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The world population is increasing day by day and is expected to reach 8.5 billion by 2025. At the other end environmental stress (biotic and abiotic stress) destroys the overall yield of the agricultural crops. Plants are subjected to variety of abiotic stresses like high temperature, cold, drought, salinity, UV. However, amongst these stresses, salinity is considered the most limiting factor for productivity of agricultural crops. Salinity is a worldwide problem that limits distribution and production of major crops. Salt stress in soil or water is one of the major stresses especially in arid and semi-arid regions (Allakhverdier et al. 2000; Koca et al. 2007). One-fifth of irrigated agriculture is adversely affected by soil salinity (Flowers and Yeo, 1995). Salinity is responsible for the induction of primary effects like ionic stress osmotic stress which in turn induces secondary stresses such as oxidative stress. Oxidative stress leads to the accumulation of reactive oxygen species (ROS) which are very dangerous to plants and in higher concentrations leads to death of the plant cells (Ahmad and Sharma, 2008; Ahmad et al., 2008; 2010a,b,c). It causes oxidative damage to biomolecules like membrane lipids, proteins, and nucleic acids (Hernandez et al. 2001).
Plants have the mechanisms to counteract the deleterious effects of primary and secondary stresses (Devi and Prasad, 1998) through the induction of osmolytes and antioxidants (Ashraf and Foolad, 2007; Foyer et al., 1997; Ahmad et al., 2008; 2010a,b,c). The antioxidant enzymes include superoxide dismutases, peroxidases, catalases and glutathione reductases. The superoxide anion, which is most dangerous reactive oxygen species, is scavenged in plants by superoxide dismutase, which converts superoxide anion to hydrogen peroxide (Alscher et al., 2003). Hydrogen peroxide (H2O2) is scavenged directly by catalase, which converts it to water and molecular oxygen. Measurement of activities of antioxidant enzymes can thus be used to indicate oxidative stress in plants (Geebelen et al., 2002).
Brassica juncea (mustard), a plant within the Brassicaceae family, is an important oil seed crop grown extensively in arid and semi-arid regions (Singh et al. 2001). India is second largest producer of rapeseed mustard (Afroz et al., 2005). However, Indian mustard production still remains insufficient to meet even the daily requirement of its people (Khan et al., 2002). So it becomes necessary to plant biologists to increase the production of mustard within the given circumstances. The present study was undertaken to investigate the effect of NaCl on Biomass, biochemical attributes, lipid peroxidation and the activity of some key antioxidant enzymes in mustard cultivars and lead us to generate the salt tolerant cultivars to reclamate the salt affected land.
MATERIALS AND METHODS
Seeds of Brassica juncea were sown in earthen pots containing 5 kg of peat, perlite, and sand (1:1:1, v/v/v). After germination they were transferred to one plant per pot and grown for further three weeks under natural photoperiod of 12-13 h and temperature of 28Â±40C. The plants were treated with different concentrations of NaCl : 100 and 200 mM. The experiments was laid out in a completely randomized design with five replicates. The samples were harvested at 45 days after treatment.
Growth, gas exchange, Chlorophyll fluorescence and pigment concentration
For dry weight (DW) determination, leaves and roots were separated and were dried at 700C for 48 h and weighed.
Measurements of net CO2 assimilation rate (A), stomatal conductance (gs) and transpiration (E) rate were made on a fully expanded youngest leaf of each plant using an open system LCA-4 ADC portable infrared gas analyzer (Analytical Development Company, Hoddesdon, England). These measurements were made from 10:30 to 12:00 hours.
Chl fluorescence measurements were made in attached leaves in the growth chamber with a PAM 2000 apparatus (H. Walz, Effeltrich, Germany). The maximum efficiency of PSII photochemistry under dark-adapted (Fv/Fm), quantum yield of PSII (Î¦psII), nonphotochemical quenching (NPQ) and photochemical quenching coefficient (qP) were calculated according to the method of Li et al. (2007).
