Slama et al 2015

Annals of Botany 115: 433–447, 2015
doi:10.1093/aob/mcu239, available online at www.aob.oxfordjournals.org
REVIEW: PART OF A SPECIAL ISSUE ON HALOPHYTES AND SALINE ADAPTATIONS
Diversity, distribution and roles of osmoprotective compounds accumulated in
halophytes under abiotic stress
Inès Slama1, Chedly Abdelly1, Alain Bouchereau2, Tim Flowers3 and Arnould Savouré4,*
1
Received: 13 August 2014 Returned for revision: 19 September 2014 Accepted: 21 October 2014 Published electronically: 5 January 2015
Background and Aims Osmolytes are low-molecular-weight organic solutes, a broad group that encompasses a
variety of compounds such as amino acids, tertiary sulphonium and quaternary ammonium compounds, sugars and
polyhydric alcohols. Osmolytes are accumulated in the cytoplasm of halophytic species in order to balance the osmotic potential of the Naþ and Cl accumulated in the vacuole. The advantages of the accumulation of osmolytes
are that they keep the main physiological functions of the cell active, the induction of their biosynthesis is controlled
by environmental cues, and they can be synthesized at all developmental stages. In addition to their role in osmoregulation, osmolytes have crucial functions in protecting subcellular structures and in scavenging reactive oxygen
species.
Scope This review discusses the diversity of osmolytes among halophytes and their distribution within taxonomic
groups, the intrinsic and extrinsic factors that influence their accumulation, and their role in osmoregulation and
osmoprotection. Increasing the osmolyte content in plants is an interesting strategy to improve the growth and yield
of crops upon exposure to salinity. Examples of transgenic plants as well as exogenous applications of some osmolytes are also discussed. Finally, the potential use of osmolytes in protein stabilization and solvation in biotechnology, including the pharmaceutical industry and medicine, are considered.
Key words: Abiotic stress, genetic engineering, halophytes, osmolytes, osmoregulation, osmoprotection, osmotic
adjustment, salinity stress, salt tolerance.
INTRODUCTION
Environmental stress (an environmental perturbation causing a
response in the plant; Cheeseman, 2013) factors such as
drought, salinity, chilling, freezing and high temperatures affect
plant growth and generate a threat to sustainable agriculture. In
field conditions, crops and other plants are routinely subjected
to a combination of different abiotic stresses (Boyer, 1982;
Ahuja et al., 2010; Atkinson and Urwin, 2012). For example,
heat stress is often accompanied by water deficiency and
drought by salinity. This has become an important issue due to
concerns about the effects of climate change on plant resources,
biodiversity and global food scarcity (with almost 70 papers
published between 2010 and early 2013 containing the words
‘food’, ‘climate’ and ‘change’ in their title). Soil salinization is
a serious soil degradation, which can arise from natural causes
and human-mediated activity such as forest clearance or irrigation with low-quality water (Pitman and Läuchli, 2004), especially in regions with high rates of evapotranspiration.
Salt-affected soils are found in more than 100 countries of the
world, especially those in arid and semi-arid zones, with a variety of extents, types and properties (Rengasamy, 2006).
Approximately 20 % of the irrigated lands in the world suffer
from salinization and it has been estimated that the world as a
whole has been losing at least 05–1 % of irrigated area every
year (Munns and Tester, 2008).
Under saline conditions, salt-sensitive glycophytes (plants of
sweet or fresh water and which do not qualify as halophytes), a
group of plants to which the majority of crops belong, have to
cope with osmotic and ionic stresses. The former affects plants
as a rise in salt levels around the roots leads to inhibition of
water uptake, cell expansion and lateral root development
(Munns and Tester, 2008). The latter develops when ions such
as Naþ and Cl accumulate in excess in plants, particularly in
leaves, leading to a decrease in the activity of primary metabolism, including photosynthesis, with an increase in chlorosis
and cell death (Glenn et al., 1999).
Physiological and molecular genetic studies have led to
increasing knowledge of the protective mechanisms that plants
use to cope with the detrimental effects of salinity (Zhu, 2002;
Munns and Tester, 2008; Horie et al., 2009). During salt stress,
Naþ and Cl enter cells and their over-accumulation can
modify the lipid and protein composition of plant cell plasma
membranes as well as the function and regulation of important membrane proteins, such as water channels and Naþ
C The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Laboratoire des Plantes Extremophiles, Centre de Biotechnologie de Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050,
Tunisia, 2UMR 1349 IGEPP, INRA/Agrocampus Ouest/Université de Rennes 1, Domaine de la Motte, BP 35327, 35653 Le
Rheu Cedex, France, 3School of Life Sciences, University of Sussex, Falmer, Brighton, Sussex BN1 9QG, UK and 4Sorbonne
Universités, UPMC Université Paris 06, Adaptation de Plantes aux Contraintes Environnementales, URF5, Case 156, 4 place
Jussieu, F-75252 Paris cedex 05, France
* For correspondence. E-mail [email protected]
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Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
CONTRIBUTION OF SODIUM TO OSMOTIC
ADJUSTMENT IN HALOPHYTES
External sodium chloride can impose on plants an initial water
deficit, injury to plasma membranes, accumulation of reactive
oxygen species (ROS), impairment of photosynthesis and
changes in respiration and nitrogen assimilation (Munns and
Tester, 2008). In addition, by effectively replacing Kþ (a cofactor in a number of enzymatic processes) in the cytoplasm with
Naþ, critical biochemical processes such as protein synthesis
are inhibited. Halophytes have evolved mechanisms for selective Naþ and Cl sequestration into the vacuole, allowing the
cytoplasm to be maintained at substantially lower concentrations of these ions, avoiding their inhibitory effects on metabolic processes (Flowers et al., 1986; Flowers and Colmer,
2008; Flowers et al., 2014).
Accumulation of Naþ, often associated with that of Cl, is
prominent in shoots of most halophytic dicotyledonous plants,
particularly those of the Amaranthaceae (Chenopodiaceae;
Flowers, 1985; Albert et al., 2000). Chenopodiaceous
halophytes growing in 100–200 mM NaCl can contain some
500 mM Naþ and 340 mM Cl (Flowers, 1985), concentrations
that contribute significantly to the internal osmotic potential
and to turgor generation [although the latter may be compromised by salt in the cell walls (Clipson et al., 1985; Flowers
et al., 2014)]. When freely available in the soil solution, Naþ
and Cl constitute readily available solutes provided that, at the
cell level, they are efficiently sequestered in vacuoles. Such
compartmentalization is hypothesized to avoid their damaging
effects on the cytosol and its organelles. This model of the cellular basis of salt tolerance implies the ability to maintain a sufficient uptake of Kþ in order to maintain a high cytosolic Kþ/
Naþ ratio, one of the key determinants of plant salt tolerance.
In spite of the wide availability of Naþ and Cl, their contribution to osmotic adjustment in halophytic species varies
among families and appears to be related to the concentrations
of accumulated organic solutes (Flowers et al., 1977; Flowers
and Colmer, 2008).
WHAT IS AN OSMOLYTE?
One of the metabolic consequences of osmotic stress is the
accumulation of osmolytes, low-molecular-weight organic
compounds, also known as compatible solutes, that are highly
soluble and do not interfere with normal metabolic reactions
because they are non-toxic even at high cellular concentrations
(Flowers et al., 1977; Wyn Jones et al., 1977; Yancey, 2005).
Osmotic adjustment has traditionally been accepted to be the
primary function of osmolytes in plants (Hasegawa et al.,
2000), but this is not always the case, especially in glycophytes
exposed to salt. For example, proline accumulation in
Arabidopsis thaliana is too low to play a role in the osmotic
adjustment of the cells (Liu and Zhu, 1997; Ghars et al., 2008).
