Responses of Camellia sinensis to Drought and Rehydration

Abstract

The effects of drought and rehydration on tea seedlings were significant. After five days of drought imposition the contents of chlorophylls, carotenoids, ascorbate and glutathione, and activities of guaiacol peroxidase and glutathione reductase decreased. Simultaneously, contents of proline, H2O2 and superoxide anion, lipid peroxidation and activities of catalase and superoxide dismutase increased. These parameters recovered to different degrees during subsequent rehydration.

Full text

BIOLOGIA PLANTARUM 48 (4): 597-600, 2004
597
BRIEF COMMUNICATION
Responses of Camellia sinensis to drought and rehydration
H. UPADHYAYA* and S. K. PANDA**,1
Plant Biochemistry Laboratory, Department of Life Science, Assam (Central) University, Silchar-788011, India*
Research Institute for Bioresources, Okayama University, Kurashiki-7100046, Japan**
Abstract
The effects of drought and rehydration on tea seedlings were significant. After five days of drought imposition the
contents of chlorophylls, carotenoids, ascorbate and glutathione, and activities of guaiacol peroxidase and glutathione
reductase decreased. Simultaneously, contents of proline, H2O2 and superoxide anion, lipid peroxidation and activities of
catalase and superoxide dismutase increased. These parameters recovered to different degrees during subsequent
rehydration.
Additional key words: ascorbate, carotenoid, catalase, chlorophyll, glutathione, guaiacol peroxidase, superoxide anion, superoxide
dismutase.

Tea plant being perennial shrubs can grow under diverse
climatic conditions and is always subjected to
environmental stress. Plant may suffer either from
excessive soil moisture or moisture deficit. Drought being
an important limitation for plant impairs severely growth,
crop yield and various morphological, anatomical,
physiological and biochemical processes (Kefei et al.
1997, Egert and Tevini 2002). The drought resistance
mechanisms can be categorised as 1) drought avoidence,
2) dehydration tolerance, and 3) dehydration postpone-
ment (Kramer and Boyer 1995). Plant may perceive
osmotic adjustment as a survival mechanism, which
enable physiological activity to be maintained at a lower
level throughout a period of water deficit (Turner 1997).
Drought tolerance mechanisms have been compared in
the clones of different plant species (e.g. in Coffea
canephora; DaMatta et. al. 2003). The post drought
recovery by the plant is also a subject of much concern.
Drought is known to cause oxidative damage in plants
as a result of production of reactive oxygen species
(ROS) like, superoxide radical, hydroxyl radical,
hydroperoxide radical, alkoxyl radical, and hydrogen
peroxide, which are inevitable products of natural redox
reactions occurring in various cellular compartments
(Zhang and Kirkham 1994, Alscher et al. 1997, Panda
2002). However, plants possess both enzymic
>superoxide dismutase (SOD), catalase (CAT), ascorbate
peroxidase (APX) guiacol peroxidase (GPX), and
glutathione reductase (GR)@ and non enzymic (carote-
noids, ascorbate, glutathione, D-tocopherol) antioxidants
to overcome the toxic effect of ROS.
The level of osmolytes or osmoprotectants are
increased in plant subjected to drought. Increase in total
free amino acids and free proline were reported in wheat
(Levitt 1980, Kathju et al. 1988) and in tea (Handique
and Manivel 1990), respectively. The molecular
mechanism of quenching of ROS by proline under
stresses, which includes water stress is well reviewed by
Matysik et al. (2002). Besides the contents and compo-
sition of osmolytes the antioxidant property also varies
between the drought susceptible and drought resistant
plants. The present experiment was undertaken to
understand the drought imposed damage and its recovery
in the developing clonal tea plant like other crop plant.
Healthy and uniform 1-year-old clonal seedlings of
tea [Camellia sinensis (L.) O. Kuntze] were procured from
Tocklai Tea Research station, Silchar, and grown under a
natural light in a greenhouse. Drought was induced by

Received 19 September 2003, accepted 12 May 2004.
Abbreviations: APX - ascorbate peroxidase; CAT - catalase; GPX - guaiacol peroxidase; GR - glutathione reductase; PDR - post-
drought rehydration; ROS - reactive oxygen species; RWC - relative water content; SOD - superoxide dismutase;
TBA - thiobarbituric acid; TBARS - thiobarbituric acid reactive substance; TCA - trichloroacetic acid.
1 Corresponding author, fax: (+81) 86 434 1210, e-mail: drskp@rib.okayama-u.ac.jp

