Pages From Handbook of Plant and Crop Stress Third Ed 2011 Titlu

THIRD EDITION
HANDBOOK OF
PLANT AND
CROP STRESS
BOOKS IN SOILS, PLANTS, AND THE ENVIRONMENT
Editorial Board
Agricultural Engineering Robert M. Peart, University of Florida, Gainesville
Crops Mohammad Pessarakli, University of Arizona, Tucson
Environment Kenneth G. Cassman, University of Nebraska, Lincoln
Irrigation and Hydrology Donald R. Nielsen, University of California, Davis
Microbiology Jan Dirk van Elsas, Research Institute for Plant Protection,
Wageningen, The Netherlands
Plants L. David Kuykendall, U.S. Department of Agriculture, Beltsville,

Maryland
Kenneth B. Marcum, Arizona State University, Tempe
Soils Jean-Marc Bollag, Pennsylvania State University, University Park

Tsuyoshi Miyazaki, University of Tokyo, Japan
Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson

Soil Biochemistry, Volume 2, edited by A. D. McLaren and J. Skujins
Soil Biochemistry, Volume 3, edited by E. A. Paul and A. D. McLaren
Soil Biochemistry, Volume 4, edited by E. A. Paul and A. D. McLaren
Soil Biochemistry, Volume 5, edited by E. A. Paul and J. N. Ladd
Soil Biochemistry, Volume 6, edited by Jean-Marc Bollag and G. Stotzky
Soil Biochemistry, Volume 7, edited by G. Stotzky and Jean-Marc Bollag
Soil Biochemistry, Volume 8, edited by Jean-Marc Bollag and G. Stotzky
Soil Biochemistry, Volume 9, edited by G. Stotzky and Jean-Marc Bollag
Organic Chemicals in the Soil Environment, Volumes 1 and 2, edited by C. A. I. Goring
and J. W. Hamaker
Humic Substances in the Environment, M. Schnitzer and S. U. Khan
Microbial Life in the Soil: An Introduction, T. Hattori
Principles of Soil Chemistry, Kim H. Tan
Soil Analysis: Instrumental Techniques and Related Procedures, edited by
Keith A. Smith
Soil Reclamation Processes: Microbiological Analyses and Applications, edited by
Robert L. Tate III and Donald A. Klein
Symbiotic Nitrogen Fixation Technology, edited by Gerald H. Elkan
9 Role of Proline in Plant
Response to Drought
and Salinity
Bruria Heuer
Contents
9.1 Introduction........................................................................................................................... 213
9.2 Osmoregulation or Osmotic Adjustment............................................................................... 214
9.2.1 Plant Response to Drought and Salinity.................................................................... 215
9.2.2 Role of Proline in Water- and Salt-Stressed Plants.................................................... 216
9.3 Proline Metabolism............................................................................................................... 217
9.3.1 Proline Synthesis....................................................................................................... 217
9.3.2 Proline Catabolism.................................................................................................... 218
9.3.3 Endproduct Inhibition................................................................................................ 218
9.3.4 Genetic Manipulations, Overexpression of Genes, and Transcriptional
Regulation of Genes Involved in Proline Synthesis.................................................. 219
9.3.5 Impact of Environmental Stresses on Proline........................................................... 220
9.4 Proline Accumulation as an Osmoregulatory Response....................................................... 220
9.4.1 Osmotic Adaptation of Bacteria and Algae............................................................... 221
9.4.2 Accumulation of Proline in Callus Cultures and Isolated Cells................................ 222
9.4.3 Seed Germination and Proline Content under Stress................................................ 223
9.4.4 Osmotic Adjustment in Halophytes...........................................................................224
9.4.5 Negative Response..................................................................................................... 225
9.5 Exogenous Proline Application............................................................................................. 226
9.6 Proline Content as an Indicator for Breeding Programs....................................................... 228
9.7 Conclusions............................................................................................................................ 229
References....................................................................................................................................... 229
9.1 Introduction
Plants face frequent periods of environmental stress that impairs their growth and reproductive
capacity. Restriction of plant growth cannot be attributed to one single process, because plant growth
is the result of many integrated and regulated physiological and biochemical processes. To survive
and maintain minimal growth potential, plants must conform to extreme environmental condi-
tions entailing adaptive changes in metabolism and cell composition. Of the various mechanisms
enabling plants to cope with water stress, the most common is the accumulation of intracellular sol-
utes, such as sugars and free amino acids. The most frequent nitrogen-containing compounds that
accumulate in plants subjected to drought and salinity are amides, amino acids, and polyamines.
The accumulation of proline on dehydration due to water deficit or increasing osmotic pressure
is the most recent information concerning the osmoregulatory role of proline in environmentally
stressed plants.
213
214 Handbook of Plant and Crop Stress
Salinity is a permanent threat to crops, especially in countries where irrigation is an essential aid
to agriculture. It is estimated that about 20% of cultivated lands and 33% of irrigated agricultural
lands worldwide are afflicted by high salinity. Moreover, the salinized areas are increasing at a
rate of 10% annually. Droughts are an inevitable and recurring feature of world agriculture, and,
despite our improved ability to predict their onset and modify their impact, they remain the single
most important factor affecting world food security and the conditions and stability of the land
resource from which that food is derived. Drought involves osmotic stress, while salt stress involves
both, osmotic stress, by limiting absorption of water from soil, and ionic stress, resulting from
high concentrations of potentially toxic salt ions within plant cells. In glycophytes, high salinity
reduces growth by inhibiting important physiological processes, such as photosynthesis and nutri-
ent supply to the growing tissues that are directly affected by the reduction of leaf water potential.
Nutrient disturbances under both drought and salinity reduce plant growth by affecting the avail-
ability, transport, and partitioning of nutrients. Drought reduces both nutrient uptake by the roots
and transport from the roots to the shoots, because of restricted transpiration rates and impaired
active transport and membrane permeability (Alam, 1999). Under salinity, reduced plant growth is
related to specific ion toxicities such as Na+ and Cl− and ionic imbalances acting on biophysical
and/or metabolic components of plant growth (Grattan and Grieve, 1999). Although increasing
the supply of nutrients to the growth medium under drought or saline conditions can alleviate the
adverse effects of either stress on plant growth, it is generally accepted that such increases will not
improve plant growth when the nutrient is already present in the soil in sufficient amounts and the
drought or salt stress is severe. A variety of protective mechanisms have evolved in plants to allow
them to acclimatize to these unfavorable environmental conditions for survival and growth. The
synthesis and accumulation of low molecular weight metabolites, known as compatible solutes, is
a ubiquitous mechanism for osmotic adjustment in plants. Their main role is to increase the ability
of cells to retain water without affecting the normal metabolism. Amino acids, sugars, polyols, and
various quaternary ammonium compounds are reported to accumulate as compatible solutes in
many plant species. Betaines, quaternary ammonium compounds occurring naturally in a variety of
plants, animals, and microorganisms and proline are among the most common nitrogen containing
compatible compounds. Both have been reported to be involved in the stabilization of proteins and
cell structures and/or in scavenging of free radicals, and might serve to store nitrogen that can be
used by the plant when the stress is relieved.
9.2 Osmoregulation or Osmotic Adjustment

The osmotic potential of cytoplasm is a dynamic cell property that can change when plants are
subjected to saline conditions or dehydration, as reported in an early work by Eaton (1927) and
subsequently by many other investigators (Morgan, 1984). Salinized plants take up salt from the soil
in quantities that decrease the osmotic potential of the cells. Bernstein (1961) termed this change
“osmotic adjustment” because the cell osmotic potential paralleled the lowered water potential of
the salt-affected soil, thus maintaining a favorable osmotic force for water uptake and a high turgor
pressure in the cells. A similar adjustment was later discovered when plants underwent dehydra-
tion (Meyer and Boyer, 1972). The term osmotic adjustment is now applied both to dehydrated and
salt-affected plants, and is defined as an accumulation or de novo synthesis of solute per cell that
causes a change in the cell osmotic potential. It excludes changes in cell water content that also can
alter cell osmotic potential. Water deficits have been shown to induce a lowering of the osmotic
potential in crops as a means of maintaining their turgor (Boggess et al., 1976). This decline in the
osmotic potential as a response to water deficit can be achieved by solute accumulation within the
plant cell or by a decreased cell volume leading to an increased concentration of osmotic solutes
as water leaves from the vacuole. These phenomena are described as osmoregulation and osmotic
adjustment. Osmoregulation has been defined as “the regulation of osmotic potential within a
cell by the addition or removal of solutes from solution until the intracellular osmotic potential is
Role of Proline in Plant Response to Drought and Salinity 215
approximately equal to the potential of the medium surrounding the cell” (Turner and Jones, 1980).
Osmotic adjustment refers to the lowering of the osmotic potential due to the net accumulation of
solutes in response to water deficits or salinity (Turner and Jones, 1980). Osmotic adjustment is an
important mechanism in drought tolerance, because it enables (1) a continuation of cell expansion
(Hsiao et al., 1976; Wyn Jones and Gorham, 1983), (2) stomatal and photosynthetic adjustments
(Ludlow, 1980), (3) better plant growth, and (4) yield production (Morgan, 1983). The compounds
involved in osmotic adjustment are mainly soluble sugars, potassium, organic acids, chloride, and
free amino acids (Cutler and Rains, 1978; Jones et al., 1980). The degree of the osmoregulatory pro-
cesses is affected by the rate of stress, stress preconditioning, the organ type, and the genetic varia-
tion between and within species (Morgan, 1984). It is accepted that the nontoxic compatible organic
solutes accumulate in the cytoplasmic compartment of cells and inorganic ions toxic to metabolic
processes are restricted to the vacuolar compartment. Considerable research has been conducted to
characterize the accumulation of proline, a compound known to contribute to the osmotic adjust-
ment and tolerance of plants exposed to unfavorable environmental conditions. How much of a role
it plays is still controversial and is discussed in detail in the following sections.
9.2.1 Plant Response to Drought and Salinity

Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes
that adversely affect plant growth and productivity (Wang et al., 2001). Drought and/or saliniza-
tion are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion
distribution in the cell (Serrano et al., 1999; Zhu, 2001). Plants respond to osmotic stress caused by
drought or salinity at the morphological, anatomical, cellular, and molecular levels (Greenway and
Munns, 1980; Bohnert et al., 1995; Yeo, 1998; Hasegawa et al., 2000). These include, for exam-
ple, morphological and developmental changes such as life cycle, inhibition of shoot growth and
enhancement of root growth, adjustment in ion transport through uptake, extrusion and sequestra-
tion of ions, and metabolic changes such as carbon metabolism, the synthesis of compatible solutes.
The deleterious effects of salinity on plant growth are associated with low osmotic potential of
soil solution, nutritional imbalance, specific ion effect, or a combination of these factors (Ashraf,
1994). Some of these responses are trigged by the primary osmotic stress signals, whereas others
may result from secondary stresses/signals caused by the primary signals. These secondary signals
can be phytohormones, mainly abscisic acid (ABA), reactive oxygen species (ROS), and intracel-
lular second messengers such as phospholipids. Inadequate response at one or several steps in the
signaling and gene activation may ultimately result in irreversible changes of cellular homeostasis
and in the destruction of functional and structural proteins and membranes, leading to cell death
mechanisms (Wang et al., 2001). In general, plant responses are of three kinds: maintenance of
homeostasis, detoxification of harmful elements, and recovery of growth. Plant modification for
enhanced tolerance is mostly based on the manipulation of genes that protect and maintain the func-
tion and structure of cellular components. ABA signaling plays a vital role in plant stress responses
as evidenced by the fact that many of the drought-inducible genes studied to date are also induced
by ABA. ABA levels rise within one hour of an imposition of water stress (Bensen et al., 1988) and
salt stress (He and Cramer, 1996).
Compatible solutes, or osmolytes, accumulate in organisms in response to osmotic stress. The
primary function of the compatible solutes is to maintain cell turgor and thus the driving gradient
for water uptake. Recent studies indicate that compatible solutes can also act as free-radical scaven-
gers or chemical chaperones by directly stabilizing membranes and/or proteins (Hare et al., 1998;
McNeil et al., 1999; Diamant et al., 2001). The compatible solutes generally found in higher plants
are low molecular weight sugars (Garcia et al., 1997; Goddijn and van Dun, 1999), organic acids,
polyols (Smith and Phillips, 1980; Abebe et al., 2003), and nitrogen containing compounds such as
amino acids, amides such as glutamine and asparagine, imino acids, ectoine, proteins and quaternary
ammonium compounds (Rabe, 1990; Rhodes and Hanson, 1993; Pareek et al., 1997; Mansour, 2000;
Carillo et al., 2008). Overexpression of compatible solutes in transgenic plants can result in improved
stress tolerance (Sakamoto and Murata, 2002; Mattana et al., 2005; Pasquali et al., 2008).
9.2.2 Role of Proline in Water- and Salt-Stressed Plants

Although its role in plant osmotolerance remains controversial, proline is thought to contribute to
osmotic adjustment, detoxification of ROS, and protection of membrane integrity. Proline accu-
mulation is believed to play adaptive roles in plant stress tolerance and has been proposed to act
as a compatible osmolyte and to be a way to store carbon and nitrogen. Salinity and drought are
known to induce oxidative stress. Early in vitro studies showed that proline can be a ROS scavenger.
Proline has also been proposed to function as molecular chaperone stabilizing the structure of pro-
teins, and proline accumulation can provide a way to buffer cytosolic pH and to balance cell redox
status. Finally proline accumulation may be part of the stress signal influencing adaptive responses.
Proline may act as a component of signal transduction pathways that regulate stress responsive
genes in addition to its previously described osmoprotective roles, thereby improving the tolerance
to salt stress. Proline has been thought to have an adaptive role in mediating osmotic adjustment and
protecting subcellular structure in stressed plants. Synthesis, degradation, and transport of proline
cooperatively control its endogenous level in higher plants in response to environmental conditions.
The physiological functions of proline are shown in Figure 9.1.
In Pancratium maritimum L., salt stress resulted in a decrease in the amount of ubiquitin con-
jugates, (a small protein targeting damaged proteins for degradation via the proteasome), particu-
larly in the roots, and this effect was reversed by exogenous proline (Khedr, 2003). Severe salt
stress resulted in an inhibition of the antioxidative enzymes, catalase and peroxidase, as revealed
by spectrophotometric assays and activity gels, but the activity of these enzymes was also main-
tained significantly higher in the presence of proline. Salt stress also upregulated several dehydrin
proteins, analyzed by western blotting, even in nonstressed plants. Thus, proline could act as a com-
ponent of signal transduction pathways that regulate stress responsive genes in addition to its pre-
viously described osmoprotective roles, thereby improving the tolerance to salt stress. In ice plant
(Mesembryanthemum crystallinum), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.)
proline has a bifunctional role in the acclimation to high salt stress; an osmoregulant role in the
light, and a substrate for dark respiration to supply energy for compartmentation of ions into vacu-
ole in the dark (Sanada et al., 1995). In Cordyline fruticosa, proline plays an osmoregulatory role,
increasing in roots not only when NaCl concentration in the nutrient solution is too high but also
Asymmetric catalysts Protein Promoting activity

for aldol reactions constituent for collagen synthesis
Osmoregulation Inhibition of dehydration

