Research Article

Drought Induced Physiological and Biochemical Changes in Leaves of Developing Seedlings of Tea [Camellia sinensis (L) O Kuntze ] Cultivars  

Upadhyaya H.1 , Dutta B.K.2 , Panda S.K.3
1 Department of Botany and Biotechnology, Karimganj College, Karimganj-788710, Assam, India
2 Microbial and Agricultural Ecology Laboratory, Department of Ecology and Environmental Sciences, Assam (Central) University, Silchar, 788011, India
3 Plant Biochemistry and Molecular Biology Laboratory, School of Life sciences, Assam (Central) University, Silchar, 788011, India
Author    Correspondence author
Journal of Tea Science Research, 2016, Vol. 6, No. 4   doi: 10.5376/jtsr.2016.06.0004
Received: 28 Oct., 2015    Accepted: 15 Dec., 2015    Published: 28 Jan., 2016
© 2016 BioPublisher Publishing Platform
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 [Camellia sinensis (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, an attempt 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 in proline 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 leaf relative growth rate. Thus, drought induced a range of physiological and biochemical alterations causing membrane damage and loss in 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 Introduction
Drought is one of the more important environmental stresses affecting agricultural productivity around the world and may result in considerable yield reduction (Boyer 1982). ‘Drought’ is a meteorological term that denotes a period without rains during which soil water content is reduced and plants suffer from lack of water. Drought affects on the morphology anatomy and physiology of the plants. Physiological, biochemical, and anatomical responses however occur much earlier than the usual symptoms of wilting, which may be permanent or temporary depending on the availability of soil moisture.

 

The physiological, biochemical and molecular  mechanism involved  in cellular and whole plant responses to drought  therefore generate considerable 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 Panda 2004; Reddy et al. 2004;  Jeyaramraja et al. 2005; Sakuma et al. 2006; Ohashi et al. 2006). Water stress results in stomatal closure and reduced transpirations rate, a decrease in water potential of plant tissues, decrease in photosysthesis and growth  inhibition (Tahi et al. 2007), accumulation of abscisic acid (ABA), proline, mannitol, sorbitol, formation  of radical scavenging compounds (ascorbate, glutathione, tocopherol etc) and systhesis of proteins. Decrease in photosysthesis is due to the limitted CO2 assimilation caused by stomatal closure and decrease is total chlorophyll content. Osmotic adjustment has been considered as beneficial to drought tolerant mechanism in field crop species. Na+ and K+ content of leaf affect and regulates the osmotic potential of the plant during stress conditions and hence Na+/ K+ ratio should be adequate for stress acclimatization by plant through osmotic adjustment .

 

However, there is no report on the changes of these ions in response to drought in Camellia sinensis. The lowering of osmotic potential by adjustment also minimizes the opportunity for significant water loss to occur from leaf tissue. This helps cells of higher plants to withstand water deficit by maintaining sufficient turgor to proceed (Grima and  Krieg 1992 a,b) . Water stress induced pigment degradation, gross decline in protein level, increased proline content, carbohydrate status, lipid peroxidation,  in plants have also been reviewed (Kar 2002). Tea is second only to water as the most consumed beverage in the world. It has been used medicinally for centuries in India and China. Green tea is comparatively healthier than black and olong tea. The active constituents in green tea are powerful antioxidants called polyphenols (catechins) and flavonols. Research shows that tea consumption is healthy and help fighting various health problems which has also been reviewed by many authors (Kabir 2002; Higdon and Frei, 2003; Cabrera et al., 2006).

