Effect of Salicylic Acid on Salinity-induced Changes in Brassica juncea

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Seeds of Indian mustard (Brassica juncea (L.) Czern. et Coss.) were exposed to 0, 50, 100 and 150 mmol/L NaCl for 8 h and seeds were sown in an earthen pot. These stressed seedlings were subsequently sprayed with 10 μmol/L salicylic acid (SA) at 30 d
  Journal of Integrative Plant Biology   2008,  50  (9): 1096–1102 Effect of Salicylic Acid on Salinity-induced Changesin  Brassica juncea Mohammad Yusuf  1 , Syed Aiman Hasan 1 , Barket Ali 1 , Shamsul Hayat 1 ∗ , Qazi Fariduddin 1 and Aqil Ahmad 2 ( 1 Plant Physiology Section, Department of Botany, Aligarh Muslim University  , Aligarh 202002, India; 2 Department of Applied Sciences, Higher College of Technology  , Al-Khuwair, Sultanate of Oman) Abstract Seeds of Indian mustard ( Brassica juncea  (L.) Czern. et Coss.) were exposed to 0, 50, 100 and 150mmol/L NaCl for 8h andseeds were sown in an earthen pot. These stressed seedlings were subsequently sprayed with 10 µ mol/L salicylic acid (SA)at 30d and were sampled at 60d to assess the changes in growth, photosynthesis and antioxidant enzymes. The seedlingsraised from the seeds treated with NaCl had significantly reduced growth and the activities of carbonic anhydrase, nitratereductase and photosynthesis, and the decrease was proportional to the increase in NaCl concentration. However, theantioxidant enzymes (catalase, peroxidase and superoxide dismutase) and proline content was enhanced in response toNaCl and/or SA treatment, where their interaction had an additive effect. Moreover, the toxic effects generated by the lower concentration of NaCl (50mmol/L) were completely overcome by the application of SA. It was, therefore, concluded that SAameliorated the stress generated by NaCl through the alleviated antioxidant system.Key words:  carbonic anhydrase; catalase; mustard; net photosynthetic rate; nitrate reductase; peroxidase; salicylic acid; salinity; superoxidedismutase.  Yusuf M, Hasan SA, Ali B, Hayat S, Fariduddin Q, Ahmad A  (2008). Effect of salicylic acid on salinity-induced changes in  Brassica juncea .  J. Integr.Plant Biol.  50 (9), 1096–1102. Available online at www.jipb.net Soil salinity has become a serious environmental problemwhich affects the growth and productivity of many crops. Ap-proximately 20% of the world’s cultivated land area and 50% of allirrigatedlandsareaffectedbysalinity(RhoadesandLoveday1990). High salt content in the soil affects the soil porosityand also decreases the soil water potential that results in aphysiological drought (Hopkins 1995). High salt content alsoaffects the physiology of plants, both at the cellular as well aswhole plant levels (Murphy and Durako 2003). Ionic imbalanceoccurs in the cells due to excessive accumulation of Na + andCl − ions and reduced uptake of mineral nutrients such as K + ,Ca 2 + and Mn 2 + (Hasegawa et al. 2000). An excessive amountof sodium ions in cells also cause enzyme inhibition suchas those of nitrogen metabolism (Soussi et al. 1998), nitratereductase (Shafea 2003), rubisco and phosphoenolpyruvate Received 30 Jan. 2008 Accepted 24 Mar. 2008 ∗  Author for correspondence.Tel: + 91 941 232 8593;Fax: + 91 571 270 2885;E-mail: < hayat_68@yahoo.co.in > . C  2008 Institute of Botany, the Chinese Academy of Sciencesdoi: 10.1111/j.1744-7909.2008.00697.x (PEP) carboxylase (Soussi et al. 1999), and metabolic dys-function (Booth and Beardall 1991) such as degradation of photosynthetic pigments (Abdullah and Ahmed 1990; Soussietal.1999).Photosynthesisisoneofthemostseverelyaffectedprocesses during salinity stress (Sudhir and Murthy 