Environmental and Experimental Botany 59 (2007) 206216

Roles of glycine betaine and proline in improving

plant abiotic stress resistance
M. Ashraf a , M.R. Foolad b,
a

Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan

Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA

Received 13 May 2005; received in revised form 15 September 2005; accepted 20 December 2005

Abstract
Glycine betaine (GB) and proline are two major organic osmolytes that accumulate in a variety of plant species in response to environmental
stresses such as drought, salinity, extreme temperatures, UV radiation and heavy metals. Although their actual roles in plant osmotolerance remain
controversial, both compounds are thought to have positive effects on enzyme and membrane integrity along with adaptive roles in mediating
osmotic adjustment in plants grown under stress conditions. While many studies have indicated a positive relationship between accumulation
of GB and proline and plant stress tolerance, some have argued that the increase in their concentrations under stress is a product of, and not
an adaptive response to stress. In this article, we review and discuss the evidence supporting each of these arguments. As not all plant species
are capable of natural production or accumulation of these compounds in response to stress, extensive research has been conducted examining
various approaches to introduce them into plants. Genetically-engineered plants containing transgenes for production of GB or proline have thus far
faced with the limitation of being unable to produce sufficient amounts of these compounds to ameliorate stress effects. An alternative shot-gun
approach of exogenous application of GB or proline to plants under stress conditions, however, has gained some attention. A review of the literature
indicates that in many, but not all, plant species such applications lead to significant increases in growth and final crop yield under environmental
stresses. In this review article, numerous examples of successful application of these compounds to improve plant stress tolerance are presented.
However, to streamline useful and economic applications of these compounds, further investigations are needed to determine the most effective
concentrations and number of applications as well as the most responsive growth stage(s) of the plant. All these factors may vary from species to
species. Furthermore, a better understanding of the mechanisms of action of exogenously applied GB and proline is expected to aid their effective
utilization in crop production in stress environments.
2006 Published by Elsevier B.V.
Keywords: Compatible solutes; Drought stress; Environmental stress; Induced tolerance; Osmoprotection; Osmoregulation; Salt stress; Stress tolerance

1. Introduction
Several environmental factors adversely affect plant growth
and development and final yield performance of a crop. Drought,
salinity, nutrient imbalances (including mineral toxicities and
deficiencies) and extremes of temperature are among the major
environmental constraints to crop productivity worldwide. It
is estimated that less than 10% of the worlds arable lands
may be free of major environmental stresses (Dudal, 1976),
with drought and salinity stresses being the most widespread
(Ashraf, 1994). For instance, up to 45% of the world agricultural lands are subject to continuous or frequent drought,

Corresponding author. Tel.: +1 814 865 5408; fax: +1 814 863 6139.
E-mail address: mrf5@psu.edu (M.R. Foolad).

0098-8472/$ see front matter 2006 Published by Elsevier B.V.

doi:10.1016/j.envexpbot.2005.12.006

wherein 38% of the world human population resides (Bot et

al., 2000), and worldwide area mapped as being affected by
salinity is more than 3 106 km2 , or approximately 6% of
the total land area (Flowers et al., 1977). Also, 19.5% of the
irrigated agricultural lands are considered saline (Flowers and
Yeo, 1995). Furthermore, each year there is a deterioration of
2 million ha (about 1%) of world agricultural lands to salinity,
leading to reduced or no crop productivity (Syverstein et al.,
1989; Tanji, 1990; Kalaji and Pietkiewica, 1993; Szabolcs, 1994;
Choukr-Allah, 1995). Low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and
poor cultural practices are among the major contributors to the
increasing salinity. Secondary salinization, in particular, exacerbates the problem where once productive agricultural lands
are becoming unfit to cultivation due to poor quality irrigation
water.

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Changes in ambient temperature occur more rapidly than

changes in stress factors such as drought, salinity and nutrient
imbalances. Furthermore, temperature extremes aggravate the
adverse effects of other stresses, including drought and salinity, on crop production and quality. For instance, heat stress
adversely affects grain quality and final crop yield in 40% of the
irrigated wheat growing area of the world (Fischer and Byerlee,
1991). The impact of global warming differs regionally, and it is
envisaged that developing countries will be affected to a greater
extent, thereby resulting in increased food insecurity therein
(Rosenzweig and Parry, 1994). Cold stress, although seasonal,
has some similarities to drought stress, because as water freezes
it creates concentrated solutions of solutes, thereby subjecting
plants to a shortage of liquid water.
Tolerance to abiotic stresses is very complex at the whole
plant and cellular levels (Foolad, 1999a,b; Foolad et al., 2003a,b;
Ashraf and Harris, 2004). This is in part due to the complexity of
interactions between stress factors and various molecular, biochemical and physiological phenomena affecting plant growth
and development (Zhu, 2002). Currently, there are no economically viable technological means to facilitate crop production
under stress conditions. However, development of crop plants
tolerant to environmental stresses is considered a promising
approach, which may help satisfy growing food demands of
the developing and under-developed countries. Development of
crop plants with stress tolerance, however, requires, among others, knowledge of the physiological mechanisms and genetic
controls of the contributing traits at different plant developmental stages. In the past 2 decades, biotechnology research has
provided considerable insights into the mechanism of abiotic
stress tolerance in plants at the molecular level (Holmberg and
Bulow, 1998; Kasuga et al., 1999; Serrano et al., 1999; Hasegawa
et al., 2000; Zhu, 2001a; Prabhavathi et al., 2002; Rontein et
al., 2002). For example, though stress tolerance mechanisms
may vary from species to species and at different developmental stages (Ashraf, 1994; Foolad, 1999b; Foolad and Lin, 2001),
basic cellular responses to abiotic stresses are conserved among
most plant species (Zhu, 2001a,b, 2002). Furthermore, different abiotic stress factors may provoke osmotic stress, oxidative
stress and protein denaturation in plants, which lead to similar
cellular adaptive responses such as accumulation of compatible
solutes, induction of stress proteins, and acceleration of reactive
oxygen species scavenging systems (Zhu, 2002).
One of the most common stress responses in plants is overproduction of different types of compatible organic solutes
(Serraj and Sinclair, 2002). Compatible solutes are low molecular weight, highly soluble compounds that are usually nontoxic
at high cellular concentrations. Generally, they protect plants

