Variation Of Physiological And Antioxidative Responses

Acta Physiol Plant DOI 10.1007/s11738-008-0143-9

ORIGINAL PAPER

Variation of physiological and antioxidative responses in tea cultivars subjected to elevated water stress followed by rehydration recovery Hrishikesh Upadhyaya Æ Sanjib Kumar Panda Æ Biman Kumar Dutta

Received: 27 June 2007 / Revised: 20 January 2008 / Accepted: 22 January 2008 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2008

Abstract Water stress is a major limitation for plant survival and growth. Several physiological and antioxidative mechanisms are involved in the adaptation to water stress by plants. In this experiment, tea cultivars (TV-1, TV-20, TV-29 and TV-30) were subjected to drought stress by withholding water for 20 days followed by rehydration. An experiment was thus performed to test and compare the effect of dehydration and rehydration in growing seedlings of tea cultivars. The effect of drought stress and post stress rehydration was measured by studying the reactive oxygen species (ROS) metabolism in tea. Water stress decreased nonenzymic antioxidants like ascorbate and glutathione contents with differential responses of enzymic antioxidants in selected clones of Camellia sinensis indicating an oxidative stress situation. This was also apparent from increased lipid peroxidation, O2- and H2O2 content in water stress imposed plants. But the oxidative damage was not permanent as the plants recovered after rehydration. Comparatively less decrease in antioxidants, higher activities of POX, GR, CAT with higher phenolic contents suggested better drought tolerance of TV-1, which was also visible from the recovery study, where it showed lower ROS level and higher recovery of antioxidant property in

Communicated by W. Filek. H. Upadhyaya (&)
S. K. Panda Plant Biochemistry and Molecular Biology Laboratory, School of Life Sciences, Assam (Central) University, Silchar 788011, India e-mail: [email protected] B. K. Dutta Microbial and Agricultural Ecology Laboratory, Department of Ecology and Environmental Sciences, Assam (Central) University, Silchar 788011, India

response to rehydration, thus proving its better recovery potential. On the other hand, highest H2O2 and lipid peroxidation with decrease in phenolic content during stress in TV-29 suggested its sensitivity to drought. The antioxidant efficiency and biochemical tolerance in response to drought stress thus observed in the tested clones of Camellia sinensis can be arranged in the order as TV-30 [ TV-1 [ TV29 [ TV-20. Keywords Water stress
Rehydration
Antioxidant
Physiological
Camellia sinensis Abbreviations RWC Relative water content GR Glutathione reductase POX Peroxidase H2O2 Hydrogen peroxide MDA Malondialdehyde ROS Reactive oxygen species SOD Superoxide dismutase

Introduction Water stress is a major limitation on plant survival and growth. In many natural locations the shortage of water is an important environmental factor limiting plant productivity, which is often called drought. This hinders the metabolic processes of plant, which ultimately retards growth and yield (Araus et al. 2002). Several studies suggested that plants respond to different kinds of stress, including water stress or drought at biochemical, molecular and cellular as well as physiological levels. Expression of variety of genes induced by these stresses

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and the role of its products in stress tolerance, regulation of gene expression and stress signal transduction have also been demonstrated by many authors (Supronova et al. 2004; Neil and Burnett 1999). Biological mechanism of stress response in plant has also been well reviewed (Griffiths and Pary 2002; Shinozaki et al. 2002; Francois Tardieu 2003; Yordanav et al. 2003). Water stress induces changes in oxidative enzymes’ activity (Mukherjee and Choudhury 1981), water use efficiencies, growth, Na+ and K+ accumulation (Li 2000; Martinez et al. 2003; Medici et al. 2003) and antioxidant defense system in plants (Srivalli et al. 2003; Zgallaı¨ et al. 2006). Drought or water stress in plant is a physiologically complex phenomenon. The genetic mechanism of adaptive responses to drought stress in plant has also been reviewed. Drought-modulated genes (dr1,dr2 and dr3) have also been identified in Camellia sinensis L. (O) Kuntze (Sharma and Kumar 2005). Drought stress caused imbalance between the generation and quenching of reactive oxygen species (ROS). ROS, such as superoxide radicals (O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (_OH), are highly reactive and in the absence of effective protective mechanism, can seriously damage plants by lipid peroxidation, protein degradation, breakage of DNA and cell death (Hendry 1993; Thambussi et al. 2000). To cope with the increased ROS level plants possess well-developed antioxidative systems which are composed of non-enzymic defence such as ascorbate, glutathione, tocopherol, etc., and enzymic scavengers such as superoxide dimutases, peroxidases, glutathione reductase and catalases, etc. (Asada 1994; Jebara et al. 2005; Lei et al. 2007) Being a perennial crop, tea plant is subjected to different environmental stresses, drought being one of the important factors among them. Drought stress induces oxidative damage in tea plant and affects antioxidant systems, altering different physiological and biochemical processes (Upadhyaya and Panda 2004b; Jeyaramaraja et al. 2005) that cause significant crop loss. Antioxidant efficiency also varies in different clonal varieties of tea (Upadhyaya and Panda 2004a) and thus varies the responses to water stress in different clones of tea (Chakraborty et al. 2002). Understanding the physiological and biochemical effects of post drought rehydration in tea is equally important and will give better insight into the mechanism of drought stress responses and tolerance as well as recovery potential of the plant. In North East India, generally, tea plants suffer from drought during November to April. In this region irrigation is increasingly used as an insurance against drought to increase tea yield during this period. The influence of irrigation on the potential yield of tea in this region has also been studied (Panda et al. 2003). Though some adequate measures against drought have been suggested by

