1 23

Acta Physiologiae Plantarum ISSN 0137-5881Volume 33Number 4 Acta Physiol Plant (2011)33:1385-1397DOI 10.1007/s11738-010-0672-x

Modification of chromium (VI)phytotoxicity by exogenous gibberellicacid application in Pisum sativum (L.)seedlings

Savita Gangwar, Vijay Pratap Singh,Prabhat Kumar Srivastava & JagatNarayan Maurya

1 23

Your article is protected by copyright and

all rights are held exclusively by Franciszek

Górski Institute of Plant Physiology, Polish

Academy of Sciences, Kraków. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you

wish to self-archive your work, please use the

accepted author’s version for posting to your

own website or your institution’s repository.

You may further deposit the accepted author’s

version on a funder’s repository at a funder’s

request, provided it is not made publicly

available until 12 months after publication.

ORIGINAL PAPER

Modification of chromium (VI) phytotoxicity by exogenousgibberellic acid application in Pisum sativum (L.) seedlings

Savita Gangwar • Vijay Pratap Singh •

Prabhat Kumar Srivastava • Jagat Narayan Maurya

Received: 11 August 2010 / Accepted: 7 December 2010 / Published online: 24 December 2010

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Effects of exogenous gibberellic acid (GA; 10

and 100 lM) application on growth, protein and nitrogen

contents, ammonium (NH4?) content, enzymes of nitrogen

assimilation and antioxidant system in pea seedlings were

investigated under chromium (VI) phytotoxicity (Cr VI;

50, 100 and 250 lM). Exposure of pea seedlings to Cr and

100 lM GA resulted in decreased seed germination, fresh

and dry weight and length of root and shoot, and protein

and nitrogen contents compared to control. Compared to

control, Cr and 100 lM GA led to the significant alteration

in nitrogen assimilation in pea. These treatments decreased

root and shoot nitrate reductase (NR), glutamine synthetase

(GS) and glutamine 2-oxoglutarate aminotransferase

(GOGAT) activities (except 50 lM Cr alone for GOGAT)

while glutamate dehydrogenase (GDH) activity and NH4?

content increased. Compared to control, the root and shoot

activities of superoxide dismutase (SOD) and ascorbate

peroxidase (APX) increased (except APX activity at

250 lM Cr ? 100 lM GA) while catalase (CAT), gluta-

thione reductase (GR) and dehydroascorbate reductase

(DHAR) activities were decreased (except GR at 100 lM

GA alone) following exposure of Cr and 100 lM GA.

Total ascorbate and total glutathione in root and shoot

decreased by the treatments of Cr and 100 lM GA while

their levels were increased by the application of 10 lM GA

compared to Cr treatments alone. It has been reported that

application of 10 lM GA together with Cr alleviated

inhibited levels of growth, nitrogen assimilation and anti-

oxidant system compared to Cr treatments alone. This

study showed that application of 10 lM GA counteracts

some of the adverse effects of Cr phytotoxicity with the

increased levels of antioxidants and sustained activities of

enzymes of nitrogen assimilation; however, 100 lM GA

showed apparently reverse effect under Cr phytotoxicity.

Keywords Antioxidants � Chromium (VI) phytotoxicity �Gibberellic acid � Nitrogen assimilation �Pisum sativum (L.)

Abbreviations

APX Ascorbate peroxidase

CAT Catalase

DHAR Dehydroascorbate reductase

GDH Glutamate dehydrogenase

GOGAT Glutamine 2-oxoglutarate aminotransferase

GR Glutathione reductase

GS Glutamine synthetase

NR Nitrate reductase

SOD Superoxide dismutase

Introduction

Chromium (Cr) contamination of soil and water is one of

the most serious environmental threats leading to losses in

crop yield and hazardous effects on health when it enters

the food chain (Vernay et al. 2007). Chromium is

Communicated by G. Klobus.

S. Gangwar (&) � J. N. Maurya

Department of Plant Science, MJP Rohilkhand University,

Bareilly 243006, India

e-mail: gangwar.savi@gmail.com

V. P. Singh � P. K. Srivastava

Ranjan Plant Physiology and Biochemistry Laboratory,

Department of Botany, University of Allahabad,

Allahabad 211002, India

123

Acta Physiol Plant (2011) 33:1385–1397

DOI 10.1007/s11738-010-0672-x

Author's personal copy

continuously being released in the environment from lea-

ther tanning, steel plating and dyeing industries and thus

has lead to a continuous increase in available Cr in water

and soil (Shanker et al. 2004). Chromium possesses

valence numbers ranging from 0 to 6, of which the trivalent

chromium (Cr, III) and hexavalent chromium (Cr, VI) are

more common in Cr-polluted substrates (Kotas and Stasi-

cka 2000). Hexavalent Cr is highly toxic because of its

ability to cross plasma membrane with powerful oxidizing

capability (Shanker et al. 2004). Chromium (VI) phyto-

toxicity inhibits seed germination, growth and photosyn-

thesis, decreases uptake of nutrient elements, alters water

balance, and affects nitrogen and sulfur metabolism

(Shanker et al. 2004; Vernay et al. 2007; Kumar and Joshi

2008; Schiavon et al. 2008). It has been reported that toxic

property of Cr(VI) originates from the formation of reac-

tive oxygen species (ROS), i.e. superoxide radical (O2�-),

hydrogen peroxide (H2O2) and hydroxyl radical (�OH), and

these ROS present in higher concentrations produce cyto-

toxic effects due to their ability to oxidize lipids, proteins

and nucleic acids (Shanker et al. 2004; Panda 2007; Pandey

et al. 2009b). In order to mitigate deleterious effects of

ROS, plants possess complex defense mechanisms. Such

defense mechanisms involve both enzymatic and non-

enzymatic antioxidants (Panda 2007). The simultaneous

action of various antioxidant enzymes viz. superoxide

dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC

1.11.1.6), glutathione reductase (GR, EC 1.6.4.2), dehy-

droascorbate reductase (DHAR, EC 1.8.5.1), ascorbate

peroxidase (APX, EC 1.11.1.11), etc. is essential for reg-

ulation of ROS levels within cell (Shanker et al. 2004;

Panda 2007). Non-enzymatic antioxidants such as ascor-

bate and glutathione also play important role in preventing

oxidative stress (Noctor and Foyer 1998).

To fulfill the food demand of increasing population,

alternative strategies are needed to alleviate the adverse

effects of metal phytotoxicity on crops. Chemical appli-

cation and agronomical crop management practices have

been used to alleviate the metal phytotoxicity with a little

bit of success. Exogenous application of plant hormones

may be an alternative strategy to induce the capability

within plants to face successfully the detrimental situation

of metal phytotoxicity. Plant hormones are a group of

chemical messengers that regulate plant growth and

development. Gibberellic acid (GA), one of the key plant

hormones influences seed germination, stem elongation,

leaf expansion and reproductive development (Hooley

1994; Matsuoka 2003). Studies have shown that exogenous

application of GA provides protection to plants against

abiotic stresses and increases crop yield (Tuna et al. 2008;

Wen et al. 2010). However, Fuchs and Lieberman (1968),

Mori and Schroeder (2004) and Celik et al. (2007) have

shown that if GA is applied in excess, it results in increased

ethylene production, ROS generation and alteration in

defense mechanisms, respectively, which, in turn, cause

tissue damage and retard growth. Though there have been

many studies which show effects of exogenous application

of GA on plants and animals, however, a lot of contra-

dictory results can be found in the literature. Very few

studies have been carried out to investigate plants respon-

ses to exogenous application of GA during their early stage

of growth under stress conditions. Therefore, we have

undertook this study to investigate the effects of exogenous

GA application on growth, nitrogen assimilation and anti-

oxidant system in Pisum sativum L. seedlings during their

early stage of growth under Cr(VI) phytotoxicity. This

paper is mainly focused on the mechanisms by which GA

influences pea seedling responses under Cr(VI)

phytotoxicity.

