar cultivar showed minimum decrease in shoot length, root length,

leaf area and plant dry mass, while RH30 showed maximum decrease in these

parameters (Figiires 2-7). The reduction in the growth of these cultivars could also be

due to suppression of the elongation growth rate of cells, because of an irreversible

inhibition exerted by Cd on the proton pump responsible for the process (Aidid and

Okamoto 1993). It has also been reported previously that species and cultivars display

marked differences for Cd tolerance in Triticum aestivum (Zhang et al. 2002, Khan et

al. 2006), Hordeum vulgare (Wu et al. 2003), Gossypium hirsutum (Wu et al. 2004),

Brassica juncea (Qadir et al. 2004), Pisum sativum (Metwally et al. 2005), Oryza

sativa (Wu et al. 2006, He et al. 2006), Vigna radiata (Wahid and Ghani 2007,

Anjum et al. 2008c) and Brassica campestris (Anjum et al. 2008d). Cadmium

treatments inhibited shoot and root growth and dry weight of two Triticum

aestivum cultivars, C-1252 and Balcali-85. The decrease was more distinct in

cultivar C-1252 (Ozturk et al. 2003). The presence of Cd decreased the seedling

growth of Zea mays, Triticum aestivum, Cucumis sativus and Sorghum bicolor in

terms of root and shoot growth (An 2004). Ghnaya et al. (2005) reported that Cd

severely inhibited Mesembryanthemum crystallinum growth even at low

concentration. The process of tolerance in plants is exhibited at different structural

and functional levels, viz., molecular, biochemical, cellular and the whole plant. At

the whole plant level it is possible that the tolerance mechanism is connected to the

87

pattern of Cd distribution as well as the response of components of antioxidant

system.

Analyzing the effect of Cd on many plant species, Vassilev and Yordanov

(1997) concluded that Cd diminishes relative growth rate through inhibiting mainly

net assimilation rate, and to a lesser extent, by restricting leaf area ratio. Inhibition of

net assimilation rate is caused by disturbances of dark respiration and photosynthesis.

Decreased leaf area ratio in Cd-treated plants is a consequence of the decrease of

turgor potential and cell wall elasticity, resulting in smaller size of leaf cells formed

with smaller intercellular space area,

5.1.2 Photosynthetic characteristics

Cd induces changes in the physiological processes such as respiration, photosynthesis

and gas exchange of the plants (Lagriffoul et al. 1998, Hegedus et al. 2001, Mobin

and Khan 2007). Chloroplasts comprise only about one percent of the total Cd

accumulated by a plant. Despite its very low relative concentration in chloroplast, it

seriously blocks the activity of photosynthetic processes at different routes

(Ghoshrony and Nadakavukaren 1990, Siedlecka and Krupa 1999). As a visible

symptom the preferential degradation of chlorophyll and carotenoids content can be

used to monitor the metal-induced damage in leaves (Horvath et al. 1996, Sanita di

Toppi et al. 1998, Oncel et al. 2000).

In the reported study, chlorophyll and carotenoids content decreased with the

increase in Cd levels at all growth stages in all Brassica juncea cultivars. The extent

of decrease in chlorophyll and carotenoids content was maximum in RH30 while it

was minimum in Alankar (Figures 9-10,14). It has been reported earlier that Cd

decreased the chlorophyll and carotenoids content in Helianthus annuus (Di Cagno et

al. 2001), Hordeum vulgare (Wu et al. 2003, Vassilev et al. 2004), Oryza sativa (Kuo

and Kao 2004), Glycine max (Drazic et al. 2004), Sedum alfredii (Zhou and Qiu

2005), Matricaria chamomilla (Kovacik et al. 2006), Brassica juncea (Mobin and

Khan 2007), Triticum aestivum (Khan et al. 2007a), Brassica campestris (Anjum et

al. 2008a) and Vigna mungo (Singh et al. 2008a). Decreased photosynthesis can be

caused by the decrease in the level of photosynthetic pigments, breakdown or the

inhibition of its synthesis (Padmaja et al. 1990, Mobin and Khan 2007). Cadmium

interferes with chlorophyll formation, with functional SH-group of sulfhydryl

requiring enzymes such as ALA synthetase, ALA dehydratase and

protochlorophyllide reductase. Apart from inhibition of biosynthetic enzymes of

88

chlorophyll formation, the increased level of free radicals of fatty acids produced

from polyunsaturated fatty acids due to the higher activity of lipooxygenase may also

contribute to the decreased level of chlorophyll with Cd treatments (Klein et al. 1984,

Somashekaraiah et al. 1992). Cd might have replaced the central Mg from chlorophyll

molecules and thereby reduced the photosynthetic light harvesting ability of plants

(Kupper et al. 1996). The decrease in chlorophyll content may also be correlated with

the adverse effect of Cd on the uptake and accumulation of other essential nutrients in

plants viz. Fe, Mg, Ca and K (Greger and Ogrer 1991, Greger and Lindberg 1987,

Ouzounidou et al. 1997), since both Fe and Mg are essential elements required for the

formation of chlorophyll. Earlier reports also suggest that the change in Fe:Zn may be

responsible for the reduced chlorophyll content in plants (Root et al. 1975).

Carotenoids are synthesized in the chloroplast of plants. Carotenoids are

important as they absorb light at wavelength between 400 and 550nm and transfer it

to the chlorophylls (an accessory light-harvesting role) (Siefermann-Harms 1987) and

protect the photosynthetic apparatus by quenching a triplet sensitizer (Chi ), singlet

oxygen and other harmfiil free radicals which are naturally formed during

photosynthesis (an antioxidant function) (OelmuUer 1989, Thiele et al. 1996, Havaux

et al. 2000). Moreover, they are important for the photosystem (PSl) assembly and

the stability of light harvesting complex proteins as well as thylakoid membrane

stabilization (a structural role) (Mayfield and Taylor 1984, Siefermann-Harms 1987,

Wrischer et al. 1998, Niyogi et al. 2001, Kim et al. 2004). The decrease of

carotenoids content in seedlings of Raphanus sativus, Ulva lactuca, Triticum aestivum

and Oryza sativa growing in the presence of Cd was reported by Naguib et al. (1982).

Panda and Khan (2003) reported decrease in carotenoids content due to heavy metal

treatments. In my experiments, carotenoids content decreased with the increase in Cd

concentration in all the cultivars at all growth stages. When the level of carotenoids

was decreased by Cd treatments (Khudsar et al. 2001, Khan et al. 2007a, Singh et al.

2008a), the protective functions of carotenoids could not be maintained.

Consequently, the oxidative degradation of chlorophyll and rapid destruction of

photosynthetic membranes occurred.

Photosynthesis is an integral process with a high degree of self regulation.

Inhibition of photosynthetic rate could be due to structural and ftmctional disorders.

Cadmium may interact with the photosynthetic apparatus at various levels of

organization and architecture (Mysliwa-Kurdziel and Strzalka 2002). Besides

89

affecting the functions of chloroplast membranes, Cd can also alter the components of

photosynthetic electron transport chain, particularly the PSI and PSII (Siedlecka and

Krupa 1996).

In the present study, increasing concentration of Cd in the soil leads to reduced

photosynthetic characteristics in all Brassica juncea cultivars at all growth stages.

Maximum decrease in these characteristics was noted with 150mg Cd kg"' soil. The

decrease in net photosynthetic rate, stomatal conductance and carbonic anhydrase

activity varied among cultivars and RH30 exhibited maximum decrease while

Alankar showed minimum decrease (Figures 11-13,15-16). Earlier investigations have

also demonstrated a marked reduction in the overall rate of photosynthesis by Cd in

different plant species (Arduini et a/. 2004, Wojcik and Tukiendorf 2005, Jing et al.

2005, Khan et al. 2006, 2007, Hayat et al. 2007, Mobin and Khan 2007, Anjum et al.

2008a, Singh et al. 2008a). Deleterious effects of Cd on various facets of

photosynthesis such as, gas exchange, biosynthesis of chlorophyll, functioning of

photochemical reactions and the activities of the enzymes of Calvin cycle have been

studied (Stobart et al. 1985, Weigel 1985, Padmaja et al. 1990, de Fillippis and

Zeigler 1993, Chugh and Sawhney 1999, Verma and Dubey 2002, Vassilev et al.

2005, Mobin and Khan 2007). In addition, conductance and index of stomata,

transpiration and net CO2 uptake are greatly reduced with elevated Cd levels in the

growth media (Bindhu and Bera 2001, Balakhnina et al. 2005). Both the PSI and PSII

have been shown to be affected by Cd. Photosystem II is highly sensitive to

deleterious effect of Cd and its functioning is inhibited to a greater extent than that of

PSI (Chugh and Sawhney 1999). Inhibition of PSII might be due to Cd-induced

alterations in the level of magno-protein (Mg-protein) in the water splitting system.

Cd restricts the PSII-related electron transport (Baszinsky et al. 1980) probably as a

result of the structural and functional changes in thylakoid membranes, the reduced

ferredoxin-NADP* oxidoreductase activity, and the arrested plastoquinine synthesis

(Krupa and Baszynski 1995). The Cd stress mostly causes metal inhibition of

Rubisco, an important enzyme of the Calvin cycle (Vassilev et al. 2005, Mobin and

Khan 2007). As observed in the study, the decrease in net photosynthetic rate has also

been attributed to the reduction in carbonic anhydrase activity under Cd stress

(Siedlecka and Krupa 1999, Mobin and Khan 2007, Singh et al. 2008a). The

inhibitory effects of Cd might be either due to its interaction with -SH group, as two

molecules of -SH group are found in enzyme center (on cys 173 and cys 456)

90

essential for normal activity of Rubisco (Lorimer 1981), and/or metal substitution

property of Cd leading to the decrease in ratio of combined activity of

carboxylation/oxygenation.

In Brassica juncea cultivars, 150mg Cd kg"' soil maximally reduced the

stomatal conductance and the maximum decrease was noted in RH30 and minimum in

Alankar (Figure 16). The loss of water from crop plants is controlled mainly by the

stomata on leaves that have been shown sensitive to Cd (Barcelo et al. 1986a,b,). To

minimize the water loss the process of stomatal closure is adopted by plants exposed

to various stresses affecting plant-water status. It has been identified as an early event

in plant response to Cd-induced water deficiency leading to limitations to C uptake by

leaves (Barcelo et al. 1986a,b, Poschenreider et al. 1989). Cd-induced alteration in

mineral nutrients may also contribute to the reductions in stomatal conductance which

in turn lead to suppression of major physiological processes and metabolic activities

as well.