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Handique (1992), there is dearth of information of oxidative stress management in relation to drought acclimatization on various tea clones cultivated in this region. There are few studies on water stress effects and rehydration response (Upadhyaya and Panda 2004b; Kamoshita et al. 2004; Siopongco et al. 2006; Xu and Zhou 2007) and enhancement of recovery by hormone treatment and other methods (Vomacka and Pospisilova 2003; Pospisilova and Batkova 2004). Therefore, the present investigation was undertaken for understanding the mechanism of drought stress induced oxidative damage on dehydration and its recovery on rehydration in selected clone of Camellia sinensis L (O) Kuntze. The ability of varieties to recover and resume rapid growth following drought imposition and subsequent rehydration is important for crop yield. Drought tolerance in tea plant can be assessed through some physiological, biochemical parameters under moisture stress and these parameters can be used as selection criteria for drought tolerance breeding programme of tea. Thus, we conclude by considering physiological and antioxidative responses of tea plant that confer an adaptive advantage in drought and in recovery after rewatering and the implications for improvement and selection of better tea cultivars.

Materials and methods Plant material and growth conditions Four clonal varieties of Camellia sinensis L. (O) Kuntze (viz. TV-1, TV-20, TV-29 and 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 nearby tea Garden of Durgakona and were 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. The soil used contained 23.46% moisture content. The mineral content was estimated as (mg/100 g DW): K, 54.37; Na, 55; Ca, 1945; B, 29.39). After 10–15 days of acclimatization, drought was imposed by withholding water for 20 days. Well-watered plant was considered as control. After 20 days, plants were rehydrated. Sampling for recovery analysis was done after every 10 days of rehydration for 30 days. 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% in the morning and afternoon,

Acta Physiol Plant

respectively. All the leaf samplings were done during morning hours between 8 a.m. to 9 a.m. All the experiments were performed during Jan–June, 2004. For each experiment, four plants were used for each point and each experiment was repeated thrice.

Soil moisture content Soil moisture content was determined by noting the differences between fresh and dry mass of soil (100 g), expressed as percentage using gravimetric method (Gupta 1999) 100 g of soil is taken from the middle of the pot without disturbing root and oven dried at 105°C for 48 h. Gravimetric moisture content was determined as difference between fresh and dry mass of soil, expressed as percentage.

Fresh mass, dry mass and RWC 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. Relative water content (RWC) was measured by following the methods of Barrs and Weatherly (1962).

yeast-GR Type III (Sigma Chemicals, USA). The change in absorbance at 412 nm was followed at 25 ± 2°C until the absorbance reached 5 U. For the extraction and estimation of ascorbate, the method of Oser (1979) was used. The reaction mixture consisted of 2 ml 2% Na-molybdate, 2 ml .15 N H2SO4, 1 ml 1.5 mM Na2HPO4 and 1 ml tissue extract. It was mixed and incubated at 60°C in water bath for 40 min. Then it was cooled, centrifuged at 3,000g for 10 min and absorbance was measured at 660 nm.

Proline content 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 up to 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 toulene was added and after cyclomixing it, absorbance was read at 570 nm.

H2O2 and lipid peroxidation Total sugar and total phenolic content Total sugar and phenolics were extracted from tea leaves in 80% (v/v) ethanol. Total phenolics were estimated as per the method of Mahadevan and Sridhar (1982) using Follin Ciocalteau reagent and Na2CO3. Aliquots from 80% ethanol extract were taken for the estimation of the total soluble sugar by Anthrone reagent (Yoshida et al. 1972).

Extraction and assay of glutathione and ascorbate Glutathione was extracted and estimated as per the method of Griffith (1980). Leaf tissue was homogenised in 5% (w/v) sulfosalicylic acid and homogenate was centrifuged at 10,000 g for 10 min. The supernatent (1 ml) was neutralised with 0.5 ml of 0.5 M potassium phosphate buffer (pH 7.5). Total glutathione was measured by adding 1 ml neutralized to a standard solution mixture consisting of 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.5) containing 0 EDTA, 0.2 ml of 6 mM 5,5 -dithio-bis (2-nitrobenzoic acid), 0.1 ml of 2 mM NADPH and 1 ml of 1-U ml-1

H2O2 was extracted in 5% trichloroacetic acid from tea leaves using (0.2 g) fresh leaf samples. The homogenate was used for the estimation of total peroxide content (Sagisaka 1976). The tissue homogenate was centrifuged at 17,000g at 0°C for 10 min. The reaction mixture contained 1.6 ml of the supernatant, 0.4 ml TCA (50%), 0.4 ml ferrous ammonium sulphate and 0.2 ml potassium thiocyanate. The absorbance was then recorded at 480 nm. 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 ll (4% in ethanol) BHT (butylated hydroxytolune) were added. The mixture was heated at 95°C for 30 min in a water bath and then cooled in rice. The contents were centrifuged at 10,000g for 15 min and the absorbancy was measured at 532 nm and corrected for 600 nm. The concentration of MDA was calculated using extinction coefficient of 155 m M-1 cm-1.

