Ch 3 Molecular Mechanism Salt Tolerance

8/6/2019 Ch 3 Molecular Mechanism Salt Tolerance

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CHAPTER 3

Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants, edited by KazuoShinozaki and Kazuko Yamaguchi-Shinozaki. 1999 R.G. Landes Company.

Molecular Mechanisms of SalinityToleranceHans J. Bohnert,Hua Suand 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 mechanismscan be classified into two categories: One includes developmental, morphological, andphysiological mechanisms; the other includes biochemical mechanisms. Developmental,morphological, and physiological mechanisms are usually complex and require the functionsof many gene products. Examples of complex changes initiated by stress are the switchfrom the C3 photosynthetic pathway to Crassulacean acid metabolism (CAM) inMesembryanthemum crystallinum following salt stress,1 the development of salt glands inLimoniumsp.,2 salt-storing epidermal bladder cells in Mesembryanthemum crystallinum3,4

and changes leading to increased water use efficiency in the development of the C4photosynthetic pathway.5

Biochemical mechanisms, in contrast, are relatively simple, typically involving theaction 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 forextending 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 ofgene products.10,11 With the current knowledge of plant genetics and biochemistry, thegenetic engineering of biochemical mechanisms is possible, but the engineering of morecomplex traits is still beyond our capabilities. Once all relevant genes are known andfunctionally characterized, it should be possible to manipulate complex developmental

mechanisms, such as flower development, vegetative growth or seed formation. This goalis within reach for the genetic make-up ofArabidopsis thalianaat least,12 but the understandingof how the 21,000 Arabidopsisgenes function and their biochemical and physiologicalinteractions lies far in the future. In this review, we will focus mainly on biochemicalmechanisms that lead to cellular and whole-plant adaptations caused by the combinedosmotic and ionic disturbance of metabolism resulting from salt stress.

Over evolutionary time, plants colonized most places on earth, being excluded onlyfrom high latitudes, the highest mountains and true deserts, which are cold and/or lackwater completely. Xerophytic and halophytic adaptations evolved in response to long-termclimate changes which allowed plants to tolerate all but the most extreme habitats. Plants

which have adapted to stressful environments provide paradigms for biochemical andphysiological tolerance mechanisms, and they provide genes for pathways that couldbecome incorporated into crop plants, which are typically stress-sensitive, having originatedfrom species in subtropical or tropical areas.13-15 Species adapted to extreme habitats arenot equally distributed among all orders or families of the angiosperm lineage. They


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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants30

appear more frequently in orders which include few crop species but many speciesrestricted to stressful environments. These orders can be considered quarries for obtainingnovel genes for alternative biochemical pathways, and paradigms for understanding howthese pathways interact physiologically.

What constitutes tolerance or resistance to salinity stress has many facets, but issurprisingly simple in principle. For both growth and development of reproductiveorgans, plants must have water for photosynthesis to continue under stress; each one ofthe many diverse mechanisms which evolved in an order-, family- or species-specific fashionmust be subordinate to this essential goal. We will review the molecular mechanisms forwhich 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 (forrecent reviews, see refs. 11, 14-20, and other articles in this volume). Biophysical andbiochemical principles that govern stress and plant stress responses are outlined by Levitt.21

Osmolytes, Osmoprotectants, Compatible Solutes, OsmoticAdjustment

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, ifstress 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 ofcarbon through the photorespiratory cycle.14,15 Eventually, carbon and nitrogen allocation andstorage require readjustment, reactions that lead to the consumption of reducing powerbecome favored, and development and growth may become altered. During the past years,the complex interrelationship of biochemical pathways that change during salt stress hasbecome appreciated, although we are far from understanding this complexity.

The accumulation of metabolites, acting as osmolytes, in response to external changesin osmolarity is probably universal.23-25 The generally accepted view is that osmolytes mustbe compatible,26,27 not inhibiting normal metabolic reactions, and that their accumulation

leads to osmotic adjustment as the major element in accomplishing tolerance.6,7,24,28Typically, compatible solutes are hydrophilic, giving rise to the view that they couldreplace water at the surface of proteins, protein complexes, or membraneswe might callthem osmoprotectants in this case. The terms carry physiological meaning, but do notexplain the biochemical function(s) such solutes carry out. There may be more than onefunction 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 preventdissociation of the oxygen-evolving complex of photosystem II.41 One argument oftenraised against these studies is that the effective concentration necessary for protection invitro is very high, approximately 500 mM, concentrations which are usually not found invivo. However, when we consider the high concentration of proteins in cells, the concentrationnecessary for protection can, we think, be much lower than that required for protection inin vitro assays. In addition, it may not be the solute concentration in solution that is


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31Molecular Mechanisms of Salinity Tolerance

important. Glycine betaine (which may be present in high or low amounts), for example,protects thylakoid membranes and plasma membranes against freezing damage or heatdestabilization,42-44 indicating that the local concentration on membranes or proteinsurfaces may be more important than the absolute concentration.

Two theoretical models have been proposed explaining protective or stabilizingeffects of compatible solutes on protein structure and function. The first is termed thepreferential exclusion model45 which assumes the solutes are largely excluded from thehydration shell of proteins. Exclusion leaves a water shell around proteins which stabilizesprotein structure, or promotes or maintains protein/protein interactions. In this model,the solutes would not disturb the native hydration shell of proteins, but would interactwith the bulk water phase in the cytosol. The preferential interaction model, in contrast,emphasizes interactions between solute and proteins.46 During water deficit, compatiblesolutes 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 factbe explained by both models. The structures of different solutes could accommodatehydrophobic, van-der-Waals interactions, as well as electrostatic interactions, butadditional biophysical studies will be necessary to gain a better insight into the stabilizingeffects documented by in vitro experiments.

