CH 3 Molecular Mechanism Salt Tolerance

CHAPTER 3
Molecular Mechanisms of Salinity

Tolerance
Hans J. Bohnert, Hua Su and Bo Shen
P lants have evolved complex mechanisms allowing for adaptation to osmotic stress caused
by drought and to osmotic and ionic stress caused by high salinity. These mechanisms
can be classified into two categories: One includes developmental, morphological, and
physiological mechanisms; the other includes biochemical mechanisms. Developmental,
morphological, and physiological mechanisms are usually complex and require the functions
of many gene products. Examples of complex changes initiated by stress are the switch
from the C3 photosynthetic pathway to Crassulacean acid metabolism (CAM) in
Mesembryanthemum crystallinum following salt stress,1 the development of salt glands in
Limonium sp.,2 salt-storing epidermal bladder cells in Mesembryanthemum crystallinum3,4
and changes leading to increased water use efficiency in the development of the C4
photosynthetic pathway.5
Biochemical mechanisms, in contrast, are relatively simple, typically involving the
action of only a few gene products. For example, the accumulation of compatible solutes,
such as glycine betaine, proline, ectoine or polyols, only requires one to three enzymes for
extending a main metabolic pathway into the branch pathway of metabolite accumulation.6-9
Similarly, adjustments in ion uptake seem to be controlled by an equally small number of
gene products.10,11 With the current knowledge of plant genetics and biochemistry, the
genetic engineering of biochemical mechanisms is possible, but the engineering of more
complex traits is still beyond our capabilities. Once all relevant genes are known and
functionally characterized, it should be possible to manipulate complex developmental
mechanisms, such as flower development, vegetative growth or seed formation. This goal
is within reach for the genetic make-up of Arabidopsis thaliana at least,12 but the understanding
of how the 21,000 Arabidopsis genes function and their biochemical and physiological
interactions lies far in the future. In this review, we will focus mainly on biochemical
mechanisms that lead to cellular and whole-plant adaptations caused by the combined
osmotic and ionic disturbance of metabolism resulting from salt stress.
Over evolutionary time, plants colonized most places on earth, being excluded only
from high latitudes, the highest mountains and true deserts, which are cold and/or lack
water completely. Xerophytic and halophytic adaptations evolved in response to long-term
climate changes which allowed plants to tolerate all but the most extreme habitats. Plants
which have adapted to stressful environments provide paradigms for biochemical and
physiological tolerance mechanisms, and they provide genes for pathways that could
become incorporated into crop plants, which are typically stress-sensitive, having originated
from species in subtropical or tropical areas.13-15 Species adapted to extreme habitats are
not equally distributed among all orders or families of the angiosperm lineage. They
Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants, edited by Kazuo
Shinozaki and Kazuko Yamaguchi-Shinozaki. ©1999 R.G. Landes Company.
30 Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants
appear more frequently in orders which include few crop species but many species
restricted to stressful environments. These orders can be considered quarries for obtaining
novel genes for alternative biochemical pathways, and paradigms for understanding how
these pathways interact physiologically.
What constitutes tolerance or resistance to salinity stress has many facets, but is
surprisingly simple in principle. For both growth and development of reproductive
organs, plants must have water for photosynthesis to continue under stress; each one of
the many diverse mechanisms which evolved in an order-, family- or species-specific fashion
must be subordinate to this essential goal. We will review the molecular mechanisms for
which the evidence is clear:
1. Scavenging of radical oxygen species,
2. Controlled ion uptake,
3. The “burning” of accumulated reducing power, and
4. Adjustments in carbon/nitrogen allocation.
From the confusing multitude of physiological data, a few principles emerge (for
recent reviews, see refs. 11, 14-20, and other articles in this volume). Biophysical and
biochemical principles that govern stress and plant stress responses are outlined by Levitt.21
Osmolytes, Osmoprotectants, Compatible Solutes, Osmotic

Adjustment
High salinity disturbs uptake and conductance of water. Salt stress and other environ-
mental factors that affect water supply lead to changes in stomatal opening which can, if
stress persists, set in motion a chain of events originating from a decline in the leaf-internal
CO2 concentration, consecutively inhibiting the carbon reduction cycle, light reactions,
energy charge, and proton pumping.22 Other pathways are affected by increased shuttling of
carbon through the photorespiratory cycle.14,15 Eventually, carbon and nitrogen allocation and
storage require readjustment, reactions that lead to the consumption of reducing power
become favored, and development and growth may become altered. During the past years,
the complex interrelationship of biochemical pathways that change during salt stress has
become appreciated, although we are far from understanding this complexity.
The accumulation of metabolites, acting as osmolytes, in response to external changes
in osmolarity is probably universal.23-25 The generally accepted view is that osmolytes must
be compatible,26,27 not inhibiting normal metabolic reactions, and that their accumulation
leads to “osmotic adjustment” as the major element in accomplishing tolerance.6,7,24,28
Typically, compatible solutes are hydrophilic, giving rise to the view that they could
replace water at the surface of proteins, protein complexes, or membranes—we might call
them osmoprotectants in this case. The terms carry physiological meaning, but do not
explain the biochemical function(s) such solutes carry out. There may be more than one
function for a particular solute29,30 and, based on results from in vitro experiments,31-34
different compatible solutes seem to have different functions.
The main function of compatible solutes may be stabilization of proteins, protein
complexes or membranes under environmental stress. In in vitro experiments, compatible sol-
utes at high concentrations have been found to reduce the inhibitory effects of ions on
enzyme activity,24,35-37 to increase thermal stability of enzymes,38-40 and to prevent
dissociation of the oxygen-evolving complex of photosystem II.41 One argument often
raised against these studies is that the effective concentration necessary for protection in
vitro is very high, approximately 500 mM, concentrations which are usually not found in
vivo. However, when we consider the high concentration of proteins in cells, the concentration
necessary for protection can, we think, be much lower than that required for protection in
in vitro assays. In addition, it may not be the solute concentration in solution that is
Molecular Mechanisms of Salinity Tolerance 31
important. Glycine betaine (which may be present in high or low amounts), for example,
protects thylakoid membranes and plasma membranes against freezing damage or heat
destabilization,42-44 indicating that the local concentration on membranes or protein
surfaces may be more important than the absolute concentration.
Two theoretical models have been proposed explaining protective or stabilizing
effects of compatible solutes on protein structure and function. The first is termed the
“preferential exclusion model”45 which assumes the solutes are largely excluded from the
hydration shell of proteins. Exclusion leaves a water shell around proteins which stabilizes
protein structure, or promotes or maintains protein/protein interactions. In this model,
the solutes would not disturb the native hydration shell of proteins, but would interact
with the bulk water phase in the cytosol. The “preferential interaction model”, in contrast,
emphasizes interactions between solute and proteins.46 During water deficit, compatible
solutes may interact directly with hydrophobic domains of proteins and prevent their
destabilization, or they may substitute for water molecules in the vicinity of such regions.
While the two models seem to be mutually exclusive at first, the actual function may in fact
be explained by both models. The structures of different solutes could accommodate
hydrophobic, van-der-Waals interactions, as well as electrostatic interactions, but
additional biophysical studies will be necessary to gain a better insight into the stabilizing
effects documented by in vitro experiments.
Cellular Mechanisms of Salt Tolerance—the Fungal Model

Osmotic Adjustment
The unicellular eukaryotic Saccharomyces cerevisiae, baker’s yeast, is an ideal model
for studying cellular and molecular mechanisms of salt tolerance in higher plants. Its small
genome, which has been sequenced,47-49 adds to several other advantages. First, yeast is salt
tolerant and the cells have stress responses similar to halophytic plants. Yeast cells
accumulate compatible solutes, mainly glycerol and some trehalose, to counteract high
external osmolarity during salt stress;36,50-53 this is similar to the reactions of higher plants.
For example, halophytic plants, such as Plantago maritima and Mesembryanthemum
crystallinum, accumulate high concentrations of sorbitol and methylated inositols,
respectively, under salt stress.54,55 Second, both yeast and plants use proton gradients as
the driving force of secondary transport systems which control ion fluxes under stress.56, 57
Many ion flux mechanisms are highly conserved in yeasts and plants. In fact, a number of
plant membrane proteins, such as the potassium, amino acid, and sugar transporters, have
been isolated by functional complementation of yeast mutants.58-61 Third, the accumulation
of glycerol is essential for salt tolerance52,53 and glycerol-deficient mutants are available for
evaluating the functions of other sugar polyols in stress tolerance. Finally, yeast provides
an unsurpassed system for genetic analysis, transformation and functional characterization of
cell-specific functions, especially in light of the recent completion of the yeast genome
sequence.48,62
Yeast cells employ two main mechanisms for adaptation to salt stress: accumulation
of a polyol, glycerol, and maintenance of ion homeostasis. When exposed to NaCl the cells
experience both osmotic stress and ion toxicity. To respond to a low external osmotic
potential, the accumulating glycerol seemingly compensates for the difference between the
extra- and intra-cellular water potential.36 For reducing sodium toxicity, yeast cells have to
maintain low cytosolic Na+ concentrations and this is achieved by several mechanisms: by
restricting Na+ influx, rapidly extruding Na+ and/or efficiently compartmentalizing
sodium into vacuoles. The genetic evidence indicates both mechanisms are essential for
yeast salt tolerance.63-65
Glycerol Accumulation
Yeast cells accumulate glycerol as the major compatible solute when exposed to high
ion concentrations (Fig. 3.1).36 High osmolarity is perceived as a signal by two membrane
osmosensors: the protein products of Sln1 and Sho1. The signal is then transferred via a
MAP-kinase cascade66-68 and finally enhances the expression of the glycerol biosynthetic
pathway. Glycerol is synthesized from dihydroxyacetone phosphate. The first reaction is
catalyzed by glycerol-3-phosphate dehydrogenase which is encoded by two genes, GPD1
and GPD2. The second reaction converts glycerol-3-phosphate to glycerol by glycerol-3-
phosphatase, encoded by GPP1 and GPP2.52,53,69 The osmotic induction of both GPD genes
is mediated by the HOG-MAP kinase signaling pathway. In addition to induced glycerol
production, yeast cells may decrease membrane permeability to glycerol, which leads to an
increased retention of glycerol in the cells under osmotic stress. In fact, the salt-tolerant
yeast Zygosaccharomyces rouxii achieves glycerol accumulation by increased retention of
glycerol within the cell, and probably by active uptake of glycerol rather than by increased
production of glycerol during osmotic stress.36 In contrast, Saccharomyces cerevisiae
appears to increase glycerol production, while it fails to significantly alter membrane
permeability for glycerol retention during osmotic stress. To maintain high glycerol
concentrations in the cell requires a high energy cost, which seems to limit further
increases in salt tolerance in Saccharomyces cerevisiae. A glycerol transport protein (FPS1)
which shows homology with MIP-like water channel proteins has been recently isolated.
The expression of FPS is not regulated by the HOG-MAP kinase signaling pathway.70
Replacing Glycerol in Yeast

Although the correlation between accumulation of glycerol and yeast osmotolerance
has been established, and although the essential role of glycerol in the adaptation to
osmotic stress has been demonstrated by analysis of mutants deficient in glycerol
production,52,53,71 the mechanism(s) by which glycerol can confer such tolerance is not
clear. One obvious possibility is that glycerol is involved in osmotic adjustment to
maintain water flux into the cell. To test whether osmotolerance could be generated by the
presence of polyols other than glycerol, which would support the osmotic adjustment
concept, we introduced the coding regions of genes encoding enzymes for mannitol
and sorbitol production into a glycerol-deficient mutant (Fig. 3.1). However, accumulation of
either sorbitol or mannitol was not able to replace glycerol function (Shen B, Hohmann S,
Bohnert HJ, unpublished). Both foreign polyols accumulated to approximately the same
concentration as glycerol in wild type, and both were retained by the cell better than
glycerol, but both polyols provided only marginal protection. Growth inhibition of 50%
(I50) was 0.6 M NaCl for sorbitol/ mannitol producers in comparison to 0.4 M for the
mutant and 1.2 M for wild type. By reintroducing one of the deleted yeast GPD genes, a
significant increase in tolerance resulted, and the I50 increased from 0.4 M to 0.9 M
NaCl.72 If osmotic adjustment through glycerol is sufficient for salt adaptation, an equal
concentration of sorbitol or mannitol would be expected to confer very similar protection.
The results suggest that the concept underlying the term “osmotic adjustment” may not be
valid, or valid only if the synthesis of a metabolite for osmotic adjustment fullfills species-
specific requirements. The consequence of our results, then, is that glycerol might have specific
protective functions which mannitol and sorbitol cannot replace. Evolutionary adaptations
might have altered yeast proteins such that glycerol, but not other osmolytes, could exert a
protective role. Alternatively, the pathway through which an osmolyte is produced could be
more important than the end-product. Finally, the minimal protection by mannitol or sorbitol
could be caused by a difference in their intracellular distribution compared to glycerol.
Fig. 3.1. Genes involved in yeast osmotic stress signal transduction, and replacement of glycerol
synthesis by foreign osmolytes. The schematic drawing of a yeast cell includes the membrane
osmosensors (Sln1 and Sho1) which transmit signals to a MAP kinase cascade. Specific
transcription is initiated, which leads to the synthesis of several proteins, among them glycerol-
3-phosphate dehydrogenase (GPD) and glycerol phosphatase (GPP). This results in glycerol
synthesis and accumulation. The glycerol facilitator protein, FPS1, a MIP-type channel, is less
permeable under stress than under normal growth conditions. Replacement of both genes
encoding GPD by mannitol-6-P dehydrogenase or sorbitol-6-P dehydrogenase leads to the
accumulation of mannitol or sorbitol to a concentration approximately equal to glycerol
accumulation, but the two foreign polyols only marginally improve salt tolerance of the cells. 72
Whether and how these polyols are compartmentalized in yeast cells is not known, but
glycerol seems to be evenly distributed.73
A major difference exists between glycerol and mannitol/ sorbitol synthesis and
accumulation with respect to energy expenditure. Glycerol biosynthesis, which is also a
requirement for the removal of excess NADH during anaerobiosis,71 is more costly than
mannitol/ sorbitol generation. While NADH oxidation is, in principle, also accomplished
by the mannitol and sorbitol metabolic pathways, which leads to NAD+ increase, the cost
is different. During salt stress, more than 95% of the glycerol produced leaked from the
cells and accumulated in the medium. In contrast, sorbitol and mannitol did not significantly
exit from the cells. We calculated that glycerol biosynthesis under stress conditions
consumed at least 10 times more carbon and NADH than sorbitol/ mannitol biosynthesis.72
Thus, it may be that “burning” of excess reducing power via glycerol biosynthesis is as
important as the increasing osmotic potential provided by the steady-state glycerol
concentration in the cytosol.
Osmosensing and Signaling

