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ORIGINAL ARTICLE
Alterations in photosynthetic pigments, protein and osmoticcomponents in cotton genotypes subjected to short-termdrought stress followed by recovery
Asish Kumar Parida Æ Vipin S. Dagaonkar ÆManoj S. Phalak Æ G. V. Umalkar ÆLaxman P. Aurangabadkar
Received: 10 September 2006 / Accepted: 7 December 2006 / Published online: 2 February 2007� Korean Society of Plant Biotechnology and Springer 2007
Abstract In order to assess drought tolerance mech-
anism in cotton, short-term drought-induced bio-
chemical responses were monitored in two cotton
(Gossypium hirsutum L.) genotypes contrasting their
tolerance to water deficit. The seeds of two genotypes,
namely GM 090304 (moderately drought tolerant) and
Ca/H 631 (drought sensitive), were sown in pots con-
taining soil, sand and peat in the ratio of 1:1:1, and
irrigated every alternate day up to 45 days after sowing
when each genotype was subjected to a cycle of water
stress by withholding irrigation for 7 days. The stress
cycle was terminated by re-watering the stressed plants
for 7 days. The leaf of the drought tolerant genotype
(GM 090304) maintained higher relative water content
under water stress than that of the drought sensitive
genotype (Ca/H 631). The levels of biochemical com-
ponents, such as chlorophylls, carotenoids, total pro-
tein, free proline, total free amino acids, sugars, starch
and polyphenols, were measured during the stress as
well as the recovery periods. The chlorophylls, carot-
enoids, protein and starch contents decreased in
drought stressed plants as compared to control and
tended to increase when the plants were recovered
from stress. The degree of decrease in chlorophylls,
carotenoids and protein contents under drought
was higher in the sensitive genotype (Ca/H 631) as
compared to the moderately tolerant genotype
(GM 090304). However, proline, total free amino
acids, total sugars, reducing sugars and polyphenol
contents were increased in drought stressed plants and
tended to decrease during the period of recovery.
Drought-induced increases in total free amino acids,
proline, sugars and polyphenols were significantly
higher in the moderately tolerant genotype (GM
090304) than in the sensitive genotype (Ca/H 631).
These results suggest that proline, sugars and po-
lyphenols act as main compatible solutes in cotton in
order to maintain osmotic balance, to protect cellular
macromolecules, to detoxify the cells, and to scavenge
free radicals under water stress condition.
Keywords Carotenoids � Chlorophylls � Drought
stress � Osmolytes � Polyphenols � Proline
AbbreviationsChl Chlorophyll
DAS Days after sowing
kDa Kilo Dalton
RWC Relative water content
TCA Trichloroacetic acid
Introduction
Drought or water deficit stress is the major environ-
mental factor that negatively impacts agricultural yield
throughout the world, particularly when the stress
occurs during reproductive growth, affecting produc-
tion whether it is for subsistence or economic gain
(Selote and Khana-Chopra 2004). The plant response
A. K. Parida (&) �V. S. Dagaonkar �M. S. PhalakG. V. Umalkar � L. P. AurangabadkarBiotechnology Division,Ankur Agricultural Research Laboratory,27 New Cotton Market Layout,Nagpur 440018, Maharashtra, Indiae-mail: [email protected]
123
Plant Biotechnol Rep (2007) 1:37–48
DOI 10.1007/s11816-006-0004-1
to drought consists of numerous processes that must
function in coordination to alleviate both cellular hy-
perosmolarity and ion disequilibrium. To cope with
drought stress, plants respond with physiological and
biochemical changes. These changes aim at the reten-
tion of water in spite of the high external osmoticum
and the maintenance of photosynthetic activity, while
stomatal opening is reduced to counter water loss.
Accumulation of low molecular compounds, such as
glycinebetaine, sugars, sugar alcohols and proline, is a
mechanism aimed at balancing water potential fol-
lowing drought (Pilon-Smits et al. 1995). In addition to
synthesis of these osmolytic compounds, specific pro-
teins and translatable mRNA are induced and in-
creased by drought stress (Reviron et al. 1992).