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Superoxide anion The estimation of O2- was done as suggested by Elstner and Heupal (1976) by monitoring the nitrate formation from hydroxylamine with some modifications. The plant materials were homogenised in 3.0 ml of 65 mM phosphate buffer (pH 7.8) and centrifuged at 5,000g for 10 min. The reaction mixture contained 0.9 ml of 65 mM phosphate buffer, 1.0 ml of 10 mM hydroxyl amine hydrochloride and 1.0 ml of the supernatent plant extract. After incubation at room temperature (25°C) for 20 min, 1.0 ml of 17 mM sulphanilanide and 1.0 ml of 7 mM a-napthyl were added. After reactions at 25°C, 1.0 ml of diethyl ether was added and centrifuged at 1,500g for 5 min and absorbency was read at 530 nm. A standard curve with NO2 was established to calculate the production rate of O2 from the chemical reaction of O2 and hydroxylamine.

Extraction and estimation of enzyme activities Leaf tissues were homogenized with potassium phosphate buffer pH 6.8 (0.1 M) containing 0.1 mM EDTA, 1% PVP and 0.1 mM PMSF in pre-chilled mortar pestle. The extract was centrifuged at 4°C for 15 min at 17,000g in a refrigerated cooling centrifuge. The supernatant was used for the assay of the following: catalase (CAT), peroxidase (POX), polyphenol oxidase (PPO), superoxide dismutase (SOD), and glutathione reductase (GR).

Catalase, peroxidase and polynophenol oxidase activities Catalase activity was assayed according to Chance and Maehly (1955). The 5.0 ml mixture comprised of 3.0 ml phosphate buffer (pH 6.8), 1.0 ml (30 mM) H2O2, 1.0 ml enzyme extract. The reaction was stopped by adding 10 ml of 2% H2SO4 after 1 min incubation at 20°C. The acidified reaction mixture was titrated against .01 N KMnO4 to determine the quantity of H2O2 utilized by the enzyme. The CAT activity was expressed as lmole H2O2 destroyed min-1 g fr wt. POX and PPO were assayed using pyrogallol as substrate according to Kar and Mishra (1976) with minor modifications, 5.0 ml of assay mixture contained 300 lM H2O2 and 1.0 ml of enzyme extract. 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. For PPO assay reaction mixture was same except that H2O2 was not added. One unit of enzyme activity is defined as that amount of enzyme, which forms 1 lmol of purpurogallin formed per minute under the assay conditions.

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Superoxide dismutase and glutathione reductase activities The activity of SOD was measured using the method of Giannopolitis and Reis (1977). About 3.0 ml assay mixture for SOD contains 79.2 mM Tris–HCI buffer (pH 8.9), containing 0.12 mM EDTA and 10.8 mM tetra ethylene diamine, bovine serum albumin (3.3 9 10-3%), 6 mM nitroblue tetrazolium (NBT), 600 lM riboflavin in 5 mM KOH and 0.2 ml enzyme extract. Reaction mixture was illuminated by placing the test tubes in between two fluorescent lamps (Philips 20 W). By switching the light on and off, the reaction mixture was illuminated and terminated. The increase in absorbance due to formazan formation was read at 560 nm. The increase in absorbance in the absence of enzyme was taken as 100, and 50% initial was taken an equivalent to 1 unit of SOD activity. Glutathione reductase (GR) was assayed by the method of Smith et al. (1988). The reaction mixture contained 1.0 ml of 0.2 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 0.5 ml of 3 mM DTNB (5, 5dithiobis-2 nitrobenzoicacid) in 0.01 M potassium phosphate buffer (pH 7.5), 0.1 ml of 2 mM NADPH, 0.1 ml enzyme extract and distilled water to make up a final volume of 2.9 ml. Reaction was initiated by adding 0.1 ml of 2 mM GSSG (oxidised glutathione). The increase in absorbance at 412 nm was recorded at 25°C over a period of 5 min spectrophotometrically. The activity is expressed as absorbance change (DA412) g. fresh mass-1 s-1.

