Plant Growth Regul (2011) 63:7–12
DOI 10.1007/s10725-010-9506-5
ORIGINAL RESEARCH
Modulation of endogenous levels of some key organic metabolites
by exogenous application of glycine betaine in drought stressed
plants of sunflower (Helianthus annuus L.)
Naeem Iqbal • Yasin Ashraf • Muhammad Ashraf
Received: 4 January 2010 / Accepted: 22 July 2010 / Published online: 29 August 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The present study was conducted to examine
the changes in some key metabolites in drought-stressed
sunflower plants supplied with glycine betaine externally.
Imposition of drought stress at the vegetative or repro-
ductive growth stages decreased the plant dry matter pro-
duction and increased the accumulation of organic solutes
(glycine betaine, proline, soluble proteins, free amino acids
and soluble sugars) in two sunflower lines, i.e., Glushan-98
and Suncross. In general, decrease in dry matter production
and increase in the endogenous levels of organic solutes,
were more pronounced when drought stress applied at the
vegetative stage than at the reproductive stage. Glycine
betaine applied as a pre-sowing seed treatment was not
found to be effective in reducing the negative effects of
drought stress in sunflower plants. Foliar application of GB
further enhanced the leaf endogenous levels of GB, soluble
proteins and total soluble sugars in drought stressed plants
without exerting any negative effects on other osmotica.
However, this GB-induced increase in endogenous levels
N. Iqbal
Department of Botany, GC University, Faisalabad, Pakistan
e-mail: naeemgc@yahoo.com
Y. Ashraf
Nuclear Institute for Agriculture & Biology (NIAB),
Faisalabad, Pakistan
e-mail: niabmyashraf@gmail.com
M. Ashraf (&)
Department of Botany, University of Agriculture,
Faisalabad, Pakistan
e-mail: ashrafbot@yahoo.com
M. Ashraf
King Saud University, Riyadh, Saudi Arabia
of organic solutes was found to be not associated with plant
dry matter production under stress conditions.
Keywords Stress tolerance
Glycine betaine
Sunflower
Introduction
Plants undergo a myriad of morphological and physiolog-
ical alterations to acclimatize themselves to various envi-
ronmental adversaries. One such mechanism, more
promising and prevalent in plants, is the accumulation of
certain organic metabolites of low molecular weight, col-
lectively known as compatible solutes. These include
sugars, polyols, amino acids, proline, and quaternary
ammonium compounds (Ashraf and Foolad 2007).
Glycine betaine (GB), an important quaternary ammo-
nium compound, is considered to be one of the most pre-
dominant and effective osmoprotectants. It is well
established that its exogenous application might have some
advantages as it improves drought tolerance in plants
(Agboma et al. 1997; Gorham et al. 2000; Iqbal et al. 2008;
Mahmood et al. 2009). It has been also reported earlier that
rate and timing of GB application significantly affects
drought tolerance ability of sunflower (Iqbal et al. 2008,
2009). This shot-gun approach of exogenous application of
GB has been used to increase the drought tolerance in some
other crop species (Agboma et al. 1997; Makela et al. 1998;
Gorham et al. 2000).
Sunflower (Helianthus annuus L.) is an important oil-
seed crop grown in different parts of the world. It is mostly
cultivated in arid and semi-arid regions of the world. Like
other crops, water stress has also a marked adverse effect on
physiological and yield attributes of sunflower (Iqbal et al.
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8 Plant Growth Regul (2011) 63:7–12
2008). Although many studies on drought tolerance of
sunflower have been carried out, little information regarding
the effect of exogenously applied GB on changing the
endogenous levels of organic metabolites in sunflower is
available. Thus, it was hypothesized that exogenous GB
application as a foliar spray or pre-sowing seed treatment
could alter the endogenous levels of some key organic sol-
utes in drought stressed sunflower plants. The present study
therefore, was conducted to examine whether and up to what
extent exogenous application of GB could alter the endog-
enous levels of different organic osmotica such as GB,
proline, soluble sugars and soluble proteins which play a
vital role in drought stress tolerance of most plant species.
Materials and methods
Two sunflower lines, viz. Gulshan-98 and Suncross were
used for the present study. Seeds of both the lines were
obtained from the regional office of Pakistan Seed Council,
Faisalabad, Pakistan. The experiment was conducted at the
