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Differential response of indica rice genotypes to NaClstress in relation to physiological and biochemicalparameters

Online Publication Date: 01 October 2007To cite this Article: Kumar, Vinay, Shriram, Varsha, Jawali, Narendra and Shitole,Mahadev Ganpat (2007) 'Differential response of indica rice genotypes to NaClstress in relation to physiological and biochemical parameters', Archives ofAgronomy and Soil Science, 53:5, 581 - 592To link to this article: DOI: 10.1080/03650340701576800URL: http://dx.doi.org/10.1080/03650340701576800

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© Taylor and Francis 2007

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Differential response of indica rice genotypes to NaCl stressin relation to physiological and biochemical parameters

VINAY KUMAR1, VARSHA SHRIRAM1, NARENDRA JAWALI2, &

MAHADEV GANPAT SHITOLE1

1Department of Botany, University of Pune, Ganeshkhind, and 2Bhabha Atomic Research Centre,

Molecular Biology Division, Trombay, Mumbai, India

(Received 8 May 2007; accepted 17 July 2007)

AbstractThe effect of NaCl stress was studied in indica rice cultivars, Panvel-3 (tolerant), Kalarata (moderatelytolerant) and Karjat-3 (sensitive) under various levels (50 – 300 mM) of salinity besides control (0 mM).Salinity stress decreased germination percentage, plant growth, biomass production and chlorophyllpigments, and increased malondialdehyde level (lipid peroxidation) and free proline accumulation.Salinity-induced decrease in germination percentage, biomass production, chlorophyll and total proteincontents were significantly higher in Karjat-3 than Panvel-3. The free proline content at all the levels aswell as the magnitude of increase in accumulation with increasing salinity was also highest in Panvel-3,as at 300 mM NaCl concentration, it was 8 times more than control, while it was about 5 times more inKarjat-3 and around 4 times in Kalarata. The level of lipid peroxidation was lowest at all levels of salinityin Panvel-3 as compared with Karjat-3, however, Kalarata showed intermediate results. Results showedsalinity tolerance of Panvel-3 was manifested by lower decrease in germination, plant growth andchlorophyll pigments and associated with higher proline accumulation and lower lipid peroxidationunder high salinity stress.

Keywords: NaCl stress, proline, lipid peroxidation, chlorophyll, biomass

Introduction

Soil salinity is a major abiotic stress problem around the globe especially in arid and semi-arid

regions and irrigation areas. Salinity affects nearly 20% of the world’s cultivated area and

about half of the world’s total irrigated lands (Zhu 2001; FAO 2006). In Asia alone, 21.5

million ha of land area is thought to be salt-affected, with India having 8.6 million ha of such

area which constitutes a major part of problem soils in India (Sahi et al. 2006).

Salinity adversely affects the quantity and quality of crop produce (Sahi et al. 2006). Salt

stress affects almost every aspect of plant physiology at both whole plant and cellular levels

through osmotic and ionic stress (Hasegawa et al. 2000; Murphy & Durako 2003). Salinity is

Correspondence: Prof. Mahadev Ganpat Shitole, PhD, Department of Botany, University of Pune, Ganeshkhind, Pune-411007,

India. E-mail: mgs@unipune.ernet.in

Archives of Agronomy and Soil Science

October 2007; 53(5): 581 – 592

ISSN 0365-0340 print/ISSN 1476-3567 online � 2007 Taylor & Francis

DOI: 10.1080/03650340701576800

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7 detrimental to the various processes of crops such as seed germination, seedling growth and

vigour, vegetative growth, flowering and fruit set and ultimately it causes diminished

economic yield and also quality of produce (Sairam & Tyagi 2004).

Rice is the world’s most important cereal crop (Bajaj & Mohanty 2005) with around

610 million tonnes production globally (IRRI 2005). Rice with its relatively small genome size

(*430 Mb), ease of transformation, well developed and known detailed genetics (Sahi et al.