(a compatible solute) O or ice nucleation
C OH
N H
Scavenging of reactive H Protein and membrane
oxygen species L-Proline stabilization
Lowering of
nucleic acid Tm
Figure 9.1 The physiological functions of proline. Proline has been shown to exhibit multiple functions
other than osmoprotection in vitro. However, the mechanism by which proline confers stress tolerance in vivo
remains poorly understood. Although proline is accumulated in many bacterial and plant cells in response to
osmotic stress, proline levels are not increased under various stresses in S. cerevisiae. (From Takagi, H., Appl.
Microbiol. Biotechnol., 81, 211, 2008.)
when it is too low (Plaza et al., 2009). In some cases, proline as a free radical scavenger may be more
important in overcoming stress than in acting as a simple osmolyte (Hong et al., 2000). Mattioli
et al. (2008) suggested that proline plays a key role in flower transition, bolting, and coflorescence
formation. The considerable increase in free proline content in the leaves of acclimated barley
plants was of a minor importance for its tolerance to dehydration (Bandurska, 2001), suggesting that
proline accumulation alone is not a sufficient factor in the development of leaf dehydration tolerance
to severe water deficit. The role of proline in Halimium halimifolium and Pistacia lentiscus, two
contrasted species of Mediterranean shrubs, seems to be more related to a protective action in cases
of severe stress conditions rather than as an osmotic agent with an osmotic potential depressing
activity (Ain-Lhout et al., 2001). In Sesbania sesban, proline fully alleviated the salt stress induced
enhancement of ribulose-1, 5-bisphosphate oxygenase activity, suggesting that proline accumulation
in plants under stress must be assisting plants in maintaining the photosynthetic efficiency and the
overall productivity (Sivakumar et al., 2000). Proline synthesis and its subsequent accumulation in
aquatic plants is a consequence of intracellular ionic adjustments that take place under salt stress to
keep the metabolic activities going on (Rout and Shaw, 1998).
9.3 Proline Metabolism

Proline metabolism is a typical mechanism of the biochemical adaptation in living organisms subjected
to stress conditions. Mitochondria have an important role regarding proline homeostasis during water
stress. In many plants, proline accumulates in response to water stress and rapidly disappears upon
recovery, due to variations in its cytosolic synthesis and mitochondrial degradation rates (Kiyosue
et al., 1996). Moreover, since proline synthesis from glutamate requires two molecules of cytosolic
NADPH, and its reverse oxidative degradation in mitochondria generates FADH2 and NADH, it has
been suggested that glutamate–proline cycling between cytosol and mitochondria could have a major
role in redox homeostasis and metabolism (Hare and Cress, 1997). Proline metabolism is regulated
by feedback inhibition mechanism (Hare and Cress, 1997; Hong et al., 2000). The synthesis of proline
may have an additional effect. Intermediates in proline biosynthesis and catabolism such as glutamine
and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated
genes in rice (Oryza sativa L.), including salT and dhn4 (Iyer and Caplan, 1998). Proline as a free
radical scavenger may be more important in overcoming stress than in acting as a simple osmolyte.
9.3.1 Proline Synthesis

The pathway of proline biosynthesis was first elucidated in Escherichia coli in 1952, but only con-
firmed in 1987 (Leisinger, 1987). Proline biosynthesis is initiated with the ATP-dependent phos-
phorylation of glutamic acid by the γ-glutamyl kinase (γ-GK), encoded by the proB gene. The
product of γ-GK is reduced to glutamic-semialdehyde (GSA) by the γ-glutamyl phosphate reductase
(γ-GPR) encoded by the proA gene (Turchetto-Zolet et al., 2009). GSA cyclizes spontaneously to
form P5C, which is finally reduced to proline by P5C reductase (P5CR, encoded by the proC gene).
The proline biosynthetic route in plants resembles the bacterial pathway and uses either glutamic
acid (Stewart, 1981) or ornithine (Mestichelli et al., 1979) as substrates. In plants, proline synthesis
is regulated by end-product inhibition (Noguchi et al., 1968; Boggess et al., 1976). Under normal
physiological conditions, both pathways contribute to proline synthesis in the cytosol. Under stress-
ful conditions, proline is synthesized preferentially from glutamic acid (Delauney and Verma, 1993)
via two intermediates: GSA and P5C. In common reed plants, free proline content and pyrroline-5-
carboxylate synthetase (P5CS) activity increased with salt stress treatment, while ornithine amino-
transferase (OAT) activity appeared to be unaffected, suggesting that the glutamate pathway, rather
than the ornithine pathway, plays a vital role in proline accumulation during osmotic regulation
(Zhen and Ma, 2009). Proline synthesis in plants can be manipulated by eliminating feedback regu-
lation of the key regulatory enzyme of the pathway, P5CS.
9.3.2 Proline Catabolism

Proline accumulation and catabolism play significant roles in adaptation to a variety of plant
stresses including osmotic stress, drought, temperature, freezing, UV irradiation, heavy metals, and
pathogen infection. The ability of plants to degrade proline through an oxidation process has been
shown clearly (Stewart, 1981). Accumulated proline is rapidly oxidized back to glutamate when
osmotic stress is relieved. The first step of this process is catalyzed by a mitochondrial proline dehy-
drogenase (oxidase) (Sweetlove et al., 2007). Studies in Arabidopsis have shown rapid transcript
response of proline oxidase to changing osmotic pressure, suggesting that it is a regulatory link in
proline homeostasis in plants. The subsequent step in this pathway is catalyzed by delta1-pyrroline-
5-carboxylate dehydrogenase. The expression of this mitochondrial enzyme is regulated by osmotic
stress in Arabidopsis (Deuschle et al., 2001) and has recently been shown that it can act in tandem
with the stress-related gene transcript to generate two types of small interfering RNAs (siRNAs)
which participate in a complex manner to regulate salt tolerance (Borsani et al., 2005). The occur-
rence of two separate translocators for proline in the mitochondria, namely a carrier solely for
proline and a proline/glutamate antiporter en route to its catabolism was shown in durum wheat
(Di Martino et al., 2006). Proline (accumulated/synthesized in the cytosol) enters mitochondria via
its own uniporter; inside the mitochondria Pro catabolism takes place at a rate exceeding that of the
transport, as shown by the lack of accumulation of proline in the matrix; Glu is then exported out-
side mitochondria in exchange for further Pro via the Pro/Glu antiporter. The catabolism of proline
in the mitochondria has been suggested to play multiple roles in plant physiology. It can raise the
energetic status of the cell following the relief of stress conditions in addition to acting as an avail-
able reservoir of carbon and nitrogen (Hare and Cress, 1997). Recently, association between proline
and programmed cell death has been reported with proline-treated plant cells undergoing a necrotic
response with features consistent with the production of ROS and programmed cell death (Deuschle
et al., 2004). It has been suggested to be important in the protection of catalase, peroxidase, and
Complex II activities (Hamilton and Heckathorn, 2001; Ozturk and Demir, 2002). In all instances,
it would appear likely that the mitochondrially localized enzymes responsible for the degradation
of proline play a prominent regulatory role in these processes. The activity of the proline degrading
enzyme, proline dehydrogenase (PDH), decreased under salt stress in common reed plants, indicat-
ing that proline catabolism may be responsible for proline accumulation in response to salt stress
(Zhen and Ma, 2009).
9.3.3 Endproduct Inhibition

Proline manifests a conspicuous ability to control its own biosynthesis. Exogenous application to
plant tissues of an amount of proline sufficient to increase the endogenous pools enhanced the rate
of proline oxidation as a result of a feedback inhibition process (Stewart, 1972; Boggess et al., 1976).
It is known that proline oxidase, one of the enzymes involved in proline degradation, can be induced
by high concentrations of proline (Boggess et al., 1976; Adams and Frank, 1980). Feedback inhibi-
tion of proline synthesis does not occur under water-stress conditions. In sorghum and chickpea,
feedback inhibition of P5CS by proline during moisture stress was observed (Dalvi et al., 2007). In
bacteria, proline biosynthesis has been shown to be regulated by the end-product inhibition of γ-GK
activity (Smith et al., 1984). A similar situation was also observed in plants. The γ-GK activity of
Vigna P5CS was inhibited by proline, but its GSA dehydrogenase activity was not affected, suggest-
ing that the γ-GK is the rate-limiting step in proline biosynthesis in plants (Zhang et al., 1995). The
P5CS is the rate-limiting enzyme in proline (Pro) biosynthesis in plants which is encoded by two
highly homologous genes in Arabidopsis and many other plants, and is subject to feedback inhibi-
tion by proline (Yoshiba et al., 1997; Fujita et al., 1998; Ginzberg et al., 1998). It is controlled at the
level of P5CS transcription and through feedback inhibition of P5CS by proline (Zhang et al., 1995;
Hong et al., 2000).
9.3.4 G
enetic Manipulations, Overexpression of Genes, and Transcriptional
Regulation of Genes Involved in Proline Synthesis
In plants, many key enzymes of metabolic pathways are generally encoded by redundant genes,
which may be generated by gene duplication events during the evolutionary history of the organ-
isms. Gene duplication produces two functionally identical copies that act in a totally redundant
way immediately following the duplication event, and are often followed by sequence alterations
causing changes in transcriptional regulation and contributing to evolution of functional divergence.
The genetic manipulations of proline metabolic pathways in plants are summarized in Figure 9.2.
Effects of genetic manipulation of proline synthesis can be plant species specific. Overexpressing
soybean P5CR gene in transgenic tobacco did not increase osmotolerance (Szoke et al., 1992). The
overexpression of the P5CS encoding gene in transgenic tobacco plants resulted in increased proline
production and conferred tolerance of these plants to osmotic stress, confirming that P5CS is of key
importance for the biosynthesis of proline in plants (Kishor et al., 1995). Field trials in South Africa
with P5CR transgenic soybean lines supported improved drought performance and higher heat tol-
erance compared to wild type cultivars (de Ronde et al., 2004). However, mutants displaying higher
proline accumulation can also be salt hypersensitive (Lui and Zhu, 1997).
It has been shown that transcriptional control of the P5CS gene is important for the regulation
of accumulation of proline during osmotic stress in plants. Studies of transcriptional regulation
of genes involved in proline synthesis confirmed developmental regulation. In young Arabidopsis
plants, beta-glucuronidase (GUS) analysis of AtP5R (Arabidopsis P5CR gene) promoter revealed
high expression in apical meristem and young leaf, in root meristem, secondary root primordia and
root vascular cylinder. In young leaf high P5R expression could be detected all over the leaf blade,
[1,2,3] [4,5]
P5CS Spontaneous P5CR
L-Glu GSA P5C L-Pro
P5CDH PDH
[9,10] [6,7,8]
Proline synthesis (cytosol and plastids)
Proline degradation (mitochondria)
Figure 9.2 Genetic manipulations of proline metabolic pathways in plants. The main precursor of proline
synthesis is l-glutamic acid (l-Glu). l-Glu is first reduced to glutamate semialdehyde, which spontaneously
cyclizes to pyrroline-5-carboxylate (P5C), by P5C synthase (P5CS). The second reduction, of P5C to proline,
is catalyzed by P5C reductase (P5CR). This pathway is found in the cytosol and in plastids. Proline is catabo-
lized to Glu in mitochondria by proline dehydrogenase (PDH) and P5C dehydrogenase (P5CDH). Examples
of genetic manipulations of proline metabolic pathway and impact on osmotolerance: [1] Overexpression
of mothbean P5CS in tobacco plants. Those transgenics were better salt tolerant (Kishor et al., 1995); [2]
Overexpression of P5CS in antisense orientation in Arabidopsis (Nanjo et al., 1999a) and [3] p5cs1 inser-
tional mutation (Székely et al., 2008) resulted in reduced osmotolerance; [4] Overexpression of soybean P5CR
gene in tobacco (Szoke et al., 1992) did not modify osmotolerance; [5] Overexpression of Arabidopsis P5CR
(AtP5R) gene in soybean improved drought and heat stress (de Ronde et al., 2000, 2004); [6] Overexpression
of PDH did not change osmotolerance in Arabidopsis, except in the presence of exogenously supplied Pro. In
these conditions PDH-sense lines were better osmotolerant than WT. [7] Higher salt tolerance was observed in
Arabidopsis plants overexpressing PDH in antisense orientation by Nanjo et al. (1999b), but not by Mani et al.
(2002). Study of modifications of Pro catabolism in Arabidopsis in relation to P5/GSA and/or proline toxicity:
[6] Overexpression of PDH decreased sensitivity to externally supplied proline; [7] decrease of PDH activity
by antisense strategy (Mani et al., 2002); or [8] knock-out mutation (Nanjo et al., 2003) increased sensitivity
to Pro; [9] P5CDH overexpression decreased sensitivity to externally supplied proline (Deuschle et al., 2004);
[10] p5cdh knock-out mutants were hypersensitive to proline (Deuschle et al., 2004) (From Verbruggen, N.
and Hermans, C., Amino Acids, 35, 753, 2008.)
while in old leaves, expression was restricted to the veins, hydathodes, guard cells and base of tri-
chomes (Hua et al., 1997). In flowering plants, high AtP5R expression could be detected in rapidly
dividing cells, such as root meristem, and cells or tissues undergoing changes in water potential,
such as hydathode, guard cell, ovule, developing seed, and pollen grain (Hua et al., 1997). AtHAL3a,
an HAL3 homolog gene in Arabidopsis thaliana overexpressing transgenic plants exhibit improved
salt and sorbitol tolerance (Ikuko et al., 2004). Overexpression of NtHAL3a improved salt, osmotic,
and lithium tolerance in cultured tobacco cells. Proline degradation can also be manipulated and
depends on prior proline transport to mitochondria. Overexpression of PDH in A. thaliana did not
result in morphological abnormalities, probably because proline homeostasis relies on regulated
transport between cell compartments (Nanjo et al., 1999; Mani et al., 2002).
9.3.5 Impact of Environmental Stresses on Proline

