variation of physiological and antioxidative responses

8/14/2019 Variation of physiological and antioxidative responses

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O R I G I N A L P A P E R

Variation of physiological and antioxidative responsesin tea cultivars subjected to elevated water stress followed

by rehydration recovery

Hrishikesh Upadhyaya Sanjib Kumar Panda Biman Kumar Dutta

Received: 27 June 2007 / Revised: 20 January 2008 / Accepted: 22 January 2008

Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2008

Abstract Water stress is a major limitation for plant

survival and growth. Several physiological and antioxida-tive 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 antioxi-

dants 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 activ-

ities 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

response to rehydration, thus proving its better recovery

potential. On the other hand, highest H2O2 and lipid per-oxidation 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 sin-

ensis can be arranged in the order as TV-30[TV-1[TV-

29[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 andgrowth. In many natural locations the shortage of water is

an important environmental factor limiting plant produc-

tivity, 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, molec-

ular and cellular as well as physiological levels.

Expression of variety of genes induced by these stresses

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

123

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DOI 10.1007/s11738-008-0143-9


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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 andChoudhury 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; Zgalla 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-), hydrogenperoxide (H2O2) and hydroxyl radicals (

_OH), are highly

reactive and in the absence of effective protective mech-

anism, 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 anti-

oxidative 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

Handique (1992), there is dearth of information of oxida-

tive 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; Po-spisilova and Batkova 2004). Therefore, the present

investigation was undertaken for understanding the mech-

anism 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 impor-

tant for crop yield. Drought tolerance in tea plant can be

assessed through some physiological, biochemical param-

eters under moisture stress and these parameters can be

used as selection criteria for drought tolerance breeding

programme of tea. Thus, we conclude by consideringphysiological 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 uni-

form 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 seed-

lings 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 esti-

mated as (mg/100 g DW): K, 54.37; Na, 55; Ca, 1945; B,

29.39).

After 1015 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

25.132.3C and 12.524.7C max/min, respectively. The

average relative humidity during the experiment period

was 8896% and 3867% in the morning and afternoon,

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respectively. All the leaf samplings were done during

morning hours between 8 a.m. to 9 a.m. All the experi-

ments were performed during JanJune, 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 dif-

ferences 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 105C 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 mea-

surement same leaves were oven dried at 80C for 48 h and

expressed as g leaf-1. Relative water content (RWC) was

measured by following the methods of Barrs and Weath-

erly (1962).

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 neutra-

lised with 0.5 ml of 0.5 M potassium phosphate buffer (pH

7.5). Total glutathione was measured by adding 1 ml neu-

tralized to a standard solution mixture consisting of 0.5 ml

of 0.1 M sodium phosphate buffer (pH 7.5) containing

EDTA, 0.2 ml of 6 mM 5,50

-dithio-bis (2-nitrobenzoic

acid), 0.1 ml of 2 mM NADPH and 1 ml of 1-U ml-1

yeast-GR Type III (Sigma Chemicals, USA). The change in

absorbance at 412 nm was followed at 25 2C 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 60C 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 fol-

lowing 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 to10 ml with sulfosalicylic acid and 2.0 ml of filtrate was

incubated with 2.0 ml glacial acetic acid and 2.0 ml nin-

hydrin reagent and boiled in a water bath at 100C 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

H2O2 was extracted in 5% trichloroacetic acid from tea

leaves using (0.2 g) fresh leaf samples. The homogenatewas used for the estimation of total peroxide content

(Sagisaka 1976). The tissue homogenate was centrifuged at

17,000g at 0C for 10 min. The reaction mixture contained

1.6 ml of the supernatant, 0.4 ml TCA (50%), 0.4 ml fer-

rous 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 centri-

fuged 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 hy-

droxytolune) were added. The mixture was heated at 95C

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 mate-

rials 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 phosphatebuffer, 1.0 ml of 10 mM hydroxyl amine hydrochloride and

1.0 ml of the supernatent plant extract. After incubation at

room temperature (25C) for 20 min, 1.0 ml of 17 mM

sulphanilanide and 1.0 ml of 7 mM a-napthyl were added.