In addition, the concentration (expressed on the basis of tissue
mass or tissue water) of osmolytes can be significantly lower
than that of inorganic solutes in some halophytes (Gagneul
et al., 2007). These osmolytes presumably have other functions
(Bartels and Sunkar, 2005; Hare et al., 1998; Szabados et al.,
2011). Indeed some of these compounds can modify the solvent
properties of water, stabilize the internal osmotic potential, increase the thermodynamic stability of folded proteins and protect macromolecular structures (Yancey, 2005). Because some
of these solutes also protect cellular components from dehydration injury, they are commonly referred to as osmoprotectants.
In halophytes several types of osmolyte can often be accumulated simultaneously, although the abundance of a particular
osmolyte may depend on cell compartment, organ, developmental stage and environmental conditions (Murakeözy et al.,
2003; Gagneul et al., 2007). This raises the question of their individual or combined (possibly synergistic) mode of action in
protecting cells against the environment (Yancey, 2005).
Accumulation processes that determine the mode of action of
osmolytes are the result of the coordinated regulation of biosynthetic and catabolic pathways. Among osmolytes, some are
subject to rapid and short-term fluctuations (accumulation/degradation, e.g. proline), while others accumulate for longer
periods (e.g. betaines) (Gagneul et al., 2007). Metabolic
dynamics also has adaptive significance to the extent that the
metabolic pathway involved in the production or consumption
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transporters (Horie and Schroeder, 2004; Ward et al., 2009) and
signalling molecules (Zhu, 2002). Halophytes, extremophiles
found on salt marshes, in saline depressions (sebkhas), in saline
inland deserts and on sand dunes or rocky coasts, have evolved
a number of adaptive traits expressed at various levels of organization that allow them to germinate, grow and achieve their
complete cycle of development under such conditions (Flowers
and Colmer, 2008). Foremost amongst these is the control of
ion concentrations in the plants and their cells.
Osmoregulation and/or osmoprotection and ion homeostasis
through compartmentalization are part of the main adaptive
mechanisms found in halophytes subjected to salinity (Flowers
and Colmer, 2008). Osmotic adjustment in halophytes is
achieved through the accumulation of energetically cheap
inorganic ions, such as Naþ and Cl, and low-molecular-weight
organic solutes, collectively known as osmolytes (Yeo, 1983;
Glenn et al., 1999). The ions are primarily located in the vacuoles and the osmolytes (amino acids, tertiary sulphonium
compounds, quaternary ammonium compounds, sugars and
polyhydric alcohols) in the cytoplasm. In addition, osmolytes
protect macromolecular sub-cellular structures and mitigate
oxidative damage caused by free radicals produced in response
to these abiotic stresses. Elevated levels of osmolyte accumulation in plant cells have been correlated with enhanced stress tolerance through the scavenging of free radicals and protecting
enzymes (Szabados et al., 2011).
This review discusses the diversity of osmolyte accumulation
in halophytes, the intrinsic and extrinsic factors that influence
their accumulation and their roles in osmoregulation and osmoprotection. The high structural diversity and metabolic origins
of osmolytes combined with their multifunctionality and the
seasonal flexibility of metabolism in plants facing multiple
stresses are also discussed. Since the engineering of osmolyte
metabolism can lead to opportunities to improve plant tolerance
of environmental stresses, examples of transgenic plants engineered to have enhanced osmolyte content as well as the effects
of exogenous applications of some osmolytes will also be
described.
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
DIVERSITY OF OSMOLYTE COMPOUNDS IN
THE PLANT KINGDOM
A wide range of osmoprotective compounds has been identified, including mono-, di-, oligo- and polysaccharides, such as
glucose, fructose, sucrose, trehalose, raffinose and fructans;
sugar alcohols (polyols) such as sorbitol, mannitol, glycerol,
inositol and methylated inositols; amino acids, such as proline,
pipecolic acid; methylated proline-related compounds, such as
methyl-proline, proline betaine and hydroxyproline betaine;
other betaines, such as glycine betaine, b-alanine betaine,
choline O-sulphate; and tertiary sulphonium compounds, such
as dimethylsulphoniopropionate (DMSP) (Rhodes et al., 2002;
Ashraf and Foolad, 2007) (Fig. 1). In the following sections,
the most common osmolytes in halophytes are described in
detail.
Amino acids
It has been reported that amino acids, such as proline, alanine, arginine, glycine, amides such as glutamine and asparagine, and the non-protein amino acids c-aminobutyric acid,
pipecolic acid, citrulline and ornithine are accumulated in
higher plants under conditions of abiotic stress (Ahmad et al.,
1981; Mansour, 2000). These compounds are synthesized from
the primary metabolic pathways that lead to metabolically inactive (safe) molecules (Rhodes et al., 2002).
Proline is known to occur widely in higher plants and can be
accumulated in considerable amounts in response to salt stress
and drought (Kavi Kishor et al., 2005). In plants, proline is
synthesized mainly from glutamate, which is reduced to glutamate semialdehyde by pyrroline-5-carboxylate synthetase
(P5CS) (a key enzyme in proline biosynthesis) and spontaneously converted to pyrroline-5-carboxylate (P5C) (Szabados
and Savouré, 2010). P5C reductase (P5CR) further reduces the
P5C intermediate to proline. Catabolism of proline occurs in
mitochondria via the sequential action of proline dehydrogenase or proline oxidase (ProDH), producing P5C from proline,
and P5C dehydrogenase (P5CDH), which converts P5C to glutamate (Szabados and Savouré, 2010; Servet et al., 2012).
In addition to glutamate, the other precursor for proline biosynthesis is ornithine, which is transaminated to P5C by a mitochondrial ornithine-d-aminotransferase (Armengaud et al.,
2004; Verbruggen and Hermans, 2008). Proline is also involved
in the alleviation of cytoplasmic acidosis and sustaining
NADPþ/NADPH ratios at the levels required for metabolism
(Hare and Cress, 1997). Proline accumulated under stress
conditions might therefore serve as a sink for excess reductants, providing the NADþ and NADPþ necessary for maintenance of respiratory and photosynthetic processes. High
H
O
OH
OH
N
H
N
H
O
Proline
+
H3C
N
O
CH3
OH
O
O
Choline-O -sulphate
OH
OH
OH
OH
OH
OH
HO
OH
OH
HO
OH
OH
OH
OH
Inositol
OH
OH
HO
O
+
N
OH
OH
Pinitol
−
O−
HO
H3C
O
S
Glycine betaine
OH
−
O
N+
H3C
Proline betaine
O
S
Dimethylsulphoniopropionate
O
H3C
+
H3 C
O
Pipecolic acid
O−
H3C
H3C
Mannitol
FIG. 1. Structures of common osmolytes identified in halophytes.
OH
Sorbitol
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of a compatible osmolyte can be as important as the terminal
product itself. For example, the biosynthesis and catabolism of
proline contributes to rapid and efficient consumption or release
of reducing power (Szabados and Savouré, 2010).
Not only the biochemical/metabolic nature of the osmolyte
but also the seasonal pattern of osmolyte accumulation show
significant species-specific fluctuations. In addition, the cellular
levels of osmolytes can change according to growth period,
developmental stage, organ and environmental parameters
(Hare et al., 1998; Murakeözy et al., 2003). Sub-cellular partitioning of osmolytes between cell compartments has also been
demonstrated to be modulated according to growing conditions,
nutritional and environmental challenges (Aubert et al., 2009,
Gagneul et al., 2007).
435
436
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
concentrations of NADPþ are necessary for the pentose phosphate pathway for the regeneration of NADPH and to supply
ribose-5-phosphate for the synthesis of purines (Kavi Kishor
et al., 2005).
long-term response to salinization deriving from several methylation steps of proline (Trinchant et al., 2004).