Although an adaptive role for organic osmolytes in
mediating osmotic adjustment and protecting subcel-
lular structure has become a central dogma in stress
physiology, the evidence in favor of this hypothesis is
largely correlative (Hare et al. 1998). Transgenic plants
engineered to accumulate proline (Zhang et al. 1997),
mannitol (Thomas et al. 1995), fructans (Pilon-Smits
et al. 1995), trehalose (Romero et al. 1997), or glycine
betaine (Rhodes and Hanson 1993) exhibit marginal
improvements in salt and/or drought tolerance. While
these studies do not dismiss causative relationships
between osmolyte levels and stress tolerance, the
absolute osmolyte concentrations in these plants are
unlikely to mediate osmotic adjustment. Metabolic
benefits of osmolyte accumulation may augment the
classically accepted roles of these compounds. In re-
assessing the functional significance of compatible
solute accumulation, it is suggested that proline and
glycine betaine synthesis may buffer cellular redox
potential (Hare et al. 1998). Disturbances in hexose
sensing in transgenic plants engineered to produce
trehalose, fructans or mannitol may be an important
contributory factor to the stress-tolerance phenotype
observed. Associated effects on photoassimilate allo-
cation between root and shoot tissues may also be in-
volved. Whether or not osmolyte transport between
subcellular compartments or different organs repre-
sents a bottleneck that limits stress tolerance at the
whole plant level is presently unclear. Nonetheless, if
osmolyte metabolism impinges on hexose or redox
signaling, then it may be important in long-range signal
transduction (Hare et al. 1998).
Cotton is one of the most important economy crops
in world. It is regarded highly by the governments not
only in relation to people’s lives, but also to the income
of cotton farmers and the economic development of
cotton planting zones, as well as to national textile
supply and foreign exchange income. Many people
consider cotton to be the purest fiber on earth, or the
‘‘fabric of our lives’’. Drought stress affects the cotton
plants by limiting fiber yield and lint quality. There are
scanty reports (De Ronde et al. 2000) on biochemical
mechanisms involved in cotton to counter water stress.
In the present study, we have analyzed the biochemical
responses involved in two contrasting cotton genotypes
to cope with drought stress. Such study will provide
valuable information that can be used for genetic basis
of improvement of cotton to enhance yield and fiber
quality under optimum and stress conditions.
Materials and methods
Plant materials and culture conditions
Seeds of two cotton genotypes (Gossypium hirsutum
L.), namely GM 090304 (moderately drought tolerant)
and Ca/H 631 (drought sensitive), were germinated in
pots [size c. 38 cm · 38 cm (15† · 15†)] containing soil,
peat and sand in the ratio of 1:1:1, and grown under
green house conditions. Temperatures in the green
house were 30 ± 2�C during day and 25 ± 2�C at night,
with relative humidity ~50% and a photoperiod of
14 h. Metal halide illumination lamps (1,000 W) were
used to supplement natural radiation. Light radiation
reached a maximum of 1,500 lmol m–2 s–1 at the top of
canopy at midday. Seeds of each genotype were sown
in 60 pots. Four seeds were sown per pot. After 2
weeks of emergence, seedlings were thinned to one
plant per pot. The plants were irrigated every alternate
day with normal tap water. After 45 days from sowing
(immediately after flower initiation), a cycle of drought
was induced by stopping irrigating the potted plants for
7 days. A control set was maintained by irrigating the
potted plants regularly. After 7 days of drought
induction, the drought-stressed plants were re-irrigated
for 7 days for recovery. The leaf samples were col-
lected from control and treated plants after 7 days of
drought as well as after 7 days of recovery for esti-
mations of various biochemical parameters. Each of
the estimation included the tissue from leaves from five
plants occupying the same position.
