transformation and compatible solutes

Transformation and compatible solutes

Hans J. Bohnert*, Bo Shen1

Department of Biochemistry, The University of Arizona, Biosciences West,

Tucson, AZ 85721-0088, USA

Abstract

Plants are frequently exposed to environmental stresses that result in water deficit, sodium

toxicity, ion deficiency, and photoinhibition. Plants deal with these factors according to their genetic

makeup through responses which, although present in all species, have evolved to different

complexity in individual plant families. A nearly universal reaction is the accumulation of

`compatible solutes', many of which are osmolytes (i.e., metabolites whose high cellular

concentration reduces the osmotic potential significantly) considered to lead to osmotic adjustment.

Recent observations indicate that compatible solutes may have other functions as well, namely in

the protection of enzyme and membrane structure and in scavenging of radical oxygen species.

Plant transformation leading to the presence of compatible solutes has resulted in significant

increases in whole plant tolerance to osmotic stress, but the increases will be marginal in a field

situation. Considering the progress in our understanding of mechanisms that lead to stress tolerance,

multigene transfer is possible with present plant transformation technologies. We propose that

targeting of different compatible solutes to different subcellular locations, and to different organs

for different purposes can lead to additive increases in plant stress tolerance. # 1999 Elsevier

Science B.V. All rights reserved.

Keywords: Compatible solute; Transgenic plants; Radical oxygen scavenging; Ion homeostasis;

Salt stress tolerance; Genetic engineering

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

2. Engineering regulatory circuits or engineering metabolic functions? . . . . . . . . . . . . . . . . . . . . . 240

3. Compatible solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Scientia Horticulturae 78 (1999) 237±260

* Corresponding author.1 Present address: Pioneer HiBred, Johnston, IA, USA.

0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 2 3 8 ( 9 8 ) 0 0 1 9 5 - 2

4. Functions of compatible solutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

5. Compatible solutes and water movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

6. Replacement of a compatible solute in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

7. Analysis of transgenic plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

8. Transfer of multiple genes into model species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

9. Models for cellular stress tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1. Introduction

The success of plants in dealing with fluctuating, (seasonally) increasing, orpermanently high salinity is largely determined by their ability to carry out threereactions: handling sodium exclusion or partitioning; taking up water that may beeither plentiful (e.g. in salt marshes) or scarce (e.g. in drought-stricken areas)against an osmotic gradient; and maintaining homeostasis with respect toessential ions. The generally accepted view is that osmotic adjustment andcompartmentation are central to accomplishing these tasks: the accumulation ofcompatible solutes is often an essential component of this process. Many essentialreactions, including those that attempt to control ion influx to a species-specificacceptable value, are not salt stress-specific, but apply similarly to droughtconditions: this allows us to deal with both salinity and drought stresssimultaneously (Bohnert et al., 1995; Bray, 1997; Jain and Selvaraj, 1997),especially when discussing a prevalent reaction to both stress conditions, theuptake or synthesis of specific solutes.

Most organisms increase the cellular concentration of osmotically activecompounds, termed compatible solutes, when in danger of becoming desiccatedby either drought or external lowering of the osmotic pressure accompanying, forexample, increases in soil salinity (Yancey et al., 1982; Le Rudulier et al., 1984;McCue and Hanson, 1990; Delauney and Verma, 1993). The accumulatingcompounds are `compatible' with normal cellular metabolism at high concentra-tions (Brown and Simpson, 1972). Typically, compatible solutes are hydrophilicgiving rise to the view that they could replace water at the surface of proteins,protein complexes, or membranes. Compatible solute is simply a term; it carries aphysiological meaning which does not explain the function(s) such solutes carryout. The biochemical mechanisms through which compatible solutes protect are

238 H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260

still unknown, but this does not necessarily preclude working on the generation oftransgenic plants in which accumulation of a metabolite is enhanced. Rather thanfocusing on such aspects, our concern is more with mechanisms of compatiblesolute action and how compatible solutes are integrated into a whole plant stressresponse that includes maintenance of ion homeostasis and water relations,carbon/nitrogen partitioning, reserve allocation, or storage (and possibly diffusion)of reducing power (Bieleski, 1982; Blomberg and Adler, 1992; Bohnert et al.,1995; Niu et al., 1995). As we will discuss, there may be more than one functionfor a particular solute (see, Shen et al., 1997a, b) and, based on results from invitro experiments (Smirnoff and Cumbes, 1989; Halliwell and Gutteridge, 1990;Orthen et al., 1994), different compatible solutes may have different functions.