2006), availability of a dense physical map and molecular markers (Wu et al. 2002),

full-genome transcription profiling using high density oligonucleotide tiling microarrays

(Li et al. 2006) together with its complete genome sequence (Sasaki et al. 2005) is considered

as a model monocot system for various biotechnological, metabolic, genetic engineering and

functional genomics development studies worldwide (Shimamoto & Kyozuka 2002; Bajaj &

Mohanty 2005; Ge et al. 2006). In addition, rice shares extensive synteny among the other

cereals thereby increasing the utility of this system.

Although India is second only to China with more than 136 million tonnes rice production

from 43 million ha lands (FAOSTAT 2005), the average yield in India is very low (3 t/ha) as

compared to some other Asian rice-producing countries like China, Korea and Japan. The

main constraints for the low yield are dependency on monsoon (floods and draughts),

improper irrigation and drainage facilities, overuse of fertilizers and soil-related problems

including salinity and alkalinity. The yield of rice especially Asian rice (sativa) is susceptible to

salinity (Flowers & Yeo 1981; Anil et al. 2005).

Increased salt tolerance of crops including rice is needed to sustain food production in

various regions of the world (Munns et al. 2006). As saline soils and saline waters are

common around the world, great effort has been devoted to understand physiological aspects

of tolerance to salinity in plants, as a basis for plant breeders to develop salinity-tolerant

genotypes. In spite of this great effort, only a small number of cultivars, partially tolerant to

salinity, have been developed. Interaction of salt with the plants may depend upon salt

concentration and genotype. Therefore, screening for salt tolerance of genotypes that could

withstand extreme soil salinity will ensure future crop production (Jogeswar et al. 2006).

In the present investigation, we have studied the differential response of indica rice cultivars

to NaCl stress in terms of physiological and biochemical parameters including plant

germination and growth, chlorophyll contents, proline, total proteins and lipid peroxidation.

Materials and methods

Plant material

Indica rice (Oryza sativa L.) cultivars namely Karjat-3, Kalarata and Panvel-3 were selected

for this study and certified seeds were obtained from The Regional Rice Research Station,

Karjat, Maharashtra, India and The Saline Land Research Station, Panvel, Maharashtra,

India.

Salinity treatment and culture conditions

Seeds of all three cultivars were surface sterilised with 0.1% mercuric chloride for 10 min and

then washed several times with sterile distilled water. Twenty seeds of each cultivar (cv.) were

sown in a Petri dish (10 cm diameter) containing germination paper and the experiment was

carried out in triplicate. Every day 10 ml Yoshida’s nutrient solution (with or without varying

NaCl levels, i.e. 50, 100, 150, 200, 250 and 300 mM) was applied per Petri dish and all the

observations were recorded on the 14th day after sowing.

582 V. Kumar et al.

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7 Plant growth analysis

The germination percentage, root length, shoots length, root/shoot ratio, fresh weight (FW)

and dry weight (DW) of seedlings were recorded on the 14th day of germination. The DW

was observed by heating the samples in a hot air oven at 608C for 48 h.

Biochemical analysis

Determination of chlorophyll. Chlorophyll contents were estimated by extracting 100 mg of the

fresh leaf material in 10 ml dimethyl sulfoxide (DMSO) (Hiscox & Israelstam 1979). The

samples were heated at 658C for 4 h and than the absorbance of extract recorded at 665 and

645 nm on UV-VIS spectrophotometer (Shimadzu- 1601, Japan). Chlorophyll contents were

calculated as per standard method (Arnon 1949).

Determination of lipid peroxidation. The level of lipid peroxidation was measured in terms of

malondialdehyde (MDA) contents (Heath & Packer 1968). Fresh shoot and root samples

(500 mg) were homogenized in 10 ml of 0.1% trichloro-acetic acid (TCA). The

homogenate was centrifuged at 15,000 g for 5 min. To 2 ml of aliquot of the supernatant,

4 ml of 0.5% thiobarbituric acid (TBA) in 20% TCA was added. The mixture was heated

at 958C for 30 min and then quickly cooled in ice bath. This was followed by centri-

fugation at 10,000 g for 10 min to remove suspended turbidity and then the absorbance

was recorded at 532 nm. The value for non-specific absorption at 600 nm was

subtracted and the MDA content was calculated using its absorption coefficient of

155 mmol71 cm71.