The signaling pathways by which yeast cells respond to external osmolarity changes
has been identified (Fig. 3.1). High osmolarity is perceived as a signal by two membrane
osmosensors which are the protein products of Sln1 and Sho1. The Sln1 protein contains an
extracellular sensor domain, a cytoplasmic histidine kinase domain, and a receiver domain.
YPD and Ssk1 receive sensor signals in the cytosol. Sln1 and YPD/Ssk1 function like bacterial
two-component systems.67,74,75 Sho1 is a transmembrane protein containing a cytoplasmic
SH3 domain which can directly activate a MAP kinase kinase, Pbs2, by interaction between
its SH3 domain and a proline-rich motif of Pbs2.68 The signal from Sln1 is transmitted via a
MAP kinase cascade encoded by MAP kinase kinase kinase (Ssk2/22), MAP kinase kinase
(Pbs2), and MAP kinase (Hog1).66-68 The cascade initiates the expression of the glycerol
biosynthetic pathway including the GPD1 and GPP2 (phosphatase) genes.52,69 In addition
to glycerol biosynthesis, genes for other stress responses, such as CCT1 encoding catalase T76
and HSP12 encoding a small heat-shock protein,77,78 are induced by this signaling pathway.
In contrast to hyperosmotic stress, hypoosmotic stress initiates a second MAP kinase
cascade called the protein kinase C1 (PKC1) pathway. The MAP kinase in this PKC1
pathway is phosphorylated when cells are transferred from high osmolarity to low osmolarity.
Protein kinases downstream of PKC1 include BCK1/SLK1, MKK1/MKK2 and MPK1/SLT2.79
PKC1 mutants exhibited a lytic phenotype due to defects in cell wall biosynthesis. The
lytic phenotype can be suppressed by the addition of osmolytes like sorbitol into the
medium.80 How the two osmosensing pathways are coordinated remains to be determined.
Ion Relations
The yeast genome contains approximately 5,800 genes which potentially encode proteins.48
About 250 genes show significant similarity to membrane transport proteins characterized in
yeast and other organisms. Among those, a (partial) functional characterization existed
for only about 60 genes prior to the completion of the sequence, which amply documents
both the value of sequencing projects and our relative ignorance of membrane transport
processes in general.81 Many of these membrane transport proteins are involved in ion
transport and carry out essential functions in salt tolerance.
Potassium Transport
Potassium plays an important role in yeast salinity tolerance. The osmotic potential
generated by high internal potassium concentrations (e.g., in halobacteria) can alleviate
sodium toxicity.36 Three membrane proteins are involved in potassium transport across
the plasma membrane, TRK1, TRK2, and TOK1.82-84 The TRK proteins are involved in K+
influx and TOK1 controls K+ efflux. TRK1 and TRK2 are required for high affinity and
low affinity potassium uptake, respectively. Importantly, TRK proteins can also transport
Na+ but both have a higher affinity for K+. Under high external Na+ concentrations, Na+
can inhibit K+ uptake and enter the cell through the potassium channels. The capacity for
transporting potassium into cells and restricting sodium influx by increased
K+discrimination over Na+ is an essential element for salt tolerance acquisition.85-87
Because the high affinity K+ transport system shows a higher K+/Na+ discrimination than
the low affinity system, under salt stress yeast cells may shift from low to high affinity K+
uptake, allowing the cells to accumulate more K+ than Na+ and to maintain a low Na+/K+
ratio.85,88 Increased K+/Na+ discrimination of a high affinity potassium transporter (HKT1)
from wheat has been shown to increase salt tolerance of yeast strains deficient in potassium
uptake.86
Two halotolerance genes, HAL1 and HAL3, have been isolated by screening for genes
that enhance salt tolerance when overexpressed.89,90 Both have been implicated in the
regulation of K+ concentrations. Overexpression of HAL1 and HAL3 resulted in a stronger

accumulation of K+ under salt stress and increased salt tolerance. The beneficial effects are
specific for NaCl stress, and cannot moderate osmotic stress by sorbitol or excess KCl,
suggesting that high K+ in a physiological range may specifically alleviate sodium toxicity.
Yeast double mutants with deletions of the high affinity K+ transporter (TRK1) and the
Na+-ATPase (ENA1) genes are sensitive to Na+ because of poor Na+/K+ discrimination
and decreased Na+ efflux.85
Similarly, long-term salt-adapted tobacco cells showed increased capacity for K+
uptake compared to wild type cells,91 suggesting better Na+/K+ discrimination by the K+-
uptake system as a significant element for salt tolerance. Likely in the same category,
Arabidopsis sos1 mutants were hypersensitive to salt stress due to a defect in the high affinity K+
uptake system, highlighting the important role of K+ for salt tolerance in plants.92
H+-ATPases
Plasma membrane and vacuolar proton ATPases are essential for generating and
maintaining membrane proton gradients and for pH regulation in yeast and plants.93-96
They must be able to sense and respond to external acidification. The yeast plasma membrane
H+-ATPases (P-ATPase), encoded by the gene PMA1, is predominantly responsible for
proton gradient maintenance, while the product of the PMA2 gene is induced at low pH
when the PMA1 protein cannot function properly.97 Regulation of activity by calcium-
dependent protein kinases, in response to glucose levels, weak organic acids, heat shock
and salt stress, has been shown.98,99 Mutants hypersensitive to the immunosuppressants
cyclosporin A and FK506 were shown to be defective in assembly of the vacuolar H+-ATPase
(V-ATPase). Their characterization indicated involvement of the calcineurin signal
transduction pathway in synthesis, endomembrane transport, assembly and activity
regulation.98,100,101
Sodium Transport Across Membranes

Maintaining low intracellular sodium amounts during salt stress is essential for yeast.
Low sodium concentrations in the cytosol could be achieved by decreased Na+ uptake,
increased Na+ efflux, transport of Na+ into vacuoles or a combination of such activities. In
S. cerevisiae, a Na+-ATPase encoded by a family of 4 or 5 ENA genes has been shown to be
involved in sodium efflux. The expression of the ENA1 gene is induced by salt stress while
the other genes are expressed constitutively and weakly. Mutants defective in the ENA
function are sensitive to sodium and lithium.65,102,103 Also, an Na+/H+ antiport protein,
encoded by Nha1, was found during sequencing of the genome81 and later functionally
characterized.104 NHA1 seems to play a minor role in sodium efflux, but it may be important in
an environment of acidic external pH which would affect the transmembrane proton
gradient.
Based on the results with yeast it was surprising, however, that in Schizosaccharomyces
pombe and Zygosaccharomyces rouxii the major sodium efflux is via such a Na+/H+-
antiporter encoded by the SOD2 gene. SOD2 was initially identified by selection for
increased LiCl tolerance in fission yeast105 and the homologous SOD2 was isolated from
Z. rouxii.106 Functional expression of ENA1 in a sod2 mutant of S. pombe restored Na+
efflux and salt tolerance. Recently, the activity of the SOD2 Na+/H+ antiporter was con-
firmed using microphysiometry, indicating reversible sodium transport, dependent on the
Na+ and H+ gradient across the membrane.107 Based on this property, it should be possible
to utilize SOD2 to transport Na+ into vacuoles by targeting the protein to the tonoplast in
higher plants.
Although mechanisms of Na+ uptake in yeast are still not understood, mutant
analysis has clearly demonstrated an essential role for membrane-located processes.
Disruption of the LIS1/ERG6 gene, encoding a SAM-dependent methyltransferase of the
ergosterol pathway, resulted in increased sodium uptake and decreased salt tolerance. The
mutation seems to affect cation transport indirectly by changing membrane composition.108
Several other uncharacterized mutants showing high internal sodium were salt sensitive
despite normal glycerol accumulation.63,64 Halophytic plants usually sequester Na+ into
vacuoles to lower the concentration of Na+ in the cytoplasm.3 Whether such a mechanism
exists in yeast is unknown, but evidence exists for the vacuole’s important role in salt
tolerance. Mutants defective in vacuole morphology and vacuolar protein targeting are
salt-sensitive.109 A mutant in subunit C of the vacuolar ATPase shows increased sensitivity
to Na+ and Li+.85 The essential function may be associated with both compartmentation
of ions and osmoregulation.
Calcineurin Signaling
The signal transduction pathway regulating ion homeostasis remains unknown in
detail, but it is known to be different from the HOG pathway. Recent studies revealed that
calcineurin and protein phosphatase PPZ seem to be involved in the regulation of ion
fluxes.88,110-112 Calcineurin, a protein phosphatase 2B consisting of a catalytic subunit (CNA)
and a regulatory subunit (CNB), requires Ca2+ and calmodulin for activity.113 Null
mutants of calcineurin fail to recover from G1-arrest in the presence of α-pheromone, but
show normal growth rates under normal growth conditions. Under salt stress, however,
the mutants exhibited a salt-sensitive phenotype,88,110 caused by reduced expression of the
ENA1 gene which is regulated by calcineurin. Also, calcineurin mutants cannot shift from
low- to high-affinity potassium transport under salt stress.88 In contrast, deletions of genes
for the protein phosphatases PPZ1 and PPZ2 increased salt-tolerance due to enhanced
expression of ENA1, suggesting an essential role of these phosphatases in yeast ion
homeostasis.112 At low salt concentrations, the HOG-MAP kinase pathway appears to be
involved in regulation of ion fluxes, while at high salt concentrations ion balance is mainly
controlled by calcineurin.114 Interestingly, calcineurin signaling seems to interact with the
MAP kinase pathway. Disruption of the calcineurin gene (Ppb1) in fission yeast resulted in
sensitivity to chloride. High copy number of the Pmp1 gene, encoding a phosphatase,
suppresses this sensitivity to chloride. The PMP1 phosphatase dephosphorylates PMK1,
the third MAP kinase in fission yeast. As expected, deletion of Pmk1 also suppresses the
chloride sensitivity of calcineurin mutants. 115 Other components in the calcineurin
signaling pathway remain to be identified.
In addition to calcineurin and PPZ, HAL1 and HAL3 are involved in the regulation of
intracellular Na+ and K+ concentrations.90,116 The effect of HAL1 and HAL3 on intracellular
Na+ is mediated by expression of the ENA1 gene. Calcineurin plays a role in the induction
of ENA1 expression by sodium, while HAL1, HAL3 and PPZ determine the basal level of
ENA1. HAL1 and HAL3 are then required for the maximal expression of ENA1 under salt
stress. Overexpression of HAL1 or HAL3 partially suppressed the salt sensitivity of
mutants with a non-functional calcineurin. Increased K+ by overexpression of HAL1 is
independent of the action of TRK1 and TOK1, probably due to decreased export of K+
during salt stress.116 Clearly, multiple regulatory pathways and control circuits govern ion
responses in a complex interaction depending on external signals.
In higher plants, a calcineurin-like protein phosphatase activity has been found in
the regulation of the K+-channel in guard cells of fava bean.117 FK506 and cyclosporin
A, immunosuppressants which bind to cellular receptors, are strong and specific
inhibitors of calcineurin.118 When FK506-receptor complexes were added to guard cells,
the Ca 2+ -induced inactivation of K + channels was inhibited. A Ca 2+ -dependent

phosphatase activity which is sensitive to complexes of FK506 and its binding protein
and to cyclophilin-cyclosporin A was also identified in guard cells. 117 The gene which
encodes cyclophilin has been cloned,119 suggesting an important role of calcineurin in
higher plants. In addition, by complementation of yeast calcineurin mutants, two cDNAs
(STO and STZ) which suppress the calcineurin deficient phenotype in yeast have been
isolated from plants, but the predicted protein sequence did not show significant
homology to phosphatases. 120 Until now, the plant calcineurin gene has not been
identified. The important function of calcineurin in salt tolerance has recently been
demonstrated by overexpression of a truncated yeast calcineurin in transgenic tobacco.
Constitutive expression of this yeast calcineurin increased salt tolerance of transgenic
tobacco plants (Bressan RA, Pardo J, personal communication).121
Molecular Mechanisms of Salt Tolerance in Plants

Metabolite Accumulation
Accumulation of compatible solutes during osmotic stress is a ubiquitous biochemical
mechanism, present in all organisms from bacteria, fungi and algae to vascular plants and
animals.24,36 The accumulating metabolites include amino acids, their derivatives (proline,
glycine betaine, β-alanine betaine, proline betaine), tertiary amines, sulfonium compounds
(choline o-sulfate, dimethylsulfoniopropionate), the raffinose series of sugars, and polyols
(glycerol, mannitol, sorbitol, trehalose, fructans, and methylated inositols).6,14,15, 122,123
Enzymes from halophytes do not show remarkably higher salt resistance than those
from glycophytes, nor do they require sodium for optimal activities. In fact, the activity of
enzymes from both is generally strongly inhibited by high concentrations of either NaCl
or KCl.3 Although halophytes and glycophytes use similar compatible solute strategies to
deal with osmotic stress,124 they use different strategies to cope with ion toxicity. Halophytes
take up sodium and sequester ions into the vacuole. High osmotic potential in vacuoles is
balanced by accumulating compatible solutes in the cytoplasm. Because the cytoplasmic
volume is relatively small compared to the large volume of the vacuole, low concentrations
of compatible solutes suffice to reach the same osmotic potential in the cytoplasm. In
contrast, glycophytes usually attempt to limit sodium uptake or transport sodium to old
leaves as an alternative way to extrude sodium out of plants.3,125,126 The halobacteria deviate
from the general compatible solute strategy, accumulating K+ as the osmolyte rather than
organic solutes to counteract high external osmotic potential. Halobacterial enzymes
require high ion concentration for their optimal activity.36 This adaptation required changes
in protein structure. During evolution, this type of stress adaptation was abandoned,
possibly because it proved inflexible to changing environments, and mechanisms became
favored which utilized organic solutes, likely because they could be synthesized through
pathways attached to basic metabolism.
Several common features characterize the different compatible solutes. First, they can
easily be synthesized from compounds diverted from basic metabolism by novel
enzymatic or regulatory reactions. For example, glycine betaine in higher plants is
synthesized from choline via two reactions catalyzed by choline monooxygenase and
betaine aldehyde dehydrogenase,6 and pinitol is synthesized from myo-inositol in two
reactions catalyzed by inositol o-methyltransferase and ononitol epimerase.4,8,15 Both
choline and myo-inositol are high-flux metabolites and are tightly regulated during growth.
Second, accumulation of compatible solutes under osmotic stress is an active process, rather
than an incidental consequence of other stress-induced metabolic changes. The biosynthetic
pathway for a particular osmolyte is coordinately up-regulated during osmotic stress. For
Table 3.1 Transgenes with Effects on Salt-, Drought- and Low Temperature
Tolerance
ROS Scavenging Enzymes 1991 SOD, catalase, GST/GSX overexpression

leading to enhanced stress tolerance.20,181,232,233,236,238
Mannitol Synthesis 1992 Protection against salt stress.251,252,253
Fructan Accumulation 1995 Enhanced drought tolerance.254
Proline Accumulation 1995 Enhanced salt stress tolerance.135
Glycine betaine Synthesis 1997 Enhanced temperature stress, salt stress tolerance.255
LEA Protein Synthesis 1996 Salinity and drought stress protection.256
Potassium Transporter 1994 Enhanced Na+/K+-discrimination in yeast.86,207
Trehalose Synthesis 1996 Enhanced drought tolerance.141
Glutathione Cycle 1997 Altered redox control, salt and low temperature
Enhancement protection.192
Mannitol as a Hydroxyl 1997 Enhanced salt tolerance, mannitol synthesis in