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from stress through different courses, including contribution

to cellular osmotic adjustment, detoxification of reactive oxygen species, protection of membrane integrity, and stabilization
of enzymes/proteins (Yancey et al., 1982; Bohnert and Jensen,
1996). Furthermore, because some of these solutes also protect
cellular components from dehydration injury, they are commonly referred to as osmoprotectants. These solutes include
proline, sucrose, polyols, trehalose and quaternary ammonium
compounds (QACs) such as glycine betaine, alaninebetaine,
prolinebetaine, choline O-sulfate, hydroxyprolinebetaine, and
pipecolatebetaine (Rhodes and Hanson, 1993). Although much
effort has been devoted to genetically engineer plants for overproduction of various osmoprotectants, there has been little
success in achieving the desired protective levels of these
osmolytes in plants (discussed below). Alternatively, in some
plants increased resistance to abiotic stresses has been achieved
by exogenous application of various organic solutes. This
approach, which may significantly contribute to increased crop
production in stress environments, however, has not received
sufficient consideration in the literature. In this article, the roles
of glycine betaine and proline as well as their exogenous applications in increasing plant stress tolerance, in particular in response
to drought, salt, and temperature stresses, are reviewed and discussed.
2. Glycine betaine
2.1. Natural production and accumulation
Among the many quaternary ammonium compounds known
in plants, glycine betaine (GB) occurs most abundantly in
response to dehydration stress (Venkatesan and Chellappan,
1998; Mansour, 2000; Mohanty et al., 2002; Yang et al., 2003).
GB is abundant mainly in chloroplast where it plays a vital role
in adjustment and protection of thylakoid membrane, thereby
maintaining photosynthetic efficiency (Robinson and Jones,
1986; Genard et al., 1991). In higher plants, GB is synthesized in
chloroplast from serine via ethanolamine, choline, and betaine
aldehyde (Hanson and Scott, 1980; Rhodes and Hanson, 1993).
Choline is converted to betaine aldehyde, by choline monooxygenase (CMO), which is then converted to GB by betaine aldehyde dehydrogenase (BADH) (Fig. 1). Although other pathways
such as direct N-methylation of glycine is also known, the pathway from choline to glycine betaine has been identified in all
GB-accumulating plant species (Weretilnyk et al., 1989).
GB is known to accumulate in response to stress in many
crop plants, including sugar beet (Beta vulgaris), spinach (Spinacia oleracea), barley (Hordeum vulgare), wheat (Triticum aes-

Fig. 1. Biosynthetic pathway of glycine betaine in higher plants.

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tivum), and sorghum (Sorghum bicolor) (Weimberg et al., 1984;

Fallon and Phillips, 1989; McCue and Hanson, 1990; Rhodes
and Hanson, 1993; Yang et al., 2003). In these species, tolerant genotypes normally accumulate more GB than sensitive
genotypes in response to stress. This relationship, however,
is not universal. For example, no significant correlation was
observed between accumulation of GB and salt tolerance in various species of Triticum, Agropyron, and Elymus (Wyn Jones et
al., 1984), and even higher concentrations of choline and betaine
were observed in salt-sensitive than salt-tolerant lines of Egyptian clover (Trifolium alexandrinum) under salt stress (Varshney
et al., 1988). Also, cell lines of lemon (Citrus limon) selected
under salt stress did not exhibit a significant increase in GB
when compared with non-selected (control) cells (Piqueras et
al., 1996). However, it is likely that the relationship between GB
accumulation and stress tolerance is species- or even genotypespecific.
Some plant species such as rice (Oryza sativa), mustard (Brassica spp.), Arabidopsis (Arabidopsis thaliana) and
tobacco (Nicotiana tabacum) naturally do not produce GB under
stress or non-stress conditions (Rhodes and Hanson, 1993). In
these species, transgenic plants with over-production of GBsynthesizing genes exhibited increases in the production of GB
and an enhancement in tolerance to salt, cold, drought or high
temperature stress (Rhodes and Hanson, 1993). The accumulated GB in these species, however, was much lower than what
is naturally found in GB-accumulating plant species under stress
conditions (Rhodes and Hanson, 1993). The limitation in production of GB in high quantities in transgenic plants is reportedly
due to either low availability of substrate choline or reduced
transport of choline into the chloroplast where GB is naturally
synthesized (Nuccio et al., 1998; Huang et al., 2000; McNeil et
al., 2000). Thus, when engineering plants for over-production
of osmolytes such as GB, other factors such as substrate avail-