 

Tea is one of the most important economic crops in Barak/Brahmaputra valley/ Dooars and other hilly terrains of India (i.e. Darjeeling, Himachal, Nilgiri and Uttaranchal). Tea plant being perennial crops is subjected to different environmental stress, drought being one of the important amongst them. In N.E. India, generally tea suffers from drought during November to April. In this region irrigation is increasingly used as an insurance against drought to maintain the productivity of tea during this period. The influence of irrigation on the potential yield of tea in this region has also been studied (Panda et al., 2003). Thus the present investigation was undertaken for understanding the mechanism of drought stress induced physiological and biochemical alterations in selected clone of Camellia sinensis L (O) Kuntze. Drought tolerance in tea can be assessed through some physiological and biochemical parameters under moisture stress and these parameters can be used as selection criteria for drought tolerance in the selection and breeding programs of tea (Handique and  Manivel 1990). In the present experiment, the field soil was used in the pot. The aim was to test the responses of selected clones to drought stress imposed by withholding water in the pot. Such pot experiments have been used to asses the drought tolerance in plants (Chakraborty et al., 2002; Sharma and Kumar, 2005, Xu and Zhou, 2007, etc).

 

However, the field conditions were different from pot as the tea is a deep rooted plant and soil area is not limited it can penetrate the soil deep and it is obvious that it can withstand more days of dehydration stress in  the field condition. During the period of natural drought plant encounters long period without rain as it is grown in rainfed ecosystem. Thus it is quite relevant to evaluate drought responses of plant like tea in potted conditions and correlate its responses in relation to field performance as because all the plants were grown in the same size pots and under same environmental conditions.  

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

The seedlings grown in field soil in polyethene sleeves were procured from the nursery of near by tea Garden of Durgakona and brought to the laboratory. The seedlings were potted after removing polyethene sleeves and adding field soil. The plants were acclimatized for 10-15 days in laboratory conditions and were grown under natural light with well irrigation.

As tea is a shade loving plant, the seedlings were grown in a shed where the intensity of light ranges from 150-300 mol m2 s1 and 200-400 mol m2 s1 inside and outside the shed respectively. All control and treated plants were kept in similar growth  conditions during acclimatization and treatment imposition.

After 10-15 days of acclimatization, drought is imposed by withholding water for 20 days. Well watered plant is considered as control.  The level of stress was quantified by measuring changes in soil moisture and RWC of leaf in stressed plant relative to control. After dehydration, soil moisture content decreased to (12.88 ± 1.34)% and (3.55 ± 0.28)% after 10 and 20d of stress imposition respectively relative to control  (23.46 ± 1.62)%. The average temperature range during experimental period was noted as 25.1 – 32.3°C and 12.5 – 24.7°C max/min respectively. The average relative humidity during the experiment period was 88-96% and 38-67% morning/afternoon respectively. All the leaf samplings were done during morning hours between 8 am to 9 am. 

Fresh mass of leaf was measured in three replicates using five leaves and expressed as g leaf -1. For dry mass measurement same leaves were oven dried at 80°C for 48 h and expressed as g leaf -1. The relative gain in leaf total fresh mass was determined as relative growth rate (RGR, [g d-1] of leaf or the increase in total leaf fresh mass per unit of existing mass per unit time), and calculated according to the formula:

 

 

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

Tea leaves were sampled, oven dried and  digested in a HNO3-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 by determining leakage of electrolytes from leaf discs placed in 20 ml of deionised water for 24 h at room temperature and measuring the electrical conductivity before and after autoclaving the samples and Electrolytic leakage was determined as described by Dionisio Sese and Tobita (1998) and calculated using formula.
 

EL = EC1/EC2 X 100
Where - EC1   is the initial conductivity of the sample before autoclaving and EC2 is the final conductivity measured after autoclaving the sample.
 
Leaves were extracted in cold with 80% acetone. The chlorophyll and carotenoid contents were estimated as per the methods of Arnon (1949).