Determination of proline. The proline content was determined separately in shoots and roots

following the method of Bates et al. (1973). Samples (500 mg) were homogenized in 5 ml of

sulphosalicylic acid (3%) using mortar and pestle. Two ml aliquot of supernatant was mixed

with an equal volume of glacial acetic acid and acid ninhydrin. The reaction mixture was

boiled in water bath at 1008C for 30 min. The reaction was terminated in an ice bath

following by addition of 4 ml toluene and then transferring to a separating funnel. After

thorough mixing, the chromophore containing toluene was separated and absorbance was

recorded at 520 nm in spectrophotometer against toluene blank. Concentration of proline

was estimated by referring to a standard curve of proline.

Protein estimation. Proteins were estimated using Lowry et al. (1951) method. Fresh

samples (250 mg) were homogenized in 2.5 ml of phosphate buffer (pH 7.0). The extract

was centrifuged at 5000 g for 15 min at 48C and the supernatant was transferred to a

tube containing a mixture of 20 ml acetone and 14 ml b-Mercaptoethanol for pre-

cipitation of protein. The sample tubes were stored at 08C for 5 h and then centrifuged at

10,000 g for 20 min. The supernatant was discarded and the pellet was dissolved in 2.5 ml

1 N sodium hydroxide solution. Aliquot of 0.2 ml from this sample was used to prepare

the reaction mixture. The intensity of blue colour developed was recorded at 660 nm and

protein concentration was measured using bovine serum albumin as standard.

Statistical analysis. Each Petri dish was considered as replicate and all of the treatments

were repeated three times and data are expressed as+ standard error (SE). The data was

subjected to analysis of variance (ANOVA) to detect significant difference between means.

The level of significance was also checked for least significant differences (LSD) at

Response of rice genotypes to NaCl stress 583

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7 p� 0.05. All the statistical analyses were done by using MSTAT-C statistical software

package.

Results

Effect of NaCl stress on germination and growth parameters

The seed germination decreased with increasing salt concentration from 50 – 300 mM NaCl.

However, the effect of salt stress on seed germination varied between the cultivars (see

Table I). Up to 50 mM NaCl, 100% germination was observed in all the cultivars; however,

beyond that level noticeable reduction in germination percentage was evident, with more

pronounced reduction at more than 100 mM NaCl concentrations. In Panvel-3, germination

was 100% up to 150 mM NaCl and was comparably least affected at higher salt

concentrations. The increase in salinity decreased the shoot and root lengths considerably

in all the cultivars, with more reduction in shoot growth than the roots. Rice genotypes

Panvel-3 and Kalarata showed lesser decline in shoot length while Karjat-3 showed more

stunted shoots under high salinity stress (see Table I). The root/shoot ratio was increased

markedly with salinity in Karjat-3 followed by Kalarata; however, contrasting to these results,

there was a decline in root/shoot ratio beyond 100 mM NaCl and at 300 mM NaCl, it was

even less than the control. The results of the root length shoot length and root/shoot ratio

makes it clear that Panvel-3 was less affected by salinity stress followed by Kalarata, while

these parameters were markedly affected in Karjat-3.

Table I. Effect of NaCl stress on germination percentage, root length, shoot length and root/shoot ratio in rice

cultivars. Results are mean of three replicates+ standard error (SE). LSD for cultivars and treatments were

significant at p�0.05.

Cultivar

NaCl stress

(mM)

Germination

percentage (%)