Radical Scavenger chloroplast.29,30
Inducible Ononitol 1997 Enhanced drought and salt tolerance; inducibility

Accumulation based on changes in substrate amounts.138
Extreme Sorbitol 1998 High accumulation, >600 mM sorbitol, leading to

Accumulation necrotic lesions in sink leaves.227
example, two key genes, Inps1 and Imt1, are transcriptionally enhanced by salt stress, and
higher enzyme amounts lead to increased carbon flux through myo-inositol into pinitol
biosynthesis in stressed Mesembryanthemum.8,127-129 Genes involved in the degradation of
compatible solutes are down-regulated under osmotic stress. This is, for example, the case
for proline oxidase in Arabidopsis thaliana. Stress-dependent lower expression of this
enzyme, at least in part, may explain the increases in proline during salinity and drought
stress.130 Third, many accumulating compounds are end-products of a branch pathway
rather than active intermediates in so far as one enzyme in the pathway catalyzes only the
forward reaction. Examples for this point are DMSP synthesis in marine algae,9,131 pinitol
synthesis in Mesembryanthemum4,55 and glycinebetaine synthesis.6,132-134 Equally, proline
biosynthesis has received much attention, because proline accumulation is a nearly universal
reaction of plants to osmotic stress.135-137 Its true role in stress protection is, however, not
clear—we consider the accumulation of proline a consequence of the necessity for
readjusting carbon nitrogen balance under stress.138 The biosynthesis of ectoine (tetra-
hydropyrimidine and derivatives), an accumulating osmolyte in bacteria, has received
Fig. 3.2. Pathways for the synthesis of selected compatible solutes. Biochemical pathways originating
from glucose-6-P or sorbitol-6-P whose presence in some stress-tolerant species or after gene
transfer into transgenic tobacco is correlated with increased osmotic stress tolerance. Genes/
enzymes used in transgenic experiments are PGM (phosphoglucomutase), INPS (myo-inositol
1-P synthase), IMT (myo-inositol O-methyltransferase), GPDH (sorbitol-6-P dehydrogenase),
MtlDH (mannitol-1-P dehydrogenase), TPS (trehalosephosphate synthase). Pase indicates
unspecific phosphatases. OEP (ononitol epimerase) is found in Mesembryanthemum, but the
gene has not yet been cloned. IMP (myo-inositol monophosphatase) is not regulated in
Mesembryanthemum during stress and has not been included in transgenic plants.
attention recently.130,140 Expression of the three enzymes leading to ectoine in bacteria

confers significant salinity tolerance. Figure 3.2 shows schematically selected pathways that
lead to the synthesis of polyols (mannitol, sorbitol, ononitol and pinitol) and to trehalose
synthesis.141 Apart from the pinitol biosynthetic pathway,8,11 the pathways shown are
engineered pathways (Table 3.1) and may be different from pathways existing in some
plant species naturally. The scheme indicates clearly how the addition of a single gene can
be exploited for metabolic engineering.
Water Channels
Water channels, aquaporins (AQP), are found in all organisms as members of a
super-family of membrane proteins, 26-30 kDa in size, termed MIP (major intrinsic
protein).142,143 The proteins are characterized by six membrane-spanning domains and a
pore-domain with a characteristic sequence signature, NH3-NPAXT-COOH. Aquaporins
enhance membrane permeability to water in both directions depending on osmotic pressure
differences across a membrane, but other members of the gene family in yeast and vertebrates
encode glycerol-facilitators.143 Other MIPs, animal and plant—among them a nodulation-
specific protein, may mediate ion transport and transport of other neutral metabolites, such as
urea.144,145
Complexity of Plant Isoforms

In human DNA, five MIP genes have been characterized among a total of seven MIP-like
genes. They are expressed in different tissues, most highly in erythrocytes, kidney cells and
the brain. In contrast, Arabidopsis contains at least 23 MIP-like coding regions.146 Sequence
signatures of the Arabidopsis MIP indicate two large sub-families of 10 to 12 proteins each
whose members are either plasma membrane-located (PIP) or tonoplast-located (TIP),
and one MIP which diverges from the others has not been characterized.146 While some of
the genes might encode facilitators for diverse small metabolites or ions, eight MIP
proteins have already been identified as aquaporins. Why are there so many plant
aquaporins? We discuss four possibilities which might explain the high number.
1. MIP-intrinsic functional variations might allow AQP to be active at different
membrane osmotic potentials. Yet, all we know is that the intrinsic water permeability
distinguishes four human AQP and one glycerol facilitator by a factor of ~100,147 and
that plant AQP can be either sluggish or effective water transporters when
expressed in Xenopus oocytes.143 There is no report about a functional plant model
that would allow mechanistic studies on AQP. By antisensing with a plasma membrane
AQP coding region148 which supposedly targeted all expressed PIP, the decrease in
AQP amounts led to a decline in water uptake in plants. Such antisense AQP
transgenics increased the root to shoot ratio, suggesting a feedback mechanism
between water uptake and root mass (Kaldenhoff R, personal communication).
Protoplasts from the antisense-expressing plants did not burst as fast as wild type
cells when transferred to hypoosmotic solutions.148
2. Functional differences could have evolved for fine tuning water flux through the
plant—with high conductance AQP located in the root cortex and vascular tissues
which accommodate bulk fluxes and low conductance channels between
mesophyll cells, for example, or even within the cell cytosol and organelles and the
vacuole.
3. Without assuming functional diversification, the number of AQP arising through
gene duplications could have changed gene and protein expression, half-life, and
turnover such that AQP amount shows a gradient that follows the water transport
gradient. In this scenario, the gene number—requiring different promoters, RNA-
stability and translation characteristics and protein half-life regulation—would be
determined by the necessity of cell-specific differences in accommodating water
flux and not by the water transport function per se. This explanation is similar to
the following one, and both find precedence in the presence of, for example, a
large number of genes encoding plasma membrane H+-ATPases, AHA, which are
differentially expressed throughout the plant.95,149,150 Deletion of several AHA genes
did not produce a phenotype under normal growth conditions, but affected growth
significantly under diverse growth and stress conditions, low temperature, salt stress
and external high acidity, for example (Sussman MR, personal communication).
4. Last, AQP/MIP multiplications and diversifications could have been dictated by
the need for a flexible response to environmental changes in water supply or
evaporation, demanding the presence of several sets of AQP. This assumes evolution of
one set of AQP genes for stress responses and that this set is different from others.
It is conceivable that a set of Mip genes exists to take care of the business of cell
expansion following meristematic activity—and this function (missing from animals)
might require regulatory circuits different from those necessary in genes that perform
housekeeping (set 2) and stress-response functions (set 3). Although the data are
not complete with respect to AQP protein expression and cell-specificity, alignments
of sequences indicate that sub-families of two to four closely related sequences
exist146,151 which might represent the three sets of genes. MIP associated with cell
expansion,152 developmental specificity 153,154 and stress functions151,155-158,257 have
been described.
Mechanisms of Regulation
Most important to the topic here is how MIP gene expression, protein amount and
aquaporin activity are controlled during development and under environmental stress.
Regulation is by gene expression and protein amount, and possibly also by post-translational
modification—but we have very little information on mechanistic details in plants.
Weig et al146 used quantitative PCR amplification for the 23 Arabidopsis MIP and
found differences in mRNA amounts spanning several orders of magnitude. Differences
in RNA amounts for each MIP in roots, leaves, bolts and the flowers and siliques were
equally pronounced. No signals were detected for at least three MIP, suggesting that these
might be expressed under conditions not found during normal growth or that they are
expressed in a few cells only or at very low levels. The analysis of such a large gene family,
once all genes are known, can best be done by in situ hybridization, immunocytology with
specific antibodies and DNA microarray analysis through which the amount, location and
regulation of the genes during development and under different environmental conditions
can be monitored. For several MIP in a number of organisms, salt stress altered mRNA
amounts have been reported. AQP expression also responds to drought and low temperature,
hormone treatment (ABA, cytokinine, GA), light, and pathogen infection.143,157,158
Promoter studies have been performed with several MIP, but cell-specificity is
most likely the essential distinguishing factor between AQP and must receive more
attention in order to understand water transport in plants. The promoter for Rb7a159 from
tobacco conveys root-specificity, leads to differential expression in the root in a cell-
specific manner and is induced by nematode feeding.156,159 The Mesembryanthemum MipB
promoter showed highest expression of the gene in roots;151 after transfer into tobacco
and observation of GUS expression, broader specificity was observed, with highest expression
in all meristematic cells and in vascular tissues.160
Even less complete is the information about protein amount, localization and changes
during development and under stress conditions. One essential consideration is that the
large number of genes and high sequence identity among PIP and TIP, respectively,
require excellent controls for avoiding cross-hybridizations between transcripts and
immunological cross-reactivity between antisera. For example, generation of anti-peptide
antibodies against six Mesembryanthemum MIP resulted in distinguishable signals to
different cells.161 However, in the absence of probes for all MIP for this species, it cannot
be excluded that some of the antibodies react to more than one MIP whose sequence is not
yet known, but shares homology with the selected peptide domain.
Regulation has been documented at the level of post-translational modification, mostly
in animal systems. Salt stress conditions in kidney cells lead to changes in protein expres-
sion, which may be controlled by oligomerization, glycosylation, or phosphoryla-
tion.162,163 In addition, the presence in the cell membrane and the half-life of AQP is
determined by the hormone vasopressin in animal cells. Increased vasopressin leads to the
deposition of AQP from internal stores, endosome vesicles, to the outer membrane, and
lower hormone levels lead to cycling of membrane patches through endosomes.164 Clearly,
such traffic and its control would constitute the fastest, most economic way of regulating
water flux. Similar observations remain to be made with plant MIP, but patches of invaginated
plasma membrane regions, termed “plasmalemmasomes” that contain abundant AQP
protein have been found in plants,165 possibly the functional equivalent of animal
endosomes. Our preliminary experiments indicate that PIP from Mesembryanthemum
sediments in different gradient fractions depending on whether salt-stressed or unstressed

cells were used,161 which might indicate that similar membrane shuttle mechanisms exist
in plant cells.
Evidence for plant AQP regulation comes from studies which measured AQP phos-
phorylation.153,166 Regulatory sites for phosphorylation have been mapped in several
MIP/AQP. 143 Also, effects of pharmaceutical agents on water flow in Chara cells, for
example, point towards an association of water flux and the integrity of the cytoskeleton (see
ref. 143). Spinach leaf PIP are reversibly phosphorylated in response to the apoplastic
water potential and calcium.166
The discovery and preliminary characterization of AQP in plants has provided more
questions than answers. Their existence cannot be questioned and they act as water
channels. It is then intuitively obvious that control over their action should be important
under stress conditions. Although there are few data available, it is equally clear that regulation
during stress is complex, involving transcriptional and post-translational controls which
seem to involve synthesis, membrane traffic and reversible insertion into membranes,
complex assembly and MIP protein half-life.
Salt Stress and Radical Scavenging
Reactive Oxygen Species and Radical Scavenging Systems

Production of Reactive oxygen species (ROS) is an unavoidable process in photosynthetic
tissues, but ROS are also produced in mitochondria and cytosol. ROS including singlet
oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals react with and can damage
proteins, membrane lipids, and other cellular components.33,167,168 Some ROS also serve
as signaling molecules, 20 for example, in the initial recognition of attack by
fungal pathogens and the transmission of signals after a primary infection.169,170 Focusing on
chloroplasts, superoxide is abundantly produced from photoreduction of oxygen. Oxygen
concentration as high as 300 mM can be photoreduced to superoxide by photosystem I via
a Mehler reaction.171,172 The production of superoxide has been estimated to be approximately
30 mmol (mg chl)-1 h-1 in intact chloroplasts,173 and the rate of production in isolated
thylakoids was increased 1.5-fold by the addition of ferredoxin and decreased 50% by
addition of NADP+.174 Most of this thylakoid lumen-produced superoxide diffuses to the
stroma.173 H2O2 in chloroplasts is predominantly generated by disproportionation of
superoxide by SODs. In peroxisomes, H2O2 originates directly from glycollate oxidase
activity. Hydroxyl radicals derive from an interaction between hydrogen peroxide and
superoxide or directly from hydrogen peroxide in the presence of transition metals such as
Fe+2 and Cu+ by a Fenton- or Haber-Weiss-reaction. The oxidized metal ions can be
re-reduced by superoxide, glutathione, or ascorbate. Trace amounts, lower than the amount
present in chloroplasts, of metal ions are needed to catalyze the Fenton reaction.168,173 It
has in fact been shown that elevated amounts of iron lead to increased oxidative stress.175
These Reactive oxygen species are scavenged by resident enzyme systems and
nonenzymatic antioxidants. 176 Non-enzymatic detoxification mechanisms include
morphological features such as waxy surfaces and leaf or chloroplast movement, non-
photochemical quenching processes by various compounds, for example, the violaxanthin-
zeaxanthin cycle, and photorespiration. Non-enzymatic antioxidants include flavonones,
anthocyanins, α-tocopherol, ascorbate (at a concentration of ~10 mM in chloroplasts),
glutathione, carotenoids, phenolics and polyols.20,32,168,177 Botanical sources of such
antioxidants not only play important roles in plant stress adaptation, but also retard aging
and diseases related to oxidative damage in animals.178
The enzyme systems involved include SODs which catalyze the reaction from superoxide
to hydrogen peroxide, and ascorbate peroxidases (APX) responsible for the conversion of
hydrogen peroxide to water. Both SOD and APX are represented by isoforms localized to
the stroma and the thylakoid membrane. Ascorbate can be regenerated by the ascorbate-
glutathione cycle. The level of reduced glutathione is maintained by glutathione reductase
using NADPH.168,179,180 In addition, catalase has recently been demonstrated as a sink for
H2O2 in C3 plants.181 In contrast to the detoxification systems for H2O2 and O2-, an
enzyme system that could deal with the short-lived, extremely toxic hydroxyl radical has
not been identified and, in fact, might not have evolved.167,168,179,180 The best way of
detoxifying hydroxyl radicals is to prevent their formation by reducing the concentration
of H2O2 and free metal ions. Once produced, however, protection depends on the presence
of antioxidants in the vicinity of the formation site. Together these systems provide sufficient
protection under normal growth conditions; in fact, the scavenging systems are able to
handle moderate increases of ROS, unless long-term stress exceeds the detoxification
capacity.20,179,182 In chloroplasts, oxidative damage includes first a decline in CO2 fixation,
and then inhibition of photochemical apparatus, loss of pigments, oxidation of proteins,
and lipid peroxidation.183,184
ROS and Environmental Stress