Carbonic anhydrase activity in all Brassica juncea cultivars also decreased in

the similar manner as photosynthesis and stomatal conductance. Maximum decrease

was noted with 150mg Cd kg"' soil. Among cultivars the decrease in its activity was

maximum in RH30, whereas, Alankar experienced minimum decrease (Figure 15). As

observed in this study, the decrease in carbonic anhydrase activity was also reported

previously in other Cd treated plants (Aravind and Prasad 2004, Hasan et al. 2007,

Khan et al. 2007a, Mobin and Khan 2007, Singh et al. 2008a). Siedlecka and Krupa

(1999) also reported Cd-induced decrease in carbonic anhydrase activity and other

elements of Rubisco activation system. Cadmiimi (like several other heavy metals) is

known to affect the structure and functioning of enzymes (Van Assche and Clijsters

1990) through peroxidation and production of ROS (Dietz et al. 1999) which would

affect the functioning of the enzymes and proteins. ROS induce fragmentation of

protein and impose oxidative modification, rendering cells susceptible to enzymatic

proteolysis and hydrolysis. Carbonic anhydrase is in fact a Zn metalloprotein

catalyzing reversible inter-conversion of HCO3" and CO2 (Xin Bin et al. 2001, Khan

et al. 2004); represents 1-2% of total soluble protein in leaf (Okabe et al. 1984) and is

related to photosynthesis in higher plants (Khan 1994, Henry 1996). Cd can readily

inhibit most of the Zn-dependent processes, either by displacement or by occupying

the active sites of the Zn metalloproteins (Nieboer and Richardson 1980, Siedlecka

1995). Aravind and Prasad (2004) noted toxic effect of Cd on carbonic anhydrase

91

activity in Ceratophyllum demersum which proved the concept that Cd occupies the

active sites of important Zn-metalloproteins. The study also showed a marked

reduction in Zn content and its substitution by Cd impaired the structure as well as the

activity of carbonic anhydrase in Ceratophyllum demersum. Consequently, this leads

to nonfunctioning of carbonic anhydrase and hence the associated photosynthetic

processes were impaired.

5.1.3 Cadmium accumulation

In the present study, plants grown with increasing levels of Cd exhibited an increase

in Cd-concentrations in root and leaf of all Brassica juncea cultivars at all growth

stages. Among cultivars, RH30 accumulated maximum Cd in root and leaf, whereas,

Alankar accumulated less (Figures 17-19). The results obtained in this study suggest

that roots are efficient barriers to Cd translocation to the aboveground plant parts.

Accumulation of heavy metals by plants and their restriction at root level is a

widespread phenomenon. The retention of Cd in roots may be due to cross linking of

Cd to carboxyl groups of cell wall proteins (Barcelo and Poschenreider 1990) and/or

an interaction of Cd ions with the thiol groups of soluble proteins and non-protein

thiols operating as a tolerance mechanism in root cells (Chaoui et al. 1997a). In some

cases, Cd ions may also be bound by pectic sites and hystidyl groups of cell wall

(Leita et al. 1995). Ranieri et al. (2005) suggested that retention/immobilization of

high amount of Cd in root tissues typical of several plants can be regarded as an

important protection mechanism against the diffusion of the metal in plants. For this

reason, Cd-concentration in roots can reach to an average of 75-80% of the total metal

taken up (Wojcik and Tukiendorf 1999). The movement of Cd fi-om roots to shoots is

also likely to occur via the xylem and is driven by the transpiration from the leaves. In

this regard the closure of stomata may reduce the accumulation or retention of Cd in

root (Sah et al. 1995). In addition, reduced movement of Cd from roots to shoots in

plants is believed to result from barrier function of root endodermis and mechanism

involving sequestration and decreased xylem loading of Cd (Hart et al. 1998).

However, the immobilization of Cd at root level is primarily dependent on the

concentration of Cd supplied and the plant species (Sanita di Toppi and Gabbrielli

1999). Due to disturbed root function(s) and/or the reduction in the selectivity of

plasma membrane root-Cd may be translocated to the aboveground parts (Vassilev et

al. 1998). In the present study, the translocation of Cd from root to leaf was found Cd-

dose dependent and higher in cultivar RH30 than in Alankar (Figuresl7-18). In fact,

92

restriction of heavy-metal transport from root to shoot has been thought as a

mechanism of plant tolerance to Cd. The lower translocation of Cd from root to leaf

may, therefore, be a strategy of Alankar to protect its photosynthetic function from

Cd-induced oxidative stress which is in close conformity with Dixit et al. (2001),

Anjum et al. (2008a) and Singh et al. (2008a).

The uptake of Cd by plants varies not only among plant species but also

among cultivars (Salt et al. 1995, Arao et al. 2003, Metwally et al. 2005, Khan et al.

2006, Anjum et al. 2008c). The difference in the ability of cultivars to accumulate Cd

may also be related to differences in their root morphology (An 2004). Das et al.

(1997) suggested that plant with numerous thin roots would accumulate more metals

than one with few thick roots. The transport of Cd in plants has been found highly

species dependent. Cucumis sativus retained greatest amount of Cd in roots followed

by Triticum aestivum and Zea mays (An 2004). Differences in the net uptake of Cd in

these cultivars may be the result of differences in the electrochemical gradient that is

created by ion exchange reactions in the membrane structures as reported in the foliar

uptake of metals by Martin and Juniper (1970). A lower electrochemical gradient in

Alankar (tolerant cultivar) than in the RH30 (non-tolerant) could, therefore, be one of

the reasons for lesser uptake of Cd in Alankar.

5.1.4 Yield characteristics

Cadmium stress affects growth and yield through disturbances in several morpho-

physiological processes and nutrient uptake. Reduction in growth and yield with

increased levels of Cd in growth media arises because of increased leaf rolling and

chlorosis of leaf and stem (Ghani and Wahid 2007) and reduced photosynthetic rate

(Chugh and Sawhney 1999, Khan et al. 2006, 2007a). Like growth and photosynthetic

characteristics, yield and its attributes were decreased in mustard cultivars with Cd

treatments. The maximum reduction was noted with 150mg kg'' soil in all the

cultivars. The tolerant cultivar, Alankar exhibited minimum decrease in yield

characteristics (number of siliqua per plant, number of seeds per siliqua, seed yield

per plant and 1000 seed weight) while maximum decrease in these characteristics was

observed in non-tolerant cultivar RH30 (Figures 20-21). In the present study, decrease

in yield and its attributes in Brassica juncea cultivars treated with Cd is possibly the

fall out of the poor growth and photosynthesis. The decrease in photosynthesis may be

considered as one of the important factors responsible for reduced plant growth and

productivity under Cd stress (Khan et al. 2007a). Inhibition of photosynthetic pigment

93

and its biosynthesis is one of the primary events in plants under Cd stress condition.

As a consequence of delay in the assembly of the photosynthetic apparatus, lower

photosynthetic efficiency, slower plant growth and decreased biomass production

occurs that leads to the reduced yield.

Wahid and Ghani (2007) reported significant reduction in number of pods per

plant and seeds per pod, 100 seed weight, seed yield and harvest index of Vigna

radiata genotypes as a result of Cd toxicity. It has been suggested that although

varietal difference exists, the accumulated Cd is mainly toxic to the mesophyll tissue,

most probably by interfering with the uptake of essential nutrients, thereby reducing

growth and yield at various stages. Cadmium significantly reduced number of ear, ear

weight, ear length, spikelet number, grains per ear, 1000 grain weight and grain yield

of Triticum aestivum cultivars and the decrease was correlated with photosynthetic

capacity of the cultivars (Khan et al. 2006, 2007a). Genotypic differences on the basis

of biomass production, yield and yield components were observed in Triticum

aestivum and it was noted that Cd significantly reduced the root and stem biomass and

spikes per plant but grains per ear and grain weight were not significantly reduced

(Zhang et al. 2002). Liu et al. (2007) noted variation among Oryza sativa cultivars in

their tolerance to soil Cd stress with respect to tillering, plant height, leaf area, dry

matter accumulation and grain yield. The relative change in the number of grains per

panicle showed a strong positive correlation with relative change in grain yield and, of

the grain yield components measured (panicles per pot, grains per panicle, filled grain

percentage, weight per grain) and the reduction of grains per panicle was noticed as

the main cause of grain yield loss under soil Cd stress. Wu et al. (2004) reported

reduction in yield of three Gossypium hirsutum genotypes under Cd stress and found

that the reduction in yield was proportional to Cd accumulation. Among the

Gossypium hirsutum cultivars tested, the cultivar Simian 3 showed higher Cd

concentration and greater decrease in lint yield than the other two genotypes

(Zhongmian 16 and Zhonmian 16-2).

Taken together, the present study revealed that all the Brassica juncea

cultivars responded differentially to Cd stress and the severity of Cd stress in terms of

decrease in the growth, photosynthetic and yield characteristics was minimum in

cultivar Alankar followed by Varuna, Pusa Bold, Sakha and RH30. Contrarily, in

terms of the accumulation of Cd in root and leaf, RH30 accumulated maximum

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followed by Sakha, Pusa Bold, Varuna and Alankar. On the basis of overall

performance of all Brassica juncea cultivars under Cd stress, Alankar proved as Cd-

tolerant and showed lesser decrease in the characteristics, whereas, RH30 emerged as

Cd non-tolerant and suffered maximum decrease.

5.2 Alleviation of Cadmium-toxicity by Sulfur and Nitrogen in Alankar (Cd

tolerant) and RH30 (Cd non-tolerant) Cultivars of Mustard

Increasing evidences suggest that mineral nutrient status of plants plays significant

role in increasing plant resistance to heavy metal stress (Marschner 1995, Astolfi et

al. 2004, Ahmad et al. 2005, Vassilev et al. 2006, Hassan et al. 2005a,b, 2008a,b,

Bouranis et al. 2008, Anjum et al. 2008a,b). Among the plant nutrients, S and N are

of great importance and are required by plants for maintaining normal growth and

development (Marschner 1995). Several studies have established regulatory

interactions between assimilatory sulfate and nitrate reduction in plants (Takahashi

and Saito 1996, Ahmad et al. 1999). The two pathways are very well coordinated

(Brunold 1993, Koprivova et al. 2000) and the deprivation of one leads to reduction in

the metabolism of the other (Reuveny et al. 1980, Prosser et al. 2001).