Statistical analysis Each experiment was repeated three times and data presented are mean ± standard errors (SE). The results were subjected to ANOVA and Tukey test was used for comparison between pairs of treatments. The data analyses were carried out using statistical package SPSS 7.5

Results Soil moisture content A significant decrease in gravimetric soil moisture content was observed. As a result of dehydration, soil moisture content decreased to 12.88 ± 1.34 and 3.55 ± 0.28 after 10 and 20 days of stress imposition, respectively as compared to control (23.46 ± 1.62). However, the average soil moisture content of 23.85 ± 1.73, 25.03 ± 1.09 and 25.21 ± 1.16 was maintained in all the pots after 10 days (PDRI), 20 days (PDRII) and 30 days (PDRIII) of rehydration, respectively, in rehydrated plants (Fig. 1).

Acta Physiol Plant

A decrease in fresh and dry mass of leaf was observed in all the stressed plants. Decrease in fresh mass was highest in TV-1 (51.85%) whereas TV-20 (20.01%) showed least decrease over control after 20 days of stress (Table 1). On rehydration, an increase in fresh mass was observed in all the tested clones with the progress of days of rehydration (Table 2).

ROS and lipid peroxidation

Fig. 1 Effect of drought and post-drought rehydration on soil moisture content. Well-watered pots (control), 10D and 20D (after 10 and 20 days of drought imposition), PDR I, PDR II and PDRIII (after 10, 20 and 30 days of rehydration in post-drought recovery phase when sampling of leaf was done). Soil from two pots for each clone was taken and the observation was repeated thrice. Data presented are mean with ±SE. To obtain mean pots with all the four types of clones were considered. *Significant mean difference from control at P = 0.05 was determined with multiple comparison by Tukey test

Growth and RWC of leaf A uniform decrease in RWC was observed as compared to control in all the tested clones of Camellia sinensis. Maximum decrease in RWC was observed in case of TV-30 (53.07%) after 20 days of stress imposition as compared to control, whereas TV-1(42.03%) showed less decrease (Table 1). After rehydration, plants recovered RWC and maintained highest content in TV-1 (91.22%).

Superoxide anion (O2-) generation in the plant increased with increased stress imposition. Increase in O2- content was highest in TV-1 (170.38%) followed by TV-29 (140.93%), TV-30 (109.85%) and TV-20 (66.7%) after 20 days of stress imposition when compared with control (Fig. 2d). But after 20 days of stress imposition O2content was highest in TV-20 followed by TV-30, TV-1 and TV-29 (Fig. 2d). However, after rehydration treatment O2- content decreased with maximum decrease in TV-1 followed by TV-30, TV-29 and TV-20 as compared to stressed plant (Fig. 4d). H2O2 content was high in all stressed plants, being highest in TV-29 (85.83%) and lowest in TV-1 with 38.77% increase (Fig 2e). On rehydration H2O2 content decreased in different recovery phases (PDR I, PDR II and PDR III) (Fig 4e). Lipid peroxidation measured in terms of MDA was higher in all the stressed plants after 10 and 20 days of drought imposition. MDA content was highest in TV-29 (420.41%), which could be attributed to the higher H2O2 content in the same, consequently accelerating lipid

Table 1 Changes in leaf fresh mass and dry mass, relative water content (RWC %), proline, total sugar and total phenolics content in four clonal varieties of Camellia sinensis subjected to drought Dry mass (mg leaf-1)

Clones

Treatments

Fresh mass (mg leaf-1)

TV-1

Control

910.83 ± 28.01

10D TV-20

TV-29

TV-30

a

593.13 ± 23.33

a

10D 20D

20D Control

RWC (%)

300.5 ± 32.93 a

213.68 ± 11.75

a

77.53 ± 3.62

Total sugar (mg g-1 FW)

Total phenolics (lg g-1 FW)

4.56 ± 0.78

14.24 ± 0.73

5466.36 ± 39.71

13.46 ± 0.28

5198.59 ± 43.34a

5.58 ± 0.20

139.78 ± 20.37 181.29 ± 15.25

a

52.26 ± 1.21 92.21 ± 2.83

10.69 ± 1.48 1.08 ± 0.03

674.3 ± 23.25a

176.62 ± 19.47

65.21 ± 7.81a

2.26 ± 0.09

a

576.38 ± 44.29

a

171.81 ± 9.63

a

52.87 ± 1.08

3.11 ± 0.08

438.53 ± 49.94 720.58 ± 27.06

a

90.15 ± 1.24

Proline (lmol g-1 FW)

a

a

a

13.37 ± 0.18 11.33 ± 0.37

4049.08 ± 37.11a 5464.81 ± 43.78

10.00 ± 0.53

4041.24 ± 36.52a

a

8.61 ± 0.38

3408.49 ± 50.39a

Control

760.11 ± 30.16

282.03 ± 10.62

87.57 ± 6.11

.784 ± 0.03

10.15 ± 0.80

4397.96 ± 37.86

10D

529.66 ± 26.94a

261.24 ± 27.15

74.89 ± 6.15a

2.69 ± 0.06

9.99 ± 0.91

4016.23 ± 19.06a

20D

a

a

a

a

9.80 ± 0.60

3159.77 ± 51.49a

.547 ± 0.01

12.64 ± 0.97

5315.02 ± 37.37

2.17 ± 0.29

11.56 ± 0.35

4497.73 ± 34.86a

520.01 ± 55.27

210.81 ± 20.96

44.99 ± 0.43

3.68 ± 0.26

Control

822.27 ± 40.38

307.19 ± 18.96

86.64 ± 4.92

10D 20D

660.83 ± 31.49a

287.38 ± 38.01

64.20 ± 6.79a

548.09 ± 19.59a

270.97 ± 11.57a

40.66 ± 1.03a

3.43 ± 0.29a

a

8.76 ± 0.65a

4196.74 ± 57.12a

Control plants were watered daily. 10D, 20D indicates 10 days and 20 days of drought imposition a