Materials and methods

Plant materials and culture conditions

Uniform sized pea seeds (Pisum sativum L. cv. Azad P-1)

were surface sterilized in 10% (v/v) sodium hypochlorite

solution for 5 min. Then they were washed thoroughly and

soaked for 2–4 h in distilled water. The imbibed seeds were

placed in Petri plates (radius 7.3 cm and surface area

167.42 cm2:150 mm RivieraTM). Bottom of each Petri

plate was lined with Whatman No-1 filter paper which was

moistened with either 20 ml of 0.5 strength Hoagland’s

solution only (control) or with 20 ml of each selected

concentration of Cr and GA alone as well as in combina-

tion prepared in 0.5 strength Hoagland’s solution (Arditti

and Dunn 1969). Petri plates were kept in a growth

chamber for seed germination for 3 days under dark con-

ditions (relative humidity of 50–60% at 25 ± 2�C). After

3 days, seedlings of each Petri plate were exposed with a

light irradiance of 150 lmol photons m-2 s-1 for next

8 days in same growth chamber (in which seeds were

germinated) under same humidity and temperature condi-

tions. The photoperiod was 12/12 h (day/night regime)

daily. After 11 days, root and shoot samples from control

and treated seedlings were harvested and different param-

eters were analyzed immediately. In the present study,

following combinations of Cr and GA were made: only 0.5

strength Hoagland’s solution (control), 50 lM Cr, 100 lM

Cr, 250 lM Cr, 10 lM GA, 100 lM GA, 50 lM Cr ?

10 lM GA, 100 lM Cr ? 10 lM GA, 250 lM

Cr ? 10 lM GA, 50 lM Cr ? 100 lM GA, 100 lM

Cr ? 100 lM GA and 250 lM Cr ? 100 lM GA.

Therefore, 12 Petri plates were arranged one for each

combination. Each Petri plate contained 25 healthy and

uniform sized seeds. These selected combinations were

1386 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

given four times during 11 days growth of pea seedlings at

an interval of 3 days, i.e. first treatment was given during

seed sowing (on the 1st day), second on the 3rd day, third

on the 6th day and fourth on the 9th day in 0.5 strength

Hoagland’s solution. All the Petri plates were kept wet by

the addition of 0.5 strength Hoagland’s solution daily or as

per requirement to avoid limitation of nutrients.

Seed germination and growth parameters

Seed germination (%) was determined by counting the

number of germinated seeds per 25 seeds. Growth param-

eters (fresh and dry weight, and length of root and shoot)

were measured after harvesting control and treated pea

seedlings randomly.

Determination of total protein, total nitrogen and NH4?

contents

Root and shoot total protein was determined by following

the method of Lowry et al. (1951) using bovine serum

albumin as standard. Total nitrogen content of each sample

was quantified using Kjeldahl method (Lang 1958). For the

measurement of root and shoot NH4? content, Nessler’s

reagent method of Molins-Legua et al. (2006) was used.

Assays of nitrogen assimilating enzymes

Nitrate reductase activity was assayed according to the

method of Debouba et al. (2006). One unit of enzyme

activity is defined as 1 nmol NO2- formed/mg protein/h.

glutamine 2-oxoglutarate aminotransferase activity also

called glutamate synthase was measured by the method of

Singh and Srivastava (1986). One unit of enzyme activity is

defined as 1 nmol NADH oxidized/mg protein/min. For

measurement of glutamine synthetase activity the method

of Lillo (1984) was used. One unit of enzyme activity is

defined as 1 nmol c-glutamylhydroxamate formed/mg

protein/min. Glutamate dehydrogenase activity was

assayed by the method of Singh and Srivastava (1983). One

unit of enzyme activity is defined as 1 nmol NADH oxi-

dized/mg protein/min.

Assays of antioxidant enzymes

Fresh root and shoot samples (1 g) from control and treated

seedlings were homogenized in ice cold mortar and pestle

using 10 ml of 50 mM potassium phosphate buffer (pH

7.0) containing 1 mM EDTA and 1% (w/v) polyvinylpyr-

rolidone. In case of APX and DHAR activities, 1 mM

ascorbic acid and 2 mM 2-mercaptoethanol were added

into above buffer, respectively. After centrifugation

(20,000g for 10 min at 4�C), the supernatant was used for

determination of SOD, APX, GR, CAT and DHAR

activities.

Superoxide dismutase activity was assayed by the

nitroblue tetrazolium (NBT) reduction method of Gian-

nopolitis and Reis (1977). One unit of SOD activity is

defined as the amount of enzyme which is required to cause

50% inhibition in the reduction of NBT. Ascorbate per-

oxidase activity was assayed using the method of Nakano

and Asada (1981). One unit of enzyme activity is defined as

1 nmol ascorbate oxidized/mg protein/min. Glutathione

reductase activity was estimated by recording decrease in

absorbance at 340 nm due to the oxidation of NADPH

(Schaedle and Bassham 1977). One unit of enzyme activity

is defined as 1 nmol NADPH oxidized/mg protein/min.

Catalase activity was determined by the method of Aebi

(1984). One unit of enzyme activity is defined as 1 nmol

H2O2 decomposed/mg protein/min. Dehydroascorbate

reductase activity was determined by measuring the

increase in absorbance at 265 nm due to the reduction of

dehydroascorbate (DHA) into reduced ascorbate (AsA)

(Nakano and Asada 1981). One unit of enzyme activity is

defined as 1 nmol DHA reduced/mg protein/min.

Measurements of total ascorbate and total glutathione

levels

Total ascorbate in control and treated seedlings was

determined by the method of Gossett et al. (1994). Total

ascorbate was calculated using standard curve prepared

with L-ascorbic acid. Total glutathione was determined by

the enzyme-recycling method of Brehe and Burch (1976).

The amount of total glutathione was calculated using a

standard curve prepared with reduced glutathione (GSH).

Statistical analysis

All the data were subjected to analysis of variance and

expressed as mean ± SE of three independent experi-

ments. Statistical significance of the means was compared

by the Duncan’s multiple range test at P \ 0.05 signifi-

cance level using SPSS-10 software. In each experiment,

ten seedlings were taken randomly for the measurements of

fresh and dry weight and length of root and shoot while for

other parameters three seedlings were used.