Positive interaction between S and N results in increased crop productivity

(Abdin et al. 2003). Brassica genotypes require higher amounts of S in addition to N

for optimum growth and yield (Abdin et al. 2003, Aulakh 2003, Khan et al. 2005).

Application of S along with N leads to enhanced biomass production and increased

leaf area as both nutrients are involved in biosynthesis of proteins and several other

molecules. Nitrogen is a basic constituent of proteins and with the increase in the rate

of N application, the N availability increases. Similarly, increased S supply increases

seed yield with higher protein content. Combined application of S and N promotes the

uptake of S and N, which leads to significant enhancement in seed protein and oil

content in Brassica juncea and Brassica campestris (McGrath and Zhao 1996,

Kachroo and Kumar 1997, Ahmad and Abdin 2000a,b, Abdin et al. 2003). Sulfur and

N relationship in terms of crop productivity has also been established in many studies

(Singh and Bairathi 1980, Sachdev and Deb 1990, Lakkineni and Abrol 1992,

McGrath and Zhao 1996, Ahmad et al. 1998, Fismes et al. 2000).

Experiments 2 and 3 were conducted where two cultivars of Brassica Juncea

namely, Alankar (Cd tolerant) and RH30 (Cd non-tolerant) (screened out from the

Experiment 1) were given S alone and in combination with N to ameliorate Cd-

95

induced toxicity. In Experiment 2 both the cuhivars of Brassica juncea were treated

with 0, 50 and 150mg Cd kg'' soil and supplemented with 0, 50 and lOOmg S kg"'

soil. Application of 50mg S kg'' soil to 50mg Cd kg'' soil-treated Alankar plants

overcome the Cd-induced toxicity whereas, in RH30, S-supplementation only lowered

the severity of Cd toxicity. Therefore, in Experiment 3, 50mg Cd kg'' soil-treated

cultivars were given 50mg S kg'' soil along with different levels of N (0, 40, 80 or

120mg N kg'' soil) to strengthen the S induced tolerance. In Experiment 3, it was

found that combined application of 50mg S kg'' soil and 80mg N kg'' soil completely

overcome the ill effects of 50mg Cd kg'' soil in Alankar (tolerant) whereas, in RH30

(non-tolerant), this combination only lowered the Cd-induced toxicity. The following

section discuses major results of the Experiments 2 and 3 with relevant supporting

citations. In addition, the participation of S and N in plant tolerance mechanisms has

also been discussed.

5.2.1 Growth characteristics

In Experiment 2, application of 50mg S kg'' soil ameliorated the Cd-induced toxicity

and improved the growth characteristics of tolerant cultivar Alankar but lowered the

reductions in non-tolerant cultivar RH30. The application of 50mg S kg'' soil nullified

the effects of Cd, to a lesser extent, in the plants fed with higher concentrations of Cd

(150mg Cd kg'' soil) but completely, if given low level of Cd (50mg Cd kg''). In

particular, the application of 50mg S kg'' soil proved most effective in improving

growth characteristics of both the Brassica juncea cultivars treated with 50mg Cd kg''

soil. A significant increase in root length, shoot length, plant dry mass and leaf area

was observed with 50mg S kg'' soil which varied greatly between cultivars grown

with 50mg Cd kg"' soil (Figures 22-25). Thus, application of S was instrumental in

mitigating to some extent the toxic effects of Cd on plant growth characteristics. This

indicates that S application reduces the toxic effects of Cd on plant through

improvement in the tolerance capacity of the plant. S supplementation to plants has

been shown to result in greater biomass production under normal (Ahmad et al. 2005)

and Cd stress conditions (Hassan et al. 2005b, Anjum et al. 2008a). However, the

mitigating effect by S nutrition was found dependent on Cd dose and on Brassica

juncea cultivar. Between the cultivars, the ameliorative effect of S nutrition was found

maximum in Alankar while minimum effect was noted in RH30. Our findings on

ameliorative effect of S in Cd-induced growth reductions are in agreement with

Anjum et al. (2008a). Anjana et al. (2006) reported that S could alleviate the Cd

96

induced impairment of biochemical and anatomical features of the Brassica

campestris plant. Hassan et al. (2005b) also showed that S application ameliorated the

Cd-induced inhibition of growth parameters in two Oryza saliva cultivars and the

effect of S treatment on these characteristics varied greatly with Cd level and between

cultivars.

In the present study, combined application of S and N further increased the

growth characteristics of Cd-treated Brassica juncea cultivars Alankar (tolerant) and

RH30 (non-tolerant) (Figures 48-51). A combination of 50mg S kg'' soil plus 80mg N

kg'' soil proved superior in enhancing the growth characteristics of both the cultivars

treated with 50mg Cd kg'' soil (Figures 50-51). S and N are essential macro nutrients

required for normal growth and development of plants (Marschner 1995) and have

been found to play a pivotal role in the protection of plants against environmental

stresses, including Cd toxicity (Pankovic et al. 2000, Hassan et al. 2005a,b, 2006,

2008a,b, Anjum et al. 2008a,b). It has also been found in previous studies that the

coordination of S and N results in enhanced crop yield and quality of S-requiring

Brassica (Aulakh et al. 1980, Lakkineni and Abrol 1992, 1994, Abdin et al. 2003,

Aulakh 2003). In the present study, the application of S along with N led to enhanced

biomass production and increased leaf area of Alankar than RH30 under Cd stress. It

is suggested that combined application of S and N promotes the uptake of S and N,

which leads to significant enhancement in growth of plants (Ahmad and Abdin

2000a,b, Abdin et al. 2003). Another explanation is that both nutrients are involved in

the synthesis of amino acids, proteins and various other cellular components,

including thiol compounds and the so-called secondary S compounds which have a

significant role in the protection of plants under stressed conditions. Moreover, N is a

basic constituent of proteins and the N availability increases with the increase in the

rate of N application. It has been shown in Hordeum vulgare that high levels of nitrate

and ammonium can induce a high affinity sulfate transporter gene and hence sulfate

uptake in N-fed plants suggesting that N metabolite may affect sulfate transporter

gene expression (Vidmar et al. 1999) and hence leads to enhancement in growth

characteristics.

5.2.2 Photosynthetic characteristics

All the Cd levels used in the present study significantly reduced the photosynthetic

characteristics. The effect of Cd stress on these characteristics also varied between

cultivars. Non-tolerant cultivar RH30 suffered maximum decrease in photosynthetic

97

characteristics, whereas, tolerant cultivar Alankar showed minimum decrease (Figures

26-31). More severe decreases of photosynthetic characteristics in RH30 than in

Alankar under Cd stress may partially account for the differential responses of growth

parameters between these two cultivars grown under Cd stress condition. Earlier

studies have also showed that photosynthetic functions are highly sensitive to heavy

metals including Cd (Zhang et al. 2003). Cd inhibits the enzymes of the Calvin cycle,

biosynthesis of chlorophyll and accessory pigments and declines the activity of

Rubisco (Siedlecka et al. 1997, Mobin and Khan 2007). In the present study, Cd

treatment declined the net photosynthetic rate, stomatal conductance and carbonic

anhydrase activity with the increase in the concentrations of Cd in the soil (Figures

27-28, 30-31). Moreover, Cd-induced reductions in photosynthetic responses have

been found to involve both stomatal and non-stomatal limitations (Mobin and Khan

2007). Contrarily, Di Cagno etal. (2001) observed no change in stomatal conductance

but noted significant reductions in CO2 assimilation rate and Rubisco activity in Cd-

exposed Helianthus annuus. Ali et al. (2000) reported that the decrease in the rate of

photosynthesis was associated with decreased stomatal conductance and transpiration

in in vitro grown Bacopa monniera plants treated with Cd. In the present

investigation, Cd significantly decreased both the net photosynthetic rate and stomatal

conductance in tolerant (Alankar) and non-tolerant (RH30) cultivars; suggests that

stomatal functions limit the photosynthesis.

Application of 50mg S kg"' soil ameliorated the Cd-induced toxicity and

improved the photosynthetic characteristics of both the tolerant (Alankar) and non-

tolerant (RH30) cultivars (Figures 26-28). In comparison to lOOmg S kg'' soil,

application of 50mg S kg'' soil ameliorated the Cd-induced (50mg Cd kg'' soil)

reductions in photosynthetic characteristics of both the cultivars (Figures 29-31). In

Triticum aestivum, an efficient S assimilation and antioxidative system was helpful in

protecting the photosynthetic ability and maintaining high yield potential under Cd

stress (Khan et al. 2007a). Resurreccion et al. (2001) reported that S application

increased the chlorophyll and Rubisco content which led to increased photosynthesis

of Oryza sativa. Khan et al. (2005) has also reported that S application increased the

relative growth rate, plant growth rate, net assimilation rate and carbon dioxide

exchange rate of Brassica juncea plants. Anjana et al. (2006) reported that the

reductions caused by lower level of Cd in leaf dry weight, chlorophyll, sugar and

protein content were reversed by S treatment in Brassica campestris. At the cellular

98

level S starvation reduces the mesophyll cell number per cm^ and the chlorophyll

content per chloroplast. The photosynthetic CO2 uptake by intact leaves,

photoreduction of ferricyanide, cyclic and non-cyclic photophosphorylation of

isolated chloroplast and the rate of CO2 assimilation by Rubisco may decrease with

the decreased total S content (Terry 1976), showing the importance of S in plant

metabolism. Chloroplasts contain proteins rich in S (Hanson et al. 1941), and

chloroplast morphology is considerably affected by S (Repka et al. 1971, Whatley

1971, Hall et al. 1972). The decreased sink strength in turn could lead to a feedback

inhibition of photosynthetic rate and a decrease in Rubisco synthesis (Krapp and Stitt

1995, Sexton et al. 1997). Further, the stresses usually were shown to enhance the

process of degradation of cellular proteins, also reduce Rubisco (Sheoran et al.