Leaf area and leaf relative water content
Total green leaf area per plant was measured in both
control, stress-induced and stress-recovered plants of
both the genotypes in five replicates following the
method of Parida et al. (2004a). The relative water
content (RWC) of leaves was measured according
to Barrs and Weatherley (1962). Immediately after
38 Plant Biotechnol Rep (2007) 1:37–48
123
sampling, leaves were weighed and then immersed in
distilled water for 4 h at room temperature. The leaves
were then blotted dry and weighed prior to oven drying
at 80�C for 48 h. The leaf relative content was calcu-
lated using the following formula: RWC = [(FW –
DW)/(TW – DW)] · 100, where FW is the fresh
weight, DW the dry weight, and TW is the turgid
weight (weight after the leaf was kept immersed in
distilled water for 4 h).
Extraction and estimation of photosynthetic
pigments
Fresh leaves (0.5 g) were thoroughly homogenized in
chilled 80% acetone in a mortar and pestle in the dark
at 4 �C and the homogenates were centrifuged at
10,000 g for 10 min. The supernatants were collected
and the absorbances of the acetone extracts were
measured at 663, 646 and 470 nm using a UV–visible
spectrophotometer (Spectra Max Plus; Molecular De-
vices, USA). The Chl a, Chl b, total chlorophylls, Chl a/
b ratios and total carotenoids content were calculated
following the equations of Lichtenthaler (1987).
Extraction and estimation of total leaf protein
Total leaf protein was extracted by the acetone-TCA
precipitation method as described by Parida et al.
(2004b) and estimated following the method of Lowry
et al. (1951) using defatted bovine serum albumin
(fraction V, Sigma) as standard. The protein concen-
trations in the unknown samples were expressed as
gram per dry weight of tissue.
SDS-PAGE analysis of protein
Protein profiles of control, drought-stressed and
drought-recovered samples of both the genotypes were
analyzed by SDS polyacrylamide gel electrophoresis
(PAGE) following the procedure of Laemmli (1970).
A 10% separating gel was prepared and 40 lg of pro-
tein solubilized with sample buffer [62.5 mM Tris–HCl,
pH 6.8, 20% (w/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-
mercaptoethanol and 0.01% (w/v) bromophenol blue]
was loaded in each lane of the gel. Electrophoresis was
accomplished at 35 mA for 3 h using Bio-Rad, Protein
II electrophoresis system. The gels were stained with
0.25% Coomassie Brilliant Blue R-250 (Sigma) in 50%
(v/v) methanol and 10% (v/v) acetic acid for 2 h and
destained with 50% (v/v) methanol and 10% (v/v)
acetic acid until the background was clear. The gels
were photographed and scanned using a densitometer
(GS-800; Bio-Rad, USA) and analyzed with Quantity
one software from Bio-Rad. Precision plus protein
standards from Bio-Rad were used for the determina-
tion of molecular weight.
Estimation of total free amino acids
Total free amino acids were extracted and determined
following the method of Sugano et al. (1975) with
slight modifications. The leaf (0.5 g) was homogenized
in 70% ethanol in a pestle and mortar. The homoge-
nate was centrifuged at 5,000 g for 10 min and the
supernatant was taken. The extraction was repeated
four to five times and the supernatants were combined.
An appropriate volume (5–10 ml) of this ethanolic
extract was evaporated to dryness on a boiling water
bath and the residue was dissolved in 5 ml of 0.2 M
citrate buffer (pH 5.0). The above sample (2 ml) was
taken in a test tube and 1 ml of ninhydrin reagent (4%
ninhydrin in methyl cellosolve and 0.2 M acetate buf-
fer in the ratio of (1:1) was added to it. The samples
were boiled for 20 min and cooled; the volume was
made up to 10 ml with distilled water. Absorbance was
noted at 570 nm. Total free amino acids were calcu-
lated from a standard curve prepared against glycine
(0–100 lg).