field area of Department of Botany, University of Agri-
culture, Faisalabad, Pakistan. The detail on crop husbandry
and climatic conditions has been given earlier (Iqbal et al.
2008). The experiment was laid out in a split plot design
with four replications of each experimental unit. Seeds
(10 kg ha-1) of each sunflower line were hand drilled with
row to row distance (75 cm) and plant to plant distance
(30 cm). Thinning of plants was done 15 days after ger-
mination and each replicate was allotted two rows having
six plants in each row. Drought stress (water supplied up to
2,250 m3) was imposed at the vegetative or reproductive
stage of plant growth along with normal irrigation (water
supplied up to 3,000 m3) in control plots for each time. The
first irrigation was applied to all the field plots 15 days
after seed emergence. The second irrigation was applied
25 days after the first one except the plants subjected to
drought stress at the vegetative stage. The third irrigation
was applied at the time of head formation (25 days after the
second one) except the plants subjected to drought stress at
the reproductive stage.
Glycine betaine (M. Wt. 117) of Sigma–Aldrich, Japan
was used for the present study. There were three levels of
GB (0, 50 and 100 mM), applied before sowing (pre-
sowing seed soaking) and at the time of initiation of stress
treatment (foliar application) at the vegetative or repro-
ductive stage. Carboxymethyl cellulose (5% solution) was
used as a sticking agent for seed treatment and seed were
soaked for 15 h in the respective treatment, whereas
Tween-20 (0.1% solution) was used as a surfactant for
foliar spray.
After 20–25 days of each drought stress and GB treat-
ment (45–50 days after the application of last irrigation to
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stressed plants), two plants were uprooted, properly washed
the plant parts, shoots and roots were separated, and shoots
dried in an oven at 65°C for 1 week for recording shoot dry
mass. Remaining three plants were used for recording yield
at maturity.
Fresh leaf material (1.0 g) was ground in 10 mL of
distilled water and GB was estimated according to Grieve
and Grattan (1983) at 365 nm. For proline analysis, fresh
leaf tissue (0.5 g) was homogenized in 10 mL of 3% sulfo-
salicylic acid and filtered. The proline contents of the fil-
trate were determined according to Bates et al. (1973) at
520 nm using toluene as a blank. Total soluble proteins
were determined using the method of Lowry et al. (1951).
Fresh leaf material was extracted in phosphate buffer of pH
7.0 and centrifuged. The supernatant was then treated with
NaOH, Na2CO3, CuSO4 and sodium potassium tartarate.
The treated material was then reacted with Folin-Phenol
reagent and absorbance read at 620 nm. Total amino acids
were estimated according to the method of Moor and Stein
(1948). Fresh leaf material was extracted in citrate buffer
(pH 5.0), incubated at room temperature and centrifuged.
The supernatant was then treated with ninhydrin solution,
heated for 20 min and cooled. The optical density was read
at 570 nm. Total soluble sugars were determined according
to the method of Yemm and Willis (1954) using anthrone
reagent. The reducing sugars were determined in an aliquot
of 80% alcoholic extract of dry sample according to
Somogyi (1952). The non-reducing sugars were estimated
according to the method of Loomis and Shull (1937).
Statistical analysis of the data for each attribute was
carried out using the MSTAT-C computer program
(MSTAT Development Team 1989). The pooled data for
GB treatments and sunflower hybrids are reported in the
results and Figs.
Results
The data recorded at different GB levels were pooled.
Drought stress caused a marked reduction in shoot dry
matter of both sunflower lines. The inhibitory effect of
drought stress on shoot dry matter was more pronounced
when it was imposed at the vegetative rather than at the
reproductive stage or pre-sowing (Fig. 1). At the vegetative
stage, drought stressed plants produced 38% less dry bio-
mass as compared with normally irrigated plants. Con-
versely, imposition of drought stress at the reproductive
stage caused only 6% reduction in shoot dry matter of
stressed plants as compared with the control plants.
Application of GB at pre-sowing and at different growth
stages showed non-significant effects on shoot dry matter
production (Fig. 1). The two sunflower lines exhibited
significant (P B 0.01) differences with respect to shoot dry
Plant Growth Regul (2011) 63:7–12 9