1990a,b, Stiborova et al. 1986), the major source of S-concentration in the chloroplast

(Ferreira and Teixeira 1992). Thus, Cd usually interacts with -SH group of Rubisco

and degrades them (Majumdar et al. 1991). Ferreira and Teixeira (1992) have shown

lesser number of-SH groups, the major form of reduced S in Rubisco, isolated from

S-deprived plants. Further, Cd-induced reductions in the rate of photosynthesis in S-

deprived plants might also be due to restricted use of ATP and NADPH by the Calvin

cycle enzymes (Dietz and Heilos 1990) and decrease in phosphorylation capacity

(Becerril et al. 1989). Nitrogen is one of the essential nutrient elements with a

remarkable effect on crop growth and productivity (Marshner 1995). Sugiharto et al.

(1990) found a significant positive correlation between the photosynthetic capacity of

leaves and their leaf N concentration suggesting that most of the N is used for the

synthesis of components of the photosynthetic apparatus. In particular, Rubisco, the

leaf protein playing the major role in carbon assimilation, was strongly affected by N

deficiency (Seemann et al. 1987).

The decrease in stomatal conductance was possibly due to the fact that cell

growth depends primarily on turgor potential as the driving force of expansion, and

Cd affects both turgor potential and cell wall elasticity (Barcelo et al. 1986b). On the

other hand, S may help in maintaining the water status of the plant by enhancing the

root growth and thereby reducing the stress condition imposed by Cd. S availability

regulates N utilization efficiency of plants and, thus photosynthesis, growth and dry

mass accumulation of crops since photosynthates accumulation has a close

relationship with N and S assimilation (Tandon 1995, Kopriva et al. 2002, Khan et al.

2005).

99

5.2.3 Sulfur and nitrogen metabolism

In the present study the parameters of S and N assimilation responded differentially to

Cd stress. Cd stress increased the ATP-sulftirylase activity and S content, whereas,

NR activity and N content were decreased in both tolerant (Alankar) and non-tolerant

(RH30) cultivars at all growth stages (Figures 32-35). ATP-sulfurylase activity and S

content were higher in Alankar than in RH30. Cd-induced increase in ATP-

sulfurylase could be a possible consequence of an increased requirement of

glutathione for the biosynthesis of phytochelatins. The capacity of plants to survive in

a polluted environment is partially linked to the efficiency of their reductive sulfate

assimilation pathway and its induction has been reported in several plants exposed to

heavy metals (Tukiendorf and Rauser 1990). Expression of genes involved in

reductive sulfate assimilation pathway and enzyme activities are stimulated by Cd

(Ernst et al. 2008). Cd-induced induction of enzymes of sulfate assimilation pathway

has been observed in Arabidopsis thaliana (Harada et al. 2002, Herbette et al. 2006),

Chlamydomonas reinhardtii (Dominguez et al. 2003) and Triticum aestivum (Khan et

al. 2007a). Nussbaum et al. (1988) reported that the accumulation of Cd increased

ATP-sulfurylase activity in Zea mays seedlings. Ruegsegger et al. (1990) also showed

that APS reductase activity is induced coordinately with glutathione synthetase in Cd-

treated Pisum sativum plants. Cd-induced decrease in the NR activity of both the

cultivars is in conformity with Gouia et al. (2000), Chaffei et al. (2003), Anjana et al.

(2006), Ghnaya et al. (2007), Wahid et al. (2007) and Wang et al. (2008). Cd reduces

absorption of nitrate and its transport from root to shoot by inhibiting NR activity in

shoots (Hernandez et al. 1996). Wahid et al. (2007) reported that Cd-induced

inhibition of growth was mainly due to the damaged photosynthetic apparatus and

disruption of the coordination between C and N metabolism in Vigna radiata. In the

present study a consistent relationship between photosynthesis and NR activity was

observed in both the cultivars. Since deprivation of CO2 causes inactivation of NR

(Kaiser and Behnisch 1991), it is not surprising that Cd decreased the NR activation

state. On the other hand, Gouia et al. (2000) have reported that Cd not only decrease

the uptake and transport of water and nitrate but also cause a decrease in NR

activation state and 80% decrease in NR protein level in short term exposure (24h) in

Phaseolus vulgaris plants. Barcelo and Poschenrieder (1990) provided direct reasons

for Cd-induced changes in N-metabolism showing that Cd inhibits the transport and

uptake of water. They proved that the accumulation of Cd in roots alters the process

100

of uptake and transport of water and nitrate to the shoot. Furthermore, the uptake of

nitrate declined under Cd stress (Hernandez et al. 1996, 1997, Ouariti et al. 1997) that

possibly lowered NR activity and disturbed N metabolism.

The application of S further increased the ATP-sulfurylase activity and S

content of Cd-treated plants but the extent of increase was greater in the tolerant

cultivar Alankar than non-tolerant cultivar RH30. Maximum significant increase was

noted in plants supplemented with 50mg S kg'' soil in 50mg Cd kg'' soil treated

plants (Figures 32-35). Application of 50mg S kg'' soil with different levels of N (40,

80 and 120mg N kg"' soil) again increased the ATP-sulfurylase activity and S content

in both the cultivars treated with 50mg Cd kg'' soil at all growth stages (Figures

58,60). Our results suggest that maximum activity of ATP-sulfurylase can only be

achieved at a suitable S and N supply (50mg S kg'' soil + 80mg N kg'' soil) to the

plants. The involvement of S in Cd-tolerance mechanism was reported in Arabidopsis

thaliana (Dominguez-Solis et al. 2001, Harada et al. 2002), Brassica Juncea (Zhu et

al. 1999a,b), Nicotiana tabacum (Harada et al. 2001), Triticum aestivum (Khan et al.

2007a) and Brassica campestris (Anjana et al. 2006, Anjum et al. 2008a). A good part

of S incorporated into organic molecules in plants is located in thiol (-SH) groups in

proteins (cys-residues) or non-protein thiols, glutathione (Noji and Saito 2003, Tausz

et al. 2003, De Kok et al. 2005, Anjum et al. 2008a). Chen and Huerta (1997) showed

that S is a critical nutritional factor for reduction of Cd toxicity. Popovic et al. (1996)

reported a positive effect of S nutrition on Cd-detoxification in Beta vulgaris. In fact

Cd stress provokes plants to enhance the biosynthesis of glutathione for the synthesis

of phytochelatins (Herbette et al. 2006). Besides, it has been previously studied that

good S nutrition diminished the toxicity of Cd by restoring a new steady state of the

glutathione level earlier than in plants grown at low S supply (McMahon and

Anderson 1998). Hence, the improved S nutrition allows a more adequate plant

defense response to Cd toxicity and also prevents S deficiency. It has been found that

at sub-optimal S nutrition, Cd exposed plants preferably allocate S to phytochelatins

synthesis. Sulfur being a component of phytochelatins may play an important role in

their synthesis and ultimately in detoxification of Cd through the formation of Cd-

binding peptides (CdBP) (Cobbett 2000a,b, Harada et al. 2002, Cobbett and

Goldsbrough 2002). Exposing the heavy metal-accumulator Brassica juncea to Cd

was reported to induce a rapid synthesis of phytochelatins, which appeared to be

sufficient to chelate all the Cd taken up, presumably resulting in a complete

101

preservation of the photosynthetic activities (Haag-Kerwer et al. 1999). Several

studies have also demonstrated the critical role of phytochelatins in Cd detoxification

and tolerance of plants to Cd (Inouhe et al. 2000, Harada et al. 2001, 2002, Ebbs et al.

2002, Srivastava et al. 2004, Wojcik et al. 2005, Zhang et al. 2008, Anjum et al.

2008b).

The reductions in NR activity and N content were reversed by S application in

Cd treated Alankar (tolerant) and RH30 (non-tolerant) cultivars (Figures 33-35).

Lesser decrease in NR activity and N content was observed when plants were given

50mg S kg"' soil plus 50mg Cd kg'' soil in comparison to 50mg Cd kg'' soil alone.

The combined application of 80mg N kg'' soil plus 50mg S kg"' soil to 50mg Cd kg''

soil-treated plants maximally lowered the reductions in NR activity and N content in

both the cultivars at all growth stages (Figures 59,61). The negative effects of heavy

metals on NR activity may be due to interaction of metals with thiol/histidyl groups of

proteins, thereby slowing down their catalytic activity and/or completely inhibiting

their functions (Wang and Evangelou 1995). As a resuh, enzymatic reactions may be

blocked, leading to accumulation of nitrate in plants and disturbing the normal plant

metabolism. Also, it may be due to interference with the uptake of nutrients and/or

induction of leakage of nutrients by damaging plasma membrane and protein

alterations due to heavy metal toxicity in leaves (Brune et al. 1994). However, the

beneficial effect of S on NR activity is primarily due to its being a component of

various enzyme proteins, cofactors, and metabolites (Sairam et al. 1995). S

application results in adequate N uptake and in high protein yields in Brassica juncea

and Triticum aestivum (Purakayastha and Nad 1997). Therefore, plants supplied with

S showed an increased NR activity and improved N assimilation as a result of

balanced supply of both N and S, ultimately leading to increased protein formation. It

may also be due to the role of S in increasing S-amino acids (Met and Cys) (Bapet et

al. 1986). N metabolism, therefore, may act as a marker for the study of the response

of plants to Cd toxicity. Upon exposure to Cd, plants often synthesize a set of N-

containing metabolites through N metabolism, such as proline, glutathione and

phytochelatins, which are well known for their significant roles in Cd tolerance of

plants (Sharma and Dietz 2006). Accordingly, plants might exhibit a higher Cd

tolerance by the maintaining the normaJ N metabolism levels under Cd stress

(Gussarsson et al. 1996). The ameliorative effect of S in recovering the NR activity as

102

obtained in our results, are in agreement with the findings of Friedrich and Schrader

(1978), Prosser et al. (2001) and Astolfi et al. (2004).

Combined application of S and N increased the activity of ATP-sulfurylase

while improved the NR activity of Cd treated plants.

