Drought induced physiological and biochemical changes in leaves
of developing seedlings of tea [Camellia sinensis (L) O Kuntze ]
cultivars
H. Upadhyaya1*, B. K. Dutta2, S. K. Panda3
1
Department of Botany and Biotechnology, Karimganj College , Karimganj-788710,Assam, India
2
Microbial and Agricultural Ecology Laboratory, Department of Ecology and Environmental
Sciences, Assam (Central) University , Silchar 788011.
3
Plant Biochemistry and Molecular Biology Laboratory, School of Life sciences,
Assam ( Central ) University ,Silchar 788011.
*Corresponding Email: [email protected]
Abstract
Drought is one of the important environmental stresses affecting agricultural
productivity around the world. In this study, an attempt has been made to understand drought
induced biochemical alterations in different clones of Camellia sinensis [TV-1, TV-20, TV29 and TV-30]. Drought stress induced decrease in total chlorophyll and carotenoid,
phenolics concentration and increases in proline concentration, lipid peroxidation and
polyphenols oxidase activity as a consequent of decrease in leaf relative water content
(RWC). Decreased Na+ and K+ concentration caused osmotic stress in leaves decreasing NR
activity, and ultimately reducing leaf relative growth rate. Thus, drought induced a range of
physiological and biochemical alterations causing membrane damage and loss in cellular
functions ultimately leading to reduction in growth of one of the most important economic
crop like tea. In comparison , TV-1 showed better drought tolerance by maintaining higher
endogenous K+ and proline content and a balance Na+/K+ ratio in leaves.
Key words : Drought. relative growth rate(RGR). chlorophyll. phenols.proline. lipid peroxidation.
Camellia sinensis.
Introduction
Drought is one of the more important environmental stresses affecting agricultural
productivity around the world and may result in considerable yield reduction (Boyer 1982).
‘Drought’ is a meteorological term that denotes a period without rains during which soil
water content is reduced and plants suffer from lack of water. Drought affects on the
morphology anatomy and physiology of the plants. Physiological, biochemical, and
anatomical responses however occur much earlier than the usual symptoms of wilting, which
may be permanent or temporary depending on the availability of soil moisture. The
physiological, biochemical and molecular mechanism involved in cellular and whole plant
responses to drought therefore generate considerable interest and are frequently reviewed
(Ingram and Bartel 1996; Chakraborty et al . 2002; Kar 2002; Shinozaki et al. 2002;
Yordanav et al. 2003; Francois Tardieu 2003; Upadhyaya and Panda 2004; Reddy et al.
2004; Jeyaramraja et al. 2005; Sakuma et al. 2006; Ohashi et al. 2006).
Water stresses
results is stomatal closure and reduced transpirations rate, a decrease is water potential of
plant tissues, decrease in photosysthesis and growth
inhibition (Tahi et al. 2007),
accumulation of abscisic acid (ABA), proline, mannitol, sorbitol, formation
of radical
scavenging compounds (ascorbate, glutathione, tocopherol etc) and systhesis of protein.