Cellular Mechanisms of Salt Tolerancethe Fungal Model

Osmotic Adjustment

The unicellular eukaryotic Saccharomyces cerevisiae, bakers yeast, is an ideal model

for studying cellular and molecular mechanisms of salt tolerance in higher plants. Its smallgenome, which has been sequenced,47-49 adds to several other advantages. First, yeast is salttolerant and the cells have stress responses similar to halophytic plants. Yeast cellsaccumulate compatible solutes, mainly glycerol and some trehalose, to counteract highexternal 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 Mesembryanthemumcrystallinum, accumulate high concentrations of sorbitol and methylated inositols,respectively, under salt stress.54,55 Second, both yeast and plants use proton gradients asthe 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 ofplant membrane proteins, such as the potassium, amino acid, and sugar transporters, havebeen isolated by functional complementation of yeast mutants.58-61 Third, the accumulationof glycerol is essential for salt tolerance52,53 and glycerol-deficient mutants are available forevaluating the functions of other sugar polyols in stress tolerance. Finally, yeast providesan unsurpassed system for genetic analysis, transformation and functional characterization ofcell-specific functions, especially in light of the recent completion of the yeast genomesequence.48,62

Yeast cells employ two main mechanisms for adaptation to salt stress: accumulationof 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 osmoticpotential, the accumulating glycerol seemingly compensates for the difference between theextra- and intra-cellular water potential.36 For reducing sodium toxicity, yeast cells have tomaintain low cytosolic Na+ concentrations and this is achieved by several mechanisms: byrestricting Na+ influx, rapidly extruding Na+ and/or efficiently compartmentalizingsodium into vacuoles. The genetic evidence indicates both mechanisms are essential for

yeast salt tolerance.63-65


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Glycerol AccumulationYeast 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 membraneosmosensors: the protein products ofSln1 and Sho1. The signal is then transferred via aMAP-kinase cascade66-68 and finally enhances the expression of the glycerol biosyntheticpathway. Glycerol is synthesized from dihydroxyacetone phosphate. The first reaction iscatalyzed by glycerol-3-phosphate dehydrogenase which is encoded by two genes, GPD1and GPD2. The second reaction converts glycerol-3-phosphate to glycerol by glycerol-3-phosphatase, encoded byGPP1 and GPP2.52,53,69 The osmotic induction of both GPDgenesis mediated by the HOG-MAP kinase signaling pathway. In addition to induced glycerolproduction, yeast cells may decrease membrane permeability to glycerol, which leads to anincreased retention of glycerol in the cells under osmotic stress. In fact, the salt-tolerant

yeast Zygosaccharomyces rouxiiachieves glycerol accumulation by increased retention of

glycerol within the cell, and probably by active uptake of glycerol rather than by increasedproduction of glycerol during osmotic stress.36 In contrast, Saccharomyces cerevisiaeappears to increase glycerol production, while it fails to significantly alter membranepermeability for glycerol retention during osmotic stress. To maintain high glycerolconcentrations in the cell requires a high energy cost, which seems to limit furtherincreases 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 osmotolerancehas been established, and although the essential role of glycerol in the adaptation toosmotic stress has been demonstrated by analysis of mutants deficient in glycerolproduction,52,53,71 the mechanism(s) by which glycerol can confer such tolerance is notclear. One obvious possibility is that glycerol is involved in osmotic adjustment tomaintain water flux into the cell. To test whether osmotolerance could be generated by thepresence of polyols other than glycerol, which would support the osmotic adjustmentconcept, we introduced the coding regions of genes encoding enzymes for mannitoland sorbitol production into a glycerol-deficient mutant (Fig. 3.1). However, accumulation ofeither sorbitol or mannitol was not able to replace glycerol function (Shen B, Hohmann S,

Bohnert HJ, unpublished). Both foreign polyols accumulated to approximately the sameconcentration as glycerol in wild type, and both were retained by the cell better thanglycerol, 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 themutant and 1.2 M for wild type. By reintroducing one of the deleted yeast GPD genes, asignificant increase in tolerance resulted, and the I50 increased from 0.4 M to 0.9 MNaCl.72 If osmotic adjustment through glycerol is sufficient for salt adaptation, an equalconcentration 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 bevalid, 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 specificprotective functions which mannitol and sorbitol cannot replace. Evolutionary adaptationsmight have altered yeast proteins such that glycerol, but not other osmolytes, could exert aprotective role. Alternatively, the pathway through which an osmolyte is produced could bemore important than the end-product. Finally, the minimal protection by mannitol or sorbitolcould be caused by a difference in their intracellular distribution compared to glycerol.


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Whether and how these polyols are compartmentalized in yeast cells is not known, butglycerol seems to be evenly distributed.73

A major difference exists between glycerol and mannitol/ sorbitol synthesis andaccumulation with respect to energy expenditure. Glycerol biosynthesis, which is also arequirement for the removal of excess NADH during anaerobiosis,71 is more costly thanmannitol/ sorbitol generation. While NADH oxidation is, in principle, also accomplishedby the mannitol and sorbitol metabolic pathways, which leads to NAD+ increase, the costis different. During salt stress, more than 95% of the glycerol produced leaked from thecells and accumulated in the medium. In contrast, sorbitol and mannitol did not significantlyexit from the cells. We calculated that glycerol biosynthesis under stress conditionsconsumed 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 asimportant as the increasing osmotic potential provided by the steady-state glycerolconcentration in the cytosol.

