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Annals of Botany 82 : 703–710, 1998
Article No. bo980731
BOTANICAL BRIEFING
New Molecular Approaches to Improving Salt Tolerance in Crop Plants
I. W I N I C O V*
Departments of Microbiology and Biochemistry, UniŠersity of NeŠada Reno, Reno, NV 89557, USA
Received : 5 May 1998
Returned for revision : 6 June 1998
Accepted : 21 July 1998
The last century has seen enormous gains in plant productivity and in resistance to a variety of pests and diseases
through much innovative plant breeding and more recently molecular engineering to prevent plant damage by insects.
In contrast, improvements to salt and drought tolerance in crop and ornamental plants has been elusive, partially
because they are quantitative traits and part of the multigenic responses detectable under salt\drought stress
conditions. However, the rapidly expanding base of information on molecular strategies in plant adaptation to stress
is likely to improve experimental strategies to achieve improved tolerance. Recently studies of salinity tolerance in
crop plants have ranged from genetic mapping to molecular characterization of salt\drought induced gene products.
With our increasing understanding of biochemical pathways and mechanisms that participate in plant stress responses
it has also become apparent that many of these responses are common protective mechanisms that can be activated
by salt, drought and cold, albeit sometimes through different signalling pathways. This review focuses on recent
progress in molecular engineering to improve salt tolerance in plants in context of our current knowledge of metabolic
changes elicited by salt\drought stress and the known plant characteristics useful for salt tolerance. While it is
instructive to draw parallels between molecular mechanisms responsive to salt-stress with accumulating evidence from
studies of related abiotic stress-responses, more data are needed to delineate those mechanisms specific for salt
tolerance. Also discussed is the alternative genetic strategy that combines single-step selection of salt tolerant cells in
culture, followed by regeneration of salt tolerant plants and identification of genes important in the acquired salt
tolerance. Currently, transgenic plants have been used to test the effect of overexpression of specific prokaryotic or
plant genes, known to be up-regulated by salt\drought stress. The incremental success of these experiments indicates
a potentially useful role for these stress-induced genes in achieving long term tolerance. In addition, it is possible that
enhanced expression of gene products that function in physiological systems especially sensitive to disruption by salt,
could incrementally improve salt tolerance. Current knowledge points towards a need to reconcile our findings that
many genes are induced by stress with the practical limitations of overexpressing all of them in a plant in a tissue
specific manner that would maintain developmental control as needed. New approaches are being developed towards
being able to manipulate expression of functionally related classes of genes by characterization of signalling pathways
in salt\drought stress and characterization and cloning of transcription factors that regulate the expression of many
genes that could contribute to salt\drought tolerance. Transcription factors that regulate functionally related genes
could be particularly attractive targets for such investigations, since they may also function in regulating quantitative
traits. Transgenic manipulation of such transcription factors should help us understand more about multigene
regulation and its relationship to tolerance.
# 1998 Annals of Botany Company
Key words : Salt tolerance, salt stress, molecular engineering, cellular selection, transgenic plants, gene regulation,
transcription factors.
INTRODUCTION
Improving salinity and drought tolerance of crop plants by
genetic means has been an important but largely unfulfilled
aim of modern agricultural development. As more land
becomes salinized through poor local irrigation practice, the
regional impact of salinity on crop production is becoming
increasingly important world-wide (Tanji, 1990 ; Flowers
and Yeo, 1995) creating a pressing need for improved salt
tolerant plants. At the genetic level, salinity tolerance has
been considered to be a quantitative trait (Foolad and
Jones, 1993) and has been generally resistant to improvements by plant breeding. Since quantitative traits influence
maximal plant yield and productivity, introducing a trait
that improves tolerance to saline growth conditions may
* Fax (702) 784-1620, e-mail winicov!unr.edu
0305-7364\98\120703j08 $30.00\0
actually lower the potential yield under normal conditions.
Thus, the need to balance productivity with salinity\drought
tolerance has become a contested point of discussion that is
likely to be resolved in favour of ‘ relative ’ yield only in
areas of limited arable land. However, rapid progress in
understanding biochemical mechanisms that may participate in plant stress responses and salt tolerance, as well as
the molecular cloning of genes involved in the various
metabolic pathways that respond to salt stress, offer new
approaches to solving this persistent problem (Bohnert and
Jensen, 1996 ; Winicov and Bastola, 1997). Accordingly, this
review will focus on recent progress in molecular engineering
and cellular selection to improve salt-tolerance in plants.
The rapid expansion of our knowledge of the diverse
genes that are induced and repressed by dehydration (Ingram
and Bartels, 1996 ; Bray, 1997 ; Shinozaki and YamaguchiShinozaki, 1997) includes the concept that most of these
# 1998 Annals of Botany Company
704
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
T     1. Examples of differential actiŠation of NaCl inducible genes by dehydration, cold and ABA
Function*
Gene
Alfin1
ARSK1
ATCDPK1
ATCDPK2
Atmyb2
AtP5CS
AtPLC1
cor6.6
kin1
mlip15
MsPRP2
OsBZ8
PKABA1
rd22
rd29A (COR78)
rd29B
Alfalfa
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Maize
Alfalfa
Rice
Wheat
Arabidopsis
Arabidopsis
Arabidopsis
DNA-binding
Prot. kinase
Prot. kinase
Prot. kinase
DNA-binding
Proline biosyn.
Phospholipase C
Antifreeze prot.
Antifreeze prot.
DNA-binding
Cell wall prot.
DNA-binding
Protein kinase
Seed protein
NaCl
Dehdr.
