drought and tea plant

Journal of Tea Science Research. 2016, Vol. 6, No. 4, 1-11http://jtsr.biopublisher.ca

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Research Article Open Access

Drought Induced Physiological and Biochemical Changes in Leaves ofDeveloping Seedlings of Tea [ Camellia sinensis (L) O Kuntze ] CultivarsUpadhyaya H.1, , Dutta B.K.2, Panda S.K.31 Department of Botany and Biotechnology, Karimganj College , Karimganj-788710, Assam, India2 Microbial and Agricultural Ecology Laboratory, Department of Ecology and Environmental Sciences, Assam (Central) University , Silchar, 788011, India3 Plant Biochemistry and Molecular Biology Laboratory, School of Life sciences, Assam ( Central ) University ,Silchar, 788011, India

Corresponding author email: [email protected] of Tea Science Research, 2016, Vol.6, No.4 doi: 10.5376/jtsr.2016.06.0004Received: 28 Oct., 2015Accepted: 15 Dec., 2015Published: 28 Jan., 2016© 2016 Upadhyaya et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.Preferred citation for this article:Upadhyaya H., Dutta B.K., and Panda S.K., 2016, Drought induced physiological and biochemical changes in leaves of developing seedlings of tea [Camelliasinensis (L) O Kuntze] cultivars, Journal of Tea Science Research, 6(4), 1-11 (doi: 10.5376/jtsr.2016.06.0004)

Abstract Drought is one of the important environmental stress affecting agricultural productivity around the world. In this study, anattempt has been made to understand drought induced biochemical alterations in different clones of Camellia sinensis [TV-1, TV-20,TV-29 and TV-30]. Drought stress induced decrease in total chlorophyll and carotenoid, phenolics concentration and increases inproline concentration, lipid peroxidation and polyphenols oxidase activity as a consequent of decrease in leaf relative water content(RWC). Decreased Na+ and K+ concentration caused osmotic stress in leaves decreasing NR activity, and ultimately reducing leafrelative growth rate. Thus, drought induced a range of physiological and biochemical alterations causing membrane damage and lossin cellular functions ultimately leading to reduction in growth of one of the most important economic crop like tea. In comparison ,TV-1 showed better drought tolerance by maintaining higher endogenous K+ and proline content and a balance Na+/K+ ratio in leaves.Keywords Drought. relative growth rate(RGR); chlorophyll; phenols.proline; lipid peroxidation; Camellia sinensis.

1 IntroductionDrought is one of the more important environmentalstresses affecting agricultural productivity around theworld and may result in considerable yield reduction(Boyer 1982). ‘Drought’ is a meteorological term thatdenotes a period without rains during which soil watercontent is reduced and plants suffer from lack of water.Drought affects on the morphology anatomy andphysiology of the plants. Physiological, biochemical,and anatomical responses however occur much earlierthan the usual symptoms of wilting, which may bepermanent or temporary depending on the availabilityof soil moisture. The physiological, biochemicaland molecular mechanism involved in cellular andwhole plant responses to drought therefore generateconsiderable interest and are frequently reviewed(Ingram and Bartel, 1996; Chakraborty et al.2002; Kar 2002; Shinozaki et al. 2002; Yordanav et al.2003; Francois Tardieu 2003; Upadhyaya and Panda2004; Reddy et al. 2004; Jeyaramraja et al. 2005;Sakuma et al. 2006; Ohashi et al. 2006). Water

stress results in stomatal closure and reducedtranspirations rate, a decrease in water potential ofplant tissues, decrease in photosysthesis and growthinhibition (Tahi et al. 2007), accumulation of abscisicacid (ABA), proline, mannitol, sorbitol, formation ofradical scavenging compounds (ascorbate, glutathione,tocopherol etc) and systhesis of proteins. Decrease inphotosysthesis is due to the limitted CO2 assimilationcaused by stomatal closure and decrease is totalchlorophyll content. Osmotic adjustment has beenconsidered as beneficial to drought tolerantmechanism in field crop species. Na+ and K+ contentof leaf affect and regulates the osmotic potential of theplant during stress conditions and hence Na+/ K+ ratioshould be adequate for stress acclimatization by plantthrough osmotic adjustment . However, there is noreport on the changes of these ions in response todrought in Camellia sinensis. The lowering ofosmotic potential by adjustment also minimizes theopportunity for significant water loss to occur fromleaf tissue. This helps cells of higher plants to