Fig. 3.1. Genes involved in yeast osmotic stress signal transduction, and replacement of glycerolsynthesis by foreign osmolytes. The schematic drawing of a yeast cell includes the membraneosmosensors (Sln1 and Sho1) which transmit signals to a MAP kinase cascade. Specifictranscription is initiated, which leads to the synthesis of several proteins, among them glycerol-3-phosphate dehydrogenase (GPD) and glycerol phosphatase (GPP). This results in glycerolsynthesis and accumulation. The glycerol facilitator protein, FPS1, a MIP-type channel, is lesspermeable under stress than under normal growth conditions. Replacement of both genesencoding GPD by mannitol-6-P dehydrogenase or sorbitol-6-P dehydrogenase leads to theaccumulation 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


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Osmosensing and SignalingThe 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 membraneosmosensors which are the protein products ofSln1 and Sho1. The Sln1 protein contains anextracellular 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 bacterialtwo-component systems.67,74,75Sho1 is a transmembrane protein containing a cytoplasmicSH3 domain which can directly activate a MAP kinase kinase, Pbs2, by interaction betweenits SH3 domain and a proline-rich motif ofPbs2.68 The signal from Sln1 is transmitted via aMAP 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 glycerolbiosynthetic pathway including the GPD1 and GPP2(phosphatase) genes.52,69 In additionto glycerol biosynthesis, genes for other stress responses, such as CCT1 encoding catalase T76

and HSP12encoding a small heat-shock protein,

77,78

are induced by this signaling pathway.In contrast to hyperosmotic stress, hypoosmotic stress initiates a second MAP kinasecascade called the protein kinase C1 (PKC1) pathway. The MAP kinase in this PKC1pathway 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. Thelytic phenotype can be suppressed by the addition of osmolytes like sorbitol into themedium.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 inyeast and other organisms. Among those, a (partial) functional characterization existedfor only about 60 genes prior to the completion of the sequence, which amply documentsboth the value of sequencing projects and our relative ignorance of membrane transportprocesses in general.81 Many of these membrane transport proteins are involved in iontransport and carry out essential functions in salt tolerance.

Potassium TransportPotassium plays an important role in yeast salinity tolerance. The osmotic potential

generated by high internal potassium concentrations (e.g., in halobacteria) can alleviatesodium toxicity.36 Three membrane proteins are involved in potassium transport acrossthe 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 andlow affinity potassium uptake, respectively. Importantly, TRK proteins can also transportNa+ 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 fortransporting potassium into cells and restricting sodium influx by increasedK+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 potassiumuptake.86

Two halotolerance genes, HAL1 and HAL3, have been isolated by screening for genesthat enhance salt tolerance when overexpressed.89,90 Both have been implicated in the


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regulation of K+ concentrations. Overexpression of HAL1 and HAL3 resulted in a strongeraccumulation of K+ under salt stress and increased salt tolerance. The beneficial effects arespecific 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 theNa+-ATPase (ENA1) genes are sensitive to Na+ because of poor Na+/K+ discriminationand 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

+

-ATPasesPlasma membrane and vacuolar proton ATPases are essential for generating andmaintaining 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 membraneH+-ATPases (P-ATPase), encoded by the gene PMA1, is predominantly responsible forproton gradient maintenance, while the product of the PMA2gene is induced at low pHwhen the PMA1 protein cannot function properly.97 Regulation of activity by calcium-dependent protein kinases, in response to glucose levels, weak organic acids, heat shockand salt stress, has been shown.98,99 Mutants hypersensitive to the immunosuppressantscyclosporin A and FK506 were shown to be defective in assembly of the vacuolar H+-ATPase

(V-ATPase). Their characterization indicated involvement of the calcineurin signaltransduction pathway in synthesis, endomembrane transport, assembly and activityregulation.98,100,101

Sodium Transport Across MembranesMaintaining 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. InS. cerevisiae, a Na+-ATPase encoded by a family of 4 or 5 ENA genes has been shown to beinvolved 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 ENAfunction are sensitive to sodium and lithium.65,102,103 Also, an Na+/H+ antiport protein,encoded byNha1, was found during sequencing of the genome81 and later functionallycharacterized.104 NHA1 seems to play a minor role in sodium efflux, but it may be important inan environment of acidic external pH which would affect the transmembrane protongradient.

Based on the results with yeast it was surprising, however, that in Schizosaccharomycespombe 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 forincreased LiCl tolerance in fission yeast105 and the homologous SOD2 was isolated from

Z. rouxii.106 Functional expression of ENA1 in a sod2mutant of S. pomberestored 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 theNa+ and H+ gradient across the membrane.107 Based on this property, it should be possibleto utilize SOD2 to transport Na+ into vacuoles by targeting the protein to the tonoplast inhigher plants.


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Although mechanisms of Na+ uptake in yeast are still not understood, mutantanalysis has clearly demonstrated an essential role for membrane-located processes.Disruption of the LIS1/ERG6 gene, encoding a SAM-dependent methyltransferase of theergosterol pathway, resulted in increased sodium uptake and decreased salt tolerance. Themutation seems to affect cation transport indirectly by changing membrane composition.108

Several other uncharacterized mutants showing high internal sodium were salt sensitivedespite normal glycerol accumulation.63,64 Halophytic plants usually sequester Na+ intovacuoles to lower the concentration of Na+ in the cytoplasm.3 Whether such a mechanismexists in yeast is unknown, but evidence exists for the vacuoles important role in salttolerance. Mutants defective in vacuole morphology and vacuolar protein targeting aresalt-sensitive.109 A mutant in subunit C of the vacuolar ATPase shows increased sensitivityto Na+ and Li+.85 The essential function may be associated with both compartmentationof ions and osmoregulation.

Calcineurin SignalingThe signal transduction pathway regulating ion homeostasis remains unknown in

detail, but it is known to be different from the HOG pathway. Recent studies revealed thatcalcineurin and protein phosphatase PPZ seem to be involved in the regulation of ionfluxes.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 Nullmutants of calcineurin fail to recover from G1-arrest in the presence of-pheromone, butshow 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 fromlow- to high-affinity potassium transport under salt stress.88 In contrast, deletions of genesfor the protein phosphatases PPZ1 and PPZ2 increased salt-tolerance due to enhancedexpression of ENA1, suggesting an essential role of these phosphatases in yeast ionhomeostasis.112 At low salt concentrations, the HOG-MAP kinase pathway appears to beinvolved in regulation of ion fluxes, while at high salt concentrations ion balance is mainlycontrolled by calcineurin.114 Interestingly, calcineurin signaling seems to interact with theMAP kinase pathway. Disruption of the calcineurin gene (Ppb1) in fission yeast resulted insensitivity 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 thechloride sensitivity of calcineurin mutants.115 Other components in the calcineurinsignaling pathway remain to be identified.