Chlorophyll content was determined by the method of Hiscox and Israelstam (1979). Fresh material (100 mg) was kept in an extraction reagent dimethyl sulfoxide (DMSO). Tubes were kept in oven at 650C for 40 min. 1 ml aliquot was mixed with 2ml DMSO and then vortexed. Absorbance was determined spectrophotometerically at 480, 510, 645, 663 nm (Beckman 640 D, USA) using DMSO for a blank.
RWC, electrolyte leakage% and Proline content
Leaf relative water content (RWC) was measured in fully expanded leaves of four plants per replicate. Five leaf discs of 10 mm diameter were excised from the interveinal areas of each Leaf. For each replicate, twenty discs were pooled and their FW determined. The leaf discs were floated on deionised water for 7 h under low irradiance and then the turgid weight (TW) recorded. Then the samples were dried at 80 oC for 24 h to determine the (DW). The tests showed that complete hydration of the leaf discs occurred within 4 h. The relative water content (RWC) was calculated using the following formula (Smart and Bingham, 1974):
RWC (%) = (FW-DW/TW-DW) X 100
The total inorganic ions leaked out in the leaves under salt stress were measured as described by Dionisio-Sese and Tobita (1998). Twenty leaf discs were taken in a boiling tube containing 10Â ml of deionized water and electrical conductivity (EC) was measured (ECa). The contents were heated at 50 and 60Â Â°C for 25Â min each in a water bath and EC was measured (ECb). Later, the contents were boiled at 100Â Â°C for 10Â min and the EC again recorded (ECc). The electrolytic leakage was calculated using the formula:
Proline concentration in the leaves was detemined spectrophotometerically following Bates et al. (1973). Fresh material (300 mg each sample) was homogenized in 10 ml of 3% aqueous sulfosalicylic acid. The homogenate was centrifuged at 12000 rpm for 15 min. 2 ml aliquot of the supernatant was mixed with an equal volume of acetic acid and acid ninhydrin and incubated for 1 h at 100 0C. The reaction was terminated in an ice bath and extracted with 4 ml of toluene. The extract was vortexed for 20 s. The chromatophore-containing toluene was then aspirated from the aqueous phase, and its absorbance determined spectrophotometerically at 520 nm (Beckman 640 D, USA) using toluene for a blank.
Determination of H2O2 content and lipid peroxidation (MDA)
The hydrogen peroxide content was determined according to Velikova et al. (2000). Fresh plant material (500 mg) was homogenized with 5 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000 rpm for 15 min, and 0.5 ml of the supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide (KI). The absorbance of the supernatant was measured at 390 nm. The content of H2O2 was calculated by comparison with a standard calibration curve, previously plotted by using different concentrations of H2O2.
Lipid peroxidation was determined by measuring malondialdehyde (MDA) formation according to Madhava Rao and Sresty (2000). Fresh leaves (500 mg) were homogenized with 2.5 mL of 0.1% trichloroacetic acid (TCA). The homogenate was centrifuged for 10 min at 10,000 rpm. For every 1mL of the aliquot, 4 mL of 20% TCA containing 0.5% thio barbituric acid (TBA) were added. The mixture was heated at 95 oC for 30 min and then cooled promptly on an ice bath. Afterwards, the mixture was centrifuged for 15 min at 10,000 rpm and the absorbance of the supernatant was measured at 532 nm. Measurements were corrected for unspecific turbidity by substracting the absorbance at 600 nm. The concentration of MDA was calculated using an extinction coefficient of 155 mMâˆ’1 cmâˆ’1.