Tertiary sulphonium compounds
Quaternary ammonium compounds
Sugars
Sugars such as sucrose and trehalose have been shown to be
accumulated in plants in response to abiotic stresses (Briens
and Larher, 1982; Yuanyuan et al., 2009). Besides their possible roles in osmotic adjustment and in stabilizing membranes
upon water stress (Lokhande and Suprasanna, 2012), these
compounds may play several other important regulatory functions in stressed plants. Sugars not only sustain the growth of
sink tissues, but also affect sugar-sensing systems that regulate
the expression, either positively or negatively, of a variety of
genes involved in photosynthesis, respiration and the synthesis
and degradation of starch and sucrose (Hare et al., 1998).
Trehalose, a non-reducing disaccharide, is highly soluble but
chemically unreactive, making it compatible with cellular metabolism even at high concentrations. Trehalose is present in
significant concentrations in several bacteria and fungi but rare
in vascular plants (Lunn et al., 2014, Fernandez et al., 2010).
In plants, the accumulation of trehalose was first demonstrated
in resurrection plants upon desiccation, but remains, to our
knowledge, to be demonstrated in halophytes.
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The quaternary ammonium compounds accumulated in
plants are glycine betaine, b-alanine betaine, proline betaine,
choline-O-sulphate, hydroxyproline betaine and pipecolate betaine (Ashraf and Harris, 2004).
Glycine betaine is accumulated by numerous organisms, including bacteria, cyanobacteria, algae, fungi and animals
(Türkan and Demiral, 2009). Among the variety of quaternary
ammonium compounds, glycine betaine is one of the most
abundantly occurring in plants exposed to dehydration due to
salinity, drought, heat and cold stress (Hanson et al., 1991; Guo
et al., 2009; Lokhande and Suprasanna, 2012). Glycine betaine
is synthesized mainly from choline, which is converted to betaine aldehyde and then to glycine betaine through the sequential
enzymatic action of choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Although
other pathways, such as the direct N-methylation of glycine, are
also known, the choline pathway to glycine betaine has been
identified in all glycine betaine-accumulating plant species
(Ashraf and Foolad, 2007; Fitzgerald et al., 2009).
b-Alanine betaine is synthesized by the S-adenosylmethionine-dependent N-methylation of b-alanine via N-methyl
b-alanine and N,N-dimethyl b-alanine. In Limonium species,
purified N-methyltransferase (NMTase) is trifunctional, methylating b-alanine, N-methyl b-alanine and N,N-dimethyl
b-alanine (Rathinasabapathi et al., 2001). Nevertheless, the
metabolic origin of b-alanine in b-alanine betaine-producing
species remains speculative since multiple routes have been
proposed deriving from aliphatic polyamines, propionate or
uracil. These pathways may cooperate for b-alanine and
b-alanine betaine production in Limonium latifolium (Duhaze
et al., 2003). It has been proposed by Hanson et al. (1991) that
b-alanine betaine is a more suitable osmoprotectant than glycine betaine under saline hypoxic conditions because the first
step in glycine betaine synthesis requires molecular oxygen.
Furthermore, b-alanine betaine accumulation was proposed to
be an evolutionary strategy to avoid metabolic competition for
choline (Hanson et al., 1994), because b-alanine betaine is synthesized from the ubiquitous primary metabolite b-alanine
(Duhaze et al., 2003).
Proline betaine, a dimethyl proline also called stachydrine,
which accumulates in non-halophytic Citrus and Medicago species (Nolte et al., 1997; Trinchant et al., 2004), has a much
more scattered distribution than glycine betaine in halophytes.
Methylated proline has been shown to accumulate in a few halophytic species of the Plumbaginaceae, Capparidaceae,
Myrtaceae, Rutaceae, Labiatae, Compositae and Leguminosae
(Wyn Jones and Storey, 1981; Rhodes and Hanson, 1993;
Hanson et al., 1994; Naidu, 2003; Carter et al., 2005). In bacteria, the osmoprotective role of proline betaine has been clearly
established, proline betaine being a more effective osmoprotectant than proline (Hanson et al., 1994). Partial investigations in
plants indicate that proline betaine accumulation represents a
Tertiary sulphonium compounds such as DMSP contain a
fully methyl substituted sulphur atom (Larher et al., 1977).
Dimethylsulphoniopropionate is synthesized in many algae, in
contrast to plants, where this compound is only found in a few
salt marsh grasses of the genus Spartina, in sugar canes
(Saccharum spp.) and in Wollastonia biflora (Otte et al., 2004).
Plants and algae that produce DMSP synthesize this compound
from methionine, but the pathways from methionine to DMSP
differ between plant groups and species (Hanson et al., 1994;
Kocsis and Hanson, 2000). In algae, methionine is first transaminated to form 4-methylthio-2-oxobutyrate, but in higher
plants it is first methylated to form S-methyl methionine, which
is then converted directly to dimethylsulphoniopropionaldehyde
(DMSP-ald) without formation of dimethylsulphoniopropylamine, most likely via a transamination/decarboxylation mechanism. Interestingly, grasses such as Spartina alterniflora have
evolved specific enzymes that mediate the conversion of
S-methyl methionine to DMSP-ald (Kocsis and Hanson, 2000).
Although many functions of DMSP have been suggested, such
as a detoxifier of excess sulphur, an antioxidant and a herbivore
deterrent, the possible involvement of DMSP in osmoregulation
has received by far the most attention. This is partly due to the
structural similarity of this tertiary sulphur compound to quaternary ammonium compounds such as glycine betaine. In
S. alterniflora, DMSP is produced in high concentration in
green tissues, where it can represent up to 86 % of the total concentration of sulphur, but independently of the external salinity.
Dimethylsulphoniopropionate could be involved in osmoregulation without changes in its concentrations at the tissue level
by movement between the cytoplasm and the vacuoles within
the cells, depending on the osmotic potential of the cytoplasm
(Otte et al., 2004).
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
Sugar alcohols
DISTRIBUTION OF OSMOLYTES AMONG
HALOPHYTES
The diversity and threshold level of metabolites in plants can
vary considerably according to species and to environmental
conditions (Sanchez et al., 2008; Lugan et al., 2010; Szabados
et al., 2011). Although increased accumulation of osmolytes by
plants exposed to abiotic stresses has been reported, as
mentioned above, not all plant species synthesize all kinds of
osmolyte; some species synthesize and accumulate very low
quantities of these compounds while some plant species not do
so at all (e.g. Ashraf and Foolad, 2007). In general, osmolyte
accumulation is reported in species that are continuously
exposed to abiotic stresses; the synthesis of these osmolytes is
an energy-dependent process that consumes large numbers of
ATP molecules (Larher et al 1977; Rhodes et al., 2002;
Flowers and Colmer, 2008).
Halophytes of the three groups listed above (in the section
What is an osmolyte?), usually accumulate one dominant
osmolyte, which can be proline, glycine betaine, sorbitol,
b-alanine betaine, choline-O-sulphate or sugar (Tipirdamaz
et al., 2006; Arbona et al., 2010; Lugan et al., 2010). However,
some halophytes accumulate more than one compatible solute
(Gagneul et al., 2007). Based on their glycine betaine and
proline accumulation potential, Tipirdamaz et al. (2006)
categorized the halophytes from an inland salt marsh in
Turkey. Their studies showed that the species that behaved as
glycine betaine accumulators appeared to be poor proline
accumulators and vice versa, although the correlation between
the concentrations of the two solutes was not significant (our
analysis). The level of glycine betaine accumulation reported in
the halophytes analysed by Tipirdamaz et al. (2006) was
15–400 mmol g1 dry weight and some of the highest glycine
betaine-accumulating halophytes were members of the
Amaranthaceae (Chenopodiaceae): on average, the concentration in the chenopods (21 analyses) was 3.3-fold that in the
other species (18) analysed. Osmolyte contents in representative families of halophytes are described in the following sections and in Table 1; more detail for individual species can be
found in Supplementary Data Table S1.