Decrease in photosysthesis is due to the limitted CO2 assimilation caused by stomatal closure
and decrease is total chlorophyll content. Osmotic adjustment has been considered as
beneficial to drought tolerant mechanism in field crop species. Na+ and K+ content of leaf
affect and regulates the osmotic potential of the plant during stress conditions and hence
Na+/ K+ ratio should be adequate for stress acclimatization by plant through osmotic
adjustment . However, there is no report on the changes of these ions in response to drought
in Camellia sinensis. The lowering of osmotic potential by adjustment also minimizes the
opportunity for significant water loss to occur from leaf tissue. This helps cells of higher
plants to withstand water deficit by maintaining sufficient turgor to proceed (Grima and
Krieg 1992 a,b) . Water stress induced pigment degradation, gross decline in protein level,
increased proline content, carbohydrate status, lipid peroxidation, in plants have also been
reviewed ( Kar 2002 ) . Tea is second only to water as the most consumed beverage in the
world. It has been used medicinally for centuries in India and China. Green tea is
comparatively healthier than black and olong tea. . The active constituents in green tea are
powerful antioxidants called polyphenols (catechins) and flavonols. Research shows that tea
consumption is healthy and help fighting various health problems which has also been
reviewed by many authors ( Kabir 2002; Higdon and Frei, 2003; Cabrera et al. 2006). Tea is
one of the most important economic crops in Barak/Brahmaputra valley/ Dooars and other
hilly terrains of India (i.e. Darjeeling, Himachal, Nilgiri and Uttaranchal). Tea plant being
perennial crops is subjected to different environmental stress, drought being one of the
important amongst them. In N.E. India, generally tea suffers from drought during November
to April. In this region irrigation is increasingly used as an insurance against drought to
maintain the productivity of tea during this period. The influence of irrigation on the
potential yield of tea in this region has also been studied (Panda et al. 2003). Thus the
present investigation was undertaken for understanding the mechanism of drought stress
induced physiological and biochemical alterations in selected clone of Camellia sinensis L
(O) Kuntze. Drought tolerance in tea can be assessed through some physiological and
biochemical parameters under moisture stress and these parameters can be used as selection
criteria for drought tolerance in the selection and breeding programs of tea ( Handique and
Manivel 1990). In the present experiment, the field soil was used in the pot. The aim was to
test the responses of selected clones to drought stress imposed by withholding water in the
pot. Such pot experiments have been used to asses the drought tolerance in plants
(Chakraborty et al., 2002; Sharma and Kumar, 2005, Xu and Zhou, 2007, etc). However, the
field conditions were different from pot as the tea is a deep rooted plant and soil area is not
limited it can penetrate the soil deep and it is obvious that it can withstand more days of
dehydration stress in
the field condition. During the period of natural drought plant
encounters long period without rain as it is grown in rainfed ecosystem. Thus it is quite
relevant to evaluate drought responses of plant like tea in potted conditions and correlate its
responses in relation to field performance as because all the plants were grown in the same
size pots and under same environmental conditions.
Materials and methods
Four commonly growing clonal varieties of Camellia sinensis L. (O) Kuntze ( viz. TV-1,
TV-20, TV-29 & TV-30 ) seedlings of uniform age, one and half year old were procured
from. Tocklai Tea Research Station, Silcoori, Silchar.
The seedlings grown in field soil in polyethene sleeves were procured from the
nursery of near by tea Garden of Durgakona and brought to the laboratory. The seedlings
were potted after removing polyethene sleeves and adding field soil. The plants were
acclimatized for 10-15 days in laboratory conditions and were grown under natural light with
well irrigation.
As tea is a shade loving plant, the seedlings were grown in a shed where the intensity of light
ranges from 150-300 mol m-2 s-1
and 200-400 mol m-2 s-1 inside and outside the shed
respectively. All control and treated plants were kept in similar growth conditions during
acclimatization and treatment imposition.
After 10-15 days of acclimatization, drought is imposed by withholding water for 20
days. Well watered plant is considered as control. The level of stress was quantified by
measuring changes in soil moisture and RWC of leaf in stressed plant relative to control.
After dehydration, soil moisture content decreased to (12.88 ± 1.34)% and (3.55 ± 0.28 )%
after 10 and 20d of stress imposition respectively relative to control (23.46 ± 1.62)%. The
average temperature range during experimental period was noted as 25.1 – 32.3 0 C and 12.5
– 24.7 0 C max/min respectively. The average relative humidity during the experiment period
was 88-96 % and 38-67 % morning/afternoon respectively. All the leaf samplings were
done during morning hours between 8 am to 9 am.
Fresh mass of leaf was measured in three replicates using five leaves and
expressed as g leaf-1 . For dry mass measurement same leaves were oven dried at 80
0
C
for 48 h and expressed as g leaf –1. The relative gain in leaf total fresh mass was determined
as relative growth rate (RGR, [g d-1] of leaf or the increase in total leaf fresh mass per unit
of existing mass per unit time), and calculated according to the formula:
where M2 was the final total fresh mass (g), M1 was the initial total fresh mass (g), t2
was
the number of days since initiation of experiment and t1 was day 0. Relative water content
(RWC) was measured by following the methods of Barrs and Weatherly (1962).
