Reviews and Opinions
73
Mechanisms of Salt Tolerance in Rice Plants:
Compatible Solutes and Aquaporins
Ching Huei Kao *
Department of Agronomy, National Taiwan University, Taipei 10617, Taiwan ROC
ABSTRACT
Decrease in water potentials in the
environment can impose osmotic stress to plants.
Soil salinity is one of the major causes of osmotic
stress of plants. Rice is known to be sensitive to
salt stress. Therefore, soil salinity is a major
abiotic stress limiting growth and productivity of
rice plants. The accumulation of compatible
solutes is often considered as a basic strategy for
protection of plants from salt stress. Aquaporins
(AQPs) are water channel proteins that facilitate
the water movement across membrane. The
mechanisms underlying salt stress tolerance of
rice plants are far from being completely
understood. The present review summarizes the
major research advances in elucidating the roles
of compatible solutes and AQPs in salt tolerance
of rice plants.
Key words: Aquaporins, Compatible solutes,
Rice, Salt stress.
水稻耐鹽之機制：相容性溶質與水通道蛋白
高景輝*
國立臺灣大學農藝系
摘要
降低環境之水勢可造成植物滲透逆境。
土壤高鹽分是引起植物滲透逆境之主因之
一。水稻對鹽分逆境非常敏感， 因此鹽分逆
境是限制水稻生長與產量之一項主要的非生
* 通信作者, [email protected]
投 稿 日 期： 2015 年 5 月 6 日
接 受 日 期： 2015 年 5 月 23 日
作 物 、 環境 與生 物 資 訊 12:73-82 (2015)
Crop, Environment & Bioinformatics 12:73-82 (2015)
189 Chung-Cheng Rd., Wufeng District, Taichung City
41362, Taiwan ROC
物性逆境。相容性溶質之累積被認為是克服
鹽分逆境之一種策略。水通道蛋白可幫助水
分透過膜之運送。到目前為止，水稻耐鹽分
逆境機制尚未被充分瞭解，本文說明相容性
溶質與水通道蛋白在水稻耐鹽機制所扮演角
色之主要研究進展。
關鍵詞︰水通道蛋白、相容性溶質、水稻、
鹽分逆境。
INTRODUCTION
It is estimated that more than 800 million
hectares of land throughout the world, which
account for more than 6% of the world’s total land
area, have been salt-accumulated (Hasegawa 2013,
Munns and Tester 2008). NaCl is the most soluble
and abundant salt released to soil solution and is
the major cause for salt stress on plants. Salt
accumulation in arable soils is mainly derived
from irrigated water that contains trace amounts
of NaCl and from seawater (Deinlein et al. 2014,
Flowers and Yeo 1995). Plants that can withstand
salt stress are classified as halophytes, whereas
those cannot withstand salt stress are
glycophytes.
When growing in saline soils, roots have to
cope with two types of stresses, osmotic stress
and ionic stress (Lin and Kao 2001). The former
stress causes an inhibition of water uptake,
whereas the ionic stress results in excess Na+
influx (Horie et al. 2012). In addition, elevated
levels of Na+, a major ion in saline environment,
can induce deficiency of the essential element K+,
imposing toxic effect by disturbing K+-dependent
processes (Horie et al. 2012). In response to salt
stress, the production of reactive oxygen species
(ROS), such as superoxide radical and hydrogen
peroxide, is enhanced (Apel and Hirt 2004,
Yamane et al. 2012). Salt-induced ROS production
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Crop, Environment & Bioinformatics, Vol. 12, June 2015
can lead to oxidative damages in various cellular
components such as DNA, protein, and lipids,
disrupting cellular functions of plants.
Basically, the possible routes for water
movement in plant tissue are the apoplastic, the
symplastic, and the transcellular route. The
transcellular route is defined as the transport of
water across the plasma membrane and across the
vacuolar membrane of each cell without the
involvement of plasmodesmata. The transmembrane water transport is accomplished by the
diffusion of water molecules across the lipid
bilayer of membranes and/or with the aid of
aquaporins (AQPs). AQPs are water channel
proteins of vacuolar and plasma membranes,
which facilitate the passive movement of water
molecules down a water potential gradient
(Kjelborm et al. 1999). Thus, osmotic adjustments
(by means of the accumulation of compatible
solutes) and AQPs are important for the water
movement under salt stress.
