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Transcript of 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
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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
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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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