Several lines of evidence support the toxicity of ROS during drought,20 chilling stress184
and salt stress.29,30 First, superoxide production is enhanced, as detected by EPR signals in
drought stressed wheat and sunflower.185,186 Equally, H2O2 content increased about three-fold
during drought and low temperature.187-189 Enhanced production of ROS resulted in an
increase in lipid peroxidation, as documented by a more than 5-fold increase of
malonaldehyde production in wheat.190 Second, the concentration of free transition iron
increased under drought stress,190,191 which stimulated production of hydroxyl radicals in
the presence of high concentrations of H 2O 2 via a Fenton reaction. Compared to
superoxide and H2O2, hydroxyl radicals oxidize a variety of molecules at near diffusion-
controlled rates. Finally, levels of non-enzymatic radical scavengers, such as ascorbate,
carotenoids, flavonoids, sugar polyols, and proline,183 increase and may complement
enzyme protection systems.
Excellent evidence for a protective effect of ROS scavenging systems has recently been
provided by the overexpression of an enzyme with the combined activities of glutathione
S-transferase, GST, and glutathione peroxidase, GPX.192 By doubling the GST/GSX activity in
transgenic tobacco, the seedlings and plants showed significantly faster growth than wild
type during chilling and salt stress episodes. The increased enzyme activities resulted in
higher amounts of oxidized glutathione (GSSG) in the stressed plants, indicating that the
oxidized form could provide an increased sink for reducing power.
Another set of experiments shed light on the relationships between ROS and the
accumulation of polyols. When a bacterial gene (mtlD) encoding mannitol-1-phosphate
dehydrogenase was modified so that the enzyme was expressed in chloroplasts, transgenic
tobacco contained approximately 100 mM mannitol in the plastids. Using transgenic plants,
freshly prepared cells and a thylakoid in vitro system, the protective effect exerted by
mannitol on photosynthesis characteristics could be shown.29, 30 The presence of mannitol
resulted in increased resistance to oxidative stress generated by methylviologen, and cells
exhibited significantly higher CO2 fixation rates than controls during stress. After
impregnation of tissue and cells with dimethyl sulfoxide, a hydroxyl radical generator,
mannitol-containing cells showed a lower rate of methane sulfinic acid production than
wild type, indicating that mannitol acted specifically as a hydroxyl radical scavenger. It
could be shown that the primary damage was to enzymes of the Calvin cycle and not to
components of the light harvesting and electron transfer systems,30 a confirmation of

earlier reports.22 At present, the interpretation which we favor is that mannitol interferes
with either hydroxyl radical production or damage, but it is unknown whether the
protective mechanism is by exclusion of hydroxyl radicals from protein surfaces, a chemical
interaction between mannitol and hydroxyl radicals, or by inhibiting or reducing the
amount of hydroxyl radicals produced in the Fenton reaction.
Plant Ion Uptake and Compartmentation
H+-ATPases and Vacuolar Pyrophosphatase

Plasma membrane and vacuolar proton transporters play essential roles in plant
salinity stress tolerance by maintaining the transmembrane proton gradient that assures
control over ion fluxes and pH regulation (Fig. 3.3).101,193 Three proteins/protein complexes
exist for this purpose: the plasma membrane (H+)-ATPase (P-ATPase) and two vacuolar
transport systems, a (H+)-ATPase (V-ATPase) and a pyrophosphatase (PPiase).
The plant P-ATPase is represented by a gene family of more than 10, encoding
proteins of ~100 kDa, with homology to the yeast PMAs.95,150 As the main proton pump
in the outer cell membrane it is essential for many physiological functions.194 Increased
activity of the proton pump has been shown to accompany salt stress. Halophytic plants
have been shown to increase pump activity under salt stress conditions more drastically
than glycophytes,56,195 but little is know about the regulatory circuits that lead to either
increased protein amount or activity during salt stress.
The V-ATPase, a multi-subunit complex homologous to organellar, yeast (VMA) and
bacterial F0F1-ATPases, has already been shown to be important in plant salinity tolerance.
Electrophysiological studies revealed increased activity of this ATPase when cells or tissues
from stressed plants were analyzed.196,197 Transcripts for several subunits of the V-ATPase
are upregulated following salt shock.198,199 In Mesembryanthemum, V-ATPase activity
increases several-fold following stress.200,201 In a Mesembryanthemum cell culture model it
has now been shown, based on immunological data, that the V-ATPase (and possibly the
P-ATPase) activity does not increase due to more protein being present, but an unknown
mechanism stimulates activity 2 to 3-fold.201 The response is specific for NaCl and could
not be elicited by mannitol-induced osmotic stress.
PPiase genes and tonoplast-located PPiase proteins have been characterized in de-
tail.94 Contrary to previous assumptions, the enzyme has now been authenticated as
also residing in the plasma membrane.202 Its function, if any, under salt stress conditions
is little known. A few reports have indicated that PPase activity declines under salt stress in
some species,203,204 but increases in others.205
Potassium Transporters and Channels

One possible passage for sodium across the plasma membrane is through transport
systems for other monovalent cations. Among those the most significant is the uptake
system for potassium, the most abundant cation in the cytosol, with important roles in
plant nutrition, development and physiological regulation. Many studies have focused on
identifying components involved in K+-transport. Physiological observations indicating a
biphasic uptake of K+ into roots206 gave rise to the assumption that two uptake entities
should be involved, a high-affinity system functioning at µM concentrations of external
K+ and a low-affinity system active in the mM range of potassium. Several plant K+
transporter and K+ channel genes have been isolated by functional complementation of
yeast mutants deficient in K+ uptake58,59,207 or by sequence homology with known K+
transporter or channel genes. 208-210 Electrophysiological studies in heterologous
Fig. 3.3. Transport proteins implicated in plant salinity stress tolerance. The schematic depiction
of a plant cell includes the vacuole, chloroplast (cp), mitochondrion (mt) and cell wall (shaded).
Transmembrane proton gradients established by proton-ATPases and pyrophosphatase are
indicated (+/-). Under NaCl stress, Na+ and Cl- are sequestered to the vacuole, and K+ and
osmolytes are present in high concentrations in the cytosol. Symbols for several membrane-located
transporters and channels are identified by the ion or proton transported and by the direction of
movement. For organelles (mt and cp) no transporters have been characterized through
molecular techniques. A Na+-ATPase, included in the plasma membrane is hypothetical, and a
Na+/H+-antiporter in the plasma membrane has not been detected.
expression systems, such as Xenopus oocytes or yeast cells, indicated that some of them
may function at both affinity ranges.211
Inward-rectifying potassium channels function in the mM range, following the
electrochemical gradient at the plasma membrane and are categorized as low-affinity
systems. 212,231 The AKT1- and KAT1-types of plant channels, similar to the Shaker
channels in animals, contain a pore-forming region conferring ion selectivity. In contrast
to earlier assumptions, these channels are highly selective against Na+,213 and evidence is
lacking for specific regulation under salt stress. We think that the potassium channels play
a minor role in salinity tolerance.
In contrast, K+ transporters which operate at low external potassium may mediate
entry of sodium in saline soil. A high-affinity K+-transporter is known from yeast.214 Some
of the cloned transporters take up potassium with dual-affinity.209,211 A high-affinity K+
transporter from wheat, HKT1, was indicated as a K+/Na+ symporter86 with high-affinity
binding sites for both K+ and Na+. Point mutations, which increased K+ selectivity over
Na+, in one of the 12 transmembrane domains of HKT1 conferred increased salt tolerance
of yeast. Another line of evidence for the involvement of high-affinity K+ uptake system in
salt tolerance came from the study of salt-sensitive mutants. The sos1 mutant of Arabidopsis
thaliana was characterized as hypersensitive to Na+ and Li+ and was unable to grow on low
potassium.92 86Rb uptake experiments showed that sos1 was defective in high-affinity
potassium uptake, and it became deficient in potassium when treated with NaCl. Interestingly
but not surprisingly, expression of the wheat Hkt1 in sos1 mutant plants alleviated the salt-
sensitive phenotype (Schroeder JI, Zhu J-K, personal communication). Further support is
provided by the expression characteristics of a rice homolog of wheat HKT1 in two
varieties that are distinguished by their salinity tolerance. The tolerant variety decreased
expression of the root-specific HKT1 and efficiently excludes sodium, while a salt-sensitive
variety maintained high expression of the HKT1 in the presence of high NaCl.210,215
Irrespective of the indices pointing to the involvement of HKT1-type transporters, or
high-affinity potassium uptake systems in general, in salt tolerance, there are other equally
likely scenarios. First, the presence of sodium is known to interfere with potassium uptake,
as shown for several of the cloned transport proteins, and protective effects exerted by
increased potassium might be based on the nutritional value, and not on a sodium
exclusion mechanism. High sodium sensitivity, as for example shown by the sos1 mutant,
might be due to growth interference when K+ uptake is reduced by the presence of
sodium. In this respect, the improved selectivity of K+ transport systems may increase salt
tolerance, while it is not involved in Na + detoxification or osmotic adjustment. Other
transport systems, finally, might act in sodium uptake. How, for example, the calcium-
regulated outward-rectifying K+-channel KCO1,216 or the regulation of other channels
and transporters, react under sodium stress conditions is unknown. It has been suggested
that sodium might enter through outward-rectifying cation channels.217 Among the many
possibilities, evidence for significant sodium currents through a calcium transporter, LCT1,
exists,218 and hexose and amino acid transporters may also let sodium pass.
Sodium Transport Systems

How sodium enters plant cells, how it enters the plant circulatory system to be
selectively transported over long distances, and how it is partitioned to the vacuole is not
known in detail. Most information is available for the last step in this series: sodium
transport from cytosol to vacuole is accomplished by a sodium/proton antiporter. A
protein of approximately 170 kD219 is a candidate for this tonoplast-located antiporter
based on immunological studies and inhibition of the ameloride-regulated antiport
activity in the presence of the antibody. It will be important to characterize the protein in
detail and to obtain the gene(s), because, when judged by protein size, the putative antiporter
seems to be different from the proteins in bacteria, yeast and vertebrate organisms.
Increased sodium/proton antiport activity during salt stress has been measured in several
model systems, tissues, cells and isolated vacuoles.96,220,221 The increase parallels an
increase in the V-ATPase activity.96,200
Our own data indicate that yet another pathway for sodium uptake may exist. When
analyzing the induction of myo-inositol synthesis in Mesembryanthemum, a surprising
decline of the rate-limiting INPS (myo-inositol-1-phosphate synthase) enzyme in roots
was observed, but the concentration of myo-inositol remained constant in the roots.128,129
This is due to drastically enhanced transport of myo-inositol from the leaves through the
phloem. In addition, myo-inositol is recycled to the leaves through the xylem and the
myo-inositol amount in xylem vessels is correlated with sodium amounts.129 We have cloned
a transcript with homology to vertebrate sodium/myo-inositol and yeast proton/myo-inositol
symporters222 and characterized its activity by complementation of a yeast mutant
defective in myo-inositol uptake.223 It seems possible that such a symporter is responsible
for the excretion of sodium into the xylem, but it is equally possible that sodium/myo-
inositol symport internalizes sodium from the apoplast of the root. The detection of such a
symport mechanism is particularly attractive, considering that the passage of myo-inositol
through the plant circulatory system connects photosynthesis competence with sodium
uptake and transport to mesophyll cells of the leaf.
The Essentiality of Calcium

Increasing calcium improves salinity tolerance of crop plants. Physiological
experiments indicated that the effect is mediated through an increase of intracellular calcium,
changes in vacuolar pH and activation of the vacuolar Na+/H+-antiporter.224, 225 The strict
control over calcium concentrations in the cytosol and calcium storage in a number of
locations (vacuole, mitochondria, endoplasmic reticulum) assign a crucial role to calcium
in plant salinity stress responses.
Recently, an Arabidopsis mutant, sos3, with hypersensitivity to NaCl has been
characterized. The mutant is different from other salt-sensitive mutants92 in that the
phenotype can be masked by the external addition of calcium.226 This phenotype represents
the first mutant with an altered response to calcium in higher plants. The phenotype
reveals the link between calcium and salinity stress tolerance, although the mechanism
through which hypersensitivity and remediation by calcium are connected is not known.
One attractive hypothesis is that a signaling system that responds to calcium spikes at low
calcium concentrations—for example a homolog of the yeast calcineurin-type system—is
defective, and that at higher calcium concentrations a second sensing system can support
the signal and elicit stress defense responses (Zhu JK, personal communication).
Metabolic Engineering of Glycophytic Plants for Increased