Root length

(cm) Mean+SE

Shoot length

(cm) Mean+SE

Root/shoot

ratio

Karjat-3 0 (Control) 100+ 0.5 5.40+ 0.18 4.06+0.18 1.33

50 100+ 1.1 5.18+ 0.30 3.93+0.13 1.32

100 96+ 1.5 6.10+ 0.42 3.13+0.09 1.95

150 90+ 1.5 4.72+ 0.22 2.42+0.29 1.95

200 75+ 1.1 2.72+ 0.15 1.66+0.16 1.64

250 70+ 1.3 1.88+ 0.04 0.78+0.03 2.39

300 25+ 1.5 1.48+ 0.12 0.41+0.02 3.56

Kalarata 0 (Control) 100+ 0.9 6.65+ 0.39 4.28+0.31 1.55

50 100+ 1.4 7.52+ 0.36 4.18+0.08 1.58

100 96+ 1.2 7.03+ 0.53 2.51+0.28 1.75

150 94+ 1.3 4.47+ 0.34 1.07+0.16 1.74

200 90+ 1.1 3.14+ 0.17 0.81+0.07 1.88

250 90+ 2.1 1.67+ 0.23 0.92+0.10 1.76

300 73+ 1.6 1.67+ 0.20 0.90+0.10 1.68

Panvel-3 0 (Control) 100+ 1.0 6.13+ 0.56 5.21+0.13 1.18

50 100+ 0.9 6.10+ 0.20 4.55+0.12 1.34

100 100+ 1.2 6.27+ 0.31 4.47+0.13 1.40

150 100+ 1.1 5.10+ 0.26 3.83+0.16 1.33

200 96+ 1.3 4.14+ 0.22 3.33+0.14 1.24

250 90+ 1.6 2.16+ 0.14 1.93+0.10 1.12

300 85+ 1.9 2.03+ 0.24 1.75+0.05 1.16

584 V. Kumar et al.

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7 A negative correlation between biomass production and salinity stress was evident from the

results obtained (Table II) as FW and DW gradually decrease in all the cultivars with increase

in NaCl concentration. The reduction in biomass under salinity stress was noticeably higher

in Karjat-3, and lowest in Panvel-3, while intermediate results were obtained in the cv.

Kalarata.

Chlorophyll content

The Chl a, b and total Chl content were influenced significantly by NaCl stress and these

pigments were gradually decreased in leaves with increasing salt concentrations in all the

cultivars irrespective of their salt tolerance (see Table III). Comparably higher Chlorophyll

contents were observed in Karjat-3 cv. under control conditions than other genotypes.

However, the decrease under salt stress was also higher in Karjat-3. The rate of decrease in

Chl with increase in salinity stress was lowest in Panvel-3 and highest in Karjat-3.

Lipid peroxidation

The results depicted in Figure 1 clearly revealed that increased lipid peroxidation (in terms of

MDA content) was observed with increasing NaCl concentrations in all the cultivars. When

exposed to 300 mM NaCl, MDA level in Karjat-3 was about 5 times higher than control, in

Karjat-3, it was 3.5, while in contrast to these genotypes, lesser extent of lipid peroxidation

was noticed in Panvel-3, where about 2.5 times more MDA content than control was

recorded at 300 mM NaCl. This indicates that Panvel-3 is able to tolerate salinity-induced

oxidative damage better than other two cultivars.

Proline content

Amongst the three rice cultivars, comparably very high proline content was observed in

Panvel-3 than the other two cultivars, at non-saline condition. NaCl stress induced an

elevation in proline contents in all cultivars irrespective of their salt stress tolerance, as evident

from the results presented in Figure 2. There was a steep increase in proline content in shoots

of Panvel-3 under salt stress ranging from 50 – 300 mM and 36.47 m mol g71 fresh weight

proline was recorded at 300 mM salinity level, i.e. about 8 times more than the non-saline

conditions followed by approximately 5 times more proline at this salinity concentration

Table II. Effect of NaCl stress on fresh and dry weights of shoots of rice cultivars. Results are mean of three

replicates+ standard error (SE). LSD for cultivars and treatments were significant at p�0.05.

NaCl (mM)

Fresh weight of shoots (mg) in rice

cultivars (Mean+SE)

Dry weight of shoots (mg) in rice

cultivars (Mean+SE)