Tea leaves were sampled, oven dried and digested in a HNO3-HCl (3:1, v/v)
mixture and Na+ and K+ concentrations were determined by Flame Photometer (Systronics,
India) [ Jackson 1973]
The stability of leaf membranes, was assessed by determining leakage of electrolytes
from leaf discs placed in 20 ml of deionised water for 24h at room temperature and
measuring the electrical conductivity before and after autoclaving the samples and
Electrolytic leakage was determined as described by Dionisio Sese and Tobita (1998) and
calculated using formula.
EL = EC1/EC2 X 100
Where - EC1 is the initial conductivity of the sample before autoclaving and EC2 is
the final conductivity measured after autoclaving the sample.
Leaves were extracted in cold with 80 % acetone. The chlorophyll and carotenoid
contents were estimated as per the methods of Arnon (1949).
Proline concentration in tea leaves was determined following the method of Bates et
al. ( 1973 ). Leaf sample ( 0.5g ) was homogenized with 5 ml of sulfosalicylic acid ( 3 % )
using mortar and pestle and filtered through Whatman No. 1 filter paper. The volume of
filtrate was made upto 10 ml with sulfosalicylic acid and 2.0 ml of filtrate was incubated
with 2.0 ml glacial acetic acid and 2.0 ml ninhydrin reagent and boiled in a water bath at 100
0
C for 30 min. After cooling the reaction mixture, 6.0 ml of toluene were added and after
cyclomixing it, absorbance was read at 570 nm.
Total phenolics were extracted from tea
leaves in 80 % ( v/v ) ethanol and were estimated as per the method of Mahadevan and
Sridhar ( 1982 ) using Follin Ciocalteau reagent and Na2CO3.
Lipid peroxidation was measured as the amount of TBARS determined by the
thiobarbituric acid ( TBA ) reaction as described by Heath and Packer ( 1968 ). The leaf
tissues ( 0.2 g ) were homogenised in 2.0 ml of 0.1 % ( w/v trichloroacetic acid ( TCA ).
The homogenate was centrifuged at 10,000g for 20 min. To 1.0 ml of the resulting
supernatent, 1.0 ml of TCA ( 20 % ) containing 10.5 % ( w/v ) of TBA and 10l ( 4% in
ethanol ) BHT (butylated hydroxytolune) were added. The mixture was heated at 950C for
30min in a water bath and then cooled in rice. The contents were centrifuged at 10,000 g for
15 min and the absorbancy was measured at 532 nm and corrected for 600 nm. The
concentration of MDA were calculated using extinction coefficient of 155m M-1cm-1.
Leaf tissues were homogenized with potassium phosphate buffer pH 6.8 ( 0.1M )
containing 0.1 mM EDTA,1 % PVP and 0.1 mM PMSF in prechilled mortar pestle. The
extract was centrifuged at 4 0C for 15 min at 17000 g in a refrigerated cooling centrifuge.
The supernatant was used for the assay of polyphenol oxidase (PPO).
PPO were assayed
using pyrogallol as substrate in 1.0 ml of enzyme extract. according to Kar and Mishra (
1976 ) . After incubations at 25 0C for 5 min, the reaction was stopped with additions of 1.0
ml of 10 % H2SO4. The purpurogallin formed was read at 430 nm. 1 unit of enzyme activity
is defined as that amount of enzyme which forms 1 mole of purpurogallin formed per
minute under the assay conditions. Total soluble protein content was estimated as per the
method of Bradford (1976) using BSA as standard.
Nitrate Reductase ( NR ) activity in tea leaves was extracted and estimated as per
the methods of Singh and Mallik ( 1980 ). The enzyme extracted in 0.1M phosphate buffer
(pH 7.2 ) and the homogenate was centrifuged in cooling centrifuge at 10,000 g at -4 0C.