Rice is the most salt sensitive among cereals,
especially at the seedling stage (Pearson et al.
1966). Soil salinity is one of major abiotic stresses
that limit rice productivity (Todaka et al. 2012). To
improve the yield under salt stress, it is essential
to understand fundamental mechanisms behind
salt stress tolerance in rice plants. Due to the
continuous efforts of investigators and the
improvement of technologies, our knowledge
concerning the mechanisms of rice salt tolerance
is rapidly expanding. In this review, we discuss
research advances on the roles of compatible
solutes and AQPs that are involved in salt
tolerance of rice plants.
COMPATIBLE SOLUTES
Water flux is positively correlated with the
product of water potential difference (∆) and
hydraulic permeability or hydraulic conductivity
(Lp), i.e., Lp (∆). In case of water uptake in root
cells, ∆ is the difference of water potential
between extracellular solution and intracellular
sap solution. Under non-stress conditions,
intracellular water potential is generally more
negative than that of the soil solution, resulting in
water influx into roots according to the water
potential gradient. Osmotic adjustments by means
of accumulations of compatible solutes inside the
cell are essential to reduce the cellular osmotic
potential, which eventually restore the uptake of
water into roots during salt stress (Greenway and
Munns 1980). Compatible solutes are organic
compounds that are highly soluble and non-toxic
even when they accumulate in the cytosol,
contribute to the decrease of cytoplasmic water
potential, and act as osmoprotectants or
osmolytes (Bohnert et al. 1995). Compatible
solutes are also known to function as a chaperone
protecting enzymes and membrane structures,
and as a scavenger reducing ROS under salt stress
(Bohnert and Shen 1999).
1. Proline
Proline is an essential amino acid for primary
metabolism in plants. The accumulation of proline
in plant cells exposed to salt is a widespread
phenomenon (Chandler and Thorpe 1986). Proline
is believed to protect plant tissues against stress
by acting as an osmoprotectant (Greenway and
Munns 1980), ROS scavenger (Matysik et al. 2004,
Smirnoff and Cumbes 1989), and reservoir of
nitrogen and carbon sources (Fukutaku and
Yamada 1984).
Proline is synthesized from glutamic acid via
∆1-pyrroline-5-caroxylate (P5C) by two enzymes,
P5C synthetase (P5C) and P5C reductase (P5CR)
(Yoshiba et al. 1997). It has been shown from
labelling experiments that ornithine can also serve
as a precursor to proline synthesis in higher
plants (Brown and Fowden 1966). Enzyme
ornithine-δ-aminotransferase (OAT) participates
in proline biosynthesis by producing P5C from
ornithine and 2-oxoglutarate in plants (Delauney
and Verma 1993). Arginine can also contribute to
proline accumulation, and the pathway from
arginine proceeds via ornithine as a result of
catalytic activity of arginase (Brown and Fowden
1966). Proline is degraded to glutamic acid via
P5C by two enzymes, proline dehydrogenase
(PDH) and P5C dehydrogenase (P5C DH)
(Yoshiba et al. 1997). In rice, Roy et al. (1992)
reported that proline accumulation in rice caused
by NaCl is related to an increase in P5CR activity
and to a decrease in PDH activity. Using two rice
cultivars differing in salt tolerance, Lutts et al.
(1999) demonstrated that the accumulation of
proline in salt-sensitive rice cultivar results from
an increase in OAT activity and an increase in
Mechanisms of Salt Tolerance in Rice Plants: Compatible Solutes and Aquaporins
endogenous pool of its precursor glutamic acid.
Lin et al. (2002) also showed that proline
accumulation in rice leaves caused by NaCl is
related to an increase in the activity of OAT and
the increase in the contents of the precursors of
proline biosynthesis, glutamic acid, ornithine, and
arginine.