5.2.4 Cadmium accumulation

In the present study, Cd content in the root and leaf of both the cultivars increased

with the increasing Cd concentration in soil. Non-tolerant cultivar RH30 accumulated

more Cd in root and leaf than tolerant cultivar Alankar. In both the cultivars,

increased accumulation of Cd in root and leaf due to 50mg Cd kg"' soil was lowered

with the application of 50mg S kg'' soil alone and in combination with 40, 80 and

120mg N kg"' soil at all growth stages. However, combined application of 50mg S kg"

' soil and 80mg N kg'' soil to 50mg Cd kg"' soil-treated plants maximally lowered the

Cd content in root and leaf of both the tolerant (Alankar) and non-tolerant (RH30)

cultivars at all growth stages (Figures 36-37,62-63).

The differences in Cd accumulation among cultivars have previously been

reported (Salt et al. 1995, Arao et al. 2003, Metwally et al. 2005, Khan et al. 2006,

Anjum et al. 2008c). In the present study, roots of both the cultivars accumulated

more Cd than leaves. The retention of high amount of Cd in the root tissues typical of

several plants can be regarded as an important protection mechanism against the

diffusion of the metal (Ranieri et al. 2005). For this reason, Cd-concentration in roots

can reach an average of 75-80% of the total metal taken up (Wojcik and Tukiendorf

1999). Ranieri et al. (2005) have reported that the highest level of Cd was retained in

roots. In the present study, S application reduced the uptake and accumulation of Cd

in both the plant parts. A combined application of S and N also reduced the

concentration of Cd in root and leaf of both the cultivars. Pankovic et al. (2000) found

the lowest inhibition of photosynthetic activity by Cd at optimal N supply, when an

investment in soluble proteins and Rubisco were at their maximum and that higher N

supplies did not alleviate the toxic Cd effects. A proper N supply has positive effects

in overcoming the adverse effects caused by Cd toxicity in Oryza sativa (Hassan et al.

2005a, Pankovic et al. 2000). In addition, S may reduce Cd availability in nutrient

solution through binding with Cd and detoxify Cd in plant cells through enhancing

synthesis of glutathione, which is considered as a first line of defense against Cd

toxicity (Hassan et al. 2005b, Anjum et al. 2008a).

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5.2.5 Cadmium-induced oxidative stress

Heavy metals, in general, cause toxicity and cellular disruption in plant species

through induction of oxidative stress via increased generation of ROS, like O2, O2 ,

OH' and H2O2 (Gratao et al. 2005, Singh et al. 2008b). In the present study, Cd-

induced oxidative stress in Brassica juncea cultivars is evident from the significant

increases in TBARS and H2O2 contents with increasing Cd concentration. However,

compared to the non-tolerant cultivar RH30, tolerant cultivar Alankar showed a much

lower H2O2 content in leaves and TBARS production, which are indicative of lower

oxidative stress in Alankar. Less Cd accumulation and lower level of H2O2 content,

together with the reduced formation of TBARS, thus explain the higher potential of

Alankar than RH30 (Figures 36-39). Malondialdehyde, as the decomposition product

of polyxmsaturated fatty acids of bio-membranes showed greater accumulation under

Cd stress in other studies also (Qadir et al. 2004, Metwally et al. 2005, Liu et al.

2007, Anjum et al. 2008a, Singh et al. 2008a). Cell membrane stability has been

widely used to differentiate stress tolerant and susceptible cultivars of some crops and

that higher membrane stability can be correlated with Cd stress tolerance (Mobin and

Khan 2007). The enhanced cellular damage in RH30 seems to reflect the deterioration

on the equilibrium between generation of ROS and defense mechanisms towards

removal of ROS. Increase in TBARS with increasing Cd concentration has also been

reported in germinating Phaseolus vulgaris seedlings (Somashekaraiah et al. 1992).

This was related to restriction of electron flow in PSII by metal ions that led to the

formation of excited chlorophyll which in tum caused the production of free radicals

(Kato and Shimizu 1985). Cd-induced increase in lipid peroxidation has also been

found earlier in different plants by Dixit et al. (2001), Chien et al. (2001), Groppa et

al. (2001), Wu et al. (2003), Balestrasse et al. (2004), Metwally et al. (2005), Guo et

al. (2007), Ahsan et al. (2007), Razinger et al. (2007), Mobin and Khan (2007),

Noriega et al. (2007), Liu et al. (2007), Ammar et al. (2007), Filek et al. (2007),

Anjum et al. (2008a) and Singh et al. (2008a).

H2O2 is produced at high flux rates by two processes associated with

photosynthesis, the Mehler reaction and the Glycolate oxidase reaction of

photorespiration (Foyer and Noctor 2000). In addition, there are number of other

enzymes in leaves that are capable of producing significant amounts of H2O2,

including peroxidases, NADPH oxidases and oxalate oxidase (Bema and Bernier

1999, Bolwell 1999, Sagi and Fluhr 2001). H2O2 is a relatively stable molecule that

104

can pass freely across the membranes and is capable of spreading damage

(Azpilicueta et al. 2007). Metabolic modeling has also suggested that the loss in

antioxidative defense was due to H2O2 accumulation (Polle 2001, Schutzendubel and

PoUe 2002). H2O2 generation is induced in plants following exposure to a wide

variety of stresses including Cd (Dixit et al. 2001, Schutzendubel et al. 2002,

Romero-Puertas et al. 2004, Cho and Seo 2005, Balestrasse et al. 2006b, Zawoznik et

al. 2007, Yang et al. 2007, Filek et al IQQl, Mobin and Khan 2007, Singh et al.

2008a). Also a pronounced increase in the activity of superoxide dismutase in RH30

with the increase in Cd levels possibly generated higher level of superoxide radicals

and resulted in higher cellular damage in comparison to Alankar. Gossett et al. (1994)

reported that higher superoxide dismutase activity without complementary increase in

the ability to scavenge the formed H2O2 can result in the increased cellular damage.

In the present study, application of 50mg S kg'' soil maximally lowered the

TBARS and H2O2 content of 50mg Cd kg'' soil-treated plants. Application of 50mg S

kg"' soil with different levels of N (40, 80 and 120mg N kg'' soil) further lowered the

contents of TBARS and H2O2 of both the cultivars grown with 50mg Cd kg'' soil and

enhanced the effectiveness of S application (Figures 38-39,64-65). These results on

the significant reduction in TBARS content with S application is in conformity with

the findings of Anjum et al. (2008a) in Brassica campestris leaves. In addition, net

photosynthetic rate and plant dry mass were strongly and positively correlated with

the contents of ascorbate and glutathione. It was concluded that S may ameliorate Cd

toxicity and protects growth and photosynthesis of mustard involving ascorbate and

glutathione. Vassilev et al. (2005) also reported that supply of (NH4)2S04, a well

knovm fertilizer consisting both S and N nutrients, could reduce Cd toxicity in

Hordeum vulgare plants by lowering the TBARS content.

5.2.6 Components of antioxidant system

5.2.6.1 Enzymatic antioxidants

Oxidative stress is a central factor in abiotic stress phenomena that occurs when there

is a serious imbalance in any cell compartment between the production of ROS and

antioxidant defense, leading to physiological changes (Foyer and Noctor 2000,

Agrawal and Mishra 2008). The presence of high concentration of ROS causes

oxidative damage to photosynthetic pigments, bio-molecules such as lipids, proteins

and nucleic acids, leakage of electrolytes via lipid peroxidation resulting in the

disruption of cellular metabolism (Asada 1994, Mobin and Khan 2007). Thus, it is

105

important for cells to have tight control on the concentration of ROS (Schutzendubel

and PoUe 2002). Plants are in fact endowed with a wide array of defense strategies to

protect the photosynthetic apparatus and cellular functions from ROS (Foyer et al.

1994). Plant antioxidant defense system comprises antioxidant enzymes such as

superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and low

molecular weight antioxidants (ascorbate; glutathione; proline; carotenoids, a-

tocopherols and phenolics etc.) (Foyer and Noctor 2003). These components help

plants to cope with the menace of ROS, thus minimizing the oxidative damages,

during exposure to stress factors including Cd (Foyer et al. 1994, Mobin and Khan

2007, Anjum et al. 2008a, Singh et al. 2008a,b). Changes in antioxidant enzyme

activities play an important role in metal tolerance. Though, the expression for

antioxidant enzymes is altered under stress conditions, their upregulation has a key

role in combating the abiotic stress-induced oxidative stress.

In my study, Cd treatments increased superoxide dismutase activity in both

cultivars, the extent of increase was higher in RH30 than Alankar (Figures 40,42).

Mobin and Khan (2007) also reported that higher superoxide dismutase activity in

Brassica juncea cv. RH30 was responsible for higher cellular damage due to

excessive accumulation of H2O2. It has been suggested that high SOD activity may be

harmful for plants due to high H2O2 production, which in turn might inhibit other

enzymes such as ascorbate peroxidase and catalase (Asada 1994, Agrawal and Mishra

2008). Cd-dependent induction of superoxide dismutase activity has also been

observed in earlier studies in other plant species (Dixit et al. 2001, Schutzendubel et

al. 2001, Vitoria et al. 2001, Qadir et al. 2004, Cho and Seo 2005, Metwally et al.

2005, Wu et al. 2006, Krantev et al. 2007, Ekmekci et al. 2008, Hasan et al. 2008,

Agrawal and Mishra 2008, Singh et al. 2008a). Increased superoxide dismutase

activity has also been shown to confer increased protection from oxidative damage in

transgenic plants (Allen et al. 1997). The activity of superoxide dismutase is of great

relevance in metal stress studies for the maintenance of overall defense system of

plants subjected to oxidative damage (Slooten et al. 1995).

Sulfur supplementation to Alankar (tolerant) and RH30 (non-tolerant) treated

with Cd lowered the superoxide dismutase activity (Figures 40,42). The application of

50mg S kg"' soil plus 80mg N kg"' soil further lowered the Cd-induced (50mg Cd kg"'

soil) increase in superoxide dismutase activity (Figures 66-68). The decrease in 50mg

Cd kg"' soil activity due to S and N application conferred to less formation of ROS.