In addition to calcineurin and PPZ, HAL1 and HAL3 are involved in the regulation ofintracellular Na+ and K+ concentrations.90,116 The effect of HAL1 and HAL3 on intracellularNa+ is mediated by expression of the ENA1 gene. Calcineurin plays a role in the inductionof ENA1 expression by sodium, while HAL1, HAL3 and PPZ determine the basal level ofENA1. HAL1 and HAL3 are then required for the maximal expression of ENA1 under saltstress. Overexpression of HAL1 or HAL3 partially suppressed the salt sensitivity ofmutants 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 ionresponses in a complex interaction depending on external signals.

In higher plants, a calcineurin-like protein phosphatase activity has been found inthe regulation of the K+-channel in guard cells of fava bean.117 FK506 and cyclosporinA, immunosuppressants which bind to cellular receptors, are strong and specificinhibitors of calcineurin.118 When FK506-receptor complexes were added to guard cells,


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the Ca2+-induced inactivation of K+ channels was inhibited. A Ca2+-dependentphosphatase activity which is sensitive to complexes of FK506 and its binding proteinand to cyclophilin-cyclosporin A was also identified in guard cells.117 The gene whichencodes cyclophilin has been cloned,119 suggesting an important role of calcineurin inhigher plants. In addition, by complementation of yeast calcineurin mutants, two cDNAs(STO and STZ) which suppress the calcineurin deficient phenotype in yeast have beenisolated from plants, but the predicted protein sequence did not show significanthomology to phosphatases.120 Until now, the plant calcineurin gene has not beenidentified. The important function of calcineurin in salt tolerance has recently beendemonstrated by overexpression of a truncated yeast calcineurin in transgenic tobacco.Constitutive expression of this yeast calcineurin increased salt tolerance of transgenictobacco 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 biochemicalmechanism, present in all organisms from bacteria, fungi and algae to vascular plants andanimals.24,36 The accumulating metabolites include amino acids, their derivatives (proline,glycine betaine,-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 ofenzymes from both is generally strongly inhibited by high concentrations of either NaClor KCl.3 Although halophytes and glycophytes use similar compatible solute strategies todeal with osmotic stress,124 they use different strategies to cope with ion toxicity. Halophytestake up sodium and sequester ions into the vacuole. High osmotic potential in vacuoles isbalanced by accumulating compatible solutes in the cytoplasm. Because the cytoplasmicvolume is relatively small compared to the large volume of the vacuole, low concentrationsof compatible solutes suffice to reach the same osmotic potential in the cytoplasm. Incontrast, glycophytes usually attempt to limit sodium uptake or transport sodium to oldleaves 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 thanorganic solutes to counteract high external osmotic potential. Halobacterial enzymesrequire high ion concentration for their optimal activity.36 This adaptation required changesin protein structure. During evolution, this type of stress adaptation was abandoned,possibly because it proved inflexible to changing environments, and mechanisms becamefavored which utilized organic solutes, likely because they could be synthesized throughpathways attached to basic metabolism.

Several common features characterize the different compatible solutes. First, they caneasily be synthesized from compounds diverted from basic metabolism by novelenzymatic or regulatory reactions. For example, glycine betaine in higher plants is

synthesized from choline via two reactions catalyzed by choline monooxygenase andbetaine aldehyde dehydrogenase,6 and pinitol is synthesized from myo-inositol in tworeactions catalyzed by inositol o-methyltransferase and ononitol epimerase.4,8,15 Bothcholine 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, ratherthan an incidental consequence of other stress-induced metabolic changes. The biosyntheticpathway for a particular osmolyte is coordinately up-regulated during osmotic stress. For


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example, two key genes, Inps1 and Imt1, are transcriptionally enhanced by salt stress, andhigher enzyme amounts lead to increased carbon flux through myo-inositol into pinitolbiosynthesis in stressed Mesembryanthemum.8,127-129 Genes involved in the degradation ofcompatible solutes are down-regulated under osmotic stress. This is, for example, the casefor proline oxidase in Arabidopsis thaliana. Stress-dependent lower expression of thisenzyme, at least in part, may explain the increases in proline during salinity and droughtstress.130 Third, many accumulating compounds are end-products of a branch pathwayrather 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 pinitolsynthesis in Mesembryanthemum4,55 and glycinebetaine synthesis.6,132-134 Equally, prolinebiosynthesis has received much attention, because proline accumulation is a nearly universalreaction of plants to osmotic stress.135-137 Its true role in stress protection is, however, notclearwe consider the accumulation of proline a consequence of the necessity forreadjusting carbon nitrogen balance under stress.138 The biosynthesis of ectoine (tetra-hydropyrimidine and derivatives), an accumulating osmolyte in bacteria, has received

Table 3.1 Transgenes with Effects on Salt-, Drought- and Low Temperature

Tolerance

ROS Scavenging Enzymes 1991 SOD, catalase, GST/GSX overexpressionleading 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 inRadical Scavenger chloroplast.29,30

Inducible Ononitol 1997 Enhanced drought and salt tolerance; inducibilityAccumulation based on changes in substrate amounts.138

Extreme Sorbitol 1998 High accumulation, >600 mM sorbitol, leading toAccumulation necrotic lesions in sink leaves.227


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attention recently.130,140 Expression of the three enzymes leading to ectoine in bacteriaconfers significant salinity tolerance. Figure 3.2 shows schematically selected pathways thatlead to the synthesis of polyols (mannitol, sorbitol, ononitol and pinitol) and to trehalosesynthesis.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 someplant species naturally. The scheme indicates clearly how the addition of a single gene canbe exploited for metabolic engineering.