The resulting supernatant was used for the assay of enzyme activity. The assay mixture
comprised of 1.0 ml phosphate buffer (0.1M) (pH 7.2), 0.5 ml NADH (1.5 mM), 0.5 ml
distilled water and 1.0 ml enzyme extract. It is then incubated at 25 0C. The enzyme reaction
was initiated by adding 0.5 ml KNO3 ( 0 .1 M ) and incubated for 30 min. Then 0.8 ml zinc
acetate (0 .1 M ) was added and centrifuged and 3.0 ml of the supernatant was retained. To
this 2.0 ml [ 1 % sulfanilamide in 1.5 N HCl and 0.2 %
N-naphthalene diamine
hydrochloride ( NEDH )], mixed in equal volume was added. After 10 min absorbency was
read at 540 nm. NR activity was expressed as moles NO2 min-1.g-1 FW.
Each experiment was done in triplicate and repeated thrice and data presented are
mean  standard error ( SE ). The results were subjected to ANOVA using GLM factorial
model on all the parameters. Tukey test was used for comparision between pairs of
treatments . For relationship between relative water content and proline accumulation , K+
content of leaf and its RWC , lipid peroxidation and solute leakage of leaf tissue, lipid
peroxidation and decrease in RWC of leaf , relative growth rate of leaf and changes in total
chlorophyll content a linear regression was performed . The data analysis were carried out
using statistical package SPSS 7.5.
Results
Relative growth rate (RGR) of leaf showed uniformly decline trend with progressive
soil moisture stress in all the tested clones (Fig. 1B). Changes in RGR of leaf due to10d of
stress imposition was minimum in TV-30 and TV-1 but after 20d of stress RGR changes
among the clones was not significant though decrease growth was about 95%. The amount
of solute leakage is an index of membrane damage. Drought induced increase in solute
leakage in all the tested clones of Camellia sinensis (Fig.1A). After 20d of stress, in
comparison with control plants solute leakage increases to 130.84, 89.75, 150 and 153.92%
in TV-1, TV-17, TV-20, TV-29 and TV-30 respectively.
However, TV-1 and TV-20
showed comparatively lesser amount of solute leakage during stress conditions. Relative
water content (RWC) of leaf uniformly decreased with decreasing soil moisture content
imposed by 20 d of water withholding (Fig. 1C). After 20 d of drought, RWC of leaf was
found to be 56.26±1.21, 52.87±1.08, 44.99±0.43 and 52.66±1.03% relative to control with
92.15±1.24, 90.21±2.83, 87.57± 6.11 and 91.64± 4.92 % in TV-1, TV-20, TV-29 and TV30.
The contents of Na+ ion increased with the progress of water stress imposition,
apparently showing highest Na+ content in TV-29(41.73%) with lowest in TV-1 as a result
of 20 d of drought stress in comparision with control plants (Fig 2A) . On the other hand,
water stress induced decrease in K accumulation was observed in leaves of Camellia sinensis
, but highest K+ content was maintained by TV-1(27.16%) and TV-29(13.85%) even after
20 d of drought imposition as depicted in Fig 2B. There was increase in Na+/ K+ ratio with
progressive days of water withholding
in TV-20(105.80%), TV-29(64.58%) & TV-
30(283.25%) , but with exception TV-1(37.09%) showed decrease in Na+/ K+ ratio with the
increasing intensity of drought stress (Fig 2C).
Photosynthetic pigments (chlorophyll and carotenoid ) content was found to be
decreased with increasing days of
stress imposition.
Drought induced degradation of
chlorophyll and carotenoid was maximum in TV-29(57.91 & 82.38%) and TV-20 (55.10 &
86.13%) respectively as depicted in Fig.3A & B. Phenolic compounds are widely distributed
in plants and are mainly produced to protect plants from stress, ROS, wounds, UV light,
disease and herbivores (Dixon and Paiva 1995). Total
phenolics content in tea includes
catechins and polyphenols, which was found to be decreased with the imposition of drought
(Fig3C). Maximum decrease in phenolics content was observed in TV-20(37.63%) and TV2928.15%).