The importance of compatible solute accumulation, interpreted as `osmoticadjustment', had been recognized long ago (e.g. Brown and Simpson, 1972;Borowitzka and Brown, 1974; Levitt, 1980). A correlation between compatiblesolute amount and tolerance has been documented (e.g. Storey and Wyn, 1977;Flowers and Hall, 1978; Bohnert et al., 1995 and references therein). Planttransformation had to be developed before experiments could be designed toreplace correlative relationships by proofs. The logical next step has been theengineering of plants to express enzymes that lead to the synthesis of compatiblesolute and subsequent physiological analysis of these plants. A discussion of whatwe have learned from such experiments will be presented.

Another discussion topic addresses recent experiments which replaced thespecific compatible solute found in one organism by a different compatible solutenot normally present in that organism. Preliminary results indicate that suchsimple replacement might not yield similar protection (Shen et al., 1998).Formulated as a hypothesis, it could be that a particular compatible solute and thecell or tissue in which this solute is found must be `compatible' in a differentsense (see below), namely that the proteins/structures that need to be protectedunder stress have evolved to `fit' a particular compatible solute. While thishypothesis might be falsifiable, the important issue is the question about tissue- ororgan-specificity of the presence of compatible solute. Are there requirements forprotection of, for example, root tissue that are different from the requirementsprotecting leaves or flower structures?

Compatible solute specificity and the elements that constitute stress tolerancemay be compared to interdigitated chainlinks. If the two parents in a crossshowed enhanced resistance to two different aspects of the stress, and sensitivityin other aspects, their progeny might exhibit a weakest link that is different fromthose of the parents (Garcia et al., 1995). As it has been abundantly documented,tolerance of water stress is governed by many genes that act synergistically andadditively (Hickok et al., 1991; Dvorak et al., 1994). Flowers and Yeo (1995)suggested pyramiding of physiological traits as a practical means of generatingsalt-resistance at the present time. They considered a task for the distant future

H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260 239

the suggestion that pyramiding might be accomplished by gene transformation(Bohnert and Jensen, 1996a). We include a recommendation for multigenetransfer, which is possible with present genetic engineering technology, in whichseveral different putative stress resistance mechanisms can be targetedsimultaneously. The problems that stand in the way of technically feasiblemultigene transfers are, however, not to be underestimated. A significant problemis that our understanding of salinity tolerance has increased only with respect tocellular aspects. In contrast, our knowledge is insufficient about the integration ofwhole plant responses in a developmental context. Also, we are still largelyignorant about the choice of appropriate gene control elements for theengineering of stress tolerance.

2. Engineering regulatory circuits or engineering metabolic functions?

Two schools of thought approach crop stress tolerance engineering withfundamentally different concepts. One strategy focuses on biochemical aspects,i.e., engineering downstream reactions that are at the end of signal transductionchains. In this view, what should be engineered are pathways or end-points of bio-chemical pathways and, also, pathways that are absent in the target crop shouldbe introduced. Modifying a species through either overexpression or antisensesuppression of individual proteins, typically enzymes for a desired biochemicalreaction, are frequently used schemes which have become routine; mosttransgenic experiments through which stress tolerance has been analyzed utilizedthis strategy (Tarczynski et al., 1992, 1993; Kishor et al., 1995; Pilon-Smits et al.,1995; HolmstroÈm et al., 1996; Shen et al., 1997a, b; Sheveleva et al., 1997, 1998).

A second approach attempts crop engineering through the alteration of stressperception and signaling. Ideally, this would utilize components of theendogenous stress-relieving mechanisms in the target species. Constitutive orinducible elicitation of a water-stress perceiving and responding signaling systemcould be accomplished that would result in the enhancement or induction ofstress-relieving functions. Variations of this strategy can be imagined, e.g., forstress-sensitive species which lack the appropriate connection between signal andresponse modules. The stress-response itself could also be engineered to behyper-inducible by the stress. All plants include a genetic makeup for stressresponses, and these responses are, likely, coordinately regulated following therecognition of stress. In this scenario, the overexpression of a single signaltransduction pathway intermediate would be sufficient to induce global stressresponses. In higher plants, a (water) stress-related phosphorylation cascadeanalogous to the HOG (`high osmolarity glycerol') pathway for osmotic stresssignaling in yeast seems to act in a very similar fashion (Jonak et al., 1994; Ruisand Schuller, 1995; Shinozaki and Yamaguchi-Shinozaki, 1997). Overexpression


of a stress-related protein kinase or protein phosphatase of this MAP (`mitogen-activated protein')-HOG kinase cascade, for example, would lead to theactivation of the pathway or pathways controlled by this particular stress-signaling intermediate.