Proline accumulation is a common physiological response in many plants in response to a wide
range of biotic and abiotic stresses. Extensive research in this area has revealed proline accumula-
tion as a universal response of plants under stress. Proline accumulation in dehydrated plants is
generally due to the concerted activation of proline biosynthesis in the cytosol and inhibition of its
catabolism in the mitochondria (Kiyosue et al., 1996). Proline accumulation due to water or salt
stress results from a stimulated synthesis in the tissue, an inhibited oxidation, or an impaired incor-
poration of proline into proteins. Clearly, under such conditions, the mechanisms of feedback inhi-
bition cannot function. Indeed, this was proven by Boggess et al. (1976) who showed that proline
does not inhibit its own synthesis in wilted barley and tobacco leaves. The increased synthesis of
proline due to wilting seems to be related to the first step of this synthesis. On the other hand, water
stress and salinization inhibit proline oxidation in the mitochondria and alter the permeability of
the mitochondrial membrane (Miller et al., 1971; Nir et al., 1971; Sells and Koeppe, 1980). Further,
the incorporation of proline into protein is inhibited by water stress, thereby also leading to proline
accumulation under stress conditions (Stewart et al., 1977). The step affected in protein synthesis is
probably the translation step (Hsiao, 1973); however, this process is not obligatory, because proline
accumulation was observed when protein synthesis inhibitors, such as cycloheximide, were applied
(Boggess and Stewart, 1980). The state of the hormonal balance in plants is suggested to play a con-
siderable role in mediation of proline accumulation under water or salt stress (Boggess and Stewart,
1980). The key role in the osmotic adaptation of plants has been ascribed to ABA. The reduction in
turgor is accepted as the primary trigger of proline accumulation in plants subjected to conditions of
drought and salinity. The loss of turgor activates a complex sequence of adaptive events correlated
to the level of stress, plant tolerances, and plant growth.
9.4 Proline Accumulation as an Osmoregulatory Response

Proline interacts with enzymes to preserve protein structure and activity within the cell. In vitro
studies have shown that high concentrations of proline reduce enzyme denaturation attributable to
heat, freeze-thaw cycles, and high NaCl (Pollard and Wyn Jones, 1979; Rajendrakumar et al., 1994).
Moreover, proline protects proteins and membranes from damage by inactivating hydroxyl radicals
or other highly reactive chemical species that accumulate when stress inhibits electron-transfer
processes (Smirnoff and Cumbes, 1989).
The accumulation of proline seems to be associated with adaptation to temperature stress. Free
proline concentrations increased under both high (Ashraf et al., 1994) or low temperature (Tarnizi
and Marziah, 1995; Wang and Cui, 1996), and proline could serve as a stress indicator in plants
exposed to these unfavorable growth conditions. Heavy metals or herbicides are other environ-
mental factors that induce proline accumulation in plants. Vicia faba plants grown in hydrocarbon-
polluted soils accumulated high levels of proline (Malallah et al., 1996). High-Pb concentrations
reduced sunflower plants’ biomass but increased the concentration of free proline (Kastori et al., 1996).
The leaf proline content also increased in pea plants (Kamenova-Yukhimenko et al., 1995) or Vigna
radiata (Arora and Saradhi, 1995) grown in nutrient solutions containing high-Cd concentrations.
Chlormequat given to soybean plants increased the leaf proline content (El-Kheir et al., 1994). The
foliar application of paraquat and diquat to Parthenium hysterophorus resulted in a reduction of
the proline level 48 h after application followed by an increase 72 h after spraying (Kore and Patil,
1995). The foliar application of fomesan, imazaquin, metobromuron, and mettolachlor to soybean
plants resulted in reduced levels of proline and some other amino acids, probably because of a
disturbed protein synthesis (Stoirnenova and Stoyanova, 1995). Chlorsulfuron, norflurazon, and tri-
allate increased the proline content in pea and V. faba (Fayez and Kristen, 1996). Increased levels of
proline were also found in barley plants infested with aphids because of low water potentials (Fayez
and Kristen, 1996). It is suggested that the changes in proline could be considered as a protective/
adaptive mechanism against expected injuries resulting from pollution or herbicides.
Proline accumulation in dehydrated plant tissues was first noted in 1954 by Kemble and
Mac-Pherson. Further experiments showed that the de novo synthesis of proline was involved, as
the amounts were greater than those released by proteolysis. Proline accumulation was observed
in many species as a result of exposure to water or salt stresses. Several reports correlate this phe-
nomenon with stress resistance indicating that a better performance and survival can be expected
in species that accumulate proline. Much evidence is available to corroborate the proline action as
a compatible solute regulating and reducing water losses from dehydrated cells. The increase in
the proline content during water stress is inversely proportional to the initial proline concentration
in plant organs (Venekamp and Koot, 1988). It was found that the generative parts of bean plants
contained considerably lower concentrations of proline than the vegetative parts after withholding
water. The accumulation of proline during tissue dehydration is time dependent (Rajagopal et al.,
1977). The accumulated proline is lost rapidly as a result of recovery from water stress but not as an
immediate response to salt removal from the media.
All the above-mentioned points are addressed in detail in the following sections.
9.4.1 Osmotic Adaptation of Bacteria and Algae

Generally, microbes respond to water potentials by accumulating intracellular compounds that are
compatible with the cellular metabolic function. There is a natural ranking or differing osmolyte
preferences among species of bacteria (Csonka, 1989). The accumulation of compatible solutes in
soil bacteria cells subjected to increased salinity depends either on direct uptake from the soil
solution (or the rhizosphere) or on biosynthesis via the uptake of a direct precursor such as cho-
line (Dupont et al., 2003). Both mechanisms occur simultaneously in a majority of bacteria, as in
Sinorhizobium meliloti. Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneu-
moniae, which are adversely affected by high osmotic strength, show osmotic adjustment when
exposed to these conditions. A. chroococcum ZSM4 accumulates trehalose and glutamate, proline
and glutamate in A. brasilense SHS6, and trehalose and proline in K. pneumoniae (Madkour et al.,
1990). Glycine betaine was accumulated in all strains. This chapter focuses on betaine transport and
glycine betaine biosynthesis in this bacterium, a typical representative of the rhizosphere and the
microsymbiont of lucerne root nodules. Also covered are the salt tolerance of various Rhizobiaceae
strains and their capacity to use choline and glycine betaine as osmoprotectants. Partial alleviation
of salt stress was obtained by adding low concentrations of proline or betaine to the growth medium
of Thiobacillus ferroxidans (Kieft and Spence, 1988). Similar results were obtained for Rhizobium
species (Gloux and Le Rudulier, 1989). In response to increased salt concentrations, algae also
synthesize osmoregulatory solutes to counterbalance the low water potential of the growth media.
Some nitrogen-fixing cyanobacteria (blue-green algae) display remarkable abilities to survive water
stress, experienced either as salinity or as drought. Survival during water stress is achieved by
regulation of ion fluxes for immediate osmotic adjustment to rapidly prevent turgor loss, and by a
long-term adaptation plan, facilitated through selective expression of “tolerance” genes and novel
stress proteins. Such osmotic adaptation comprises of accumulation of compatible solutes conducive
to metabolism and repair of oxidative damage caused by excess of free radicals. A variety of osmo-
lytes are synthesized such as sugars, polyols, and betaines, respectively conferring low, medium,
and high level of tolerance (Apte, 2001). Proline plays a key role in the osmoregulatory mecha-
nism of Chlorella autotrophica (a marine microalga) when exposed to high salinities (Ahmad and
Hellebust, 1984). In this alga, proline accumulation begins immediately after an osmotic shock
without a lag phase. This alga is not dependent on protein synthesis and requires light, because
photosynthesis supplies the required energy for proline biosynthesis.
9.4.2 Accumulation of Proline in Callus Cultures and Isolated Cells

Compatible solutes were involved in the response of Thellungiella holophila and A. thaliana calli
to salt stress (Zhao et al., 2009). Exogenous proline increases the cadmium accumulation in callus
and regenerated shoots of Solanum nigrum (Xu et al., 2009). Endogenous free proline content in
tomato calli increased gradually in response to elevating polyethylene glycol (PEG) concentrations
(Shtereva et al., 2008). PEG-induced osmotic stress tolerance was found to be associated with the
accumulation of free proline in 1-month-old calli of two indica rice genotypes (Ahmad et al., 2007).
Proline increased significantly in date palm calli in response to salinity (Al Mansoori et al., 2007).
During the induction stage, the increase in endogenous free proline content was more pronounced
in the progeny of the cultivar which exhibited higher percentage of callus induction. Therefore, the
better dedifferentiation process could be related to proline content, which adjusts the intracellular
osmotic pressure between the cytoplasm and the vacuole.
Callus cultures of rice, adapted to grow under increasing levels of NaCl, accumulated consid-
erable amounts of free proline compared with unadapted cells (Kishor, 1988). This trait was not
lost when the cells were transferred through 10 passages in the absence of selection pressure and
regrown on salt. On the other hand, mature embryo-derived calli from rice cultivars differing in
their salt tolerance also accumulated proline when exposed to NaCl, KCl, or mannitol (Lutis et al.,
1996). This accumulation did not depend on the nature of the stressing agent or the stress intensity
and did not appear to be involved in osmotic adjustment; therefore, it was considered to be a symp-
tom or injury in the stressed rice calli and not an indicator or resistance. In an embryogenic callus
culture of lemon, selected for resistance to salinity, the proline concentration significantly increased
as compared with control cells (Piqueras et al., 1996). The transfer of salt-tolerant cell cultures of
alfalfa to NaCl-containing medium resulted in a tenfold increase in proline concentrations (Petrusa
and Winicov, 1997). In Brassica napus, both unselected and tolerant calli responded to water stress
by osmotic adjustment and proline concentration (Chandler and Thorpe, 1987). Increases in proline
concentrations were approximately linear in tolerant calli, reaching a maximum of 175 dry weight
and 520 μM in selected calli. This accumulation was correlated with growth inhibition and nega-
tively correlated with the culture age for tolerant calli. Callus cultures of Medicago sativa accumu-
lated proline in response to NaCl (Shah et al., 1990). This accumulation was enhanced by calcium
and was positively correlated with salt tolerance. The salt tolerance of sugar beet calli was also
accompanied by a significant increase in the proline concentration under conditions of high salinity
(Le Dily, 1991). The proline content in a callus culture of pearl millet grown in 1% NaCl increased
more than 20-fold compared with nonsalinized controls (Das et al., 1990). Exposure of tobacco
callus cultures to osmotic shock greatly enhanced the proline accumulation in proportion to the
amount of absorbed sorbitol (Eberhardt and Wegmann, 1989). NaCl-resistant cell lines of tobacco
increased the proline content within 5–10 h after transplantation to a selective medium. In the wild
strain, the proline content remained unchanged over a 24 h period (Watad et al., 1983; Kiryan and
Shevyakova, 1984). A correlation between the viability of cells in a saline environment and the
proline content was observed. The intracellular proline concentration was positively correlated with
the osmotic potential indicating that proline was a component of the osmotic adjustment of the cells
(Binzel et al., 1987). Glutamate was the main source of the newly produced proline, and the relative
contribution of the catabolic pathway was small. Cell lines selected for resistance to salt stress
responded to water stress by accumulating markedly more proline than the wild type (Watad et al.,
1983). This response was stable through at least eight generations and was fully reversible. Similar
results were obtained with cultured cells of a salt marsh grass (Distichlis spicata L.) (Ketchum
et al., 1991). Most of the accumulated proline effective in osmoregulation was found in the cyto-
plasm. Therefore, it plays a major role in the osmotic adjustment of the vacuole. Cells maintained a
cytoplasmic proline concentration at least one order of magnitude greater than that of the vacuole.
Proline accumulation was inhibited by cycloheximide but not by actinomycin D. This indicated that
mRNA translation, not mRNA transcription, is required before proline production. Reports are also
available on a preferential accumulation of proline in nontolerant cells, as opposed to tolerant cells,
indicating a dependence on a salinity threshold (Jain et al., 1987).
A significantly higher level of free proline content was observed in sugarcane calli grown under
salt stress (Patade et al., 2008). An inverse correlation was found between the salt-tolerance and
NaCl-induced proline accumulation in intact plants and cell cultures of Salicornia europeae L.,
Chenopodium album L. and in two cotton (Gossipium hirsutum L.) cultivars, although the accumu-
lation of proline in leaves was of no importance for adaptation or survival of the plants (Shevyakova
et al., 1998).
Cultured cells of sorghum exposed to water stress by the addition of PEG to the growing media
increased proline content significantly (Bhaskaran et al., 1985). The magnitude of this increase was
not correlated with the drought tolerance of the individual varieties ruling out the role of proline as
an osmoprotectant in sorghum, as well as in other cereal crops.
9.4.3 Seed Germination and Proline Content under Stress