Extraction of the enzymes
All experiments were performed at 4Â Â°C. The 10Â g leaf were homogenized with 50 volumes of 100Â mM Tris-HCl (pH 7.5) containing 5Â mM DTT, 10Â mM MgCl2, 1Â mM EDTA, 5Â mM magnesium acetate and 1.5% PVP-40. After filtration in cheesecloth the homogenate was centrifuged at 10,000 rpm for 15Â min. The supernatant collected was used as a source of enzyme. Serine and cysteine proteinase inhibitors (1 mM PMSF + 1 Î¼g/ml aproptinin) was also added in the extraction buffer. For measuring APX activity the tissue was separately ground in homogenizing medium containing 2.0 mM ascorbate in addition to other ingredients. The soluble protein content was determined by Bradford (1976) with standard curves prepared using bovine serum albumin.
Superoxide dismutase (SOD, EC 126.96.36.199) activity was determined by the method of Van Rossun et al. (1997) by following the photoreduction of nitroblue tetrazolium. The reaction mixture contained: 50Â mM phosphate buffer (pH 7.8), 0.1Â mM EDTA, 13Â mM methionine, 75Â Î¼M nitroblue tetrazolium (NTB), 2Â Î¼M riboflavin and 100Â Î¼l of the supernatant. The reaction was initiated by placing the tubes under two 15Â W fluorescent lamps. The reaction was terminated after 10Â min by removing the reaction tubes from the light source. Non-illuminated reactions mixture served as a blank. The absorbance of the reaction products were measured at 560Â nm. SOD activity was expressed as Unit mg-1 protein. One unit of SOD was defined as the amount of protein causing a 50% decrease of the SOD-inhibitable NBT reduction.
A method of Luck (1974) was employed for the assay of catalase (CAT, EC 188.8.131.52). The enzyme extract (50Â Î¼l) was added to 3Â ml of 20 mM hydrogen peroxide and 50 mM phosphate buffer (pH 7.0) solution. The decrease in absorbance was measured at 240 nm. The enzyme activity was calculated using the extinction coefficient of 36x103 mM-1 cm-1 and expressed as unit mg-1 protein.
Ascorbate peroxidase (APX, EC184.108.40.206) was spectrophotometrically assayed following a decrease in the absorbance at 265Â nm (Nakano and Asada, 1981). The 3 ml assay mixture contained: 0.1 mM EDTA, 0.5Â mM ascorbate and 0.1Â mM H2O2 in 50Â mM potassium phosphate buffer (pH 7.0) with 0.1 ml of the enzyme extract. The H2O2 dependent oxidation of ascorbate was followed by a decrease in the absorbance in 290 nm. APX activity was expressed as unit mg-1 protein.
Glutathione reductase (GR, EC 220.127.116.11) activity was determined by following the rate of NADPH oxidation as measured by the decrease in the absorbance at 340Â nm (Carlberg and Mannervik, 1985). The assay mixture (1Â ml) contained: 0.75 Î¼l potassium phosphate buffer (pH 7) with 2Â mM EDTA, 75Â Î¼M NADPH (2 mM), 75Â Î¼l GSSG (20 mM). Reaction was initiated by adding 0.1 ml enzyme extract to the mixture and the decrease in absorbance was measured at 340 nm for 2 min. GR activity was calculated with the extinction coefficient of NADPH of 6.2 mM-1 cm-1 and expressed as Î¼molNADPH oxidized min-1 (Units mg-1 protein).
Data for each variable were subjected to one way analysis of variance (ANOVA). Duncan's Multiple Range Test (DMRT) at 5% probability was employed for assessing the significant differences among the mean values of different attributes. The values are means of five replications.
Salt stress decreases the biomass of the mustard cultivars and the results are depicted in fig.1(A-D). Shoot and root length was observed to decrease but the decrease was more pronounced in Rohini as compared to RH-30 and Varuna. A significant decrease in shoot dw of 31.4%, 37.3% and 45.7% was observed in Varuna, RH-30 and Rohini respectively at 200 mM of NaCl stress as compared to control. Root dw also showed the same decreasing trend (fi. 1, C&D).
CO2 assimilation rate (A) decreases from 14.5% to 21.8% at 100 and 200 mM NaCl stress in Varuna respectively. Same decreasing trend was observed in RH-30 (26.8%) and Rohini (42.2%) at 200 mM NaCl stress (fig.2, A).