Results

Seed germination, fresh and dry weight, and length of root

and shoot were used to assess the impact of exogenous GA

application on growth of pea seedling under Cr phytotox-

icity. As shown in Figs. 1 and 2, Cr (50, 100 and 250 lM)

and GA (100 lM) decreased seed germination, fresh and

Acta Physiol Plant (2011) 33:1385–1397 1387

123

Author's personal copy

dry weight, and length of root and shoot compared to

control. Moreover, it was noticed that combined applica-

tion of Cr and 100 lM GA further decreased seed germi-

nation and growth of pea seedlings. Maximum decrease in

seed germination and growth parameters was noticed under

the treatment of 250 lM Cr ? 100 lM GA (seed germi-

nation decreased by 44%, root and shoot fresh weight

decreased by 59 and 54%, root and shoot dry weight

decreased by 62 and 58%, and length of root and shoot

decreased by 56 and 45%, respectively, compared to con-

trol). On the contrary, it was reported that addition of

10 lM GA together with Cr increased the germination of

seeds (equal to the control), and alleviated inhibited levels

of growth in pea seedlings compared to Cr treatments alone

(Figs. 1, 2). Moreover, it was observed that 10 lM GA

alone did not significantly influence length of root; how-

ever, length of shoot was significantly increased by this

dose compared to control (Fig. 2).

Results shown in Fig. 3 indicated that exposure of pea

seedlings to Cr and 100 lM GA decreased both total

protein and total nitrogen in root and shoot compared to

control. Exposure of pea seedlings to 250 lM

Cr ? 100 lM GA decreased protein content in root and

shoot by 51 and 38% and nitrogen content by 50 and 45%,

respectively, compared to control. On the contrary, sup-

plementation of 10 lM GA together with Cr increased both

total protein and total nitrogen compared to Cr treatments

alone (Fig. 3).

As shown in Fig. 4, treatments of pea seedlings with Cr

and 100 lM GA alone as well as in combination decreased

NR activity compared to control. However, application of

10 lM GA together with Cr increased NR activity com-

pared to Cr treatments alone (Fig. 4). Results pertaining to

the NH4? content are shown in Fig. 5. Exposure of pea

seedlings to single and combined treatments of Cr and

100 lM GA increased NH4? content in root and shoot

compared to control as it was increased by 79 and 74%,

respectively, under the treatment of 250 lM Cr ? 100 lM

GA. However, 10 lM GA alone did not significantly

influence NH4? content in root and shoot compared to

control. Besides, when 10 lM GA was applied together

with Cr, it appreciably lowered NH4? content in root and

shoot compared to Cr treatments alone (Fig. 5). Results

related to the enzymes of NH4? assimilation are depicted

in Fig. 5. Results showed that Cr and GA alone as well as

in combination increased GDH activity in root and shoot

compared to control. On the contrary, Cr and 100 lM GA

alone as well as in combination decreased GOGAT and GS

activities (except 50 lM Cr alone for GOGAT activity) in

root and shoot compared to control. Reduction in GOGAT

and GS activities was higher in roots than shoots. Exposure

of pea seedlings to 250 lM Cr ? 100 lM GA led to a

decrease in root and shoot GOGAT activity by 44 and 35%

and in GS activity by 41 and 37%, respectively, compared

to control. However, supplementation of 10 lM GA

together with Cr either increased GOGAT and GS activi-

ties over control values or restored them over Cr treatments

alone (Fig. 5).

Results for antioxidant enzyme activities are shown in

Figs. 6 and 7. Exposure of pea seedlings to Cr and GA

alone as well as in combination increased SOD and APX

activities (except root and shoot APX activity at 250 lM

Cr ? 100 GA treatment; decreased by 28 and 19%,

respectively) in root and shoot compared to control

(Fig. 6). On the contrary, exposure of pea seedlings to Cr

and 100 lM GA led to the decrease in CAT, GR and

DHAR activities (except GR activity at 100 lM GA alone)

compared to control (Fig. 7). Treatment of pea seedlings

with 250 lM Cr ? 100 lM GA produced decrease in root

and shoot CAT activity by 44 and 36%, in GR activity by

33 and 25% and in DHAR activity by 36 and 31%,

respectively, compared to control. On the contrary, under

Cr phytotoxicity, addition of 10 lM GA increased CAT,

GR and DHAR activities in root and shoot even above their

respective control values (Fig. 7). Though CAT, GR and

DHAR activities did not show much variation in

Cr ? 10 lM GA treatments however, they were signifi-

cantly higher than Cr treatments alone (Fig. 7).

Ascorbate and glutathione were tested due to their

involvement in several useful metabolic processes and their

capability to reduce ROS. As shown in Fig. 8, treatments

of pea seedlings with Cr and 100 lM GA alone as well as

in combination decreased total ascorbate and total gluta-

thione levels in root and shoot compared to control.

However, glutathione was more affected than ascorbate. It

was noticed that exposure of pea seedlings to 250 lM

Cr ? 100 lM GA declined root and shoot ascorbate level

by 51 and 46% and glutathione level by 58 and 54%,

respectively, compared to control. On the contrary, supply

Seed

ger

min

atio

n

(%

)

0

30

60

90

120

150

+ 10 µM GA + 100 µM GA

a a a a aabc

f

c cdef

g

0 µM Cr50 µM Cr

100 µM Cr

250 µM Cr

- GA

Fig. 1 Effect of exogenous GA application on pea seed germination

under Cr phytotoxicity. Data are mean ± SE of three independent

experiments. Mean values followed by different letters on bars are

significantly different (P \ 0.05) according to the Duncan’s multiple

range test

1388 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

Fre

sh w

eigh

t(m

g/se

edlin

g)

0

100

200

300

400

Fre

sh w

eigh

t(m

g/se

edlin

g)

0

50

100

150

200

Dry

wei

ght

(mg/

seed

ling)

0

10

20

30

40

Dry

wei

ght

(mg/

seed

ling)

0

7

14

21

28

Len

gth

(mm

/see

dlin

g)

0

10

20

30

40

50

60

Len

gth

(mm

/see

dlin

g)

0

5

10

15

20a a

abb bcc cc

d

cd

e

ab

cbd d

e

cde

f f g

Root

Root Shoot

Root Shoot

ab

e

g

a ab

dec cd

f

h

b bc

ef

h

ab

cfg

c cdg

i

abc

e

g

a ac

ed d

f

h

bbc

d

e

a b

cd

bccde

f

0 µM Cr50 µM Cr

100 µM Cr

250 µM Cr

+ 10 µM GA + 100 µM GA + 10 µM GA + 100 µM GA

Shoot

- GA - GA

Fig. 2 Effect of exogenous GA application on fresh and dry weight,

and length of root and shoot of pea seedlings grown for the 11 days

under Cr phytotoxicity. Data are mean ± SE of three independent

experiments. Mean values followed by different letters on bars are

significantly different (P \ 0.05) according to the Duncan’s multiple

range test

T

otal

pro

tein

(m

g/g

dry

wei

ght)

0

20

40

60

80

100

120

T

otal

pro

tein

(m

g/g

dry

wei

ght)

0

30

60

90

120

150

T

otal

nitr

ogen

(m

g/g

dry

wei

ght)

0

10

20

30

40

50

T

otal

nitr

ogen

(m

g/g

dry

wei

ght)

0

15

30

45

60

75

90

Root Shoot

Root Shoot

ab

ce

a ab

c c cdd

f

ab

d e

a a

bcd cdd e f

ab

df

a abc

d cdde

g

bc

de

bc

d d dde

f

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

+ 10 µM GA + 100 µM GA + 10 µM GA + 100 µM GA

a

- GA- GA

Fig. 3 Effect of exogenous GA application on total protein and total

nitrogen in root and shoot of pea seedlings grown for the 11 days

under Cr phytotoxicity. Data are mean ± SE of three independent

experiments. Mean values followed by different letters on bars are

significantly different (P \ 0.05) according to the Duncan’s multiple

range test

Acta Physiol Plant (2011) 33:1385–1397 1389

123

Author's personal copy

of 10 lM GA together with Cr increased total ascorbate

and total glutathione levels compared to Cr treatments

alone (Fig. 8).