Most of the research on proline as an osmoregulatory compound has been carried out on the veg-
etative parts of plants. Little attention has been paid to the reproductive organs, especially seeds.
Information on the osmotic adjustment of seeds under stress conditions is available. Drought-
induced accumulation of soluble sugars, soluble proteins, free amino acids, and proline in seeds
of V. faba L. cultivars was seen when exposed to different levels of PEG (Al Tayeb, 2006). Proline
was used as an indicator of temperature stress in bean seeds (Machado et al., 2004). Proline content
was used to predict the rate of deterioration in corn seed that were treated with ascorbic acid and
germinated on NaCl stress condition (Anwar and Widajati, 2002). A close relationship between
proline content and quality of the corn seed was found; the higher the proline content of the seeds,
the lower the seed quality. Although there was a significant effect of salt stress on proline contents
of all the different parts of germinating seeds of spring wheat cultivars, there was no consistent pat-
tern of proline accumulation with time (Riaz, 2001). Salinity led to an accumulation of proline and
total soluble sugar in the radicals of the sensitive genotype of Cicer arietinum (Soussi et al., 2001).
Contents of proline, saccharides, and soluble proteins decreased in the germinating seeds during
3-d osmotic and salt stress of mung bean (V. radiata) (Zayed and Zeid, 1998). An approximately
fourfold increase in free proline was observed prior to radical emergence in A. thaliana seeds
(Hare et al., 2003). A dose-dependent inhibition of Arabidopsis seed germination by millimolar
concentrations of proline capable of feedback inhibition of proline synthesis reinforced the role of
proline synthesis in promoting germination. The ability of the artificial oxidants methylene blue
and phenazine ethosulphate to overcome the inhibitory effects of proline suggests a functional link
between elevated proline synthesis and increased oxidative pentose phosphate pathway activity and
the importance of coupling of both pathways in stimulating germination. During germination of
peanut seeds subjected to NaCl salinity stress, proline, and glycine–betaine concentrations in the
embryonic axis increased continuously (Girija et al., 2002). Two enzymes play an important role
in controlling the level of proline. Proline oxidase catalyzes the conversion of proline to gluta-
mate, thus reducing the concentration of proline. Another enzyme, gamma-glutamyl kinase, plays
an important role in the synthesis of proline. Addition of calcium chloride to NaCl-stressed peanut
seedlings lowered the proline concentration by increasing the level of proline oxidase and decreas-
ing gamma-glutamyl kinase activities. Salinity stress, in the absence of calcium, increased proline
due to reduced proline oxidase activity and increased gamma-glutamyl kinase activity both in the
cotyledons and embryonic axis of peanut seedlings. Thus calcium ions increase glycine–betaine
production but decrease proline levels in NaCl stressed peanut seedlings. Water absorption and
germination rate of the seeds were negatively correlated with the salt stress (Zhi and Li, 2009).
The contents of soluble sugar, soluble protein, and proline of the seedlings were higher in high salt
stress than that in low salt stress. Germination rates of seeds of Glycine max and Phaseolus vulgaris
were affected by exposure to NaCl, with critical thresholds at 9 and 12 g/L, respectively, while for
Mucuna poggei, Vigna unguiculata, and Phaseolus adenanthus, critical thresholds were more than
21 g/L (Taffouo et al., 2008). Germination rate of Pennisetum Rich. was decreased significantly
with increasing contents of PEG, but the radical/embryonic bud was increased (Jiao et al., 2009).
Drought resistance in annual medics (Medicago spp.) plants is gained by their capability of proline
accumulation (Ghamari-Zare et al., 2009). Seed germination of Dianthus japonicus was suppressed
at over 200 mM NaCl and proline content was increased in adversely affected plants (Heo et al.,
2007). Proline substantially accumulated in the seedlings of wheat cultivar (Doha) exposed to vari-
ous levels of salt stress (Yasseen et al., 2006). There was a linear relationship between accumulation
and the increase in NaCl concentration. Salt concentration of 50–100 mM increased seed germi-
nation and growth of Elaeagnus angustifolia, while at 300 mM, it was reduced by 50% (Yunus
et al., 2006). Proline content was insignificantly changed at low salinity but remarkably increased
at severe stress. There was strong negative correlation between germination capacity and increase
in proline content in germinating embryos of sorghum (Thakur and Sharma, 2005). Germination
percentage decreased in cultivars of sugarcane due to salinity, while proline content increased by
two- to threefold over the controls (Vasantha and Rao, 2005). Increased antioxidant enzyme activ-
ity coincided with enhanced proline content in Cassia angustifolia (Agarwal and Pandey, 2004).
Increase of membrane permeability of Hedysarum scoparium, H. fruticosum var. laeve, Caragana
korshinskii and Onobrychis viciifolia seedlings caused by salt stress was negatively correlated with
salt concentrations, while the cumulative amount of proline had no direct correlation with salt tol-
erance (An et al., 1995). Time at which proline accumulation peaked indicated the sensitivity of
plants to salt. A decrease in germination percentage, root length, shoot length, and fresh mass of
Phaseolus mungo was noticed with an increase in NaCl concentration (Dash and Panda, 2001). Salt
stress increased proline accumulation in the cotyledons and roots of germinating groundnut seeds
(Satakopan and Rajendran, 1989). Proline accumulated in the endosperm and radicals of germinat-
ing barley seeds with increasing NaCl concentrations in the growing media (Yasseen et al., 1989).
This proline probably originated from the degradation of stored protein in the endosperm. Under
increasing levels of salinity, germinating seeds from salt-tolerant cultivars of rice contained higher
levels of free amino acids than salt-sensitive cultivars (Dubey and Rani, 1989). In contrast, irrigation
of Cajeme wheat with saline water resulted in a continuous decrease in free proline in the grains
(Devitt et al., 1987). This decrease was associated with increased protein concentrations suggesting
either the rate of protein incorporation was accelerated under salinity or that free proline accumula-
tion was stunted in other parts of the plant.
9.4.4 Osmotic Adjustment in Halophytes

Halophytes are plants capable of growing and reproducing in highly saline environments. These
plants usually absorb large amounts of NaCl, which is believed to be sequestered in the cell vacu-
oles; otherwise, enzyme activity would be impaired. Although halophytes are naturally adapted
to salinity, their salt-tolerance limits are greatly influenced by their provenance and developmen-
tal stage. Halophytes are characterized by considerable asymmetry in the distribution of osmotica
within their cells, particularly between the vacuole and the cytoplasm (Yeo, 1981). The tolerance
of all halophytes to salinity relies on controlled uptake and compartmentalization of Na+, K+, and
Cl− and the synthesis of organic compatible solutes, even where salt glands are operative (Flowers
and Colmer, 2008). Proline, known to be accumulated in the cell cytoplasm to balance the osmotic
potential of the accumulated salt in the vacuole, does not play a role in the osmotic regulation of halo-
phytes. For example, in halophytic Chenopodiaceae, such as Suaeda monoica, Atriplex spongiosa
and Arthrocnemum fructicosum, proline accumulation was observed only at high inhibitory salini-
ties, promoting normal growth (Doddema et al., 1986; Storey and Wyn Jones, 1997). Similar results
were obtained with the halophyte M. crystallinum growth at 400 mM NaCl (Demming and Winter,
1986). Proline accumulation preceded the shift of crassulacean acid metabolism (CAM) in these
plants, but only under light (Sanada et al., 1995). The results suggested that in Mesembryanthemum,
proline has a bifunctional role in the acclimation to high salt stress: an osmoregulatory role in the
light and as a substrate for dark respiration to supply energy for compartmentation of ions into
vacuoles in the dark. Proline concentration in Spartina varied with N availability, although the
higher accumulation could not alleviate the growth inhibition caused by the high salinity level
(van Diggelen et al., 1986). Sporobolus virginicus showed high salt tolerance due to its ability to
accumulate high sodium and the secretion of salt through leaves for osmotic adjustment to reduce
the osmotic potential of the cell to avoid dehydration and death (Somsri and Arunee, 1994). All
accessions of Atriplex canescens responded to salinity by increasing their uptake of Na, which
is the primary mechanism of osmotic adjustment to salinity in this species (Glenn et al., 1996). It
is suggested that differences in tendency to accumulate Na or K among A. canescens genotypes
are related to their specialization for saline or xeric habitats, respectively. Exposure of Saticornia
europaea to short-term osmotic shock resulted in a rapid increase in free Mg2, followed by a five-
fold increase in sugar concentration, either of which could have provided adequate osmoticum to
prevent excessive water stress if mostly confined to the cytoplasm (McNulty, 1985). Subsequent
adjustment can only be explained by a reapportionment of compatible osmotica within the cells.
Such reapportionment would permit the osmotic adjustment in halophytes to occur primarily by
the absorption of extraneous ions. Salvadora persica, an evergreen perennial halophyte capable of
growing under extreme conditions, accumulates Na+ as a primary osmoticum (Maggio et al., 2000).
The concentration of Na+ in leaves of salinized plants was approximately 40-fold greater than that
measured in nonsalinized controls, and this was associated with significant reductions in leaf K+
and Ca2+ concentrations. In addition, a significant accumulation of proline, probably associated with
osmotic adjustment and protection of membrane stability, occurred in roots of salinized plants. The
salt tolerance of the annual halophyte Cakile maritima depends on the growth stage and severity of
the salt stress (Megdiche et al., 2007). C. maritima plants, subjected to a high salt stress (particu-
larly at 400 mM NaCl) accumulated high concentrations of proline and total soluble carbohydrates.
9.4.5 Negative Response

Despite the abundance of reports on proline accumulation in plants growing under drought or
salinity, a few others demonstrate a negative response as far as osmoregulation is concerned.
For example, the contribution of proline to osmotic adjustment in legumes is of minor impor-
tance (Larher et al., 1987). No significant accumulation of proline in the leaves of green gram
seedlings (Garg, 1987) or maize plants (Garg and Garg, 1982) was observed when grown in
sodium bicarbonate or sodium carbonate salts. Increased concentrations of chloride or sulfate
salts or PEG could not effectively stimulate proline accumulation in sugar cane leaves of a salt-
sensitive variety (Naik and Joshi, 1983). Similarly, pigeon pea plants failed to accumulate proline
at high-salinity level (Joshi, 1984). The lack of this adaptive mechanism may explain the failure
to develop salt tolerance in cultivated pigeon pea or sugar cane. In a more recent work, however,
salt tolerance could be found in wild relatives of pigeon pea belonging to the genera Atylosia,
Dunbaria, and Rynchosia but without correlation between salinity tolerance and proline accumu-
lation (Subbarao et al., 1990). In Andropogon glomeratus, a C 4 nonhalophytic salt marsh grass,
proline played no role in osmotic adjustment, since very high levels of salinity are required to
increase its concentrations (Bowman, 1988). One of the adaptive mechanisms suggested as being
associated with osmotic adjustment is a restriction in cell expansion (van Volkenburgh and Boyer,
1985). Extensin, a major plant cell wall glycoprotein in dicots, was found to be a hydroxyproline-
rich glycoprotein (Cassab and Vamer, 1988). Therefore, some work was carried out to deter-
mine changes in cell wall protein induced by salt stress and expressed as changes in proline and
hydroxyproline concentrations. No significant effect of stress on the proline and hydroxyproline
contents was found in a purified cell wall fraction of sunflower (Golan-Goldhirsh et al., 1990).
Therefore, changes in the physicochemical properties of the cell wall accompanying osmotic
adjustment appear to lie in other posttransitional modifications of extension. Cell membrane sta-
bility of four Arachis cultivars exposed to PEG was tested in response to drought tolerance (Deb
et al., 1996). It was found that proline was not effective in controlling the physiological status of
the cell membrane and its stability. Water-stressed pea seedlings did not significantly accumulate
proline (Generozova et al., 2009). Significant negative correlations were observed in barley geno-
types between Na+-induced K+ efflux (an indicator of salt tolerance) and leaf glycine–betaine and
proline (Chen et al., 2007), suggesting that hyperaccumulation of known major compatible solutes
in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of
salt-susceptibility. The small (twofold) increase of proline content in salinity stressed plants indi-
cates proline does not play a significant role in olive stress response (Rejskova et al., 2007). Other
reports are also cautioning its use (Lin and Kao, 2001; Hare et al., 2002; Heuer, 2003). Different
plant species, proline concentrations, and treatment stages may explain the discrepancy between
the effect of exogenous application of proline and its negative effects (Ashraf and Foolad, 2007).
9.5 Exogenous Proline Application