Karjat-3 Kalarata Panvel-3 Karjat-3 Kalarata Panvel-3

0 (Control) 139.3+ 3.5 133.7+ 5.8 135.2+3.9 25.2+ 1.0 24.4+ 3.0 24.5+2.0

50 121.5+ 4.0 129.0+ 1.2 135.5+4.7 24.5+ 1.2 24.3+ 1.1 24.7+1.5

100 111.6+ 3.0 122.5+ 2.3 122.7+2.2 22.7+ 1.6 20.3+ 1.0 23.5+1.7

150 93.8+ 2.0 121.2+ 4.2 121.5+2.6 19.0+ 1.2 19.7+ 0.8 20.3+1.0

200 85.6+ 2.5 103.5+ 2.8 115.8+1.9 17.3+ 1.1 17.2+ 1.5 18.9+0.7

250 82.5+ 2.8 88.4+ 4.0 101.9+2.5 13.5+ 1.0 18.1+ 1.7 18.5+0.5

300 31.3+ 2.0 80.9+ 3.0 95.5+1.8 9.1+ 0.9 16.3+ 1.3 17.2+0.6

Response of rice genotypes to NaCl stress 585

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7

Tab

leII

I.E

ffec

to

fN

aClst

ress

on

(a)

chlo

rop

hyl

l-a

(Ch

l-a)

con

ten

ts,(b

)ch

loro

ph

yll-

b(C

hl-

b)

con

ten

ts,an

d(c

)to

talch

loro

ph

yllco

nte

nts

inle

aves

of

rice

cult

ivar

s.R

esu

lts

are

mea

no

fth

ree

rep

lica

tes+

stan

dar

der

ror

(SE

).L

SD

for

cult

ivar

san

dtr

eatm

ents

wer

esi

gn

ifica

nt

atp�

0.0

5.

NaC

l(m

M)

Ch

loro

ph

yll

‘a’

inri

cecu

ltiv

ars

(Mea

n+

SE

)C

hlo

rop

hyl

l‘b

’in

rice

cult

ivar

s(M

ean+

SE

)T

ota

lch

loro

ph

yllin

rice

cult

ivar

s(M

ean+

SE

)

Kar

jat-

3K

alar

ata

Pan

vel-

3K

arja

t-3

Kal

arat

aP

anve

l-3

Kar

jat-

3K

alar

ata

Pan

vel-

3

0(C

on

tro

l)2

.27+

0.0

92.0

0+

0.0

62

.13+

0.0

90

.61+

0.0

30

.52+

0.0

40

.54+

0.0

12

.87+

0.0

92

.52+

0.0

32

.67+

0.0

9

50

1.8

5+

0.0

31.7

8+

0.0

21

.89+

0.0

50

.53+

0.0

20

.45+

0.0

30

.47+

0.0

12

.38+

0.0

12

.23+

0.0

22

.36+

0.0

6

10

01

.55+

0.0

41.6

2+

0.0

21

.82+

0.0

20

.41+

0.0

10

.37+

0.0

20

.41+

0.0

21

.95+

0.0

41

.98+

0.0

32

.23+

0.0

2

15

01

.03+

0.0

31.2

5+

0.0

31

.60+

0.0

60

.35+

0.0

20

.33+

0.0

30

.37+

0.0

31

.38+

0.0

31

.58+

0.0

41

.97+

0.0

7

20

00

.75+

0.0

30.9

9+

0.0

61

.40+

0.0

30

.28+

0.0

20

.30+

0.0

10

.34+

0.0

31

.03+

0.0

51

.29+

0.0

61

.74+

0.0

4

25

00

.55+

0.0

50.8

0+

0.0

31

.13+

0.0

30

.22+

0.0

20

.28+

0.0

20

.30+

0.0

40

.78+

0.0

21

.08+

0.0

11

.43+

0.0

4

30

00

.32+

0.0

20.5

6+

0.0

10

.91+

0.0

50

.20+

0.0

10

.22+

0.0

10

.29+

0.0

10

.52+

0.0

10

.78+

0.0

81

.20+

0.0

3

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in Karjat-3 and more than 4 times higher in Kalarata. These results clearly indicated that

Panvel-3 has higher salt-induced proline accumulation than other two cultivars, which may

have played a role in salt stress tolerance.

Figure 1. Effect of different concentrations of NaCl stress on lipid peroxidation in rice cultivars. Each value represents

mean of three replications and vertical bars indicate+SE. Data are statistically significant at p50.05.

Figure 2. Effect of different concentrations of NaCl stress on proline content in rice cultivars. Each value represents

mean of three replications and vertical bars indicate+SE. Data are statistically significant at p50.05.