2004)whichismediatedthroughadecreaseofstomatalconductance,internal CO 2  partial pressure (Sultana et al. 1999) and stomatalclosure that affect the gaseous exchange (Bethkey and Drew1992). The decrease in photosynthesis under saline conditionsis considered as one of the most important factors responsiblefor reduced plant growth and productivity (Ball et al. 1987).Some quick and effective measures urgently need to beworked out so that the stress factors deteriorating crops, suchas salinity, can be countered successfully. The applications of plant growth regulators have been found to play an importantrole in plant responses to stress (Chakrabarti and Mukherjee2003). Salicylic acid (SA) is one of the strong candidates for stress ameliorators that have recently been recognized as aplant hormone (Hayat and Ahmad 2007). It plays diverse physi-ological roles in plants, which include plant growth, thermogen-esis, flower induction, nutrient uptake, ethylene biosynthesis,stomatal movements, photosynthesis and enzyme activities(HayatandAhmad2007).DiseaseresistanceandabioticstresstolerancearetheotherrolesassignedtoSA(Jandaetal.2007).  Effect of SA and salinity in  Brassica  1097 Among abiotic stresses, SA has been reported to counter water stress (Singh and Usha 2003), low temperature (Tasgin et al. 2003), high temperature (He et al. 2005) and salinity stress (Khodary 2004; El Tayeb 2005). The present study was designed to study the effect of SAon changes in growth, photosynthesis and activities of someenzymes, in mustard ( Brassica juncea  L.) plants subjected toNaCl stress. Results The growth (length of root and shoot; leaf area and the freshand dry mass/plant) was significantly affected by the treatments(Table 1). The treatment of plants with NaCl significantlyreduced the growth parameters and the decrease was pro-portional to the concentration of the NaCl used. The high-est concentration (150mmol/L) was the most deleterious anddecreased the length of root by 28.73%, length of shoot by45.45%, leaf area by 22.78%, fresh mass by 47.69% anddry mass by 59.18% as compared to the control. However,all the above parameters were significantly enhanced by SA.The toxic effect generated by NaCl (50mmol/L) was completelyovercome by the application of SA whereas the effects of ahigher concentration (100 or 150mmol/L) of NaCl were reducedpartially by SA.The activity of nitrate reductase (NR) and that of carbonicanhydrase (CA) was significantly decreased by the NaCl treat-ment,irrespectiveoftheconcentrationofthesaltused(Table2).However,theSAtreatmentoftheplantsgrownunderstress-freeconditions significantly elevated the activities of the enzymeswheretheNRwas27.21%andCAwas46.15%higherthanthatof the control. SA treatment also improved the activities of theseenzymes in the plants that were subjected to salinity stress. Theactivities of enzymes NR and CA in the plants receiving bothNaCl (50mmol/L) and SA were 4.7% and 9.0% higher thanthose receiving NaCl (50mmol/L) alone, respectively.The level of proline increased in the plants receiving NaCl or SA, compared with the control. Moreover, the plants exposed Table 1.  Effect of salicylic acid on salinity-induced changes in length of shoot (cm) and root (cm), leaf area (cm 3 ), and fresh and dry mass (g) of  Brassica juncea  at 60d after sowing ( ± standard error)Treatment Shoot length Root length Leaf area Fresh mass of plant Dry mass of plantControl 60.9 ± 1.1 13.2 ± 1.70 38.80 ± 1.77 13.0 ± 0.83 4.9 ± 1.01SA (10 − 5 mol/L) 82.1 ± 3.6 18.9 ± 1.72 67.81 ± 1.49 16.6 ± 1.50 6.7 ± 1.19NaCl (50mmol/L) 58.9 ± 3.6 10.7 ± 1.45 35.50 ± 1.68 11.8 ± 0.83 4.2 ± 0.80NaCl (100mmol/L) 52.2 ± 2.3 9.4 ± 1.40 33.35 ± 0.95 7.0 ± 0.80 3.1 ± 0.94NaCl (150mmol/L) 43.4 ± 2.4 