ability and metabolic flux must also be considered. However,

an alternative, and may be quicker, approach to increasing plant
stress tolerance through manipulation of osmolytes is exogenous
application of such compounds, as discussed in the following
section.
2.2. Exogenous application of GB
In many crop plants the natural accumulation of GB is lower
than sufficient to ameliorate the adverse effects of dehydration caused by various environmental stresses (Wyn Jones and
Storey, 1981; Yancey, 1994; Subbarao et al., 2001). Exogenous
application of GB to low-accumulating or non-accumulating
plants may help reduce adverse effects of environmental stresses
(Agboma et al., 1997a,b,c; Makela et al., 1998a; Yang and
Lu, 2005). Externally-applied GB can rapidly penetrate through
leaves and be transported to other organs, where it would contribute to improved stress tolerance (Makela et al., 1998a).
The efficiency of penetration can be improved by using various surfactants such as kinetic, lus-50 and sito+ (Subbarao et
al., 2001). Furthermore, because naturally produced GB does
not normally break down in plants (Bray et al., 2000), it can
easily be collected as a relatively inexpensive by-product from
high-producing plants such as sugar beets (Rhodes and Hanson,
1993). This may make extraction and exogenous application of
GB an economically feasible approach to counteract adverse
effects of environmental stresses on crop productivity.
There are many reports demonstrating positive effects of
exogenous application of GB on plant growth and final crop
yield under drought stress; examples include those in tobacco,
wheat, barley, sorghum, soybean (Glycine max) and common
beans (Phaseolus vulgaris) (Table 1). In common beans, for
example, GB-treated plants exhibited a slower decrease in leaf
water potential during drought stress and developed wilting

Table 1
Summary of the effects of exogenous application of glycine betaine on different plant species
Plant Species

Stress

Effect of exogenous GB

Reference

Nicotiana tabacum

Drought

Agboma et al. (1997b)

Phaseolus vulgaris

Drought

Glycine max
Triticum aestivum

Drought
Drought

Brassica napus
Zea mays
Lycopersicon esculentum

Drought
Drought
Salt and high temperature

GB induced improvement in growth and yield of

water-stressed plants
GB-treated plants showed a slower decrease in leaf
water potential
GB improved growth
(i) GB improved growth
(ii) GB did not improve growth
GB did not improve growth
GB improved growth of stressed plants
GB improved growth of stressed plants

Oryza sativa

Salt

Harinasut et al. (1996) and Lutts (2000)

Lutts (2000) and Rahman et al. (2002)
Lutts (2000) and Rahman et al. (2002)

Arabidopsis thaliana

Freezing temperatures

Solanum tuberosum

Low temperature

(i) GB improved growth of salt-stressed plants

(ii) GB improved shoot growth but not root growth
(iii) GB-treated salt-stressed plants had lower Na+
and higher K+ in the shoot
GB improved freezing tolerance (reduced freezing
temperature from 3.1 to 4.5 C)
GB improved growth of stressed plants

Gossypium hirsutum

Drought

(i) GB improved growth and yield of stressed plants

(ii) GB did not improve growth and yield of stressed
plants

WeiBing and Rajashekar (1999)

Agboma et al. (1997c)
Borojevic et al. (1980)
Agboma et al. (1997a)
Makela et al. (1996)
Agboma et al. (1997a)
Makela et al. (1998a,b)

WeiBing and Rajashekar (2001)

Somersalo et al. (1996)
Naidu et al. (1998) and Gorham et al. (2000)
Meek et al. (2003)