Plants growing in saline environments usually accumulate large amounts of NaCl in their tissue.
Because Na and Cl are inhibitory to a large number of enzymes, their presence in the cytoplasm
should be minimal. Evidence of the compartmentation of electrolytes between the cytosol and the
vacuole is available. The necessary osmotic balance between the two compartments is achieved
through the accumulation of organic solutes in the cytoplasm. Proline is one of these compatible sol-
utes. Besides playing an osmotic role, it should protect enzymes against denaturation or inhibition of
activity. This could be determined easily by adding exogenous proline to the assay media or to crude
extracts. Contradictory reports for differing plant species are known. For example, full protection
of phosphoenolpyruvate (PEP) carboxylase against NaCl inhibition was obtained in two Poaceae
species with a proline concentration between 200–800 mM, and proline behaved as a competitive
inhibitor in Chenopodiaceae (Manetas et al., 1986). Similar results were obtained from the activities
of NAD-malate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydroge-
nase, and glyceraldehyde phosphate dehydrogenase (Schwab and Gaff, 1990). Almost full protec-
tion of enzyme activities was obtained when proline was added at a molar ratio of 2:1 (protectant to
salt) simultaneously with the addition of salt to the reaction media. No protection was found when
proline was added after 1 h preincubation of enzyme extracts with high salt concentration. Extracts
from air-dried leaves recovered almost fully after more than 5 h of preincubation in 1 M proline.
Exogenous proline supplied to radish seedlings reduced tissue Hg levels owing to the inhibition of
Hg uptake (Khanna et al., 1995).
One approach to assess the metabolic consequences of proline accumulation in response to
stress is to examine its exogenous application to whole organism or tissues. Exogenously applied
proline stimulated the growth of bacteria subjected to osmotic stress (Britten and McClure, 1962;
le Redulier and Valentine, 1982). Proline has also been applied to higher plants to determine its
ability to counteract the inhibitory effects of environmental stress, mainly water or salt stress. The
exogenous application of proline has been suggested to be an effective approach in improving crop
salt tolerance in groundnut (Jain et al., 2001), melon (Kaya et al., 2007), and tobacco (Hoque et al.,
2007). The addition of 100 mM proline to a Hoagland solution containing 120 mM NaCl neutralized
the effect of salinity on pea plants (Bar-Nun and Poljakoff-Mayber, 1977). Incubation of Commelina
communis epidermal tissue in proline inhibited stomatal opening (Klein and Itai, 1989). The addi-
tion of 10 mM proline to cultured barley embryos increased shoot elongation under saline condi-
tions (Lone et al., 1987). This effect was attributed to the ability of proline to decrease the leaf
salt load. Proline allowed an enhanced K/Na discrimination in transport to the shoots and a better
salt exclusion from the shoots with retention in the roots. The callus lines of C. arietinum grown
in a medium containing 100 mM NaCl and 10 mM proline increased their fresh and dry weights
(Pandey and Ganapathy, 1985). Optimal concentrations of proline increased the cellular levels of
K and decreased Na and Cl levels. Spraying cotton plants grown under conditions of low soil water
potential with proline solutions counteracted the effects of stress, especially at moderate and high
stresses (Gadallah, 1995).
Exogenous proline had no effect on the germination of tomato seeds under water or salt stress
(Wu, 1987) or on the ribosome stability in the presence of 250 or 500 mM KCl (Brady et al., 1984),
but it increased pollen germination when exposed to brief temperature stress (Thind and Malik,
1994). The presence of 1 or 10 mM proline in media containing 100 or 200 mM NaCl had little
effect on the growth of the salt-adapted callus of rice (Kishor, 1989). Some concentrations sig-
nificantly increased the growth of salt-unadapted callus. Exogenous proline has been reported to
protect plants under stress. It improved the tolerance of somatic embryos of celery (Apium gra-
veolens L. cv. SB 12) to partial dehydration (Saranga et al., 1992). Okuma et al. (2000) found that
exogenous proline improved the growth of salt-stressed tobacco cell cultures and the improvement
was attributed to the role of proline as an osmoprotectant for enzymes and membranes against
salt inhibition rather than as a compatible solute. Exogenous proline partly alleviated growth of
Pancratium seedlings reduced by salt stress (Khedr et al., 2003). Proline induces the expression of
salt-stress-responsive proteins and may improve the adaptation of P. maritimum L. to salt stress.
The ability of exogenous proline to maintain higher water content in severely stressed seedlings
might be attributed to its contribution to osmotic adjustment both directly by increasing the internal
proline content and indirectly by increasing the internal contents of other amino acids. Exogenous
proline increased the protein content of the shoot and root at all levels of salt stress. Changes in
growth and protein contents as a result of exogenous proline correlated with increases in the internal
content of proline suggesting that proline was taken up into the roots and transported to the shoots.
Ion fluxes across the plasma membrane may be regulated by low concentrations of proline in barley
roots (Cuin and Shabala, 2005).
On the other hand, despite its protective role under a variety of stress conditions, external sup-
ply of proline was also found to be toxic to plant and animal cells (Hellman et al., 2000; Heuer,
2003). High concentrations of proline (50 and 100 mM) inhibited the growth of NaCl-stressed
as well as NaCl-nonstressed callus cultures of mung bean (Kumar and Sharma, 1989). Proline
(10 mM) inhibited the growth of salt grass suspension cultures in the presence of 260 mM NaCl
(Rodriguez and Heyser, 1988). Exogenous [13C] proline inhibited the normal biosynthesis of pro-
line that would have occurred in suspensions grown at this salinity level. It has been proposed
that proline-induced damage is mediated by P5C/GSA, which, if not metabolized rapidly, might
induce cell death. Exogenous proline was found to reduce the growth of roots of rice seedlings and
the amount of inhibition increased with increasing the concentration of proline from 1 to 4 mM.
This inhibition was attributed to the observation that proline-induced cell wall bound peroxidases
which resulted in increased lignin synthesis, thereby stiffening the cell wall and reducing expan-
sion (Lin and Kao, 2001). It has become apparent that an excess of free proline has negative or
side effects on cell growth or protein functions. Intracellular proline accumulation significantly
represses several genes involved in the synthesis of other amino acids or normal morphogenesis
in Arabidopsis plants (Nanjo et al., 2003). It is also suggested that the proportion of proline to the
total amino acids, rather than the concentration, influences the growth of petunia plants (Yamada
et al., 2005). Furthermore, the toxicity of proline and its metabolite P5C have been considered to
function as signals in stress-induced cell death (Hellman et al., 2000; Maxwell and Davis, 2000;
Morita et al., 2002; Deuschle et al., 2004; Nomura and Takagi, 2004). In S. cerevisiae, Maggio
et al. (2002) found that intracellular proline accumulation is associated with reduced growth rate. It
has also been reported that excess proline might be toxic to yeast cells only when it accumulates
in the cytosol (Morita et al., 2002) or might delay yeast cell growth in the presence of ethanol
(Takagi et al., 2007). Even at the enzyme level, proline overaccumulation to a concentration as low
as 100 mM suppresses the activity of the major chloroplastic enzyme ribulose 1, 5-bis-phosphate
carboxylase in higher plants (Sivakumar et al., 1998). These results suggest that intracellular pro-
line must be present at an appropriate level in order to confer stress tolerance. Foliar application
of proline was an effective way to improve the salt tolerance of cucumber (Huang et al., 2009).
The enhanced salt tolerance could be partially attributed to the improved water status and peroxi-
dase enzyme activity in the leaves.
The method of using exogenous proline proved that mechanism of feedback inhibition of proline
synthesis exists in fully turgid plant tissues but not in stressed tissues (Boggess et al., 1976). At this
point, enhanced proline oxidation also cannot be ignored. Moreover, this technique emphasized the
role of proline as a compatible solute involved in the process of the osmotic adjustment of living
organisms.
9.6 Proline Content as an Indicator for Breeding Programs

