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ORIGINAL ARTICLE
Moisture stress induced increases in the activity of enzymesof osmolytes biosynthesis are associated with stress tolerancein wheat genotypes
Aradhna Kumari • R. K. Sairam
Received: 9 October 2012 / Accepted: 9 April 2013 / Published online: 24 September 2013
� Indian Society for Plant Physiology 2013
Abstract Moisture stress is one of the most important
factors limiting the survival and growth of plants in semi
arid tropics. Osmotic adjustment is an important adaptive
response to moisture stress in wheat (Triticum aestivum L.).
The objective of this study was to determine the relation-
ships between osmolyte accumulation, activity of enzymes
involved in osmolyte biosynthesis with moisture stress
tolerance in different wheat genotypes. An experiment was
conducted to study the role of osmolytes in imparting
moisture stress tolerance in five selected wheat genotypes,
two tolerant and three susceptible. Contents of proline,
glycine betaine and trehalose accumulated in all the
genotypes under moisture stress in comparison to control.
Moisture stress tolerant genotypes like C 306 and HD 2987
maintained higher level of these osmolytes in comparison
to susceptible genotypes HD 2733, PBW 343 and HD 2967
under moisture stress condition. Studies on the activities of
enzymes involved in osmolyte biosynthesis showed that
comparatively greater increases in the activity of trehalose-
6-phosphate synthase, betaine aldehyde dehydrogenase and
pyrroline-5-carboxylate synthetase under moisture stress
lead to increased biosynthesis of various osmolytes in
tolerant genotypes C 306 and HD 2987, which ultimately
resulted in improved moisture stress tolerance as compared
to susceptible genotypes HD 2733, PBW 343 and HD
2967.
Keywords Glycine-betaine � Moisture stress �Osmolytes � Proline � Trehalose � Wheat
Introduction
Plants respond to various types of stresses, such as drought,
salinity, and high and low temperatures by a number of
physiological and developmental changes. During moisture
stress, plant cells can undergo changes in concentrations of
solutes, in cell volume and in the shape of cell membranes,
as well as disruption of gradients in water potential, loss of
turgor, disruption of membrane integrity and the denatur-
ation of proteins. Compatible osmolytes are potent osmo-
protectants that play important role in counteracting the
effects of osmotic stress. Compatible solutes are overpro-
duced under osmotic stress aiming to facilitate osmotic
adjustment (Hasegawa et al. 2000; Shao et al. 2005; Zhu
2000). The osmotic potential inside the cell is lowered by
the accumulation of osmolytes in the cytosol. It has been
suggested that osmolytes/compatible solutes do not inter-
fere with normal biochemical reactions and act as osmo-
protectants during osmotic stress (Yoshu et al. 1997).
Osmotic adjustment (OA) is considered to be an important
component of drought tolerance mechanisms in plants
(Zhang and Oweis 1999). Compatible solutes include some
amino acids (e.g., proline), sugar alcohols (e.g., mannitol,
pinitol, inositol), other sugars (e.g., sucrose, trehalose), and
quaternary ammonium compounds (e.g., glycine betaine),
which accumulate at high concentrations without the dis-
ruption of protein functions.
Soluble sugars (Kameli and Losel 1995, 1996; Rekika
et al. 1998) and proline (Ali-dib et al. 1994; Rascio et al.
1994; Mattioni et al. 1997) have been shown to increase
under water stress and are potentially important contributors
A. Kumari � R. K. Sairam (&)
Division of Plant Physiology, Indian Agricultural Research
Institute, Pusa, New Delhi 110012, India
e-mail: [email protected]
Present Address:
A. Kumari
Department of Plant Physiology, College of Agriculture, Sub
Campus JNKVV, Ganj Basoda, MP, India
123
Ind J Plant Physiol. (July–September 2013) 18(3):223–230
DOI 10.1007/s40502-013-0032-0
to OA. Proline has been shown to have a key role in stabi-
lizating cellular proteins and membranes under osmotic
stress (Errabii et al. 2006). Vendruscolo et al. (2007) found
that proline is involved in tolerance mechanisms against
oxidative stress and this was the main strategy of plants to
avoid detrimental effects of water stress. Higher proline
content in wheat plants under water stress has been reported
by Vendruscolo et al. (2007) and Chandrasekhar et al.