Cold
j
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j
p
nd
j
j
j
j
j
j
j
j
k
nd
j
j
j
j
p
nd
j
k
k
k
j
j
j
j
j
nd
p
j
nd
j
k
ABA Reference
k
j
k
k
j
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j
j
j
j
k
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p
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Bastola et al., 1998 a
Hwang and Goodman, 1995
Urao et al., 1994
Ibid
Urao et al., 1993
Yoshiba et al., 1995
Hirayama et al., 1995
Wang et al., 1995
Ibid
Kussano et al., 1995
Deutsch and Winicov, 1995
Nakagawa, Ohmiya and Hattori, 1996
Holappa and Walker-Simmons, 1995
Iwasaki, Yamaguchi-Shinozaki and Shinozaki, 1995
Yamaguchi-Shinozaki and Shinozaki, 1994
Ibid
nd, Not determined.
p, Weak or delayed response.
* Function shown or implied by sequence similarity with proteins of known function.
genes also respond to salt stress. In addition, it is becoming
increasingly apparent that among the genes that respond
to salinity stress and drought, some, but not all, respond to
cold stress (Shinozaki and Yamaguchi-Shinozaki, 1996).
Table 1 gives selected examples of a variety of genes that are
induced by salt stress, but differ in their responses to
dehydration, cold and the plant hormone abscisic acid
(ABA), which accumulates under these same abiotic stress
conditions. The commonality of responses may indicate
similar functions of these gene products in detoxification
and cellular maintenance for plants under stress conditions
involving water deficit. They also suggest interacting signal
perception and transduction pathways. In contrast, genes
that are induced in a stress specific manner emphasize the
likely existence of several signalling pathways and increase
the complexity of adjustments that may need to be made for
engineering tolerance traits in plants.
At present, we do not know to what extent acute stress
responses can best be utilized at the molecular level to
achieve significant long-term salt tolerance, nor which
responses hold the key to tolerance. Adaptive mechanisms
leading to increased long-term salt tolerance may utilize
either the gene products accumulated under short-term salt
stress or use other means for increased resilience. It is
possible that improved salt tolerance may be achieved by
the maintenance, activation or enhanced function of
physiological systems that are especially sensitive to disruption by increased levels of salt. Overexpression or
activation of the limiting components of such systems
would overcome the system failure under salt stress. A
number of such physiological systems could contribute
individually to a specific aspect of salt tolerance and so
provide both incremental and additive improvements in salt
tolerance (Winicov, 1994). Recently, it has been reported
that overexpression of the cold regulated gene (COR)
Binding Factor 1 (CBF1), which normally regulates COR
genes turned on by the cold acclimation response, leads to
enhanced freezing tolerance (Jaglo-Ottosen et al., 1998).
This example indicates that some adaptive responses can
provide increased tolerance by activation of a subset of
genes. Other data on temporal analysis of gene activation in
both wheat and barley have shown that while coordinated
induction of genes responsive to salt occurs within 2 h,
many transcripts decline in abundance within 24 h and
others disappear after about 6 d (Robinson, Tanaka and
Hurkman, 1990 ; Gulick and Dvor) a! k, 1992). These results
emphasize the transient nature of the early response to salt
stress, but do not address the potential of their use long
term. Our comparisons of salt inducible polypeptides and
mRNAs between salt-tolerant and salt-sensitive cells within
the same alfalfa genotype demonstrated different salt
inducible classes of genes for tolerant and sensitive cell lines
(Winicov et al., 1989). Since the tolerant cells were selected
at mutational frequencies from the salt-sensitive cells, these
results support the concept that different short-term and
long-term responses each contribute to salt tolerance. As we
become increasingly able to evaluate the results from
experimentally constructed transgenic plants harbouring
different trans-genes, the efficacy of these temporal responses
will become clarified in terms of long-term salt tolerance.
SALT TOLERANCE—THE GENERAL AIM
Optimistic discussions of future salt tolerance in plants have
sometimes invoked the unrealistic vision of crop plants
growing as halophytes on severely salt affected land or being
irrigated with sea water. This scenario appears unlikely,
since centuries of plant breeding have developed desirable
agronomic traits for most crop and ornamental plants.
Also, there are major morphological and physiological
differences between halophytes and most cultivated plants.
For the purposes of this discussion, the qualitative term of
‘ improved salt tolerance ’ will imply continued survival and
productive growth of a salt-tolerant plant under conditions
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
where a similar salt-sensitive genotype shows severely
inhibited growth or dies.
The varietal utilization of genetic information has long
been recognized as a potential source of beneficial traits for
salt tolerance. Native varietal halotolerance was exploited
to characterize differences between salt-sensitive and salttolerant barley varieties (Hurkman, Fornari and Tanaka,
1989). Examples from other plant species that show varietal
differences in salt tolerance are tomato (Tal, Heikin and
Dehan, 1978), rice (Flowers and Yeo, 1981) and alfalfa
(Smith and McComb, 1981), demonstrating that the genetic
repertoire within each species can provide enhanced salt
tolerance characteristics for improving general salt tolerance
of plants. The potential for augmenting this repertoire with
exogenous genetic information lacking in salt-sensitive
plants, or enhancing the utilization of endogenous genes
remains intriguing and is currently being explored in a
number of laboratories.
An alternate strategy for salt tolerance improvement
adopted by our laboratory is to utilize the regeneration of
alfalfa and rice with heritable improved salt tolerance after
selection of salt tolerant cells in culture (Winicov, 1991,
1996). The cell culture selection and regeneration protocol
has been undertaken in a number of other laboratories with
limited success. Many of the selected variants cannot be
evaluated, since we lack information about the heritability
of the trait. For others, the tolerance trait has been epigenetic
in nature and has yielded many albino plants as in the case
of rice (KrishnaRaj and SreeRangasamy, 1993) or produced
dwarf plants with limited fertility as shown in rice (Yano,
Ogawa and Yamada, 1982) or no fertility as in alfalfa
(McCoy, 1997). It is likely, however, that the early problems
encountered with this method were due to prolonged
selection on NaCl in culture and lack of secondary screening
of an adequate number of the regenerated plants (Winicov,
1996).