Amaranthaceae
The Amaranthaceae (which include the former Chenopodiaceae) is a relatively large family, having about 174 genera and
2050 species, and represents the most species-rich lineage
within the Caryophyllales. The largest number of halophytic
species is found in this family. All the species are reported to
accumulate glycine betaine, with the exceptions of C. quinoa
(Tables 1 and Supplementary Data Table S1, and Yokoishi and
Tanimoto, 1994; Ruffino et al., 2010) and Noaea mucronata
(Tipirdamaz et al., 2006). It is clear that, where salinities are
above the optimal for growth, the concentrations of proline and
sugars increase, e.g. in Atriplex halimus (Bajji et al., 1998);
Kochia sieversiana (Yang et al., 2007); Suaeda fruticosa
(Hameed et al., 2012). The low glycine betaine and high proline
concentrations reported for Atriplex hastata (Tipirdamaz et al.,
2006) probably reflect that this species grows under such conditions. In a relatively early report, Guy et al. (1984) noted that,
where glycine betaine was measured in shoots of Salicornia
europaea grown at different salinities, the concentration of glycine betaine appeared to fall when expressed on a dry weight
basis but plateaued when expressed on the basis of organic dry
matter, highlighting the importance of the basis of expression
of concentrations.
In addition the glycine betaine content may vary according
to the type of photosynthesis, e.g. C3 type, dual-celled (Kranz)
C4 type and the single-celled C4 type discovered in
Amaranthaceae (Voznesenskaya et al., 2001). A general trend,
pointed out by Larher et al. (2009), is that glycine betaine contents decreased from C3 plants to single-cell C4 plants, the C4
plants exhibiting Kranz anatomy being at an intermediate position. This observation suggests that different concentrations of
glycine betaine might be needed to achieve osmoprotection according to the type of photosynthesis. However, these preliminary observations on glycine betaine content could also reflect
variability in the level of glycine betaine according to the genus
or the species selected for the study of Larher et al. (2009).
Plumbaginaceae
The Plumbaginaceae consist of 15 genera and 500–700 species. The family as a whole is tolerant of saline or dry conditions, although there is some specificity in the types of
environmental cues (habitats) to which different groups in the
family are adapted (Labbe, 1962). The biochemical diversity of
organic osmolytes accumulated by Plumbaginaceae is rarely
met in other halophytes (Larher and Hamelin, 1975; Rhodes
and Hanson, 1993; Gagneul et al., 2007). Such metabolic diversity has been used as an example to explore the selective
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In general, sugar alcohols are divided between acyclic (e.g.
mannitol) and cyclic (e.g. pinitol) polyols. The accumulation
of polyols may have dual functions: facilitating osmotic
adjustment and supporting redox control. Mannitol, which is
not widely accumulated in halophytes, is synthesized from
fructose-6-phosphate by subsequent enzymatic action of mannose-6-phosphate isomerase (phosphomannose isomerase),
mannose-6-phosphate reductase and mannose-1-phosphate
phosphatase (Loescher et al., 1992). Although the function of
this osmolyte has not been well investigated in halophytes, a
possible role in Prosopis strombulifera is stabilizing macromolecular structures and promoting scavenging systems for reactive oxygen species (Llanes et al., 2013).
The most frequently reported form of cyclic polyol in
halophytes is pinitol, which is derived from the methylation of
myo-inositol and epimerization of ononitol by the actions of
inositol-O-methyltransferase and ononitol epimerase (Sengupta
et al., 2008). Myo-inositol is the most common of six isomers
of inositol that occur in plant tissues and methylated derivatives
of all these isomers have been isolated, with presumed
important functions as protective and signalling compounds
(Sureshan et al., 2009). The co-occurrence in Limonium species
of chiro-inositol and pinitol raises questions about an alternative
way of synthesizing pinitol in which epimerization from myoinositol may precede the methylation step (Gagneul et al.,
2007).
437
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
438
TABLE 1. Osmolyte distribution in halophyte family among
angiosperms
Order
Family
Monocotyledoneae
Poales
Cyperaceae
Juncaceae
Poaceae
Osmolytes
Sucrose
Sucrose
Sucrose, proline, low level of glycine
betaine
Dicotyledoneae
Alismatales
The most common osmolytes in a given family are indicated in bold.
advantages that the different solutes could have depending on
the nature of the constraints and habitats (Hanson et al., 1994;
Gagneul et al., 2007). Hanson et al. (1994) claimed that glycine
betaine was dominant in species of the Plumbaginaceae adapted
to dry environments and that this compound is not co-accumulated with b-alanine betaine in these species. Although multiple
quaternary ammonium compounds are found (Table 1), most
members of this family accumulate choline-O-sulphate and
b-alanine betaine as osmoprotective compounds (Larher and
Hamelin, 1975; Rathinasabapathi et al., 2001; Gagneul et al.,
2007). Specifically, choline-O-sulphate can function in sulphate
storage as well as in osmoprotection, b-alanine betaine may be
superior to glycine betaine in hypoxic saline (oxygen-depleted)
conditions, and proline-derived betaines may be beneficial in
chronically dry environments. Hanson et al. (1994) have shown
that Aegialitis, a species of mangrove, accumulates cholineO-sulphate and have proposed that its biosynthesis serves to
detoxify sulphate, a major anion in seawater that can be inhibitory at high concentrations (Hanson et al., 1994). A detailed
metabolic study in L. latifolium under control and salt-stress
conditions revealed that the contribution of betaines to osmotic
potential remained low compared with sugars and cyclitols
(Gagneul et al., 2007). The study of Gagneul et al. (2007) also
indicated that long-term saline treatment did not induce (or suppress) the production of these solutes. Surprisingly, the uncommon betaines of L. latifolium were not found to be significantly
osmoregulated (Gagneul et al., 2007).
While some families of halophytes produce high levels of
nitrogenous compounds, others preferentially accumulate sugars and polyols. Sorbitol has been described as the most abundant soluble carbohydrate in all Plantago species, including
taxa with different degrees of salt tolerance (Ahmad et al.,
1979; Königshofer, 1983; Koyro, 2006; Pommerrenig et al.,
2007). In addition, rhamnose was found to be abundant in
Plantago maritima roots (Briens and Larher, 1982).
From the study of Nadwodnik and Lohaus (2007), among
Plantago species only sea plantain has a higher sorbitol concentration in the vacuoles of its mesophyll cells (by about 7-fold)
than in the cytosol. The high sorbitol concentration in vacuoles
of P. maritima indicates that sorbitol may have functions in this
species additional to serving as a transport form of carbon, as
postulated in non-halophytes. Contrasting results were obtained
between P. maritima collected from inland salty areas surrounding a lake and those collected in coastal salt marshes
(Ahmad et al., 1979; Tipirdamaz et al., 2006); surprisingly, proline and glycine betaine were found to accumulate in the lake
group while these compounds were not detected in the saltmarsh plants. This discrepancy may be linked to plant metabolic adaptation to oxygen deficiency conditions as it occurs in
marine salt marshes (Tipirdamaz et al., 2006). This needs to be
further addressed.
Aizoaceae
In the Aizoaceae, Lokhande et al. (2011) found a large increase in proline concentration in Sesuvium portulacastrum
when callus and axillary shoot cultures were exposed to salt
and drought alone or under iso-osmotic stress conditions of
NaCl and polyethylene glycol (PEG). High proline accumulation has also been shown in S. portulacastrum plants exposed
to various abiotic constraints, including salinity, drought and
heavy metals (Messedi et al., 2004; Ghnaya et al., 2007; Slama
et al., 2008). Furthermore, S. portulacastrum exposed to salinity
exhibited increased synthesis of proline and polyalcohols such
as myo-inositol, D-ononitol and D-pinitol (Ishitani et al., 1996;
Lokhande et al., 2010). Salt induces the accumulation of
methylated
inositols,
D-ononitol
and D-pinitol in
Mesembryanthemum crystallinum, as shown by the results of
coordinate induction of several genes of the inositol synthesis
pathway (Paul and Cockburn, 1989; Ishitani et al., 1996).