Stomatal conductance (gs) and transpiration rate (E) also decreases in all the cultivars at all stress regimes but more decrease was observed in Rohini and RH-30 as compared to Varuna (fig. 2, B&C).
A non-significant decrease was observed in Fv/Fm in all the cultivars of mustard at 100 mM NaCl stress. But at higher concentration of salt (200 mM NaCl) a decrease in Fv/Fm, Î¦psII and qP accompanied with increase in NPQ was observed (table 1).
Salt stress decreases chlorophyll content 'a', 'b' and 'a/b' ratio in all the cultivars of mustard. A significant (pâ‰¤ 0.5) decrease in chlorophyll 'a' was noticed in all the cultivars and more pronounced decrease (28.9%) was shown by Rohini at 200 mM NaCl stress (fig. 3, A).
A significant decrease in chlorophyll 'b' was observed between Rohini and Varuna but non significant decrease was observed between RH-30 and Rohini (fig. 3, B). same decreasing trend was also observed in chl. a/b ratio (fig. 3, C).
The results related to the effect of NaCl on RWC is presented in fig. 3, D. As the concentration of NaCl increase the RWC decreases and the decrease was more pronounced in Rohini (46.8%) than RH-30 (40%) and Varuna (26.3%) at 200 mM NaCl stress.
A very high electrolyte leakage was observed in all the three cultivars of mustard especially at 200 mM NaCl stress. A decrease of 62.9%, 67% and 67.5% was observed in Varuna, RH-30 and Rohini respectively at 200 mM NaCl stress (fig. 3, E).
Proline was observed to increase at all stress levels in all the cultivars of mustard. The order of increase in proline was 59.1%, 53.8% and 46.8% by Varuna, RH-30 and Rohoni respectively at 200 mM NaCl stress (fig. 3, F).
Varuna showed increase of H2O2 from 41.1% to 51.2%, RH-30 55.4% and 64.9% and Rohini showed 55.3% to 64.4% during 100 and 200 mM NaCl stress respectively (fig. 4, A).
The MDA concentration was observed more in Rohini (45.9%) and RH-30 (45.7%) than Varuna (34.6%) (fig. 4, B).
SOD activity in the shoots of all three cultivars increased due to salt stress and the cultivars differed significantly for this attribute. Cultivar Varuna had the highest SOD activity followed by cvs. RH-30, but the lowest SOD activity was recorded in cv. Rohini (fig. 4, C).
A significant increase in shoot catalase activity was observed in all three cultivars under salt stress. However, cvs. Rohini had the lowest shoot catalase activity under saline conditions. In contrast, RH-30 and Varuna were the highest in catalase activities under salt stress (fig. 4, D).
A significant increase in shoot APX activity was observed in all three cultivars under salt stress. Cultivars differed significantly in peroxidase activity under both control and saline conditions. Highest activity of the enzyme was recorded in cvs. Varuna and RH-30 under both saline and control conditions, but this increase was not marked in cvs. Rohini under saline conditions (fig. 4, E).
All the three cultivars of mustard showed increased activity of GR under saline conditions. Highest GR activity was observed in Varuna and RH-30 as compared to Rohini under saline conditions (fig. 4, F).
Our results of decreasing biomass corroborates with the findings of Zribi et al. (2009) who showed that increase in salt concentration decreases the biomass of tomato plants. Similar results have been reported by Akram et al. (2009) in sunflower, Mohammad et al. (1998) in tomato, Chartzoulakis and Klapaki (2000) in pepper, Imada and Tamai (2009) in populous alba, Keutgen and Pawelzik (2009) in strawberry and Ahmad and Sharma (2010) in mulberry. The decreased biomass in the high-salt treatment resulted in the smaller accumulation of nutrients. Increased salt levels under natural conditions may thus lead to severe growth reductions and discourage establishment of the species. Cell division and cell elongation is reduced due to NaCl stress which in turn inhibits growth (Yasseen et al., 1987).