7.2 ± 1.00 29.96 ± 1.94 6.8 ± 1.00 2.0 ± 0.60NaCl (50mmol/L) + SA 10 − 5 mol/L 75.8 ± 3.1 13.8 ± 1.02 44.75 ± 1.55 12.5 ± 1.42 5.1 ± 0.92NaCl (100mmol/L) + SA 10 − 5 mol/L 70.4 ± 1.6 13.0 ± 1.40 41.33 ± 0.77 10.2 ± 0.85 3.9 ± 0.78NaCl (150mmol/L) + SA 10 − 5 mol/L 64.8 ± 4.3 11.4 ± 1.86 36.23 ± 1.24 9.7 ± 0.47 3.2 ± 1.02LSD at 5% 5.9 2.3 4.7 1.6 0.96LSD, least significant difference; SA, salicylic acid. to the SA as well as salinity possessed the largest quantities of proline which increased further with an increase in the level of NaCl. The highest level of proline were recorded in the plantsthat received NaCl (150mmol/L) and SA. Here, the values were98.8% higher than the control. The relative water content inthe leaves decreased with the increasing concentration of NaCl(Table 3), whereas the subsequent treatment of SA significantlyincreased its level over the control and also overcome the toxiceffect generated by NaCl. All the photosynthetic parameters (i.e. stomatal conductance,internal CO 2  concentration, water use efficiency and net photo-syntheticrate)weresignificantlyreducedbytheNaCltreatment.The maximum inhibition was recorded at the highest concentra-tionofNaCl(150mmol/L),wherethestomatalconductancewasreducedby13.8%,internalCO 2  by21.15%,wateruseefficiencyby 43.91% and net photosynthetic rate by 48.87%, compared tothecontrol.ThetreatmentoftheplantswithSAincreasedalltheabove parameters over the control. However, when the NaCl-treated plants received SA as spray, it overcome the inhibitiongenerated by NaCl treatment. SA treatment was most effectiveon the plants receiving 50 and 100mmol/L of NaCl. The valueswere comparable to water soaked control (Table 4).The activities of antioxidant enzymes (i.e. catalase, peroxi-daseandsuperoxidedismutase)weresignificantlyenhancedbyNaCl and/or SA treatments (Table 3). Control plants possessedthe minimum values. The activity of enzymes increased withan increase in the level of NaCl. Moreover, the spray of SA to the NaCl-treated plants had an additive effect on theenzyme activity, where the interaction of SA with the highestconcentration (150mmol/L) of NaCl was the most effective andthe activity of superoxide dismutase (SOD) was increased by44.34%, catalase (CAT) by 14.07% and peroxidase (POX) by63.28% over the control. Discussion Thegrowthparameters(freshanddrymassofrootsandshoots,their lengths and the leaf area) decreased progressively with  1098  Journal of Integrative Plant Biology   Vol. 50 No. 9 2008 Table 2.  Effectofsalicylic acidon salinity-inducedchangesin leafnitrate reductaseactivity (nmol NO 2 · kg − 1 (leaf fresh mass) · s − 1 ), carbonicanhydraseactivity (molCO 2 · kg − 1 (leaf fresh mass) · s − 1 ) and SPAD chlorophyll value of   Brassica juncea  at 60d after sowing ( ± standard error)Treatment Nitrate reductase Carbonic anhydrase SPAD chlorophyll valueControl 463 ± 3.05 2.6 ± 0.65 35.8 ± 1.85SA (10 − 5 mol/L) 589 ± 6.50 3.8 ± 0.70 38.6 ± 1.34NaCl (50mmol/L) 439 ± 2.08 2.2 ± 0.70 35.0 ± 1.59NaCl (100mmol/L) 410 ± 3.00 1.9 ± 0.36 33.7 ± 1.28NaCl (150mmol/L) 373 ± 2.08 1.4 ± 0.39 32.8 ± 1.92NaCl (50mmol/L) + SA 10 − 5 mol/L 460 ± 1.52 2.4 ± 0.75 36.2 ± 1.41NaCl (100mmol/L) + SA 10 − 5 mol/L 441 ± 2.51 2.5 ± 0.70 33.5 ± 1.81NaCl (150mmol/L) + SA 10 − 5 mol/L 422 ± 2.05 2.1 ± 1.00 33.1 ± 1.98LSD at 5% 17.6 0.53 1.06LSD, least significant difference; SA, salicylic acid. Table 3.  Effect of salicylic acid on salinity-induced changes in relative water content (%), leaf proline (mg/gfresh mass), catalase (mmol/L H 2 O 2 decomposed/g fresh mass), peroxidase activity (units/g fresh mass) and superoxide dismutase (units/g fresh mass) of   Brassica juncea  at 60d after sowing ( ± standard error)Treatment Relative water content Proline Catalase Peroxidase Superoxide dismutaseControl 80.1 ± 4.9 8.2 ± 1.02 412 ± 1.00 12.8 ± 0.66 115 ± 0.49SA (10 − 5 mol/L) 85.2 ± 2.7 12.5 ± 0.70 472 ± 2.08 18.0 ± 1.50 153 ± 1.21NaCl (50mmol/L) 79.6 ± 3.4 11.7 ± 1.11 431 ± 2.30 15.3 ± 0.20 128 ± 0.61NaCl (100mmol/L) 76. 