When successful, the cellular selection and regeneration
approach relies on identification of mutants optimized for
continued survival and productive growth under saline
conditions, but does not provide ready identification of the
affected genes without further study of changes in regulation
of the endogenous genetic information (Winicov and
Krishnan, 1996 ; Winicov and Bastola, 1997). However,
since the tolerant cells\plants continue productive growth
under saline conditions for months, we assume that the
changes in gene regulation are associated with the ability to
survive otherwise lethal conditions.
CHARACTERISTICS IMPORTANT IN SALT
TOLERANCE
Salt tolerance of plants depends primarily on characteristics
that can be broadly grouped in three categories : (1) physical
uptake or exclusion of salt followed by transport and
compartmentation of salt ; (2) morphological features and
biomass distribution of plant shoots and roots, which would
include rates of transpiration and stomatal closure ; (3)
physiological and metabolic events that counteract the
presence of salt at the cellular level. These characteristics
705
could be the primary targets for manipulation in engineering
of salt\drought tolerance. Plant morphology and salt
transport in the xylem depend on a complex pattern of
developmental regulation and have so far received little
attention with current molecular techniques. Similarly,
guard cell responses to environmental stimuli have been
documented (Kearns and Assmann, 1993), but currently
these responses cannot be manipulated in a heritable fashion.
General inhibition of shoot growth with continued root
growth has been considered as a morphological adaptation
to salt stress or water stress (Creelman et al., 1990 ; Saab et
al., 1990). While enhanced root development could be
beneficial in salt\drought tolerance as indicated from studies
on adaptation, molecular techniques for effectively manipulating root mass have not been developed (Aeschbacher,
Schiefelbein and Benfey, 1994). Ion uptake and transfer
across membranes has been investigated as an integral
metabolic change in salt stress and adaptation (Niu et al.,
1995). Membrane components of ion pumps could thus be
encoded by a group of genes which, when activated, would
counteract acute salt stress. This mechanism is suggested by
increased Na+ tolerance in yeast with a mutated transmembrane domain of the high affinity K+-transporter,
HKT1 (Rubio, Gassman and Schroeder, 1996). However,
much more needs to be understood about the role different
members of these multigene families play in tissue specific
function. Most progress to date has been made in
understanding biochemical mechanisms in physiological or
metabolic adaptation to salt\drought stress at the cellular
level as a means of providing potential candidate genes for
engineering improved tolerance.
ENGINEERING PHYSIOLOGIC OR
METABOLIC ADAPTATION
Physiologic or metabolic adaptations to salt stress at the
cellular level are the main responses amenable to molecular
analysis and have led to the identification of a large number
of genes induced by salt (Ingram and Bartels, 1996 ; Bray,
1997 ; Shinozaki and Yamaguchi-Shinozaki, 1997). These
genes can be classified in groups related to their physiologic
or metabolic function predicted from sequence homology
with known proteins and are summarized in Table 2. Most
of the genes in the functional groups have been identified as
salt inducible under stress conditions. Other genes have
T     2. Functional groups of genes\proteins actiŠated in
salt stress with potential for proŠiding tolerance
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Carbon metabolism and energy production\photosynthesis
Cell wall\membrane structural components
Osmoprotectants and molecular chaperons
Water channel proteins
Ion transport
Oxidative stress defences
Detoxifying enzymes
Proteinases
Proteins involved in signalling
Transcription factors
706
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
been detected by a salt-hypersensitivity assay in Arabidopsis,
which led to the identification of mutants in potassium
uptake as being critical in salt sensitivity through the
manifestation of increased salt sensitivity (Wu, Ding and
Zhu, 1996). However, other physiological systems may be
equally limiting under stress conditions and mutants in
these physiological pathways could lead to increased salt
toxicity and would affect survival in a negative manner. Our
approach has been to clone genes that are differentially
induced by salt in salt-tolerant alfalfa cells (Winicov and
Bastola, 1997) and are also regulated by salt at the whole
plant level (Winicov, 1993 ; Winicov and Deutch, 1994 ;
Deutch and Winicov, 1995 ; Winicov and Shirzadegan,
1997) with the aim of testing their relevance to the improved
salt tolerance of the selected plants. For example, one of
these genes Alfin1 encodes a transcription factor that has
been found to induce enhanced expression of the saltinducible MsPRP2 gene in callus and in plants (Bastola,
Pethe and Winicov, 1998 b). It will be interesting to
determine if Alfin1 can also influence the expression of other
salt inducible genes and if it functions in concert with other
regulatory factors under salt stress conditions.
SINGLE GENE TRANSFER TO IMPROVE
SALT TOLERANCE
While salt induced gene activation has been demonstrated
for genes belonging to all the functional groups listed in
Table 2, only a limited number of genes have been tested in
transgenic plants for their effect on stress tolerance.