106

The function of S in alleviating the stress may be attributed to its participation in the

synthesis of glutathione, an important ROS scavenger. Sulfur helped the plants to

maintain CO2 (whose absence is the major cause of 02"" production in the process of

photosynthesis) and major reductants (ascorbate, glutathione) in chloroplasts,

maintenance of the sulf-hydryl status of proteins; and hence diminished the chances of

O2' formation and ultimately provided protection from 02~. In a study conducted on

Oryza sativa cvs. Bing 97252 and Xiushui 63, Hassan et al. (2005b) found that higher

S levels caused significant reduction in superoxide dismutase activity in Cd treated

plants. They found that the reduction in superoxide dismutase activity was more

pronounced in Bing 97252 than in Xiushui 63. They concluded that high Cd and

MDA content were consistently accompanied by higher superoxide dismutase activity

and higher S levels caused a marked increase in GSH content and a reduction in SOD

activity, indicating a positive effect of S in alleviating Cd-induced oxidative stress.

Ajnum et al. (2008a) also reported that S supplementation alleviates the Cd-induced

oxidative stress in Brassica campestris plants. Hassan et al. (2008a) suggested that the

oxidative stress of Oryza sativa plants exposed to Cd toxicity could be alleviated

when (NH4)2S04 was supplied as N fertilizer. Moreover, it was also found that

(NH4)2S04-fed plants had higher GSH content under Cd stress.

Catalase activity increased with the increasing Cd concentration at all growth

stages in Alankar, whereas, in RH30, the increase was noted only at 30DAS (Figures

40-42). At later stages (60 and 90DAS), Cd significantly decreased the catalase

activity. In the present study the inhibition of catalase activity in non-tolerant cultivar

RH30 may be due to its inactivation by excess H2O2 produced by superoxide

dismutase under Cd stress. Agrawal and Rathore (2007) also reported that the decline

in catalase activity was due to more consumption of catalase to detoxify H2O2 or its

inactivation. The variable response of catalase activity has been observed under Cd

stress in different plant species. Catalase activity increased in Glycine max nodules

(Balestrasse et al. 2001), Oryza sativa leaves (Hsu and Kao 2004), Brassica

campestris leaves (Ajnum et al. 2008a), in tolerant varieties of Solarium tuberosum

(Stroinski and Kozlowska 1997), in roots ofRaphanus sativus seedlings (Vitoria et al.

2001), Brassica juncea (Mobin and Khan 2007), Triticum aestivum (Khan et al.

2007a) and Cicer arietinum (Hasan et al. 2008). However, catalase activity decreased

in Amaranthus lividus (Bhattacharjee 1998), Glycine max roots (Balestrasse et al.

2001), Phragmites australis (lannelli et al. 2002), Capsicum annuum (Leon et al.

107

2002) and Arabidopsis thaliana (Cho and Seo 2005) under Cd stress conditions.

Furthermore, catalase activity has also been found to remain unaltered under Cd stress

in Glycine max leaves (Ferreira et al. 2002).

Sulfur supplementation further increased the Cd-induced increase in catalase

activity in Alankar. In RH30, addition of S lowered the decrease in catalase activity

only at 60 and 90DAS, whereas, slight increase in its activity was noted at 30DAS. In

Alankar, the increase in catalase activity due to 50mg Cd kg'' soil was further

increased with the application of 50mg S kg'' soil alone and in combination with

80mg N kg'' soil at all growth stages (Figures 40,42,66,68). A combined application

of S and N increased the Cd-induced catalase activity in Alankar which is in close

conformity with the findings of Hassan et al. (2008a). They noted that (NH4)2S04 the

source of both S and N also increased the catalase activity of Cd treated Oryza sativa

plants.

In our study, Cd-dose dependent increase in ascorbate peroxidase activity was

observed in both the cultivars but its activity was greater in Alankar (tolerant) than

RH30 (non-tolerant) at all growth stages (Figures 41,43). Ascorbate peroxidase has an

important role in the scavenging of H2O2 under stressed conditions but its activity

depends on the Cd concentration applied (Mobin and Khan 2007, Singh et al.

2008a,b). Increased leaf ascorbate peroxidase activity under Cd stress has been

reported in Phaseolus aureus (Shaw 1995b), Phaseolus vulgaris (Chaoui et al.

1997b), Pisum sativum (Romero-Puertas et al. 1999), Ceratophyllum demersum

(Aravind and Prasad 2003), Brassica juncea (Mobin and Khan 2007), Zea mays

(Krantev et al. 2007), Triticum aestivum (Khan et al. 2007a), Brassica campestris

(Anjum et al. 2008a) and Vigna mungo (Singh et al. 2008a). The action of ascorbate

peroxidase may be correlated with the reductions in the total ascorbate content i.e.,

greater ascorbate peroxidase activity needed more substrate and hence consumed

more ascorbate as electron donor, thus causing a decline in the ascorbate

concentration in the present study. Similar variations in the ascorbate content were

observed by Schutzendubel et al. (2001) and Qadir et al. (2004) in Cd-exposed Pinus

sylvestyris and Brassica genotypes, respectively.

Sulfur supplementation further increased the ascorbate peroxidase activity of

both the cultivars and maximum significant increase in its activity was noted in plants

treated with 50mg Cd kg"' soil. Combination of 50mg S kg"' soil and 50mg Cd kg'

soil proved best in enhancing the ascorbate peroxidase activity. The increase in

108

ascorbate peroxidase activity due to 50mg Cd kg"' soil was further increased by the

application of 50mg S kg'' soil alone and in combination with 80mg N kg'' soil in

both the cultivars at all growth stages (Figures 41-42,67-68). The demand-driven

increase in the ascorbate peroxidase activity implies the existence of stress sensors

and signal transduction cascades that elicit the response of ascorbate peroxidase

activity using ascorbate as reductant. In fact, S supplementation to the Cd-exposed

plants helped to maintain the synthesis of electron providers (ascorbate and

glutathione), that improved the regeneration of ascorbate (Anjum et al. 2008a), an

electron source for ascorbate peroxidase. Thus, S alleviated the Cd stress alterations

and helped plants to continue the ascorbate peroxidase activity to maintain the

conversion of H2O2 into H2O and O2 by regenerating ascorbate and glutathione

efficiently. Role of glutathione as a signal intermediate in increasing ascorbate

peroxidase expression under metal stress has also been reported by Pekker et al.

(2002). The addition of S and N to Cd-fed plants increased the ascorbate peroxidase

activity in both the cultivars which may be correlated with the improvement in

ascorbate content by S and N.

Glutathione reductase activity of both the cultivars was increased under Cd

stress. Maximal significant increase in glutathione reductase activity was noted in

both the cultivars treated with 50mg Cd kg'' soil. Treatment of 150mg Cd kg ' soil

significantly decreased the glutathione reductase activity in non-tolerant cultivar

RH30 (Figures 41-42). The majority of the studies determining the response of

glutathione reductase to Cd exposure have shown that glutathione reductase activity

increases as part of the defense against the Cd-exposure, an alteration that has often

been dose-dependent and variable over time (Fomazier et al. 2002a,b).

The up regulation of glutathione reductase activity may resuh in the

improvement of abiotic stress tolerance by reducing oxidized glutathione (GSSG)

produced in Ascorbate-Glutathione cycle. Besides, S plays a crucial role in the

synthesis of Cys, a precursor molecule for the production of glutathione (Suter et al.

2000). Addition of 50mg S kg'' soil plus 80mg N kg"' soil further increased the

glutathione reductase activity of both the cultivars at all growth stages and extent of

increase was higher in Alankar than RH30 (Figures 41-42, 67-68). The enhancement

in glutathione reductase activity might be due to enhancement in the overexpression

of enzymes (y-ECS and GSH synthetase) in the chloroplasts (Noctor et al. I998a,b).

In almost all the biological functions, glutathione is oxidized to GSSG which should

109

be converted back to glutathione in plant cell to perform normal physiological

ftinctions. Hence, rapid recycling of glutathione is more essential rather than synthesis

of glutathione, which is a highly regulated and ATP requiring process, glutathione is

regenerated by glutathione reductase in a NADPH-dependent reaction. In this way

glutathione reductase activity maintains cellular GSH:GSSG ratio which is essential

for the control of gene expression and normal protein functions (Foyer and Noctor

2001, Noctor et al. 2002). Hence, the S-induced upregulation of glutathione reductase

activity maintained one of the important hydrophilic antioxidants, the glutathione.

Another explanation of the higher glutathione reductase activity in the present work

may be that as a component of Ascorbate-Glutathione cycle, glutathione maintains the

normal operation of Ascorbate-Glutathione cycle at a high rate in order to detoxify the

ROS; it is also essential to keep glutathione in a reduced form prior to its

incorporation into the synthesis of phytochelatins (Cobbett 2000a,b).

5.2.6.2 Non-enzymatic antioxidants

All plants can synthesize ascorbate, which can accumulate to millimolar

concentrations in both photosynthetic and non-photosynthetic tissues (Foyer et al.

1983). Ascorbate is one of the most powerful antioxidants (Noctor and Foyer 1998,

Smirnoff e/ al. 2001), reacts directly with OH', O2" and ^Oj and reduces H2O2 to H2O

via ascorbate peroxidase (Noctor and Foyer 1998, Chen and Gallie 2004). Ascorbate

is the major, probably the only, antioxidant buffer in the apoplast and is an essential

metabolite involved with vital cell ftinctions. The ascorbate content of plant tissues is

modulated by a large number of factors internal or external to plants.

In the present study, Cd treatments significantly decreased the ascorbate

content at all growth stages and the decrease was greater in non-tolerant cultivar

RH30 than tolerant cultivar Alankar (Figures 44-45). Ascorbate together with

glutathione affects plant tolerance to ROS by participation in the detoxification of

ROS in plant cells (Noctor and Foyer 1998, Wu and Zhang 2002a). The ascorbate

peroxidase activity, in the present investigation, was found enhanced in both the

cultivars. Maximum utilization of ascorbate by up-regulated ascorbate peroxidase

activity, might be one of the major reasons of reduction in ascorbate content.

Secondly, the reduction of MDHA/or DHA into the reduced ascorbate (regeneration)

form might be delayed by Cd. Other workers have also observed that Cd toxicity

reduces ascorbate content (Ozturk et al. 2003, Hsu and Kao 2004, Kuo and Kao 2004,

110

Qadir et al. 2004, Aravind and Prasad 2005, Romero-Puertas et al. l^Ql, Agrawal and

Mishra 2008, Anjum et al. 2008a).