Indicates significant mean difference from control at P = 0.05 in multiple comparison by Tukey test

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Acta Physiol Plant Table 2 Changes in leaf fresh mass and dry mass, relative water content (RWC %), proline, total sugar and total phenolics content in four clonal varieties of Camellia sinensis subjected to Post-drought rehydration Clones TV-1

Treatments

Proline (lmol g-1 FW)

Total sugar (mg g-1 FW)

Total phenolics (lg g-1 FW)

13.37 ± 0.2

4049.08 ± 37.1

438 ± 49.9

139.78 ± 20.4

52.26 ± 1.2

10.69 ± 1.5

210 ± 5.7a

69.30 ± 5.7a

80.98 ± 5.8a

1.41 ± 0.1

a

a

Control

a

319 ± 12.1 380 ± 12.1a 576.38 ± 44.3 203 ± 5.7a 298 ± 12.1

a

PDR III

378 ± 12.7

a

Control

520.01 ± 55.3

PDR II

PDRI PDR II PDR III TV-30

RWC (%)

Control

PDRI

TV-29

Dry mass (mg leaf-1)

PDRI PDR II PDR III TV-20

Fresh mass (mg leaf-1)

Control

391 ± 12.1 420 ± 12.1

a

480 ± 18.5

a

a

a

7699.44 ± 114.9

a

a

81.45 ± 5.8 91.22 ± 5.7a

1.53 ± 0.2 1.56 ± 0.1a

3.19 ± 0.1 4.46 ± 0.7a

7713.19 ± 114.9 7910.28 ± 119.5a

171.81 ± 9.6

52.87 ± 1.2

3.11 ± 0.1

8.61 ± 0.4

3408.49 ± 50.4

66.99 ± 5.7a

80.26 ± 2.9a

0.99 ± 0.1a

1.37 ± 0.1a

6860.75 ± 88.9a

74.50 ± 5.7

a

86.12 ± 5.8

a

a

a

7869.11 ± 114.9a

94.50 ± 5.7

a

90.12 ± 5.8

a

a

3.86 ± 0.1

8258.57 ± 114.8a

9.80 ± 0.6

3159.77 ± 51.5

44.99 ± 0.4

144.87 ± 7.2

a

155.4 ± 7.2

a

177.6 ± 7.2

a

1.28 ± 0.2

3.60 ± 0.1

1.42 ± 0.1 3.68 ± 0.3

79.81 ± 5.3

a

0.84 ± 0.1

a

80.65 ± 6.1

a

1.05 ± 0.1

a

84.61 ± 6.1

a

1.08 ± 0.03

a

a

7910.26 ± 114.9a

a

8070.66 ± 119.5a

a

3.64 ± 0.1

8817.69 ± 119.5a

1.92 ± 0.1

3.5 ± 0.1 a

270.97 ± 11.6

40.66 ± 1.0

3.43 ± 0.3

8.76 ± 0.6

4196.74 ± 57.1

PDRI

315 ± 12.1a

116.55 ± 5.7a

80.02 ± 5.8a

1.06 ± 0.03a

3.67 ± 0.1a

7919.42 ± 114.9a

PDR II

346 ± 12.1a

128.02 ± 7.2a

82.80 ± 5.8a

1.28 ± 0.1a

5.59 ± 0.3a

8363.52 ± 119.5a

a

a

a

a

8716.86 ± 119.5a

PDR III

548.09 ± 19.6

a

2.50 ± 0.1

105.27 ± 5.7 125.40 ± 5.7a

210.81 ± 2 a

a

394 ± 12.1

145.78 ± 7.2

89.01 ± 5.8

1.36 ± 0.1

7.07 ± 0.7

Control plants (20 days of drought imposition); PDR I, PDR II and PDR III indicates 10, 20 and 30 days of rehydration a

Indicates significant mean difference from control at P = 0.05 in multiple comparision by Tukey test

peroxidation, whereas TV-30 showed 58.95% increase over control after 20 days of drought imposition (Fig 2f) which was minimum in comparison with other clones. Lipid peroxidation was decreased after rehydration. Among the PDR III plants, MDA content was lowest in TV-1 as depicted in Fig. 4f.