Salt stress decreases all gas exchange parameters such as CO2 assimilation rate (A), transpiration rate (E), stomatal conductance (gs) significantly. Similar reports have been reported by Hameed and Ashraf (2008) in Cynodon dactylon, Akram et al. (2008-09) in sunflower, Zheng et al. (2009) in wheat and Ahmad and Sharma (2010) in mulberry. A similar report is also observed by Yang et al., (2009) in Populus cathayana and Zribi et al. (2009) in tomato. Leaf stomatal conductance decreased significantly with increasing NaCl concentration (Zribi et al. 2009; Ahmad and Sharma, 2010). A, gs and Fv/Fm decreased in parallel during salt stress. Stomatal closure might be one of the factors, which are responsible for the reduction in A. The salt induces ABA accumulation and causes stomatal closure (Aldesuguy and Ibrahim, 2001).
No significant impact on Fv/Fm was observed in the mild-stressed plant, but a significant decrease was found under severe stress and the results are in accordance with the findings of Zribi et al. (2009) in tomato. Similar results have been reported by Everard et al. (1994); Lu et al. (2003) Cha-um and Kirdmanee (2009) and Yang et al., (2009). Akram et al. (2009) also showed that salt stress reduced the quantum yield (Fv/Fm) of photosystem-II (PSII). During severe salt treatment a decrease in photochemical processes along with, a significant increase in non-photochemical quenching (NPQ) was observed in tomato (Zribi et al. 2008-2009) and wheat (Zheng et al. 2009). A sustained decrease of the Fv/Fm may indicate the occurrence of photoinhibitory damage (Maxwell and Johnson, 2000; Colom and Vazzana, 2003). A correlation of Na+ accumulation in leaves and qp, NPQ, Î¦PSII have been found by many workers and these parameters could be considered as an indicator of photosynthetic disturbance in plants under salt stress (Zribi et al. (2008-2009).
In the present study salt stress decreases the RWC in all cultivars of mustard. Yang et al., (2009) have observed the same decrease in Populus cathayana during salt stress. Decrease in RWC under salt stress was also detected in populus (Eitel et al. 2006), in olive tree (Boussadia et al. 2008) in pea (Ahmad and Jhon, 2005) and in mulberry (Ahmad and Sharma, 2010). The decrease in RWC suggests that salinity caused water deficit in plants. The negative effect on plant water relations was induced by an increase in soluble salts, which slowed down the uptake of water and nutrients, causing osmotic effects and toxicity. Duan et al. (2005) have reported that decreased leaf RWC is positively correlated with reduced photosynthetic rates.
Salt stress decreases the chlorolophyll content have been reported by many workers (Zheng et al., 2009; Hameed and Ashraf 2008; EfeoÄŸlu et al., 2009; Cha-um and Kirdmanee 2009; Yang et al., 2009; Ahmad, 2010; Ahmad and Jhon, 2005). The decrease in Chla and Chlb, might be due to either slow synthesis or fast breakdown of chlorophyll pigments under salt stress (Ashraf, 2003). Salt hampers the uptake of nutrients from the soil that might decrease the pigment concentration.
Leaf proline content increased significantly in Cynodon dactylon (Hameed and Ashraf 2008) during salt stress. Cha-um and Kirdmanee (2009) also showed that increasing concentration of salt increases the proline content in sugarcane. Proline content have been reported to increase during other stresses (EfeoÄŸlu et al., 2009; Ahmad et al., 2006; Ahmad, 2010; Ahmad et al., 2010a,b; Ahmad and Sharma, 2010). The synthesis of proline is widely used by plants to stabilize membranes and maintain the conformation of proteins at low leaf water potentials. Proline is also known to be involved in reducing the photo damage in the thylakoid membranes by scavenging and/or reducing the production of 1O2 (Reddy et al., 2004).