Primarily these genes encode enzymes involved in osmoprotectant synthesis, molecular chaperons and detoxifying
enzymes involved in oxidative stress responses. Increased
osmoprotectant synthesis has been manipulated in plants by
overexpression of enzymes leading to increased mannitol
synthesis in tobacco (Tarczynski, Jensen and Bohnert, 1993)
and Arabidopsis (Thomas et al., 1995), ononitol production
in tobacco (Sheveleva et al., 1997), fructan synthesis in
tobacco (Pilon-Smits et al., 1995), trehalose synthesis in
tobacco (Holmstrom et al., 1996) and the amino acid proline
in tobacco (Kishor et al., 1995). In each case incremental
improvements in salt\drought tolerance were measured
under laboratory conditions that correlated with increased
constitutive accumulation of the manipulated solutes. The
applicability of this approach for all plant species has been
questioned (Flowers et al., 1997). In addition, the results
with transgenics accumulating mannitol and proline
provided some unexpected information. While transgenic
plants accumulating proline demonstrated that degradative
as well as synthetic pathways may need to be manipulated
if constitutively higher than normal proline levels were to be
attained, elevated basal levels of proline provided immediate
protection against stress. These results are consistent with
observations in maize, where the osmoprotectant glycinebetaine accumulation has been shown to correlate with Bet1
gene copy number and improved salt tolerance (Saneoka et
al., 1995) and with our findings that salt-tolerant alfalfa
plants rapidly double their proline concentration in the
roots, while salt-sensitive plants had a delayed response
(Petrusa and Winicov, 1997). In the case of mannitol, it has
been proposed that this solute may provide protection by
reducing oxidative damage in the chloroplast (Shen, Jensen
and Bohnert, 1997) since cytoplasmic mannitol concentrations were considered to be too low to provide sufficient
osmotic adjustment.
Late embryogenesis abundant (LEA) proteins are thought
to play a role in dessication tolerance in seed development
and in response to dehydration, salinity and cold stress
(Close, 1997). Rice plants transformed with the barley LEA
gene, HVA1, have shown increased tolerance to water
deficit and salt stress (Xu et al., 1996) while improved salt
and freezing tolerance has been observed in yeast transformed with a tomato LEA-class gene (Imai et al., 1996). At
present these proteins are thought to preserve the structural
integrity of the cell, but the extent of their utility in
improving salt tolerance needs to be further explored.
Oxidative stress has been considered to be a common
component of both biotic and abiotic stress in plants. Many
of the genes for antioxidant enzymes have been cloned and
expressed in transgenic plants to test their role in plant
protection against oxidative and other stresses (Foyer,
Descourvieres and Kunert, 1994 ; Allen, 1995). Transgenic
alfalfa overexpressing superoxide dismutase has been
reported to show reduced injury from water deficit stress
along with field performance (McKersie et al., 1996) and
oxidative stress tolerance was shown in Fe-superoxide
dismutase overproducing chloroplasts (Van Camp et al.,
1996). Increased tolerance to oxidative stress has also been
reported in tobacco plants that overexpress cytosolic
ascorbate peroxidase (APX) but not those that express a
chloroplast targeted isoform (Torsethaugen et al., 1997).
Overexpression of glutathione S-transferase\glutathione
peroxidase provided some protection against cold and salt
stress in tobacco (Roxas et al., 1997). The complexity of the
interacting pathways, cellular compartmentalization and
differential responses of antioxidant gene expression
(Conklin and Last, 1995) make it somewhat difficult to
compare results obtained from different systems at the
present time and will require further elucidation of the
extent of tolerance that can be obtained with single gene
transfer. In addition, most expression of the transferred
genes has been ubiquitous under the direction of the
CAMV 35 S promoter, which may overlook the necessary
tissue specificity for regulation in order to achieve optimal
protection.
MECHANISMS OF COORDINATE
REGULATION OF STRESS RELATED
GENES
Signalling pathways
Multigenic cellular adaptation to increases in the ionic
environment implies integrated changes in regulation of
gene expression for groups of functionally related genes.
The phenomenon of specific and coordinate mRNA
accumulation in response to salt and water stress has been
documented in a variety of plants (Bohnert, Nelson and
Jensen, 1995 ; Ingram and Bartels, 1996 ; Bray, 1997)
indicating that creating transgenic plants overexpressing a
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
gene encoding a single function may not be sufficient to lead
to optimal adaptation to saline environments. The signalling
pathways and molecular mechanisms responsible for this
coordinate transcript accumulation have been reviewed
recently (Bray, 1997 ; Shinozaki and Yamaguchi-Shinozaki,
1997). Although an increasing number of kinases and
phosphatases have been identified that respond to both salt
and drought stress (see Table 1), the pathways themselves
have remained generally unresolved. Since the level of the
plant hormone abscisic acid (ABA) increases with salt,
drought and cold stress, it has been postulated to play a
central role in signalling for these stress responses besides
playing an important role in seed production. Exogenous
ABA can activate transcription of many of the genes
induced by salt\drought stress, while other salt\drought
inducible genes are not activated by ABA, suggesting both
ABA-dependent and ABA-independent signalling pathways
(Bray, 1997 ; Shinozaki and Yamaguchi-Shinozaki, 1997).
Signal perception and transduction pathways certainly make
attractive targets for signal manipulation to coordinate gene
regulation, but it is currently difficult to design definitive
experiments in this area because of the cascade characteristics of signalling systems and the likely multiplicity of
intersecting signalling pathways (Ishitani et al., 1997).
Transcriptional regulators
Another attractive target category for manipulation and
coordinate gene regulation is the small group of transcription factors that have been identified to bind to
promoter regulatory elements in genes regulated by salt\
drought stress Shinozaki and Yamaguchi-Shinozaki, 1997 ;
Winicov and Bastola, 1997). As recently shown for the cold
acclimation response (Jaglo-Ottoson et al., 1998), overexpression of the transcription factor CBF1 that regulates
numerous COR genes was able to enhance demonstrable
freezing tolerance in Arabidopsis, while previous overexpression of just one of the COR genes gave much more
subtle results. This suggests that transcriptional activation
of salt\drought induced genes might be possible in transgenic plants overexpressing one or more transcription
factors that recognize promoter regulatory elements of these
genes. Many of the salt\drought stress regulated genes may
depend on a combinatorial activation by several transcription factors as suggested by the requirement of a
coupling element for stress regulation of the barley HVA22
gene containing the ABRE (PyACGTGGC) element (Shen
et al., 1996). It will be a challenge to identify those factors
that may be limiting in the overall response and to
manipulate their expression in a tissue targeted manner.