Total sugar and proline contents Water stress induced uniform decrease of total sugar content was observed in all tested clones of Camellia sinensis. The decrease in total sugar content was minimum in TV-29(3.45%) and TV-1(6.11%) followed by TV-20(24%) and TV-30(30.69%) as compared to control plant (Table 1). However, comparing with other clones, TV-1 maintained higher sugar content even after 20 days of stress imposition. But after rehydration, plants recovered sugar contents slowly, maximum recovery being shown by TV-30 (Table 2). Proline plays important role as osmoprotectant during water stress. An increase in proline content was observed in all the clones after water stress imposition as compared to well-irrigated plant. TV1 (134.43%) showed highest proline content with maximum after 20 days of drought, whereas TV-20 (109.26%) showed lowest content (Table 1). However, during recovery, proline contents were maintained almost same

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levels as that of the control, which indicated the importance of osmotic regulation for recovering growth in these plants (Table 2).

Total phenolics, ascorbate and glutathione contents Total phenolic content in tea leaves decreases with increasing water stress. The decrease in phenolic contents was maximum in TV-20 (37.63%) followed by TV-29 (28.15%), whereas TV-30 (21.04%) and TV-1 (25.92%) showed minimum decrease over control after 10 and 20 days of water stress (Table 1). On rewatering, the phenolic content of stressed plant was increased. Increase in phenolic contents due to rehydration was maximum in TV-29 followed by TV-30, TV-20 and TV-1 in PDR III plants (Table 2). Increasing water stress resulted in a significant decrease of non-enzymic antioxidant (ascorbate and glutathione) content in all the clonal seedlings of tea. Decrease in ascorbate content was maximum in TV-1 (39.17%) with minimum content in TV-30 (7.29%) (Fig. 3a) compared to control. Ascorbate content initially decreased and then increasing trend was observed with the progressive rehydration treatments. Glutathione decreases to its maximum in TV-30 (50.14%) and TV-1 (47.75%). Comparatively, glutathione content was highest in TV-1 and TV-20, which was maintained even after stress

Acta Physiol Plant Fig. 2 Changes in superoxide dismutase (SOD) (a), polyphenol oxidase (PPO) (b), Peroxidase (POX) (c), activities, superoxide anion (O2-) (d), peroxide (H2O2) (e), and malondialdehyde (MDA) (f). Content in four clonal varieties of Camellia sinensis (TV-1, TV20, TV-29 and TV-30) subjected to drought control (open rectangle). About 10 days of drought (thin shaded rectangle) 20 days of drought (darkly shaded rectangle) imposition. Data presented are mean ± SE (n = 3). *Significant mean difference from control at P = 0.05 in multiple comparison by Tukey test

imposition (Fig. 3b). The glutathione content varied within clones in response to rehydration. Glutathione significantly increased in PDR III plants only for TV-30 (Fig. 5b).

Antioxidant enzymes SOD activity decreased in TV-1(26.78%) with increasing water stress, whereas other clones [TV-29(51.98%) and TV-30(68.14%)] showed an increase in SOD activity. TV20(57.73%) showed highest SOD activity after 10 days of water stress (Fig. 2a). Rehydration caused decrease in SOD activities when compared with stressed plants, but TV-29 showed increase SOD activities in PDR III plants (Fig. 4a). There was a significant increase in GR activity in all the tested clones subjected to stress condition. Increase in GR activity in stressed plant was maximum in TV-1 (579.04%) and TV-29 (373.01%), followed by TV-30 (298.23%) and

TV-20 (278.01%) (Fig. 3c). Post-stress rehydration treatments showed drastic decrease in GR activities in all the tested clones (Fig. 5c). POX activity was increased in the stressed plant as compared to control after 10 and 20 days of dehydration, with maximum activity in TV-1 (951.98%) and TV-20 (489.63%) after 20 days of stress imposition, while in TV30 (340.71%) and TV-29 (448.17%) POX activity was lower (Fig. 2c). PPO is also one of the important enzymes that have potent role in tea phenol metabolism. The activity of this enzyme was found to be increased with increasing dehydration stress in almost all the tested clones, in the order of TV-29 (458.82%) [TV-1 (424.91%) [ TV-30 (206.51%) [ TV-20 (95.37%) (Fig. 2b). With the increasing duration of rehydration, POX activities increased with maximum POX activities shown by TV-1 in PDR III plants (Fig. 4c). PPO activities also showed similar trend, except for TV-29 where PPO activities decreased with the progress of rehydration treatments (Fig. 4b).