(2000).
Trehalose, a non-reducing disaccharide of glucose,
serves as a protectant in response to multitude of stress
conditions and also as reserve of carbohydrate. It has been
suggested that the combined accumulation of sucrose and
trehalose might be sufficient to protect the plant(s) against
the adverse effects caused by desiccation (Goddijn and
Dun 1999). Trehalose can stabilize dehydrated biological
structures, such as membranes or enzymes, more effec-
tively than other sugars (Paiva and Panek 1996).
Glycine-betaine is a quaternary ammonium compound
synthesized naturally in a wide variety of organisms. Many
studies indicate that GB might play an important role in
enhancing plant tolerance to individual drought stress and
heat stress (Khan et al. 2009). Glycine-betaine has been
shown to stabilize enzymes and membranes (Zhao et al.
1992) and the photosystem II protein-pigment complex
under stress (Murata et al. 1992; Papageorgiou and Mura-
rata 1995).
The increased synthesis of osmolytes is achieved by
modulating activities of enzymes of the osmolyte biosyn-
thetic pathway. For instance, simultaneous up-regulation of
pyrroline-5-carboxylate synthase (P5CS) and down regu-
lation of the proline dehydrogenase (ProDH) leads to
proline accumulation during water stress (Yoshiba 1997).
Betaine aldehyde dehydrogenase (BADH) activity has been
shown to be induced by salt or water stress. In salt- or
water-stressed leaves of barley (Ishitani et al. 1995),
Amaranth (Legaria et al. 1998), and sugar beet (McCue and
Hanson 1992) the rise in BADH activity paralleled the
accumulation of betaine, and was accompanied by an
increase of the levels of BADH protein and mRNA. A
sharp increase in trehalose-6-phosphate synthase (TPS)
activity has been reported under drought stress conditions,
and greater increase was observed in roots, which reached
to three to four times of its activity under control condition.
The increase in the activity of TPS showed parallelism with
trehalose accumulation under stress condition (El-Bashiti
et al. 2005). It is known that abiotic stress is the primary
cause of crop loss worldwide, causing average yield losses
of more than 50 % for major crops (Boyer 1982, 2000).
The objective of present investigation was to study the role
of osmolytes accumulation, activity of enzymes responsi-
ble for their synthesis with moisture stress tolerance in
wheat genotypes.
Materials and methods
An experiment was conducted in the pot culture, Division of
Plant Physiology, Indian Agricultural Research Institute,
Pusa, New Delhi during the rabi (winter) seasons of
2011–2012 with two tolerant genotypes, C 306 and HD 2987
and three susceptible genotypes PBW 343, HD 2733, HD
2967 in earthen pots of uniform size (30 9 30 cm), filled
with 10 kg mixture of air dried soil and farm yard manure in
3:1 ratio. Moisture stress was given to plants at 50 days after
sowing (DAS) and anthesis through withdrawal of irrigation.
Measurement of water stress was done through tensiometer,
which records moisture tension. A calibration curve was
made with the help of pressure plate apparatus at different
pressure from 0 to 15 bars for the same soil and farm yard
mixture. One bar soil water potential condition was opti-
mized for taking wheat samples, which is nearly 67–70 %
moisture of field capacity. Schedule routine of irrigation was
practiced for control plants and for treated plants when they
were not subjected to water stress, throughout the crop
growth period. Each treatment was replicated 20 times for all
the genotypes in the form of pots.
The plants were sampled and observations were taken
for total soluble sugar, proline, glycine betaine, trehalose
content and enzymes related to their synthesis at (1) veg-
etative (60 days after sowing), and (2) at anthesis stages.