H. UPADHYAYA, S.K. PANDA
598
withholding the watering for five days under controlled
conditions. On the sixth day leaves from the control and
drought imposed tea seedlings were sampled for various
biochemical analysis and plants were rehydrated for
another five days. On the sixth day of rehydration leaves
were again sampled.
Leaves were extracted in cold 80 % acetone and
chlorophylls and carotenoids were extracted and
estimated by spectrophotometer type 106, (Systronics,
India) as per the methods of Arnon (1949). Extraction
and estimation of H2O2 content was done according to
Sagisaka (1976). Lipid peroxidation was measured as the
amount of thiobarbituric acid reactive substance
(TBARS) determined by the thiobarbituric acid (TBA)
reaction as described by Heath and Packer (1968).
Glutathione was extracted and estimated according to
Griffith (1980). For the extraction and estimation of
ascorbate method of Oser (1979) was used. Proline
content in leaves was determined following the method of
Bates et al. (1973). Presence of superoxide anion (O2˙Ǧ)
was determined as described by Elstner and Heupel
(1976). Relative water content (RWC), defined as water
content of tissue as a percentage of that in water saturated
leaf tissue, was determined by the method of Weatherley
(1950). Samples of fresh tissue were floated in distilled
water at 25 r 1 qC for 4 h.
The leaf tissues were homogenised with phosphate
buffer pH 6.8 (0.1M) in prechilled mortar and pestle. The
extract was centrifuged at 4 qC for 15 min at 17 000 g in
a cooling centrifuge. The supernatant was used for the
assay of CAT, GPX, SOD, and GR. Extractions and
assay of CAT, GPX, GR, and SOD were done as per the
methods described in Chance and Maehly (1955),
Fig. 1. Changes in contents of proline, H2O2, thiobarbituric aci
d
reactive substances (TBARS) (A), ascorbate (Asc), glutathione
(Glu) and superoxide anion (SA) (B) in Camellia sinensis
leaves subjected to drought stress and rehydration (PDR).
Means of 5 separate experiments r SE.
Giannopolitis and Reis (1977) and Smith et al. (1988)
respectively. Each experiment was repeated five times
and data presented are means r SE.
RWC decreased with drought stress, but only a slight
increase in RWC was observed on rehydration for 5 d. A
decrease in chlorophyll (Chl) and carotenoid (Car)
contents (42 and 51.96 % of that in control plants,
respectively) was observed after 5 d of drought treatment.
Such observation suggested a drought induced pigment
degradations (Baisak et. al. 1994) and/or inhibition of
their synthesis. Decrease in net photosynthetic rate by
water stress in tea seedlings was observed by Sobrado
(1996) and Yordanov et al. (2000). After rehydration, Chl
and Car contents were 34.18 and 7.25 %, respectively,
less than those in the control.
Fig. 2. Changes in chlorophyll (Chl) and carotenoid (Car)
contents (A), and superoxide dismutase (SOD), catalase (CAT),
guaiacol peroxidase (GPX) and glutahione reductase (GR)
activities (B), and relative water content (RWC) (C) in
Camellia sinensis leaves subjected to drought stress an
d
rehydration (PDR). Means r SE, n = 5.
An increase in proline content (251.64 %) was
observed, whereas after rehydration it decreased to
108.07 % of that in control. Such proline accumulation in
response to water deficit stress was reported in wheat
(Kathju et al. 1988, Levitt 1980) and in tea (Handique
and Manivel 1990). However, this accumulation may not
be sufficient to reduce completely the damaging effect of
dehydration on membrane disintegration or enzyme
inactivation (Bohnert and Jenson 1996).
TBARS content is the measure of lipid peroxidation.
It significantly increased in drought treated plants, but
rehydration showed decrease of the same. An increase in

EFFECTS OF DROUGHT AND REHYDRATION
599
H2O2 content with simultaneous increase in lipid
peroxidation in drought imposed tea plant suggested a
loss of membrane function and induction of oxidative
damage (Zhang and Kirkham 1994, Baisak et al. 1994,
Sairam et al. 1997, Fu and Huang 2001, Egert and Tevini
2002). But post drought recovery analysis suggested that
rehydration minimizes the negative effect of drought, as
was evidenced by 44.57 % decreased lipid peroxidation
and 7.8 % decrease in H2O2 content observed after
rewatering the drought imposed plant.
Although an increase in SOD activity was visible with
simultaneous increase in CAT activity, significant
decrease in GPX and GR activities and a decrease in
contents of non-enzymic antioxidants, ascorbate and
glutathione suggested the incomplete ability of tea
seedlings to overcome a drought induced oxidative stress.
This is in agreement with results of Mukherjee and
Choudhuri (1983), Jagtap and Bhargava (1995), Egert
and Tevini (2002), and Fu and Huang (2001). The post
drought recovery analysis suggested a marked increase in
GPX, GR and CAT activities after rehydration of drought
imposed tea seedlings with significant decrease in SOD
activity. The increased amount of superoxide anion in
drought imposed tea seedlings and its little recovery on
subsequent rehydration also confirmed oxidative stress
In conclusion, imposition of drought caused serious
damage in tea seedlings and only moderate recovery was
noticed upon rehydration.
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