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withstand water deficit by maintaining sufficientturgor to proceed (Grima and Krieg 1992 a,b).Water stress induced pigment degradation, grossdecline in protein level, increased proline content,carbohydrate status, lipid peroxidation, in plantshave also been reviewed ( Kar 2002 ) . Tea is secondonly to water as the most consumed beverage in theworld. It has been used medicinally for centuries inIndia and China. Green tea is comparatively healthierthan black and olong tea. The active constituents ingreen tea are powerful antioxidants called polyphenols(catechins) and flavonols. Research shows that teaconsumption is healthy and help fighting varioushealth problems which has also been reviewed bymany authors ( Kabir 2002; Higdon and Frei, 2003;Cabrera et al. 2006). Tea is one of the most importanteconomic crops in Barak/Brahmaputra valley/ Dooarsand other hilly terrains of India (i.e. Darjeeling,Himachal, Nilgiri and Uttaranchal). Tea plant beingperennial crops is subjected to different environmentalstress, drought being one of the important amongstthem. In N.E. India, generally tea suffers from droughtduring November to April. In this region irrigation isincreasingly used as an insurance against drought tomaintain the productivity of tea during this period.The influence of irrigation on the potential yield of teain this region has also been studied (Panda et al. 2003).Thus the present investigation was undertaken forunderstanding the mechanism of drought stressinduced physiological and biochemical alterations inselected clone of Camellia sinensis L (O) Kuntze.Drought tolerance in tea can be assessed through somephysiological and biochemical parameters undermoisture stress and these parameters can be used asselection criteria for drought tolerance in the selectionand breeding programs of tea (Handique andManivel 1990). In the present experiment, thefield soil was used in the pot. The aim was to test theresponses of selected clones to drought stress imposedby withholding water in the pot. Such pot experimentshave been used to asses the drought tolerance in plants(Chakraborty et al., 2002; Sharma and Kumar, 2005,Xu and Zhou, 2007, etc). However, the fieldconditions were different from pot as the tea is a deeprooted plant and soil area is not limited it canpenetrate the soil deep and it is obvious that it can

withstand more days of dehydration stress in thefield condition. During the period of natural droughtplant encounters long period without rain as it isgrown in rainfed ecosystem. Thus it is quite relevantto evaluate drought responses of plant like tea inpotted conditions and correlate its responses inrelation to field performance as because all the plantswere grown in the same size pots and under sameenvironmental conditions.

2 Materials and MethodsFour commonly growing clonal varieties of Camelliasinensis L. (O) Kuntze ( viz. TV-1, TV-20, TV-29 &TV-30 ) seedlings of uniform age, one and half yearold were procured from. Tocklai Tea Research Station,Silcoori, Silchar.

The seedlings grown in field soil in polyethenesleeves were procured from the nursery of near by teaGarden of Durgakona and brought to the laboratory.The seedlings were potted after removing polyethenesleeves and adding field soil. The plants wereacclimatized for 10-15 days in laboratory conditionsand were grown under natural light with well irrigation.

As tea is a shade loving plant, the seedlings weregrown in a shed where the intensity of light rangesfrom 150-300 mol m- 2 s- 1 and 200-400 mol m- 2 s-1

inside and outside the shed respectively. All controland treated plants were kept in similar growthconditions during acclimatization and treatmentimposition.

After 10-15 days of acclimatization, drought isimposed by withholding water for 20 days. Wellwatered plant is considered as control. The levelof stress was quantified by measuring changes in soilmoisture and RWC of leaf in stressed plant relative tocontrol. After dehydration, soil moisture contentdecreased to (12.88 ± 1.34)% and (3.55 ± 0.28 )%after 10 and 20d of stress imposition respectivelyrelative to control (23.46 ± 1.62)%. The averagetemperature range during experimental period wasnoted as 25.1 – 32.3°C and 12.5 – 24.7°C max/minrespectively. The average relative humidity during theexperiment period was 88-96% and 38-67%morning/afternoon respectively. All the leaf samplings were


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doneduring morning hours between 8 am to 9 am.