Sulfur supplementation lowered the Cd-induced reduction in ascorbate content

of both the cultivars at all growth stages. The decrease in ascorbate content due to

50mg Cd kg"' soil was lowered by the application of 50mg S kg'' soil alone and in

combination with 80mg N kg"' soil in both the cultivars at all growth stages (Figures

44-45,70-71). Sulfur application under Cd stress has shown important role in the

alleviation of Cd-induced oxidative stress (Hassan et al. 2005b, Anjum et al. 2008a,

Khan et al. 2008a). In the present study, S application improved the ascorbate content

which probably reacted with Oj', 'O2 (directly), H2O2 (enzymatically through

ascorbate peroxidase) and thereby assisted in maintaining these potential toxicants.

The Cd-induced changes in the levels of cellular antioxidants severely affected and

impaired the functioning of the Ascorbate-Glutathione cycle (Zhang and Kirkham

1996). The Ascorbate-Glutathione cycle is a major H2O2 scavenging antioxidant

pathway that operates both in chloroplasts as well in cytosol (Zhang and Kirkham

1996).

The tripeptide (y-GluCysGly) glutathione (GSH) plays a central role in several

physiological processes, including regulation of sulfate transport, signal transduction,

conjugation of metabolites, detoxification of xenobiotics (Xiang et al. 2001) and the

expression of stress-responsive genes (MuUineaux and Rausch 2005). The reduced

form of glutathione, GSH, is an abundant compound in plant tissue that exists

interchangeably with the oxidized form, GSSG. GSH has been associated with several

growth and development related events in plants, including cell differentiation, cell

death and senescence, pathogen resistance and enzymatic regulation (Ogawa 2005,

Rausch and Wachter 2005) and its content is affected by S nutrition (Blake-Kalff et

al. 2000, Anjum et al. 2008a,b, Khan et al. 2008a). In addition, GSH is a substrate for

glutathione peroxidase (GPX) and glutathione-S-transferases (GST), which are also

involved in the removal of ROS (Noctor et al. 2002). GSH is a precursor of

phytochelatins, which are crucial in controlling cellular heavy metal concentrations in

plants (Wojcik et al. 2005, Herbette et al. 2006, Zhang et al. 2008, Anjum et al.

2008a,b).

Cd dose-dependent increase in GSH content was observed in both the cultivars

but its content was greater in Alankar (tolerant) than RH30 (non-tolerant) at all the

growth stages (Figures 44-45). Environmental stresses trigger an increase in ROS

111

levels in plants and the response of GSH can be crucial for adaptive responses.

Antioxidant activity in the leaves and chloroplast of Phragmites australis Trin. (cav.)

ex Steudel was associated with a large pool of GSH, protecting the activity of many

photosynthetic enzymes against the thiophilic bursting of Cd and exerting a direct

important protective role in the presence of Cd (Pietrini et al. 2003). Increased

concentration of GSH has been observed with the increasing Cd concentration in

Pisum sativum (Gupta et al. 2002), Lactuca sativa (Maier et al. 2003), Phragmites

australis (Pietrini et al. 2003), Brassica juncea (Qadir et al. 2004), Pisum sativum

(Metwally et al. 2005), Sedum alfredii (Sun et al. 2007), Oryza sativa (Hassan et al.

2008a).

Sulfur supplementation further increased the glutathione content of both the

cultivars and maximum significant increase in its content was noted in plants grown

with 50mg S kg'' soil at all growth stages. The increase in glutathione content due to

50mg Cd kg"' soil was further increased by the application of 50mg S kg'' soil alone

and in combination vvith 80mg N kg'' soil in both the cultivars at all growth stages

(Figures 44-45,70-71). S is required for the synthesis of various compounds, such as

thiols (GSH), sulpholipids and secondary S compounds, which play an important role

in the metabolism of plants, and in the protection and adaptation of plants against

stresses. The content of the secondary S compounds is strongly dependent on the

stage of development of the plant, temperature, water availability, and the level of S

and N nutrition (Randle et al. 1993, 1995, Randle 2000, Randle and Lancaster 2002,

Coolong and Randle 2003a,b). Fitzgerald et al. (1999) reported that when plants

receive adequate S and N, they develop substantial reserves of sulfate in the root and

glutathione in the leaves. Glutathione besides performing critical functions in

regulating plant growth and adaptation to abiotic stresses, acts as an important S sink

in the plant system (Leustek et al. 2000, Maughan and Foyer 2006). The synthesis of

glutathione is mainly regulated by the availability of its constituent amino acids Cys,

Glu and Gly along with transcriptional regulation of enzymes of glutathione

biosynthesis, y-glutamylcysteine synthase and GSH synthetase (Tomaszewska 2002).

Further, the S-supplementation might help plants to improve the content of GSH by

enhancing y-ECS enzymes as shown by Schneider and Bergmaim (1995) and Strohm

et al. (1995). Cadmium is known to rapidly induce the synthesis of phytochelatins,

thiol-based complexing substances through the up-regulation of glutathione

biosynthesis (Xiang and Oliver 1998, Rauser 2000). As phytochelatins are Glu- and

112

Cys-rich peptides, S and N assimilation pathways are most likely involved in their

synthesis: enzymes of the S metabolism are required for the synthesis of Cys,

enzymes of N metabolism are required for the synthesis of Glu. The glutathione

synthesis in plant cells is dependent on and regulated by the S-supply of the plants. In

addition, glutathione contains three moles of N per mole of S and glutathione

biosynthesis may depend on the availability of N precursors and thus N nutrition of

plants. However, the sink strength of GSH biosynthesis for N may be low compared

with the other major N sinks such as the synthesis of proteins or nucleotides (Kopriva

and Rennenberg 2004). Our results are in close conformity with the observations of

Hassan et al. (2005a,b, 2008a) and Anjum et al. (2008a). Hassan et al. (2005b)

reported that the higher S level in the growing medium alleviate the oxidative stress of

the Oryza sativa plants exposed to Cd. Anjum et al. (2008a) found strong and positive

correlation between net photosynthetic rate and plant dry mass with the contents of

ascorbate and glutathione. In addition, application of S to Cd-treated plants showed

increase in the contents of ascorbate and glutathione which reduced the Cd and

TBARS content in leaves and restored the growth and photosynthesis of Brassica

campestris. Hassan et al. (2008a) reported that addition of (NH4)2S04 to Cd-fed plants

showed increase in glutathione content which reduces the oxidative stress in Oryza

sativa plants that was related to more S supply for this N form. It has also been

suggested that (NH4)2S04 is a better fertilizer because of the presence of both S and N

for use in Cd-contaminated soil (Hassan et al. 2008a,b).

5.2.7 Yield characteristics

Yield is the final manifestation of growth, photosynthesis and biochemical traits of a

plant which are strongly regulated by several environmental factors. In our study, Cd

treatments alone significantly decreased the yield characteristics and the extent of

decrease was greater in RH30 than Alankar. The reductions in yield characteristics

due to 50mg Cd kg"' soil were lowered with the application of 50mg S kg"' soil alone

and in combination with 40, 80 and 120mg N kg"' soil in both the cultivars. The

application of 50mg S kg"' soil plus 80mg N kg"' soil maximally lowered the Cd-

induced (50mg Cd kg"' soil) reduction in yield characteristics of both the cultivars

(Figures 46-47,72-73). The improvement in yield characteristics due to S and N

application to Cd-treated plants can be correlated with the enhancement in growth,

photosynthesis, and biochemical characteristics. Furthermore, S and N application

reduced the Cd uptake and H2O2 and TEARS content along with significant increase

U3

in the enzymatic and non-enzymatic components of antioxidant defense system which

in turn reduced the Cd-induced oxidative stress and hence provided tolerance to plants

and maintained the yield. S and N supplementation to the Cd-stressed plants

ameliorated the ill effects of Cd on yield and its components by greater increase in

siliqua per plant, seeds per siliqua, 1000 seed weight and seed yield per plant of

Alankar (tolerant) than RH30 (non-tolerant) cultivars. Sulfur supplementation to

plants has also been shown as a result of greater biomass production under normal

(Abdin et al. 2003, Ahmad et al. 2005, Bouranis et al. 2008, Bimbraw 2008) and Cd

stress conditions (Hassan et al. 2005b, Anjum et al. 2008a).

Studies have shown a marked influence of applied S on the yield of pulses,

oilseeds and other crops (Pasricha and Aulakh 1991, Tandon 1991, Ahmad et al.

1998, Aulakh and Pasricha 1986, 1998, Singh 2001, Abdin et al. 2003, Bimbraw

2008). Sulfur fertilization is useful not only for increasing crop production, oil

content, and protein content, but also in improving soil conditions for crop growth

(Abdin et al. 2003). Besides it was suggested that an adequate S fertilization to

Brassica is a feasible technique to suppress the uptake of Cd by plants, and to enhance

the uptake efficiency of several essential elements which resulted in increased crop

production and improved quality. Nitrogen is critical in plant growth and development

and is an essential component of amino acids, proteins, nucleic acids, and many

enzymes. Plants grown under limited N levels have reduced Chi a and Chi b

pigments, resulting in stimted plants and characteristic leaf chlorosis (Marschner

1995). As observed also in the present study, increased additions of N usually resuh in

increased yield of crop plants (Mills and Jones 1996, Hochmuth et al. 1999). N

increases yield by influencing a number of growth parameters such as branches and

flowers per plant and by producing more vigorous growth and development (Allen

and Morgan 1972, Taylor et al. 1991). Wright et al. (1988) reported that N prolongs

the life of leaves, improves leaf area duration after flowering and increases overall

crop assimilation, thus contributing to increased seed yield. Therefore, the combined

application of S and N will improve the yield of the crop.

Hassan et al. (2008a) reported that the addition of NH4(S04)2 to Cd-fed plants

improved the yield characteristics of Oryza sativa. They suggested that the

improvement in yield characteristics was due to the ameliorative action of N and S in

the NH4(S04)2 on growth and components of antioxidant defense system of Cd-treated

plants.