At each stage samples were collected in triplicate from 3
pots. At vegetative stage upper most expanded leaf i.e.,
third leaf from the top and at anthesis flag leaf was used for
recording observations. For estimation of total soluble
sugars and glycine betaine content leaf material were dried
in an oven at 60 �C and ground to fine powder using a
grinding machine. Proline and activities of various
enzymes were estimated in fresh leaf samples. For treha-
lose leaf sample were collected, dipped in liquid nitrogen
and stored in -20 �C refrigerator.
Leaf relative water content (RWC) was estimated by
recording the turgid weight of 0.5 g fresh leaf samples by
keeping in water for 4 h, followed by drying in hot air oven
till constant weight was achieved (Weatherley 1950).
RWC ¼ ½ðFresh wt.�Dry wt.Þ=ðTurgid wt.�Dry wt.Þ�� 100
For estimation of total sugar samples were extracted as
per McCready et al. (1950) and estimation was done
according to Sadasivam and Manickam (1992). Estimation
of glycine-betaine was done as per Greive and Grattan
(1983), proline by the protocol of Bates et al. (1973) and
trehalose by the method of Ferreira et al. (1997).
D-Pyrroline-5-carboxylate synthetase activity was
assayed by recoding the decrease in optical density due to
NADPH at 340 nm (Garcia-Rios et al. 1997). BADH
activity was assayed as per the protocol described by Xiao
224 Ind J Plant Physiol. (July–September 2013) 18(3):223–230
123
et al. (1997). Trehalose 6-phosphate synthase activity was
measured using the coupled assay described by Hottiger
et al. (1987).
Results
Relative water content
There was decline in RWC under moisture stress condition
in all the genotypes at vegetative stage and anthesis, and
the mean decline was 19 and 16 % respectively (Table 1).
HD 2987 and C 306 showed least reductions at both the
stages over control, while PBW 343, HD 2967 and HD
2733 showed greater declines.
Total sugar content
Total sugar contents increased significantly under moisture
stress condition at both the stages, and all the genotypes
(Fig. 1A). The sugar content was found to be highest at
anthesis stage of the plant. The increases were considerably
higher in tolerant genotype C 306, followed by HD 2,987 at
both vegetative and anthesis stages. Across all the stages
and stress treatments, average soluble sugar content in the
leaves was higher in C 306. At vegetative stage tolerant
genotypes accumulated 6–8 times more sugar, while sus-
ceptible genotypes accumulated 3–4 times of their
respective controls. Similar trend in sugar accumulation
was observed at anthesis stage.
Proline content
Proline content increased in all the genotypes at vegetative
and anthesis stages under water stress treatments (Fig. 1B).
There was significantly greater increase in proline content
in C 306, HD 2687 and PBW 343 at both the stages under
water stress treatments. Though total proline content was
more at anthesis stage, the percent increase in proline
content under water stress was more at vegetative stage. At
vegetative and anthesis stages C 306 and HD 2987 showed
10 and fourfold, and 6 and threefold increases, respec-
tively, in proline content under water stress. HD 2733 and
HD 2967 showed very less increase in proline content
under water stress at both the stages, which ranged from 24
to 49 %. PBW 343 showed intermediate response in pro-
line accumulation under moisture stress condition with
about twofold increase at both vegetative and anthesis
stages. HD 2733 showed minimum proline content among
all the genotypes under moisture stress condition at both
vegetative and anthesis stages.
Glycine betaine content
Glycine betaine content increased under moisture stress in
all the genotypes at all the stages (Fig. 1C). Under moisture
stress, HD 2987 maintained highest GB accumulation of
190 and 211 % at the vegetative and anthesis stages,
respectively. C 306 also showed higher GB accumulation,
though slightly less than HD 2987, viz., 160 and 126 %
increases at vegetative and anthesis stages, respectively,
under water stress. PBW 343, HD 2733 and HD 2967
showed 57, 76 and 72 % increases, respectively under
water stress at vegetative stages and 51, 70 and 93 %
increases, respectively at anthesis stage.