Increase in compatible solutes like proline, Glycine betain etc., is the characteristic
feature of plants acclimatizing stress conditions. In this study proline accumulation during
water stress condition was maximum in TV-1 (Fig. 4A). Drought stress induced significant
increase in PPO activity was observed in all the tested tea cultivars, maximum increase being
shown by TV-29 (Fig 4B). Highest PPO activity was shown by TV-29(458.82%) and TV1(424.91%) .
MDA content was maximum in TV-29(420.41%) after 20 d of stress
imposition, whereas TV-30(58.95%) showed minimum
increase as compared to control
plant (Fig. 4C).
As indicated in Fig 5A , NR activity decreased due to drought in four clones of
tea , where minimum decrease was observed in TV-1(62.59%) and TV-30(72.03%), that
could be correlated with stress induced decrease in total soluble protein content in tested
clones of Camellia sinensis (Fig.5B).
The interesting aspect in this study is that there was no significant correlation
between relative water content and changes in proline content of leaf (r2 = 0.15, ns) [ Fig. 6
A]. In our study, increase in Na+ content is followed by significant decrease in K+ content of
leaf which is significantly correlated with decrease in RWC of leaf (r2 = 0.50, p<0.01) as
depicted in Fig. 6 B. Concomitant with the increase in MDA content of leaf amount of
solute leakage also increased which was evident from the significant correlation (r2 = 0.66,
p<0.001) between increase in lipid peroxidation and amount of solute leakage as shown in
Fig. 6 C. Increase in MDA content is significantly correlated with decrease in RWC of leaf
(r2 = 0.39, p<0.05) as depicted in Fig 6D. There was significant correlation between
chlorophyll degradation and RGR of leaf (r2 = 0.40, p<0.05) [Fig. 6 E].
Discussion
Drought imposition resulted in significant changes of
growth and biochemical
responses in various clones of Camellia sinensis. The contents of Na+ ion increased with the
progress of water stress imposition, apparently showing highest Na+ content in TV-29 with
lowest in TV-1 as a result of 20 d of drought stress in comparision with control plants. On
the other hand, water stress induced decrease in K accumulation was observed in leaves of
Camellia sinensis , but highest K+ content was maintained by TV-1 even after 20 d of
drought imposition. This is in contradiction with the findings of Osmond et al. (1980) and
Martinez et al. (2003) who reported involvement of Na+ and K+ accumulation in the
osmotic adjustment of leaf tissues to low external water potential in Artiplex species during
water stress. Sodium contribution to osmotic adjustment may be indirectly by triggering
osmolyte synthesis independent of osmotic stress was however demonstrated by Subbarao et
al. (2001). In our study, increase in Na+ content is followed by significant decrease in K+
content of leaf which is significantly correlated with decrease in RWC of leaf (r2 = 0.50, p<
.01) as depicted in Fig. 6 B. K+ nutrition in plants is known to enhanced drought resistance
, water use efficiency and growth under drought condition (Egilla et al. 2001 ). Thus it
suggested adequate K+ fertilizer application of tea plant may facilitate osmotic adjustment
improving its drought tolerance potential. There was increase in Na+/ K+ ratio with
progressive days of water withholding in TV-20, TV-29 & TV-30, but with exception TV-1
showed decrease in Na+/ K+ ratio with the increasing intensity of drought stress (Fig 2C).
As K+ is the main ion regulating osmotic adjustment in leaves , increase in Na+/K+ ratio
suggested occurrence of osmotic stress in three tea cultivars (TV-20, TV-29 & TV-30) but
exceptional decreased Na+/ K+ ratio in TV-1 is the indication of its comparatively better
drought acclimatizing potential.
Water deficit stress, often called drought affects the
growth and productivity of plants growing in natural or agricultural field conditions. In the
present study, relative growth rate (RGR) of leaf showed uniformly decline trend with
increasing drought stress. The growth of leaf is directly or indirectly related with the various
metabolism dependent on its osmotic potential which is regulated by potassium content of
leaf.
The amount of solute leakage is an index of membrane damage. Drought induced
increase in solute leakage in all the tested clones of Camellia sinensis. However, TV-1 and
TV-30 showed comparatively lesser amount of solute leakage during stress conditions.