Water Channels

Water channels, aquaporins (AQP), are found in all organisms as members of asuper-family of membrane proteins, 26-30 kDa in size, termed MIP (major intrinsicprotein).142,143 The proteins are characterized by six membrane-spanning domains and apore-domain with a characteristic sequence signature, NH3-NPAXT-COOH. Aquaporinsenhance 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 vertebratesencode glycerol-facilitators.143 Other MIPs, animal and plantamong them a nodulation-specific protein, may mediate ion transport and transport of other neutral metabolites, such asurea.144,145

Fig. 3.2. Pathways for the synthesis of selected compatible solutes. Biochemical pathways originatingfrom glucose-6-P or sorbitol-6-P whose presence in some stress-tolerant species or after genetransfer into transgenic tobacco is correlated with increased osmotic stress tolerance. Genes/enzymes used in transgenic experiments are PGM (phosphoglucomutase), INPS (myo-inositol1-P synthase), IMT (myo-inositol O-methyltransferase), GPDH (sorbitol-6-P dehydrogenase),

MtlDH (mannitol-1-P dehydrogenase), TPS (trehalosephosphate synthase). Pase indicatesunspecific phosphatases. OEP (ononitol epimerase) is found in Mesembryanthemum, but thegene has not yet been cloned. IMP (myo-inositol monophosphatase) is not regulated inMesembryanthemum during stress and has not been included in transgenic plants.


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Complexity of Plant IsoformsIn 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 andthe brain. In contrast, Arabidopsiscontains at least 23 MIP-like coding regions.146 Sequencesignatures of the ArabidopsisMIP indicate two large sub-families of 10 to 12 proteins eachwhose 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 ofthe genes might encode facilitators for diverse small metabolites or ions, eight MIPproteins have already been identified as aquaporins. Why are there so many plantaquaporins? We discuss four possibilities which might explain the high number.

1. MIP-intrinsic functional variations might allow AQP to be active at differentmembrane osmotic potentials. Yet, all we know is that the intrinsic water permeabilitydistinguishes 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 whenexpressed in Xenopusoocytes.143 There is no report about a functional plant modelthat would allow mechanistic studies on AQP. By antisensing with a plasma membraneAQP coding region148 which supposedly targeted all expressed PIP, the decrease inAQP amounts led to a decline in water uptake in plants. Such antisense AQPtransgenics increased the root to shoot ratio, suggesting a feedback mechanismbetween water uptake and root mass (Kaldenhoff R, personal communication).Protoplasts from the antisense-expressing plants did not burst as fast as wild typecells 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 tissueswhich accommodate bulk fluxes and low conductance channels betweenmesophyll cells, for example, or even within the cell cytosol and organelles and thevacuole.

3. Without assuming functional diversification, the number of AQP arising throughgene duplications could have changed gene and protein expression, half-life, andturnover such that AQP amount shows a gradient that follows the water transportgradient. In this scenario, the gene numberrequiring different promoters, RNA-stability and translation characteristics and protein half-life regulationwould bedetermined by the necessity of cell-specific differences in accommodating water

flux and not by the water transport function per se. This explanation is similar tothe following one, and both find precedence in the presence of, for example, alarge number of genes encoding plasma membrane H+-ATPases, AHA, which aredifferentially expressed throughout the plant.95,149,150 Deletion of several AHA genesdid not produce a phenotype under normal growth conditions, but affected growthsignificantly under diverse growth and stress conditions, low temperature, salt stressand external high acidity, for example (Sussman MR, personal communication).

4. Last, AQP/MIP multiplications and diversifications could have been dictated bythe need for a flexible response to environmental changes in water supply orevaporation, 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 cellexpansion following meristematic activityand this function (missing from animals)might require regulatory circuits different from those necessary in genes that performhousekeeping (set 2) and stress-response functions (set 3). Although the data arenot complete with respect to AQP protein expression and cell-specificity, alignmentsof sequences indicate that sub-families of two to four closely related sequences


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exist146,151 which might represent the three sets of genes. MIP associated with cellexpansion,152 developmental specificity153,154 and stress functions151,155-158,257 havebeen described.

Mechanisms of RegulationMost 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-translationalmodificationbut we have very little information on mechanistic details in plants.

Weig et al146 used quantitative PCR amplification for the 23 ArabidopsisMIP andfound differences in mRNA amounts spanning several orders of magnitude. Differencesin RNA amounts for each MIP in roots, leaves, bolts and the flowers and siliques wereequally 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 areexpressed 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 withspecific antibodies and DNA microarray analysis through which the amount, location andregulation of the genes during development and under different environmental conditionscan be monitored. For several MIP in a number of organisms, salt stress altered mRNAamounts 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 ismost likely the essential distinguishing factor between AQP and must receive more

attention in order to understand water transport in plants. The promoter for Rb7a159

fromtobacco conveys root-specificity, leads to differential expression in the root in a cell-specific manner and is induced by nematode feeding.156,159 The MesembryanthemumMipBpromoter showed highest expression of the gene in roots;151 after transfer into tobaccoand observation of GUS expression, broader specificity was observed, with highest expressionin all meristematic cells and in vascular tissues.160

Even less complete is the information about protein amount, localization and changesduring development and under stress conditions. One essential consideration is that thelarge 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-peptideantibodies against six Mesembryanthemum MIP resulted in distinguishable signals todifferent cells.161 However, in the absence of probes for all MIP for this species, it cannotbe 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 isdetermined by the hormone vasopressin in animal cells. Increased vasopressin leads to the

deposition of AQP from internal stores, endosome vesicles, to the outer membrane, andlower 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 regulatingwater flux. Similar observations remain to be made with plant MIP, but patches of invaginatedplasma membrane regions, termed plasmalemmasomes that contain abundant AQPprotein have been found in plants,165 possibly the functional equivalent of animalendosomes. Our preliminary experiments indicate that PIP from Mesembryanthemum


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sediments in different gradient fractions depending on whether salt-stressed or unstressedcells were used,161 which might indicate that similar membrane shuttle mechanisms existin 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 severalMIP/AQP.143 Also, effects of pharmaceutical agents on water flow in Chara cells, forexample, point towards an association of water flux and the integrity of the cytoskeleton (seeref. 143). Spinach leaf PIP are reversibly phosphorylated in response to the apoplasticwater potential and calcium.166