5 ± 2.5 14.2 ± 1.59 463 ± 2.08 18.3 ± 0.81 141 ± 1.74NaCl (150mmol/L) 60.3 ± 4.2 16.3 ± 1.30 470 ± 1.52 20.9 ± 0.70 166 ± 0.98NaCl (50mmol/L) + SA 10 − 5 mol/L 81.8 ± 2.9 12.8 ± 1.28 509 ± 1.00 19.1 ± 0.95 172 ± 1.13NaCl (100mmol/L) + SA 10 − 5 mol/L 80.2 ± 2.2 15.5 ± 1.73 540 ± 2.08 20.0 ± 1.30 191 ± 2.27NaCl (150mmol/L) + SA 10 − 5 mol/L 73.8 ± 4.6 17.4 ± 2.10 549 ± 4.58 24.9 ± 1.27 122 ± 0.80LSD at 5% 2.1 0.94 17.5 2.7 6.9LSD, least significant difference; SA, salicylic acid. the rise of stress level, compared with the control (Table 1).These results are in agreement with those of  Ghoulam et al.(2002), who showed that salinity caused a marked reduction ingrowthparametersofsugarbeetplants.TheplantssubjectedtoNaClandsubsequentlytreatedwithSA,possessedhigherfreshand dry mass compared to those grown without SA treatment(Table 1). This indicates that SA application on mustard plantexhibited an increase in salt tolerance. Gutierrez-Coronadoet al. (1998) have also reported a similar increase in the growthof shoots and roots of soybean plant under normal conditionsin response to SA treatment. Similarly, SA treatment enhancedthe growth of wheat plants under water stress (Singh and Usha2003), maize (Khodary 2004) and barley (El Tayeb 2005) under NaCl stress.Salt stress is reported to damage the photosynthetic machin-ery at multiple levels, such as pigments, stomatal functioningand gaseous exchange, structure and function of thylakoidmembrane, electron transport and enzymes (Sudhir and Murthy2004). Excess salt causes the closure of stomata, thereby de-creasing the partial CO 2  pressure (Bethkey and Drew 1992) aswell as internal CO 2  concentration (Table 4) and consequentlythe activity of CA (Table 2) because its activity is largelyregulated by the CO 2  concentration (Tiwari et al. 2005). SA,on the other hand, increased stomatal conductance as wellas the internal CO 2  concentration, both in stress-exposed aswell as stress-free plants (Table 4). Therefore, the level of CA in SA-treated plants was higher than those which did notreceive SA treatment. Fariduddin et al. (2003) also observed anincrease in the activity of this enzyme in mustard plants, grownunder stress-free conditions. SA treatment also increased thelevel of chlorophyll in the present investigation (Table 2), whichis well supported by the earlier observations in wheat and/or mung bean seedlings under stress-free conditions (Moharekar et al. 2003; Hayat et al. 2005) as well as under water stress(Singh and Usha 2003). In addition to this, the role of SA in theactivation of rubisco and PEP carboxylase under stress is alsowell documented (Singh and Usha 2003). The improvement inall these processes ultimately resulted in an increase in thephotosynthetic rate of mustard plants subjected to NaCl stress(Table 4). Likewise, Khan et al. (2003) also noted an increasein photosynthesis of corn and soybean. The damage caused bysalt stress can also be attributed to the water stress or a kind  Effect of SA and salinity in  Brassica  1099 Table 4.  