Igarashi et al. (1997) compared the content of
proline and expression of the P5CS gene in
‘Dee-gee-woo-gen (DGWG)’, a salt tolerant rice
cultivar, and ‘IR28’, a salt sensitive rice cultivar
under salt conditions. They observed that the
expression of the P5CS gene and the accumulation
of proline in ‘DGWG’ steadily increase, whereas
those in ‘IR28’ increase slightly. When P5CS gene
was overexpressed in the transgenic rice plants,
an increased production of proline coupled with
salt tolerance were noted (Zhu et al. 1998). Su and
Wu (2004) used stress-inducible promoter and a
constitutive promoter, to drive the expression of
P5CS gene. They found that under salt stress
conditions, the growth is significantly better in
transgenic rice plants harboring the P5CS gene
driven by a stress-inducible promoter as
compared to those plants harboring a constitutive
promoter. Moreover, rice seedlings of a knockout
mutation in OsP5CS2 were observed to be more
sensitive to salt stress than those of wild-type
controls (Hur et al. 2004). All these results suggest
that proline accumulation is a cause of salt
tolerance. However, Lutts et al. (1996) reported
that proline accumulation of salt tolerant rice
cultivars was lower than that of salt sensitive ones.
Furthermore, Garcia et al. (1997) and Lin and Kao
(1996) demonstrated that exogenously applied
proline increases the salt-induced injuries.
Recently, Nounjan et al. (2012) reported that
exogenous proline has no effect on growth
inhibition of rice seedlings during salt stress, but
promotes recovery of rice seedlings from salt
stress. Deivanai et al. (2011) demonstrated that
rice seedlings from seeds pretreated with 1 mM
but not 10 mM proline are effective in improving
growth of rice seedlings under high level of salt
(300 and 400 mM). Using GC-MS, Zhao et al. (2014)
identified metabolites in leaves and roots,
respectively, of ‘FL478’ (salt tolerance cultivar)
and ‘IR64’ (salt sensitive cultivar). They observed
that leaf proline content increases in both cultivar
under stress, whereas proline content in roots
75
decreases in ‘IR64’ but increases in ‘FL478’ and
concluded that proline accumulation in roots is an
indicator of stress tolerance, not a symptom of
injury. It seems that debate regarding the function
of proline in salt stress tolerance can be partially
resolved.
2. Glycinebetaine
Glycinebetaine, a representative member of
betaines, is a quaternary ammonium compound,
i.e., glycine derivative in which the nitrogen atom
is fully methylated. In higher plants choline is
first oxidized to betaine aldehyde by choline
monooxygenase (CMO) (Sakamoto and Murata
2000). Betaine aldehyde is then oxidized to
glycinebetaine
by
betaine
aldehyde
dehydrogenase (BADH) (Sakamoto and Murata
2000). CMO and BADH are localized in the
stroma of chloroplasts. In Escherichis coli,
glycinebetaine is synthesized by choline
dehydrogenase (CDH) in combination with
BADH (Sakamoto and Murata 2000). In contrast
to these two pathways, soil bacterium Arthrobacter
globiformis synthesizes glycinebetaine by one
single enzyme choline oxidase, a H2O2-generating
enzyme (Sakamoto and Murata 2000).
Nakamura et al. (1997) found that rice plants
possess the ability to take up exogenously added
betaine aldehyde through the roots and convert it
to glycinebetaine, resulting in an enhancement of
salt tolerance. Foliar application of glycinebetaine
has been shown to increase salt tolerance in rice
seedlings (Harinasut et al. 1996). Other reports
also demonstrated the positive effect of
exogenous glycinebetaine on the photosynthesis
and ultrastructure of rice seedlings exposed to salt
stress (Cha-Um and Kirdmanee 2010, Rahmam et
al. 2002). The fact that rice plants cannot
synthesize glycinebetaine due to the lack of an
upstream enzyme, the CMO, leads to the
proposals that it is possible to increase salt stress
tolerance by genetic manipulation of biosynthesis
of glycinebetaine (Nakamura et al. 1997).