Pinitol represented 71 % of the soluble carbohydrate fraction
and 97 % of dry weight when plants were exposed to 400 mM
NaCl (Paul and Cockburn, 1989). In contrast, Arabidopsis does
not show upregulation of these genes or increases in these inositol-derived compounds in response to salt stress, which suggests
an important role for these compounds in salt tolerance.
Salt-stimulated levels of glycine betaine in S. portulacastrum
have also been observed (Ramesh Kannan et al., 2013). Paul
and Cockburn (1989) found that irrigation of M. crystallinum
plants with 400 mM NaCl to induce crassulacean acid metabolism was accompanied by the accumulation of pinitol. Pinitol
may function as a compatible solute in the cytosol and especially in the chloroplasts to counteract the presence of high concentrations of Naþ and Cl ions in the vacuoles. Such a
pronounced accumulation of osmolytes and their physiological
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Cymodoceaeceae Proline, glycine, inositol
Juncaginaceae
Proline, pipecolate, fructose, maltose,
sucrose
Posidoniaceae
Proline, glycine, sucrose
Zosteraceae
Proline, sucrose
Asterales
Asteraceae
Proline, glycine betaine, myo-inositol,
sucrose
Brassicales
Brassicaceae
Proline, sucrose
Caryophyllales Aizoaceae
Proline, myo-inositol, ononitol, pinitol,
glycine betaine
Amaranthaceae Glycine betaine or proline, methylated
onium compounds, sugars
Frankeniaceae
Glycine betaine
Plumbaginaceae b-Alanine betaine, choline-O-sulphate,
proline, pipecolate, sucrose, glycine
betaine in a few species
Portulaceae
Proline
Fagales
Casuarinaceae
Proline
Lamiales
Acanthaceae
Glycine betaine
Malpighiales Rhizophoraceae Proline, sucrose
Myrtales
Combretaceae
Mannitol
Myrtaceae
Proline analogues, methylated proline
Plantaginales Plantaginaceae Sorbitol, proline, rhamnose
Rosales
Fabaceae
Proline, glycine betaine, pinitol, sugars
Solanales
Solanaceae
glycine betaine
Plantaginaceae
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
role in osmotic adjustment and anti-oxidative defence may
have contributed to the success of these species in growing under adverse environmental stresses.
Poaceae
Brassicaceae
Proline accumulation is widely observed in halophytes of
the Brassicaceae (Table 1 and Supplementary Data Table S1)
and salt-induced expression of proline biosynthetic genes have
been reported frequently. For instance, the proline concentration increased in Eutrema salsugineum (Koch and German,
2013; formerly Thellungiella salsuginea, an arabidopsis relative and a model species for abiotic stress tolerance studies)
under salt stress to reach a very high level (1600 mmol g1 dry
weight) at 500 mM NaCl (Inan et al., 2004; Ghars et al.,
2008). A comparative metabolomic approach between
Arabidopsis and Thellungiella under salt-stress conditions revealed that Thellungiella appears to be able to cope with low
water potentials by sustaining a water potential gradient with
the environment, due to passive reduction in water content and
constitutive over-concentration of osmolytes (Lugan et al.,
2010). Megdiche et al. (2007) showed that the coastal halophyte Cakile maritima accumulates proline and soluble carbohydrates in its leaves during the period of intensive leaf
growth. These organic compounds likely play a role in leaf
osmotic adjustment and in the protection of membrane stability at severe salinity. A study reporting seasonal changes in
the levels of compatible osmolytes in three halophytic species
from inland saline vegetation in Hungary revealed that
the high cytoplasmic concentration of proline observed in
Lepidium crassifolium allows a decrease in osmotic potential
of 50 % (Murakeözy et al., 2003). Although detected in several Brassicaceae species (Selvaraj et al., 1995; Xing and
Rajashekar 2001; Jdey et al., 2014), glycine betaine has never
been found to play any osmotic role.
Other families
Increased accumulation of osmolytes is also observed in
halophytes belonging to other families (Table 1), where accumulation of various sugars and polyols can also be involved in
osmoregulatory mechanisms.
Conclusion
Osmolytes, including carbohydrates, quaternary ammonium
compounds, proline and sulphonium compounds, are widely
distributed among orders of flowering plants, reflecting both
phylogeny and functional needs (Flowers and Colmer, 2008;
Flowers et al., 2010). From Table 1, it can be seen that sucrose
is more frequently accumulated in monocots than in dicots.
Grasses usually accumulate low amount of glycine betaine
when this compound is present. In general, species accumulate
either quaternary ammonium compounds or proline in response
to salinity, but rarely both. Glycine betaine serves as an osmoticum in response to salt stress in most halophytes within the
Amaranthaceae. However, the concentration of compatible solutes does not increase with increasing external salinity in all
cases, suggesting that the synthesis of these compounds is constitutive in some species; e.g. glycine betaine in S. fruticosa
(Khan et al., 1998) and b-alanine betaine (at least partially) in
L. latifolium (Gagneul et al., 2007). In addition, the high concentrations of DMSP in the tissues of Spartina species,
W. biflora and Saccharum species would contribute to a high
baseline osmotic potential, thus giving constitutive tolerance of
salinity-related stress (Otte and Morris, 1994; Stefels, 2000).
HOW DO OSMOLYTES PROTECT CELL
FUNCTION UPON ABIOTIC STRESS?
The accumulation of osmoprotective compounds represents a
specific metabolic response that is important in withstanding
harmful conditions (Szabados et al., 2011). Despite the enormous amount of information accumulated in recent decades,
the exact function of low-molecular-weight protective
compounds in adaptation to extreme environmental conditions
is still not completely understood. In this section some important roles of osmoprotective compounds and the advantages of
the organic osmolyte system are detailed.
Osmoregulation and sub-cellular compartmentation of osmolytes
From the thermodynamic point of view, osmolytes reduce
the osmotic potential of water and thereby the water potential
in cells in which they are present (Borowitzka, 1981). In this
way, they are able to contribute to osmotic adjustment. The
ability for osmotic adjustment via solute accumulation—i.e. an
increase in the concentration of a solute in a cell due to an increase in its content rather than a decrease in the water content—has been reported for many plants (Borowitzka, 1981;
Yancey et al., 1982). Lowering the water potential is important
in the maintenance of tissue water content in the face of low
external water potentials generated in saline conditions (Niu
et al., 1995). It is clear, however, that osmotic adjustment to the
external water potential experienced by halophytes cannot, for
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Many grasses accumulate glycine betaine (Supplementary
Data Table S1), although this was not detected in Puccinellia
nuttalliana (Guy et al., 1984). Soluble sugars are also commonly reported as components of the solute potential, which
was the case for sucrose in Agrostis stolonifera and Phragmites
communis growing in a sulphate-dominated soil in Austria:
glycine betaine increased in the autumn in A. stolonifera
(Hutterer and Albert, 1993). Proline, which is also commonly
reported to make only a small contribution to the osmotic potential of Distichlis spicata (Fan et al., 1993; Marcum, 1999),
appears at high NaCl concentrations in Sporobolus virginicus
(Marcum and Murdoch, 1992). Although DMSP has been reported in species of Spartina (Supplementary Data Table S1),
3-DMSP was not detected in Spartina townsendii by AdrianRomero et al. (1998). Salt induces the synthesis of pinitol in
Porteresia coarctata, a halophytic wild rice, in contrast to its
absence in domesticated rice (Sengupta et al., 2008). These authors (Sengupta et al., 2008) suggested that the enhanced synthesis of pinitol in P. coarctata under stress may be one of the
adaptive features employed by the plant in addition to its
known salt excretion mechanism.
439
440
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
chloroplast in mesophyll and bundle sheath cells of Kranz-type
species. Species having C4 single cells have dimorphic chloroplasts in individual chlorenchyma cells, which are considered
specialized for functioning in C4 photosynthesis, analogous to
mesophyll and bundle sheath cells in the Kranz C4 system.