Fig. 1 Effect of exogenous Control Seed treatment Spray of GB at Veg. stage Spray of GB at Rep. stage
glycine betaine application on

ot dry wt (g/plant)
600 LSD 5% (D x T x L x S) = ns
shoot dry weight, yield, total
free amino acids and total 500
soluble proteins in two 400
sunflower lines under different 300
water regimes (Mean ± S.E; 200
n = 4)

Shoo
100
0

LSD 5% (D x T x L x S) = ns
120
100

Yield/plant (g) 80
60
40
20
0

LSD 5% (D x T x L x S) = ns
2500
Tottal free amino acids

2000
(µg/g f.wt.)

1500

1000

500

LSD 5% (D x T x L x S) = ns
Total soluble sugars

120
(mg/g d.wt)

100
s

80
60
40
20
0
Normal Irrigation at Water deficit at Veg. Normal irrigation at Water deficit at Rep.
Veg. stage stage Rep. stage stage

biomass production. The sunflower line, Suncross pro-
duced 14% higher shoot biomass than that of Gulshan-98.
Drought stress applied at both vegetative and repro-
ductive stages significantly decreased the yield of sun-
flower plants. Maximum reduction was observed when
drought was imposed at the vegetative stage (Fig. 1).
However, exogenously applied GB at all growth stages
had a significant increasing effect on yield per plant of
both sunflower lines under drought stress conditions
(Fig. 1). Achene yield was improved when GB applied
through different modes at the reproductive stage under
water deficit conditions imposed at the vegetative or
reproductive stage. Increase in yield due to foliar applied
GB at the vegetative stage in plants which experienced
drought stress at the vegetative stage while the reverse
was true when water stress imposed at the reproductive
stage (Fig. 1).
Drought stress treatment resulted in a significant
(P B 0.01) increase in the leaf free amino-acids of both
lines. The increase in total free amino-acids was more
pronounced in plants subjected to drought stress at the
vegetative stage (61%) than that at the reproductive stage
(54%). Exogenous application of GB did not affect the
concentration of total free amino-acids in sunflower plants
under drought stress or normal irrigation (Fig. 1).
Imposition of drought stress at different growth stages
significantly (P B 0.01) increased total soluble sugars in
leaves of the two sunflower lines. Water deficit imposed at
the vegetative stage increased total soluble sugars by 23%
in the leaves of the drought stressed plants as compared to
the control plants (Fig. 1). A 22% increase in total soluble
sugars of drought stressed plants as compared to non-
stressed plants was observed when water deficit was
imposed at the reproductive stage.

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10 Plant Growth Regul (2011) 63:7–12

Fig. 2 Effect of exogenous Control Seed treatment Spray of GB at Veg. stage Spray of GB at Rep. stage
glycine betaine application at
450 LSD 5% (D x T x L x S) = 31.58
different growth stages on leaf
A
proline, glycine betaine and 400 AB AB B AB AB A

g/g f.wt.)
AB
total soluble proteins in two 350 CD CD
C C
CD CD
sunflower lines under different 300
D D

Leaf proline (µg

water regimes (Mean ± S.E;
250
n = 4). Bars with different
letters (A–J) differ significantly 200
at P B 0.05 150
100
50
0

LSD 5% (D x T x L x S) = 58.47
aine (µg/g f.wt.)

1000
900 A AB
800 C BC
C
700 D D
600 E E
Leaf glycine beta

500
F
400 FG FGH
HI GHI
300 I HI

200
100
0
oteins (µg/g f.wt.)