Information to date on transcriptional regulation in
response to salt\drought stress is relatively gene specific.
Much of the current information focuses on cis-acting
elements of the genome involved in ABA induced gene
expression and the trans-acting bZIP proteins that recognize
these elements (Bray, 1997 ; Shinozaki and YamaguchiShinozaki, 1997). The broader function of ABRE elements
in plant gene regulation has been recently investigated by
overexpression in tobacco of the EmBP-1 gene (ABRE
707
binding protein from wheat embryo) and its truncated form
containing the DNA binding and dimerization domains.
While overexpression of EmBP did not alter the plant
response to water stress, the truncated form acted as a
dominant negative inhibitor, revealing an important developmental function of this protein in vegetative tissues
(Eckardt, McHenry and Guiltinan, 1998). ABA-mediated
inducible expression of genes that are also induced by salt
and drought stress has been recently linked to several myband myc-related transcriptional activators, two classes of
transcription factors associated with cellular proliferation.
Atmyb2 from Arabidopsis (Urao et al., 1993) and cpm10 and
cpm7, which have been cloned from Cereterostigma
(Iturriaga et al., 1996), encode MYB type transcription
factors and are induced by dehydration stress. The rd22BP1
gene from Arabidopsis has been shown to encode a myc type
transcription factor (Abe et al., 1997) expressed in seeds, but
not induced under stress conditions. Both myc- and mybtranscriptional activators belong to multigene families and
the myc (CANNTG) and myb (PyAACPyN) recognition
sites can be found in many salt\drought stress activated
gene promoters, including rd22 (Abe et al., 1997) and the
alfalfa MsPRP2 promoter (Bastola, Pethe and Winicov
1998 a). Recent transient transactivation experiments with
Arabidopsis leaf protoplasts have shown that the Arabidopsis
MYC (rd22BP1 ) and MYB (ATMYB2 ) proteins function
as transcriptional activators in ABA and dehydration
inducible expression using a 67 bp region of the rd22 gene
promoter containing the myc and myb DNA recognition
elements (Abe et al., 1997). These results further show the
multiplicity of factors likely to be involved in salt\drought
stress regulation. The ABA-independent salt\drought inducible DRE element (TACCGACAT) was initially identified in Arabidopsis (Yamaguchi-Shinozaki and Shinozaki,
1994) and is recognized by the transcription factor CBF1
(Stockinger, Gilmour and Thomashow, 1997). While CBF1
overexpression has been shown to increase COR gene
transcription and provide increased cold tolerance (JagloOttosen et al., 1998), no data are currently available on the
salt\drought tolerance of these transgenic plants.
We have documented coordinate gene regulation in longterm acquired salt tolerance in alfalfa and rice (Winicov,
1991, 1996) and have focused on the role for a putative
transcriptional regulator Alfin1 in altered gene expression in
salt tolerant alfalfa (Winicov, 1993 ; Winicov and Bastola,
1997). Alfin1 cDNA encodes a novel member of zinc finger
family of proteins. It contains sequence information for one
Cys and one His\Cys zinc finger domain, and an acidic
%
$
region characteristic of DNA binding proteins that are
likely to interact with other proteins. Alfin1 appears to be a
unique or a low copy gene in the alfalfa genome and shows
conservation among diverse plants, such as rice and
Arabidopsis as demonstrated by southern analysis (Winicov
and Bastola, 1997). Alfin1 is expressed primarily in roots
and binds DNA in a sequence specific manner (Winicov and
Bastola, 1997 ; Bastola et al., 1998 a). Elements of this GC
rich binding sequence are found in promoter fragments of
the salt inducible gene MsPRP2 that is also primarily
expressed in roots (Winicov and Deutch, 1994 ; Deutch and
Winicov, 1995). We have transformed alfalfa with Alfin1
708
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
cDNA and obtained expression with the 35S CAMV
promoter in callus and regenerated plants. The regenerated
plants appear normal despite the ubiquitous expression of
Alfin1. Interestingly, we have found that Alfin1 overexpression in transgenic alfalfa leads to enhanced levels of
MsPRP2 transcript accumulation in callus and in roots
(Bastola et al., 1998 b), indicating that Alfin1 can act as
transcriptional regulator on endogenous genes when transformed into alfalfa. Particularly interesting is our finding
that Alfin1 overexpression can induce the MsPRP2 gene,
which is also induced by salt. These results suggest that
Alfin1 may be an important transcription factor involved in
gene regulation in our salt tolerant alfalfa and an excellent
target of gene manipulation for improved salt tolerance.
CONCLUSIONS
Salt tolerance is a complex trait in plants, but molecular and
genetic approaches are beginning to characterize the diverse
biochemical events that occur in response to salt stress. In
the short term, it will remain a challenge to manipulate the
essential protective mechanisms in plants and to utilize our
biochemical knowledge for optimal molecular engineering
of salt tolerance in plants. A major unresolved question is
the extent and importance of both short-term and long-term
stress responses for sustained tolerance and their effects on
agriculturally desirable traits in crop plants.