Thus, it is logical that plants that utilize glycine betaine as an
osmoprotectant synthesize it in both types of chloroplast (single-cell C4 and Kranz-type C4 plants), where it is directly
available for protecting chloroplasts and for export to protect
other parts of the cell (Park et al., 2009).
Chemical chaperones
One important function of osmoprotective compounds is the
stabilization of macromolecular structures (proteins, membranes) under conditions that lead to protein denaturation.
Unfortunately, our knowledge of the structure of the cytosol is
poor, although it is clear that it is not a dilute solution of proteins and inorganic ions and organic solutes (Cheeseman,
2013). Protective osmolytes improve the thermodynamic stability of proteins by hydrogen bonding, which, in contrast to hydrophobic interactions, does not affect other cellular functions
during stress (Kumar, 2009). However, not all osmolytes are
equal in the stabilization of proteins (Yancey, 2005). Szabados
and Savouré (2010) concluded that proline functions as a molecular chaperone able to protect protein integrity and enhance
the activities of different enzymes; examples of such roles included the prevention of protein aggregation and stabilization
of M4 lactate dehydrogenase during extreme temperatures
(Rajendrakumar et al., 1994). This was attributed to the ability
of proline to form hydrophilic colloids in aqueous media with a
hydrophobic backbone interacting with protein. In addition,
proline protects nitrate reductase during heavy metal and
osmotic stress (Sharma and Dubey, 2005) and stabilizes ribonucleases and proteases upon arsenate exposure (Mishra and
Dubey, 2006). Glycine betaine accumulation protects cytoplasm from ion toxicity, dehydration and temperature stress by
stabilizing macromolecule structures, protecting chloroplasts
and photosystem II, by stabilizing the association of the extrinsic photosystem II complex proteins and indirectly interacting
with phosphatidylcholine moieties) to alter their thermodynamic properties (Subbarao et al., 2001).
Polyol hydroxyl groups effectively replace water in
establishing hydrogen bonds in the case of cellular dehydration,
thus helping to maintain enzyme activities and to protect
membrane structures (Noiraud et al., 2001). Trehalose, for
example, has the remarkable ability to stabilize membranes
and proteins in the dry state (Crowe, 2007). Trehalose was proposed to act as a water replacement, conferring on membranes
and proteins physical properties that resemble those seen in the
fully hydrated state. Trehalose stabilizes macromolecules and
membrane bilayer structure. This effect is achieved via hydrogen bonding between trehalose and polar groups on membrane
lipids or proteins, thus maintaining the phase separation
of the bilayer and preventing leakage through the membranes.
Nevertheless, depending upon concentrations and solvent
conditions, naturally occurring osmolytes can also have
destabilizing effects on proteins (Singh et al., 2011). For example, trehalose at high concentrations (>1 M) is toxic to
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energetic reasons, be achieved by organic solutes alone (Yeo,
1983).
For halophytes in particular, in which a considerable proportion of osmotic adjustment is achieved by ion accumulation, the
process seems to be largely dependent on their capacity to compartmentalize ions in the vacuole and to accumulate compatible
solutes in the cytoplasm (Flowers et al., 1977, 1986; Wyn
Jones et al., 1977; Glenn et al., 1999). Although ion accumulation in the vacuoles is relatively simple to establish (Flowers
et al., 1986), osmolyte accumulation in the cytosol is more
difficult to demonstrate, largely because osmolytes are more
difficult to visualize. Glycine betaine has been localized to the
cytoplasm of Suaeda maritima by staining with iodoplatinic
acid (Hall et al., 1978), but histochemical procedures are not
available for most solutes. Based on non-aqueous fractionation,
Gagneul et al. (2007) revealed that b-alanine betaine and
proline were preferentially accumulated in the cytosol in
L. latifolium after salt treatment, although a vacuolar pool of
both solutes could be detected. In addition, isolated protoplasts
and vacuoles of cells of D. spicata treated with 200 mM NaCl
revealed a cytosolic proline concentration estimated to be
>230 mM (Ketchum et al., 1991).
An added complication in the evaluation of the role of osmolytes in the adjustment of cytosolic water relations is the possibility that compatible organic solutes move between the
cytoplasm and the vacuoles within cells, depending on the osmotic potential of the cytoplasm, without changes in their
concentrations at the tissue level (Greenway and Munns, 1980).
A model for intracellular compartmentation of inorganic ions
and other osmolytes in salinized tissues of higher plants has
emerged from the pioneering works of Flowers (1972),
Greenway and Osmond (1972), Stewart and Lee (1974) and
Wyn Jones et al. (1977). These authors expected that, in plants
coping with salinity, the distribution of osmolytes between cell
compartments would provide osmotic balance between the cytosol and the vacuole on the one hand and between the cytosol
and the apoplast on the other hand. These approaches have
shown that some typical compatible solutes and ions such as
Naþ and Cl were indeed preferentially compartmentalized in
the extravacuolar compartments and the vacuoles, respectively.
However, according to Wyn Jones and Gorham (2002), these
statements should now be regarded with caution since great
plasticity occurs at both the tissue and the subcellular level.
These authors suggested that vacuoles are not inert balloons,
but that some of them might be filled with inorganic ions while
others might contain large amounts of organic osmolytes.
The prevailing model for the allocation of osmolytes at the
sub-cellular level might also be modulated by the degree of
vacuolation of plant cells, which depends on cell expansion and
changes induced by salinity in the volume fraction of the
vacuoles (Chang et al., 1996). Compartmental analysis of
proline and b-alanine betaine in L. latifolium leaf tissues
demonstrated that these solutes, mainly located in vacuoles
under non-saline conditions, could be partly directed to the
cytosol in response to salinization (Gagneul et al., 2007).
Partitioning of proline pools in Pringlea antiscorbutica was
also shown to be split between the cytoplasm and vacuolar
compartments (Aubert et al., 1999). Studies on the C3 chenopods spinach and sugar beet indicate that CMO and BADH are
located in the chloroplasts. C4 plants have two types of
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
Hydroxyl radical-scavenging activity of osmolytes
Accumulation of ROS is the result of various abiotic stresses,
including drought and salinity. The protective function of osmolytes can include the suppression of oxygen radical production,
scavenging ROS directly, or contributing to the protection of the
enzymes involved in the antioxidant system (Ozgur et al., 2013).
Mannitol may scavenge hydroxyl radicals to shield susceptible
thiol-regulated enzymes such as phosphoribulokinase plus thioredoxin, ferredoxin and glutathione from inactivation by ROS
(Shen et al., 1997). Interestingly, proline was found to be an effective hydroxy radical scavenger (Smirnoff and Cumbes, 1989).
Proline can act as a singlet oxygen quencher and as a scavenger
of OH radicals (Alia et al., 1997). Thus, proline is an effective
quencher of ROS formed under salt, metal and dehydration stress
in all plants, including algae (Alia and Sardhi, 1991). Activities
of the enzymes catalase, peroxidase and polyphenoloxidase were
promoted and stabilized by proline in vivo (Paleg et al., 1984).
Proline is also involved in protecting thylakoid membranes
against free radical-induced photodamage upon salt stress
(Sivakumar et al., 2000). Glycine betaine accumulation in chloroplast protects photosynthetic and oxygen-evolving complex of
photosystem II activities by preventing high salt (Naþ and Cl)induced dissociation of the extrinsic proteins (Yang et al.,
2008b). Glycine betaine has also been implicated in reducing
lipid peroxidation in plants (reviewed by Cushman, 2001).
Dimethylsulphoniopropionate is certainly able to act as an antioxidant from a chemical point of view; this has been demonstrated in the algae Thalassiosira pseudonana and Emiliana
huxleyi (Sunda et al., 2002), but not yet explored in higher
plants. Dimethylsulphoniopropionate and its breakdown products, e.g. dimethyl sulphide, acrylate, dimethylsulphoxide and
methane sulphinic acid, readily scavenge hydroxyl radicals and
other ROS, and thus may serve as an antioxidant system regulated in part by enzymatic cleavage of DMSP (Sunda et al.,
2002).