After reactions at 25C, 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 4C for 15 min at 17,000g in a refrig-

erated 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 20C. 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 pyro-

gallol 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 incu-

bations at 25C 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 mix-

ture 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.

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 TrisHCI 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 mMnitroblue 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 fluo-

rescent lamps (Philips 20 W). By switching the light on

and off, the reaction mixture was illuminated and termi-

nated. 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, 5-

dithiobis-2 nitrobenzoicacid) in 0.01 M potassium phos-

phate 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 25C 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 pre-

sented are mean standard errors (SE). The results were

subjected to ANOVA and Tukey test was used for com-

parison 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 com-

pared 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 rehy-

dration, respectively, in rehydrated plants (Fig. 1).

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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%).

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

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 O2-

content was highest in TV-20 followed by TV-30, TV-1

and TV-29 (Fig. 2d). However, after rehydration treat-

ment 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 rehy-

dration 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 H2O2content in the same, consequently accelerating lipid

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

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

Clones Treatments Fresh mass

(mg leaf-1)

Dry mass

(mg leaf-1)

RWC (%) Proline

(lmol g-1 FW)

Total sugar

(mg g-1 FW)

Total phenolics

(lg g-1 FW)

TV-1 Control 910.83 28.01 300.5 32.93 90.15 1.24 4.56 0.78 14.24 0.73 5466.36 39.71

10D 593.13 23.33a 213.68 11.75a 77.53 3.62a 5.58 0.20 13.46 0.28 5198.59 43.34a

20D 438.53 49.94a

139.78 20.37a

52.26 1.21a

10.69 1.48a

13.37 0.18a

4049.08 37.11a

TV-20 Control 720.58 27.06 181.29 15.25 92.21 2.83 1.08 0.03 11.33 0.37 5464.81 43.78

10D 674.3 23.25a

176.62 19.47 65.21 7.81a

2.26 0.09 10.00 0.53 4041.24 36.52a

20D 576.38 44.29a 171.81 9.63a 52.87 1.08a 3.11 0.08a 8.61 0.38a 3408.49 50.39a

TV-29 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 520.01 55.27a 210.81 20.96a 44.99 0.43a 3.68 0.26a 9.80 0.60a 3159.77 51.49a

TV-30 Control 822.27 40.38 307.19 18.96 86.64 4.92 .547 0.01 12.64 0.97 5315.02 37.37

10D 20D 660.83 31.49a 287.38 38.01 64.20 6.79a 2.17 0.29 11.56 0.35 4497.73 34.86a

548.09 19.59a 270.97 11.57a 40.66 1.03a 3.43 0.29a 8.76 0.65a 4196.74 57.12a

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

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

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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 mini-

mum 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 recov-ered 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. TV-

1 (134.43%) showed highest proline content with maxi-

mum after 20 days of drought, whereas TV-20 (109.26%)

showed lowest content (Table 1). However, during

recovery, proline contents were maintained almost same

levels as that of the control, which indicated the impor-

tance 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 (ascorbateand 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

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 Treatments Fresh mass

(mg leaf-1)

Dry mass

(mg leaf-1)

RWC (%) Proline

(lmol g-1 FW)

Total sugar

(mg g-1 FW)

Total phenolics

(lg g-1 FW)