Fresh mass of leaf was measured in three replicatesusing five leaves and expressed as g leaf -1. Fordry mass measurement same leaves were oven dried at80°C for 48 h and expressed as g leaf -1. The relativegain in leaf total fresh mass was determined as relativegrowth rate (RGR, [g d-1] of leaf or the increase intotal leaf fresh mass per unit of existing mass per unittime), and calculated according to the formula:

RGR=(InM2-InM1)/(t2-t1)where M2 was the final total fresh mass (g), M1 wasthe initial total fresh mass (g), t2 was the number ofdays since initiation of experiment and t1 was day 0.Relative water content (RWC) was measured byfollowing the methods of Barrs and Weatherly (1962).

Tea leaves were sampled, oven dried and digested in aHNO3-HCl (3:1,v/v) mixture and Na+ and K+

concentrations were determined by Flame Photometer(Systronics, India) (Jackson, 1973)

The stability of leaf membranes, was assessed bydetermining leakage of electrolytes from leaf discsplaced in 20 ml of deionised water for 24 h at roomtemperature and measuring the electrical conductivitybefore and after autoclaving the samples andElectrolytic leakage was determined as described byDionisio Sese and Tobita (1998) and calculated usingformula.

EL = EC1/EC2 X 100Where - EC1 is the initial conductivity of the samplebefore autoclaving and EC2 is the final conductivitymeasured after autoclaving the sample.

Leaves were extracted in cold with 80 % acetone. Thechlorophyll and carotenoid contents were estimated asper the methods of Arnon (1949).

Proline concentration in tea leaves was determinedfollowing the method of Bates et al. (1973). Leafsample (0.5 g)was homogenized with 5 ml ofsulfosalicylic acid (3%) using mortar and pestle andfiltered through Whatman No. 1 filter paper. Thevolume of filtrate was made upto 10 ml withsulfosalicylic acid and 2.0 ml of filtrate was incubatedwith 2.0 ml glacial acetic acid and 2.0 ml ninhydrin

reagent and boiled in a water bath at 100°C for 30 min.After cooling the reaction mixture, 6.0 ml of toluenewere added and after cyclomixing it, absorbance wasread at 570 nm. Total phenolics were extracted fromtea leaves in 80 % (v/v) ethanol and were estimated asper the method of Mahadevan and Sridhar (1982)using Follin Ciocalteau reagent and Na2CO3.

Lipid peroxidation was measured as the amount ofTBARS determined by the thiobarbituric acid (TBA)reaction as described by Heath and Packer (1968). Theleaf tissues (0.2 g) were homogenised in 2.0 ml of0.1% ( w/v trichloroacetic acid (TCA). The homogenatewas centrifuged at 10,000g for 20 min. To 1.0 ml of theresulting supernatent, 1.0 ml of TCA (20%) containing10.5% (w/v) of TBA and 10L (4% in ethanol) BHT(butylated hydroxytolune) were added. The mixturewas heated at 950C for 30min in a water bath and thencooled in rice. The contents were centrifuged at10,000 g for 15 min and the absorbancy was measuredat 532 nm and corrected for 600 nm. The concentration ofMDA were calculated using extinction coefficient of155mM-1cm-1.

Leaf tissues were homogenized with potassiumphosphate buffer pH 6.8 (0.1M) containing 0.1 mMEDTA, 1% PVP and 0.1 mM PMSF in prechilledmortar pestle. The extract was centrifuged at 40C for15 min at 17000 g in a refrigerated cooling centrifuge.The supernatant was used for the assay of polyphenoloxidase (PPO). PPO were assayed using pyrogallol assubstrate in 1.0 ml of enzyme extract. according toKar and Mishra (1976).After incubations at 250C for 5min, the reaction was stopped with additions of 1.0 mlof 10 % H2SO4. The purpurogallin formed was read at430 nm. 1 unit of enzyme activity is defined asthat amount of enzyme which forms 1 mole ofpurpurogallin formed per minute under the assayconditions. Total soluble protein content wasestimated as per the method of Bradford (1976) usingBSA as standard.

Nitrate Reductase (NR) activity in tea leaves wasextracted and estimated as per the methods of Singhand Mallik (1980). The enzyme extracted in 0.1Mphosphate buffer (pH7.2) and the homogenate wascentrifuged in cooling centrifuge at 10,000 g at -4°C.