With the recognition that the enhanced expression of a
number of functionally related genes may be required for
optimal improvements in salt tolerance, molecular engineering has been expanded to include proposals for
multiple gene transfers to enhance salt tolerance (Bohnert
and Jensen, 1996). An equally promising approach to
manipulating many genes may emerge as we learn more
about the specificity of signalling pathways that turn on
transcription of related genes that counteract salt stress at
the cellular level. Redundancy of the intersecting signalling
pathways and communication between the different pathways, however, is likely to create difficulties in using this
information in a directed approach at improving salinity
tolerance in the near future. Transcriptional regulation is
another new area with potential for coordinate regulation of
genes relevant to tolerance, but will require identification of
factors limiting the sustained response so that their
expression may be manipulated in a tissue targeted manner.
Overall, we are likely to see continued significant progress in
our understanding and ability to modify salt tolerance by
molecular engineering using both model and crop plants
based on knowledge of how salinity affects plant biochemistry and physiology through gene expression.
A C K N O W L E D G E M E N TS
My thanks for the helpful comments of the reviewers and to
Dr M. J. Guiltinan for providing the EmBP results, in press.
This work was supported in part by a Hatch grant from
NAES, NSF EPSCoR WISE Program and NRICGP
9401235 grant to I.W.
LITERATURE CITED
Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D,
Shinozaki K. 1997. Role of Arabidopsis MYC and MYB homologs
in drought- and abscisic acid-regulated gene expression. The Plant
Cell 9 : 1859–1868.
Aeschbacher RA, Schiefelbein JW, Benfey PN. 1994. The genetic and
molecular basis of root development. Annual ReŠiew of Plant
Physiology and Plant Molecular Biology 45 : 25–45.
Allen RD. 1995. Dissection of oxidative stress tolerance using transgenic
plants. Plant Physiology 107 : 1049–1054.
Bastola DR, Pethe VV, Winicov I. 1988 a. Alfin1, a novel zinc-finger
protein in alfalfa roots that binds to promoter elements in the saltinducible MsPRP2 gene. Plant Molecular Biology 38 (in press).
Bastola DR, Pethe VV, Winicov I. 1998 b. Alfin1, a transcription factor
that regulates the salt inducible MsPRP2 gene in alfalfa roots.
Plant Biology ’98 798a.
Bohnert HJ, Jensen RG. 1996. Metabolic engineering for increased salt
tolerance – the next step. Australian Journal of Plant Physiology
23 : 661–667.
Bohnert HJ, Nelson DE, Jensen RG. 1995. Adaptations to environmental stresses. The Plant Cell 7 : 1099–1111.
Bray EA. 1997. Plant responses to water deficit. Trends in Plant Science
2 : 48–54.
Close T. 1997. Dehydrins : A commonality in the response of plants to
dehydration and low temperature. Physiologia Plantarum 100 :
291–296.
Conklin PL, Last RL. 1995. Differential accumulation of antioxidant
mRNAs in Arabidopsis thaliana exposed to ozone. Plant Physiology
109 : 203–212.
Creelman RA, Mason HS, Bensen RJ, Boyer JS, Mullet JE. 1990.
Water deficit and abscisic acid cause differential inhibition of
shoot versus root growth in soybean seedlings. Plant Physiology
92 : 205–214.
Deutch CE, Winicov I. 1995. Post-transcriptional regulation of a saltinducible alfalfa gene encoding a putative chimeric proline-rich
cell wall protein. Plant Molecular Biology 27 : 411–418.
Eckardt AN, McHenry L, Guiltinan MJ. 1998. Overexpression of
EmBP, a dominant negative inhibitor of G-box-dependent
transactivation, alters vegetative development in transgenic
tobacco. Plant Molecular Biology 38 (in press).
Flowers TJ, Yeo AR. 1981. Variability in the resistance of sodium
chloride salinity within rice (Oryza satiŠa L) varieties. The New
Phytologist 88 : 363–373.
Flowers TJ, Yeo AR. 1995. Breeding for salinity resistance in crop
plants : where next ? Australian Journal of Plant Physiology 22 :
875–884.
Flowers TJ, Garcia A, Koyarna M, Yeo AR. 1997. Breeding for salt
tolerance in crop plants—the role of molecular biology. Acta
Physiologiae Plantarum 19 : 427–433.
Foolad MR, Jones RA. 1993. Mapping salt-tolerance genes in tomato
(Lycopersicon esculentum) using trait-based marker analysis.
Theoretical Applied Genetics 87 : 184–192.
Foyer CH, Descourvieres P, Kunert KJ. 1994. Protection against
oxygen radicals : an important defence mechanism studied in
transgenic plants. Plant, Cell and EnŠironment 17 : 507–523.
Gulick PJ, Dvor) a! k J. 1992. Coordinate gene response to salt stress in
Lophopyrum elongatum. Plant Physiology 100 : 1384–1388.
Hirayama T, Ohto C, Mizoguchi T, Shinozaki K. 1995. A gene encoding
a phosphatidylinositol-specific phospholipase C is induced by
dehydration and salt stress in Arabidopsis thaliana. Proceedings of
the National Academy of Sciences of the USA 92 : 3903–3907.
Holappa LD, Walker-Simmons MK. 1995. The wheat abscisic acidresponsive protein kinase mRNA, PKABA1, is up-regulated by
dehydration, cold temperature, and osmotic stress. Plant Physiology 108 : 1203–1210.
Holmstrom K-O, Mantyla E, Welin B, Mandal A, Paiva ET, Tunnela
OE, Londesborough J. 1996. Drought tolerance in tobacco. Nature
379 : 683–684.
Hurkman WJ, Fornari CS, Tanaka CK. 1989. A comparison of the
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
effect of salt on polypeptides and translatable mRNAs in roots of
a salt-tolerant and a salt-sensitive cultivar of barley. Plant
Physiology 90 : 1444–1456.
Hwang I, Goodman HM. 1995. An Arabidopsis thaliana root-specific
kinase homolog is induced by dehydration, ABA, and NaCl. The
Plant Journal 8 : 37–43.