Trehalose content
Trehalose content increased with the decrease in water
availability in all the eight genotypes at both vegetative and
anthesis stages (Fig. 1D). But the increases were very
Table 1 Effect of moisture stress on relative water content (%) in wheat genotypes at different growth stages
Genotypes Vegetative Anthesis
Control Moisture stress Mean % Decrease Control Moisture stress Mean % Decrease
C 306 70.52 (88.91) 58.95 (73.43) 61.22 (81.17) 16.41 (17.41) 65.14 (82.35) 57.58 (71.29) 61.36 (76.82) 11.60 (13.43)
HD 2987 69.07 (87.26) 57.83 (71.69) 60.31 (79.48) 16.27 (17.85) 64.13 (81.00) 56.72 (69.92) 60.42 (75.46) 11.56 (13.68)
PBW 343 71.39 (89.84) 54.30 (65.98) 57.18 (77.55) 23.94 (25.96) 64.51 (81.51) 51.17 (60.71) 57.84 (71.11) 20.68 (25.52)
HD 2733 67.07 (84.86) 53.61 (64.84) 57.49 (74.85) 20.07 (23.60) 63.07 (79.52) 51.76 (61.73) 57.42 (70.63) 17.92 (22.37)
HD 2967 66.91 (84.66) 54.72 (66.67) 61.22 (75.66) 18.23 (21.25) 63.92 (80.72) 53.12 (64.02) 58.52 (72.37) 16.90 (20.69)
Mean 68.99 (86.96) 55.88 (68.52) 59.02 (77.74) 64.15 (81.02) 54.07 (65.53) 59.11 (73.28)
Stage (S) Treatments (T) S 9 T Genotypes (G) S 9 G T 9 G S 9 T 9 G
SEm± 0.058 0.058 0.081 0.091 0.129 0.129 0.182
LSD (P B 0.05) 0.165 0.165 0.233 0.26 0.368 0.368 0.521
Data shown in table is transformed value, original data is written in parentheses which are in percentage
Ind J Plant Physiol. (July–September 2013) 18(3):223–230 225
123
drastic at anthesis stage in comparison to at vegetative
stage. Under moisture stress C 306 showed maximum
trehalose accumulation at vegetative stage and HD 2987 at
anthesis stage, while HD 2733 showed minimum trehalose
accumulation at vegetative stage and HD 2967 at anthesis
stage. At vegetative stage C 306 and HD 2987 showed 2.8
and threefold increases, respectively in trehalose content
under water stress. PBW 343, HD 2733 and HD 2967
showed lesser trehalose accumulations, which ranged from
42 to 60 % at vegetative stage. At anthesis stage C 306 and
HD 2987 showed 10 and sevenfolds increases, respec-
tively, in trehalose accumulation under moisture stress
condition. PBW 343, HD 2733 and HD 2967 also
performed comparatively better at anthesis stage, with
more than threefolds increase under stress condition.
Pyrroline-5-carboxylate synthetase activity
D-Pyrroline-5-carboxylate synthetase activity increased in
all the genotypes under moisture stress treatment (Fig. 2A).
There was 4–4.5 fold increase in P5CS activity in C 306
and HD 2987 at vegetative stage and 3.2–3.5 folds increase
at anthesis stage under moisture stress compared to control.
PBW 343, HD 2733 and HD 2967 showed 2–2.4 fold
increases in activity at vegetative and anthesis stages under
moisture stress condition. At anthesis, the moisture stress
0
50
100
a
b
c
d
150
200
250
300
To
tal s
olu
ble
su
gar
(mg
g-1
dry
wt.
)
Control Water stress
0
500
1000
1500
2000
2500
3000P
rolin
e c
on
ten
t(µ
g g
-1d
ry w
t.)
0
300
600
900
1200
1500
1800
Gly
cin
e b
etai
ne
co
nte
nt
(µg
g-1
dry
wt.
)
0
300
600
900
C 3
06
HD
298
7
PB
W 3
43
HD
273
3
HD
296
7
C 3
06
HD
298
7
PB
W 3
43
HD
273
3
HD
296
7
Tre
hal
ose
co
nte
nt
(µm
ol g
-1d
ry w
t.)