Estimation of free proline
Free proline content was estimated following the
method of Bates et al. (1973). Fresh leaves (0.5 g) were
extracted in 3% sulphosalicylic acid and the homo-
genates were centrifuged at 10,000 g for 10 min. A
2 ml of the supernatant was reacted with 2 ml of acid
ninhydrin reagent and 2 ml of glacial acetic acid in a
test tube for 1 h at 100�C and the reaction terminated
in an ice bath. The reaction mixture was extracted with
4 ml of toluene and mixed vigorously with a vortex
mixture for 15–20 s. The chromophore containing tol-
uene was aspirated from the aqueous phase, warmed to
room temperature and the absorbance measured at
510 nm using toluene as blank. Proline concentration
was calculated from a standard curve using 0–100 lg L-
proline (Sigma).
Extraction and estimation of total soluble sugars,
reducing sugars and starch
Total soluble sugars, reducing sugars and starch con-
tents were estimated in 20 ml of 80% (v/v) ethanol
extract at 95 �C for 1 h from 100 mg of leaf powder
frozen in liquid nitrogen. After centrifugation at
10,000 g for 10 min, starch was measured in the pellet
according to Jarvis and Walker (1993). Total soluble
Plant Biotechnol Rep (2007) 1:37–48 39
123
sugars were analyzed by reacting 0.25 ml of the
supernatant with 3 ml freshly prepared anthrone re-
agent [0.06% (w/v) anthorone in 95% H2SO4] and
placing in boiling water bath for 10 min. After cooling
to room temperature, the absorbance at 625 nm was
measured and total sugar was quantified according to
Irigoyen et al. (1992). Starch content was determined
according to Murata et al. (1968).
Reducing sugars were estimated following alkaline
copper method as described by Parida et al. (2002)
using arsenomolybdate reagent. Absorbance was re-
corded at 510 nm and reducing sugar content was
determined from a standard curve prepared against
pure glucose (0–50 lg).
Estimation of total polyphenol
Total polyphenols were determined according to the
procedures of Chandler and Dodds (1983). Fresh
leaves (0.5 g) were homogenized in 5 ml of 80% eth-
anol using a chilled pestle and mortar with subsequent
centrifugation at 10,000 g for 20 min. The supernatant
was preserved and residue re-extracted with 2.5 ml of
80% ethanol, centrifuged and the supernatants were
pooled and evaporated to dryness. The residue was
dissolved in 5 ml of distilled water. In a test tube 3 ml
of aliquots were taken, 0.5 ml Folin-Ciocalteau’s re-
agent (1 N) was added and kept for 3 min. Then 2 ml
of 20% freshly prepared Na2CO3 solution was added to
each tube and mixed thoroughly. The solution was
boiled in a water bath for exactly 1 min, cooled and
then the absorbance was measured at 650 nm against a
reagent as a blank. A standard curve was prepared
using 10–100 lg of catechol (Sigma). From the stan-
dard curve, the concentrations of phenols in the un-
known samples were calculated.
Statistical analysis
Standard errors were computed from the values of two
independent experiments with replicates. Statistical
analysis of the results from two different experiments
was carried out according to Duncan’s multiple range
tests. Data were subjected to a two-way analysis of
variance (ANOVA) and the LSD at P £ 0.01 was
determined (Sokal and Rohlf 1995).
Results
As drought was induced in cotton plants during the
early stages of flowering (45 days after sowing) under
pot culture, the plants of both the genotypes (GM
090304 and Ca/H 631) wilted within 7 days of drought
induction. The degree of wilting was more in the sen-
sitive genotype (Ca/H 631) than in the moderately
tolerant genotype (GM 090304). More than 7 days of
water stress was found to be lethal. When the water-
stressed plants were recovered from drought, the
plants became normal within 7 days of recovery. Thus,
two data points (7 days after drought induction and
7 days after recovery) were selected in order to
investigate the short-term effects of drought in cotton
genotypes.
Leaf area and Relative water content of leaf
There were 24% and 29% decreases in leaf area in GM
090304 and Ca/H 631 genotypes, respectively, in
drought-stressed plants as compared to the controls
(Fig. 1). However, during the re-watering period the
plants were able to resume growth in terms of leaf
area.