114

Conclusively, nutrients are known to decipher essential role in plant

metabolism and augmenting growth and productivity of crops. However, a nutrient

package is required for sustainable agriculture under varied environmental conditions.

Crop cultivars display their inherent potential. The cultivar Alankar surpassed other

cultivars tested and tolerated Cd stress to a significant degree. The cultivar RH30 was

weak in performance and least tolerant to Cd among the cultivars tested. Therefore,

Alankar exhibited lesser decreases in growth, photosynthetic and yield characteristics

under Cd stress. Correspondingly, Alankar also showed lesser oxidative stress and

increased antioxidant system than RH3Q to protect photosynthetic machinery and

consequent effects on other attributes. Sulfur proved significant potential in the

alleviation of Cd stress in both the cultivars. The decreases in the characteristics

observed due to Cd stress were lowered by S application and 50mg S kg'' soil proved

effective in alleviation of Cd stress than the high doses of S. The coordination of S

and N proved most effective in nullifying the Cd stress effects. The potential of 50mg

S kg"' soil in the alleviation of Cd stress was substantially enhanced by the

simultaneous application of 80mg N kg"' soil. The combined application of 50mg S

kg"' soil plus 80mg N kg"' soil not only resulted in restricting the decrease caused by

Cd but also nullified the Cd effects and even enhanced the characteristics values over

the control. A package of 50mg S kg"' soil plus 80mg N kg"' soil appears to be most

effective in the cultivation of mustard under Cd stress. The effect of this combination

was due to coordination of S and N in maintaining plant metabolism and alleviating

Cd stress.

5.3 New Report in the Thesis and Future Prospects

Mineral nutrient status of plants plays a critical role in increasing plant resistance to

enviroimiental stress factors. Of the mineral nutrients, S and N are major

macronutrients necessary for the plant life cycle. The uptake and assimilation of S and

N in higher plants are the crucial factors determining plant growth and vigor and crop

yield. The present study showed that a balanced S and N supply can mitigate the

inhibitory effects of Cd on mustard metabolism. The individual effects of S and N and

other nutrients have been studied. However, no effort has been made to study

combined application of S and N in tolerance of plants, although coordinated

functions of these nutrients are well known. For sustainable agriculture a nutrient

protocol with balanced S and N is necessary in the growth and development of crops

under Cd stress. The results reported in this thesis have confirmed this. The effect of

115

combined application of S and N on oxidative stress and antioxidants under Cd stress

has been reported for the first time. Efforts have been made for the first time to

develop S and N protocol for alleviation of Cd stress in mustard.

Further, the efforts should be focused to dissect the mechanism(s) of

regulation of sulfate and nitrate assimilation at the molecular level in crop plants

under varied environmental conditions. Strategies should also aim to manipulate steps

of pathways leading to the production of thiols and their products in plants through

overexpressing Ser acetyl transferase (SAT), y-ECS and GSHS enzymes of S and N

nutrition under Cd stress. More detailed studies are required to understand the Cd-

induced stress response modulated by S and N metabolism at molecular and

mechanistic levels. This would help to develop an effective strategy to raise

transgenic species for stress resistance.

116

SUMMARY

SUMMARY Chapter 6

The present thesis entitled "Coordination of sulfur and nitrogen in the protection of

photosynthesis, growth and yield of cadmium-treated mustard {Brassica juncea)"

comprises of six chapters.

The importance of the problem and justifications for the present work

imdertaken were emphasized in Chapter 1.

Chapter 2 is the review of literature. It includes literature available on the

aspects of the physiological analysis of various growth-, photosynthetic- and,

biochemical-characteristics, antioxidant studies and yield of various crop plants under

Cd stress. The importance of S and N nutrients in the alleviation of stress and in the

regulation of plant growth and development under stress condition were also

reviewed. The chapter has been divided in to sections and sub-sections for better

understanding of the work of other research reports in this field of study. In addition,

the critical appraisal of the review of literature has also been included to identify the

gap in the field of study.

The details of the material used in the study and methodology adopted to

determine various characteristics recorded in the three experiments have been

described in the Chapter 3. In addition the Chapter 3 also mentions the relevant

information on the location of the study and the environmental conditions during the

data sampling times.

Chapter 4 includes the results on the response of mustard to cadmium, sulfur

and nitrogen treatments in three Experiments. The data obtained were statistically

analyzed and the significance was determined at;7<0.05.

The results obtained in the Experiments have been discussed in Chapter 5 in

the light of observations recorded and supported with the earlier findings, if available.

This chapter also presents the possible explanations of the data obtained to reach a

conclusion and possible future prospects.

Chapter 6 present the summary of the work reported in this thesis.

The results of the Experiments are summarized below:

Experiment 1

The Experiment was conducted to study the effect of five concentrations of Cd viz., 0,

25, 50, 100 and 150mg kg'' soil on Cd accumulation in root and leaf, growth,

photosynthetic and yield characteristics of five mustard cultivars {Brassica juncea L.)

117

namely, Alankar, Varuna, Pusa Bold, Sakha and RH30. The treatments in this

Experiment were arranged in a factorial randomized block design. Cadmium

accumulation, growth and photosynthetic characteristics were studied at pre-flowering

(30DAS), flowering (60DAS) and post-flowering (90DAS) growth stages, while yield

characteristics were noted at harvest (120DAS). Tolerance index of the five cultivars

of mustard was calculated and the cultivars were designated as Cd tolerant and Cd

non-tolerant on the basis of their performance under Cd stress. The effect of Cd on

growth, photosynthetic and yield characteristics was found significant at all sampling

times. The interaction of Cd treatments and cultivars was also found significant. For

growth characteristics, the interaction effect was found non-significant at 30 DAS.

The increase in Cd levels decreased the growth, photosynthetic and yield

characteristics of all the five cultivars at all sampling stages. The observations

recorded at the three sampling stages showed similar pattern of cultivar response to

Cd treatments. Maximum reduction in growth, photosynthesis and yield

characteristics was noted with 150mg Cd kg"' soil followed by 100, 50 and 25mg Cd

kg'' soil.

Among cultivars, Alankar showed lesser decrease in growth, photosynthetic

and yield characteristics followed by Varuna and Pusa Bold, whereas, RH30 and

Sakha exhibited greater reduction in growth characteristics under Cd stress. The

tolerance index of cultivars was: Alankar >Varuna >Pusa Bold >Sakha >RH30.

Experiment 2

Experiment 2 was conducted on the basis of findings of Experiments 1. As observed

in Experiment 1, Alankar emerged as Cd-tolerant and RH30 as Cd non-tolerant

cultivar. Among Cd levels, 150mg Cd kg"' soil was found most toxic and caused

maximum reductions in the characteristics studied. In the present Experiment, the aim

was to study the alleviation potential of elemental S levels (50 and lOOmg S kg"' soil)

on the effects of 50 and 150mg Cd kg"' soil. The treatments in this Experiment were

arranged in a factorial randomized block design. The assessment was done by

studying the changes in Cd accumulation in root and leaf, growth and photosynthetic

characteristics, S and N assimilation, components of antioxidant defense system and

yield characteristics of tolerant (Alankar) and non-tolerant (RH30) cultivars. The

sampling stages for Cd accumulation, growth and photosynthetic characteristics, S

and N assimilation, components of antioxidant defense system were pre-flowering

118

(SODAS), flowering (60DAS) and post-flowering (90DAS). The yield characteristics

were noted at harvest (120DAS).

The growth and photosynthetic characteristics and N assimilation decreased

maximally in both the cultivars treated with 150mg Cd kg'' soil. Contrarily, there was

significant increase in the Cd accumulation, S assimilation, H2O2 and TBARS content

and components of enzymatic antioxidant system and glutathione content was found.

Non-tolerant cultivar RH30 exhibited greater Cd effects than tolerant cultivar

Alankar. In comparison to lOOmg S kg'' soil, the application of 50mg S kg'' soil

maximally alleviated the toxic effect of 50mg Cd kg'' soil and improved growth,

photosynthesis, S and N assimilation and components of enzymatic and non-

enzymatic antioxidant system thus yield characteristics of both the cultivars. Further,

no ameliorative effect of lOOmg S kg'' soil was observed in plants treated with 150mg

Cd kg"' soil. Alleviation effects of S were higher in Alankar than RH30.

Experiment 3

Experiment 3 was conducted on the basis of the findings of Experiments 2. In

Experiment 2, it was observed that the application of 50mg S kg"' soil to plants treated

with 50mg Cd kg"' soil alleviated Cd-induced toxicity in both the cultivars.

Supplementation of 50mg S kg"' soil maximally overcome the toxic effects of 50mg

Cd kg"' soil in Alankar (tolerant cultivar), while, the same combination lowered the

Cd-induced toxic effects in RH30 to some extent. The present experiment was aimed

to study the effect of supplementation of different N levels (40, 80 and 120mg N kg"'

soil) to 50mg S kg"' soil for alleviation of adverse effects of 50mg Cd kg"' soil. The

treatments in this Experiment were arranged in a factorial randomized block design.

The assessment was done by analyzing the changes in Cd accumulation in root and

leaf, growth and photosynthetic characteristics, S and N assimilation, components of

antioxidant defense system at 30, 60 and 90DAS and yield characteristics at harvest.

The alterations in growth, photosynthesis, S and N assimilation, components

of antioxidant system and yield characteristics caused by 50mg Cd kg"' soil were

alleviated by 50mg S kg"' soil in tolerant (Alankar) and non-tolerant (RH30) cultivars,

but the alleviation potential of S varied between the cultivars. Application of different

levels of N (40, 80 and 120mg N kg"' soil) further enhanced the alleviation effect of S

(50mg S kg"' soil) in both the cultivars. Among the N levels used, the application of

80mg N kg"' soil proved superior to the other N levels. A combined application of S

119

and N (50mg S kg'' soil plus 80mg N kg"' soil) not only ameliorated the Cd-induced

(50mg Cd kg"' soil) effects but also increased the growth, photosynthesis, S and N

assimilation and components of enzymatic and non-enzymatic antioxidant system

thus yield characteristics in Alankar. In RH30, this combination (50mg S kg"' soil

plus 80mg N kg"' soil) only lowered the adverse effects of Cd.

The present chapter is followed by an up-to-date bibliography of the literature

cited in the text.

120

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