Proline concentration in tea leaves was determined following the method of Bates et al. (1973). Leaf sample (0.5 g) was homogenized with 5 ml of sulfosalicylic acid (3%) using mortar and pestle and filtered through Whatman No.1 filter paper. The volume of filtrate was made upto 10 ml with sulfosalicylic acid and 2.0 ml of filtrate was incubated with 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 toluene were added and after cyclomixing it, absorbance was read at 570 nm. Total phenolics were extracted from tea leaves in 80% (v/v) ethanol and were estimated as per the method of Mahadevan and Sridhar (1982) using Follin Ciocalteau reagent and Na2CO3

Lipid peroxidation was measured as the amount of TBARS determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer (1968). The leaf tissues (0.2 g) were homogenised in 2.0 ml of 0.1% (w/v trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000g for 20 min. To 1.0 ml of the resulting supernatent, 1.0 ml of TCA (20%) containing 10.5% (w/v) of TBA and 10 L (4% in ethanol) BHT (butylated hydroxytolune) were added. The mixture was heated at 95°C for 30min in a water bath and then cooled in rice. The contents were centrifuged at 10,000 g for 15 min and the absorbancy was measured at 532 nm and corrected for 600 nm. The concentration of MDA were calculated using extinction coefficient of 155 mM-1cm-1.

Leaf tissues were homogenized with potassium phosphate buffer pH 6.8 (0.1M) containing 0.1 mM EDTA, 1% PVP and 0.1 mM PMSF in prechilled mortar pestle. The extract was centrifuged at 40C for 15 min at 17000 g in a refrigerated cooling centrifuge. The supernatant was used for the assay of polyphenol oxidase (PPO). PPO were assayed using pyrogallol as substrate in  1.0 ml of enzyme extract.  according to Kar and Mishra (1976). After incubations at 25°C for 5 min, the reaction was stopped with additions of 1.0 ml of 10 % H2SO4. The purpurogallin formed was read at 430 nm. 1 unit of enzyme activity is defined as that amount of enzyme which forms 1 u mole of purpurogallin formed per minute under the assay conditions. Total soluble protein content was estimated as per the method of Bradford (1976) using BSA as standard.

Nitrate Reductase (NR) activity in tea leaves was extracted and estimated as per the methods of Singh and Mallik (1980). The enzyme extracted in 0.1M phosphate buffer (pH7.2) and the homogenate was centrifuged in cooling centrifuge at 10,000 g at -4°C. The resulting supernatant was used for the assay of enzyme activity. The assay mixture comprised of 1.0 ml phosphate buffer (0.1M) (pH7.2), 0.5 ml NADH (1.5 mM), 0.5 ml distilled water and 1.0 ml enzyme extract. It is then incubated at 25°C. The enzyme reaction 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 the supernatant was retained. To this 2.0 ml [1% sulfanilamide in 1.5 N HCl and 0.2% N-naphthalene diamine hydrochloride (NEDH)], mixed in equal volume was added. After 10 min absorbency was read at 540 nm. NR activity was expressed as moles NO2 min-1.g-1 FW. 


Each experiment was done in triplicate and repeated thrice and data presented are mean standard error (SE). The results were subjected to ANOVA using GLM factorial model on all the parameters. Tukey test was used for comparision between pairs of treatments. For relationship between relative water content and proline 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 total chlorophyll content a linear regression was performed. The data analysis were carried out using statistical package SPSS 7.5.

 

 
Figure 1 Changes in solute leakage, relative growth rate(RGR) of leaf  and relative water content(RWC) 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). Different lower case letters indicates differences between drought treatment in the same variety, while * indicates  differences within the varieties at P< 0.05 according to multiple comparision  by  Tukey test.

 

3 Results 

Relative growth rate (RGR) of leaf showed uniformly decline trend with progressive soil moisture stress in all the tested clones (Figure 1B). Changes in RGR of leaf due to10 d of  stress  imposition was minimum in TV-30 and TV-1 but after 20d of stress RGR changes among  the clones was not significant though decrease growth was about 95%.The amount of solute leakage is an index of membrane damage. Drought induced increase in solute leakage in all the tested clones of Camellia sinensis (Figure 1A). After 20d of stress, in comparison with control plants solute leakage 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 comparatively lesser amount of solute leakage during stress conditions. Relative water content (RWC) of leaf uniformly decreased with decreasing soil moisture content imposed by 20 d of water withholding (Figure 1C). After 20 d of drought, RWC of leaf was found to be 56.26±1.21, 52.87±1.08, 44.99±0.43 and  52.66±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 of water stress imposition, apparently showing highest Na+ content in TV-29 (41.73%) with lowest in TV-1 as a result of 20 d of drought stress in comparision with control plants (Figure 2A). On the other hand, water stress induced decrease in K accumulation was observed in leaves of Camellia sinensis , but highest K+  content was maintained by TV-1 (27.16%) and TV-29 (13.85%) even after 20 d of drought imposition as depicted in Figure 2B. There was  increase in Na+/ K+ ratio with  progressive days of water withholding 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 the increasing intensity of drought stress (Figure 2C).