Drought induced membrane damage
could be due to
enhanced membrane lipid
peroxidation which was evident from increased MDA content with the progress of stress
imposition, observed in four clones of tea. MDA content was maximum in TV-29 after 20 d
of stress imposition, whereas TV-30 showed minimum
increase as compared to control
plant. Concomitant with the increase in MDA content of leaf amount of solute leakage also
increased which was evident from the significant correlation (r2 = 0.66, p< .001) between
increase in lipid peroxidation and amount of solute leakage as shown in Fig. 6 C. Thus, it
apparently suggests that increase in leakage of solute could be due to drought induced lipid
peroxidation of membrane disturbing the structural and functional integrity of cell causing
ultimate reduction in growth of tea plant. Photosynthetic pigments (chlorophyll and
carotenoid ) content was found to be decreased with increasing days of stress imposition.
Drought induced degradation of chlorophyll and carotenoid was maximum in TV-29 and
TV-20 respectively. There was significant correlation between chlorophyll degradation and
RGR of leaf (r2 = 0.40, p< .05). Decrease in chlorophyll and carotenoid
in response to
water stress was reported earlier (Upadhyaya and Panda, 2004). Such degradation of
chlorophyll pigments may eventually decrease photosynthetic efficiency in plants which
might be one of the potent causes of reduction in growth of plant. However, a very recent
study also provides evidence for a significant positive relationship between transpiration
efficiency and leaf chlorophyll concentration in plant (Sheshshayee et al. 2006). During
prolonged periods of drought, the decrease in water availability for transport-associated
processes leads to changes in the concentrations of many metabolites, followed by
disturbances in amino acid and carbohydrate metabolism. For example, there is an increase
in the synthesis of compatible solutes such as special amino acids (e.g. Proline), sugars and
sugar-alcohols, and Gly betaine . Acclimation to drought requires responses that allow
essential reactions of primary metabolism to continue and enable the plant to tolerate water
deficits. In the complex interplay of natural conditions, simple water deficits are unlikely to
occur, since they intrinsically affect the acquisition of essential nutrients such as N etc. The
reduction of NO3− to NO2− catalyzed by NR is considered to be the rate-limiting step of N
assimilation. NR activity is coordinated with the rate of photosynthesis Indeed, droughtinduced N deficiency was found to limit recovery of photosynthesis in plants. In situations of
water deprivation, maximal foliar extractable NR activity has been found to decrease in
some cases (Foyer et al. 1998). In the present study, NR activity decreased due to drought
in four clones of tea , where minimum decrease was observed in TV-1 and TV-30, that
could be correlated with stress induced decrease in total soluble protein content in tested
clones of Camellia sinensis. Similar results were already reported in other plants (Foyer et
al. 1998; Panda 2002; Xu and Zhou 2006).
Increase in compatible solutes like proline,
Glycine betain etc., is the characteristic feature of plants acclimatizing stress conditions. In
this study proline accumulation during water stress condition was maximum in TV-1.
Proline acts as an osmoprotectant and greater accumulation of proline in TV-1 suggested
genotypic tolerance of tea to water deficit stress, as proline accumulation helps in
maintaining the water relations, prevents membrane distortion and acts as a hydroxyl radical
scavenger. Drought induced biochemical modifications and proline metabolism has also
been studied in other plants (Sanker et al. 2007). The molecular mechanism of quenching of
reactive oxygen species proline has also been well reviewed ( Matysik et al. 2001).
However, Wu et al. (2007) reported osmotic adjustment in Citrus seedling colonized by
Glomus versiforme subjected to drought stress did not correlate with proline but with ions
like K+, Ca2+ , etc and carbohydrates. Thus, similarly K+ might play critical role in providing
osmotic adjustment in response to drought stress in tea cultivars as evidenced by significant
correlation between changes in endogenous K+ content and RWC of leaves in stress imposed
tea cultivars (r2 = 0.50, p< .01). Also higher K+ content of TV-1 in stressed condition
showed better drought tolerance in comparision with other clones.