TV-1 Control 438 49.9 139.78 20.4 52.26 1.2 10.69 1.5 13.37 0.2 4049.08 37.1

PDRI 210 5.7a 69.30 5.7a 80.98 5.8a 1.41 0.1 a 2.50 0.1 a 7699.44 114.9 a

PDR II 319

12.1

a

105.27

5.7

a

81.45

5.8

a

1.53

0.2

a

3.19

0.1

a

7713.19

114.9

a

PDR III 380 12.1a 125.40 5.7a 91.22 5.7a 1.56 0.1a 4.46 0.7a 7910.28 119.5a

TV-20 Control 576.38 44.3 171.81 9.6 52.87 1.2 3.11 0.1 8.61 0.4 3408.49 50.4

PDRI 203 5.7a 66.99 5.7a 80.26 2.9a 0.99 0.1a 1.37 0.1a 6860.75 88.9a

PDR II 298 12.1a 74.50 5.7a 86.12 5.8a 1.28 0.2a 3.60 0.1a 7869.11 114.9a

PDR III 378 12.7a 94.50 5.7a 90.12 5.8a 1.42 0.1 3.86 0.1a 8258.57 114.8a

TV-29 Control 520.01 55.3 210.81 2 44.99 0.4 3.68 0.3 9.80 0.6 3159.77 51.5

PDRI 391 12.1a 144.87 7.2a 79.81 5.3a 0.84 0.1a 1.92 0.1a 7910.26 114.9a

PDR II 420 12.1a 155.4 7.2a 80.65 6.1a 1.05 0.1a 3.5 0.1a 8070.66 119.5a

PDR III 480 18.5a 177.6 7.2a 84.61 6.1a 1.08 0.03a 3.64 0.1a 8817.69 119.5a

TV-30 Control 548.09 19.6 270.97 11.6 40.66 1.0a

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

PDR III 394 12.1a

145.78 7.2a

89.01 5.8a

1.36 0.1 7.07 0.7a

8716.86 119.5a

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

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

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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. TV-

20(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 treat-

ments 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 TV-

30 (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 sim-

ilar trend, except for TV-29 where PPO activities decreased

with the progress of rehydration treatments (Fig. 4b).

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

ofCamellia sinensis (TV-1, TV-

20, 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

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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 TV-

30) 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 ofCamellia

sinensis (TV-1, TV-20, TV-29and TV-30) subjected to post-

drought 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

Acta Physiol Plant

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9/12

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, suggestingphotosynthetic arrest in almost all the tested clones, but it

was not able to induce permanent damage to photosyn-

thetic 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 indi-

cates 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 TV30 of PDR III treatments. Prolineaccumulation in response to drought stress was maximum

in TV-1 and minimum in TV-20. Such proline accumula-

tion 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 osmo-

protectant and greater accumulation of proline in TV-1

suggested genotypic tolerance of tea to water deficit stress

as proline accumulation helps in maintaining water rela-

tions, 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 rehy-

dration, endogenous proline content was optimized and

plant osmotic potential might be regulated by net accu-mulation of other carbohydrates and ionic solutes as

reported by Wu et al. (2007) in citrus.

H2O2 and other active oxygen species OH,1O2 and O2

-

are known to be responsible for lipid peroxidation (Douglas

1996) and oxidative damage leading to disruption of met-

abolic 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 betteradaptation 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 respon-

sible for most of the plain black tea attributes. Catechins

are the most abundant polyphenols present in tea 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 TV-

30) 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

Acta Physiol 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 aresubjected 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 antioxi-

dant 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 max-

imum in TV-1 with least in TV-30, the post droughtrecovery (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, syn-

thesis of antioxidants like ascorbate and glutathione,

though induced by the water stress as a means of adapta-

tion, 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 anti-

oxidant 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. Glu-

tathione is widely used as a marker of oxidative stress to

plants, although its role in plant metabolism is a multi-

faceted 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 seques-

tration 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

with the increasing water stress in all the clones except TV-

1. Increase in SOD activities in stressed plants was indic-

ative 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. How-

ever, decrease in SOD activities after rehydration in tea was

reported earlier (Upadhyaya and Panda 2004b). Decrease inSOD 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 over-

coming 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 hydro-

gen peroxide formed in the photorespiratory pathway and

thereby reduced photorespiration rate (Jeyaramraja et al.

2003). Considering this fact, comparatively higher CATactivities 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 com-

ponent 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 o-

diquinones, 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 phenyl-

propanoid 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 differ-ential recovery potential during rehydration observed in the

tested clones can be arranged in order of TV-30[TV-

1[TV-29[TV-20.

Conclusion

In conclusion, it is assuemed that decrease in non-enzy-

mic 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 stressedplant 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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