AnthesisVegetative
Fig. 1 Effect of water stress on
the contents of soluble sugar (a),
proline (b), glycine-betaine
(c) and trehalose (d) in wheat
genotypes. LSD significant
(P B 0.05). Vertical bars
show ± SE of mean
226 Ind J Plant Physiol. (July–September 2013) 18(3):223–230
123
induced up regulation in activity was less in all the geno-
types compared to vegetative stage. C 306 and HD 2987
maintained higher moisture stress induced P5CS activity at
both the stages.
Betaine aldehyde dehydrogenase activity
Moisture stress treatment led to an increase in BADH
activity in all the genotypes (Fig. 2B). All genotypes
showed comparatively higher enzyme activity at anthesis
stage compared to at vegetative stage, except C 306, which
showed similar increase in activity at both the stages under
moisture stress. Under moisture stress condition HD 2967
and PBW 343 showed lowest BADH activity at vegetative
and anthesis stages, respectively, C 306 showed highest
activity at both the stages. C 306 and HD 2987 showed
120–180 % increases at both the stages under water stress
condition, while PBW 343, HD 2733 and HD 2967 showed
only 14–91 % increases under moisture condition.
Trehalose -6-phosphate synthase activity
Significant stress induced up regulation in the activity
T6PS was observed under moisture stress at both the stages
(Fig. 2C). But the higher increase in activity was observed
at anthesis stage as compared to vegetative stage. PBW
343, HD 2733 and HD 2967 showed comparatively lesser
increases in enzyme activity under moisture stress condi-
tion compared to C 306 and HD 2987. At vegetative stage
HD 2733 and at anthesis stage PBW 343 showed lowest
level of enzyme activity under water stress. At both the
stages HD 2987 maintained highest activity under water
stress treatment.
Discussion
The results obtained in the present study, conducted with
two tolerant genotypes, C 306 and HD 2987 and three
0
5
10
15
20
25
30a
b
c
Pyr
rolin
e 5
carb
oxy
late
sy
nth
etas
e ac
tivi
ty(u
nit
mg
-1p
rote
in m
in-1
) Control Water stress
0
10
20
30
40
50
60B
etai
ne
ald
ehyd
e d
ehyd
rog
enas
e ac
tivi
ty(n
mo
l mg
-1p
rote
in m
in-1
)
0
5
10
15
20
25
C 3
06
HD
298
7
PB
W 3
43
HD
273
3
HD
296
7
C 3
06
HD
298
7
PB
W 3
43
HD
273
3
HD
296
7
Tre
hal
ose
6 p
ho
sph
ate
syn
thas
e ac
tivi
ty(µ
mo
l N
AD
H m
g-1
pro
tein
m
in-1
)
Vegetative Anthesis
Fig. 2 Effect of water stress on
the activity of pyrolline-5-
carboxylate synthetase (a),
betaine aldehyde dehydrogenase
(b) and trehalose-6-phosphate
synthase (c) in wheat genotypes.
LSD significant (P B 0.05).
Vertical bars show ± SE of
mean
Ind J Plant Physiol. (July–September 2013) 18(3):223–230 227
123
susceptible genotypes PBW 343, HD 2733, HD 2967 for
osmolyte content and related enzyme activity revealed
differential response to moisture stress. Tolerant genotypes
C 306 and HD 2987 were able to retain higher RWC under
moisture stress as compared to susceptible genotypes.
It has been suggested that sugars are the best osmolytes
to accumulate under moisture stress. Results obtained from
this experiment also showed very high accumulation of
sugar under water stress to cope the situation. The tolerant
genotypes showed much greater increase in total sugar
content under moisture stress condition than the susceptible
genotypes. Sugars play role in OA, as evidenced by
increase in soluble sugars accumulation under moisture
stress (Johari-Pireivatlou et al. 2010). A central role of
sugars depends not only on direct involvement in the
synthesis of other compounds, production of energy but
also on stabilization of membranes (Hoekstra et al. 2001).
Higher amount of soluble sugars and a lower amount of
starch were found under water stress conditions in maize
plants (Mohammadkhani and Heidari 2008).