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symptoms much later than untreated plants. In addition, GBtreated bean plants showed a better ability to recover from
wilting following the removal of the stress, a very important
characteristic for growing plants under water deficit conditions.
Furthermore, GB treatment fully alleviated the adverse effects
of water deficit on CO2 absorption and chlorophyll fluorescence while it had little or no effect on shoot biomass or pod
yield.
GB also mitigates adverse effects of salinity stress in some
plant species (Table 1). For example, foliar application of GB
resulted in a significant improvement in salt tolerance of rice
plants (Harinasut et al., 1996; Lutts, 2000), and exogenous application of GB on tomato plants subjected to either salt stress or
high temperatures resulted in about 40% increase in fruit yield
compared with untreated plants (Makela et al., 1998a; Makela
et al., 1998b). In a comprehensive study, ameliorating effects of
exogenous application of GB on salt-induced shoot- and rootgrowth inhibition and ultra-structural damages were studied in
rice seedlings (Rahman et al., 2002). Briefly, while GB treatment alleviated salt-induced inhibition of shoot growth, it did
not affect root growth. Moreover, under salt stress, GB-treated
plants had significantly lower Na+ and higher K+ concentrations
in the shoots, compared with untreated plants. Furthermore, saltinduced ultra-structural damages to the leaf, including swelling
of thylakoids, disintegration of grana stacking and intergranal
lamellae, and destruction of mitochondria, were prevented by
GB pre-treatment. These beneficiary effects in the shoot were
suggested to be due to GB-induced production of additional
vacuoles in root cells, which resulted in a greater accumulation
of Na+ in the root and a decrease in its transportation to the
shoot. In the root, the most frequently observed ultra-structural
change due to salt stress was an increase in the number and
aggregation of mitochondria in the cytoplasm of the root tip
and root cap cells. In a similar study, exogenous application of
GB to rice plants growing under salt stress resulted in reduced
Na+ accumulation and maintenance of K+ concentration in the
shoot (Lutts et al., 1999). In maize (Zea mays, L.), exogenously
applied GB improved growth, leaf water content, net photosynthesis, and the apparent quantum yield of photosynthesis of the
salt-stressed plants (Yang and Lu, 2005). GB application, however, did not affect maximum efficiency of PSII photochemistry
(Fv/Fm). The improvement in photosynthesis of salt-stressed
maize plants by GB application was suggested to be associated
with improvements in stomatal conductance and actual PSII efficiency.
Exogenous application of GB is also reported to ameliorate adverse effects of low temperatures on growth of different
plant species (Table 1). For example, foliar spray of GB on
Arabidopsis plants decreased the freezing temperature from
3.1 to 4.5 C (WeiBing and Rajashekar, 2001). Similarly,
tolerance to low temperatures was improved in two potato
cultivars by exogenous application of GB (Somersalo et al.,
1996). In strawberry (Fragaria x ananassa), exogenous application of GB induced cold tolerance in unhardened as well
as cold-hardened plants (Rajashekar et al., 1999). Specifically,
application of 2 mM of GB to unhardened strawberry plants
increased their cold tolerance by almost two-fold within 72 h

209

of application, suggesting practical use of GB for field production of strawberries under low temperatures (Rajashekar et al.,
1999).
In addition to its direct protective roles, either through positive effects on enzyme and membrane integrity or as an osmoprotectant, GB may also protect cells from environmental stresses
indirectly via its role in signal transduction. For example, GB
may have a role in Na+ /K+ discrimination, which substantially
or partially contributes to plant salt tolerance. Ion homeostasis
in plants is governed by various membrane transport systems.
Recently, significant progress has been made in the characterization of cation transporters that maintain ion homeostasis during
salt stress in plants, of which SOS (salt overly sensitive) is a
novel signaling pathway (Chinnusamy et al., 2005). This pathway is somewhat regulated by MAP kinases, expressions of
which are highly affected by GB. Also, some physiological
studies of GB-treated turfgrass and Arabidopsis plants indicate that GB up-regulates expression of many genes (360
genes), of which more than 6% are known to be related to signal
transduction (John, 2002). Examples are lipoxygenase, monodehydroascorbate reductase, osmotin, putative receptor kinase,
calmodulin, protein kinase, and receptor protein kinase. These
and other evidence have led some investigators to suggest that
GB contributes to plant salt tolerance through its role in signal
transduction and ion homeostasis (John, 2002; Yilmaz, 2004).
However, knowledge of how GB affects expression of genes
responsible for, or related to, plant salt tolerance is scarce.
Elucidation of the roles of GB in regulating genes of signaling pathways used by plants to respond to environmental
stresses may lead to devising approaches to improve plant stress
tolerance.
Although most investigations attest to positive effects of
exogenous application of GB on plant stress tolerance, there
are a few reports suggesting lack of such positive effects or even
apparent negative effects of exogenous GB on plants growing
under stress conditions. Presence of such contrasting effects can
be seen from examples listed in Table 1. For instance, foliar
applications of GB on plants of turnip (Brassica rapa), rapeseed (Brassica napus) or spring cereals did not improve their
growth under drought stress (Makela et al., 1996). Similarly,
foliar application of GB did not affect yield components, physiological processes, or endogenous levels of GB in cotton plants
grown under drought stress (Meek et al., 2003). These apparent
contrasting effects however, could be due to either experimental
conditions or real differences among plant species and genotypes in their responses to exogenous application of GB. In
either case, they suggest the need for further research to examine effects of exogenous application of GB across plant species
and under different environmental conditions. Furthermore, at
present, the mechanism(s) by which exogenously applied GB
exerts its effects at the cellular and molecular levels is not fully
understood. A recent study of two GB non-accumulating crucifer species, B. napus (canola) and Arabidopsis, suggested that
exogenous GB had destabilizing effects on photorespiration via
competitive effects with glycine at the mitochondrial step of the
glycolate pathway (Sulpice et al., 2002). However, to implement
economic and routine application of GB to improve crop stress