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The resulting supernatant was used for the assay ofenzyme activity. The assay mixture comprised of 1.0ml phosphate buffer (0.1M) (pH7.2), 0.5 ml NADH(1.5 mM), 0.5 ml distilled water and 1.0 ml enzymeextract. It is then incubated at 25°C. The enzymereaction was initiated by adding 0.5 ml KNO3 (0.1 M)and incubated for 30 min. Then 0.8 ml zinc acetate(0.1 M) was added and centrifuged and 3.0 ml of thesupernatant was retained. To this 2.0 ml [1 %sulfanilamide in 1.5 N HCl and 0.2 % N-naphthalenediamine hydrochloride (NEDH)], mixed in equalvolume was added. After 10 min absorbency was readat 540 nm. NR activity was expressed as moles NO2min-1.g-1 FW.

Each experiment was done in triplicate and repeatedthrice and data presented are mean standard error(SE). The results were subjected to ANOVA usingGLM factorial model on all the parameters. Tukey testwas used for comparision between pairs of treatments.For relationship between relative water content andproline accumulation, K+ content of leaf and its RWC,lipid peroxidation and solute leakage of leaf tissue,lipid peroxidation and decrease in RWC of leaf ,relative growth rate of leaf and changes in totalchlorophyll content a linear regression was performed.The data analysis were carried out using statisticalpackage SPSS 7.5.

3 ResultsRelative growth rate (RGR) of leaf showed uniformlydecline trend with progressive soil moisture stress inall the tested clones (Figure 1B). Changes in RGR ofleaf due to10d of stress imposition was minimum inTV-30 and TV-1 but after 20d of stress RGR changesamong the clones was not significant though decreasegrowth was about 95%.The amount of soluteleakage is an index of membrane damage. Droughtinduced increase in solute leakage in all the testedclones of Camellia sinensis (Figure 1A). After 20d ofstress, in comparison with control plants soluteleakage increases to 130.84, 89.75, 150 and 153.92%in TV-1, TV-17, TV-20, TV-29 and TV-30 respectively.However, TV-1 and TV-20 showed comparativelylesser amount of solute leakage during stressconditions. Relative water content (RWC) of leafuniformly decreased with decreasing soil moisture

Figure 1 Changes in solute leakage, relative growth rate(RGR)of leaf and relative water content (RWC) in four clonalvarieties of Camellia sinensis ( TV-1, TV-20, TV-29 & TV-30 )subjected to drought. Control (white); 10 days of drought(gray) ; 20 days of drought (black) imposition. Data presentedare mean ± SE (n=3). Different lower case letters indicatesdifferences between drought treatment in the same variety ,while * indicates differences within the varieties at P< .05according to multiple comparision by Tukey test.

content imposed by 20 d of water withholding (Figure1C). After 20 d of drought, RWC of leaf wasfound to be 56.26±1.21, 52.87±1.08, 44.99±0.43 and52.66±1.03% relative to control with 92.15±1.24,90.21±2.83, 87.57± 6.11 and 91.64± 4.92 % in TV-1,TV-20, TV-29 and TV-30.

The contents of Na+ ion increased with the progress ofwater stress imposition, apparently showing highestNa+ content in TV-29(41.73%) with lowest in TV-1 asa result of 20 d of drought stress in comparision withcontrol plants (Figure 2A). On the other hand,water stress induced decrease in K accumulation


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was observed in leaves of Camellia sinensis , buthighest K+ content was maintained by TV-1 (27.16%)and TV-29 (13.85%) even after 20 d of droughtimposition as depicted in Fig 2B. There was increasein Na+/ K+ ratio with progressive days of waterwithholding in TV-20 (105.80%),TV-29 (64.58%)& TV-30 (283.25%), but with exception TV-1(37.09%) showed decrease in Na+/ K+ ratio with theincreasing intensity of drought stress (Figure 2C).

Photosynthetic pigments (chlorophyll and carotenoid)content was found to be decreased with increasingdays of stress imposition. Drought induceddegradation of chlorophyll and carotenoid wasmaximum in TV-29 (57.91 & 82.38%) and TV-20(55.10 & 86.13%) respectively as depicted in Fig.3A& B. Phenolic compounds are widely distributed inplants and are mainly produced to protect plants fromstress, ROS, wounds, UV light, disease and herbivores(Dixon and Paiva 1995). Total phenolics content in teaincludes catechins and polyphenols, which was foundto be decreased with the imposition of drought (Figure3C). Maximum decrease in phenolics content wasobserved in TV-20(37.63%) and TV-2928.15%).