 

 
Figure 2 Changes in Na+, K+ content and Na+/ K+ ratio 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 significant differences are shown as in (Figure 1)

 

Photosynthetic pigments (chlorophyll and carotenoid) content was found to be decreased with increasing days of stress imposition. Drought induced degradation of chlorophyll and carotenoid was maximum in TV-29 (57.91 & 82.38%) and TV-20 (55.10 & 86.13%) respectively as depicted in Figure 3A & B. Phenolic compounds are widely distributed in plants and are mainly produced to protect plants from stress, ROS, wounds, UV light, disease and herbivores (Dixon and Paiva 1995). Total phenolics content in tea includes catechins and polyphenols, which was found to be decreased with the imposition of drought (Figure 3C). Maximum decrease in phenolics content was observed in TV-20(37.63%) and TV-2928.15%).

 

 
Figure 3 Changes in total chlorophyll, carotenoid and phenolic contents  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 significant differences are shown as in Figure 1

 

Increase in compatible solutes like proline, Glycine betain etc., is the characteristic feature of plants acclimatizing stress conditions. In this study proline accumulation during water stress condition was maximum in TV-1 (Figure 4A). Drought stress induced significant increase in PPO activity was observed in all the tested tea cultivars, maximum increase being shown by TV-29 (Figure 4B). Highest PPO activity was shown by TV-29 (458.82%) and TV-1 (424.91%). MDA content was maximum in TV-29 (420.41%) after 20 d of stress imposition, whereas TV-30(58.95%) showed minimum   increase as compared to control plant (Figure 4C). 
 

 
Figure 4 Changes in proline content, polyphenol oxidase (PPO) activity and MDA content  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 significant differences are shown as in Figure 1

 

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

 
Figure 5 Changes in nitrate reductase (NR) activity and total soluble protein content  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 significant differences are shown as in Figure 1.

 

The interesting aspect in this study is that there was no significant correlation between relative water content and changes in proline content of leaf (r2 = 0.15, ns) (Figure 6A). In our study, increase in Na+ content is followed by significant decrease in K+ content of leaf which is significantly correlated with decrease in RWC of leaf (r2=0.50, p<0.01) as depicted in Figure 6B. Concomitant with the increase in MDA content of leaf amount of solute leakage also increased which was evident from the significant correlation (r2=0.66, p<0.001)  between increase in lipid peroxidation and amount of solute leakage as shown in Figure 6C. Increase in MDA content is significantly correlated. with decrease in RWC of leaf (r2 = 0.39, p<0.05) as depicted in Figure 6D. There was significant correlation between chlorophyll degradation  and RGR of leaf (r2 = 0.40, p<0.05) (Figure 6E).  

 

 

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

 