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Acta Physiol Plant Fig. 3 Changes in ascorbate (a), glutathione (b) content and activities of glutathione reductase (GR) (c) and catalase (CAT) (d) in four clonal varieties of Camellia sinensis (TV-1, TV-20, TV-29 and TV30) subjected to drought control (open rectangle); 10 days of drought (thin shaded rectangle); 20 days of drought (darkly shaded rectangle) imposition. Data presented are mean ± SE (n = 3). *Significant mean difference from control at P = 0.05 in multiple comparison by Tukey test

Fig. 4 Changes in superoxide dismutase (SOD) (a), polyphenol oxidase (PPO) (b), Peroxidase (POX) (POX) activities superoxide anion (O2-) (d), total peroxide (H2O2) (e), and (MDA) (f) content in four clonal varieties of Camellia sinensis (TV-1, TV-20, TV-29 and TV-30) subjected to postdrought rehydration. Control (filled rectangle). [20 days of drought]; PDRI (mesh filled rectangle). [10 days of rehydration], PDR II(thin shaded rectangle) [20 days of rehydration]; PDR III (open and filled rectangle) [10 days of rehydration]. Data presented are mean ± SE (n = 3). *Significant mean difference from control at P = 0.05 in multiple comparison by Tukey test

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Acta Physiol Plant Fig. 5 Changes in ascorbate (a), glutathione (b) content and activities of glutathione reductase (GR) (c) and catalase (CAT) (d) in four clonal varieties of Camellia sinensis (TV-1, TV-20, TV-29 and TV30) subjected to post-drought rehydration. Control (filled rectangle) [20 days of drought]; PDR I (mesh filled rectangle) [10 days of rehydration], PDR II (thin shaded rectangle) [20 days of rehydration]; PDR III (open and filled rectangle) [10 days of rehydration]. Data presented are mean ± SE (n = 3). *Significant mean difference from control at P = 0.05 in multiple comparison by Tukey test

Discussion RWC of leaves decreased in all the cultivars due to drought but decrease in RWC was least in TV-1. Maintenance of high RWC in drought-resistant cultivars has been reported to be an adaptation to water stress in several crop species (Farooqui et al. 2000). However, after rehydration, RWC gradually increased to pre-stress level. Fresh and dry mass of leaves decreased with increasing stress, suggesting photosynthetic arrest in almost all the tested clones, but it was not able to induce permanent damage to photosynthetic system. After rehydration, growth resumed in plants and photosynthetic activity started. Such photosynthetic recovery of the plant after different period of soil drought has also been reported recently (Xu and Zhou 2007). Decreased total sugar content in stressed plants also indicates loss of photosynthetic rate due to drought, with least decrease in TV-1 and TV-30 comparatively, suggesting better stress tolerance in these clones. Total sugar content slowly increased with the progress of rehydration showing maximum content in TV–30 of PDR III treatments. Proline accumulation in response to drought stress was maximum in TV-1 and minimum in TV-20. Such proline accumulation in response to water deficit stress was reported in wheat (Kathju et al. 1988; Levitt 1980) and in tea (Handique and Mannivel 1990). 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 water relations, prevents membrane distortion and acts as a hydroxyl

radical scavenger (Yoshiba et al. 1997; Matysik et al. 2002). Osmotic adjustment involves the lowering of the osmotic potential due to a net solute accumulation in response to drought stress (Chimenti et al. 2006). Thus, a high proline level might help plant to survive drought stress and recover from stress. However, with progressive rehydration, endogenous proline content was optimized and plant osmotic potential might be regulated by net accumulation of other carbohydrates and ionic solutes as reported by Wu et al. (2007) in citrus. H2O2 and other active oxygen species OH, 1O2 and O2are known to be responsible for lipid peroxidation (Douglas 1996) and oxidative damage leading to disruption of metabolic function and loss of cellular integrity at sites where it accumulates (Foyer et al. 1997). In our study, O2-, H2O2 and lipid peroxidation were increased in all the stressed plants indicating loss of membrane function and induction of oxidative damage. Increase in O2-, H2O2 content and lipid peroxidation, as a consequent of stress imposition was least in TV-1, which could be attributed to its better adaptation in comparison with other tested clones. Better stress tolerance and recovery of TV-1 and TV-30 was also supported by comparatively minimum ROS level and lipid peroxidation after rehydration. The important biochemicals in determining tea quality include the green leaf tea catechins and their oxidation products (theaflavins and thearubigins), which are responsible for most of the plain black tea attributes. Catechins are the most abundant polyphenols present in tea plant,