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tolerance, a better understanding of its mechanism of action and

the magnitude of its effects in different plant species is essential.
In spite of potential benefits, a possible disadvantage of
exogenous application of GB is the risk of pathogen attacks
on plants applied with GB, as it is a potent growth substrate
for certain plant pathogenic fungi (Rhodes and Hanson, 1993).
For example, the growth of Fusarium graminearum, a common
pathogenic fungus of wheat and other cereal crops, was found
to be stimulated by the presence of choline and GB in anthers
(Strange et al., 1974; Pearce et al., 1976; Nkongolo et al., 1993;
Engle et al., 2004). Similarly, a major factor of susceptibility
of drought- and salt-stressed barley and other cereals to aphid
attack was suggested to be higher accumulation of GB under
stress conditions (Araya et al., 1991). Therefore, it is prudent
that before any recommendation is made as to the commercial
application of exogenous GB to improve plant stress tolerance,
the extent of possible collateral damages caused by fungi and
insects are examined in different plant species.
2.3. Effective concentrations of exogenous GB
Knowledge of the concentration, timing and frequency of GB
application is a pre-requisite to its commercial use for improving
crop stress tolerance. Unfortunately, however, limited research
has been conducted to determine the effectiveness of different
concentrations of GB in different plant species. In one study,
effects of exogenous application of GB on rooting and weaning
of apple micro-cuttings obtained from tissue culture were studied (Uosukainen et al., 2000). Under non-stress conditions, foliar
applications of 0.1, 0.2 or 0.3 M GB on new plantlets increased
shoot growth by 3076% over non-treated plants. A higher concentration of GB (e.g. 0.5 M) was detrimental to the plants. It
was also determined that the effect of one application of GB
lasted for at least 8 weeks. In rice, application of 1.0 mM GB
through nutrient solution was effective in improving growth and
reducing Na+ accumulation in plants grown under salt stress,
and the same concentration did not have any deleterious effect
on non-stressed plants (Lutts, 2000). In cotton (Gossypium hirsutum), application of 15 kg/ha of GB, either as seed treatment
or foliar application, to plants grown under field drought-stress
conditions was effective to improve stress tolerance and crop
yield (Naidu et al., 1998). The most efficient application, however, was seed treatment at a rate of 1.1 kg/ha GB. With a GB
cost of US$ 2025/kg, the net benefit appears to be as high as
US$ 580/ha (Naidu et al., 1998). Foliar application of similar
concentrations (24 kg/ha) of GB did not affect plant growth or
seed yield of cotton plants grown under non-stress field conditions (Meek et al., 2003). Thus, based on the limited information
available, it seems that effective and efficient doses of GB may
vary with plant species. In addition, the results may vary significantly depending on plant developmental stage, the time of

application, and environmental conditions under which plants

are grown. Therefore, there is a need for optimizing the doseresponse relationship in different plant species and at different
developmental stages and growth conditions before the use of
GB as a stresstolerance stimulator may become routine and
commercially profitable.
3. Proline
3.1. Natural production and accumulation
Amino acid proline is known to occur widely in higher plants
and normally accumulates in large quantities in response to
environmental stresses (Rains, 1989; Ashraf, 1994; Ali et al.,
1999; Rhodes et al., 1999; Ozturk and Demir, 2002; Hsu et
al., 2003; Kavi Kishore et al., 2005). In addition to its role as
an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures (e.g. membranes and proteins),
scavenging free radicals, and buffering cellular redox potential under stress conditions. It may also function as a proteincompatible hydrotrope (Srinivas and Balasubramanian, 1995),
alleviating cytoplasmic acidosis, and maintaining appropriate
NADP+ /NADPH ratios compatible with metabolism (Hare and
Cress, 1997). Also, rapid breakdown of proline upon relief of
stress may provide sufficient reducing agents that support mitochondrial oxidative phosphorylation and generation of ATP for
recovery from stress and repairing of stress-induced damages
(Hare and Cress, 1997; Hare et al., 1998). Furthermore, proline is known to induce expression of salt stress responsive
genes, which possess proline responsive elements (e.g. PRE,
ACTCAT) in their promoters (Satoh et al., 2002; Oono et al.,
2003; Chinnusamy et al., 2005).
In response to drought or salinity stress in plants, proline
accumulation normally occurs in the cytosol where it contributes
substantially to the cytoplasmic osmotic adjustment (Leigh et
al., 1981; Binzel et al., 1987; Ketchum et al., 1991). For example, in cells of Distichlis spicata treated with 200 mM NaCl,
the cytosolic proline concentration was estimated to be more
than 230 mM (Ketchum et al., 1991). In apical region of maize
roots growing at a water potential of 1.6 MPa, proline concentration reached approximately 120 mM and accounted for up
to 50% of the osmotic adjustment (Voetberg and Sharp, 1991).
Furthermore, it was determined that, in response to water deficit,
increased concentration of proline in maize root apical meristem was paralleled with increased concentration of abscisic acid
(Ober and Sharp, 1994; Sharp et al., 1994).
In plants, the precursor for proline biosynthesis is l-glutamic
acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS)
and pyrroline-5-carboxylate reductase (P5CR), play major roles
in proline biosynthetic pathway (Delauney and Verma, 1993)
(Fig. 2). Transgenic tobacco plants over-expressing P5CS have

Fig. 2. Biosynthetic pathway of proline in higher plants.