LSD 5% (D x T x L x S) = 39.7

3500
D B AB A A
3000 C E
G F F
2500
H
H H
2000
Total soluble pro

J I IJ
1500
1000
500
0
Normal Irrigation at Water deficit at Veg. Normal irrigation at Water deficit at Rep.
Veg. stage stage Rep. stage stage

Leaf proline concentration in both sunflower lines growth stages (Fig. 2). However, maximum values of leaf
increased significantly (P B 0.01) under water limited endogenous GB was recorded in plants sprayed with
conditions, imposed at the vegetative or reproductive stage. 100 mM of GB followed by 50 and 0 mM. Seed treatment
Shortage of water at the vegetative stage caused an increase with either level of this osmolyte showed almost no effect
in leaf proline concentration by 28% as compared to that in on leaf GB concentration. A gradual increase in leaf
well-watered plants. When water deficit was imposed at the internal GB was recorded in plants sprayed with 50 and
reproductive stage, drought stressed plants accumulated 100 mM GB at the vegetative or reproductive growth
24% more leaf proline than that of the non-stressed ones. stage. Under normal irrigation, leaf GB was somewhat
Exogenously applied GB at all growth stages had non- higher in plants receiving GB at the reproductive stage than
significant effect on leaf proline concentration (Fig. 2). that at the vegetative stage (Fig. 2). Exogenous (foliar)
More increase in endogenous GB concentration was application of GB was found effective when it was applied
found in plants exposed to water deficit at the vegetative at the time of the initiation of water deficit treatment at the
stage than that in plants exposed to water deficit at the vegetative or reproductive stage (Fig. 2) but pre-sowing
reproductive stage. The leaf GB concentration of the sun- treatment did not show any appreciable influence. Under
flower lines also differed significantly (P B 0.01) being drought stress, 50 and 100 mM GB applied at the vegeta-
32% higher in Suncross than that of Gulshan-98. Pooled tive stage increased leaf GB concentration by 17 and 29%,
data indicated that exogenous application of GB on its respectively with regard to controls. However, when
endogenous level was variable when applied at different drought stress was imposed at the reproductive stage,

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Plant Growth Regul (2011) 63:7–12 11