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which makes it a potent health drink. Decrease in total phenolic contents in tea cultivars in response to water stress with simultaneous decrease in glutathione and ascorbate content suggested not only the gradual loss of protection of tea seedling to overcome a drought-induced oxidative damage as reported in other plant (Dixon and Steele 1999; Battle and Munne Bosch 2003) but also decrease in quality of tea growing in drought prone areas. When plants are subjected to drought stress, it is characterized by an increase in the level of ROS, expression of antioxidant genes and activities of antioxidant system meant for ROS scavenging and these parameters result in tolerance against drought (Mano 2002). Though many stress conditions cause an increase in the total foliar antioxidants, little is known of the coordination and control of various antioxidant enzyme activities in plants, especially tea, under drought stress and during post-stress recovery period. Water stress disrupts the non-enzymic antioxidant system in plants. Though decrease in ascorbate content was maximum in TV-1 with least in TV-30, the post drought recovery (PDR) study with rehydration showed a rapid recovery in TV-1 and TV-30 owing to the highest content of the same. However, minimum decrease in glutathione content in response to water stress was observed in TV-1, which maintained the highest glutathione content during recovery process. Thus it apparently indicates that, synthesis of antioxidants like ascorbate and glutathione, though induced by the water stress as a means of adaptation, has some potent role to play during post-stress recovery as evidenced by the slow increase of the same with progressive rehydration. The role of these antioxidants in regulating active oxygen species has also been well reviewed (Noctor and Foyer 1998). Ascorbate is a key substance in the network of antioxidants that include ascorbate, glutathione, a-tocopherol, and a series of antioxidant enzymes. Ascorbate has also been shown to play multiple roles in plant growth, such as in cell division, cell wall expansion, and other developmental processes. Glutathione is widely used as a marker of oxidative stress to plants, although its role in plant metabolism is a multifaceted one. As it is a nonprotein sulphur-containing tripeptide, glutathione acts as a storage and transport form of reduced sulphur. Glutathione is related to the sequestration of xenobiotics and heavy metals and is also an essential component of the cellular antioxidative defense system, which keeps ROS under control. Antioxidative defence and redox reactions play a central role in the acclimation of plants to their environment, which made glutathione a suitable candidate as a stress marker. The dismutation of superoxide is catalysed by SODs, which are ubiquitous enzymes and constitute forefront in ROS defense and overproduction of chloroplast SODs is known to enhence stress tolerance. SOD activities increased

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with the increasing water stress in all the clones except TV1. Increase in SOD activities in stressed plants was indicative of enhanced O2- production and oxidative stress tolerance (Asada and Takahaslin 1987). Increase in SOD activities after rehydration during recovery period could be an adaptation to improve growth after rehydration. However, decrease in SOD activities after rehydration in tea was reported earlier (Upadhyaya and Panda 2004b). Decrease in SOD with increasing days of rehydration in few tested clones could be due to decrease in O2- generations. In this study, CAT appeared to be an important enzyme in overcoming drought stress imposed oxidative stress as there has been an increase in CAT activities in stressed plants. The ability of tea clones to enhance the CAT activity with increasing stress indicates that this enzyme could be the first line of defense during drought adaptation process. As tea is a C3 plant, higher CAT activity could scavenge the hydrogen peroxide formed in the photorespiratory pathway and thereby reduced photorespiration rate (Jeyaramraja et al. 2003). Considering this fact, comparatively higher CAT activities with lower O2- content and lipid peroxidation in PDR III, TV-1 showed better recovery potential. Increased GR activity in stressed clones with a maximum in TV-1 facilitates improved stress tolerance of TV-1 and has the ability to alter the redox poise of important component of the electron transport chain. Glutathione is maintained in a reduced state by GR. Increase in GR activities do not influence the glutathione content and so it seems that GSH content may be merely dependent on the synthesis, export and degradation of glutathione itself than by recycling of GSSG via GR activity (Foyer et al. 1991). However, lower GR activity after rehydration could be due to tendency of the plants to acclimatize (Loggini et al. 1999). This finding also indicates that increase in GR activities is more concerned with acclimatization during stress rather than influencing much the stress recovery process. Increase in POX and PPO activities in almost all the stressed clones could be an acclimatization step against the stress. The role of POX in oxidation of tea catechins to form theaflavin-type compounds in presence of H2O2 has been reported earlier (Sang et al. 2004). PPO plays important role in the production of theaflavins in tea. PPO is widely distributed in plants and plays a role in oxygen scavenging and defense against stress. PPO catalyses the O2- dependent oxidation of mono- and o-diphenols to odiquinones, where secondary reactions may be responsible for the defense reaction and hypersensivity response. Notably, PPO activity increased in our study suggesting its defensive response against drought stress. Moreover, it is proposed that PPO activity might regulate the redox state of phenolic compounds and become involved in phenylpropanoid pathways and thereby play an important role in phenol metabolism.

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Summarizing the findings, it can be said that imposed drought caused oxidative damage in tea plant, resulting in the decrease of its antioxidant potential with various physiological and biochemical alterations. Such damages were not permanent as the resumption of growth and physiological processes were observed after post-drought rehydration. The variation of antioxidant efficiency and biochemical tolerance in response to drought with differential recovery potential during rehydration observed in the tested clones can be arranged in order of TV-30 [ TV1 [ TV-29 [ TV-20.

Conclusion In conclusion, it is assuemed that decrease in non-enzymic antioxidant with differential response of enzymic antioxidant under drought stress in various clones of Camellia sinensis caused oxidative damage. Increase in antioxidant enzymes like SOD, CAT and GR in stressed plant throws light on the different role of each enzyme in the drought adaptation process. However, rehydration recovery showed differential response in activating and enhancing the coordinated antioxidant defense system in plant to recover and resume growth after rehydration. During the drought acclimatization as well as during the recovery process POX, PPO and CAT activities seem to play important role in resuming normal growth of the tea plant. Such study will help to understand the drought tolerance potential of various clones of tea plant better. In this process some of them can be recommended for growing in drought-prone areas and in particular, for the benefit of the tea industry at large. Acknowledgments 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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