Imai R, Chang L, Ohta A, Bray E, Takagi M. 1996. A lea-class gene of
tomato confers salt and freezing tolerance when expressed in
Saccharomyces cereŠisiae. Gene 170 : 243–248.
Ingram J, Bartels D. 1996. The molecular basis of dehydration
tolerance in plants. Annual ReŠiew of Plant Physiology and Plant
Molecular Biology 47 : 377–403.
Ishitani M, Xiong L, Stevenson B, Zhu J-K. 1997. Genetic analysis of
osmotic and cold stress signal transduction in Arabidopsis :
interactions and convergence of abscisic acid-dependent and
abscisic acid-independent pathways. The Plant Cell 9 : 1935–1949.
Iturriaga G, Leyns L, Villegas A, Gharaibeh R, Salamini F, Bartels D.
1996. A family of novel myb-related genes from the resurrection
plant Craterostigma plantagineum are specifically expressed in
callus and roots in response to ABA or desiccation. Plant
Molecular Biology 32 : 707–716.
Iwasaki T, Yamaguchi-Shinozaki K, Shinozaki K. 1995. Identification
of a cis-regulatory region of a gene in Arabidopsis thaliana whose
induction by dehydration is mediated by abscisic acid and requires
protein synthesis. Molecular General Genetics 247 : 391–398.
Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O,
Thomashow MF. 1998. Arabidopsis CBF1 overexpression induces
COR genes and enhances freezing tolerance. Science 280 : 104–106.
Kearns EV, Assmann SM. 1993. The guard cell-environment connection.
Plant Physiology 102 : 711–715.
Kishor PBK, Hong Z, Miao G-H, Hu C-A, Verma SPS. 1995.
Overexpression of ∆"-pyrroline-5-carboxylate synthetase increases
proline production and confers osmotolerance in transgenic plants.
Plant Physiology 108 : 1387–1394.
KrishnaRaj S, SreeRangasamy SR. 1993. In Šitro salt tolerance screening
in long-term anther cultures of rice (Oryza satiŠa L.) variety IR 50.
Journal of Plant Physiology 142 : 754–758.
Kusano T, Berberich T, Harada M, Suzuki N, Sugawara K. 1995. A
maize DNA-binding factor with a bZIP motif is induced by low
temperature. Molecular General Genetics 248 : 507–517.
McCoy TJ. 1987. Characterization of alfalfa (Medicago satiŠa L.)
plants regenerated from selected NaCl tolerant cell lines. Plant
Cell Reports 6 : 417–422.
McKersie BD, Bowley SR, Harjanto E, Leprince O. 1996. Water-deficit
tolerance and field performance of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiology 111 : 1177–
1181.
Nakagawa H, Ohmiya K, Hattori T. 1996. A rice bZIP protein,
designated OSBZ8, is rapidly induced by abscisic acid. The Plant
Journal 9 : 217–227.
Niu X, Bressan RA, Hasegawa PM, Prado JM. 1995. Ion homeostasis
in NaCl stress environments. Plant Physiology 109 : 735–742.
Petrusa LM, Winicov I. 1997. Proline status in salt-tolerant and saltsensitive alfalfa cell lines and plants in response to NaCl. Plant
Physiology and Biochemistry 35 : 303–310.
Pilon-Smits EAH, Ebskamp MJM, Paul MJ, Jeuken MJW, Weisbeek
PJ, Smeekens SCM. 1995. Improved performance of transgenic
fructan-accumulating tobacco under drought stress. Plant Physiology 107 : 125–130.
Robinson NL, Tanaka CK, Hurkman WJ. 1990. Time dependent
changes in polypeptide and translatable mRNA levels caused by
NaCl in barley roots. Physiologia Plantarum 78 : 128–134.
Roxas VP, Smith RK, Allen ER, Allen RD. 1997. Overexpression of
glutathione S-transferase\glutathione peroxidase enhances the
growth of transgenic tobacco during stress. Nature Biotechnology
15 : 988–991.
Rubio F, Gassmann W, Schroeder JI. 1996. Sodium-driven potassium
uptake by the plant potassium transporter HKT1 and mutations
conferring salt tolerance. Science 270 : 1660–1663.
Saab IN, Sharp RE, Pritchard J, Voetberg GS. 1990. Increased
709
endogenous abscisic acid maintains primary root growth and
inhibits shoot growth of maize seedlings at low water potentials.
Plant Physiology 93 : 1329–1336.
Saneoka H, Nagasaka C, Hahn DT, Yang W-J, Premachandra GS, Joly
RJ, Rhodes D. 1995. Salt tolerance of glycinebetaine-deficient and
containing maize lines. Plant Physiology 107 : 631–638.
Shen B, Jensen RG, Bohnert HJ. 1997. Increased resistance to oxidative
stress in transgenic plants by targeting mannitol biosynthesis to
chloroplasts. Plant Physiology 113 : 1177–1183.
Shen Q, Zhang P, Ho T-HD. 1996. Modular nature of abscisic acid
(ABA) response complexes : composite promoter units that are
necessary and sufficient for ABA induction of gene expression in
barley. The Plant Cell 8 : 1107–1119.
Sheveleva E, Chmara W, Bohnert HJ, Jensen RG. 1997. Increased salt
and drought tolerance by D-ononitol production in transgenic
Nicotiana tabacum L. Plant Physiology 115 : 1211–1219.
Shinozaki K, Yamaguchi-Shinozaki K. 1996. Molecular responses to
drought and cold stress. Current Opinion in Biotechnology 7 :
161–167.
Shinozaki K, Yamaguchi-Shinozaki K. 1997. Gene expression and signal
transduction in water-stress response. Plant Physiology 115 :
327–334.