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shown increased concentration of proline and resistance to both

drought and salinity stresses (Kishor et al., 1995). However,
whether proline accumulation in these transgenic plants resulted
in increased stress tolerance through osmotic adjustment or other
mechanisms is unknown (Sharp et al., 1996).
Accumulation of proline under stress in many plant species
has been correlated with stress tolerance, and its concentration
has been shown to be generally higher in stress-tolerant than in
stress-sensitive plants. For example, while in salt-tolerant alfalfa
plants proline concentration in the root rapidly doubled under
salt stress, in salt-sensitive plants the response was slow (Fougere
et al., 1991; Petrusa and Winicov, 1997). Similarly, salt-tolerant
ecotypes of Agrostis stolonifera accumulated more proline in
response to salinity than salt-sensitive ecotypes (Ahmad et al.,
1981). Besides positive effects of proline on improving plant
salt tolerance at the organismal level, considerable improvement
in salt tolerance has also been observed at the cellular level.
For example, in vitro studies with brown mustard (Brassica
juncea) indicated that salt-adapted calli had higher accumulation of free proline compared with non-stressed calli (Madan et
al., 1995; Gangopadhyay et al., 1997). Similarly, salt-tolerant
calli of C. limon had significantly higher proline concentration
compared with salt-sensitive calli (Piqueras et al., 1996). Proline accumulation also occurs in plants subjected to drought
stress. For example, in rice plants subjected to water deficit,
the concentration of proline was increased in the leaves (Hsu
et al., 2003). This drought-induced accumulation of proline
was related to increased contents of the precursors for proline
biosynthesis, including glutamic acid, ornithine, and arginine.
In wheat, an assessment of the effects of drought stress on proline accumulation in a drought-tolerant and a drought-sensitive
cultivar revealed that the rate of proline accumulation and utilization was significantly higher in the drought-tolerant cultivar
(Nayyar and Walia, 2003). Furthermore, in B. juncea plants
grown under stress conditions, activities of proline biosynthetic enzymes P5CR and ornithine -aminotransferase (OAT)
increased mainly in tolerant lines though the activity of proline degrading enzyme proline oxidase decreased in all lines
(Madan et al., 1995).
Although enhanced accumulation of proline in plants under
abiotic stress is well documented, information on signaling
mechanism(s) that regulates proline biosynthesis is scarce. In
light of the available information, however, it seems that proline
accumulation in plants is mediated by both ABA-dependent and
ABA-independent signaling pathways (Chiang and Dandekar,
1995; Shinozaki and Yamaguchi-Shinozaki, 1997; Hare et al.,
1999; Zhu, 2001b, 2002). ABA is known to mediate signals in
plant cells subjected to environmental stresses. These signals
can bring about expression of stress-related genes followed by
synthesis of compatible osmolytes such as proline (Thomashow,
1999; Kavi Kishore et al., 2005). Furthermore, ABA accumulation in plants in response to osmotic stress has been determined
to regulate expression of P5CS gene (Fig. 2), which is involved
in proline biosynthesis (Xiong et al., 2001). However, it has been
reported that ABA is not solely sufficient for the induction of
P5CS transcript. The role of calcium in ABA-mediated induction of P5CS gene during drought- and salt-stress has also been

211

reported (Knight et al., 1997). Furthermore, a more recent study

suggested the role of phospholipase D as a signaling component along with calcium and ABA in the regulation of proline
biosynthesis (Thiery et al., 2004). Whether MAP kinases have
any role in the regulation of proline biosynthesis is not fully
known (Kavi Kishore et al., 2005). However, it is imperative to
elucidate the role of stress-related signaling pathways in modulation of proline biosynthesis, so as to help devise effective
strategies to improve plant stress tolerance.
Despite the presence of a strong correlation between stress
tolerance and accumulation of proline in higher plants, this relationship may not be universal. For example, in rice plants grown
under salt stress, accumulation of proline in the leaf was deemed
to be a symptom of salt injury rather than an indication of salt
tolerance (Lutts et al., 1999). Similarly, assessment of proline
accumulation and distribution during shoot and leaf development in two sorghum genotypes contrasting in salt tolerance
suggested that proline accumulation was a reaction to salt stress
and not a plant response associated with tolerance (de-Lacerda
et al., 2003). And yet in another study, under salt stress, sensitive
rice cultivars accumulated greater amounts of proline than did
the tolerant genotypes (Lutts et al., 1999). However, further studies are needed to determine whether the relationship between
stress tolerance and accumulation of proline is species-specific
or if it can be altered by experimental conditions. Currently,
there is more evidence supporting the presence of a positive
relationship.
Transgenic approach to improve plant stress tolerance via
over-producing proline has had some success. For example,
engineered tobacco plants over-producing proline significantly
reduced the level of free radicals and improved tolerance to
200 mM NaCl (Hong et al., 2000). In Arabidopsis, plants engineered with an antisense proline dehydrogenase cDNA resulted
in an increase in accumulation of proline and a constitutive tolerance to freezing temperatures (as low as 7 C) as well as
salinity (up to 600 mM NaCl) (Nanjo et al., 2003). The level of
stress tolerance of these transgenic plants was comparable to that
of rd29A::DREB1A transgenics. The RD29A promoter-driven
DREB1A transgenic plants exhibited tolerance to water deficit,
salt stress, and freezing temperatures (Kasuga et al., 1999). However, an alternative, and may be quicker, approach to improving
plant stress tolerance is through exogenous application of proline, as discussed in below.
3.2. Exogenous application of proline
Exogenous application of proline can play an important role
in enhancing plant stress tolerance. This role can be in the
form of either osmoprotection (Wyn Jones and Gorham, 1983;
Handa et al., 1986) or cryoprotection (Songstad et al., 1990;
Santarius, 1992) (Table 2). For example, in various plant species
growing under saline conditions, exogenously-supplied proline
provided osmoprotection and facilitated growth (Csonka and
Hanson, 1991; Yancey, 1994). In rice, exogenous application of
30 mM proline counteracted the adverse effects of salinity on
early seedling growth, though higher concentrations of proline
resulted in reduced growth (Roy et al., 1993). Exogenous appli-