Table 1 Regression analysis showing the dependence of yield per that foliar-applied GB enhances its endogenous level under
plant on different metabolites of two sunflower (Helianthus annuus environmental stresses in GB accumulators (Gorham et al.
L.) under drought stress (R2 = 0.996)
2000). The results of our experiment strongly support this
Variables Coefficient Standard error P value view, because a significant increase in leaf GB concen-
tration was observed by its exogenous application in both
Intercept 108.1 9.74 1.489 ns
lines of sunflower, a natural GB accumulator.
Shoot dry weight 0.168 0.02 4.22 ns
Leaf proline of drought stressed plants was significantly
Leaf glycine betaine 0.016 0.008 0.066*
higher than that of normally irrigated plants. However, leaf
Leaf proline -0.02 0.11 0.854 ns
proline concentration in drought stressed and non-stressed
Total soluble protein 0.003 0.003 0.359 ns
plants of both sunflower lines was not affected by exoge-
Total soluble sugars -1.21 0.49 0.037**
nous GB application as pre-sowing or at all growth stages.
Total free amino acids -0.013 0.009 0.208 ns
Makela et al. (1998) also found that exogenous application
* Significant at 0.05 level; ** significant at 0.01 level; ns non- of GB did not alter the proline concentration in tomato
significant plants grown under stress conditions. There are, however,
some contrasting reports which indicate the non-compati-
plants sprayed with 50 and 100 mM GB at the same stage bility of exogenous GB in some plants. For example,
had 33 and 39% higher leaf GB, respectively than the Larher et al. (1995) found that exogenous GB exerts
untreated plants. inhibitory effects on osmo-induced proline accumulation in
Water deficit treatments caused a significant (P B 0.01) rape leaf discs suggesting that it is not a compatible solute.
increase in total soluble proteins in both sunflower lines In contrast, Sulpice et al. (2002) found an increase in leaf
(Fig. 2). In normally irrigated plants, the value of leaf proline concentration in response to exogenous GB appli-
soluble proteins at the reproductive stage was higher than cation in canola leaf discs. The difference between the
that at the vegetative stage. The effect of drought stress on results of the present experiment and those of the above
total soluble proteins was more pronounced when it was mentioned studies can be explained by the fact that in the
imposed at the vegetative stage than that at the reproduc- present study, GB was sprayed on intact plants, whereas
tive stage (Fig. 2). Exogenous application of GB at dif- contradictory results of some pervious studies (Larher et al.
ferent growth stages had also a significant (P B 0.01) 1995; Sulpice et al. 2002) were based on indirect evidence
effect on total soluble proteins (Fig. 2) but it was non- (in vitro).
significant in case of pre-sowing treatment of GB. The role of amino-acids in osmotic adjustment of plants
Regression analysis presented in Table 1 showed that grown under water limited environment is well docu-
improvement in yield of sunflower plants significantly was mented. A highly significant increase in total free amino
associated with the accumulation of leaf glycinebetaine and acids of drought stressed plants over control was observed
total soluble sugars. in the present study. The increase in total free amino acids
due to drought stress can be related to decrease in total
soluble proteins in both sunflower lines, because it is
Discussion reported that under water limited conditions, structural
proteins break up into component amino acids, which take
It is evident from the results of present experiment that GB an active part in osmotic adjustment under such stressful
application as pre-sowing or foliar spray at any growth environment. The present findings clearly showed non-
stage was not effective in alleviating the water deficit significant effects of exogenous pre-sowing or foliar
induced inhibition in dry matter production in both sun- application of GB on total free amino acids in sunflower
flower lines, despite the fact that leaf endogenous level of plants grown under both normally irrigated and drought
GB was enhanced by its foliar application. Makela et al. stressed regimes. In contrast, Sulpice et al. (2002) reported
(1996) also reported a non-significant role of foliar sup- a marked increase (2- to 3-fold) in free amino acids by
plied GB in alleviating the adverse effects of drought stress exogenous application of GB on detached leaf discs of
on growth of Brassica napus. Arabidopsis thaliana and Brassica napus.
The enhancement of endogenous GB level by its exog- Exogenous application of GB did not alter the concen-
enous application has been reported by many researchers in tration of total soluble proteins when applied before sowing
GB non-accumulating plants such as tomato (Makela et al. (seed treatment) or at the time of initiation of water deficit
1998), and rape (Sulpice et al. 2002) grown under stressful at the vegetative stage. Sulpice et al. (2002), however,
environment. The role of exogenous GB in increasing leaf found that exogenous application of GB decreased the total
endogenous concentration in GB accumulators is however, soluble proteins in canola and Arabidopsis thaliana. These
controversial. However, some scientists were of the view scientists were of the view that the lower level of soluble

123
12
proteins in GB treated leaf discs could be related to the
inhibitory effect of GB on protein biosynthesis, thus sug-
gesting its incompatibility for these non-accumulators. The
above mentioned study was carried out in vitro (using
detached leaf discs) with GB non-accumulators (Bassica
napus and Arabidopsis thaliana), but the present study was
carried out in vivo (using intact leaves) with a natural GB
accumulator, i.e., sunflower. Hence, the response of GB
was different in the studies reported earlier and in the
present investigation.
In the present experiment, an increase in soluble sugars
was observed due to water deficit treatments, particularly
when imposed at the vegetative stage. These results can be
related to earlier findings (Irigoyen et al. 1992), in which an
increase in soluble sugars due to drought stress in alfalfa
was also reported, suggesting the role of soluble sugars in
osmoregulation under stressful environment.
The present findings clearly indicate that leaf endoge-
nous level of GB in drought stressed plants increased by
the foliar application of this compound without decreasing
the level of other solutes. Pre-sowing seed treatment with
GB was ineffective in enhancing either growth or level of
any of the solutes analyzed in the present study.
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