Response of rice genotypes to NaCl stress 587

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7 Total proteins

Protein contents were increased in Panvel-3 and Kalarata up to 100 mM NaCl concentra-

tions and decreased thereafter. The highest amount of proteins was observed at 50 mM NaCl

stress in Panvel-3, while in Karjat-3, the protein content was decreased with increase in

salinity levels at all the concentrations of NaCl. Amongst the three cultivars, maximum

protein contents were observed in Panvel-3 at 300 mM NaCl concentration followed by

Kalarata and Karjat-3 (Figure 3).

Discussion

Seed germination is usually the most critical stage in seedling establishment, determining the

successful crop production (Almansouri et al. 2001). Understanding the responses of plants

at these stages is particularly important for elucidating the mechanisms of salt resistance or

sensitivity in plants and their survival (Mayer & Poljakoff-Mayber 1963). In the present

investigation, the severity of salinity antagonism to the normal growth of plant as indicated by

germination percentage, shoot length, root length, root/shoot ratio, fresh and dry mass

production by plants was higher in the salt-sensitive rice cultivar Karjat-3, indicating that

NaCl has a negative influence on the growth of rice seedlings comparably more on salt-

sensitive cultivars than tolerant ones. Our results clearly indicated that high levels of NaCl

(more than 50 mM) adversely affected the germination process and growth in all the three

rice cultivars.

The proportion of dry weight allocated to roots increased with increasing NaCl levels, as

shown by the responses of root/shoot ratio (Table I). Previous studies carried out in soybean

and alfalfa (Berstein & Ogata 1966) and in cotton (Meloni et al. 2001) showed that shoot

Figure 3. Effect of different concentrations of NaCl stress on total proteins content in rice cultivars. Each value

represents mean of three replications and vertical bars indicate+SE. Data are statistically significant at p50.05.

588 V. Kumar et al.

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7 growth was more inhibited by NaCl than root growth. Our results are in harmony of all these

reports and root/shoot was increased with salt concentration, with lower to higher root/shoot

ratio in sensitive to tolerant cultivars.

Likewise, the biomass production was influenced negatively by NaCl stress significantly.

However, highest fresh weight and dry weight was recorded in Panvel-3 while lowest dry

weight of was observed in Karjat-3 at 300 mM NaCl. Thus, it was clear from the results that

the Panvel-3, a salt-tolerant rice cultivar could withstand high NaCl stress comparably better

than other rice cultivars. Earlier reports by Yeo and Flowers (1983); Garcia et al. (1995) and

Anil et al. (2005) observed similar trends and reported lesser effect of NaCl stress on

germination process and better survival of salt-tolerant rice cultivars than the salt-sensitive

rice cultivars. Furthermore, the effect of salinity stress on biomass production in rice has been

described by Khan and Panda (2002). Our results are in agreement of these reports.

Chlorophyll concentration in stressed tissues has been advocated as an index of tissue

tolerance to NaCl (Lutts et al. 1996). Chlorophyll contents in our study were markedly

reduced under NaCl stress in leaves of rice cultivars at vegetative phase. Salinity-sensitive rice

genotype Karjat-3 showed significantly higher decline in Chl a, b and total Chl contents than

tolerant genotypes Panvel-3 and Kalarata under increasing salt stress. Similar observations are

reported earlier and chlorophyll contents were reduced more in salt-sensitive cultivars than

the tolerant ones. Lutts et al. (1996) observed a highly significant decrease in chlorophyll

concentration with increasing salinity in salt-sensitive rice genotypes, while a much lesser

effect was recorded in salt-tolerant genotypes. Misra et al. (1997) reported that the

chlorophyll contents were affected heavily by NaCl stress in salt-sensitive rice cultivar Jaya,

while in the salt-tolerant cultivar Damodar, more chlorophyll accumulation was observed

than the control plants. According to Choudhury and Choe (1996), a decline in photo-

synthetic rate with increased concentration of NaCl may be associated with decreased

pigmentation. Our results clearly supported this hypothesis.

Our results clearly indicated that amongst the rice cultivars, the damage to the lipid

membranes was comparatively less severe in the salt-tolerant cultivar Panvel-3. The severe

damage was observed in Karjat-3 while intermediate responses were observed in Kalarata.