Salt Tolerance
In increasing numbers, experiments are reported using transgenic plants for testing
concepts originating from the correlative evidence of physiological analyses. Table 3.1
summarizes some of these reports. The concepts tested target four aspects of tolerance
acquisition:
1. ROS scavenging,
2. Compatible solutes and osmotic adjustment—carbohydrate biosynthesis and
synthesis of charged molecules,
3. Ion balance—potassium uptake vs. sodium uptake, and
4. The synthesis of specific, putatively protective proteins.
A note of caution must be added with respect to the over-expression and accumulation
strategies that have been followed up to now. Too high an accumulation of metabolites, or
too efficient scavenging of H2O2, for example, may not be desirable. When analyzing
transgenic tobacco plants that accumulated sorbitol to extremely high concentrations in
the cytosol, we observed stunted growth and the formation of necrotic lesions that reduced
biomass production, although the plants showed increased salinity and salt stress tolerance.227
The importance of radical oxygen scavenging for preventing oxidative stress in plants
has been demonstrated by genetic engineering of several enzymes into transgenic
plants.179,180,228 Overexpression of superoxide dismutase (Cu/Zn-SOD and Mn-SOD),
ascorbate peroxidase, catalase and glutathione reductase in transgenic plants has already
been shown to lead to increased resistance to oxidative stress.181,229,230,232-238 The most
dramatic protective effect, up until now, was observed after enhancement of the glutathione
cycle.192 In contrast, overexpression of an Fe-SOD in transgenic tobacco neither enhanced
tolerance to chilling-induced photoinhibition in leaf discs nor increased tolerance to salt
stress in whole plants,240 suggesting that isoforms of SOD may have different roles.
Noctor and Foyer20 provided a lucid assessment of the relatively marginal protection
that has been observed in many transgenic plant studies, whether with respect to ROS
scavenging or otherwise. It would certainly be premature to consider the protection
provided by the overexpression of SOD, ASX, or enzymes of the ascorbate/glutathione

cycle as the final word. Protection has typically been observed in strictly controlled
environments, and protective effects have often been marginal. We would like to provide
one consideration as to why this is to be expected. In the case of ASX, at least six different
isoforms exist which are located in mitochondria, in chloroplasts (several, in different
sub-compartments/membranes), soluble in the cytosol, and in the cytoplasmic
endomembrane system.241 A similarly complex distribution has been seen for SOD isoforms
which are found in the cytosol (Cu/Zn-SOD), mitochondria (Mn-SOD) and plastids
(Fe-SOD and Cu/Zn-SOD). Transgenic modifications of single enzymes are likely to have
a minimal effect because of the multitude of compartments that require protection.
Irrespectively, these experiments have clearly shown that—in practically every study—
the engineered expressed transgene elicited some protection. It is now necessary to adopt
multi-gene transfer strategies that alter several components of the stress tolerance system:
1. Targeting, for example, ROS scavenging enzymes to several compartments;
2. Assembling gene constructs that target sodium exclusion and enhanced potassium
uptake;
3. Generating transgenomes in which different pathways are satisfied, for example,
ion homeostasis, carbon allocation, and protein protection simultaneously;
4. Generating transgenomes with strategies that take into account cell-, tissue-,
organ- and developmental specificity.
The last point is particularly important, because little attention has typically been
paid to the “when,” “where,” and “how much” of transgene expression in the presently
concluded transgenic experiments. Significantly more attention needs to be directed to
the promoter elements that drive transgenes. Most attempts have targeted the metabolic
engineering of carbon and nitrogen allocation: ectopic enzyme expression leading to the
synthesis of uncharged carbohydrates—mannitol, sorbitol, trehalose, fructan, and
ononitol—and to glycinebetaine and proline accumulation (Table 3.1). The underlying
mechanism is becoming apparent for some of these strategies, e.g., in the hydroxyl radical
scavenging function of mannitol. 29,30 The mechanisms of protection underlying the
synthesis or presence of chaperones or specific LEA proteins remain to be determined.
Within a very short time, all genes that are essential for the salt tolerance phenotype
shown by some species and all genes that support damage avoidance in sensitive species
will be available. The task remaining, however, is understanding in which metabolic and
signaling pathways the gene products function and in which developmental context stress
protection is necessary. This task will require new approaches. We consider two approaches:
1. Multi-gene transfer into model species—yeast, Arabidopsis, tobacco and rice are
our suggestions; and
2. A focus on metabolic control analysis.
The first strategy utilizes the transfer of all genes, controlled by appropriate promoter
elements, for one or several biochemical pathways to generate protection which can be ana-
lyzed. Through the second approach, a biochemical description of flux in a multitude of
pathways, we will be able to gauge the cost of enzymes/pathways that enhance tolerance in
comparison to the cost and benefits of resident pathways.
Perspectives
High salinity is a major factor responsible for the loss of crop biomass.242 Salinity
caused by irrigation affects many productive agricultural areas. The degeneration of still
productive soils will become a more severe problem in the future. Development of
drought- and salt-tolerant crops has been a major objective of plant breeding programs
for decades in order to maintain crop productivity in semiarid and saline lands. Although
several salt-tolerant varieties have been released, the overall progress of traditional
breeding has been slow and has not been successful.13 The lack of success is mainly due to
the quantitative trait character of salinity tolerance which has to be reconciled with
another multigenic trait, high productivity, which is the ultimate goal of any breeding
program. Marginal progress has equally been grounded in our poor understanding of the
mechanisms of salt tolerance, while the collected body of physiological data has focused
our attention more on details in a large variety of species and less on the principles.
This has changed over the last few years. Biochemical pathways that lead to the
production of compatible solutes such as proline, glycine betaine, DMSP, or pinitol have
been studied and most of the pathway genes have been characterized.6,7,9,14,15 We have the
first glimpses of how the resulting metabolites from such pathways function in protection.
Similarly, the principles of how radical oxygen species act and the principles, genes and
proteins which deter radical damage have emerged. Membrane channels, transporters and
pores are now available through which cells exert control over ion, carbohydrate, amino
acid or water fluxes.58,59,61,146,207,243 We owe most of this recent progress to the power of
the yeast and Arabidopsis thaliana molecular genetic systems. Finding the genes whose
disruptions generate the various mutant phenotypes becomes rapidly easier as additional
mapping data and genomic DNA sequences from Arabidopsis are made available.12
Finally, plant stress perception, and inter- and intracellular signaling of salt stress has
been advanced greatly. Mutants in signal transduction pathways and components of
several signal transduction pathways have been found and are being characterized at
present.244-249 Future studies can follow the blueprint of signaling components isolated
from yeast 10,66,68,88,250 for finding and characterizing homologs of the essential
signaling intermediates in plants. If we accept that a major objective of plant stress
research is application, transgenic crops can be engineered not only for expression of novel
biochemical characters, but also for stress signal transduction that enhances the stress
response inherent to all plants.
Acknowledgments
Because of space constraints a number of references could not be included, and we
apologize. We thank Ms. Pat Adams for help with the manuscript. Different projects have,
off and on, been supported by the US National Science Foundation (Integrative Plant
Biology and International Programs), Department of Energy (Biological Energy), and
Department of Agriculture (NRI). Additional support has been provided by the Arizona
Agricultural Experiment Station, Japan Tobacco Inc., Rockefeller Foundation (New York)
and New Energy Development Organization (Tokyo).
References
1. Cushman JC, Bohnert HJ. Molecular biology of CAM. Plant Physiol 1997; 113:667-676.
2. Shachar-Hill Y. Osmotic coupling and chloride transport in the limonium salt gland.
Proc Roy Soc London-Ser. B: Biol Sci 1990; 241:159-163.
3. Flowers TJ, Troke PF, Yeo AR. The mechanism of salt tolerance in halophytes. Annu Rev
Plant Physiol 1977; 28:89-121.
4. Adams P, Nelson DE, Yamada S et al. Growth and development of Mesembryanthemum
crystallinum (Aizoaceae). New Phytol 1998; 138:171-190.
5. Langdale JA, Nelson T. Spatial regulation of photosynthetic development in C4 plants.
Trends Genet 1991; 7:191-196.
6. McCue KF, Hanson AD. Drought and salt tolerance: Towards understanding and
application. Biotechnol 1990; 8:358-362.
7. Delauney AJ, Verma DPS. Proline biosynthesis and osmoregulation in plants. Plant J
1993; 4:215-223.
8. Ishitani M, Majumder AL, Bornhouser A et al. Coordinate transcriptional induction of