The discovery and preliminary characterization of AQP in plants has provided morequestions than answers. Their existence cannot be questioned and they act as waterchannels. It is then intuitively obvious that control over their action should be importantunder stress conditions. Although there are few data available, it is equally clear that regulation

during stress is complex, involving transcriptional and post-translational controls whichseem 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 SystemsProduction of Reactive oxygen species (ROS) is an unavoidable process in photosynthetic

tissues, but ROS are also produced in mitochondria and cytosol. ROS including singletoxygen, superoxide, hydrogen peroxide, and hydroxyl radicals react with and can damage

proteins, membrane lipids, and other cellular components.33,167,168

Some ROS also serveas signaling molecules,20 for example, in the initial recognition of attack byfungal pathogens and the transmission of signals after a primary infection.169,170 Focusing onchloroplasts, superoxide is abundantly produced from photoreduction of oxygen. Oxygenconcentration as high as 300 mM can be photoreduced to superoxide by photosystem I viaa Mehler reaction.171,172 The production of superoxide has been estimated to be approximately30 mmol (mg chl)-1 h-1 in intact chloroplasts,173 and the rate of production in isolatedthylakoids was increased 1.5-fold by the addition of ferredoxin and decreased 50% byaddition of NADP+.174 Most of this thylakoid lumen-produced superoxide diffuses to thestroma.173 H2O2 in chloroplasts is predominantly generated by disproportionation of

superoxide by SODs. In peroxisomes, H2O2 originates directly from glycollate oxidaseactivity. Hydroxyl radicals derive from an interaction between hydrogen peroxide andsuperoxide or directly from hydrogen peroxide in the presence of transition metals such asFe+2 and Cu+ by a Fenton- or Haber-Weiss-reaction. The oxidized metal ions can bere-reduced by superoxide, glutathione, or ascorbate. Trace amounts, lower than the amountpresent in chloroplasts, of metal ions are needed to catalyze the Fenton reaction.168,173 Ithas 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 andnonenzymatic antioxidants.176 Non-enzymatic detoxification mechanisms includemorphological 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, -tocopherol, ascorbate (at a concentration of ~10 mM in chloroplasts),glutathione, carotenoids, phenolics and polyols.20,32,168,177 Botanical sources of suchantioxidants not only play important roles in plant stress adaptation, but also retard agingand diseases related to oxidative damage in animals.178


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The enzyme systems involved include SODs which catalyze the reaction from superoxideto hydrogen peroxide, and ascorbate peroxidases (APX) responsible for the conversion ofhydrogen peroxide to water. Both SOD and APX are represented by isoforms localized tothe stroma and the thylakoid membrane. Ascorbate can be regenerated by the ascorbate-glutathione cycle. The level of reduced glutathione is maintained by glutathione reductaseusing NADPH.168,179,180 In addition, catalase has recently been demonstrated as a sink forH2O2 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 hasnot been identified and, in fact, might not have evolved.167,168,179,180 The best way ofdetoxifying hydroxyl radicals is to prevent their formation by reducing the concentrationof H2O2 and free metal ions. Once produced, however, protection depends on the presenceof antioxidants in the vicinity of the formation site. Together these systems provide sufficientprotection under normal growth conditions; in fact, the scavenging systems are able to

handle moderate increases of ROS, unless long-term stress exceeds the detoxificationcapacity.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 indrought stressed wheat and sunflower.185,186 Equally, H2O2 content increased about three-foldduring 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 ofmalonaldehyde production in wheat.190 Second, the concentration of free transition ironincreased under drought stress,190,191 which stimulated production of hydroxyl radicals inthe presence of high concentrations of H2O2 via a Fenton reaction. Compared tosuperoxide 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 complementenzyme protection systems.

Excellent evidence for a protective effect of ROS scavenging systems has recently beenprovided 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 intransgenic tobacco, the seedlings and plants showed significantly faster growth than wildtype during chilling and salt stress episodes. The increased enzyme activities resulted inhigher amounts of oxidized glutathione (GSSG) in the stressed plants, indicating that theoxidized form could provide an increased sink for reducing power.

Another set of experiments shed light on the relationships between ROS and theaccumulation of polyols. When a bacterial gene (mtlD) encoding mannitol-1-phosphatedehydrogenase was modified so that the enzyme was expressed in chloroplasts, transgenictobacco 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 mannitolresulted in increased resistance to oxidative stress generated by methylviologen, and cellsexhibited significantly higher CO2 fixation rates than controls during stress. Afterimpregnation of tissue and cells with dimethyl sulfoxide, a hydroxyl radical generator,mannitol-containing cells showed a lower rate of methane sulfinic acid production thanwild type, indicating that mannitol acted specifically as a hydroxyl radical scavenger. Itcould be shown that the primary damage was to enzymes of the Calvin cycle and not to


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components of the light harvesting and electron transfer systems,30 a confirmation ofearlier reports.22 At present, the interpretation which we favor is that mannitol interfereswith either hydroxyl radical production or damage, but it is unknown whether theprotective mechanism is by exclusion of hydroxyl radicals from protein surfaces, a chemicalinteraction between mannitol and hydroxyl radicals, or by inhibiting or reducing theamount of hydroxyl radicals produced in the Fenton reaction.