4 Discussion
Drought imposition resulted in significant changes of growth and biochemical responses in various clones of Camellia sinensis. The contents of Na+ ion increased with the progress of water stress imposition, apparently showing highest Na+ content in TV-29 with lowest in TV-1 as a result of 20 d of drought stress in comparision with control plants. On the other hand, water stress induced  decrease in K accumulation was observed in leaves of Camellia sinensis , but highest K+ content was maintained by TV-1 even after 20 d of drought imposition. This is in contradiction with the findings of Osmond et al. (1980) and Martinez et al. (2003) who reported  involvement of Na+ and K+ accumulation in the osmotic adjustment of leaf tissues to low external water potential in Artiplex species during water stress. Sodium contribution to osmotic adjustment may be indirectly by triggering osmolyte synthesis independent of osmotic stress was however demonstrated by Subbarao et al. (2001). In our study, increase in Na+ content is followed by significant decrease in K+ content of leaf which is significantly correlated with decrease in RWC of leaf (r2 = 0.50, p< .01) as depicted in Figure 6B. K+  nutrition in plants  is  known to enhanced drought resistance , water use efficiency  and growth under drought condition (Egilla et al., 2001). Thus it suggested adequate K+ fertilizer application of tea plant may facilitate osmotic adjustment improving its drought tolerance potential. There was increase in Na+/ K+ ratio with progressive days of water withholding in TV-20, TV-29 & TV-30, but with exception TV-1 showed decrease in  Na+/ K+  ratio with  the  increasing intensity of drought stress (Figure 2C). As K+  is the main ion regulating osmotic adjustment in leaves, increase in Na+/K+ ratio  suggested occurrence of osmotic stress in three tea cultivars (TV-20, TV-29 & TV-30) but exceptional decreased Na+/ K+ ratio in TV-1 is the indication of  its comparatively better drought acclimatizing potential.  Water deficit stress, often called drought affects  the growth and productivity  of plants growing in natural or agricultural  field conditions. In the present study, relative growth rate (RGR) of leaf showed uniformly decline trend with increasing drought stress. The growth of leaf is directly or indirectly related with the various metabolism dependent on its osmotic potential which is regulated by potassium content of leaf. The amount of solute leakage is an index of membrane damage. Drought induced increase in solute leakage in all the tested clones of Camellia sinensis. However, TV-1 and TV-30  showed comparatively  lesser amount of solute leakage during stress conditions. Drought induced membrane damage  could be due to  enhanced membrane lipid peroxidation which was evident from increased MDA content with the progress of stress imposition, observed in four clones of tea. MDA content was maximum in TV-29 after 20 d of stress imposition, whereas TV-30 showed minimum increase as compared to control plant. Concomitant with the increase in MDA content of leaf  amount of solute leakage also increased which was evident from the significant correlation (r2 = 0.66, p< .001) between increase in lipid peroxidation and amount of solute leakage as shown in Figure 6C. Thus, it apparently suggests that increase in leakage of solute could be due to drought induced lipid peroxidation of membrane disturbing the structural and functional integrity of cell causing ultimate reduction in growth of tea plant. Photosynthetic pigments (chlorophyll and carotenoid ) content was found to be  decreased with increasing days of stress imposition.Drought induced degradation of chlorophyll and carotenoid was maximum in TV-29 and TV-20 respectively. There was significant correlation between chlorophyll degradation and RGR of leaf (r2 = 0.40, p< .05). Decrease in chlorophyll and carotenoid in response to water stress was reported earlier (Upadhyaya and Panda, 2004). Such degradation of chlorophyll pigments may eventually decrease photosynthetic efficiency in plants which might be one of the potent causes of reduction in growth of plant. However, a very recent study also provides evidence for a significant positive relationship between transpiration efficiency and leaf chlorophyll concentration in plant (Sheshshayee et al. 2006). During prolonged periods of drought, the decrease in water availability for transport-associated processes leads to changes in the concentrations of many metabolites, followed by disturbances in amino acid and carbohydrate metabolism. For example, there is an increase in the synthesis of compatible solutes such as special amino acids (e.g. Proline), sugars and sugar-alcohols, and Gly betaine. Acclimation to drought requires responses that allow essential reactions of primary metabolism to continue and enable the plant to tolerate water deficits. In the complex interplay of natural conditions, simple water deficits are unlikely to occur, since they intrinsically affect the acquisition of essential nutrients such as N etc. The reduction of NO3− to NO2− catalyzed by NR is considered to be the rate-limiting step of N assimilation. NR activity is coordinated with the rate of photosynthesis Indeed, drought-induced N deficiency was found to limit recovery of photosynthesis in plants. In situations of water deprivation, maximal foliar extractable NR activity has been found to decrease in some cases (Foyer et al., 1998). In the present study, NR activity decreased  due to drought in four clones of tea, where minimum decrease  was observed in TV-1 and TV-30, that could be correlated with  stress induced decrease in total soluble protein content in tested clones of Camellia sinensis. Similar results were already reported in other plants (Foyer et al., 1998; Panda, 2002; Xu and Zhou, 2006). Increase in compatible solutes like proline, Glycine betain etc., is the characteristic feature of plants acclimatizing stress conditions. In this study proline accumulation during water stress condition was maximum in TV-1. Proline acts as an osmoprotectant and greater accumulation of proline in TV-1 suggested genotypic tolerance of tea to water deficit stress, as proline accumulation helps in maintaining the water relations, prevents membrane distortion and acts as a hydroxyl radical scavenger. Drought induced biochemical modifications and proline metabolism has also been studied in other plants (Sanker et al., 2007). The molecular mechanism of quenching of reactive oxygen  species  proline has also been well reviewed (Matysik et al., 2001). However, Wu et al., (2007) reported osmotic adjustment in Citrus seedling colonized by Glomus versiforme subjected to drought stress did not correlate with proline but with ions like K+, Ca2+, etc and carbohydrates. Thus, similarly K+ might play critical role in providing osmotic adjustment in response to drought stress in tea cultivars as evidenced by significant correlation between changes in endogenous K+ content and RWC of leaves in stress imposed tea cultivars (r2 = 0.50, p< 0.01). Also higher K+ content of TV-1 in stressed condition showed better drought tolerance in comparision with other clones. Relative water content (RWC) of leaf uniformly decreased with decreasing soil moisture content imposed by 20 d of water withholding (Figure 1C). The interesting aspect in this study is that there was no significant  correlation between relative water content and changes in proline content of leaf (r2 = 0.15, ns) (Figure 6A). 
 