myo-inositol metabolism during environmental stress. Plant J 1996; 9:537-548.
9. Gage DA, Rhodes D, Nolte KD et al. A new route for synthesis of dimethyl-sulfoniopropionate
in marine algae. Nature 1997; 387:891-894.
10. Niu X, Bressan RA, Hasegawa PM et al. Ion homeostasis in NaCl stress environments.
Plant Physiol 1995; 109:735-742.
11. Nelson DE, Shen B, Bohnert HJ. Salinity tolerance-mechanisms, models, and the metabolic
engineering of complex traits, In: Setlow JK, ed. Genetic Engineering, Principles and
Methods. New York: Plenum Press, in press. 1998; 20:153-176.
12. Bevan M, Bancroft I, Bent E et al: Analysis of 1.9 Mb of contiguous sequence from
chromosome 4 of Arabidopsis thaliana. Nature 1998; 391:485-488.
13. Flowers TJ, Yeo AR. Breeding for salinity resistance in crop plants: Where next? Aust J
of Plant Physiol 1995; 22:875-884.
14. Bohnert HJ, Jensen RG. Metabolic engineering for increased salt tolerance-the next step.
Aust J Plant Physiol 1996; 23:661-667.
15. Bohnert HJ, Jensen RG. Strategies for engineering water-stress tolerance in plants. Trends
Biotech 1996; 14:89-97.
16. Bartels D, Nelson DE. Approaches to improve stress tolerance using molecular genetics.
Plant Cell Envir 1994; 17:659-667.
17. Serrano R. Salt tolerance in plants and microorganisms: Toxicity targets and defense
responses. Intl Rev Cytol 1996; 165:1-52.
18. Jain RK, Selvaraj G. Molecular genetic improvement of salt tolerance in plants. Biotech
Annu Rev 1997; 3:245-267.
19. Zhu J-K, Hasegawa PM, Bressan RA. Molecular aspects of osmotic stress in plants. Crit
Rev Plant Sci 1997; 16:253-277.
20. Noctor G, Foyer CH. Ascorbate and glutathione: Keeping active oxygen under control.
Annu Rev Plant Physiol Plant Mol Biol 1998; 49:in press.
21. Levitt J. Responses of Plant to Environmental Stress Chilling, Freezing, and High
Temperature Stresses 2nd ed New York:Academic Press, Inc., 1980.
22. Kaiser WM. Reversible inhibition of the Calvin cycle and activation of oxidative pentose
phosphate cycle in isolated chloroplast by hydrogen peroxide. Planta 1979; 145:377-382.
23. Bieleski RL. Sugar alcohols. In: Loewus FA, Tanner W, eds. Encyclopedia of Plant Physiol
Vol 13A, Plant Carbohydrates I. Berlin:Springer Verlag. 1982:158-192.
24. Yancey PH, Clark ME, Hand SC et al. Living with water stress: Evolution of osmolyte
system. Science 1982; 217:1214-1222.
25. Ford CW. Accumulation of low molecular weight solutes in water stress tropical legumes.
Phytochem 1984; 23:1007-1015.
26. Brown AD, Simpson JR. Water relations of sugar-tolerant yeasts: The role of intracellular
polyols. J Gen Microbiol 1972; 72:589-591.
27. Borowitzka LJ, Brown AD. 1974. The salt relations of marine and halophilic species of
the unicellular green alga, Dunaliella. The role of glycerol as a compatible solute. Arch
Microbiol 1974; 96:37-52.
28. Le Rudulier D, Strom AR, Dandekar et al. Molecular biology of osmoregulation. Science
1984; 224:1064-1068.
29. Shen B, Jensen RG, Bohnert HJ. Increased resistance to oxidative stress in transgenic
plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiol 1997;
113:1177-1183.
30. Shen B, Jensen RG, Bohnert HJ. Mannitol protects against oxidation by hydroxyl radicals.
Plant Physiol 1997; 115:527-532.
31. Schobert B, Tschesche H. Unusual solution properties of proline and its interaction with
proteins. Biochem Biophys Acta 1978; 541:270-277.
32. Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes.
Phytochem 1989; 28:1057-1060.
33. Halliwell B, Gutteridge MC. Role of free radicals and catalytic metal ions in human
disease: An overview. Meth Enzymol 1990; 186:1-85.
34. Orthen B, Popp M, Smirnoff N 1994. Hydroxyl radical scavenging properties of cyclitols.
Proc Royal Soc Edinburgh 1994; 102B:269-272.
35. Pollard A, Wyn Jones RG. Enzyme activities in concentrated solutes of glycine betaine
and other solutes. Planta 1979; 144:291-298.
36. Brown AD. Microbial Water stress Physiology, Principles and Perspectives. New York:
John Wiley & Sons, 1990.
37. Solomon A, Beer S, Waisel Y et al. Effects of NaCl on the carboxylating activity of Rubisco
from Tamarix jordanis in the presence and absence of proline-related compatible solutes.
Plant Physiol 1994;. 90:198-204.
38. Back JF, Oakenfull D, Smith MB. Increased thermal stability of proteins in the presence
of sugars and polyols. Biochem 1979; 18:5191-5196.
39. Paleg LG, Douglas TJ, van Daal A et al. Proline, betaine and other organic solutes protect
enzymes against heat inactivation. Aust J Plant Physiol 1981; 8:107-114.
40. Galinski EA. Compatible solutes of halophilic eubacteria: Molecular principles,
water-solute interaction, stress protection. Experientia 1993; 49:487-496.
41. Papageorgiou G, Murata N. The unusually strong stabilizing effects of glycine betaine on
the structure and function of the oxygen-evolveing photosystem II complex. Photosyn
Res 1995; 44:243-252.
42. Coughlan SJ, Heber U. The role of glycine betaine in the protection of spinach thylakoids
against freezing stress. Planta 1982; 156:62-69.
43. Jolivet Y, Larher F, Hamelin J. Osmoregulation in halophytic higher plants: The protective
effect of glycine betaine against the heat destabilization of membranes. Plant Sci Lett
1982; 25:193-201.
44. Zhao Y, Aspinall D, Paleg LG. Protection of membrane integrity in Medicago sativa L. by
glycine betaine against effects of freezing. J Plant Physiol 1992; 140:541-543.
45. Arakawa T, Timasheff SN. The stabilization of proteins by osmolytes. Biophys J 1985;
47:411-414.
46. Schobert B. Is there an osmotic regulatory mechanism in algae and higher plants? J Theor
Biol 1977; 68:17-26.
47. Dujon B. The yeast genome project: What did we learn? Trends Genet 1996; 12:263-270.
48. Goffeau A, Barrell BG, Bussey H et al. Life with 6000 genes. Science 1996; 274:546-567.
49. Wodicka L, Dong H, Mittman M et al. Genome-wide expression monitoring in Saccha-
romyces cerevisiae. Nature Biotech 1997; 15:1359-1367.
50. Gadd GM, Chalmers K, Reed RH. The role of trehalose in dehydration resistance of
Saccharomyces cerevisiae. FEMS Microbiol Lett 1987; 48:249-254.
51. Mackenzie KF, Singh KK, Brown AD. Water stress hypersensitivity of yeast: Protective
role of trehalose in Saccharomyces cerevisiae. J Gen Microbiol 1988; 134:1661-1666.
52. Albertyn J, Hohmann S, Prior BR. Characterization of the osmotic-stress response in
Saccharomyces cerevisiae: Osmotic stress and glucose repression regulate glycerol-3-
phosphate dehydrogenase independently. Curr. Genet 1994; 25:12-18.
53. Albertyn N, Hohmann S, Thevelein JM et al. GPD1, which encodes glycerol-3-phosphate
dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae,
and its expression is regulated by the high-osmolarity osmolarity glycerol response
pathway. Mol Cell Biol 1994; 14:4135-4144.
54. Ahmad I, Larher F, Stewart GR. Sorbitol, a compatible osmotic solute in Plantago
maritima. New Phytol 1979; 82:671-678.
55. Adams P, Thomas JC, Vernon DM et al. Distinct cellular and organismic responses to
salt stress. Plant Cell Physiol 1992; 33:1215-1223.
56. Niu X, Narasimhan ML, Salzman RA et al. NaCl regulation of plasma membrane H+-ATPase
gene expression in a glycophyte and a halophyte. Plant Physiol 1995; 103:713-718.
57. van der Rest ME, Kamminga AH, Nakano A et al. The plasma membrane of Saccharomyces
cerevisiae:Structure, function, and biogenesis. Microbiol Rev 1995; 59:304-322.
58. Anderson JA, Huprikar SS, Kochian LV et al. Functional expression of a probable
Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc Natl Acad Sci
USA 1992; 89:3736-3740.
59. Sentenac H, Bonneaud N, Minet M et al. Cloning and expression in yeast of a plant
potassium ion transport system. Science 1992; 256:663-665.
60. Frommer WB, Ninnemann O. Heterologous expression of genes in bacterial, fungal,
animal, and plant cells. Annu Rev Plant Physiol Plant Mol Biol 1995; 46:419-444.
61. Rentsch D, Hirner B, Schmelzer E et al. Salt stress-induced proline transporters and salt
stress-repressed broad specificity amino acid permeases identified by suppression of a
yeast amino acid permease-targeting mutant. Plant Cell 1996; 8:1437-1441.
62. Oliver SG. From gene to screen with yeast. Curr Opin Genet Dev 1997; 7(3):405-409.
63. Yagi T, Tada K. Isolation and characterization of salt-sensitive mutants of a salt-tolerant
yeast Zygosaccharomyces rouxii. FEMS Microboil. Lett. 1988; 49:317-321.
64. Ushio K, Ohtsuka H, Nakata Y. Intracellular Na+ and K+ contents of Zygosaccharomyces
rouxii mutants defective in salt tolerance. J Fermen Bioeng 1992; 73:11-15.
65. Garciadeblas B, Rubio F, Quintero FJ et al. Differential expression of two genes encoding
isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae. Mol Gen
Genet 1993; 236:363
66. Brewster JL, De Valoir T, Dwyer ND et al. An osmosensing signal transduction pathway
in yeast. Science 1993; 259:1760-1763.
67. Maeda T, Wurgler-Murphy SM, Saito H. A two-component system that regulates an
osmosensing MAP kinase cascade in yeast. Nature 1994; 369:242-245.
68. Maeda T, Takekawa M, Saito H. Activation of yeast PBS2 MAPKK by MAPKKKs or by
binding of an SH3-containing osmosensor. Science 1995; 269:554-558.
69. Norbeck J, Pahlman A, Akhtar N et al. Purification and characterization of two isoenzymes
of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae. J Biol Chem 1996;
271:13875-13881.
70. Luyten K, Albertyn J, Skibbe WF et al. Fps1, a yeast member of the MIP family of channel
proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.
EMBO J 1995; 14:1360-1371.
71. Ansell R, Granath K, Hohmann S, Thevelein JM et al. The two isoenzymes for yeast
NAD-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have
distinct roles in osmoadaptation and redox regulation. EMBO J 1997; 16:2179-2187.
72. Shen B, Hohmann S, Jensen RG et al. Roles of sugar alcohols in osmotic stress adaptation-
Replacement of glycerol in yeast by mannitol and sorbitol. To be submitted, 1998.
73. Blomberg A, Adler L. Physiology of osmotolerance in fungi. Adv Microbiol Physiol 1992;
33:145-212.
74. Ota IM, Varshavsky A. A yeast protein similar to bacterial two-component regulators.
Nature 1993; 262:566-569.
75. Posas F, Wurgler-Murphy SM, Maeda T et al. Yeast HOG1 MAP kinase cascade is regulated
by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 two-component
osmosensor. Cell 1996; 86:865-875.
76. Schuller C, Brewster JL, Alexander MR et al. The HOG pathway controls osmotic
regulation of transcription via the stress response element (STRE) of the Saccharomyces
cerevisiae CTT1 gene. EMBO J 1994; 13:4382-4389.
77. Hirayama T, Maeda T, Saito H et al. Cloning and characterization of seven cDNAs
for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae. Mol Gen
Genet 1995; 249:127-138.
78. Varela JCS, Praekelt UM, Meacock PA et al. The Saccharomyces cerevisiae: Hsp12 gene is
activated by the high-osmolarity glycerol pathway and negatively regulated by protein
kinase A. Mol Cell Biol 1995; 15:6232
79. Davenport KR, Sohaskey M, Kamada Y et al. A second osmosensing signal transduction
pathway in yeast: Hypotonic shock activates the PKC1 protein kinase-regulated cell
integrity pathway. J Biol Chem 1995; 270:30157-351
80. Shimizu J, Yoda K, Yamasaki M. The hypo-osmolarity-sensitive phenotype of the
Saccharomyces cerevisiae hop2 mutant is due to a mutation in PCK1, which regulates
expression of β-lucanase. Mol Gen Genet 1994; 242:641-648.
81. Andre B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast

1995; 11:1575-1611.
82. Ko CH, Buckley AM, Gaber RF. TRK2 is required for low affinity K + transport in
Saccharomyces cerevisiae. Genetics 1990; 125:305-312.
83. Ko CH, Gaber RF. TRK1 and TRK2 encode structurally related K + transporters in
Saccharomyces cerevisiae. Mol Cell Biol 1991; 11:4266-4271.
84. Ketchum KA, Joiner WJ, Sellers AJ et al. A new family of outwardly rectifying potassium
channel proteins with two pore domains in tandem. Nature 1995; 376:690-695.
85. Haro R, Banuelos M, Quintero FJ et al. Genetic basis of sodium exclusion and sodium
tolerance in yeast. Plant Physiol 1993; 89:868-874.
86. Rubio F, Gassman W, Schroeder JI. Sodium-driven potassium uptake by the plant
potassium transporter HKT1 and mutations conferring salt tolerance. Science 1995;
270:1660-1663.
87. Gomez MJ, Luyten K, Ramos J. The capacity to transport potassium influences sodium
tolerance in Saccharomyces cerevisiae. FEMS Microbiol Lett 1996; 135:157-160.
88. Mendoza I, Rubio F, Rodriguez-Navarro A et al. The protein phosphatase calcineurin is
essential for NaCl tolerance of Saccharomyces cerevisiae. J Biol Chem 1994; 269:8792-
8796.
89. Gaxiola R, de Larrinoa IF, Villalba JM et al. A novel and conserved salt-induced protein
is an important determinant of salt tolerance in yeast. EMBO J 1992; 11:3157-3164.
90. Ferrando A, Kron SJ, Rios G et al. Regulation of cation transport in Saccharomyces
cerevisiae by the salt tolerance gene HAL3. Mol Cell Biol 1995; 15:5470-5481.
91. Watad AE, Reuveni M, Bressan RA et al. Enhanced net K+ uptake capacity of NaCl-
adapted cells. Plant Physiol 1991; 95:1265-1269.
92. Wu S, Ding L, Zhu J. SOS1, a genetic locus essential for salt tolerance and potassium
acquisition. Plant Cell 1996; 8:617-627.
93. Nelson N. The vacuolar H+-ATPase: One of the most fundamental pumps in nature. J
Exp Biol 1992; 172:19-27.
94. Rea PA, Poole RJ. Vacuolar H+-translocating pyrophosphatase. Annu Rev Plant Physiol
Plant Mol Biol 1993; 44:157-180.
95. Sussman MR. Molecular analysis of proteins in the plant plasma membrane. Annu Rev
Plant Physiol Plant Mol Biol 1994; 45:211-234.
96. Barkla BJ, Pantoja O. Physiology of ion transport across the tonoplast of higher plants.
Annu Rev Plant Physiol Plant Mol Biol 1996; 47:127-157.
97. Carmelo V, Bogaerts P, Sa-Correia I. Activity of plasma membrane H+-ATPase and
expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at optimal
and low pH. Arch Microbiol 1996; 166:315-320.
98. Estrada E, Agostinis P, Vendenheede JR et al. Phosphorylation of yeast plasma membrane
H+-ATPase by casein kinase I. J Biol Chem 1996; 271:32064-32072.
99. Braley R, Piper PW. The C-terminus of yeast plasma membrane H+-ATPase is essential
for the regulation of this enzyme by heat shock protein HSP30, but not for stress
activastion. FEBS Lett 1997; 418:123-126.
100. Hemenway CS, Dolinski K, Cardenas ME et al. vph6 mutants of Saccharomyces cerevisiae
require calcineurin for growth and are defective in vacuolar H+ -ATPase assembly.
Genetics 1995; 141:833-844.
101. Stevens TH, Forgac M. Structure, function and regulation of the vacuolar (H+)-ATPase.
Annu Rev Cell Dev Biol 1997; 13:779-808.
102. Haro R, Garciadeblas B, Rodriguez-Navarro A. A novel P-type ATPase from yeast
involved in sodium transport. FEBS Lett 1991; 291:189-191.
103. Wieland J, Nitsche AM, Strayle J et al. The PMR2 gene cluster encodes functionally distinct
isoforms of a putative Na + pump in the yeast plama membrane. EMBO J 1995;
14:3870-3882.
104. Prior C, Potier S, Souciet J et al. Characterization of the NHA1 gene encoding a
Na + /H + -antiporter of the yeast Saccharomyces cerevisiae. FEBS Lett 1996; 387:89-93.
105. Jia ZP, McMullough N, Martel R et al. Gene amplification at a locus encoding a putative
Na+/H+ antiport confer sodium and lithium tolerance in fission yeast. EMBO J 1992;
11:1631-1640.
106. Watanabe Y, Miwa S, Tamai Y. Characterization of Na+/H(+)-antiporter gene closely
related to the salt-tolerance of yeast Zygosaccharomyces rouxii. Yeast 1995; 11:839-838.
107. Hahnenberger KM, Jia Z, Young PG. Functional expression of the Schizosaccharomyces
pombe Na+/H+ antiporter gene, sod2, in Saccharomyces cerevisiae. Proc Natl Acad Sci USA
1996; 93:5031-5036.
108. Welihinda AA, Beavis AD, Trumbly RJ. Mutations in LIS1 (ERG6) gene confer increased
sodium and lithium uptake in Saccharomyces cerevisiae. Biochem Biophys Acta 1994;
1193:107-117.
109. Latterich M, Watson MD. Isolation and characterization of osmosensitive vacuolar
mutants of Saccharomyces cerevisiae. Mol Microbiol 1991; 5:2417-2426.
110. Nakamura T, Liu Y, Hirata D et al. Protein phosphatase type 2B (calcineurin)-mediated,
FK506-sensitive regulation of intracellular ions in yeast is an important determinant for
adaptation to high salt stress conditions. EMBO J 1993; 12:4064-4071.
111. Hirata D, Harada S, Namba H et al. Adaptation to high-salt stress in Saccharomyces
cerevisiae is regulated by Ca 2+ /calmodulin-dependent phosphoprotein phosphatase
(calcineurin) and cAMP-dependent protein kinase. Mol Gen Genet 1995; 249:257-264.
112. Posas F, Camps M, Arino J. The PPZ protein phosphatases are important determinants
of salt tolerance in yeast cells. J Biol Chem 1995; 270:13036-13041.
113. Cyert MS, Kunisawa R, Kaim D et al. 1991. Yeast has homologs (CNA1 and CNA2 gene
products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.
Proc Natl Acad Sci USA 1991; 88:7376-7380.
114. Marquez JA, Serrano R. Multiple transduction pathways regulate the sodium-extrusion
gene PMR2/ENA1 during salt stress in yeast. FEBS Lett. 1996; 382:89-92.
115. Sugiura R, Toda T, Shuntoh H et al. Pmp1(+), a suppressor of calcineurin deficiency,
encodes a novel MAP kinase phosphatase in fission yeast. EMBO J 1998; 17:140-148.
116. Rios G, Ferrando A, Serrano R. Mechanisms of salt tolerance conferred by overexpression
of the HAL1 gene in Saccharomyces cerevisiae. Yeast 1997; 13:515-528.
117. Luan S, Li W, Rusnak F, Assmann SM et al. Immunosuppressants implicate protein
phosphatase regulation of K+ channels in guard cells. Proc Natl Acad Sci USA 1993;
90:2202-2206.
118. Liu J. FK506 and cyclosporin, molecular probes for studing intracellular signal
transduction. Immunol Today 1993; 14:290-295.
119. Luan S, Lane WS, Schreiber SL. pCyP B: A chloroplast-localized, heat shock-responsive
cyclophilin from fava bean. Plant Cell. 1994; 6:885-892.
120. Lippuner V, Cyert MS, Gasser CS. Two classes of plant cDNA clones differentially
complement yeast calcineurin mutants and increase salt tolerance of wild type yeast. J
Biol Chem 1996; 271:12859-12866.
121. Mendoza I, Quintero FJ, Bressan RA et al. Activated calcineurin confers high tolerance
to ion stress and alters the budding pattern and cell morphology of yeast cells. J Biol
Chem 1996; 271:23061-23067.
122. Hanson AD, Rathinasabapathi B, Rivoal J et al. Osmoprotective compounds in the
plumbaginaceae: A natural experiment in metabolic engineering of stress tolerance. Proc
Natl Acad Sci USA 1994; 91:306-310.
123. Trossat C, Nolte KD, Hanson AD. Evidence that the pathway of dimethylsulfoniopropionate
biosynthesis begins in the cytosol and ends in the chloroplast. Plant Physiol 1996;
111:965-973.
124. Bray EA. 1997. Plant responses to water deficit. Trends Plant Sci 1997; 2:48-54.
125. Greenway H, Munns R. Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant
Physiol 1980; 31:149-190.
126. Bohnert HJ, Nelson DE, Jensen RG. Adaptations to environmental stresses. Plant Cell
1995; 7:1099-1111.
127. Vernon DM, Bohnert HJ. A novel methyl transferase induced by osmotic stress in the
facultative halophyte Mesembryanthemum crystallinum. EMBO J 1992; 11:2077-85.
128. Nelson DE, Rammesmayer G, Bohnert HJ. Cell-specific myo-inositol metabolism and
transport in plant salinity tolerance. Plant Cell 1998; 10:753-764.
129. Nelson DE, Koukoumanos M, Bohnert HJ. Myo-inositol is necessary for efficient NaCl
uptake and transport in Mesembryanthemum crystallinum. Plant Physiol 1998; in press.
130. Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K et al. A nuclear gene encoding mito-
chondrial proline dehydrogenase, an enzyme involved in proline metabolism, is
upregulated by proline but downregulated by dehydration in Arabidopsis. Plant Cell 1996;
8:1323-1335.
131. Rhodes D, Hanson AD. Quaternary ammonium and tertiary sulfonium compounds in
higher plants. Annu Rev Plant Physiol Plant Mol Biol 1993; 44:357-384.
132. Ishitani M, Nakamura T, Han SY et al. Expression of the betaine aldehyde dehydrogenase
gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol 1995;
27:307-315.
133. Saneoka H, Nagasaka C, Hahn DT et al. Salt tolerance of glycine betaine-deficient
and -containing maize lines. Plant Physiol 1995; 107:631-638.
134. Russell BL, Rathinasabapathi B, Hanson AD. Osmotic stress induces expression of
choline monooxygenase in sugar beet and amaranth. Plant Physiol 1998; 116:859-865.
135. Kishor PBK, Hong Z, Miao G et al. Overexpresion of pyrroline-5-carboxylate synthetase
increases proline production and confers osmotolerance in transgenic plant. Plant Physiol
1995; 108:1387-1394.
136. Yoshiba Y, Kiyosue T, Katagiri T et al. Correlation between the induction of a gene for
pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana
under osmotic stress. J Plant 1995; 7:751-760.
137. Verbruggen N, Hua XJ, May M et al. Environmental and developmental signals modulate
proline homeostasis: Evidence for a negative transcriptional regulators. Proc Natl Acad
Sci USA 1996; 93:8787-8791.
138. Sheveleva E, Chmara W, Bohnert HJ et al. Increased salt and drought tolerance by
D-ononitol production in transgenic Nicotiana tabacum L. Plant Physiol 1997; 115:1211-1219.
139. Malin G, Lapidot A. Induction of synthesis of tetrahydropyrimidine derivatives in
Streptomyces strains and their effect on E. coli in response to osmotic and heat stress. J
Bact 1996; 178:385-395.
140. Louis P, Galinski EA. Characterization of genes for the biosynthesis of the compatible
solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia
coli. Microbiol 1997; 143:1141-1149.
141. Holmström KO, Mäntyla E, Welin B et al. Drought tolerance in tobacco. Nature 1996;
379:683-684
142. Chrispeels MJ, Agre P. 1994. Aquaporins: Water channel proteins of plant and animal
cells. Trends Biochem Sci 1994; 19:421-425.
143. Maurel C. Aquaporins and water permeability of plant membranes. Annu Rev Plant
Physiol Plant Mol Biol 1997; 48:399-429.
144. Ishibashi K, Sasaki S, Fushimi K et al. Molecular cloning and expression of a member of
the aquaporin family with permeability to glycerol and urea in addition to water
expressed at the basolateral membrane of kidney collecting duct cells. Proc Natl Acad Sci
USA 1994; 91:6269-6273.
145. Weaver CD, Shomer NH, Louis CF et al. Nodulin 26, a nodule-specific symbiosome
embrane protein from soybean, is an ion channel. J Biol Chem 1994; 269:17858-17862.
146. Weig A, Deswarte C, Chrispeels JJ. The major intrinsic protein family of Arabidopsis
has 23 members form three distinct groups with functional aquaporins in each group.
Plant Physiol 1997; 114:1347-1357.
147. Yang B, Verkman AS. Water and glycerol permeabilities of aquaporins 1-5 and MIP
determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes.
J Biol Chem 1997; 272:16140-16146.
148. Kaldenhoff R, Källing A, Meyers J et al. The blue light-responsive AthH2 gene of
Arabidopsis thaliana is primarily expressed in expanding as well as in differentiating
cells and encodes a putative putative channel protein of the plasmalemma. Plant J
1995; 7:87-95.
149. DeWitt ND, Sussman MR. Immunocytological localization of an epitope-tagged plasma
membrane proton pump (H+-ATPase) in phloem companion cells. Plant Cell 1995;
7:2053-2067.
150. DeWitt ND, Hong B, Sussman MR et al. Targeting of two Arabidopsis H+-ATPase isoforms
to the plasma membrane. Plant Physiol 1996; 112:833-844.
151. Yamada S, Katsuhara M, Kelly WB et al. A family of transcripts encoding water channel
proteins: Tissue-specific expression in the common ice plant. Plant Cell 1995; 7:1129-1142.
152. Ludevid D, Hoefte H, Himmelblau E et al. The expression pattern of the tonoplast
intrinsic protein γ-TIP in Arabidopsis thaliana is correlated with cell enlargement. Plant
Physiol 1992; 100:1633-1639.
153. Maurel C, Kado RT, Guern J et al. Phosphorylation regulates the water channel activity
of the seed-specific aquaporin γ-TIP. EMBO J 1995; 14:3028-3035.
154. Ikeda S, Nasrallah JB, Dixit R et al. An aquaporin-like gene required for the Brassica
self-incompatibility response. Science 1997; 276:1564-1566.
155. Yamaguchi-Shinozaki K, Koizumi M, Urao S et al. Molecular cloning and characterization
of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: Sequence
analysis of one cDNA clone that encodes a putative transmembrane channel protein.
Plant Cell Physiol 1992; 33:217-224.
156. Opperman CH, Taylor CG, Conkling MA. Root-knot nematode-directed expression of a
plant root-specific gene. Science 1994; 263:221-223.
157. Phillips AL, Huttly AK. Cloning of two gibberellin-regulated cDNAs from Arabidopsis
thaliana by subtactive hybridization: Expression of the tonoplast water channel,
gamma-TIP, is increased by GA3. Plant Mol Biol 1994; 24:603-615.
158. Kaldenhoff R, Kaling A, Richter G. Regulation of the Arabidopsis thaliana aquaporin gene
AthH2 (PIP1b). J Photochem Photobiol B 1996; 36:351-354.
159. Yamamoto YT, Cheng CL, Conkling MA. Root-specific genes from tobacco and
Arabidopsis homologous to an evolutionarily conserved gene family of membrane
channel proteins. Nucl Acids Res 1990; 18:7449-7457.
160. Yamada S, Nelson DE, Ley E et al. The expression of an aquaporin promoter from
Mesembryanthemum crystallinum in tobacco. Plant Cell Physiol 1997; 38:1326-1332.
161. Kirch H-H, Michalowski CB, Bohnert HJ. Anti-MIP antibodies recognize distinct cell types
in roots and leaves from Mesembryanthemum crystallinum. Manuscript in preparation, 1998.
162. Verkman AS. Water channels. Austin:RG Landes Company, 1993.
163. King LS, Agre P. Pathophysiology of the aquaporin water channels. Annu Rev Physiol
1996; 58:619-648.
164. Nielsen S, Chou C-L, Marples D et al. Vasoprssin increases water permeability of kidney
collecting duct by inducing translocation of aquaporin-CD water channels to plasma
membrane. Proc Natl Acad Sci USA 1995; 92:1013-1017.
165. Robinson DG, Sieber H, Kammerloher W et al. PIPq aquaproins are concentrated in
plasmalemmasomes of Arabidopsis thaliana mesophyll. Plant Physiol 1996; 111:645-649.
166. Johansson I, Larsson C, Ek B et al. The major integral proteins of spinach leaf plasma
membranes are putative aquaporins and are phosphorylated in response to Ca2+ and
apoplastic water potential. Plant Cell 1996; 8:1181-1191.
167. Buxton GV, Greenstock CL, Helman WP et al. Critical review of rate constants for reactions
of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J Phys
Chem Ref Data 1988; 17:512-579.
168. Asada K. Production and action of active oxygen species in photosynthetic tissues. In:
Foyer CH and Mullineaux PM, eds. Causes of photooxidative stress and amelioration of
defense systems in plants. London: CRC Press, 1994; 77-104.
169. Tenhaken R, Levine A, Brisson LF et al. Function of the oxidative burst in hypersensitive
disease resistance. Proc Natl Acad Sci USA 1995; 92:4158-4163.
170. Lamb C, Dixon RA:The oxidative burst in plant disease resistance. Annu Rev Plant Physiol
Plant Mol Biol 1997; 48:251-275.
171. Mehler AH, Brown AH. Studies on reactions of illuminated chloroplasts. III. Simultaneous
photoproduction and consumption of oxygen studied with oxygen isotopes. Arch Biochem
Biophys 1952; 38:365-370.
172. Robinson JM. Does O2 photoreduction occur within chloroplasts in vivo? Plant Physiol
1988; 72:666-680.
173. Asada K, Takahashi M. Production and scavenging of active oxygen species in
photosynthetic tissues. In: Kyle DJ, Osmond CB, Arntzen CJ, eds. Photoinhibition.
Amsterdam: Elsevier Publ, 1994:227-287.
174. Hodgson RA, Raison JK. Superoxide production by thylakoids during chilling and its
implication in the susceptibility of plants to chilling-induced photoinhibition. Planta 1991;
183:222-228.
175. Kim CS, Jung J. The susceptibility of mung bean chloroplasts to photoinhibition is
increased by an excess supply of iron to plants: A photobiological aspect of iron toxicity
in plant leaves. Photochem Photobiol 1993; 58:120-126.
176. Elstner EF. Metabolism of activated oxygen species. In: Davies DD, ed. Biochemistry of
Metabolism, Vol 11. San Diego: Academic Press Inc, 1987:253-315.
177. Fryer MJ. The antioxidant effects of thylakoid vitamin E (α-tocopheral). Plant Cell Eviron
1992; 15:381-392.
178. Dalton DA. Antioxidant defense of plants and fungi. In: Ahmad S, ed. Oxidant-induced
Stress and Antioxidant Defenses in Biology. New York: Chapman & Hall, 1995:298-355.
179. Foyer CH, Descourvieres P, Kunert KJ. Protection against oxygen radicals: An important
defense mechanism studied in transgenic plants. Plant, Cell Environ 1994; 17:507-523.
180. Foyer CH, Lelandais M, Kunert KJ. Photooxidative stress in plants. Physiol Plant 1994;
92:696-717.
181. Willekens H, Chamnongpol S, Davey M et al. Catalase is a sink for H2 O 2 and is
indispensable for stress defence in C3 plants. EMBO J 1997; 16:4806-4816.
182. Osmond CB, Grace SC. Perspectives on photoinhibition and photorespiration in the field:
Quintessential inefficiencies of the light and dark reactions of photosynthesis? J Exp Bot
1995; 46:1351-1362.
183. Smirnoff N. The role of active oxygen in the response of plants to water deficit and
desiccation. New Phytol 1993; 125:27-58.
184. Wise RR. Chilling-enhanced photooxidation: The production, action and study of
reactive oxygen species produced during chilling in the light. Photosyn Res 1995;
45:79-97.
185. Price AH, Atherton N, Handry GAF. Plants under drought-stress generate activated oxygen.
Free Radical Research Comm 1989; 8:61-66.
186. Quartacci MF, Navari-Izzo F. Water stress and free radical mediated changes in
sunflower seedlings. J Plant Physiol 1992; 139:621-625.
187. Chowdhury SR, Choudhuri MA. Hydrogen peroxide metabolism as an index of water
stress tolerance in jute. Plant Physiol 1985; 65:503-507.
188. Prasad TK, Anderson MD, Martin BA et al. Evidence for chilling-induced oxidative stress
in maize seedling and a regulatory role for hydrogen peroxide. Plant Cell 1994; 6:65-74.
189. Prasad TK. Mechanisms of chilling-induced oxidative stress injury and tolerance in
developing maize seedlings: Changes in antioxidant system, oxidation of proteins and
lipids, and protease activities. J Plant 1996; 10:1017-1026.
190. Price AH, Handry GAF. Iron-catalysed oxygen radical formation and its possible
contribution to drought damage in nine native grasses and three cereals. Plant Cell
Environ 1991; 14:477-484.
191. Moran JF, Becana M, Iturbe-Ormaetxe I et al. Drought induces oxidative stress in pea
plants. Planta 1994; 194:346-352.
192. Roxas VP, Smith RK Jr, Allen ER et al. Overexpression of glutathione S-transferase/glutathione
peroxidase enhances the growth of transgenic tobacco seedlings during stress. Nature
Biotech. 1997; 15:988-991.
193. Guern J, Mathieu Y, Kurkdjian A et al. Regulation of vacuolar pH in plant cells. Plant
Physiol 1989; 89:27-36.
194. Michelet B, Boutry M. The plasma membrane H+-ATPase. Plant Physiol 1995; 108:1-6.
195. Weiss M, Pick U. Primary structure and effect of pH on the expression of the plasma
membrane H+-ATPase from Dunaliella acidophila and Dunaliella salina. Plant Physiol
1996, 112:1693-1702.
196. Reuveni M, Bennett AB, Bressan RA et al. Enhanced H+ transport capacity and ATP
hydrolysis activity of the tonoplast H+-ATPase after NaCl adaptation. Plant Physiol 1990;
94:524-530.
197. Ayala F, O’Leary JW, Schumaker KS. Increased vacuolar and plasma membrane H+-ATPase
activities in Salicornia bigelovii Torr. in response to NaCl. J Exp Bot 1996; 47:25-32.
198. Loew R, Rockel B, Kirsch M et al. Early salt stress effects on the differential expression
of vacuolar H+-ATPase genes in roots and leaves of Mesembryanthemum crystallinum.
Plant Physiol 1996; 110:259-265.
199. Tsiantis M, Bartholomew DM, Smith JAC. Salt regulation of transcript levels for the
c-subunit of a leaf vacuolar H+-ATPase in the halophyte Mesembryanthemum crystallinum.
Plant J 1996; 9:729-736.
200. Barkla BJ, Zingarelli L, Blumwald E, Smith JAC. Tonoplast Na+/H+ antiport activity and
its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum
crystallinum. Plant Physiol 1995; 109:549-556.
201. Vera-Estrella R, Barkla BJ, Bohnert HJ et al. Salt stress in Mesembryanthemum crystallinum
suspension cells activates adaptive mechanisms identical to those observed in the whole
plant. Planta, in press.
202. Robinson DG. Pyrophosphatase is not (only) a vacuolar marker. Trend Plant Sci 1996;
1:330.
203. Bremberger C, Luettge U. Dynamics of tonoplast proton pumps and other tonoplast
proteins of Mesembryanthemum crystallinum L. during the induction of crassulacean acid
metabolism. Plant 1992; 188:575-580.
204. Matsumoto H, Chung GC. Increase in proton-transport activity of tonoplast vesicles as
an adaptive response of barley roots to NaCl stress. Plant Cell Physiol 1988; 29:1133-1140.
205. Zingarelli L, Anzani P, Lado P. Enhanced K + -stimulated pyrophosphatase activity in
NaCl-adapted cells of Acer pseudoplatanus. Physiol Plant 1994; 91:510-516. 141.
206. Epstein E (1966) Dual pattern of ion absorption by plant cells and by plants. Nature
212:1324-1327.
207. Schachtman DP, Schroeder JI. Structure and transport mechanism of a high-affinity
potassium uptake transporter from higher plants. Nature 1994; 370:655-658.
208. Santa-Maria GE, Rubio F, Dubcovsky J et al. The HAK1 gene of barley is a member of a
large gene family and encodes a high-affinity potassium transporter. Plant Cell 1997;
9:2281-2289.
209. Kim EJ, Kwak JM, Uozumi N et al. AKUP1: An Arabidopsis gene encoding high-affinity
potassium transport activity. Plant Cell 1998; 10:51-62.
210. Golldack D, Su H, Bennett J et al. Differential expression of HKT1-type potassium
transporters in salt-sensitive and salt-tolerant rice lines. Manuscript in preparation, 1998.
211. Fu H-H, Luan S. AtKUP1: A dual affinity K+ transporter from Arabidopsis. Plant Cell
1998; 10:63-73.
212. Maathuis FJM, Verlin D, Smith FA et al. The physiological relevance of Na +-coupled
K +-transport. Plant Physiol 1996; 112:1609-1616.
213. Bertl A, Anderson JA, Slayman CL et al. Use of Saccharomyces cerevisiae for patch-clamp
analysis of heterologous membrane proteins: Characterization of Kat1, an inward-
rectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast
channels and carriers. Proc Natl Acad Sci USA 1995; 92:2701-2705
214. Gaber RF, Styles CA, Fink GR. TRK1 encodes a plasma membrane protein required for
high-affinity potassium transport in Saccharomyces cerevisiae. Mol Cell Biol 1988;
8:2848-2859.
215. Golldack D, Kamasani U, Quigley F et al. Salt stress-dependent expression of a HKT1-type