The existence in plants of quantitative variations in the physiological trait of proline accumulation in
response to water or salt stresses has suggested its possible consideration as a selection criterion for
breeding programs. This was indeed recommended for cereals growing in Mediterranean envi-
ronments (Richards et al., 1987). Research performed with 12 paddy genotypes showed a stimulation
of proline accumulation in the leaves of plants exposed to salinity (Pandey and Srivastava, 1990).
The salinity index of yield showed a significant positive association with proline accumulation,
prompting the suggestion of this physiological trait as one of the promising indices for breeding
salt-tolerant genotypes in rice. Nitrate reductase activity and proline content were much higher in
drought-resistant cultivars of paddy rice; therefore, it was suggested that these two factors may be
adopted as an indicator in the identification of drought resistance in rice breeding (Liu et al., 1993).
The magnitude of proline response also was suggested for screening alfalfa plants for salt tolerance
(Refoufi and Larher, 1989). On the other hand, Ashraf (1989) concluded that proline accumulation
cannot be used as an indicator for salt tolerance of black gram and is thus unsuitable for breeding
programs. The same is true for soybeans (Moftah and Michel, 1987) and pearl millet (Chandra and
Chauhan, 1983) although Petcu et al. (1995) stated that free proline content of the primary leaves
appeared to be a good selection criterion in breeding for drought tolerance. Final concentrations of
proline accumulated in tobacco plants are advocated as criteria to be used in selecting for drought-
tolerant tobacco genotypes as early as the F1- or F2-generations (1993). Hou et al. (1990) suggested
that increase of proline content in plants under soil water stress can be taken as an indicator of
drought resistance. Free proline and glycine–betaine accumulation in the leaves can be used as
the possible indicator for drought tolerance in maize genotypes (Moussa ana Abdel-Aziz, 2008).
Drought resistant sugarcane plants have an enhanced ability to accumulate proline when compared
with susceptible lines, making proline content a useful selection criterion (Widyasari and Sugiyarta,
1997). Proline content in storage roots of sugar beet is potentially useful as indicator of situations
that lead to decreased yield and quality of the root, such as drought stress (Monreal et al., 2007).
Proline accumulation can be used as an indicator of the salt stress imposed on citrus cell lines in
suspension cultures (Ferreira and Lima-Costa, 2006). High relative water content (RWC) and Pro
over-accumulation were recognized as beneficial drought tolerance indicators and may be used as
selection criteria in wheat breeding programs (Bayoumi et al., 2008). Correlation analysis discarded
proline as an indicator of drought tolerance (Farshadfar et al., 2008). It can be concluded that, in
general, proline accumulation is specific to a genotype, and generalization over different varieties
of a crop is not always possible.
9.7 Conclusions
The accumulation of proline in plants subjected to water or salt stress has been observed widely,
although not universally. Several possible physiological functions have been ascribed to induced
proline accumulation by water shortage. These functions include osmoregulation, a soluble N sink,
a signal of senescence, and an indicator of plant resistance to stress. Proline accumulation is a com-
mon physiological response to various stresses but is also part of the developmental program in
generative tissues. Proline may affect the solubility of various proteins, thus protecting them against
denaturation under water-stressed conditions. An increase in the proline content may be associ-
ated with either enhanced biosynthesis, with stimulated proline oxidation, or an impaired protein
synthesis. In general, proline concentrations are directly proportional to the salinity level or to the
intensity of water stress. Genotype variations are very common; however, a positive correlation
cannot always be found between proline content and a plant’s relative tolerance or susceptibility.
Restoring plants to optimal growth conditions, results in a rapid decline in the proline content.
Transgenic approaches have confirmed the beneficial effect of proline overproduction during stress,
although no consensus was achieved on the exact roles of proline accumulation. Classical gain or
loss of function strategies could not bring clear answers, probably because proline also displays the
essential role of being a protein component. Environmental stresses such as drought or salinity have
multiple targets and proline is believed to play different roles. The balance between biosynthesis and
degradation of proline is probably essential in the determination of the osmoprotective and devel-
opmental functions of proline. Additional studies are required to elucidate conclusively the role of
proline in plant adaptation to stress.
References
Abebe, T., Guenzi, A.C., Martin, B., and Cushman, J.C. (2003). Tolerance of mannitol-accumulating transgenic
wheat to water stress and salinity. Plant Physiology 131: 1748–1755.
Adams, E. and Frank, L. (1980). Metabolism of proline and the hydroxy. Annual Review of Biochemistry 49:
1005.
Agarwal, S. and Pandey, V. (2004). Antioxidant enzyme responses to NaCl stress in Cassia angustifolia.
Biologia Plantarum 48: 555–560.
Ahmad, I. and Hellebust, J.A. (1984). Osmoregulation in the extremely euryhaline marine micro alga Chlorella
autotrophica. Plant Physiology 74: 1010.
Ahmad, M.S.A., Javed, F., and Ashraf, M. (2007). Iso-osmotic effect of NaCl and PEG on growth, cations and
free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes. Plant Growth
Regulation 53: 53–63.
Ain-Lhout, F., Zunzunegui, M., Barradas, M.C.D., Tirado, R., Clavijo, A., and Novo, F.G. (2001). Comparison
of proline accumulation in two Mediterranean shrubs subjected to natural and experimental water deficit.
Plant and Soil 230: 175–183.
Al Mansoori, T.A., El-Deen, M.N.A., and Caligari, P.D.S. (2007). Evaluation of in vitro screening techniques
for salt tolerance in date palm. Proceedings of the Third International Date Palm Conference 736:
301–307.
Al Tayeb, M.A. (2006). Differential responses of pigments, lipid per-oxidation, organic solutes, catalase and
peroxidase activity in the leaves of two Vicia faba L. cultivars to drought. International Journal of
Agriculture and Biology (Pakistan) 8: 116–122.
Alam, S.M. (1999). Nutrient uptake by plants under stress conditions. In: Handbook of Plant and Crop Stress,
Pessarakli, M., ed. New York, Marcel Dekker, pp. 285–314.
An, S.Q., Yu, Z., Kang, L.J., and Chen, X.R. (1995). Study on salt tolerance in four legumes at seed germination
and seedling stages. Grassland of China 6: 29–32.
Anwar, A. and Widajati, E. (2002). Prediction the rate of deterioration of corn seed treated with ascorbic acid
and germinated on NaCl stress condition using proline content. Journal Stigma (Indonesia) 10: 196–201.
Apte, S.K. (2001). Coping with salinity/water stress: Cyanobacteria show the way. Proceedings of the Indian
National Science Academy Part B, Reviews and Tracts Biological Sciences 67: 285–310.
Arora, S. and Saradhi, P.P. (1995). Light-induced enhancement in proline in Vigna radiata exposed to environ-
mental stresses. Australian Journal Plant Physiology 22: 383.
Ashraf, M. (1989). The effect of NaCl on water relations, chlorophyll and proline contents of two cultivars of
blackgram (Vigna mungo L.). Plant Soil 119: 205.
Ashraf, M. (1994). Breeding for salinity tolerance in plants. Critical Reviews in Plant Science 13: 17–42.
Ashraf, M. and Foolad, M.R. (2007). Roles of glycine betaine and proline in improving plant abiotic stress
resistance. Environmental Experimental Botany 59: 206–216.
Ashraf, M., Saeed, M.M., and Qureshi, M.J. (1994). Tolerance to high temperature in cotton (Gossypium hir-
sutum L.) at initial growth stages. Environmental Experimental Botany 34: 275.
Bandurska, H. (2001). Does proline accumulated in leaves of water deficit stressed barley plants confine
cell membrane injuries? II. Proline accumulation during hardening and its involvement in reduc-
ing membrane injuries in leaves subjected to severe osmotic stress. Acta Physiologia Plantarum
23: 483–490.
Bar-Nun, N. and Poljakoff-Mayber, A. (1977). Salinity stress and the content of proline in roots of Pisum sati-
vum and Tamarix tetragyna. Annals of Botany 41: 173.
Bayoumi, T.Y., Eid, M.H., and Metwali, E.M. (2008). Application of physiological and biochemical indices
as a screening technique for drought tolerance in wheat genotypes. African Journal of Biotechnology
7: 2341–2352.
Bensen, R.J., Boyer, J.S., and Mullet, J.E. (1988). Water deficit induced changes in abscisic acid, growth, poly-
somes, and translatable RNA in soybean hypocotyls. Plant Physiology 88: 289–294.
Bernstein, L. (1961). Osmotic adjustment of plants to saline media. I. Steady state. American Journal of Botany
48: 909–918.
Bhaskaran, S., Smith, R.H., and Newton, R.J. (1985). Physiological changes in cultured sorghum cells in
response to induced water stress. I. Free proline. Plant Physiology 79: 266.
Binzel, M.L., Hasegawa, P.M., Rhodes, D., Handa, S., Handa, A.K., and Bressan, R.A. (1987). Solute accumu-
lation in tobacco cells adapted to NaCl. Plant Physiology 84: 1408.
Boggess, S.F. and Stewart, C.R. (1980). The relationship between water stress induced proline accumulation
and inhibition of protein synthesis in tobacco leaves. Plant Science Letters 17: 245.
Boggess, S.F., Aspinall, D., and Paleg, L. (1976). Stress metabolism. IX. The significance of end-product inhi-
bition of proline biosynthesis and of compartmentation in relation to stress induced proline accumula-
tion. Australian Journal of Plant Physiology 3: 513–525.
Bohnert, H.J., Nelson, D.E., and Jensen, R.G. (1995). Adaptations to environmental stresses. Plant Cell
7: 1099–1111.
Borsani, O., Zhu, J.H., Verslues, P.E., and Zhu, J.K. (2005). Endogenous siRNAs derived from a pair of natural
cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123: 1279–1291.
Bowman, W.D. (1988). Ionic and water relation responses of two populations of a non-halophyte to salinity.
Journal Experimental Botany 39: 97.
Brady, C.J., Gibson, T.S., Barlow, E.W.R., Speirs, J., and Wyn Jones, R.G. (1984). Ions, compatible organic
solutes and the stability of plant ribosomes. Plant Cell Environment 7: 571.
Britten, R.J. and McClure, F.T. (1962). The amino acid pool in E. coli. Bacteriology Review 26: 292.
Carillo, P., Mastrolonardo, G., Nacca, F., Paris, D., Verlotta, A., and Fuggi, A. (2008). Nitrogen metabolism in
durum wheat under salinity: Accumulation of proline and glycinebetaine. Functional Plant Biology 35:
412–426.
Cassab, G.I. and Vamer, J.E. (1988). Cell wall proteins. Annual Review of Plant Physiology and Plant Molecular
Biology 39: 321.
Chandler, S.F. and Thorpe, T.A. (1987). Characterization of growth, water relations and proline accumulation
in sodium sulfate tolerant callus of Brassica napus L. cv. Westar (Canola). Plant Physiology 184: 106.
Chandra, S. and Chauhan, R.S. (1983). Free proline in barley pearl millet and chickpea grown under soil salin-
ity stress. Indian Journal of Genetics 43: 457.
Chen, Z.H., Cuin, T.A., Zhou, M.X., Twomey, A., Naidu, B.P., and Shabala, S. (2007). Compatible solute accu-
mulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. Journal of
Experimental Botany 58: 4245–4255.
Csonka, L.N. (1989). Physiological and genetic response of bacteria to osmotic stress. Microbiological Review
53: 121.
Cuin, T. and Shabala, S. (2005). Exogenously supplied compatible solutes rapidly ameliorate NaCl-induced
potassium efflux from barley roots. Plant and Cell Physiology 46(12): 1924–1933.
Cutler, J.M. and Rains, D.W. (1978). Effects of water stress and hardening on the internal water relations and
osmotic constituents of cotton leaves. Physiologia Plantarum 42: 261.
Dalvi, U.S., Chavan, U.D., Kachare, D.P., and Naik, R.M. (2007). Proline metabolism in sorghum and chickpea
cultivars during water stress. Indian Journal of Plant Physiology 12(3): 287–289.
Das, N., Misra, M., and Misra, A.N. (1990). Sodium chloride salt stress induced metabolic changes in callus
cultures of pearl millet (Pennisetum americanum L. Leeke): Free solute accumulation. Journal of Plant
Physiology 137: 244.
Dash, M. and Panda, S.K. (2001). Salt stress induced changes in growth and enzyme activities in germinating
Phaseolus mungo seeds. Biologia Plantarum 44: 587–589.
Deb, N., Alam, B, Gupta, S.D., and Ghosh, B.C. (1996). Cell membrane stability of leaf tissues and its relation-
ship with drought tolerance in Arachis. Indian Journal of Experimental Biology 34: 1044.
Delauney, A.J. and Verma, D.P.S. (1993). Proline biosynthesis and osmoregulation in plants. Plant Journal 4:
215–223.
Demming, B. and Winter, K. (1986). Sodium, potassium, chloride and proline concentration of chloroplasts
isolated from a halophyte, Mesembryanthemum crystallinum L. Planta 168: 421.
Deuschle, K., Funck, D., Forlani, G., Stransky, H., Biehl, A., Leister, D., van der Graaff, E., Kunze, R., and
Frommer, W.B. (2004). The role of [Delta]1-pyrroline-5-carboxylate dehydrogenase in proline degrada-
tion. Plant Cell 16: 3413–3425.
Devitt, D.A., Stolzy, L.H., and Labanauskas, C.K. (1987). Impact of potassium, sodium and salinity on the
protein and free amino acid content of wheat grain. Plant Soil 103: 101.
Di Martino, C., Pizzuto, R., Pallotta, M.L., De Santis, A., and Passarella, S. (2006). Mitochondrial transport in
proline catabolism in plants: The existence of two separate translocators in mitochondria isolated from
durum wheat seedlings. Planta 223: 1123–1133.
Diamant, S., Eliahu, N., Rosenthal, D., and Goloubinoff, P. (2001). Chemical chaperones regulate molecular
chaperones in vitro and in cells under combined salt and heat stresses. Journal of Biological Chemistry
276: 39586–39591.
van Diggelen, J., Rozema, J., Dickson, D.M., and Broekman, R. (1986). ß-3-dimethylsulphoniopropionate,
proline and quaternary ammonium compounds in Sparina anglica in relation to sodium chloride, nitro-
gen and sulphur. New Phytologist 103: 573.
Doddema, H., Eddin, R.S., and Mahasneh, A. (1986). Effects of seasonal changes of soil salinity and soil
nitrogen on the N-metabolism of the halophyte Arthrocnemum fructicosum (L.). Moq. Plant Soil
92: 279.
Dubey, R.S. and Rani, M. (1989). Salinity induced accumulation of free amino acids in germinating rice seeds
differing in salt tolerance. Journal of Agronomy and Crop Science 163: 236.
Dupont, L., Boscari, A., Mandon, K., Boncompagni, E., Trinchant, J.C., and Rudulier, D. le. (2003). In:
Biotechnology in Sustainable Biodiversity and Food Security, Prasad, B.N., ed. pp. 65–77.
Eaton, F.M. (1927). The water requirement and cell-sap concentration of Australian saltbush and wheat as
related to the salinity of the soil. American Journal of Botany 14: 212–226.
Eberhardt, H.J. and Wegmann, K. (1989). Effects of abscicic acid and proline on adaptation of tobacco callus
cultures to salinity and osmotic shock. Physiologia Plantarum 76: 283.
Ehsanpour, A.A. and Fatahian, N. (2003). Effects of salt and proline on Medicago sativa callus. Plant Cell,
Tissue and Organ Culture 73: 53–56.
El-Kheir, M.S.A.A., Kandil, S.A., and Mekki, B.B. (1994). Physiological response of two soybean cultivars
grown under water stress conditions as affected by CCC treatment. Egyptian Journal of Physiological
Science 18: 179.
Farshadfar, E., Haghparast, R., and Qaitoli, M. (2008). Chromosomal localization of the genes controlling
agronomic and physiological indicators of drought tolerance in barley using disomic addition lines. Asian
Journal of Plant Sciences 7: 536–543.
Fayez, K.A. and Kristen, U. (1996). The influence of herbicides on the growth and proline content of primary
roots and on the ultrastructure of root caps. Environmental and Experimental Botany 36: 71.
Ferreira, A.L. and Lima-Costa, M.E. (2006). Metabolic responses to salt stress in cell suspension cultures of
sensitive and resistant Citrus. Journal of Horticultural Science and Biotechnology 81: 983–988.
Flowers, T.J. and Colmer, T.D. (2008). Salinity tolerance in halophytes. New Phytologist 179: 945–963.
Fujita, T., Maggio, A., Garcia-Rios, M., Bressan, R.A., and Csonka, L.N. (1998). Comparative analysis of the
regulation of expression and structures of two evolutionarily divergent genes for Delta(1)-pyrroline-5-
carboxylate synthetase from tomato. Plant Physiology 118: 661–674.
Gadallah, M.A.A. (1995). Effect of water stress, abscicic acid and proline on cotton plants. Journal of Arid
Environment 30: 315.
Garcia, A.B., Almeida-Engler, J., Lyer, S., Gerats, T., Van Montagu, M., and Caplan, A.B. (1997). Effects of
osmoprotectants upon NaCl stress in rice. Plant Physiology 115: 159–169.
Garg, B.K. (1987). Sodium carbonate and bicarbonate induced growth and some metabolic changes in green
gram seedlings. Current Agriculture 11: 41.
Garg, B.K. and Garg, O.P. (1982). Growth and some metabolic changes in maize leaves as affected by saline-alkaline
conditions due to sodium carbonate and sodium bicarbonate. Indian Journal of Plant Physiology 25: 220.
Generozova, I.P., Maevskaya, S.N., and Shugaev, A.G. (2009). The inhibition of mitochondrial metabolic activ-
ity in etiolated pea seedlings under water stress. Russian Journal of Plant Physiology 56: 38–44.
Ghamari-Zare, A., Rezvani, S., and Forootan, M. (2009). Assessment of resistance to PEG-induced drought in
annual medic using aquaculture conditions. Iranian Journal of Rangelands and Forests Plant Breeding
and Genetic Research 16: 182–197.
Ginzberg, I., Stein, H., Kapulnik, Y., Szabados, L., Strizhov, N., Schell, J., Koncz, C., and Zilberstein, A.
(1998). Isolation and characterization of two different cDNAs of Delta(1)-pyrroline-5-carboxylate syn-
thase in alfalfa, transcriptionally induced upon salt stress. Plant Molecular Biology 38: 755–764.
Girija, C., Smith, B.N., and Swamy, P.M. (2002). Interactive effects of sodium chloride and calcium chloride
on the accumulation of proline and glycinebetaine in peanut (Arachis hypogaea L.). Environmental and
Glenn, E., Pfister, R., Brown, J.J., Thompson, T.L., and O’Leary, J. (1996). Na and K accumulation and salt tol-
erance of Atriplex canescens (Chenopodiaceae) genotypes. American Journal of Botany 83: 997–1005.
Gloux, K. and Le Rudulier, D. (1989). Transport and catabolism of proline betaine in salt-stressed Rhizobium
meliloti. Archives of Microbiology 151: 143.
Goddijn, O.J.M. and van Dun, K. (1999). Trehalose metabolism in plants. Trends in Biochemical Science
4: 315–319.
Golan-Goldhirsh, A., Hankamer, B., and Lips, S.H. (1990). Hydroxyproline and proline content of cell walls of
sunflower, peanut and cotton grown under salt stress. Plant Science 69: 27.
Grattan, S.R. and Grieve, C.M. (1999). Mineral nutrient acquisition and response by plants grown in saline
environments. In: Handbook of Plant and Crop Stress, Pessarakli, M. ed. New York, Marcel Dekker,
pp. 203–229.
Greenway, H. and Munns, R. (1980). Mechanisms of salt tolerance in non-halophytes. Annual Review of Plant
Physiology 31: 149–190.
Hamilton, E.W. and Heckathorn, S.A. (2001). Mitochondrial adaptations to NaCl. Complex I is protected by
anti-oxidants and small heat shock proteins, whereas Complex II is protected by proline and betaine.
Plant Physiology 126: 1266–1274.
Hare, P.D. and Cress, W.A. (1997). Metabolic implications of stress-induced proline accumulation in plants.
Plant Growth Regulation 21: 79–102.
Hare, P.D., Cress, W.A., and van Staden, J. (1998). Dissecting the roles of osmolyte accumulation during stress.
Plant Cell Environment 21: 535–553.
Hare, P.D., Cress, W.A., and van Staden, J. (2002). Disruptive effects of exogenous proline on chloroplast and
mitochondrial ultrastructure in Arabidopsis leaves. South African Journal of Botany 68: 393–396.
Hare, P.D., Cress, W.A., and van Staden, J. (2003). A regulatory role for proline metabolism in stimulating
Arabidopsis thaliana seed germination. Plant Growth Regulation 39: 41–50.
Hasegawa, P.M., Bressan, R.A., Zhu, J.K., and Bohnert, H.J. (2000). Plant cellular and molecular responses to
high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51: 463–499.
He, T. and Cramer, G.R. (1996). Abscisic acid concentrations are correlated with leaf area reductions in two
salt-stressed rapid cycling Brassica species. Plant and Soil 179: 25–33.
Hellmann, H., Funck, D., Rentsch, D., and Frommer, W.B. (2000). Hypersensitivity of an Arabidopsis sugar
signaling mutant toward exogenous proline application. Plant Physiology 123: 779–789.
Heo, E.J., Jung, H.H., and Kim, K.S. (2007). Response of Dianthus japonicus Thunb. to NaCl stress imposed
at different growth stages. Horticulture, Environment and Biotechnology 48: 381–386.
Heuer, B. (2003). Influence of exogenous application of proline and glycinebetaine on growth of salt-stressed
tomato plants. Plant Science 165: 693–699.
Hong, Z., Lakkineni, K., Zhang, Z., and Verma, D.P.S (2000). Removal of feedback inhibition of delta(1)-
pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants
from osmotic stress. Plant Physiology 122: 1129–1136.
Hoque, M.A., Okuma, E., Banu, M.N.A., Nakamura, Y., Shimoishi, Y., and Murata, Y. (2007). Exogenous pro-
line mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant
enzyme activities. Journal of Plant Physiology 164: 553–561.
Hou, H.T., Li, B.D., and Zao, G.D. (1990). Genetic correlation of drought-resistant characters and selection
effectiveness in sorghum. Hereditas (Beijing) 12: 5–8.
Hsiao, T.C. (1973). Plant responses to water stress. Annual Review of Plant Physiology 24: 519.
Hsiao, T.C., Acevedo, F., Fereres, E., and Henderson, D. (1976). Stress metabolism, water stress, growth and
osmotic adjustment. Philosophical Transactions of the Royal Society of London (B), 273: 479.
Hua, X.J., Van de Cotte, B., Van Montagu, M., and Verbruggen, N. (1997). Developmental regulation of pyrro-
line-5-carboxylate reductase gene expression in Arabidopsis. Plant Physiology 114: 1215–1224.
Huang, Y., Bie, Z., Liu, Z., Zhen, A., and Wang, W. (2009). Protective role of proline against salt stress is
partially related to the improvement of water status and peroxidase enzyme activity in cucumber. Soil
Science and Plant Nutrition 55: 698–704.
Ikuko, Y., Kazuya, Y., Keiji, K., Atsushi, N., Hideki, N., and Atsuhiko, S. (2004). Overexpression of NtHAL3
genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured
tobacco cells. Journal of Experimental Botany 55(396): 387–395.
Iyer, S. and Caplan, A. (1998). Products of proline catabolism can induce osmotically regulated genes in rice.
Plant Physiology 116: 203–211.
Jain, R.K., Dhawan, R.S., Saarma, D.R., and Chowdhry, J.B. (1987). Salt tolerance and proline accumulation:
A comparative study in salt tolerant and wild type cultured cells of egg plant. Plant Cell Reports 6: 382.
Jain, M., Mathur, G., Koul, S. and Sarin, N.B. (2001). Ameliorative effects of proline on salt stress-induced
lipid peroxidation in cell lines of groundnut (Arachis hypogea L.). Plant Cell Reports 20: 463–468.
Jaiwal, P.K., Singh, R.P., and Gulat, A. (Eds.). Strategies for Improving Salt Tolerance in Higher Plants, Oxford
and IBH Publication Co., New Delhi, pp. 365–391.
Jiao, S.Y., Li, Y.Q., Shayila, S., and Chen, X.L. (2009). Seeds germination and seedling growth about 3 Pennisetum
ornamental grasses under drought stress. Acta Botanica Boreali Occidentalia Sinica 29: 308–313.
Jones, M.M., Osmond, C.B., and Turner, N.C. (1980). Accumulation of solutes in leaves of sorghum and sun-
flower in response to water deficit. Australian Journal of Plant Physiology 7: 193.