Fig. 1 Effects of short-termdrought stress (dehydration)and rehydration on leaf areain cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
40 Plant Biotechnol Rep (2007) 1:37–48
123
RWC of leaves were 75% and 68.4% in GM 090304
and Ca/H 631 genotypes, respectively, under control
conditions (Fig. 2), while the drought stress caused a
decrease in RWC to 72% and 63.7% in GM 090304
and Ca/H 631 genotypes, respectively. However, on re-
watering, the plants recovered fully in terms of RWC
(Fig. 2).
Changes in photosynthetic pigments
The control plants showed a slight overall increase in
total chlorophyll and carotenoid contents from 52 to
59 days after sowing. Total Chl and carotenoid contents
decreased significantly by drought induction in both the
genotypes as compared to their respective controls
(Figs. 3, 4). On re-watering, both chlorophyll and
carotenoid contents of the stressed plants tended to
increase. The total Chl expressed on unit dry weight
basis decreased by 21% and 23% in GM 090304 and Ca/
H 631 genotypes, respectively, after 7 days of drought
induction (Fig. 3). When the drought-stressed plants
were re-watered, a 32% increase in chlorophyll content
of GM 090304 and 28% increase in Ca/H 631 were
observed in recovered plants as compared to the
drought-stressed plants (Fig. 3). Similarly, after 7 days
of drought induction, a 31% decrease in carotenoids
content in GM 090304 and a 33% decrease in Ca/H 631
was observed as compared to their respective controls
(Fig. 4). Upon recovery from drought stress, carote-
noids content increased significantly in recovered plants
of both the genotypes (39% in GM 090304 and 34% in
Ca/H 631). Chl a/b ratio of drought stressed plants de-
creased significantly in both the genotypes (Fig. 5).
However, Chl a/b ratio of both the genotypes recovered
to control values after 7 days of rehydration (Fig. 5).
Changes in total leaf protein, total free amino acids
and proline
The protein contents showed a statistically significant,
but very small decrease (8% in GM 090304 and 15% in
Ca/H 631) upon drought treatment (Fig. 6). When the
Fig. 2 Effects of short-termdrought stress (dehydration)and rehydration on relativewater content (RWC) of leafin cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Fig. 3 Effects of short-termdrought stress (dehydration)and rehydration on totalchlorophyll contents of leavesin cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Plant Biotechnol Rep (2007) 1:37–48 41
123
stressed plants were recovered from drought, the pro-
tein contents of recovered plants increased significantly
as compared to stress plants and tended to be equal to
their respective controls. SDS-PAGE analysis of pro-
tein extracted from leaf revealed identical protein
profiles in control, drought-stressed and drought-
recovered samples (Fig. 7).
The free amino acid pool did not change very much
in control samples during the entire period of investi-
gation, while in drought induced plants, total free
Fig. 4 Effects of short-termdrought stress (dehydration)and rehydration on totalcarotenoid contents of leavesin cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means asat P £ 0.01
Fig. 5 Effects of short-termdrought stress (dehydration)and rehydration on Chl a/bratio of leaves in cottongenotypes (GM 090304 andCa/H 631). A stress period of7 days was terminated by a 7-day period of rehydration.The values are mean ± SE(n = 10). Different letters onthe top of the error barsindicate statistically differentmeans at P £ 0.01
Fig. 6 Effects of short-termdrought stress (dehydration)and rehydration on totalprotein contents of leaves incotton genotypes (GM 090304and Ca/H 631). A stressperiod of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
42 Plant Biotechnol Rep (2007) 1:37–48
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amino acid contents increased by 2.4-fold and 2-fold in
GM 090304 and Ca/H 631 genotypes, respectively
(Fig. 8). Upon recovery from drought, total free amino
acid contents decreased significantly and were compa-
rable to the control plants.
As compared to control, the proline contents of
leaves increased dramatically in both the genotypes
(41-fold in GM 090304 and 21-fold in Ca/H 631) after
7 days of drought induction (Fig. 9). After recovery
from drought, the proline contents of both the geno-
types decreased significantly and tended to be equal to
their respective control.