high affinity potassium transporter in rice. Plant Physiol 1997; S114:118.
216. Czempinski K, Zimmermann S, Ehrhardt T et al. New structure and function in plant
K+-channels: KCO1, an outward rectifier with a steep Ca2+ dependency. EMBO J 1997;
16:2565-2575.
217. Schachtman DP, Tyerman SD, Terry BR. The K+/Na+ selectivity of a cation channel in
the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat
species. Plant Physiol 1991; 97:598-605.
218. Schachtman DP, Kumar R, Schroeder JI et al. Molecular and functional characterization
of a novel low-affinity cation transporter (LCT1) in higher plants. Proc Natl Acad Sci
USA 1997; 94:11079-11084.
219. Barkla BJ, Apse MP, Manolson MF et al. The plant vacuolar Na+/H+ antiport. Symp Soc
Exp Biol 1994; 48:141-153.
220. Blumwald E, Poole RJ. Salt tolerance in suspension cultures of sugar beet: Induction of
Na+/H+ antiport activity at the tonoplast by growth in salt. Plant Physiol 1987; 83:884-887.
221. Garbarino J, DuPont FM. NaCl induces Na + /H + antiport in tonoplast vesicles from
barley roots. Plant Physiol 1988; 86:231-236.
222. Cammarata PR, Xu GT, Huang L et al. Inducible expression of Na + /myo-inositol
cotransporter mRNA in anterior epithelium of bovine lens: Affiliation with hypertonicity
and cell proliferation. Exp Eye Res 1997; 64:745-757.
223. Nelson DE, Bohnert HJ. Characterization of the sodium/myo-inositol symporter from
Mesembryanthemum crystallinum. Manuscript in preparation, 1998.
224. Colmer TD, Fan TWM, Higashi RM et al. Interactions of Ca2+ and NaCl stress on the
ion relations and intracellular pH of Sorghum bicolor roots tips: An in vivo 31P-NMR
study. J Exp Bot 1994; 45:1037-1044.
225. Martinez V, Lauchli A. Effects of calcium on the salt-stress response of barley roots as
observed by in vivo phosphorus-31 nuclear magnetic resonance and in vitro analysis.
Plata 1993; 190:519-524.
226. Liu J, Zhu JK. An Arabidopsis mutant that requires increased calcium for potassium
nutrition and salt tolerance. Proc Natl Acad Sci USA 1997; 94:14960-14964.
227. Sheveleva E, Marquez S, Zegeer A et al. Sorbitol dehydrogenase expression in transgenic
tobacco: High sorbitol accumulation leads to necrotic lesions in immature leaves. Plant
Physiol 1998; 117:831-839.
228. Allen RD. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol
1995; 107:1049-1054.
229. Aono M, Kubo A, Saji H et al. Resistance to active oxygen toxicity of transgenic
Nicotiana tabacum that expresses the gene for glutathione reductase from E. coli. Plant
Cell Physiol 1991; 32:691-697.
230. Aono M, Kubo A, Saji H et al. Enhanced tolerance to photooxidative stress of transgenic
Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol
1993; 34:129-135.
231. Maathuis FJM, Ichida AM, Sanders D et al. Roles of higher plant K+ channels. Plant
Physiol 1997; 114:1141-1149.
232. Bowler C, Slooten L, Vandenbranden S et al. Manganese superoxide dismutase can
reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J 1991;
10:1723-1732.
233. Bowler C, Van Montagu M, Inze D. Superoxide dismutase and stress tolerance. Annu
Rev Plant Phys Plant Mol Biol 1992; 43:83-116.
234. Gupta AS, Heinen JL, Holaday AS et al. Increased resistance to oxidative stress in
transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl
Acad Sci USA 1993; 90:1629-1633.
235. Van Camp W, Wilekens H, Bowler WH et al. Elevated levels of superoxide dismutase
protect transgenic plants against ozone damage. Biotechnol 1994;.12:165-168.
236. McKersie BD, Chen Y, de Beus M et al. Superoxide dismutase enhances tolerance of
freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol 1993; 103:1155-1163.
237. Foyer CH, Souriau N, Perret S et al. Overexpression of glutathione reductase but not
glytathione synthetase leads to increase in antioxidant capacity and resistance to
photoinhibition in poplar trees. Plant Physiol 1995; 109:1047-1057.
238. McKersie BD, Bowley SR, Harjanto E et al. Water-deficit tolerance and field performance
of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiol 1996;
111:1177-1181.
239. Pitcher LH, Zilinskas BA. Overexpression of copper/zinc superoxide dismutase in the
cytosol of transgenic tobacco confers partial resistance to ozone-induced foliar necrosis.
Plant Physiol 1996; 110:583-588.
240. Van Camp W, Capiau K, Montagu M et al. Enhancement of oxidative stress tolerance in
transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant
Physiol 1996; 112:1703-1714.
241. Jespersen HM, Kjaersgard IV, Ostergard L et al. From sequence analysis of three novel
ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of
seven types of ascorbate peroxidase. Biochem J 1997, 326:305-310.
242. Boyer JS. Plant productivity and environment. Science 1982; 218:443-448.
243. Sauer N, Stolz J. SUC1 and SUC2: Two sucrose transporters from Arabidopsis thaliana;
expression and characrerization in baker’s yeast and identification of the histidine-tagged
protein. Plant J 1994; 6:67-77.
244. Nishihama R, Banno H, Shibata W et al. Plant homologues of components of MAPK
(mitogen-activated protein kinase) signal pathways in yeast and animal cells. Plant Cell
Physiol 1995; 36:749-757.
245. Kakimoto T. CKI1, a histidine kinase homolog implicated in cytokinin signal transduction.
Science 1996; 274:982-985.
246. Ishitani M, Xiong L, Stevenson B et al. Genetic analysis of osmotic and cold stress signal
transduction in Arabidopsis: Interactions and convergence of abscisic acid-dependent and
abscisic acid-independent pathways. Plant Cell 1997; 9:1935-1949.
247. Hirt H: Multiple roles of MAP kinases in plant signal transduction. Trends Plant Sci
1997; 2:11-15.
248. Mizoguchi T, Irie K, Harashida N et al. A gene encoding a mitogen-activated protein
kinase kinase kinase is induced simultaneously with genes for a mitogen-activated
protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in
Arabidopsis thaliana. Proc Natl Acad Sci USA 1996; 93:765-769.
249. Mizoguchi T, Ichimura K, Shinozaki K. Environmental stress response in plants: The
role of mitogen-activated protein kinases. Trends in Biotech 1997; 15:15-19.
250. Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-
stress response. Plant Physiol 1997; 115:327-334.
251. Tarczynski MC, Jensen RG, Bohnert HJ. Expression of a bacterial mtlD gene in transgenic
tobacco leads to production and accumulation of mannitol. Proc Natl Acad Sci USA
1992; 89:2600-2604.
252. Tarczynski MC, Jensen RG, Bohnert HJ. Stress protection of transgenic tobacco by
production of the osmolyte, mannitol. Science 1993; 259:508-510.
253. Thomas JC, Sepahi M, Arendall B et al. Enhancement of seed germination in high salinity by
engineering mannitol expression in Arabidopsis thaliana. Plant Cell Environ 1995;
18:801-806.
254. Pilon-Smits EAH, Ebskamp MJM, Paul MJ et al. Improved performance of transgenic
fructan-accumulating tobacco under drought stress. Plant Physiol 1995; 107:125-130.
255. Hayashi H, Alia, Mustardy L et al. Transformation of Arabidopsis thaliana with the codA
gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt
and cold stress. Plant J 1997; 12:133-142.
256. Xu D, Duan X, Wang B et al. Expression of a late embryogenesis abundant protein gene,
HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice.
Plant Physiol 1996; 110:249-257.
257. Jones JT, Mullet JE. Developmental expression of a turgor-responsive gene that encodes
an intrinsic membrane protein. Plant Mol Biol 1995; 28:983-996.

CH 3 Molecular Mechanism Salt Tolerance

Enviado por

Dados do documento

Descrição original:

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

CH 3 Molecular Mechanism Salt Tolerance

Enviado por

Direitos autorais:

Formatos disponíveis

CHAPTER 3

Molecular Mechanisms of Salinity

Osmolytes, Osmoprotectants, Compatible Solutes, Osmotic

Cellular Mechanisms of Salt Tolerance—the Fungal Model

Replacing Glycerol in Yeast

Osmosensing and Signaling

regulation of K+ concentrations. Overexpression of HAL1 and HAL3 resulted in a stronger

Sodium Transport Across Membranes

the Ca 2+ -induced inactivation of K + channels was inhibited. A Ca 2+ -dependent

Molecular Mechanisms of Salt Tolerance in Plants

ROS Scavenging Enzymes 1991 SOD, catalase, GST/GSX overexpression

Mannitol Synthesis 1992 Protection against salt stress.251,252,253

Fructan Accumulation 1995 Enhanced drought tolerance.254

Proline Accumulation 1995 Enhanced salt stress tolerance.135

LEA Protein Synthesis 1996 Salinity and drought stress protection.256

Potassium Transporter 1994 Enhanced Na+/K+-discrimination in yeast.86,207

Trehalose Synthesis 1996 Enhanced drought tolerance.141

Mannitol as a Hydroxyl 1997 Enhanced salt tolerance, mannitol synthesis in

Inducible Ononitol 1997 Enhanced drought and salt tolerance; inducibility

Extreme Sorbitol 1998 High accumulation, >600 mM sorbitol, leading to

attention recently.130,140 Expression of the three enzymes leading to ectoine in bacteria

Complexity of Plant Isoforms

sediments in different gradient fractions depending on whether salt-stressed or unstressed

Salt Stress and Radical Scavenging

Reactive Oxygen Species and Radical Scavenging Systems

ROS and Environmental Stress

components of the light harvesting and electron transfer systems,30 a confirmation of

Plant Ion Uptake and Compartmentation

H+-ATPases and Vacuolar Pyrophosphatase

Potassium Transporters and Channels

Sodium Transport Systems

The Essentiality of Calcium

Metabolic Engineering of Glycophytic Plants for Increased

provided by the overexpression of SOD, ASX, or enzymes of the ascorbate/glutathione

8. Ishitani M, Majumder AL, Bornhouser A et al. Coordinate transcriptional induction of

81. Andre B. An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast

215. Golldack D, Kamasani U, Quigley F et al. Salt stress-dependent expression of a HKT1-type

Você também pode gostar