Increase in compatible solutes like proline, Glycinebetain etc., is the characteristic feature of plantsacclimatizing stress conditions. In this study prolineaccumulation during water stress condition wasmaximum in TV-1 (Figure 4A). Drought stressinduced significant increase in PPO activity wasobserved in all the tested tea cultivars, maximumincrease being shown by TV-29 (Figure 4B). HighestPPO activity was shown by TV-29(458.82%) andTV-1 (424.91%). MDA content was maximum inTV-29 (420.41%) after 20 d of stress imposition,whereas TV-30(58.95%) showed minimum increase ascompared to control plant (Figure 4C).

As indicated in Figure 5A, NR activity decreased dueto drought in four clones of tea, where minimumdecrease was observed in TV-1(62.59%) and TV-30(72.03%), that could be correlated with stress induceddecrease in total soluble protein content in testedclones of Camellia sinensis (Figure 5B).

Figure 2 Changes in Na+, K+ content and Na+/ K+ ratio in fourclonal varieties of Camellia sinensis ( TV-1, TV-20, TV-29 &TV-30 ) subjected to drought . Control (white); 10 days ofdrought (gray); 20 days of drought (black) imposition. Datapresented are mean ± SE (n=3) and significant differences areshown as in (Figure 1)

The interesting aspect in this study is that there was nosignificant correlation between relative water contentand changes in proline content of leaf (r2 = 0.15, ns)(Figure 6 A). In our study, increase in Na+ content isfollowed by significant decrease in K+ content of leafwhich is significantly correlated with decrease inRWC of leaf (r2=0.50, p<0.01) as depicted in Figure 6B. Concomitant with the increase in MDA content ofleaf amount of solute leakage also increased whichwas evident from the significant correlation (r2=0.66,p<0.001) between increase in lipid peroxidation and


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Figure 3 Changes in total chlorophyll, carotenoid and phenoliccontents in four clonal varieties of Camellia sinensis (TV-1,TV-20, TV-29 & TV-30) subjected to drought. Control (white) ;10 days of drought (gray); 20 days of drought (black)imposition. Data presented are mean ± SE (n=3) and significantdifferences are shown as in Figure 1

amount of solute leakage as shown in Figure 6C.Increase in MDA content is significantly correlatedwith decrease in RWC of leaf (r2 = 0.39, p<0.05) asdepicted in Figure 6D. There was significantcorrelation between chlorophyll degradation andRGR of leaf (r2 = 0.40, p<0.05) (Figure 6 E).

4 DiscussionDrought imposition resulted in significant changes ofgrowth and biochemical responses in various clones ofCamellia sinensis. The contents of Na+ ion increasedwith the progress of water stress imposition,apparently showing highest Na+ content in TV-29 withlowest in TV-1 as a result of 20 d of drought stress in

Figure 4 Changes in proline content, polyphenol oxidase (PPO)activity and MDA content in four clonal varieties of Camelliasinensis (TV-1, TV-20, TV-29 & TV-30) subjected to drought.Control (white ); 10 days of drought (gray) ; 20 days of drought(black ) imposition . Data presented are mean ± SE (n=3) andsignificant differences are shown as in Figure 1

comparision with control plants. On the other hand,water stress induced decrease in K accumulationwas observed in leaves of Camellia sinensis, buthighest K+ content was maintained by TV-1even after 20 d of drought imposition. This is incontradiction with the findings of Osmond et al.(1980) and Martinez et al. (2003) who reportedinvolvement of Na+ and K+ accumulation in theosmotic adjustment of leaf tissues to low externalwater potential in Artiplex species during water stress.Sodium contribution to osmotic adjustment may beindirectly by triggering osmolyte synthesis independent ofosmotic stress was however demonstrated by Subbaraoet al. (2001). In our study, increase in Na+ content is


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followed by significant decrease in K+ content of leafwhich is significantly correlated with decrease inRWC of leaf (r2 = 0.50, p< .01) as depicted in Fig. 6 B.K+ nutrition in plants is known to enhanced droughtresistance , water use efficiency and growth under