The results of the present investigation are in agreement of earlier reports, in which higher

lipid peroxidation in sensitive cultivars than the tolerant ones have been reported.

Vaidyanathan et al. (2003) and Demiral and Turkan (2005) reported greater lipid peroxi-

dation under salinity stress in salt-sensitive rice genotypes than in salt-tolerant ones. The

lower level in shoots and roots of Panvel-3 than of Karjat-3 and Kalarata suggests that it may

have better protection against oxidative damage under salt stress.

Proline is one of the most important and widely studied osmolytes found in plants and is

related with abiotic stress tolerance. Proline is generally assumed to serve as a physiologically

compatible solute that increases as needed to maintain a favourable osmotic potential between

the cell and its surroundings (Pollard & Wyn Jones 1979). In addition to this, proline is an

important osmoprotectant in plants and can protect the photosynthetic machinery against

salt-induced damage (Sivakumar et al. 2002). Rapid accumulation of free proline is a typical

response to salt stress (Demiral & Turkan 2005). When exposed to drought or a high salt

content in soil, many plants have been observed to accumulate high amounts of proline, in

some cases several times the sum of all other amino acids (Mansour 2000). In the present

investigation, the free proline content was significantly increased in the stressed plants over

control plants at all stress regimes in all the cultivars. Significantly higher accumulation of

proline was observed at 300 mM NaCl in salt tolerant cv. Panvel-3 compared to comparably

sensitive cultivars Kalarata and Karjat-3. Similar to our results, Sairam et al. (2002) in

wheat and Jogeswar et al. (2006) in sorghum observed much higher proline accumulation in

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7 the salt-tolerant genotype than the salt-sensitive genotype. Contrary to these results,

Vaidyanathan et al. (2003) and Demiral and Turkan (2005) concluded higher proline

accumulation in salt-sensitive rice cultivars than salt-tolerant cultivars under high salinity

stress. A large number of researchers have advocated the major role played by the proline in

salt stress tolerance and post-stress recovery of the plants. Proline has been found to protect

cell membranes of onion against salt injury (Mansour 1998). Furthermore, Sultana et al.

(1999) have suggested that proline accumulation in both salinized leaves and grains of rice

plants is implicated in osmotic adjustment to salinity. It is suggested that proline may be the

major source of energy and nitrogen during immediate post-stress metabolism and the

accumulated proline apparently supplies energy for growth and survival thereby inducing

salinity tolerance (Ahmad & Jhon 2005). The results of the present investigation suggested

that comparably higher amount of salt-induced synthesis and accumulation of proline in

Panvel-3 under salinity stress may play a role in combating salt stress in this cultivar.

The protein contents were increased up to 100 mM NaCl concentration than the control in

salt-tolerant cultivars Panvel-3 and Kalarata; however, the protein levels were highly reduced

at all levels of salinity from 50 mM to 300 mM NaCl in Karjat-3. Similar trends were

reported by Lutts et al. (1996) and Misra et al. (1997) and observed comparably higher

reduction in protein contents in salt-sensitive rice cultivars than the tolerant ones under NaCl

stress. However, contrary to these results, Ahmad & Jhon (2005) concluded that NaCl stress

had a positive impact on total protein contents in Pisum sativum and observed a gradual

increase in protein contents with increasing salinity.

Conclusion

From the results of the present investigation, we can conclude that NaCl stress affected

germination and plant growth significantly, with lesser affects on Panvel-3, while Karjat-3 was

affected most. Comparably higher amounts as well as accumulation of proline and lowest

peroxidation of lipid membrane under increasing salt stress in Panvel-3 as compared with

other cultivars, suggested that this cultivar may have a better antioxidant machinery to combat

the salt stress induced injuries in this cultivar.

Acknowledgements

This work was supported by grant from Board of Research in Nuclear Sciences, Department

of Atomic Energy, Government of India (Grant No. 2004/37/28/BRNS) to MGS and

research fellowship to the first author. The authors also acknowledge the facilities used for this

work at the Department of Botany, University of Pune created under ASIST and DRS-SAP

programs of University Grants Commission, India.

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