Phenolic compounds are widely distributed in plants and are mainly produced to protect plants from stress, ROS, wounds, UV light, disease and herbivores (Dixon and Paiva, 1995). Total phenolics content in tea includes catechins and polyphenols, which was found to be decreased with the imposition of drought (Figure 3C). Tea polyphenols are mostly catechins which have potential antioxidant properties that makes tea a good health drink. Polyphenol oxidases (PPO) are widely distributed in the plant and play a role in oxygen scavenging and defense against stress (Esen, 1993; Shivishankar, 1988). The antioxidant activity of phenolic compounds is mainly due to their redox properties. The medicinal value of phenolics in human health is well documented (Caia et al., 2004; Tona et al., 2004 ). In this study, drought stress induced significant increase in PPO activity was observed in all the tested tea cultivars, maximum increase being shown by TV-29. PPO catalyses the O2-dependent oxidation of mono- and o diphenols to o-diquinones, where secondary reactions may be responsible for the defense reaction and hypersensitive response (Mayer and Harel, 1991) .Moreover, it is proposed that PPO activity may regulate the redox state of phenolic compounds and become involved in the phenylpropanoid pathway (Kojima and Takeuchi 1989; Nakayama et al., 2000). 
 
In conclusion, it can be said that drought induced a range of physiological and biochemical alterations causing membrane damage and loss in cellular functions ultimately leading to reduction in growth of one of the most important economic crop like tea. In comparision TV-1 showed better drought tolerance by maintaining higher endogenous K+ and proline content and a balance Na+/K+ ratio in leaves. Hence, the present result may provide insight into underlying mechanism of tea plant in response to soil drought at the juvenile stage. However, more investigation is required in the field at different plant growth stages in the future. It can be further confirmed by studying various physiological and biochemical responses of different tea cultivars in field conditions and conducting experiments to study the  responses of those cultivars to rehydration and post stress manipulation of soil and leaf nutrients which are already initiated at our laboratory.

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

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