Introduction of spinach CMO genes or the
Arthrobacter globiformis choline oxidase into rice
plants promotes the synthesis of glycinebetaine
and enhancement of salt stress tolerance in the
transgenic rice plants (Sakamoto et al. 1998,
Shirasawa et al. 2006, Su et al. 2006). The
more-efficient maintenance of photosystem II
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Crop, Environment & Bioinformatics, Vol. 12, June 2015
activity n transgenic rice plants under salt
conditions suggests that glycinebetaine protects
against the salt-induced inactivation of the
photosystem II complex.
3. Trehalose
Trehalose is a non-reducing disaccharide in
which two glucose molecules joined together by a
glycosidic-(1-1) bond. Its chemical structure is
similar to that of sucrose but its physical
properties are very different, trehalose being only
slightly sweet and not hydrolyzing to reducing
sugars at high temperature (Lee 1980). It has been
shown that trehalose accumulates significantly in
resurrection plants (such as Selaginalla lepidophylla)
and enables to tolerate complete dehydration
(Wingler 2002). Therefore, it can function as stress
protection or stress tolerance. However, in many
other higher plants such as rice, tobacco and
potato, trehalose is nearly undetectable. Trehalase
is an enzyme that hydrolyzes trehalose into two
glucose molecules. Goddijn et al. (1997) could
demonstrate significant amounts of trehalose in
tabacco plants and potato micro-tubers cultured
in the presence of validamycin A, an inhibitor of
trehalase. Thus, the capacity to synthesize
trehalose may not be limited to certain
resurrection plants, but may exist throughout
other species of plants. The biosynthesis of
trehalose usually occurs by the formation of
trehalose-6-phosphate (T6P) from UDP-glucose
and glucose-6-phosphate, a reaction catalyzed by
the enzyme trehalose-6-phosphate synthase (TPS).
Subsequently, T6P is dephosphorylated by the
T6P phosphatase (TPP), resulting in the formation
of free trehalose (Wingler 2002).
In rice, Gracia et al. (1997) were the first to
report that trehalose plays a role in salt stress
tolerance. They observed that trehalose is able to
reduce Na+ accumulation and growth inhibition
caused by NaCl treatment and the effects are
more pronounced than proline. Recently,
Theerakulpisut and Gunnula (2012) demonstrated
that exogenous trehalose mitigates salt stress
damage in salt-sensitive cultivar ‘Khao Dawk
Mali’ but not salt-tolerant cultivar ‘Pokkali’. Due
to the presence of the enzyme trehalase, which
hydrolyzes trehalose to glucose, trehalose level is
generally low in rice plants. Thus, it is possible to
increase trehalose level by down regulating
trehalase in rice plants. Abdelgawad et al. (2014)
found that validamaycin A has a regulatory role
in increasing trehalose level and improve salt
tolerance in rice plants.
Escherichia coli otsA and otsB genes encode
TPS and TPP, respectively. Garg et al. (2002)
transformed rice plants with a gene that encodes a
bifunctional fusion enzyme (TPSP) of TPS and
TPP from E. coli otsA and otsB genes. The resultant
transgenic rice plants produced higher level of
trehalose and no visible phenotypical changes.
Overexpression of TPSP under the control of
either
tissue-specific
or
stress-dependent
promoters exhibits increased tolerance to salt
stress. Almost at the same time, Jang et al. (2003)
demonstrated that rice plants overexpressing E.
coli trehalose biosynthetic gene (otsA and otsB) as
a fusion gene, under the control of the maize
ubiquitin promoter, exhibit increase trehalose
accumulation and salt stress tolerance. Since the
increase in trehalose content is too low, it is
unlikely that the increased salt tolerance in these
transgenic rice plants could be ascribed to
trehalose acting as an osmoprotectant (Garg et al.
2002). The improved salt tolerance seemed instead
to correlate with a higher soluble sugar content
and an increased photosynthetic capacity (Garg et
al. 2002). Recently, Redillas et al. (2012) observed
that the elevated production of trehalose in rice,
through TPSP overexpression, increases the
soluble sugar content. They speculated that the
production of trehalose may play an important
role in sugar signaling, allocation, and
metabolism in a manner that ultimately favors the
tolerance of plants to salt stress.