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M. Ashraf, M.R. Foolad / Environmental and Experimental Botany 59 (2007) 206216

cation of proline to stressed plants of the halophyte Allenrolfea

occidentalis increased their growth and halted increased production of ethylene due to salt- or drought-stress (Chrominski
et al., 1989). Proline can also protect cell membranes from
salt-induced oxidative stress by enhancing activities of various
antioxidants (Yan et al., 2000). For example, growth of tobacco
suspension cells under salt stress was promoted by exogenous
application of 10 mM proline, which was proposed to be due
to proline action as a protectant of enzymes and membranes
(Okuma et al., 2000). In soybean cell cultures maintained under
salt stress, exogenous application of proline increased activities
of superoxide dismutase and peroxidase, which normally contribute to increased salt tolerance (Yan et al., 2000; Hua and Guo,
2002) (Table 2). In barley embryo cultures under saline conditions, exogenous application of proline resulted in a decrease in
Na+ and Cl accumulations and an increase in growth (Lone et
al., 1987). Such ameliorative effects of proline were indicated
to be due to plasma membrane stabilization (Mansour, 1998)
(Table 2).
In suspension cultures of halophilic D. spicata, metabolism
of 13 C-glutamic acid to proline was determined to be greater
in cells adapted to NaCl than in cells not adapted to the salt
(Heyser et al., 1989). Such metabolism, however, was inhibited
when 5.0 mM proline was added to the growth medium. This
inhibition was not due to a reduction in glutamic acid transport
in response to proline application, but rather regulated by either
feedback-inhibition of existing enzymes or repression of transcription (or translation) of genes encoding enzymes involved in
proline biosynthesis (Heyser et al., 1989). In general, accumulation of proline in the cytoplasm is associated with a reduction in
the concentration of toxic ions and an increase in the cytosolic
water volume (Cayley et al., 1992).
In contrast to the above findings on beneficial effects of
exogenous application of proline, there are a few reports cautioning its use. For example, foliar application of proline to
rice plants growing under saline conditions did not change concentrations of either Na+ or Cl in the leaves (Krishnamurthy
and Bhagwat, 1993) (Table 2). Moreover, in Arabidopsis plants

exogenous application of proline was suggested to cause damages to ultra-structures of chloroplast and mitochondria (Hare
et al., 2002). Such loss of organelles integrity in response to
exogenous proline can result in a significant increase in reactive oxygen intermediates in the chloroplast and mitochondria.
Such destructive effects were attributed to feedback inhibition
of proline biosynthesis, causing considerable reduction in photosynthetic electron acceptor pools (Hare et al., 2002). These
authors questioned the widely-accepted hypothesis that proline
is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular function. Thus, when using
exogenous proline as a tolerance-inducing agent, the effective
and beneficial concentrations must be first determined.
3.3. Effective concentrations of exogenous proline
Although exogenous application of proline to plants exposed
to abiotic stresses generally provides a stress preventing or
recovering effect, high concentrations of proline may be harmful
to plants, including inhibitory effects on growth or deleterious effects on cellular metabolisms (Ehsanpour and Fatahian,
2003; Nanjo et al., 2003). Therefore, it is essential to determine
optimal concentrations of proline that provide beneficial effects
in different plant species. In mung bean (Vigna radiata), for
example, it was determined that while addition of 2033 mM
of proline to cell cultures mitigated the adverse effects of NaCl
stress, concentrations of 50 mM or higher were inhibitory to the
growth of both salt-stressed and non-stressed cultures (Kumar
and Sharma, 1989). In this study, while cellular contents of Na+
and Cl decreased when proline reached optimal concentration,
they increased with further elevation in proline concentration. In
alfalfa (Medicago sativa) callus cultures, while 10 mM exogenous proline was very effective in alleviating the effects of salt
stress, higher concentrations were not beneficial (Ehsanpour and
Fatahian, 2003). In rice, while 30 mM proline was the most
effective concentration in improving germination and seedling
growth under salt stress, higher concentrations (40 or 50 mM)
resulted in reduced seedling growth and lowered K+ /Na+ ratio