Effect of salicylic acid on salinity-induced changes in stomatal conductance (mol · m − 2 · s − 1 ), internal CO 2  concentration (mg/L), water useefficiency and net photosynthetic rate (molCO 2 · m − 2 · s − 1 ) of   Brassica juncea  at 60d after sowing ( ± standard error)Treatment Stomatal conductance Internal CO 2  concentration (Ci) Water use efficiency Net photosynthetic rateControl 0.355 ± 0.004 0.260 ± 0.001 1.89 ± 0.40 10.74 ± 1.09SA (10 − 5 mol/L) 0.395 ± 0.007 0.270 ± 0.001 2.02 ± 0.20 14.19 ± 1.05NaCl (50mmol/L) 0.313 ± 0.002 0.232 ± 0.001 1.39 ± 0.20 10.17 ± 1.47NaCl (100mmol/L) 0.310 ± 0.001 0.218 ± 0.001 1.20 ± 0.20 8.86 ± 0.94NaCl (150mmol/L) 0.306 ± 0.002 0.205 ± 0.001 1.06 ± 0.03 6.35 ± 1.04NaCl (50mmol/L) + SA 10 − 5 mol/L 0.348 ± 0.002 0.259 ± 0.002 1.87 ± 0.30 11.12 ± 1.10NaCl (100mmol/L) + SA 10 − 5 mol/L 0.333 ± 0.002 0.245 ± 0.001 1.62 ± 0.01 10.08 ± 1.22NaCl (150mmol/L) + SA 10 − 5 mol/L 0.321 ± 0.002 0.233 ± 0.003 1.43 ± 0.18 8.17 ± 1.06LSD at 5% 0.024 0.012 0.13 1.43LSD, least significant difference; SA, salicylic acid. of physiological drought generated by NaCl (Hopkins 1995), asevident from the decrease in water use efficiency and relativewater content (Tables 3 and 4). However, the SA treatmentsignificantly antagonized such kinds of adverse impact. Theearlier studies strongly favor these observations. Such as, SAand acetyl SA proved effective in protecting tomato and bean(Senaratna et al. 2000) and wheat (Singh and Usha 2003)plantsagainstdroughtstress.Anyhow,mostoftheworkershaveattributed the increased level of abscisic acid (ABA) and prolineto the development of anti-stress reactions, induced by SA(Janda et al. 2007). Moreover, the reason that seems the mostappropriate to explain the SA-mediated elevation in the activityof nitrate reductase (NR) is that it corrects the stress-mediateddamage to the plasma membrane, as evident from an increasein the membrane stability index in wheat (Agarwal et al. 2005).Themembranecorrection/stabilizationcouldhavefacilitatedtheincreaseduptakeofnutrientsincludingthatofnitrate,whichactsasaninducerofNR(Campbell1999).Theincreaseintheuptakeof various nutrients, including NO 3  and activation of NR, under normal growth conditions is well established (Hayat et al. 2005)which strongly support our present results.One of the most promising influences of various abioticstresses including that of NaCl stress is the generation of oxida-tivestressthatresultsfromanincreasedlevelofreactiveoxygenspecies (ROS) in cells exposed to stress (Schutzendubel andPolle 2002). In order to repair the damage generated by ROS,plants evolve complex antioxidant metabolisms. This includesenzymes like CAT, POX, SOD, glutathione reductase (GR) andnon-enzymatic metabolites like ascorbate, glutathione, prolineand tocopherols. Senaratna et al. (2000) have suggested asimilar mechanism to be responsible for SA-induced multiple-stresstoleranceinbeanandtomatoplants.Theuseofadvancedmolecularapproacheshasrevealedanumberofdetoxifyingandantioxidant genes coding for proteins/enzymes that play crucialroles in defense response (Holuigue et al. 2007). The genesupregulated by SA under stress conditions include glutathioneS-transferase ( GST  ), glycosyl transferase ( GT  ), peroxiredoxin( PRX  ),peroxidase( PX  ),thioredoxin( TRX  ),glutaredoxin( GRX  )and pathogen resistance ( PR  ) genes (Vanderauwera et al.2005; Wang et al. 2005). It is also well established that SAactivates defense gene expression by triggering redox changesin components of the signal transduction pathway (Durrant andDong 2004; Fobert and Despres 2005). There are at least twocomponents of SA signal transduction pathway involved in theactivation of   PR   genes, NPR1 and the transcription factors,TGAs. Also, one of the promoter elements responsive to SAin defense genes ( as-1 -like element) is responsive to oxidativesignals (Garreton et al. 2002). One piece of concrete evidencein this regard is the use of SA-deficient rice that has higher ROS levels and reduced antioxidant capacity. They also exhibitspontaneous