Relative water content
(RWC) of leaf uniformly decreased with decreasing soil moisture content imposed by 20 d
of water withholding (Fig. 1C). The interesting aspect in this study is that there was no
significant correlation between relative water content and changes in proline content of leaf
(r2 = 0.15, ns) [ Fig. 6 A].
Phenolic compounds are widely distributed in plants and are mainly produced to
protect plants from stress, ROS, wounds, UV light, disease and herbivores (Dixon and Paiva
1995). Total phenolics content in tea includes catechins and polyphenols, which was found
to be decreased with the imposition of drought (Fig3C). Tea polyphenols are mostly
catechins which
have potential antioxidant properties that makes tea a good health drink.
Polyphenol oxidases (PPO) are widely distributed in the plant and play a role in oxygen
scavenging and defense against stress (Esen 1993; Shivishankar 1988). The antioxidant
activity of phenolic compounds is mainly due to their redox properties. The medicinal value
of phenolics in human health is well documented ( Caia et al. 2004; Tona et al. 2004 ). In
this study, drought stress induced significant increase in PPO activity was observed in all the
tested tea cultivars, maximum increase being shown by TV-29.
PPO catalyses the O2-
dependent oxidation of mono- and o diphenols to o-diquinones, where secondary reactions
may be responsible for the defense reaction and hypersensitive response (Mayer and Harel
1991) .Moreover, it is proposed that PPO activity may regulate the redox state of phenolic
compounds and become involved in the phenylpropanoid pathway (Kojima and Takeuchi
1989; Nakayama et al. 2000).
In conclusion, it can be said that drought induced a range of physiological and
biochemical alterations causing membrane damage and loss in cellular functions ultimately
leading to reduction in growth of one of the most important economic crop like tea. In
comparision TV-1 showed better drought tolerance by maintaining higher endogenous K+
and proline content and a balance Na+/K+ ratio in leaves. Hence, the present result may
provide insight into underlying mechanism of tea plant in response to soil drought at the
juvenile stage. However, more investigation is required in the field at different plant growth
stages in the future. It can be further confirmed by studying various physiological and
biochemical responses of different tea cultivars in field conditions and conducting
experiments to study the
responses of those cultivars to rehydration and post stress
manipulation of soil and leaf nutrients which are already initiated at our laboratory.
Acknowledgements
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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Control
10D
Solute leakage (%)
100
*c
80
*
b
*c
60
40
A
20D
*
b
*c
*c
*
b
a
*
b
a
a
a
20
0
Leaf RGR
DW
-1 -1
( mg g d )
6
B
*a
5
a
*a *a
*a
*a
b
*
b
4
3
2
1
c
c
c
c
0
Relative water content (RWC%)
C
120
100
80
60
a
b
b
b
c
a
a
a
b
c
c
c
40
20
0
TV-1
TV-20
TV-29
TV-30
Fig. 1. Changes in solute leakage, relative growth rate(RGR) of leaf and relative water
content(RWC) in four clonal varieties of Camellia sinensis ( TV-1, TV-20, TV-29 & TV-30
) subjected to drought . Control (
) ; 10 days of drought (
) ; 20 days of drought ( )
imposition . Data presented are mean ± SE (n=3). Different lower case letters indicates
differences between drought treatment in the same variety , while * indicates differences
within the varieties at P< .05 according to multiple comparision by Tukey test.
Control
Na+ Content ( mg g -1 DW )
20
10D
*a
*c
*b
15
A
20D
c
*c
10
*a *a
c
a a
a a
5
0
B
*a *
a
40
*c
-1
K+ Cotent mg g DW
50
*a
*a
*c
a a
30
*a
c
*
b
20
*c
10
0
C
0.9
*c
0.8
Na+/K+ Ratio
0.7
0.6
c
0.5
0.4
0.3
*c
*a
a
*c
a a
a a
b
*a
0.2
0.1
0
TV-1
TV-20
TV-29
TV-30
Fig. 2. Changes in Na+, K+ content and Na+/ K+ ratio in four clonal varieties of Camellia
sinensis ( TV-1, TV-20, TV-29 & TV-30 ) subjected to drought . Control ( ) ; 10 days
of drought ( ) ; 20 days of drought ( ) imposition . Data presented are mean ± SE (n=3)
and significant differences are shown as in Fig. 1.