REGULATION OF OSMOLYTE BIOSYNTHESIS
BY ENVIRONMENTAL CUES
Osmolyte accumulation can be induced by different stresses,
such as salinity, drought, heat and cold (Ghars et al., 2012;
Savouré et al., 1997; Slama et al., 2007b, 2008, 2011;
DeRidder and Crafts-Brandner, 2008). In order to gain a better
picture of their role in adaptation to stress, it is necessary to
identify and characterize any signalling pathways involved in
their synthesis and their redistribution in planta and/or between
subcellular compartments.
How does a plant cell sense changes in osmotic pressure or
in ion concentration? In yeast, hyperosmolarity can be sensed
by a phosphorelay signal transduction pathway composed of
the synthetic lethal of N-end rule (SLN1) His kinase, the tyrosine (Y) phosphatase-dependent phosphorelay intermediate
(YPD1) and the suppressor of sensor kinase (SSK1) response
regulator, which are homologous to bacterial two-component
signal transducers, leading to the activation of the high osmolarity glycerol (HOG1) MAPK pathway to trigger glycerol biosynthesis. The arabidopsis SLN1 homolog histidine kinase
(AtHK1) induced by salt and drought stresses has been postulated to be a plant osmosensor (Urao et al., 1999). Recently
Kumar et al. (2013) demonstrated that this osmosensor is not
involved in ABA, proline and solute accumulation in A. thaliana at low water potential, which calls into questions its role as
a main osmosensor at low water potential. Another His kinase,
cytokinin response 1 (CRE1), which was identified as a cytokinin receptor, is also able to complement the yeast sln1D mutant
in the presence of cytokinin (Inoue et al., 2001). Interestingly,
Reiser et al. (2003) reported that CRE1 perceives the osmotic
signal by turgor sensing, similarly to SLN1. It remains to be
shown whether these osmosensors are involved in the regulation of the biosynthesis of osmolytes in plants. At present, how
Naþ-specific signals are sensed remains elusive in any cellular
system. In plants, direct functional connections between members of the salt overly sensitive (SOS) signalling pathway and
the dynamics of the cytoskeleton upon salt stress have been established (Wang et al., 2007). The reorganization of the cytoskeleton is a necessary component of cell adaptation to salinity.
Further studies should be developed to determine whether the
protein(s) responsible for the Naþ-specific reorganization of the
cytoskeleton may be a constituent of the salinity sensor.
As far as regulation of the biosynthesis of osmolytes is
concerned, P5CS and ProDH are the rate-limiting enzymes for
the biosynthesis and catabolism of proline, respectively. Proline
accumulation is mainly regulated at the transcriptional level of
P5CS and ProDH genes during dehydration and rehydration
(Szabados and Savouré, 2010). Interestingly, ProDH gene expression is induced by rehydration but is repressed by dehydration.
Moreover, its expression is induced by proline and repressed by
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macromolecules, likely because high viscosity affects dynamic
interactions between chaperones and folding substrates and
stabilizes protein aggregates. Therefore, it is very likely that
cells regulate protein folding, protein disaggregation and protein–protein interactions via concentration-dependent accumulation of osmolytes, which may be an important determinant of
adaptation to stress.
By contrast to salt ions, many stabilizing solutes do not bind
to proteins; indeed, they are excluded from protein hydration
layers (Timasheff, 1992). Termed the ‘osmophobic’ effect by
Bolen and Baskakov (2001), exclusion arises from an apparent
repulsion between stabilizers and the peptide backbone, explaining how this effect can be universal. Because of this
repulsion, proteins will tend to fold more compactly, since this
will reduce exposure of the peptide-bond backbone to thermodynamically unfavourable interactions with the stabilizing solute (Yancey, 2005). It has been reported by Paleg et al. (1984)
that in Hordeum distichum proline reduces the amount of
glutamine synthase precipitated by PEG in a concentrationdependent manner. The authors suggested that these results
were consistent with the hypothesis that a protein-containing
system in which high concentrations of proline and/or betaine
are present is better protected against the biologically unfavourable consequences of dehydration-induced thermodynamic
perturbation. Among osmolytes, proline has the property of
forming hydrophilic colloids in aqueous media, with a hydrophobic backbone interacting with the protein. On the other
hand, the effects of proline might be involved in the hydration
layer surrounding phospholipids (Rajendrakumar et al., 1994).
441
442
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
osmotic stress (Yoshiba et al., 1997). In arabidopsis it was reported that the expression of P5CS is independent of ABA upon
exposure to cold and osmotic stress, even though expression of
this gene can be triggered by treatment with exogenous ABA
(Savouré et al., 1997). Recently, lipid signalling pathways have
been shown to be involved in the regulation of proline metabolism in A. thaliana (Thiery et al., 2004; Parre et al., 2007).
Interestingly, these lipid signalling pathways are regulated in the
opposite sense in T. salsuginea, revealing that common signalling
components affect the physiology of closely related glycophyte
and salt-tolerant plants differently (Ghars et al., 2012).
The allocation and partitioning of compatible osmolyte pools in
organs and tissues of halophytic plants is far from being clearly
depicted. Osmolyte functions are conceptually associated with
accumulation in specific subcellular locations at the right time,
thus requiring coordinated transport over both long and short
distances.
For polyols, their synthesis as primary products of photosynthesis occurs mainly in the leaves, so the absence of the corresponding synthetic enzymes in sink organs where polyols have
been detected is in favour of long-distance transport of these
compounds. This has been clearly shown for mannitol and sorbitol (Conde et al., 2011; Noiraud et al., 2001). Functional inositol transporters have been reported in Mesembryanthemum
species to take up myo-inositol in a manner depending on the
proton gradient, and low Naþ concentrations exerted a stimulating effect on inositol movement (Miyazaki et al., 2004).
D-Ononitol, one of the methylated myo-inositol derivatives that
accumulate in salt-stressed ice plants, is synthesized from myoinositol by a stress-induced inositol methyltransferase in a
tissue-specific reaction that includes the drastically increased
transport of myo-inositol and its derivatives from the leaves to
the root system (Nelson et al., 1998).
Proline-specific transporters (ProTs) contribute to tissue- and
organ-specific proline deposition under salt stress or during development (Schwacke et al., 1999; Ueda et al., 2007).
However, ProTs from tomato (LeProTs) were subsequently
shown to also transport glycine betaine as well as proline,
although tomato is a betaine non-accumulating plant
(Schwacke et al., 1999; Grallath et al., 2005). The barley
HvProT1 was reported to recognize proline, but not betaine
(Ueda et al., 2001). It has been demonstrated recently that four
types of Bet/ProTs could take up choline with higher affinity
than betaine, and in situ hybridization experiments in sugar
beet indicate the localization of BvBet/ProT1 in phloem and
xylem parenchyma cells (Yamada et al., 2011).
EXAMPLES OF IMPROVING PLANT
TOLERANCE TO ABIOTIC STRESS BY
INCREASING ACCUMULATION OF OSMOLYTES
Using genes from halophytes
A promising approach to avoiding or to reducing crop losses
due to the constraints imposed by drought or salinity is to use
halophytes instead of glycophytic crops (Koyro and Lieth,
2008). This approach has been long in its gestation (Rozema
Using model plants
Recently, the elucidation of the importance of osmolyte
accumulation in extremophiles became possible through comparative analysis of closely related halophyte and glycophyte
species, especially those that are related to the model plant arabidopsis (Orsini et al., 2010). The halophytes E. salsugineum
and C. maritima are close relatives of A. thaliana and can grow
under extreme saline conditions (Amtmann, 2009; Debez et al.,
2013). Salt cress, E. salsugineum, displays extreme tolerance to
high salinity, low humidity and freezing. This member of the
Brassicaceae has emerged as a model for understanding the adaptation of halophytes to abiotic stress tolerance due to its homology with the glycophyte model A. thaliana (Amtmann,
2009). Relevant variation in metabolic composition and function in response to salt or drought treatments were observed
(Lugan et al., 2010; Pedras and Zheng, 2010). For example,
proline contents in E. salsugineum and C. maritima are higher
than those in arabidopsis, even in optimal growth conditions,
and proline accumulates to higher levels when salt stress is
imposed (Ghars et al., 2012; Jdey et al., 2014). Another species
that has been frequently used as a model in salt tolerance studies is the facultative halophyte ice plant M. crystallinum, which
has bladder cells that accumulate ions (Adams et al., 1998;
Cushman and Bohnert, 2000). However, considering the
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TRANSPORT OF OSMOLYTES
and Schat, 2013), and the crops generated are often novel (e.g.