In higher plants, trehalose biosynthesis genes
encoding TPS and TPP were first identified in
Arabidopsis (Blazquez et al. 1998, Vogel et al.
1998). Among 11 OsTPS genes from rice isolated
by Zang et al. (2011), only OsTPS1 encodes an
active TPS. Li et al. (2011) demonstrated that
overexpression of OsTPS1 in rice plants enhances
salt stress tolerance by increasing the amount of
trehalose and proline and activating some abiotic
stress-related
genes.
Expression
analysis
demonstrated that OsTPP1 in rice was induced by
salt stress (Chao et al. 2005, Ge et al. 2008). Ge et al.
(2008) demonstrated that overexpression of
OsTPP1 enhanced rice tolerance to salt stress.
However, overexpression of OsTPP1 in rice did
Mechanisms of Salt Tolerance in Rice Plants: Compatible Solutes and Aquaporins
not significantly increase the trehalose content.
This may be due to the fact that trehalose is
synthesized in two steps and their study only the
second step was affected. Thus, the enhanced salt
tolerance of rice by overexpressing OsTPP1
cannot be explained by the accumulation of
trehalose. In order to investigate the mechanism
of salt stress tolerance, they investigated further
the transcript levels of some well-known abiotic
stress-induced genes in OsTPP1 overexpression
rice (Ge et al. 2008). Their results led them to
conclude that OsTPP1 may trigger the expression
of a series of abiotic response genes, which in turn
contributes to rice stress tolerance.
AQUAPORINS
CHIP28, a 28 kDa integral membrane protein
isolated from erythrocytes, was identified as the
first protein with water transport activity (Preston
and Agre 1991). CHIP28 belongs to the major
intrinsic protein (MIP, also known as AQP0) and
is now renamed as aquaporin 1 (AQP1). The
identification of several major plant membrane
proteins with high sequence homology to AQP1
and MIP led to the identification of water channel
proteins, known as AQPs in plant membranes
(Maurel 1997, Maurel et al. 1993). In 2003, the
Royal Swedish Academy of Science awarded the
Nobel Prize in chemistry for “discoveries
concerning channels in cell membranes”, with one
half of the prize to Peter Agre for the discovery of
AQPs. Based on the sequence similarity, there are
four AQP subfamilies in plants, which usually
reflect their subcellular location: (1) the plasma
membrane intrinsic proteins (PIPs), (2) the
tonoplast intrinsic proteins (TIPs), (3) the
nodulin26-like intrinsic proteins (NIPs), and (4)
the small basic intrinsic proteins (SIPs). PIPs and
TIPs are located in plasma membrane and
tonoplast, respectively, whereas the subcellular
location of NIPs and SIPs is still uncertain
(Chaumont et al. 2005). PIPs are further
subdivided into two phylogenetic subgroups:
PIP1 and PIP2 (Kaldenhoff and Fischer 2006). The
tonoplast protein from Arabidopsis (-TIP or
AtTIP1;1) was the first plant AQP identified
(Maurel et al. 1993). Plant AQPs are not only
function as water channels, but also able to
transport carbon dioxide, ammonia, boron, and
hydrogen peroxide (Tyerman et al. 2002). Hg2+ is
77
known to bind to cysteine residues in or near the
aqueous pore of the AQPs (Daniels et al. 1996,
Maggio and Joly 1995) or to induce the
conformation change in the AQP structure
(Barone et al. 1997). Addition of HgCl2 to the
growth medium, the Lp of plant roots is reduced
compared with the control (Maggio and Joly 1995,
Martinez-Ballesta et al. 2003, Tazawa et al. 1997).
This inhibition is reversible upon the addition of
-mercaptoethanol or dithiothreitol (Maggio and
Joly 1995, Martinez-Ballesta et al. 2003). These
results suggest that the main part of the water
flux in roots is limited by aquaporin-mediated
water transport.