Plant Ion Uptake and Compartmentation

H+-ATPases and Vacuolar PyrophosphatasePlasma membrane and vacuolar proton transporters play essential roles in plant

salinity stress tolerance by maintaining the transmembrane proton gradient that assurescontrol 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 vacuolartransport 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 pumpin the outer cell membrane it is essential for many physiological functions.194 Increasedactivity of the proton pump has been shown to accompany salt stress. Halophytic plantshave been shown to increase pump activity under salt stress conditions more drasticallythan glycophytes,56,195 but little is know about the regulatory circuits that lead to eitherincreased 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 tissuesfrom stressed plants were analyzed.196,197 Transcripts for several subunits of the V-ATPaseare upregulated following salt shock.198,199 In Mesembryanthemum, V-ATPase activityincreases several-fold following stress.200,201 In a Mesembryanthemum cell culture model ithas now been shown, based on immunological data, that the V-ATPase (and possibly theP-ATPase) activity does not increase due to more protein being present, but an unknownmechanism stimulates activity 2 to 3-fold.201 The response is specific for NaCl and couldnot 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 asalso residing in the plasma membrane.202 Its function, if any, under salt stress conditionsis little known. A few reports have indicated that PPase activity declines under salt stress insome species,203,204 but increases in others.205

Potassium Transporters and ChannelsOne possible passage for sodium across the plasma membrane is through transport

systems for other monovalent cations. Among those the most significant is the uptakesystem for potassium, the most abundant cation in the cytosol, with important roles inplant nutrition, development and physiological regulation. Many studies have focused on

identifying components involved in K+-transport. Physiological observations indicating abiphasic uptake of K+ into roots206 gave rise to the assumption that two uptake entitiesshould be involved, a high-affinity system functioning at M concentrations of externalK+ 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 ofyeast mutants deficient in K+ uptake58,59,207 or by sequence homology with known K+

transporter or channel genes.208-210 Electrophysiological studies in heterologous


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expression systems, such as Xenopusoocytes 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 theelectrochemical gradient at the plasma membrane and are categorized as low-affinitysystems.212,231 The AKT1- and KAT1-types of plant channels, similar to the Shakerchannels in animals, contain a pore-forming region conferring ion selectivity. In contrastto earlier assumptions, these channels are highly selective against Na+,213 and evidence islacking for specific regulation under salt stress. We think that the potassium channels playa minor role in salinity tolerance.

In contrast, K+ transporters which operate at low external potassium may mediateentry 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-affinitybinding sites for both K+ and Na+. Point mutations, which increased K+ selectivity overNa+, in one of the 12 transmembrane domains of HKT1 conferred increased salt toleranceof yeast. Another line of evidence for the involvement of high-affinity K+ uptake system insalt tolerance came from the study of salt-sensitive mutants. The sos1 mutant ofArabidopsisthalianawas characterized as hypersensitive to Na+ and Li+ and was unable to grow on low

Fig. 3.3. Transport proteins implicated in plant salinity stress tolerance. The schematic depictionof a plant cell includes the vacuole, chloroplast (cp), mitochondrion (mt) and cell wall (shaded).

Transmembrane proton gradients established by proton-ATPases and pyrophosphatase areindicated (+/-). Under NaCl stress, Na+ and Cl- are sequestered to the vacuole, and K+ andosmolytes are present in high concentrations in the cytosol. Symbols for several membrane-locatedtransporters and channels are identified by the ion or proton transported and by the direction ofmovement. For organelles (mt and cp) no transporters have been characterized throughmolecular techniques. A Na+-ATPase, included in the plasma membrane is hypothetical, and aNa+/H+-antiporter in the plasma membrane has not been detected.


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potassium.92 86Rb uptake experiments showed that sos1 was defective in high-affinitypotassium uptake, and it became deficient in potassium when treated with NaCl. Interestinglybut 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 isprovided by the expression characteristics of a rice homolog of wheat HKT1 in twovarieties that are distinguished by their salinity tolerance. The tolerant variety decreasedexpression of the root-specific HKT1 and efficiently excludes sodium, while a salt-sensitivevariety 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, orhigh-affinity potassium uptake systems in general, in salt tolerance, there are other equallylikely 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 byincreased 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 ofsodium. In this respect, the improved selectivity of K+ transport systems may increase salttolerance, while it is not involved in Na+ detoxification or osmotic adjustment. Othertransport systems, finally, might act in sodium uptake. How, for example, the calcium-regulated outward-rectifying K+-channel KCO1,216 or the regulation of other channelsand transporters, react under sodium stress conditions is unknown. It has been suggestedthat sodium might enter through outward-rectifying cation channels.217 Among the manypossibilities, 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 SystemsHow 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 notknown in detail. Most information is available for the last step in this series: sodiumtransport from cytosol to vacuole is accomplished by a sodium/proton antiporter. Aprotein of approximately 170 kD219 is a candidate for this tonoplast-located antiporterbased on immunological studies and inhibition of the ameloride-regulated antiportactivity in the presence of the antibody. It will be important to characterize the protein indetail 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 severalmodel systems, tissues, cells and isolated vacuoles.96,220,221 The increase parallels anincrease in the V-ATPase activity.96,200

Our own data indicate that yet another pathway for sodium uptake may exist. Whenanalyzing the induction of myo-inositol synthesis in Mesembryanthemum, a surprisingdecline of the rate-limiting INPS (myo-inositol-1-phosphate synthase) enzyme in rootswas observed, but the concentration ofmyo-inositol remained constant in the roots.128,129

This is due to drastically enhanced transport ofmyo-inositol from the leaves through thephloem. 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 cloneda transcript with homology to vertebrate sodium/myo-inositol and yeast proton/myo-inositolsymporters222 and characterized its activity by complementation of a yeast mutantdefective in myo-inositol uptake.223 It seems possible that such a symporter is responsiblefor 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 asymport mechanism is particularly attractive, considering that the passage ofmyo-inositol


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through the plant circulatory system connects photosynthesis competence with sodiumuptake and transport to mesophyll cells of the leaf.

The Essentiality of CalciumIncreasing 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 strictcontrol over calcium concentrations in the cytosol and calcium storage in a number oflocations (vacuole, mitochondria, endoplasmic reticulum) assign a crucial role to calciumin plant salinity stress responses.