Changes in sugars and starch
In control samples, marginal changes in both starch and
sugar contents of leaves were observed from 52 to
59 days after sowing. In drought induced plants, the total
soluble sugar contents increased by 68% and 52% in GM
090304 and Ca/H 631 genotypes, respectively, (Fig. 10)
in comparison to control. Similarly, the reducing sugar
contents of leaves increased by 2.5-fold in GM 090304
and 2.3-fold in Ca/H 631 genotype during drought
induction (Fig. 11). After recovery from drought, both
total soluble sugar and reducing sugar contents of leaves
tended to decrease in both the genotypes. On the other
hand, the starch contents of the leaves of drought-treated
plants decreased by 18 and 13% in GM 090304 and Ca/H
631 genotypes, respectively (Fig. 12). When the drought-
treated plants were recovered, the starch contents of
leaves tended to be equal to their respective controls.
Changes in polyphenols
Polyphenol contents of leaves increased by 59% and
52% in GM 090304 and Ca/H 631 genotypes, respec-
tively, upon drought induction (Fig. 13). On re-water-
ing, the polyphenol contents of stressed plants
decreased and tended to be equal to the respective
control plants.
Discussion
Plasicity in leaf area is an important means by which a
drought-stressed crop maintains control over water use
Fig. 7 Effects of short-term drought stress on protein profile ofcotton genotypes. Lane 1 represents molecular weight marker.Lanes 2, 3 and 4, respectively, represent protein samplesextracted from leaves of control, drought-stressed and drought-recovered plants, respectively, of genotype Ca/H-631. Lanes 5, 6and 7 represent protein samples extracted from leaves of control,drought stressed and drought recovered plants of genotype GM090304, respectively. Equal amount of protein (40 lg) wereloaded in each lane
Fig. 8 Effects of short-termdrought stress (dehydration)and rehydration on total freeamino acid contents of leavesin cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Plant Biotechnol Rep (2007) 1:37–48 43
123
(Blum 1996). The leaf growth was used as a physio-
logical trait to evaluate acclimation to water deficit and
the rate of leaf growth was observed to be negative
during water deficit (Fig. 1). Despite the decline in
RWC during stress cycle, leaf growth resumed after the
relieving of stress, suggesting thereby that the basic
components of leaf growth were not completely dam-
aged.
The decrease in Chl contents in drought-stressed
plants might possibly be due to changes in the lipid
Fig. 9 Effects of short-termdrought stress (dehydration)and rehydration on freeproline contents of leaves incotton genotypes (GM 090304and Ca/H 631). A stressperiod of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Fig. 10 Effects of short-termdrought stress (dehydration)and rehydration on totalsoluble sugars of leaves incotton genotypes (GM 090304and Ca/H 631). A stressperiod of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Fig. 11 Effects of short-termdrought stress (dehydration)and rehydration on reducingsugars of leaves in cottongenotypes (GM 090304 andCa/H 631). A stress period of7 days was terminated by a 7-day period of rehydration.The values are mean ± SE(n = 10). Different letters onthe top of the error barsindicate statistically differentmeans at P £ 0.01
44 Plant Biotechnol Rep (2007) 1:37–48
123
protein ratio of pigment–protein complexes or in-
creased chlorophyllase activity (Iyengar and Reddy
1996; Parida et al. 2004c). Our results agree with sev-
eral reports of decrease contents of chlorophylls and
carotenoids by drought or salt stress as reported in a
number of plant species (Logini et al. 1999; Agastian
et al. 2000). The decrease in Chl a/b ratio by drought
induction in cotton suggests that the light harvesting
complexes of thylakoid membranes are affected by
short-term drought (Parida et al. 2003).