Discussion

The problem of heavy metal contamination exists due to

the fact that metal cannot be degraded like other organic

xenobiotics but its toxicity can be minimized to a consid-

erable extent using low-cost alternative strategies. Nitrogen

assimilation is one of the most important biochemical

processes which determine growth, development and yield

of plants. However till date, scant information is available

about impact of Cr phytotoxicity on nitrogen assimilation

in pea. Our study showed that exposure of pea seedlings to

Cr and 100 lM GA decreased seed germination, fresh and

dry weight, and length of root and shoot compared to

control (Figs. 1, 2). Chromium which remains in soil for a

long time can affect germination of seeds, plant growth,

leaf water potential and turgor, and induces plasmolysis

(Vazques et al. 1987; Vernay et al. 2007). Moreover, it is

known that P and Cr compete for surface site binding

(Wallace et al. 1976). Besides, Fe, Mn and S are also

known to compete with Cr for transport (Schiavon et al.

2008). Therefore, it might be possible that Cr is competed

effectively with these elements and thus might lead to

disturbed mineral status in pea seedlings. It has been

reported that Cr significantly declined Fv/Fm ratio (maxi-

mum quantum yield of photosystem II) in Lolium perenne

L. and pea, which is frequently used to assess the health of

photosystem II (Vernay et al. 2007; Pandey et al. 2009a).

Declined Fv/Fm ratio suggests that the photosystem II

became impaired when plants were exposed to Cr. More-

over, Cr may have indirect negative effect on growth that is

resulted from oxidative damage to mitochondrial electron

transport chain and photosynthetic apparatus through pro-

duction of ROS by auto oxidation and Fenton reaction

(Vernay et al. 2007; Pandey et al. 2009b). Regarding the

impact of 100 lM GA on growth of pea seedlings, it has

been shown that exogenous application of GA in excess

induces increased ethylene production, generation of ROS

and alteration in antioxidant system (Fuchs and Lieberman

1968; Mori and Schroeder 2004; Celik et al. 2007). Celik

et al. (2007) have shown that 75 ppm of GA (equal to

216 lM) caused tissue damage by enhanced lipid peroxi-

dation and decreased levels of glutathione-S-transferase

and glutathione. Besides, it has been reported that

increased production of ethylene triggers abscisic acid

synthesis which, in turn, led to the reduction in stomatal

aperture and CO2 assimilation followed by growth inhibi-

tion and tissue senescence (Hansen and Grossmann 2000).

However, Wen et al. (2010) reported that application of

100 lM GA counteracts salinity stress to some extent. This

discrepancy might be due to the heterogeneous experi-

mental approaches, including laboratory grown conditions,

field experiments and developmental stage of studied plant

at which GA is applied. On the contrary, our results indi-

cated that application of 10 lM GA together with Cr

counteracts negative effects of Cr and increases seed ger-

mination, fresh and dry weight, and length of root and

shoot of pea seedlings compared to Cr treatments alone

(Figs. 1, 2). Beevers and Guernsey (1966) have reported

that during pea seed germination nitrogen of cotyledons

rapidly declined with an accompanying increase of nitro-

gen in the developing axis. Moreover, they showed that the

accumulation of alcohol soluble nitrogen, primarily amino

nitrogen in cotyledons and axis during germination indi-

cates that the mobilization of nitrogen is facilitated by

proteolysis and translocation of the products. Besides, they

showed that cotyledons had initially high RNA content

which declined during seed germination while RNA in the

developing axis increased. The increase in developing axis

RNA was greater than the decline in cotyledonary RNA

indicating a net nucleic acid synthesis. Our results sug-

gested that Cr and 100 lM GA might have negatively

affected mobilization of these components from cotyledons

to the developing axis that is why reduction in seed ger-

mination and growth was observed (Figs. 1, 2). However,

addition of 10 lM GA might have supported mobilization

NR

act

ivity

(u

nits

/mg

prot

ein)

0

13

26

39

52

65

78 Root

ac

e g

a bc d cd

e fh

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

N

R a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

100

200

300

400

500

600 Shoot

ab

cdf

a ab c bcdde

g

- GA + 10 µM GA + 100 µM GA

Fig. 4 Effect of exogenous GA application on activity of NR in root

and shoot of pea seedlings grown for the 11 days under Cr

phytotoxicity. Data are mean ± SE of three independent experiments.

Mean values followed by different letters on bars are significantly

different (P \ 0.05) according to the Duncan’s multiple range test

1390 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

of these components from cotyledons to the developing

axis under Cr phytotoxicity as supported by the data of

seed germination, fresh and dry weight, and length of root

and shoot (Figs. 1, 2).

Protein and nitrogen contents may be considered as

important indicators to assess growth performance of plants

under stress conditions. Chromium and 100 lM GA alone

as well as in combination decreased both total protein and

total nitrogen contents in root and shoot compared to

control (Fig. 3). Their contents were more affected in roots

than shoots. Decrease in protein content could be a con-

sequence of increased protein degradation and/or a

decrease in protein synthesis. Balestrasse et al. (2003) have

shown that under cadmium stress, decrease in protein

content was related with increased protease activity in

soybean. Similar to protein content, decreased level of

nitrogen has also been reported in range of crops under

stress conditions (Shen et al. 1994; Tuna et al. 2008).

Declined level of nitrogen might be due to the decreased

nitrate absorption efficiency of roots as evidenced from

decreased NR activity (Figs. 3, 4). In the present study,

however, addition of 10 lM GA together with Cr increased

both total protein and total nitrogen contents compared to

Cr treatments alone (Fig. 3).

Nitrogen is an essential plant macronutrient. The avail-

ability of nitrogen has a major influence on crop yield.