Table 2
Summary of the effects of exogenous application of proline on different plant species
Plant species

Stress

Effect of exogenous proline

Reference

Distichlis spicata

Salt

Heyser et al. (1989)

Glycine max

Salt

Allenrolfea occidentalis

Salt and drought

Hordeum vulgare

Salt

Allium cepa

Salt

Oryza sativa

Salt

Nicotiana tabacum

Salt

Arabidopsis thaliana

No stress

Proline accumulation was high in cells adapted to a

concentration of salt
Proline application increased production of superoxide
dismutase and peroxidase in stressed plants
Proline neutralized the increased ethylene production in
stressed plants
Proline caused a decrease in shoot Na+ and Cl
accumulation in embryo culture cells
Proline resulted in mitigating the effect of NaCl on cell
membrane disruption
Proline did not alter leaf Na+ and Cl contents in
salt-stressed plants
Proline promoted the growth of suspension cells under salt
stress without maintaining a high ratio of K+ /Na+
Proline caused damages to chloroplast and mitochondria
ultra-structures

Yan et al. (2000) and Hua and Guo (2002)

Chrominski et al. (1989)
Lone et al. (1987)
Mansour (1998)
Krishnamurthy and Bhagwat (1993)
Krishnamurthy and Bhagwat (1993)
Hare et al. (2002)

M. Ashraf, M.R. Foolad / Environmental and Experimental Botany 59 (2007) 206216

in the leaves (Roy et al., 1993). Thus, in spite of its acclaimed

protective role, the toxicity effect of proline at high concentrations may be a problem. In a study to examine toxicity effects of
proline in Arabidopsis, a mutant (pdh) was isolated with a defect
in enzyme proline dehydrogenase (AtProDH), which catalyzes
the first step of proline catabolism (Nanjo et al., 2003). While
pdh mutants showed hypersensitivity to exogenous application
of 10 mM proline, wild-type plants grew normally at this concentration. Further investigations determined a dose-dependent
elevation in internal concentration of free proline in pdh mutants
during exogenous application of proline. The results of this study
indicated toxicity effects of proline and suggested that AtProDH
was the only enzyme acting as a functional proline dehydrogenase in Arabidopsis.
The available information from different studies suggest that
optimal concentrations of proline may be species or genotype
dependent, which need to be determined a priori before commercial application of exogenous proline to improve crop stress
tolerance. Furthermore, because in most crop species stress
tolerance may vary with developmental stages (Ashraf, 1994;
Foolad, 2000), it is also as critical to determine the growth stage
at which exogenous application of proline may be the most effective.
4. Concluding remarks
Many plant species naturally accumulate GB and proline as
major organic osmolytes when subjected to different abiotic
stresses. These compounds are thought to play adaptive roles in
mediating osmotic adjustment and protecting subcellular structures in stressed plants. However, not all plants accumulate GB
or proline in sufficient amounts to help averting adverse effects
of abiotic stresses. Thus, different approaches have been contemplated to increase the concentrations of these compounds
in plants grown under stress conditions to increase their stress
tolerance. First, it is the use of traditional protocols of plant
genetics and breeding to develop cultivars with natural abilities to produce high levels of these compounds under stress
conditions. However, in spite of the presence of considerable
genetic variation in natural production of these compounds in
different plant species, little is known regarding the genetic
basis (e.g. inheritance) of these traits to warrant crop improvement via traditional breeding. A second approach has been to
engineer genetically plants that are capable of producing sufficient amounts of these compounds in response to environmental
stresses. Although some progress has been made in introducing genes for the production of these compounds in naturally
non-accumulating or low-accumulating plant species, levels of
accumulation in transgenic plants have often been low or insufficient to improve plant stress tolerance. Further research is
currently underway to improve the efficiency of this approach, in
particular by taking into account factors such as the availability
of substrates and metabolic fluxes in engineered plants, which
may affect production of these osmolytes. A third approach,
which may be considered as a short-cut method, is exogenous
application of these osmolytes to plants growing under stress
conditions to enhance their tolerance.

213

Exogenous applications of GB and proline to plants, before,

during, or after stress exposure, have been shown to increase
the internal levels of these compounds and generally enhance
plant growth and final crop yield under stress conditions. There
are, however, great variations in different plant species in
response to exogenous application of GB and proline. First,
not all plant species are responsive to external application of
these compounds. Second, the effective concentration may
vary from species to species, and over-application may be
toxic to some plants resulting in growth inhibition or yield
reduction. Third, plants may exhibit different responses to
external application of these compounds at different stages
of plant development. Therefore, for effective application
and commercial use of exogenous GB and proline as stimulators of crop stress tolerance, the mechanisms of actions
of these compounds, the most optimal concentrations, and
appropriate plant developmental stages must be carefully
determined. The current research to determine specific roles
of GB and proline in plant stress tolerance is expected to
help improving their application as exogenous treatments
to improve plant growth and productivity under stress
conditions.
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