The marginal change in protein contents and pro-
tein profiles in cotton suggests that protein synthesis
or proteolysis is affected minimally by short-term
drought stress in this plant. Several reports of alter-
ation of protein synthesis or degradation of protein in
various plant species in response to drought (Chan-
dler and Robertson 1994; Ourvard et al. 1996; Ric-
cardi et al. 1998) support our results. A drought-
induced decrease in total soluble protein has also
been reported in safflower (Carthamus mareoticus L.)
by Abdel-Nasser and Abdel-Aal (2002). We have also
reported the degradation of a 23 kDa polypeptide in
the non-secreting mangrove B. parviflora in response
to high salinity (Parida et al. 2005). However, in two
cultivars of tall fescue (Festuca arundinacea L.), levels
of 20 and 29 kDa polypeptides increased during
drought stress, and a 35 kDa polypeptide was noted in
both cultivars only when subjected to drought stress
either with or without abscisic acid treatment (Jiang
and Huang 2002). There are several reports of accu-
mulation of the dehydrin family of proteins in a wide
range of plant species under water stress varying from
9 to 200 kDa (Wood and Goldsbrough 1997; Arora
et al. 1998; Cellier et al. 1998). Our results in cotton,
contrasts with increasing evidences of drought-in-
duced accumulation of proteins and physiological
adaptations to water limitation (Bray 1997; Han and
Kermode 1996; Riccardi et al. 1998).
Total amino acid pool were increased by drought in
both the genotypes of cotton. Our results are in
accordance with many reports of increased levels of
free amino acid pool during drought in different plant
Fig. 12 Effects of short-termdrought stress (dehydration)and rehydration on starchcontents of leaves in cottongenotypes (GM 090304 andCa/H 631). A stress period of7 days was terminated by a 7-day period of rehydration.The values are mean ± SE(n = 10). Different letters onthe top of the error barsindicate statistically differentmeans at P £ 0.01
Fig. 13 Effects of short-termdrought stress (dehydration)and rehydration on totalpolyphenol contents of leavesin cotton genotypes (GM090304 and Ca/H 631). Astress period of 7 days wasterminated by a 7-day periodof rehydration. The values aremean ± SE (n = 10).Different letters on the top ofthe error bars indicatestatistically different means atP £ 0.01
Plant Biotechnol Rep (2007) 1:37–48 45
123
species (Good and Zaplachinski 1994; Mattioni et al.
1997). Thus, the drought induction in cotton showed an
increase in total amino acid pool and marginal change
in protein contents, which reflect the mode of adjust-
ment to drought in this plant.
It is well known that proline contents in leaves of
many plants are enhanced by several stresses including
drought stress (Lopez et al. 1994; Lee and Liu 1999;
Hernandez et al. 2000; De Ronde et al. 2000; Parida
et al. 2002; Abdel-Nasser and Abdel-Aal 2002). Thus,
we monitored the proline levels in leaves of cotton
genotypes during drought and recovery periods. Our
results of drought-induced dramatic increase in proline
contents in leaves of cotton agree with earlier reports
of accumulation of proline as a compatible osmolyte
during drought exposure (Lopez et al. 1994; Abdel-
Nasser and Abdel-Aal 2002). Increased accumulation
of proline in cotton might be due to the decreased
activity of proline dehydrogenase, a catabolic enzyme
of proline (Sundaresan and Sudhakaran 1995; Lee and
Liu 1999). Thus, it appears that the increase in proline
contents during drought induction is an adaptive
mechanism in cotton.