Nitrate is the common source of nitrogen available to

plants. Once nitrate is being absorbed by plants it may be

reduced in roots, stored in the vacuoles, or transferred to

the shoots before being processed. The conversion of

N

H4+

con

tent

(µm

ol/g

fres

h w

eigh

t)

0

2

4

6

N

H4+

con

tent

(µm

ol/g

fres

h w

eigh

t)

0

1

2

3

4

G

DH

act

ivity

(u

nits

/mg

prot

ein)

0

25

50

75

100

G

DH

act

ivity

(u

nits

/mg

prot

ein)

0

14

28

42

56

70

G

OG

AT

act

ivity

(un

its/m

g pr

otei

n)

0

4

8

12

16

G

OG

AT

act

ivity

(un

its/m

g pr

otei

n)

0

3

6

9a a aa

bc bccde

c d

f

a ab

a a

cbc bc ce d

f

Root Shoot

Root Shoot

Root Shoot

g fd

b

gfgef ed d

bca

g ef

cb

g ef efdcd

b a

gef

cab

fdecd c

dec c

a

gefbc

a

fgefcd bc

efbcb

a

G

S a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

7

14

21

28

G

S ac

tivity

(u

nits

/mg

prot

ein)

0

30

60

90

120

150toohStooRab a bc c

de de d

f

deeg

a a abc b

cd d d ddee

f

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

- GA- GA + 10 µM GA + 100 µM GA + 10 µM GA + 100 µM GA

e

Fig. 5 Effect of exogenous GA application on NH4? content, and

activities of GDH, GOGAT and GS in root and shoot of pea seedlings

grown for the 11 days under Cr phytotoxicity. Data are mean ± SE of

three independent experiments. Mean values followed by differentletters on bars are significantly different (P \ 0.05) according to the

Duncan’s multiple range test

Acta Physiol Plant (2011) 33:1385–1397 1391

123

Author's personal copy

nitrate into NH4? is brought about by successive and reg-

ulated action of nitrate reductase (NR, EC 1.6.6.1) and

nitrite reductase (NiR, EC 1.7.7.1) (Ogawa et al. 2000).

Ammonium is toxic to cell so it must rapidly be assimilated

(Balestrasse et al. 2003). This is achieved by the highly

regulated action of glutamine synthetase (GS, EC 6.3.1.2),

S

OD

act

ivity

(u

nits

/mg

prot

ein)

0

4

8

12

16

20

S

OD

act

ivity

(u

nits

/mg

prot

ein)

0

7

14

21

28

A

PX a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

275

550

825

1100

1375

A

PX a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

300

600

900

1200

1500

1800

Root Shoot

Root Shoot

f fe de ecdcd

bcd c

b a

fefcd

ab

edbcbc dcd

ab a

dbccdcd cdcdcdcd d

ab a

eecd

bcb

de ede ebc

ba

f

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

+ 10 µM GA + 100 µM GA + 10 µM GA + 100 µM GA- GA- GA

Fig. 6 Effect of exogenous GA application on activities of SOD and

APX in root and shoot of pea seedlings grown for the 11 days under

Cr phytotoxicity. Data are mean ± SE of three independent

experiments. Mean values followed by different letters on bars are

significantly different (P \ 0.05) according to the Duncan’s multiple

range test

C

AT

act

ivity

(u

nits

/mg

prot

ein)

0

8

16

24

32

40

48

C

AT

act

ivity

(u

nits

/mg

prot

ein)

0

10

20

30

40

50

G

R a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

4

8

12

16

20

G

R a

ctiv

ity

(uni

ts/m

g pr

otei

n)

0

6

12

18

24

DH

AR

act

ivity

(u

nits

/mg

prot

ein)

0

125

250

375

500

DH

AR

act

ivity

(u

nits

/mg

prot

ein)

0

250

500

750b b b

c

a

d cd

b

ed

ef

ab a abc

cdab

de dedee ef

Root Shoot

Root Shoot

Root Shoot

bc

efg

a a a a

d def

h

cd

efg

ababa bc

e ef

g

de efg

bcab acd cd

efg

h

cddeef f

bcab aabc

eff g

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

+ 10 µM GA + 100 µM GA- GA + 10 µM GA + 100 µM GA- GA

Fig. 7 Effect of exogenous GA application on activities of CAT, GR

and DHAR in root and shoot of pea seedlings grown for the 11 days

under Cr phytotoxicity. Data are mean ± SE of three independent

experiments. Mean values followed by different letters on bars are

significantly different (P \ 0.05) according to the Duncan’s multiple

range test

1392 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

glutamine 2-oxoglutarate aminotransferase (also called

glutamate synthase; NADH-GOGAT, EC 1.4.1.14) (Mas-

claux-Daubresse et al. 2006). Glutamine synthetase cata-

lyzes the ATP-dependent amination of glutamate, leading

to the formation of glutamine, while GOGAT performs

reductive transfer of amino groups of glutamine to 2-oxo-

glutarate to produce two molecules of glutamate (Mascl-

aux-Daubresse et al. 2006). In addition to the GS/GOGAT

cycle, NH4? may also incorporate into glutamate by the

action of glutamate dehydrogenase (GDH, EC 1.4.1.2).

Glutamate dehydrogenase catalyzes reversible NADH-

dependent amination of 2-oxoglutarate resulting in the

production of glutamate (Masclaux-Daubresse et al. 2006).

Glutamate formed as a result of NH4? assimilation acts as

donor of amino groups for synthesis of amino acids

(Skopelitis et al. 2006). As shown in Fig. 4, exposure of

pea seedlings to Cr and 100 lM GA alone and in combi-

nation decreased NR activity compared to control. The

observed decrease in NR activity in pea seedlings may be

due to the effect of these treatments on the enzyme protein

synthesis and/or activity. It is known that NR used NADH

as electron donor (Solomonson and Barber 1990). Since

sulfhydryl (–SH) are required for NADH binding and cat-

alytic activity of NR (Solomonson and Barber 1990), Cr

and 100 lM GA might affect the NR activity by binding to

functional –SH groups present in the active sites of this

enzyme (Sharma and Dubey 2005). However, addition of

10 lM GA together with Cr increased NR activity com-

pared to Cr treatments alone (Fig. 4). We reported that pea

seedlings treated with Cr and 100 lM GA showed signif-

icant increase in NH4? content in root and shoot compared

to control (Fig. 5). It is reported that NH4? is released from

plant during senescence (Loulakakis et al. 1994). Studies

have shown that Cr and excess GA induces oxidative stress

(Mori and Schroeder 2004; Shanker et al. 2004; Celik et al.

2007; Pandey et al. 2009b), and it is well known that

oxidative stress induces senescence (Sandalio et al. 2001).

Therefore, oxidative stress might be responsible for

increased NH4? content in pea seedlings treated with Cr

and 100 lM GA. Besides, altered GS/GOGAT system in

Cr and 100 lM GA treated seedlings may also be

accounted for increased NH4? accumulation (Fig. 5). On

the contrary, under Cr phytotoxicity, supply of 10 lM GA

lowered NH4? content in root and shoot compared to Cr

treatments alone suggesting its proper incorporation into

organic form (glutamate) as this fact is supported by

increased activities of NH4? assimilating enzymes (Fig. 5).