The identification and characterization of
AQP genes in rice is expected to be useful for
improving the water relations and for enhancing
slat tolerance. Several AQP genes have been
identified and characterized in rice (Li et al. 2000,
Lian et al. 2004, Liu et al. 1994, Malz and Sauter
1999, Takahashi et al. 2004). A complete set of rice
AQPs was conducted by Sakurai et al. (2005).
They identified 33 genes for AQPs in the genome
sequence of rice, 10 of which encode TIPs. Among
OsPIP and OsTIP genes, 12 and 10 genes were
expressed at higher levels in roots and leaves,
respectively. Sakurai et al. (2008) later provided
evidence to show that rice AQPs have various
water transport activities and suggest that they
may play distinct roles in facilitating water flux
and maintaining the water potential in different
tissues and cells.
Barley HvPIP2;1 is a plasma membrane AQP
(Katsuhara et al. 2002). Katsuhara et al. (2003)
demonstrated that overexpression of barley
HvPIP2;1 in rice plants substantially increased
root radial Lp, the mass ratio of shoot to roots,
and sensitivity to 100 mM NaCl. However, Guo et
al. (2006) observed that overexpression of either
an OsPIP1 or an OsPIP2 gene in Arabidopsis
enhances the tolerance to 100 mM NaCl.
Obviously, different research groups reached
different conclusions on the function of PIP
proteins.
CONCLUSIONS AND PERSPECTIVES
Soil salt stress is a serious problem in
agriculture. As mentioned above, great leaps
towards understanding of the regulation of water
homeostasis via osmotic adjustment or AQPs in
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Crop, Environment & Bioinformatics, Vol. 12, June 2015
rice plants under salt stress have been achieved in
the past 20 years. However, many challenges still
lie ahead. The accumulation of compatible solutes
in transgenic rice plants overexpressing
biosynthesis genes of compatible solutes is not
high enough to be osmotically significant. Thus,
the mechanisms of enhanced salt tolerance by
means of osmotic adjustment need further
explored and explained.
In Arabidopsis, trehalase is encoded by a
single gene AtTRE1 (Muller et al. 2001). Recently,
Van Houtte et al. (2013) reported an approach for
engineering drought stress tolerance by
modifying the endogenous trehalase activity in
Arabidopsis. Surprisingly, they observed that
AtTRE1-overexpression Arabidopsis decreases
trehalose content and recovers better after
drought stress, whereas Attre1 mutants leads to
increased trehalose content and exhibits
drought-susceptible phenotype. Clearly, these
results are contradictory to the commonly held
view that trehalose functions as a compatible
solute and stress protectant in many plants. It
appears that trehalose could be an unwanted,
perhaps deleterious, by product of T-6-P signaling,
and so the presence of high trehalase activity
would help to prevent trehalose accumulation
(Van Houtte et al. 2013). In future, it will be of
great interest to know whether manipulating the
expression of the endogenous trahalase can have
a beneficial effect on the response of rice to salt
stress.
The complicated results obtained from
transgenic rice plants overexpressing OsPIP genes
suggest that the exact physiological roles of PIPs
in salt stress tolerance are far from clear. It has
been shown that overexpression of ginseng
PgTIP1 in Arabidopsis enhances salt tolerance
(Peng et al. 2007) and loss of TIP1;1 AQP in
Arabidopsis leads to cell and plant death (Ma et al.
2004). It is not known whether overexpression of
OsTIPs or introducing antisense OsTIPs affects
rice salt tolerance. Further studies are thus
required to understand the exact roles of OsTIPs
in salt stress tolerance of rice plants. Molecular
analysis of the transgenic rice plants indeed
provides information about the relationship of
compatible solutes or AQPs with salt stress
tolerance. It will be of great interest to know how
transgenic rice plants perform in real salt stress
conditions in the field, and therefore contribute to
sustainable agriculture.
ACKNOWLEDGEMENTS
Research in the author’s laboratory has been
supported by grants from the Ministry of Science
and Technology (formerly National Science
Council) of the Republic of China.
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