Joshi, S.S. (1984). Effect of salinity stress on organic and mineral constituents in the leaves of pigeonpea
(Cajanus cajan L. var. C-11). Plant Soil 82: 69.
Kamenova-Yukhimenko, S., Georgieva, V., Georgieva, N., and Balabanova, M. (1995). Effect of Polystimulin
K on the resistance of two pea cultivars to high cadmium concentrations. Rasteniev”dni-Nauk 32: 48–50.
Kastori, R., Petrovic, N., and Petrovic, M. (1996). Effect of lead on water relations, proline concentration and
nitrate reductase activity in sunflower plants. Acta Agronomica Hungary 44: 21–28.
Kaya, C., Tuna, A.L., Ashraf, M., and Altunlu, H. (2007). Improved salt tolerance of melon (Cucumis melo L.)
by the addition of proline and potassium nitrate. Environmental and Experimental Botany 60: 397–403.
Kemble, A.R. and MacPherson, H.T. (1954). Liberation of amino acids in perennial ryegrass during wilting.
Biochemistry Journal 58: 46.
Ketchum, R.E.B., Waren, R.S., Klima, L., Gutierrez, F.L., and Nabors, M.W. (1991). The mechanism and
regulation of proline accumulation in suspension cell cultures of the halophytic grass Distichlis spicata
L. Journal Plant Physiology 137: 368.
Khanna, S., Rai, V.K., and Khanna, S. (1995). Amelioration of mercury toxicity in radish Raphanus sativus L.
seedlings by L-proline and other amino acids. Indian Journal of Experimental Biology 33: 766.
Khedr, A., Abbas, M.A., Wahid, A., Quick, W., and Abogadallah, G. (2003). Proline induces the expression
of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-
stress. Journal of Experimental Botany 54(392): 2553–2562.
Kieft, T.L. and Spence, S.D. (1988). Osmoregulation in Thiobacillus jerrooxidans: Stimulation of iron oxida-
tion by proline and betaine under salt stress. Current Microbiology 17: 255.
Kiryan, I.G. and Shevyakova, N.I. (1984). Pathways of accumulation of free proline in an NaCl-resistant cell
line of Nicoriana sylvestris. Soviet Plant Physiology 31: 561.
Kishor, P.B.K. (1988). Effect of salt stress on callus cultures of Oryza sativa L. Journal of Experimental Botany 39: 235.
Kishor, P.B.K. (1989). Salt stress in cultured rice cells: Effects of proline and abscicic acid. Plant Cell
Environment 12: 629–633.
Kishor, P.B.K., Hong, Z.L., Miao, G.H., Hu, C.A.A., and Verma, D.P.S. (1995). Overexpression of [delta]-
pyrroline-5-carboxylate synthetase increases roline production and confers osmotolerance in transgenic
plants. Plant Physiology 108: 1387–1394.
Kiyosue, T., Yoshiba, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1996). A nuclear gene encoding mito-
chondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline
but downregulated by dehydration in Arabidopsis. Plant Cell 8: 1323–1335.
Klein, A. and Itai, C. (1989). Is proline involved in stomata regulation of Commelina communis plants recover-
ing from salinity stress. Physiologia Plantarum 75: 399.
Kore, B.A. and Patil, T.M. (1995). Effect of two bipyridylium herbicides on transpiration rate, pigments, pro-
line and peroxidase enzyme in Parthenium hysterophorus Linn. Advances in Plant Science 8: 360.
Kumar, V. and Sharma, D.R. (1989). Effect of exogenous proline on growth and ion content in NaCl stressed
and non-stressed cells of mungbean, Vigna radiata var. radiata. Indian Journal of Experimental Botany
27: 813.
Larher, F., Huq, S.M.I., Gerant-Sauvage, D., and Imamu-Hug, S.M. (1987). Salt sensitivity in legumes during
the early stages of development. Coll INRA 37: 181.
Le Dily, F., Billarld, J.B., and Boucaud, J. (1991). Polyamine levels in relation to growth and NaCl concentra-
tion in normal and habituated sugarbeet callus cultures. Plant Cell Environment 14: 327.
Leisinger, T. (1987). Biosynthesis of proline in E. coli and Salmonella typhimurium. In: Cellular and molecu-
lar Biology, Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M., and Umbarger,
H.E., eds. Washington, DC, American Society for Microbiology, pp. 345–351.
Lin, C.C. and Kao, C.H. (2001). Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root
growth of rice seedlings. Plant and Soil 230: 135–143.
Liu, B.G., Li, C.M., Ren, C.F., Cai, X.G., Yang, Q.L., and Chen, X.W. (1993). A study of the physiological
basis for upland culture of paddy rice. Journal of Southwest Agricultural University 15: 477–482.
Lone, M.I., Kueh, R.G., and Wyn Jones, S.W.J. (1987). Influence of proline and glycinebetaine on salt tolerance
cultured barley embryos. Journal of Experimental Botany 38: 479.
Ludlow, M.M. (1980). Adaptive significance of stomatal responses to water stress. In: Adaptation of Plants to
Water and High Temperature, N.C. Turner and P.J. Kramer, eds. New York, Wiley.
Lui, J. and Zhu, J.K. (1997). Proline accumulation and salt-stress-induced gene expression in a salt-hypersen-
sitive mutant of Arabidopsis. Plant Physiology 114: 591–596.
Lutis, S., Kinet, J.M., and Bouharmont, J. (1996). Effects of various salts and of mannitol on ion and pro-
line accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultures. Journal of Plant
Madkour, M.A., Smith, L.T., and Smith, G.M. (1990). Preferential osmolyte accumulation: A mecha-
nism of osmotic stress adaptation in diazotrophic bacteria. Applied and Environmental Microbiology
56: 2876–2881.
Maggio, A., Reddy, M.P., and Joly, R.J. (2000). Leaf gas exchange and solute accumulation in the halophyte
Salvadora persica grown at moderate salinity. Environmental and Experimental Botany 44: 31–38.
Malallah, G., Agzal, M., Gulshan, S., Abraham, D., Kurian, M., and Dharni, M.S.I. (1996). Vicia faba as a
bioindicator of oil pollution. Environmental Pollution 92: 213–217.
Manetas, J., Petropoulu, J., and Karabourniotis, G. (1986). Compatible solutes and their effects on PEP carbox-
ylase of C4 halophytes. Plant Cell Environment 91: 145.
Mani, S., Van de Cotte, B., Van Montagu, M., and Verbruggen, N. (2002). Altered levels of proline dehydro-
genase cause hypersensitivity to proline and its analogs in Arabidopsis. Plant Physiology 128: 73–83.
Mansour, M.M.F. (2000). Nitrogen containing compounds and adaptation of plants to salinity stress. Biologia
Plantarum 43: 491–500.
Mattana, M., Biazzi, E., Consonni, R., Locatelli, F., Vannini, C., Provera, S., and Coraggio, I. (2005).
Overexpression of Osmyb4 enhances compatible solute accumulation and increases stress tolerance of
Arabidopsis thaliana. Physiologia Plantarum 125: 212–223.
Mattioli, R., Marchese, D., D’Angeli, S., Altamura, M.M., Costantino, P., and Trovato, M. (2008). Modulation
of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis. Plant
Molecular Biology 66: 277–288.
McNeil, S.D., Nuccio, M.L., and Hanson, A.D. (1999). Betaines and related osmoprotectants: Targets for meta-
bolic engineering of stress resistance. Plant Physiology 120: 945–949.
Mestichelli, L.J., Gupta, R.N., and Spenser, I.D. (1979). The biosynthetic route from ornithine to proline.
Journal of Biological Chemistry 254: 640–647.
Meyer, R.F. and Boyer, J.S. (1972). Sensitivity of cell division and cell elongation to low water potentials in
soybean hypocotyls. Planta 108: 77–87.
Miller, R.J., Bell, D.T., and Koeppe, D.E. (1971). The effect of water stress on some membrane characteristics
of corn mitochondria. Plant Physiology 48: 229.
Moftah, A.E. and Michel, B.E. (1987). The effect of sodium chloride on solute potential and proline accumula-
tion in soybean leaves. Plant Physiology 83: 238.
Monreal, J.A., Jimenez, E.T., Remesal, E., Morillo-Velarde, R., Garcia-Maurino, S., and Echevarria, C. (2007).
Proline content of sugar beet storage roots: Response to water deficit and nitrogen fertilization at field
conditions. Environmental and Experimental Botany 60: 257–267.
Morgan, J.M. (1983). Osmoregulation as a selection criterion for drought tolerance in wheat. Australian
Journal of Agricultural Research 34: 607.
Morgan, J.M. (1984). Osmoregulation and water stress in higher plants. Annual Review of Plant Physiology
35: 299–319.
Moussa, H.R. and Abdel-Aziz, S.M. (2008). Comparative response of drought tolerant and drought sensitive
maize genotypes to water stress. Australian Journal of Crop Science 1: 31–36.
Naik, G.P. and Joshi, G.V. (1983). Ineffectual role of proline metabolism in salt-stressed sugarcane leaves.
Proceeding of Indian Academy Sciences 92: 265.
Nanjo, T., Kobayashi, M., Yoshiba, Y., Sanada, Y., Wada, K., Tsukaya, H., Kakubari, Y., Yamagushi-Shinozaki,
K., and Shinozaki, K. (1999a). Biological functions of proline in morphogenesis and osmotolerance
revealed in antisense transgenic Arabidopsis thaliana. Plant Journal 18: 185–193
Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999b).
Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis
thaliana. FEBS Letters 461: 205–210.
Nanjo, T., Fujita, M., Seki, M., Kato, T., Tabata, S., and Shinozaki, K. (2003). Toxicity of free proline revealed
in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiology
44: 541–548.
Nir, I., Poljakoff-Mayber, A., and Klein, S. (1971). The effect of water stress on mitochondria of root cells.
Plant Physiology 145: 173.
Noguchi, M., Kowai, A., Yokoyama, M., and Tamaki, E. (1968). Studies on nitrogen metabolism in tobacco
plants. IX. Effect of various compounds on proline biosynthesis in the green leaves. Plant Cell Physiology
9: 35–47.
Okuma, E., Soeda., K., Tada, M., and Murata, Y. (2000). Exogenous proline mitigates the inhibition of growth of
Nicotiana tabacum cultured cells under saline conditions. Soil Science and Plant Nutrition 46: 257–263.
Ozturk, L. and Demir, Y. (2002). In vivo and in vitro protective role of proline. Plant Growth Regulators
38: 259–264.
Pandey, R. and Ganapathy, P.S. (1985). The proline enigma: NaCl-tolerant and NaCl-sensitive callus lines of
Cicer arientinum. Plant Science 40: 13.
Pandey, U.K. and Srivastava, R.D.L. (1990). Salinity index in relation to nitrate reductase activity and proline
accumulation in paddy genotypes. Indian Journal of Plant Physiology 32: 175.
Pareek, A., Singla, S.L., and Grover, A. (1997). Salt responsive proteins/genes in crop plants. In: Strategies
for Improving Salt Tolerance in Higher Plants, Jaiwal, P.K., Singh, R.P., and Gulati, A. eds. New Delhi,
Oxford and IBH Publication Co., pp. 365–391.
Pasquali, G., Biricolti, S., Locatelli, F., Baldoni, E., and Mattana, M. (2008). Osmyb4 expression improves
adaptive responses to drought and cold stress in transgenic apples. Plant Cell Reports 27: 1677–1686.
Patade, V.Y., Suprasanna, P., and Bapat, V.A. (2008). Effects of salt stress in relation to osmotic adjustment on
sugarcane (Saccharum officinarum L.) callus cultures. Plant Growth Regulators 55: 169–173.
Petcu, E., Dencescu, S., and Vladu, P. (1995). Aspects of the tolerance of some soyabean genotypes to water
stress. Probleme de Genetica Teoretica si Aplicata 27: 115–124.
Petrusa, L.M. and Winicov, I. (1997). Proline status in salt-tolerant and salt-sensitive alfalfa cell lines and plants
in response to NaCl. Plant Physiology and Biochemistry 35: 303.
Piqueras, A., Hemandez, J.A.E., Olmos, E., Hellin, E., and Sevilla, F. (1996). Changes in antioxidant enzymes
and organic solutes associated with adaptation of citrus cells to salt stress. Plant Cell, Tissue and Organ
Culture 45: 53.
Plaza, B.M., Jimenez, S., Segura, M.L., Contreras, J.I., and Lao, M.T. (2009). Physiological Stress caused by
Salinity in Cordyline fruticosa and its indicators. Communications in Soil Science and Plant Analysis
40: 473–484.
Pollard, A. and Wyn Jones, R.G. (1979). Enzyme activities in concentrated solutions of glycinebetaine and
other solutes. Planta 144: 291–298.
Rabe, B. (1990). Stress physiology: The functional significance of the accumulation of nitrogen containing
compounds. Journal of Horticultural Science 65: 231–243.
Rajagopal, V., Balasubramanian, V., and Sinha, S.K. (1977). Diurnal fluctuations in relative water content,
nitrate reductase and proline content in water-stressed and non-stressed wheat. Physiologia Plantarum
40: 69.
Rajendrakumar, C.S.V., Reddy, B.V.D., and Reddy, A.R. (1994). Proline–protein interactions: Protection of
structural and functional integrity of M4 lactate dehydrogenase. Biochemistry and Biophysical Research
Communications 201: 957–963.
le Redulier, D. and Valentine, R.C. (1982). Genetic engineering in agriculture. Trends in Biochemical Sciences
7: 431.
Refoufi, A. and Larher, F. (1989). Compatible organic solutes and salt tolerance in seedlings of three annual
Medicago species. Compted Rendus Academy Science Paris 308: 329.
Rejskova, A., Patkova, L., Stodulkova, E., and Lipavska, H. (2007). The effect of abiotic stresses on carbohy-
drate status of olive shoots (Olea europaea L.) under in vitro conditions. Journal of Plant Physiology
164: 174–184.
Rhodes, D. and Hanson, A.D. (1993).Quaternary ammonium and tertiary sulfonium compounds in higher
plants. Annual Review of Plant Physiology and Plant Molecular Biology 44: 357–384.
Riaz, S. (2001). Salt induced changes in germinating seeds of salt tolerant and salt sensitive lines of spring
wheat. Dissertation Faisalabad (Pakistan). UAF., p. 81.
Richards, R.A., Denett, C.W., Qualset, C.O., Edstein, E., Norlyn, J.D., and Winslow, M.D. (1987). Variation
in yield of grain and biomass in wheat, barley and triticale in a salt-affected field. Field Crop Research
15: 277.
Rodriguez, M.M. and Heyser, J.W. (1988). Growth inhibition by exogenous proline and its metabolism in salt-
grass (Distichlis spicata) suspension cultures. Plant Cell Reports 7: 305.
de Ronde, J.A., Spreeth, M.H., and Cress, W.A. (2000). Effect of antisense l-Δ1-pyrroline-5-carboxylate reductase
transgenic soybean plants subjected to osmotic and drought stress. Plant Growth Regulators 32: 13–26.
de Ronde, J.A., Laurie, R.N., Caetano, T., Gray Ling, M.M., and Kerepesi, I. (2004). Comparative study
between transgenic and non-transgenic soybean lines proved transgenic lines to be more drought toler-
ant. Euphytica 138: 123–132.
Rout, N.P. and Shaw, B.P. (1998). Salinity tolerance in aquatic macrophytes: Probable role of proline, the
enzymes involved in its synthesis and C4 type of metabolism. Plant Science 136: 121–130.
Sakamoto, A. and Murata, N. (2002). The role of glycine betaine in the protection of plants from stress: Clues
from transgenic plants. Plant, Cell and Environment 25: 163–171.
Sanada, Y., Ueda, H., Kuribayashi, K., Andoh, T., Hayashi, F., Tamai, N., and Wada, K. (1995). Novel light-dark
change of proline levels in halophyte (Mesembryanthemum crystallinum L.) and glycophytes (Hordeum
vulgare L. and wheat (Triticum aestivum L.) leaves and roots under salt stress. Plant and Cell Physiology
36: 965–970.
Saranga, Y, Rhodes, D., and Janick, J. (1992). Changes in amino acid composition associated with tolerance to
partial desiccation of celery somatic embryos. Journal of the American Society for Horticultural Science
117: 337–341.
Satakopan, V.N. and Rajendran, L. (1989). Changes in proline levels in germinating groundnut seeds under
different stress conditions. Current Research 18: 8–10.
Schwab, K.B. and Gaff, D.F. (1990). Influence of compatible solutes on soluble enzymes from dessication-
tolerant Sporobolus stapfianus and dessication-sensitive Sporobolus pyramidalis. Journal of Plant
Sells, G.D. and Koeppe, D.E. (1980). Proline oxidation by water-stressed corn shoot mitochondria. Plant
Physiology 65: 25.
Shah, S.H., Wainwright, S.J., and Merrett, M.J. (1990). The interaction of sodium and calcium chlorides and
light on growth, potassium nutrition and proline accumulation in callus cultures of Medicago sativa L.
New Phytologist 116: 37.
Shevyakova, N.I., Rakitin, V.Y., and Muzychko, L.M. (1998). Stress-induced accumulation of proline in relation
to salt tolerance of intact plants and isolated cells. Applied Biochemistry and Microbiology 34: 291–295.
Shtereva, L., Atanassova, B., Karcheva, T., and Petkov, V. (2008). The effect of water stress on the growth rate,
water content and proline accumulation in tomato calli and seedlings. Acta Horticulturae 789: 189–197.
Sivakumar, P., Sharmila, P., and Pardha Saradhi, P. (2000). Proline alleviates salt-stress-induced enhancement in
ribulose-1,5-bisphosphate oxygenase activity. Biochemical and Biophysical Research Communications
279: 512–515.
Smirnoff, N. and Cumbes, Q.J. (1989). Hydroxyl radical scavenging activity of compatible solutes.
Phytochemistry 28: 1057–1060.
Smith, A.E. and Phillips, D.V. (1980). Occurrence of pinitol in foliage of several forage legume species. Crop
Science 20: 75–77.
Smith, C.J., Deuth, A.H., and Rushlow, K.E. (1984). Purification and characteristics of a γ-glutamyl kinase
involved in Escherichia coli proline biosynthesis. Journal of Bacteriology 157: 545–551.
Somsri, A. and Arunee, Y. (1994). Halophyte research in Thailand. National Center for Genetic Engineering
and Biotechnology. In: Biotechnology and Biology Diversity. Bangkok (Thailand). pp. 1–13.
Soussi, M., Khadri, M., Pliego, L., Lluch, C., and Ocana, A. (2001). Germination de differents cultivars de
Cicer arietinum en conditions de salinite: profil de proteines et accumulation de solutes compatibles.
Colloques de l’ INRA (France) 100: 4198.
Stewart, C.R. (1972). Effects of proline and carbohydrates on the metabolism of exogeneous proline by excised
bean leaves in the dark. Plant Physiology 50: 551.
Stewart, C.R. (1981). Proline accumulation: Biochemical aspects. In: Physiology and Biochemistry of Drought
Resistance in Plants, Paleg, L.G. and Aspinall, D., eds. Sydney, NSW, Academic Press, pp. 243–259.
Stewart, C.R, Boggess, S.F., Aspinall, D., and Paleg, L.G. (1977). Inhibition of proline oxidation by water
stress. Plant Physiology 59: 930.
Stoirnenova, I. and Stoyanova, I. (1995). The influence of some herbicides on the quality of soybean seeds.
Bulgarian Journal of Agricultural Science 1: 235.
Storey, R. and Wyn Jones, R.G. (1997). Quaternary ammonium compounds in plants in relation to salt resis-
tance. Phytochemistry 16: 447.
Subbarao, G.V., Johansen, C., Jana, M.K., and Kumar Rao, J.V.D.K. (1990). Physiological basis of differences
in salinity tolerance of pigeonpea and its related wild species. Journal of Plant Physiology 137: 64.
Sweetlove, L.J., Fait, A., Nunes-Nesi, A., Williams, T., and Fernie, A.R. (2007). The mitochondrion: An inte-
gration point of cellular metabolism and signaling. Critical Reviews in Plant Sciences 26: 17–43.
Székely, G., Abraham, E., Cseplo, A., Rigo, G., Zsigmond, L., Csiszar, J., Ayaydin, F., Strizhov, N., Jasik, J.,
Schmelzer, E., Koncz, C., and Szabados, L. (2008). Duplicated P5CS genes of Arabidopsis play distinct
roles in stress regulation and developmental control of proline biosynthesis. Plant Journal 53: 11–28.
Szoke, A., Miao, G.H., Hong, Z., and Verma, D.P.S. (1992). Subcellular location of Δ1-py-5-carboxylate reduc-
tase in root/nodule and leaf of soybean. Plant Physiology 99: 1642–1649.
Taffouo, V.D., Meguekam, L., Kenne, M., Yayi, E., Magnitsop, A., Akoa, A., and Ourry, A. (2008). Germination
and accumulation of metabolites in seedlings of legumes grown under salt stress. Agronomie Africaine
20: 129–139.
Takagi, H. (2008). Proline as a stress protectant in yeast: Physiological functions, metabolic regulations, and
biotechnological applications. Applied Microbiology and Biotechnology 81: 211–23.
Tarnizi, A.H. and Marziah, M. (1995). The influence of low temperature treatment on growth and proline accu-
mulation in polyembryogenic cultures of oil palm (Elaeis guineensis J acq.). Elaeis 7: 107–117.
Thakur, M. and Sharma, A.D. (2005). Salt-stress-induced proline accumulation in germinating embryos:
Evidence suggesting a role of proline in seed germination. Journal of Arid Environments 62: 517–523.
Thind, S.K. and Malik, C.P. (1994). Proline protects pollen germination under adverse temperatures in Amaryllis
vittata. Journal Palynology 30: 107.
Turner, N.C. and Jones, M.M. (1980). Turgor maintenance by osmotic adjustment: A review and evaluation. In:
Adaptation of Plants to Water and High Temperature Stress, Turner, N.C. and Kramer, P.J. eds. New York,
J. Wiley & Sons, pp. 87–103.
Vasantha, S. and Rao, P.N.G. (2005). Response of sugarcane genotypes to salinity. Plant Archives 5: 193–196.
Venekamp, J.H. and Koot, J.T.M. (1988). The sources of free proline and asparagine in field bean plants, Vicia
faba L., during and after a short period of water withholding. Journal of Plant Physiology 132: 102.
Verbruggen, N. and Hermans, C. (2008). Proline accumulation in plants: A review. Amino Acids 35: 753–759.
van Volkenburgh, E. and Boyer, J.S. (1985). Inhibitory effects of water deficit on maize leaf elongation. Plant
Physiology 77: 190.
Wang, J.Q. and Cui, H.W. (1996). Variation in free proline content of cucumber (Cucumis sativus L.) seedlings
under low temperature stress. Report Cucurbit Genetics Cooperative 19: 25–26.
Wang, W.X., Vinocur, B., Shoseyov, O., and Altman, A. (2001). Biotechnology of plant osmotic stress toler-
ance: Physiological and molecular considerations. Acta Horticultura 560: 285–292.
Watad, A.A., Reinhold, L., and Lerner, H.R. (1983). Comparison between a stable NaCl-selected Nicotiana cell
line and a wild type. Plant Physiology 73: 624.
Widyasari, W.B. and Sugiyarta, E. (1997). Free proline accumulation in the leaf tissues as an indicator of
drought-resistant sugarcane varieties. Majalah Penelitian Gula 33: 1–10.
Wu, M.L. (1987). The effects of seed treatments on the germination of tomato seeds under water and salinity
stress. Journal of the Agricultural Association of China 138: 52.
Wyn Jones, R.G. and Gorham, J. (1983). Aspects of salt and drought tolerance in higher plants. In: Genetic
Engineering of Plants, an Agricultural Perspective, Kosuge, T., Meredith, C.P., Hollander, A., eds. New York,
Plenum Press, pp. 355–370.
Xu, J., Yin, H., and Li, X. (2009). Protective effects of proline against cadmium toxicity in micropropagated
hyperaccumulator, Solanum nigrum L. Plant Cell Reports 28: 325–333.
Yasseen, B.T., Shihab, E.M., and Yahaya, R.A. (1989). Cytological and physiological studies on the effect
of sodium chloride on growth processes and proline accumulation in the germinating seeds of barley.
Mesopotamia Journal of Agriculture 21: 237.
Yeo, A. (1998). Molecular biology of salt tolerance in the context of whole-plant physiology. Journal of
Yeo, A.R. (1981). Salt tolerance in the halophyte Suaeda maritima L. Dunn.: Intracellular compartmentation of
ions. Journal of Experimental Botany 32: 487–497.
Yoshiba, Y., Kiyosue, T., Nakashima, K., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1997). Regulation of
levels of proline as an osmolyte in plants under water stress. Plant Cell Physiology 38: 1095–1102.
Yunus, Q., Li, Y., Muhetaer, J., Ma, P.T., Takahashi, H., and Takahashi, S. (2006). Effects of salt stress treatment
on seed germination and seedlings physiological characteristics in Elaeagnus angustifolia L. Xinjiang
Agricultural Sciences 43: 136–139.
Zayed, M.A. and Zeid, I.M. (1998). Effect of water and salt stresses on growth, chlorophyll, mineral ions and
organic solutes contents, and enzymes activity in mung bean seedlings. Biologia Plantarum 40: 351–356.
Zhang, C.S., Lu, Q., and Verma, D.S.P. (1995). Removal of feedback inhibition of delta-1-pyrroline-5-carboxylate
synthetase, a bifunctional enzyme catalyzing the first 2 steps of proline biosynthesis in plants. Journal of
Biological Chemistry 270: 20491–20496.
Zhao, X., Tan, H.J., Liu, Y.B., Li, X.R., and Chen, G.X. (2009). Effect of salt stress on growth and osmotic
regulation in Thellungiella and Arabidopsis callus. Plant Cell, Tissue and Organ Culture 98: 97–103.
Zhen, W.B. and Ma, Q.H. (2009). Proline metabolism in response to salt stress in common reed [Phragmites
australis (Cav.) Trin. ex Steud]. Botanica Marina 4: 341–347.
Zhi, M.X. and Li, X.J. (2009). Effect of NaCl stress on germination and seedling’s physiology of maize.
Guizhou Agricultural Sciences 4: 31–32.