Figure 5 Changes in nitrate reductase (NR) activity and totalsoluble protein content in four clonal varieties of Camelliasinensis ( TV-1, TV-20, TV-29 & TV-30 ) subjected to drought .Control (white) ; 10 days of drought (gray) ; 20 days of drought(black) imposition . Data presented are mean ± SE (n=3) andsignificant differences are shown as in Figure 1.

drought condition (Egilla et al., 2001 ). Thus itsuggested adequate K+ fertilizer application of teaplant may facilitate osmotic adjustment improving itsdrought tolerance potential. There was increase inNa+/ K+ ratio with progressive days of waterwithholding in TV-20, TV-29 & TV-30, but withexception TV-1 showed decrease in Na+/ K+ ratio withthe increasing intensity of drought stress (Figure 2C).As K+ is the main ion regulating osmoticadjustment in leaves , increase in Na + /K + ra tiosuggested occurrence of osmotic stress in three teacultivars (TV-20, TV-29 & TV-30) but exceptionaldecreased Na+/ K+ ratio in TV-1 is the indication of itscomparatively better drought accl imatizingpotential. Water deficit stress, often called droughtaffects the growth and productivity of plantsgrowing in natural or agricultural field conditions. In

the present study, relative growth rate (RGR) of leafshowed uniformly decline trend with increasingdrought stress. The growth of leaf is directly orindirectly related with the various metabolismdependent on its osmotic potential which is regulatedby potassium content of leaf. The amount of soluteleakage is an index of membrane damage. Droughtinduced increase in solute leakage in all the testedclones of Camellia sinensis. However, TV-1 and TV-30showed comparatively lesser amount of solute leakageduring stress conditions. Drought induced membranedamage could be due to enhanced membrane lipidperoxidation which was evident from increased MDAcontent with the progress of stress imposition,observed in four clones of tea. MDA content wasmaximum in TV-29 after 20 d of stress imposition,whereas TV-30 showedminimum increase as compared tocontrol plant. Concomitant with the increase in MDAcontent of leaf amount of solute leakage also increasedwhich was evident from the significant correlation (r2

= 0.66, p< .001) between increase in lipidperoxidation and amount of solute leakage as shownin Fig. 6 C. Thus, it apparently suggests that increasein leakage of solute could be due to drought inducedlipid peroxidation of membrane disturbing thestructural and functional integrity of cell causingultimate reduction in growth of tea plant.Photosynthetic pigments (chlorophyll and carotenoid )content was found to be decreased with increasingdays of stress imposition.Drought induceddegradation of chlorophyll and carotenoid wasmaximum in TV-29 and TV-20 respectively. Therewas significant correlation between chlorophylldegradation and RGR of leaf (r2 = 0.40, p< .05).Decrease in chlorophyll and carotenoid in response towater stress was reported earlier (Upadhyaya andPanda, 2004). Such degradation of chlorophyllpigments may eventually decrease photosyntheticefficiency in plants which might be one of the potentcauses of reduction in growth of plant. However, avery recent study also provides evidence for asignificant positive relationship between transpirationefficiency and leaf chlorophyll concentration in plant(Sheshshayee et al. 2006). During prolonged periodsof drought, the decrease in water availability for


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transport-associated processes leads to changes in theconcentrations of many metabolites, followed bydisturbances in amino acid and carbohydrate

metabolism. For example, there is an increase in thesynthesis of compatible solutes such as special aminoacids (e.g. Proline), sugars and sugar-alcohols, and

Figure 6 Relationship between relative water content and proline accumulation (A), K+ content of leaf and its RWC (B), lipidperoxidation and solute leakage of leaf tissue (C), lipid peroxidation and decrease in RWC of leaf (D), relative growth rate of leaf andchanges in total chlorophyll content in four clonal varieties of Camellia sinensis subjected to drought . The open square, closedtriangle, open circle and closed circle denotes TV-1, TV-20, TV-29 and TV-30 variety of tea subjected to drought treatmentrespectively. *, ** and *** indicates significant correlation at P<.05,.01 and .001 respectively.