H2O2 content were much higher under salt stress than the control in wheat (Zheng et al. (2009), Yang et al., (2009) in Populus cathayana. Salt tolerant cultivars of mulberry accumulates less H2O2 as compared to salt sensitive cultivar (Ahmad et al., 2010a). Jaleel et al. (2007) also demonstrated that NaCl increased H2O2 in all parts of Catharanthus roseus plants. The increase in H2O2 in turn increases the lipid peroxidation that leads to the leakage of the membranes.
MDA content significantly increased under the salt treatment and as compared with the control in wheat (Zheng et al. (2009). Yang et al., (2009) also observed increase in MDA content in Populus cathayana. Salt sensitive cultivars shows more lipid peroxidation as compared to tolerant cultivars of mulberry (Ahmad et al., 2010a). The increases in electrolyte leakage, MDA and H2O2 content suggested that salt-stressed plants encountered cellular damage and lipid peroxidation. Similar results have been reported by Lei et al. (2007). our earlier work on pea also confirms the same results (Ahmad et al., 2008). Membrane lipid peroxidation, has often been used as a tool to assess the degree of plant sensitivity to stress.
The results related to the change in antioxidants (SOD, CAT, APX and GR) in the present study corroborates with the findings of Ashraf and Ali, 2008 in canola (Brassica napus) under saline conditions. Our increased antioxidant activity also corroborates with the findings of Yang et al., (2009) who also recorded the increased CAT, APX, GR in Populus cathayana during salt stress. Experimental results showed increase in antioxidant activity under salt stress, in pea, maize, tea, mustard and mulberry (Ahmad et al. 2008; Tuna et al. 2008; Upadhyaya et al. 2008; Ahmad, 2010; Ahmad et al., 2010a,b). Antioxidant enzymes are known to reduce the levels of superoxide and hydrogen peroxide in plants (Ali and Alqurainy, 2006). The first line of defence in oxidative stress is superoxide dismutase that converts O2âˆ’â€¢ to H2O2. Catalases are the principal scavenging enzymes which can directly dismutate H2O2 and is indispensable for ROS detoxification during stress (Van Breusegem et al. 2001). This is also due to the fact that there is proliferation of peroxisomes during stress, which might help in scavenging of H2O2 that diffuse from the cytosol (Lopez-Huertas et al. 2000). Increase in catalase activity is supposed to be an adaptive trait possibly helping to overcome the damage to the tissue metabolism by reducing toxic levels of H2O2 (Sekmen et al. 2007; Vital et al. 2008). Ascorbate peroxidase is one of the most important antioxidant enzymes of plants involved in scavenging of H2O2 in water-water and ascorbate-glutathione cycles and utilizes AsA as the electron donor. APXs reduce H2O2 to water and play an important role in the antioxidant system of plants (Kangasjärvi et al. 2008). GR catalyses the NADPH-dependent reduction of oxidized glutathione (GSSG) to its reduced form (GSH). GR activity is thought to increase the ratio of NADP+/NADPH, and the NADP+ accepts electrons from the photosynthetic electron transport chain. Thus the flow of electrons to O2 and therefore, the formation of O2âˆ’ can be minimized.
In conclusion, differences in biomass yield, leaf water status, photosynthetic performance, pigment content, proline accumulation, H2O2 content, lipid peroxidation and antioxidative enzyme activity between Brassica juncea cultivars were observed in the present study. Of all cultivars, Varuna were relatively higher in shoot fresh and dry weight, proline, SOD, APX, CAT and GR activities than the other cultivars of mustard, while RH-30 and Rohini were higher in electrolyte leakage, H2O2 and MDA levels. The differential salt tolerance of the mustard cultivars was found to be associated with higher antioxidant enzyme activities, and some other key metabolites and could be related to the difference in the mechanisms underlying oxidative stress injury and subsequent tolerance to salt. These antioxidants can be used as potential selection criteria for salt tolerance in mustard cultivars.