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THIRD EDITION

Agricultural Engineering Robert M. Peart, University of Florida, Gainesville

Crops Mohammad Pessarakli, University of Arizona, Tucson

Environment Kenneth G. Cassman, University of Nebraska, Lincoln

Irrigation and Hydrology Donald R. Nielsen, University of California, Davis

Plants L. David Kuykendall, U.S. Department of Agriculture, Beltsville,

Soils Jean-Marc Bollag, Pennsylvania State University, University Park

Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson

9.2 Osmoregulation or Osmotic Adjustment

9.2.1 Plant Response to Drought and Salinity

9.2.2 Role of Proline in Water- and Salt-Stressed Plants

Asymmetric catalysts Protein Promoting activity

Osmoregulation Inhibition of dehydration

9.3 Proline Metabolism

9.3.1 Proline Synthesis

9.3.2 Proline Catabolism

9.3.3 Endproduct Inhibition

Proline synthesis (cytosol and plastids)

Proline degradation (mitochondria)

9.3.5 Impact of Environmental Stresses on Proline

9.4 Proline Accumulation as an Osmoregulatory Response

9.4.1 Osmotic Adaptation of Bacteria and Algae

9.4.2 Accumulation of Proline in Callus Cultures and Isolated Cells

9.4.3 Seed Germination and Proline Content under Stress

9.4.4 Osmotic Adjustment in Halophytes

9.4.5 Negative Response

9.5 Exogenous Proline Application

9.6 Proline Content as an Indicator for Breeding Programs

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9.2 Osmoregulation or Osmotic Adjustment

9.2.1 Plant Response to Drought and Salinity

9.2.2 Role of Proline in Water- and Salt-Stressed Plants

9.3 Proline Metabolism

9.3.1 Proline Synthesis

9.3.2 Proline Catabolism

9.3.3 Endproduct Inhibition

9.3.5 Impact of Environmental Stresses on Proline

9.4 Proline Accumulation as an Osmoregulatory Response

9.4.1 Osmotic Adaptation of Bacteria and Algae

9.4.2 Accumulation of Proline in Callus Cultures and Isolated Cells

9.4.3 Seed Germination and Proline Content under Stress

9.4.4 Osmotic Adjustment in Halophytes

9.4.5 Negative Response

9.5 Exogenous Proline Application

9.6 Proline Content as an Indicator for Breeding Programs