Results showed that GDH activity increased in response to

Cr and GA treatments compared to control (Fig. 5). Our

results suggested that under Cr and 100 lM GA treatments,

increased GDH activity might have compensated for

decreased GOGAT and GS activities. However, under

similar treatments it seems that increased GDH activity

might not be sufficient to sustain NH4? assimilation as

indicated by increased NH4? content and poor growth of

pea seedlings (Figs. 2, 5). Under stress conditions, increase

in GDH activity has been reported in earlier studies (Ba-

lestrasse et al. 2003; Gajewska and Sklodowska 2009). It

has been shown in previous studies that GDH did not

participate in primary NH4? assimilation process in plants;

however, it has been suggested that under stress conditions

including heavy metals, increased GDH activity may play

T

otal

asc

orba

te

(nm

ol/g

fre

sh w

eigh

t)

0

40

80

120

160

200

T

otal

asc

orba

te

(nm

ol/g

fres

h w

eigh

t)

0

100

200

300

400

500

T

otal

glu

tath

ione

(n

mol

/g fr

esh

wei

ght)

0

110

220

330

440

T

otal

glu

tath

ione

(n

mol

/g fr

esh

wei

ght)

0

150

300

450

600

750

toohStooR

Root Shoot

abc

eg

ab a bd

e f gh

ab

cde

a a a

bc

de

f

ab

d

e

a a a

c d de

f

bb

cd

e

a a a

c cd

ef

0 µM Cr50 µM Cr

100 µM Cr250 µM Cr

+ 10 µM GA + 100 µM GA- GA + 10 µM GA + 100 µM GA- GA

Fig. 8 Effect of exogenous GA application on levels of total

ascorbate and total glutathione in root and shoot of pea seedlings

grown for the 11 days under Cr phytotoxicity. Data are mean ± SE of

three independent experiments. Mean values followed by differentletters on bars are significantly different (P \ 0.05) according to the

Duncan’s multiple range test

Acta Physiol Plant (2011) 33:1385–1397 1393

123

Author's personal copy

an important in relieving the pressure of accumulating

toxic amount of NH4? and also in the replenishment of

glutamate pool (Srivastava and Singh 1987; Syntichaki

et al. 1996; Skopelitis et al. 2006). Glutamine synthetase

and GOGAT both are considered prime enzymes for NH4?

assimilation. Decrease in their activities (except 50 lM Cr

alone for GOGAT) was observed following single and

combined exposure of Cr and 100 lM GA (Fig. 5). Under

similar treatments, decrease in GOGAT and GS activities

suggests impairment in NH4? assimilation as indicated by

decreased protein and nitrogen contents, and increased

NH4? content (Figs. 3, 5). Reduction in both GOGAT and

GS activities has been attributed to oxidative modifications

of these enzyme proteins (Balestrasse et al. 2006). As Cr

and excess GA both are known to stimulate generation of

ROS which might be related partially to the oxidative

destruction of these enzymes (Mori and Schroeder 2004;

Celik et al. 2007; Kumar and Joshi 2008; Pandey et al.

2009b). However, addition of 10 lM GA together with Cr

increased GOGAT and GS activities in root and shoot

compared to Cr treatments alone indicating proper incor-

poration of NH4? into glutamate (Fig. 5).

Under stress conditions, greater production of ROS

results into oxidative damage. In such conditions, the

combined action of antioxidants is critical in mitigating

ROS-mediated oxidative damage. Superoxide dismutase is

a ubiquitous metalloenzyme in aerobic organisms catalyses

disproportionation of more toxic O2�– leading to the for-

mation of comparatively less toxic H2O2 (Salin 1988).

Ascorbate peroxidase is an important enzyme of ascorbate–

glutathione cycle and decomposes H2O2 into H2O and O2

using ascorbate as electron donor (Asada 1999). It is

known that if cellular level of H2O2 is regulated, it acts as

systemic intracellular signal for induction of defense sys-

tem while its higher and un-regulated level leads to oxi-

dative stress (Hernandez et al. 2004). As shown in Fig. 6,

Cr and GA alone as well as in combination increased SOD

and APX activities in root and shoot (except APX activity

at 250 lM Cr ? 100 lM GA) compared to control.

However, in case of Cr ? 10 lM GA treatments, there was

no much variation in APX activity when compared to

control. Under Cr and 100 lM GA treatments, our results

suggested that increased SOD and APX activities might not

be sufficient to prevent oxidative stress as indicated by

reduced growth of pea seedling (Figs. 2, 6). However,

better growth of pea seedlings grown under Cr ? 10 lM

GA treatments suggested that SOD and APX activities

probably keep ROS under control mitigating oxidative

stress when compared to Cr treatments alone (Figs. 2, 6).

Similarly, Shanker et al. (2004) and Panda (2007) have

reported decreased growth despite increased SOD and APX

activities. Catalase is another key antioxidative enzyme

which decomposes H2O2 (Corpas et al. 1999). Exposure of

pea seedlings to Cr and 100 lM GA produced reduction in

CAT activity in root and shoot compared to control

(Fig. 7). Decrease in CAT activity suggested possible delay

in H2O2 scavenging which, in turn, causes oxidative

damage as judged by reduced growth (Figs. 2, 7). Romero-

Puertas et al. (2002) have reported that under cadmium

stress, decrease in CAT activity was due to the oxidative

stress-mediated modification of this enzyme protein and

subsequently oxidized protein could be the target of spe-

cific peroxisomal proteases. Therefore, Cr- and 100 lM

GA-mediated decrease in CAT activity may be attributed

to its oxidative modification. However, addition of 10 lM

GA together with Cr increased CAT activity compared to

Cr treatments alone indicated its greater efficiency in

removing of H2O2 as reflected by better growth of pea

seedlings grown under Cr ? 10 lM GA treatments when

compared with Cr treatments alone (Figs. 2, 7). Glutathi-

one reductase and DHAR are important enzymes of

ascorbate–glutathione cycle required for regenerating

reduced glutathione (GSH) and reduced ascorbate (AsA),

respectively, and maintaining cellular redox balance

(Noctor and Foyer 1998). We observed that exposure of

pea seedlings to single and combined treatments of Cr and

100 lM GA decreased GR and DHAR activities (except

100 lM GA alone for GR) in root and shoot compared to

control (Fig. 7). Under such treatments, decrease in GR

and DHAR activities suggested dys-functioning of ascor-

bate–glutathione cycle as indicated by decreased levels of

ascorbate and glutathione (Figs. 7, 8). Decrease in GR and

DHAR activities can lead to shift in cellular redox balance

which, in turn, may cause disturbances in ROS scavenging

processes. Decrease in GR activity (a member of flavoen-

zyme family) may be due to the enzyme protein damage as

reported for Brassica sp. under cadmium stress (Nouairi

et al. 2009). It has been reported that DHAR was labile in

the absence of thiols and inactivated at higher H2O2 levels

(Hossain and Asada 1984); therefore, Cr and 100 lM GA

may also cause inactivation in DHAR activity through

oxidative stress (Celik et al. 2007; Pandey et al. 2009b). On

the contrary, application of 10 lM GA together with Cr

increased GR and DHAR activities compared to Cr treat-

ments alone indicating important role of these enzymes in

preventing oxidative stress through enhanced levels of

ascorbate and glutathione (Figs. 7, 8).

Ascorbate and glutathione are two major non-enzymatic

antioxidants involved in scavenging of ROS directly or

indirectly (Noctor and Foyer 1998). It is known that under

stress conditions plants can adjust ascorbate and glutathi-

one levels by modulating the regeneration and biosynthesis

of ascorbate and glutathione. We observed that exposure of

pea seedlings to single and combined treatments of Cr and

100 lM GA decreased total ascorbate and total glutathione

levels in root and shoot compared to control (Fig. 8).

1394 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

Under similar treatments, decreased ascorbate and gluta-

thione levels would have led to weakening of the antioxi-

dant potential thereby culminating into oxidative damage

as judged by reduced growth of pea seedlings (Figs. 2, 8).