Recently, an Arabidopsismutant, sos3, with hypersensitivity to NaCl has beencharacterized. The mutant is different from other salt-sensitive mutants92 in that thephenotype 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 phenotypereveals the link between calcium and salinity stress tolerance, although the mechanismthrough 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 lowcalcium concentrationsfor example a homolog of the yeast calcineurin-type systemisdefective, and that at higher calcium concentrations a second sensing system can supportthe signal and elicit stress defense responses (Zhu JK, personal communication).

Metabolic Engineering of Glycophytic Plants for IncreasedSalt Tolerance

In increasing numbers, experiments are reported using transgenic plants for testingconcepts originating from the correlative evidence of physiological analyses. Table 3.1summarizes some of these reports. The concepts tested target four aspects of toleranceacquisition:

1. ROS scavenging,2. Compatible solutes and osmotic adjustmentcarbohydrate biosynthesis and

synthesis of charged molecules,3. Ion balancepotassium uptake vs. sodium uptake, and4. 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, ortoo efficient scavenging of H2O2, for example, may not be desirable. When analyzingtransgenic tobacco plants that accumulated sorbitol to extremely high concentrations inthe cytosol, we observed stunted growth and the formation of necrotic lesions that reducedbiomass production, although the plants showed increased salinity and salt stress tolerance.227

The importance of radical oxygen scavenging for preventing oxidative stress in plantshas been demonstrated by genetic engineering of several enzymes into transgenicplants.179,180,228 Overexpression of superoxide dismutase (Cu/Zn-SOD and Mn-SOD),ascorbate peroxidase, catalase and glutathione reductase in transgenic plants has alreadybeen 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 glutathionecycle.192 In contrast, overexpression of an Fe-SOD in transgenic tobacco neither enhancedtolerance to chilling-induced photoinhibition in leaf discs nor increased tolerance to saltstress in whole plants,240 suggesting that isoforms of SOD may have different roles.

Noctor and Foyer20 provided a lucid assessment of the relatively marginal protectionthat has been observed in many transgenic plant studies, whether with respect to ROSscavenging or otherwise. It would certainly be premature to consider the protection


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provided by the overexpression of SOD, ASX, or enzymes of the ascorbate/glutathionecycle as the final word. Protection has typically been observed in strictly controlledenvironments, and protective effects have often been marginal. We would like to provideone consideration as to why this is to be expected. In the case of ASX, at least six differentisoforms exist which are located in mitochondria, in chloroplasts (several, in differentsub-compartments/membranes), soluble in the cytosol, and in the cytoplasmicendomembrane system.241 A similarly complex distribution has been seen for SOD isoformswhich 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 havea minimal effect because of the multitude of compartments that require protection.Irrespectively, these experiments have clearly shown thatin practically every studythe engineered expressed transgene elicited some protection. It is now necessary to adoptmulti-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 potassiumuptake;

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 beenpaid to the when,where, and how much of transgene expression in the presentlyconcluded transgenic experiments. Significantly more attention needs to be directed to

the promoter elements that drive transgenes. Most attempts have targeted the metabolicengineering of carbon and nitrogen allocation: ectopic enzyme expression leading to thesynthesis of uncharged carbohydratesmannitol, sorbitol, trehalose, fructan, andononitoland to glycinebetaine and proline accumulation (Table 3.1). The underlyingmechanism is becoming apparent for some of these strategies, e.g., in the hydroxyl radicalscavenging function of mannitol.29,30 The mechanisms of protection underlying thesynthesis 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 phenotypeshown by some species and all genes that support damage avoidance in sensitive specieswill be available. The task remaining, however, is understanding in which metabolic and

signaling pathways the gene products function and in which developmental context stressprotection is necessary. This task will require new approaches. We consider two approaches:

1. Multi-gene transfer into model speciesyeast, Arabidopsis, tobacco and rice areour 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 ofpathways, we will be able to gauge the cost of enzymes/pathways that enhance tolerance incomparison to the cost and benefits of resident pathways.

PerspectivesHigh 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 stillproductive soils will become a more severe problem in the future. Development ofdrought- and salt-tolerant crops has been a major objective of plant breeding programsfor decades in order to maintain crop productivity in semiarid and saline lands. Although


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several salt-tolerant varieties have been released, the overall progress of traditionalbreeding has been slow and has not been successful.13 The lack of success is mainly due tothe quantitative trait character of salinity tolerance which has to be reconciled withanother multigenic trait, high productivity, which is the ultimate goal of any breedingprogram. Marginal progress has equally been grounded in our poor understanding of themechanisms of salt tolerance, while the collected body of physiological data has focusedour 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 theproduction of compatible solutes such as proline, glycine betaine, DMSP, or pinitol havebeen studied and most of the pathway genes have been characterized.6,7,9,14,15 We have thefirst 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 andproteins which deter radical damage have emerged. Membrane channels, transporters and

pores are now available through which cells exert control over ion, carbohydrate, aminoacid or water fluxes.58,59,61,146,207,243 We owe most of this recent progress to the power ofthe yeast and Arabidopsis thalianamolecular genetic systems. Finding the genes whosedisruptions generate the various mutant phenotypes becomes rapidly easier as additionalmapping data and genomic DNA sequences from Arabidopsisare made available.12

Finally, plant stress perception, and inter- and intracellular signaling of salt stress hasbeen advanced greatly. Mutants in signal transduction pathways and components ofseveral signal transduction pathways have been found and are being characterized atpresent.244-249 Future studies can follow the blueprint of signaling components isolatedfrom yeast10,66,68,88,250 for finding and characterizing homologs of the essential

signaling intermediates in plants. If we accept that a major objective of plant stressresearch is application, transgenic crops can be engineered not only for expression of novelbiochemical characters, but also for stress signal transduction that enhances the stressresponse inherent to all plants.

AcknowledgmentsBecause 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 PlantBiology and International Programs), Department of Energy (Biological Energy), and

Department of Agriculture (NRI). Additional support has been provided by the ArizonaAgricultural Experiment Station, Japan Tobacco Inc., Rockefeller Foundation (New York)and New Energy Development Organization (Tokyo).

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Ch 3 Molecular Mechanism Salt Tolerance

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