-1
Total chlorophyll (  g g FW)
Control
10D
A
20D
6000
5000
a a
a a
a
a
a
4000
c
3000
b
c
bc
c
2000
1000
0
B
-1
Carotenoid content ( g g FW)
2500
2000
1500
a
a
a
a
b
b
b
c
b b
c
1000
c
500
0
7000
Total Phenolics
( mol g-1 FW )
6000
C
*a
*b
a
*c
5000
a
*a
*
b
b
c
4000
b
c
*c
3000
2000
1000
0
TV-1
TV-20
TV-29
TV-30
Fig. 3. Changes in total chlorophyll, carotenoid and phenolic contents in four clonal
varieties of Camellia sinensis ( TV-1, TV-20, TV-29 & TV-30 ) subjected to drought .
Control (
) ; 10 days of drought (
) ; 20 days of drought (
) imposition . Data
presented are mean ± SE (n=3) and significant differences are shown as in Fig. 1.
14
10D
-1
PPO activity
10
8
6
*a
*a
b
4
c
b
a
2
c
b
b
a
a
0
B
250
*b
*c
200
150
c
100
* *
b b
b b
b
50
a
a
a
a
0
C
2.5
MDAmol g -1 FW
A
20D
*c
12
-1
( mol purpurogallin min g FW)
-1
Proline content (  mol g FW)
Control
c
2
c
b
1.5
1
c
a
a
c
a
a
a
0.5
a a
0
TV-1
TV-20
TV-29
TV-30
Fig. 4. Changes in proline content, polyphenol oxidase (PPO) activity and MDA content in
four clonal varieties of Camellia sinensis ( TV-1, TV-20, TV-29 &
TV-30 ) subjected
to drought . Control ( ) ; 10 days of drought ( ) ; 20 days of drought ( ) imposition .
Data presented are mean ± SE (n=3) and significant differences are shown as in Fig. 1. .
30
a
-1
-1
NR activity (  mol min g FW)
A
25
a
20
a
a
b
15
c
10
b
b
b
5
c
c
c
0
20
-1
Total soluble protein (mg g Fw)
B
a
15
10
a
a
a
b
c
b
b
b
5
c
c
c
TV-20
TV-29
TV-30
0
TV-1
Fig. 5. Changes in nitrate reductase (NR) activity and total soluble protein content in four
clonal varieties of Camellia sinensis ( TV-1, TV-20, TV-29 & TV-30 ) subjected to drought .
Control (
) ; 10 days of drought (
) ; 20 days of drought ( ) imposition . Data
presented are mean ± SE (n=3) and significant differences are shown as in Fig. 1.
C
A
MDA, content [  mol g-1FW ]
= 0.154
RWC of
leaf (%)
r2
r2 =0.664***
Solute leakage (%)
Changes in Proline content
(  mol g-1 FW)
r2 =0.386*
r2 = 0.501**
MDA, content [  mol g-1FW ]
K+ content of leaf (mg g-1 FW)
D
B
RWC of leaf (%)
RWC of leaf (%)
r2 = 0.399*
Leaf RGR ( mg g-1d-1 )
E
Total chlorophyll content
(  g g-1 FW)
Fig.6. Relationship between relative water content and proline accumulation (A), K+ content of leaf and
its RWC (B), lipid peroxidation and solute leakage of leaf tissue (C), lipid peroxidation and decrease in
RWC of leaf (D), relative growth rate of leaf and changes in total chlorophyll content in four clonal
varieties of Camellia sinensis subjected to drought . The open square ( ), closed triangle ( ), open
circle ( ) and closed circle ( ) denotes TV-1, TV-20, TV-29 and TV-30 variety of tea subjected to
drought treatment respectively. *, ** and *** indicates significant correlation at P< .05,.01 and .001
respectively.