Changes observed in total protein, free amino acid
and proline contents of several drought-stressed plant
species have been attributed to a reduction in the rates
of protein synthesis and an increase in proteolytic
activity, both of which tend to cause an increase in the
total soluble nitrogen (Shen et al. 1990). In the present
study, drought resulted in a marginal decrease in total
protein. The present data also show a significant in-
crease in free amino acids (Fig. 8). These results would
suggest that the decrease in the protein contents cannot
be related to the increase in amino acids, but could be
due to the slight reduction in protein synthesis rather
than the initiation of proteolysis as previously shown in
Brassica napus (Good and Zaplachinski 1994) and
wheat seedlings (Mattioni et al. 1997). Although the
accumulation of free amino acids showed a significant
increase under drought conditions amounting to nearly
2- to 2.5-fold in both the genotypes of cotton, only the
proline contents increased by 20- to 41-fold under
identical conditions (Fig. 9). Thus, proline was high
enough to be considered the principal solute that may
allow plants to overcome drought effect through os-
motic adjustment, and serves as storage forms of
nitrogen and carbon for future use under less stressful
conditions. A function of proline as non-protein amino
acid in osmo-adjustment has been proposed, although
there may be no cause and effect relationship between
proline accumulation and osmo-regulation in plants
grown under drought conditions and responses of
plants suggested by differences in proline concentrations
and responses of plants species to drought (Sundaresan
and Sudhakaran 1995). However, the accumulation of
proline during drought may have other functions, such
as enzyme protection (Solomon et al. 1994) and sta-
bilization of biological membranes (Van Rensburg
et al. 1993), and the degradation of proline may im-
prove the energy status of cells recovering from water
deficit (Mattioni et al. 1997).
Like other cellular constituents, starch and sugar
levels are also affected by stress (Prado et al. 2000;
Abdel-Nasser and Abdel-Aal 2002). In both the
genotypes of cotton, we observed an increase in total
soluble sugar, as well as reducing sugar contents, with
a concomitant decrease in starch contents by drought
which suggest that drought induces starch sugar inter-
conversion (Chaves 1991). A drought-induced de-
crease in starch contents may also be associated with
inhibition of starch synthesis (Geigenberger et al.
1997). Our results are supported by Abdel-Nasser and
Abdel-Aal (2002), who also reported an increase in
sucrose and decrease in starch contents in safflower
(C. mareoticus L.). There are also contradictory re-
sults on the effect of water and salt stress on sugar
accumulation. Some studies have reported the sugar
contents rose (Pilon-Smits et al. 1995; Dubey and
Singh 1999; Kerepesi and Galiba 2000) while others
have found sugar contents decreased (Hanson and
Hitz 1982) or remained constant (Morgan 1992) dur-
ing stress conditions.
Polyphenol contents were increased by drought in
both the genotypes of cotton. Increase in polyphenol
contents in different tissues under salt stress has also
been reported in a number of plants (Agastian et al.
2000; Muthukumarasamy et al. 2000). Recently, Parida
et al. (2004c) reported that increases in polyphenol in
the tissue ameliorate the ionic effect of NaCl. The
enhanced level of polyphenols in cotton under drought
stress may be an acclimatory mechanism the nature of
which has yet to be elucidated.
In summary, our results showed that, in cotton,
drought induces a decrease in total chlorophylls, car-
otenoids, proteins and starch contents and an increase
in total free amino acid, proline, sugar and polyphenol
contents. The decrease in protein contents might be
due to increased proteolytic activity. Proteins are hyr-
olysed by proteases to release amino acids for storage
and/or transport and for osmotic adjustment during
drought stress in cotton. Osmotic adjustment, protec-
tion of cellular macromolecules, storage form of
nitrogen, maintaining cellular pH, detoxification of the
cells, and scavenging of free radicals are proposed
functions of free amino acid accumulation. Sugars and
polyphenols also act as compatible solutes in cotton in
46 Plant Biotechnol Rep (2007) 1:37–48
123
the drought acclimation process. Despite the decline in
RWC during the stress cycle, leaf growth resumed after
the relieving of stress, thus suggesting that the basic
components of leaf growth were not completely dam-
aged under drought stress due to accumulation of
compatible solutes. The higher efficiency of the com-
patible solute accumulation (proline, sugars and po-
lyphenols) in the genotype GM 090304 can be
considered as one of the factors responsible for its
tolerance to drought.
Acknowledgments The authors are grateful to Prof. P. Moh-anty, Former Dean, School of Life Science, JNU, New Delhi,and Dr. A.B. Das, Senior Scientist, RPRC, Bhubaneswar, fortheir valuable suggestions during the course of this investigation.The financial assistance from Ankur Seeds, Nagpur, for this re-search is duly acknowledged.
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