It is reported that ascorbate is used for the hydroxylation of

proline residues during extensive synthesis, and implicated

in root elongation, the cell cycle regulation and cell wall

formation while physiological significance of glutathione

in plants as well as in animals is due to its involvement in

sulfur metabolism and in controlling cellular heavy metal

concentrations (Scheller et al. 1987; Arrigoni 1994; Kerk

and Feldman 1995; Cordoba-Pedregosa et al. 1996; Lap-

partient and Touraine 1996). Recently, Chao et al. (2010)

and Shan and Liang (2010) showed that increased levels of

ascorbate and glutathione protect rice and Agropyron

cristatum from oxidative stress. Therefore, their decreased

levels may cause Cr and 100 lM GA toxicity symptoms in

pea. Decrease in cellular ascorbate and glutathione levels

may primarily be associated with decreased DHAR and GR

activities as reported in the present study when seedlings

were exposed to Cr and 100 lM GA (Figs. 7, 8). More-

over, decrease in ascorbate and glutathione levels may also

be due to their increased oxidation for scavenging of ROS

and/or prevention of their formation (Shanker et al. 2004;

Pandey et al. 2009b).On the contrary, addition of 10 lM

GA together with Cr increased total ascorbate and total

glutathione levels in root and shoot of pea compared to Cr

treatments alone (Fig. 8). Thus, application of 10 lM GA

under Cr phytotoxicity reflects its important role in

inducing expression of ascorbate and glutathione to protect

pea seedlings from oxidative stress. The increased levels of

ascorbate and glutathione in Cr ? 10 lM GA treated pea

seedlings might have led to better antioxidant system to

combat Cr phytotoxicity.

Conclusions

Our results showed that exogenous application of GA (10

and 100 lM) led to different changes in pea seedlings

under Cr phytotoxicity. Our study pointed out that Cr and

100 lM GA alone as well as in combination decreased

growth and alters nitrogen assimilation in pea seedlings

compared to control. Chromium- and 100 lM GA-medi-

ated adverse effects on growth and nitrogen assimilation

may be attributed to decreased levels of antioxidants. On

the contrary, it was reported that application of 10 lM GA

together with Cr was able to alleviate Cr phytotoxicity

appreciably. This 10 lM GA-mediated amelioration of Cr

phytotoxicity may be assigned to the better antioxidant

system and sustained activities of enzymes of nitrogen

assimilation. Furthermore, these data suggest that GA may

play different roles based on its exogenous concentrations

and plant species used under specific developmental and

environmental conditions.

Acknowledgments We wish to thank The Head, Department of

Plant Science, MJP Rohilkhand University, Bareilly, India for pro-

viding necessary laboratory facilities to carry out this work. We are

also thankful to UGC, India for financial support.

References

Aebi II (1984) Catalase in vitro. Methods Enzymol 105:121–126

Arditti J, Dunn A (1969) Environmental plant physiology—experi-

ments in cellular and plant physiology. Holt, Rinehart and

Winston Inc, New York

Arrigoni O (1994) Ascorbate system in plant development. J Bioenerg

Biomemb 26:407–419

Asada K (1999) The water-water cycle in chloroplasts: scavenging of

active oxygen and dissipation of excess photons. Annu Rev Plant

Physiol Plant Mol Biol 50:601–639

Balestrasse KB, Benavides MP, Gallego SM, Tomaro ML (2003)

Effect of cadmium stress on nitrogen metabolism in nodules and

roots of soybean plants. Funct Plant Biol 30:57–64

Balestrasse KB, Gallego SM, Tomaro ML (2006) Oxidation of the

enzymes involved in ammonium assimilation plays an important

role in the cadmium-induced toxicity in soybean plants. Plant

Soil 284:187–194

Beevers L, Guernsey FS (1966) Changes in some nitrogenous

components during the germination of pea seeds. Plant Physiol

41:1455–1458

Brehe JE, Burch HB (1976) Enzymatic assay for glutathione. Anal

Biochem 74:189–197

Celik I, Tuluce Y, Isik I (2007) Evaluation of toxicity of abscisic acid

and gibberellic acid in rats: 50 days drinking water study.

J Enzym Inhib Med Chem 22:219–226

Chao YY, Hong CY, Kao CH (2010) The decline in ascorbic acid

content is associated with cadmium toxicity of rice seedlings.

Plant Physiol Biochem 48:374–381

Cordoba-Pedregosa MC, Gonzalez-Reyes JA, Sanadillas MS, Navas

P, Cordoba F (1996) Role of apoplastic and cell-wall peroxidases

on the stimulation of root elongation by ascorbate. Plant Physiol

112:1119–1125

Corpas F, Palma JM, Sandalio LM, Lopez-Huertas E, Romero-

Puertas MC, Barroso JB (1999) Purification of catalase from pea

leaf peroxisomes: identification of five different isoforms. Free

Rad Res 31:235–241

Debouba M, Gouia H, Suzuki A, Ghorbel MH (2006) NaCl stress

effects on enzymes involved in nitrogen assimilation pathway in

tomato ‘‘Lycopersicon esculentum’’ seedlings. J Plant Physiol

163:1247–1258

Fuchs Y, Lieberman M (1968) Effects of kinetin, IAA and gibberellin

on ethylene production and their interactions in growth of

seedlings. Plant Physiol 43:2029–2036

Gajewska E, Sklodowska M (2009) Nickel-induced changes in

nitrogen metabolism in wheat shoots. J Plant Physiol

166:1034–1044

Giannopolitis CN, Reis SK (1977) Superoxide dismutase. I. occur-

rence in higher plants. Plant Physiol 59:309–314

Gossett DR, Millhollon EP, Cran LM (1994) Antioxidant response to

NaCl stress in salt-sensitive cultivars of cotton. Crop Sci

34:706–714

Hansen H, Grossmann K (2000) Auxin-induced ethylene triggers

abscisic acid biosynthesis and growth inhibition. Plant Physiol

124:1437–1448

Acta Physiol Plant (2011) 33:1385–1397 1395

123

Author's personal copy

Hernandez JA, Escobar C, Creissen G, Mullineaux PM (2004) Role of

hydrogen peroxide and the redox state of ascorbate in the

induction of antioxidant enzymes in pea leaves under excess

light stress. Funct Plant Biol 31:359–368

Hooley R (1994) Gibberellins: perception, transduction responses.

Plant Mol Biol 26:1529–1555

Hossain MA, Asada K (1984) Purification of dehydroascorbate

reductase from spinach and its characterization as thiol enzyme.

Plant Cell Physiol 25:85–92

Kerk NM, Feldman LJ (1995) A biochemical model for initiation and

maintenance of the quiescent center: implications for organiza-

tion of root meristems. Plant Dev 121:2825–2833

Kotas J, Stasicka Z (2000) Chromium occurrence in the environment

and methods of its speciation. Environ Pollut 107:263–283

Kumar S, Joshi UN (2008) Nitrogen metabolism as affected by

hexavalent chromium in sorghum (Sorghum bicolor L.). Environ

Exp Bot 64:135–144

Lang CA (1958) Simple microdetermination of Kjeldahl nitrogen in

biological materials. Anal Chem 30:1692–1694

Lappartient AG, Touraine B (1996) Demand-driven control of root

ATP sulfurylase activity and sulphate uptake in intact Canola.