Smith MK, McComb JA. 1981. Use of callus cultures to detect NaCl
tolerance in cultivars of three species of pasture legumes. Australian
Journal of Plant Physiology 8 : 437–442.
Stockinger EJ, Gilmour SJ, Thomashow MF. 1997. Arabidopsis thaliana
CBF1 encodes an AP2 domain-containing transcriptional activator
that binds to the C-repeat\DRE, a cis-acting DNA regulatory
element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of
Sciences of the USA 94 : 1035–1040.
Tal M, Heikin H, Dehan K. 1978. Salt tolerance in the wild relative of
the cultivated tomato : responses of callus tissue of Lycopersicon
esculentum, L. peruŠianum and Solanum pennellii to high salinity.
Zeitschrift fuW r Pflanzenphysiologie 86 : 231–240.
Tanji KK. 1990. Agricultural salinity assessment and management. NY.
USA : Irrigation and Drainage Division, American Society of Civil
Engineers.
Tarczynski MC, Jensen RG, Bohnert HJ. 1993. Stress protection of
transgenic tobacco by production of the osmolyte mannitol.
Science 259 : 508–510.
Thomas JC, Sepahi M, Arendall B, Bohnert HJ. 1995. Enhancement of
seed germination in high salinity by engineering mannitol
expression in Arabidopsis thaliana. Plant Cell EnŠironment 18 :
801–806.
Torsethaugen G, Pitcher LH, Zilinskas BA, Pell EJ. 1997. Overproduction of ascorbate peroxidase in the tobacco chloroplast
does not provide protection against ozone. Plant Physiology 114 :
529–537.
Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. 1993. An
Arabidopsis myb homolog is induced by dehydration stress and its
gene product binds to the conserved MYB recognition sequence.
The Plant Cell 5 : 1529–1539.
Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, Hayashida
N, Shinozaki K. 1994. Two genes that encode Ca#+-dependent
protein kinases are induced by drought and high-salt stresses in
Arabidopsis thaliana. Molecular General Genetics 244 : 331–340.
Van Camp W, Capiau K, Van Montagu M, Inze D, Slooten L. 1996.
Enhancement of oxidative stress tolerance in transgenic tobacco
plants overproducing Fe-superoxide dismutase in chloroplasts.
Plant Physiology 112 : 1703–1714.
Wang H, Datla R, Georges F, Loewen M, Cutler AJ. 1995. Promoters
from kin1 and corb6.6, two homologous Arabidopsis thaliana
genes : transcriptional regulation and gene expression induced by
low temperature, ABA, osmoticum and dehydration. Plant
Molecular Biology 28 : 605–617.
Winicov I. 1991. Characterization of salt tolerant alfalfa (Medicago
satiŠa L.) plants regenerated from salt tolerant cell lines. Plant Cell
Reports 10 : 561–564.
Winicov I. 1993. cDNA encoding putative zinc finger motifs from salt-
710
WinicoŠ—New Molecular Approaches to ImproŠing Salt Tolerance in Crop Plants
tolerant alfalfa (Medicago satiŠa L.) cells. Plant Physiology 102 :
681–682.
Winicov I. 1994. Gene expression in relation to salt tolerance. In : Basra
AS, ed. Stress induced gene expression in plants. Switzerland :
Harwood Academic Publishers, 61–85.
Winicov I. 1996. Characterization of rice (Oryza satiŠa L.) plants
regenerated from salt-tolerant cell lines. Plant Science 113 :
105–111.
Winicov I, Bastola DR. 1997. Salt tolerance in crop plants : new
approaches through tissue culture and gene regulation. Acta
Physiologiae Plantarum 19 : 435–449.
Winicov I, Deutch CE. 1994. Characterization of a cDNA clone from
salt-tolerant alfalfa cells that identifies salt inducible root specific
transcripts. Journal of Plant Physiology 144 : 222–228.
Winicov I, Krishnan M. 1996. Transcriptional and post-transcriptional
activation of genes in salt-tolerant alfalfa cells. Planta 200 :
397–404.
Winicov I, Shirzadegan M. 1997. Tissue specific modulation of salt
inducible gene expression : callus versus whole plant response in
salt tolerant alfalfa. Physiologia Plantarum 100 : 314–319.
Winicov I, Waterborg JH, Harrington RE, McCoy TJ. 1989. Messenger
RNA induction in cellular salt tolerance of alfalfa (Medicago
satiŠa). Plant Cell Reports 8 : 6–11.
Wu S-J, Ding, L, Zhu J-K. 1996. SOS1, a genetic locus essential for
salt tolerance and potassium acquisition. The Plant Cell 8 :
617–627.
Xu D, Duan X, Wang B, Hong B, Ho T-HD, Wu R. 1996. Expression
of a late embryogenesis abundant protein gene, HCA, from barley
confers tolerance to water deficit and salt stress in transgenic rice.
Plant Physiology 110 : 249–257.
Yamaguchi-Shinozaki K, Shinozaki K. 1994. A novel cis-acting element
in an Arabidopsis gene is involved in responsiveness to drought,
low temperature, or high-salt stress. The Plant Cell 6 : 251–264.
Yano S, Ogawa M, Yamada Y. 1982. Plant formation from selected rice
cells resistant to salts. In : Fujiwara A, ed. Plant tissue culture.
Proceedings of the 5th International Congress of Plant Tissue and
Cell Culture, Tokyo, Japan, 495–496.
Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, YamaguchiShinozaki K, Wada K, Harada Y, Shinozaki K. 1995. Correlation
between the induction of a gene for ∆"-pyrroline-5-carboyxylate
synthetase and accumulation of proline in Arabidopsis thaliana
under osmotic stress. The Plant Journal 7 : 751–760.