In this investigation proline, glycine-betaine and treha-
lose accumulated in higher amount under moisture stress,
and the accumulation was greater in tolerant genotypes C
306 and HD 2987 compared to susceptible genotypes PBW
343, HD 2733 and HD 2967. It has been shown that proline
plays a key role in stabilizing cellular proteins and mem-
branes in the presence of high concentrations of osmoticum
(Errabii et al. 2006). These results are in accordance with
the findings of Mohammadkhani and Heidari (2008), Tatar
and Gevrek (2008) and Kameli and Losel (1996) who
reported increase in sugar and proline content under
moisture stress conditions. Higher proline content in wheat
plants after moisture stress has also been reported by
Vendruscolo et al. (2007), Poustini et al. (2007) and Tian
and Lei (2007). Vendruscolo et al. (2007) indicated that
accumulation of high proline content could be a very good
criterion for selecting tolerant genotypes.
It has been reported that the accumulation of GB in
plants is induced by stress conditions (Gorham 1995).
Many studies indicated that GB might play an important
role in enhancing plant tolerance to drought, salinity and
heat stresses (Khan et al. 2009). Accumulation of GB could
protect the chloroplast and thylakoid lamellae from damage
not only under moisture stress or heat stress separately, but
also when they were applied in combination (Wang et al.
2010). Wang et al. (2010) further reported that the
improvement of water balance and antioxidant metabolism
may be involved in GB mediated increase in photosyn-
thesis under stress condition.
Accumulation of Trehalose under drought condition has
been reported by many researchers. El-Bashiti et al. (2005)
noticed higher accumulation of trehalose in tolerant
genotypes of wheat under moisture stress compared to
susceptible genotypes. Trehalose can stabilize dehydrated
biological structures, such as lipid membranes or enzymes,
more effectively than other sugars (Colaco et al. 1995).
Activity ofD-pyrroline-5-carboxylate synthetase increased
under moisture stress in all the genotypes. The activity was
much higher in tolerant genotypes which could be the reason
for higher accumulation of proline in these genotypes.
Accumulation of proline in plants under stress is a result of the
reciprocal regulation of two pathways: increased activity of
proline synthetic enzymes and repressed activity of proline
degradation (Peng et al. 1996; Delauney and Verma 1993).
In the present study a parallel increase was observed in
glycine-betaine content and BADH activity. This indicates
that glycine-betaine accumulation in water stressed plants
is regulated via changes in the activity of the glycine-
betaine biosynthesis pathway. Similar result has been
reported by Gao et al. (2004).
The increase in trehalose content under moisture stress
was accompanied by an increase in TPS activity in all the
wheat genotypes. A sharp increase in TPS activity has been
reported under drought stress conditions, and greater
increase was observed in roots, which reached to three to
four times of its activity under control condition. Enhanced
activity of T6PS enzyme has also been reported by El-
Bashiti et al. (2005) in water stressed wheat plants.
It can thus be concluded that increase in various
osmolytes like soluble sugars, trehalose, glycine-betaine
and proline observed under drought stress conditions in all
the wheat genotypes was significantly higher in tolerant
genotypes. This study underlined the significance of
osmolytes as osmoprotectant compound in wheat geno-
types under drought stress conditions. Osmolyte accumu-
lation was achieved by increased activity of the enzymes
involved in biosynthesis of osmolytes, resulting in osmo-
lyte accumulation. OA led to stress acclimation in wheat
genotypes. There was significant positive relationship
between osmolyte accumulation and water stress tolerance.
Genotypes like C 306 and HD 2987, which exhibited
greater accumulation of soluble sugars, trehalose, proline
and glycine betaine have better osmo-regulation potential
and therefore, exhibited higher RWC under water stress, as
compared to susceptible genotypes, PBW 343, HD 2967
and HD 2733, which showed less osmo-regulation and
consequently greater decline in RWC. So, differential
accumulation of compatible solutes can be a useful trait to
select moisture stress tolerant wheat genotypes.
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