Plant Physiol 111:147–157

Lillo C (1984) Diurnal variations of nitrite reductase, glutamine

synthetase, glutamate synthase, alanine amino transferase and

aspartate amino transferase in barley leaves. Physiol Plant

61:214–218

Loulakakis KA, Roubelakis-Angelakis KA, Kanellis AK (1994)

Regulation of glutamate dehydrogenase and glutamine synthe-

tase in avocado fruit during development and ripening. Plant

Physiol 106:217–222

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein

measurement with the folin phenol reagent. J Biol Chem

193:265–275

Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K, Lelandias M,

Grandjean J, Valadier MH, Feraud M, Jouglet T, Suzuki A

(2006) Glutamine synthetase-glutamate synthase pathway and

glutamate dehydrogenase play distinct roles in the sink source

nitrogen cycle in tobacco. Plant Physiol 140:444–456

Matsuoka M (2003) Gibberellin signaling: how do plant cells respond

to GA signals? J Plant Growth Regul 22:123–125

Molins-Legua C, Meseguer-Lloret S, Moliner-Martinez Y, Campıns-

Falco P (2006) A guide for selecting the most appropriate

method for ammonium determination in water analysis. Trends

Anal Chem 25:282–290

Mori IC, Schroeder JI (2004) Reactive oxygen species activation of

plant Ca2? channels. a signaling mechanism in polar growth,

hormone transduction, stress signaling and hypothetically mech-

anotransduction. Plant Physiol 135:702–708

Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by

ascorbate specific peroxidase in spinach chloroplasts. Plant Cell

Physiol 22:867–880

Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping

active oxygen under control. Annu Rev Plant Physiol Plant Mol

Biol 49:249–279

Nouairi I, Ammar WB, Youssef NB, Miled DDB, Ghorbal MB,

Zarrouk M (2009) Antioxidant defense system in leaves of

Indian mustard (Brassica juncea) and rape (Brassica napus)

under cadmium stress. Acta Physiol Plant 31:237–247

Ogawa K, Soutome R, Hiroyama K, Hagio T, Ida S, Nakagawa H

(2000) Co-regulation of nitrate reductase and nitrite reductase in

cultured spinach cells. J Plant Physiol 157:299–306

Panda SK (2007) Chromium-mediated oxidative stress and ultra-

structural changes in root cells of developing rice seedlings.

J Plant Physiol 164:1419–1428

Pandey V, Dixit V, Shyam R (2009a) Chromium (VI) induced

changes in growth and root plasma membrane redox activities in

pea plants. Protoplasma 235:49–55

Pandey V, Dixit V, Shyam R (2009b) Chromium effect on ROS

generation and detoxification in pea (Pisum sativum) leaf

chloroplasts. Protoplasma 236:85–95

Romero-Puertas M, Palma JM, Gomez M, del Rıo LA (2002)

Cadmium causes the oxidative modification of proteins in pea

plants. Plant Cell Environ 25:677–686

Salin ML (1988) Toxic oxygen species and protective systems of the

chloroplast. Physiol Plant 72:681–689

Sandalio LM, Dalurzo HC, Gomez M, Romero-Puertas MC, del Rıo

LA (2001) Cadmium-induced changes in the growth and

oxidative metabolism of pea plants. J Exp Bot 52:2115–2126

Schaedle M, Bassham JA (1977) Chloroplast glutathione reductase.

Plant Physiol 59:1011–1012

Scheller HV, Huang B, Hatch E, Goldsbrough PB (1987) Phytochel-

atin synthesis and glutathione levels in response to heavy metals

in tomato cells. Plant Physiol 85:1031–1035

Schiavon M, Pilon-Smits EAH, Wirtz M, Hell R, Malagoli M (2008)

Interaction between chromium and sulfur metabolism in Bras-sica juncea. J Environ Qual 37:1536–1545

Shan C, Liang Z (2010) Jasmonic acid regulates ascorbate and

glutathione metabolism in Agropyron cristatum leaves under

water stress. Plant Sci 178:130–139

Shanker AK, Djanaguiraman M, Sudhagar R, Chandrashekar CN,

Pathmanabhan G (2004) Differential antioxidative response of

ascorbate glutathione pathway enzymes and metabolites to

chromium speciation stress in green gram (Vigna radiata (L.)

R. Wilczek. Cv CO 4) roots. Plant Sci 166:1035–1043

Sharma P, Dubey RS (2005) Modulation of nitrate reductase activity

in rice seedlings under aluminium toxicity and water stress:

role of osmolytes as enzyme protectant. J Plant Physiol

162:854–864

Shen Z, Shen Q, Liang Y, Liu Y (1994) Effect of nitrogen on the

growth and photosynthetic activity of salt-stressed barley. J Plant

Nutr 17:787–789

Singh RP, Srivastava HS (1983) Regulation of glutamate dehydro-

genase activity by amino acids in maize seedlings. Physiol Plant

57:549–554

Singh RP, Srivastava HS (1986) Increase in glutamate synthase

(NADH) activity in maize seedlings in response to nitrate and

ammonium nitrogen. Physiol Plant 66:413–416

Skopelitis DS, Paranychianakis NV, Paschalidis KA (2006) Abiotic

stress generates ROS that signal expression of anionic glutamate

dehydrogenases to form glutamate for proline synthesis in

tobacco and grapevine. Plant Cell 18:2767–2781

Solomonson LP, Barber MJ (1990) Assimilatory nitrate reductase;

functional properties and regulation. Annu Rev Plant Physiol

Plant Mol Biol 41:225–253

Srivastava HS, Singh RP (1987) Role and regulation of L-glutamate

dehydrogenase during defence response in maize. Phytochem-

istry 26:597–610

Syntichaki KM, Loulakakis KA, Roubelaki-Angelakis KA (1996) The

amino acid sequence similarity of plant glutamate dehydroge-

nase to the extremophilic archaeal enzyme conforms to its stress-

related function. Gene 168:87–92

Tuna AL, Kaya C, Dikilitas M, Higgs D (2008) The combined effects

of gibberellic acid and salinity on some antioxidant enzyme

activities, plant growth parameters and nutritional status in

maize plants. Environ Exp Bot 62:1–9

Vazques MD, Poschenrieder C, Barcelo J (1987) Chromium VI

induced structural and ultrastructural changes in bush bean

plants (Phaseolus vulgaris L.). Ann Bot (Lond) 59:427–438

1396 Acta Physiol Plant (2011) 33:1385–1397

123

Author's personal copy

Vernay P, Gauthier-Moussard C, Hitmi A (2007) Interaction of

bioaccumulation of heavy metal chromium with water relation,

mineral nutrition and photosynthesis in developed leaves of

Lolium perenne L. Chemosphere 68:1563–1575

Wallace A, Soufi SM, Cha JW, Romney EM (1976) Some effects of

chromium toxicity on bush bean plants grown in soil. Plant Soil

44:471–473

Wen F, Zhang Z, Bai T, Xu Q, Pan Y (2010) Proteomics reveals the

effects of gibberellic acid (GA3) on salt-stressed rice (Oryzasativa L.) shoots. Plant Sci 178:170–175

Acta Physiol Plant (2011) 33:1385–1397 1397

123

Author's personal copy