lesion formation. However, the symptoms devel-oped were suppressed by exogenous application of SA-analog,benzothiadiazole (Yang et al. 2004), which clearly indicates thatSA scavenges hydroxyl radicals and protects plant catalaseinactivation by H 2 O 2  (Durner and Klessig 1996). Hence, SAcan play a critical role in modulating the cell redox balance,thereby protecting the plants against the oxidative damage(Yang et al. 2004). A similar mechanism could be operativein NaCl-stressed plants, which is supported by our data onPOX, CAT and SOD enzymes involved in antioxidative defensemechanism (Table 3). The activity of POX in the leaves of theplant treated with SA was higher than those of plants grownunder varied salinity levels (Table 3). Consistent with this, SAapplicationwasshowntoincreasePOXactivityindifferentplantspecies subjected to various abiotic stresses (Kang and Salveit2002;Popovaetal.2003).SODactivityintheleavesofstressedplants was higher than that of the control (Table 3) which is inaccordance with Singh and Usha (2003).Prolineisanotherimportantcomponentofthedefensesystemof the plants to counter salinity. According to Hong et al.(2000), proline is synthesized by the enzyme   2  -pyrroline-5-carboxylate synthetase (   P5CS) and   2  -pyrroline-5-carboxylate reductase (  2  P5CR) and is subsequently me-tabolized by the enzyme proline dehydrogenase (Pro DH).  1100  Journal of Integrative Plant Biology   Vol. 50 No. 9 2008The activities of the enzymes,   2  P5CS and   2  P5CR (theenzymes of proline biosynthesis) are reported to be increasedand that of Pro DH decreased in cowpea grown under water stress(SumithraandReddy2004).Similarly,Kishoretal.(1995)reported an overexpression of the genes coding P5CS andrepression of those coding Pro DH in some transgenic plants.It is plausible that NaCl generated a physiological drought, asevident from decreased relative water content (Table 3) thatlead to an activation of the enzymes of proline biosynthesis andsuppression of those of its degradation and consequently theaccumulation of higher level of proline (Table 3). The previousfindings such as those of  Sultana et al. (1999) in rice, Soussi et al. (1999) in chickpea and Ghoulam et al. (2002) in sugar  beet, also suggested an increase in the level of proline, under NaCl stress. It is also noteworthy that the follow-up treatmentof the stressed plant with SA caused up to 112% increase over the control in the proline pool (Table 3). These observationsare conform with those of El Tayeb (2005) who suggestedthat proline can be considered an important component in thespectra of SA-induced protective reaction of plants to salinity.The enzyme SOD is the first line of defense to counter theO 2 ·− radical. It catalyzes the conversion of O 2 − to H 2 O 2  whichis subsequently converted to H 2 O by enzyme POX ( Alscher et al. 2002). CAT scavenges H 2 O 2  by converting it to H 2 O andfinallyO 2  andPOXreducesH 2 O 2  usingseveralreductantssuchas ascorbate, guaicol and phenolic compounds ( Apel and Hirt2004). Proline under stress conditions acts as an osmoprotec-tant (Hartzendort and Rolletscheck 2001), membrane stabilizer (Bandurska 2001) and ROS scavenger (Matysik et al. 2002). It is, therefore, inferred that the elevated level of antioxidantenzymes and proline in the plants treated with SA inducedtheir tolerance to NaCl that was reflected in the improvedgrowth, enzyme activities and photosynthetic performance. Theinduction of the stress tolerance by SA has also been reportedby a number of researchers (Dat et al. 2001; Tasgin et al. 2003;He et al. 2005).In conclusion, it can be said that SA treatment enhancedthe level of antioxidant system (CAT, POX, SOD and proline)in