Gly betaine.Acclimation to drought requires responsesthat allow essential reactions of primary metabolismto continue and enable the plant to tolerate waterdeficits. In the complex interplay of natural conditions,simple water deficits are unlikely to occur, since theyintrinsically affect the acquisition of essential nutrientssuch as N etc. The reduction of NO3− to NO2−

catalyzed by NR is considered to be the rate-limitingstep of N assimilation. NR activity is coordinated with

the rate of photosynthesis Indeed, drought-induced Ndeficiency was found to limit recovery ofphotosynthesis in plants. In situations of waterdeprivation, maximal foliar extractable NR activityhas been found to decrease in some cases (Foyer et al.,1998). In the present study, NR activity decreaseddue to drought in four clones of tea , whereminimum decrease was observed in TV-1 and TV-30,that could be correlated with stress induced decrease


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in total soluble protein content in tested clones ofCamellia sinensis. Similar results were alreadyreported in other plants (Foyer et al., 1998; Panda,2002; Xu and Zhou, 2006). Increase in compatiblesolutes like proline, Glycine betain etc., is thecharacteristic feature of plants acclimatizing stressconditions. In this study proline accumulation duringwater stress condition was maximum in TV-1. Prolineacts as an osmoprotectant and greater accumulation ofproline in TV-1 suggested genotypic tolerance of teato water deficit stress, as proline accumulation helpsin maintaining the water relations, prevents membranedistortion and acts as a hydroxyl radical scavenger.Drought induced biochemical modifications andproline metabolism has also been studied in otherplants (Sanker et al., 2007). The molecular mechanismof quenching of reactive oxygen species proline hasalso been well reviewed ( Matysik et al., 2001).However, Wu et al., (2007) reported osmoticadjustment in Citrus seedling colonized by Glomusversiforme subjected to drought stress did notcorrelate with proline but with ions like K+, Ca2+ , etcand carbohydrates. Thus, similarly K+ might playcritical role in providing osmotic adjustment inresponse to drought stress in tea cultivars as evidencedby significant correlation between changes inendogenous K+ content and RWC of leaves in stressimposed tea cultivars (r2 = 0.50, p< .01). Also higherK+ content of TV-1 in stressed condition showedbetter drought tolerance in comparision with otherclones. Relative water content (RWC) of leafuniformly decreased with decreasing soil moisturecontent imposed by 20 d of water withholding (Fig.1C). The interesting aspect in this study is that therewas no significant correlation between relative watercontent and changes in proline content of leaf (r2 =0.15, ns) ( Figure 6A).

Phenolic compounds are widely distributed in plantsand are mainly produced to protect plants from stress,ROS, wounds, UV light, disease and herbivores(Dixon and Paiva, 1995). Total phenolics content intea includes catechins and polyphenols, which wasfound to be decreased with the imposition of drought(Fig.3C). Tea polyphenols are mostly catechins whichhave potential antioxidant properties that makes tea a

good health drink. Polyphenol oxidases (PPO) arewidely distributed in the plant and play a role inoxygen scavenging and defense against stress (Esen,1993; Shivishankar, 1988). The antioxidant activity ofphenolic compounds is mainly due to their redoxproperties. The medicinal value of phenolics in humanhealth is well documented (Caia et al., 2004; Tona etal., 2004 ). In this study, drought stress inducedsignificant increase in PPO activity was observed inall the tested tea cultivars, maximum increase beingshown by TV-29. PPO catalyses the O2-dependentoxidation of mono- and o diphenols to o-diquinones,where secondary reactions may be responsible for thedefense reaction and hypersensitive response (Mayerand Harel, 1991) .Moreover, it is proposed that PPOactivity may regulate the redox state of phenoliccompounds and become involved in thephenylpropanoid pathway (Kojima and Takeuchi 1989;Nakayama et al., 2000).

In conclusion, it can be said that drought induced arange of physiological and biochemical alterationscausing membrane damage and loss in cellularfunctions ultimately leading to reduction in growth ofone of the most important economic crop like tea. Incomparision TV-1 showed better drought tolerance bymaintaining higher endogenous K+ and proline contentand a balance Na+/K+ ratio in leaves. Hence, thepresent result may provide insight into underlyingmechanism of tea plant in response to soil drought atthe juvenile stage. However, more investigation isrequired in the field at different plant growth stages inthe future. It can be further confirmed by studyingvarious physiological and biochemical responses ofdifferent tea cultivars in field conditions andconducting experiments to study the responses ofthose cultivars to rehydration and post stressmanipulation of soil and leaf nutrients which arealready initiated at our laboratory.

AcknowledgementThe authors thank Mr S.M. Bhati, General Manager,Tocklai Tea Estate, Silcoorie, Silchar for providing Teaseedlings throughout the experimental work.

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