Salicornia species) and may not be those (e.g. cereals) familiar
to most people. Consequently, others have, over the years, advocated the use of resources held in the genomes of halophytes;
for example, the contention of some is that they might be bioengineered in related crop species (Amtmann, 2009). However, it
is important to point out that wide crossing has not proved to be
a very effective route to enhanced salt tolerance of common
crops (Yeo and Flowers, 1989). Furthermore, since a large
number of genes appear to be involved in tolerance of salt
stress, the functions of many of which have yet to be investigated, the genetic engineering of tolerance by single-gene transformation is likely to be unsuccessful in field conditions
because plants have to react to a multitude of environmental
factors (Flowers and Yeo, 1995). Between 1993 and early 2013
there were nearly 40 papers in which plants were transformed
using genes from halophytes; of these papers, nine reported an
association with the production of compatible solutes. Twothirds of the papers described transformation of tobacco, but of
these only a single publication reported growth data, and even
here the plants were all grown in Murashige and Skoog medium. Of the nine reports of transformations involving compatible solutes from halophytes only a single paper, in which cotton
was transformed with choline monooxygenase from Atriplex
hortensis, demonstrated that, in a saline field, yield of the transgenics was enhanced over two consecutive years when compared with an untransformed line (Zhang et al., 2009); this
enhancement was associated with the enhanced synthesis of
glycine betaine. Alternative strategies, such as genomics-based
approaches, for halophytes should be considered because they
provide access to the genetic basis of salt tolerance and the discovery of superior exotic alleles. Introgression of favourable
exotic alleles into elite breeding lines will thereby facilitate the
speedy development of superior cultivars.
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
responses to salinity, none of these dicot plants is ideal as a halophytic model for grain crops, which belong mostly to the
Poaceae. Halophytic relatives of wheat (species of
Thinopyrum), barley (species of Hordeum) and rice (species of
Porteresia) could be investigated as models. However, molecular responses and genome sequence information on these halophyte relatives are poorly documented and need further
analysis.
443
(Crowe, 2007) and the treatment of dry eye syndrome and dry
skin in humans. Trehalose is listed as a prominent ingredient in
cosmetics, apparently because it is reputed to inhibit the oxidation of certain fatty acids in vitro that might be related to body
odour (Crowe, 2007). These various uses suggest there may be
many opportunities to exploit halophytes for the diversity of
osmolytes they contain.
CONCLUSIONS
Exogenous application of osmolytes
IMPORTANCE OF HALOPHYTES AS A SOURCE
OF OSMOLYTES FOR PHARMACEUTICAL AND
MEDICINAL APPLICATIONS
Osmolytes modulate protein folding and unfolding as well as
interactions with lipid membranes and DNA and with ligands
that associate and dissociate. The properties of osmolytes are
increasingly useful in molecular biology. Proline is currently
used in many biological kits with pharmacological and cosmological applications. Osmolytes known to stabilize protein
structure improve the crystallization of proteins (Jeruzalmi and
Steitz, 1997). Betaine improves cell-free transcription, including PCR (Henke et al., 1997; Hethke et al., 1999), and, in combination with an hyperosmotic medium, it maximizes
monoclonal antibody production by hybridoma cells (Welch
and Brown, 1996).
Many human pathologies that are linked to poor protein folding, referred to as ‘conformational diseases’, may be corrected
by osmolytes (Kim et al., 2006). Proline can protect human
cells against carcinogenic oxidative stress (Krishnan et al.,
2008). Some osmolytes can also rescue the misfolded protein
of cystic fibrosis in vitro (Yancey, 2005). Protein aggregation/
misfolding constitutes a hallmark of neurodegenerative pathologies, including Alzheimer’s, Huntington’s and Parkinson’s diseases, and if osmolytes could provide a unifying mechanism of
action this may have far-reaching consequences in developing
better therapeutic tools for the management of such diseases
(Kumar, 2009).
Trehalose has been used for the stabilization of vaccines
and liposomes, the hypothermic storage of human organs
Environmental stress factors affect plant growth and constitute
a growing threat to sustainable agriculture. Upon exposure to
abiotic stress, it is commonly observed that, concomitantly with
controlled ion uptake and sequestration of toxic ions into the
vacuole, halophytes also synthesize and accumulate metabolites
that are compatible with metabolic reactions and even stabilize
cellular structures, photosynthetic complexes, specific enzymes
and other macromolecules. Compatible osmolytes include a variety of compounds such as sugars, amino acids, tertiary sulphonium compounds, quaternary ammonium compounds and
polyhydric alcohols. These osmolytes do not have any harmful
effects on plant metabolism even at high concentrations in the
cytoplasm.
Halophytes constitute an interesting source of various osmolytes (Table 1). Interestingly, certain osmolytes, such as nitrogen compounds and carbohydrates, are typical of certain
families. Investigation of new halophytic species, especially
those growing in extreme environments, is of importance in order to find new compounds. These molecules could be very
useful in the pharmaceutical industry and in medicine due to
their ability to stabilize proteins and to protect against oxidative
damage.
Halophytes also represent an important genetic reservoir that
can be exploited for improving crops for marginal land.
Engineering osmoprotectant biosynthesis pathways is a potential way to improve stress tolerance. First-generation engineering work, much of it with single genes, has successfully
introduced osmoprotectant pathways into plants that lack them
naturally. However, engineered osmoprotectant levels are generally low and the increases in tolerance commensurately small.
For these reasons, simultaneous engineering of osmolyte accumulation and various other traits by transgenic technology is a
feasible strategy to improve abiotic stress tolerance in crops.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxfordjournals.org and consist of Table S1: osmolyte distribution in
representative halophyte species among angiosperms.
ACKNOWLEDGEMENTS
The COST action FA0901, ‘Putting Halophytes to
Work—From Genes to Ecosystems’, is acknowledged for its
financial support. Part of the work was supported by the
Tunisian–French PHC UTIQUE 2013 network (no.
29174UF). Many thanks and deep gratitude are due to
Professor F. Larher (University of Rennes 1, France) for his
very significant contribution to the advancement of knowledge
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Exogenous application of osmoprotectants has been reported
to have osmoprotective roles in abiotic stress responses and has
been suggested as an alternative approach to improving crop
productivity under abiotic stress conditions (Ashraf and Foolad,
2007). An improvement in growth after applying exogenous
osmolytes has been observed in tobacco, wheat, barley, rice,
soybean and common beans (Borojevic et al., 1980; Agboma
et al.1997; Lutts, 2000). In arabidopsis, exogenous application
of either proline or glycine betaine triggers the expression of
numerous genes involved in regulatory functions and antioxidant defence mechanisms (Oono et al., 2003; Einset et al.,
2007). In certain circumstances, however, the accumulation of
solutes after exogenous supply can cause deleterious side effects and the universal compatible property of solutes can be
questioned (Sulpice et al., 1998). It remains to be determined
what effects the application of osmolytes would have on the
growth and survival of halophytes.
444
Slama et al. — Diversity, distribution and roles of osmolytes in halophytes
concerning the distribution and production of osmoprotective
compounds in halophytic plants.
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