mustard plants under NaCl stress. The elevated antioxidantsystem counters the oxidative stress as well as other directeffects of NaCl stress, thereby improving the photosyntheticcapacity, metabolism and plant growth in mustard plants. Materials and Methods The authentic seeds of   Brassica juncea  (L.) Czern. et Coss. cv.Krishnaii obtained from the National Seed Corporation (NewDelhi, India) were surface-sterilized with a 0.01% aqueoussolution of mercuric chloride followed by repeated washingwith double-distilled water (DDW). The surface-sterilized seedswere soaked for 8h in 0, 50, 100 and 150mmol/L NaCl, withthe durations based on our preliminary study. These seedswere sown in earthen pots (0.254 meter) filled with sandyloam soil and farmyard manure (mixed to the ratio of 6:1) andlined in a net house, where the average day/night temperature,humidity and photoperiod were 25 ◦ C/20 ◦ C, 65% ± 3% and12h, respectively. At the 30d stage, plants were sprayed with0 or 10 µ mol/L of SA. Each seedling was sprinkled thrice. Thenozzle of the sprayer was adjusted in such a way that it pumpedout 1mL in one sprinkle. Therefore, each plant received 3mLof DDW or SA solution. The plants were sampled at 60d after sowing (DAS) for assessment of the following observations.The plants were uprooted and washed under running tapwateranddriedinahotairoven,runat80 ◦ C,for24h.Thesam-ples were weighed to obtain dry mass. The activity of NR wasdetermined in fresh leaf samples by the procedure explainedby Jaworski (1971). The fresh leaf samples were cut into smallpieces and transferred to plastic vials, containing phosphatebuffer (pH7.5) followed by the addition of potassium nitrate andisopropanol solutions. The reaction mixture was incubated at30 ◦ C for 2h followed by addition of sulfanilamide and  N  -1-naphthylethylenediamine dihydrochloride. The absorbance of the color was read at 540nm and was compared with that of the calibration curve. The activity of NR (nmol NO 2 · g − 1 · h − 1 )was computed on a fresh mass basis. The activity of CAwas determined following the procedure described by Dwivediand Randhawa (1974). The leaf samples were cut into smallpieces and suspended in cystein hydrochloride solution. Thesamples were incubated at 4 ◦ C for 20min. The pieces wereblotted and transferred to the test tubes, containing phosphatebuffer (pH6.8) followed by the addition of alkaline bicarbonatesolution and bromothymol blue indicator. The test tube wasincubated at 5 ◦ C for 20min. The reaction mixture was titratedagainst 0.01mol/L HCl, after addition of 0.2mL of methyl redindicator. The results were expressed as molCO 2 · kg − 1 (leaf fresh mass) · s − 1 .The proline content in fresh leaf and root samples wasdetermined by adopting the method of  Bates et al. (1973).Samples were extracted with sulfosalicylic acid and an equalvolume of glacial acetic acid and ninhydrine solutions wereadded. The samples were heated at 100 ◦ C, to which 5mL of toluene was added. The absorbance of the toluene layer wasread at 528nm on a spectrophotometer.The chlorophyll content in the leaves was measured with thehelp of a SPAD chlorophyll meter. The rate of photosynthesisand its related parameters (stomatal conductance, water useefficiency, internal CO 2  concentration and transpiration rate)were measured by using a portable photosynthetic system(LI-COR-6400, Lincoln, USA). The measurements were madein the uppermost fully expanded leaves, between 11.00 and13.00hours.For the estimation of antioxidant enzymes, the leaf tis-sue (0.5g) was homogenized in 50mmol/L phosphate buffer (pH7.0) containing 1% (w/v) soluble polyvinylpyrrolidone. The
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