A test permitting judgment about the validity of the second concept has not yetbeen reported. However, the gene encoding a subunit of the yeast proteinphosphatase, calcineurin (Di Como et al., 1995; Marquez and Serrano, 1996), hasrecently been expressed in transgenic tobacco (R.A. Bressan and P.M. Hasegawa,personal communication). In yeast, calcineurin has been shown to be involved inthe regulation of several pathways, foremost in the regulation of ion homeostasis(Cyert et al., 1991; Ferrando et al., 1995; Guerini, 1997). According to thepreliminary data, a calcineurin-regulated plant pathway exists which affects ionhomeostasis in transgenic tobacco, and overexpression of the yeast calcineurinincreases potassium uptake and sodium exclusion. Thus, a global enhancement ofexisting plant signal transduction chains for dealing with salt stress could providevaluable information which may lead to applicable protection strategies.

One concern must be raised. If a global increase in tolerance could beaccomplished by the mutation of a single gene or very few genes in a stress-relevant signaling pathway, such a mutation should have been hit upon in variousbreeding programs. Apparently, this has not happened. This might mean thatincreases in signaling-mediated tolerance must occur in more than one signal trans-duction pathway for any increase in salt tolerance to be exhibited. Alternatively,constitutive increases in stress signaling affecting many downstream reactionsmight have other detrimental effects, e.g. on productivity, so that the mutants werenot recognized and then eliminated during screening in classical breeding programs.Yet another possibility is that the permanent enhancement of stress-relievingpathways might lead to epigenetic silencing of the response in the progeny.

3. Compatible solutes

As is discussed in other contributions to this volume, different compounds canfunction as compatible solutes. Potassium, if available, serves this function inmany unicellular organisms (Serrano, 1996) and sufficient potassium in the soilleads to more efficient exclusion of sodium in higher plants (Niu et al., 1995, andreferences therein). Also amino acids and some amino acid derivatives, sugars,acyclic and cyclic polyols, fructans, and quaternary amino and sulfoniumcompounds frequently act as compatible solutes (Levitt, 1980; McCue andHanson, 1990; Delauney and Verma, 1993; Bartels and Nelson, 1994; Bohnertand Jensen, 1996b). Recently, genes have been characterized leading to ectoine(1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), a zwitterionic com-patible solute found in a number of halobacteria which shows exceptional


protection of protein function in in vitro assays (Galinski, 1993; Louis andGalinski, 1997). Typically, pathways leading to their synthesis are connected topathways in general metabolism with high flux rates (Bohnert and Jensen,1996b). Examples are the proline biosynthetic pathway (Delauney and Verma,1993), glycine betaine synthesis (McCue and Hanson, 1990), and the pathwayleading to the methylated inositol, D-pinitol (Vernon and Bohnert, 1992; Ishitaniet al., 1996; Bohnert and Jensen, 1996a, b). We present a description of pinitolbiosynthesis, highlighting essential features that seem to characterize what isrequired of compatible solutes.

Biosynthesis of D-pinitol in the halophyte Mesembryantheumum crystallinum(ice plant) requires increased flux of carbon from glucose 6-phosphate to myo-inositol 1-phosphate and then myo-inositol (Ishitani et al., 1996). The first gene inthe pathway, encoding inositol-1P synthase is transcriptionally upregulated, andincreased protein amounts can be detected (Ishitani et al., 1996; Nelson et al.,1998b). The second enzyme, inositol monophosphatase, is not regulated understress conditions in the ice plant (Mesembryantheumum crystallinum) (NelsonDE, personal communication). Utilizing increased amounts of inositol followingstress, the enzyme myo-inositol O-methyltransferase (IMT), generates D-ononitol(Vernon and Bohnert, 1992). In the ice plant, IMT is only expressed following saltstress, i.e. the protein is virtually absent in unstressed plants and increasesdramatically within one to two days of stress (Vernon and Bohnert, 1992; Nelsonet al., 1998b). Finally, D-ononitol is converted into D-pinitol by an epimerizationreaction which may include more than one enzyme. This activity, which we termOEP, has not yet been characterized biochemically or genetically. There are twosignature features of this pathway. First, the pathway is connected to inositolsynthesis and phospholipid biosynthesis, pathways which are tightly controlled inorganisms in which they have been studied (Nikoloff and Henry, 1991), andbeyond that the synthesis of the methylated inositols is connected to the majorflux of carbon in photosynthetic cells. The second feature is that the pathwayincludes additional enzymes which remove the product from general metabolism.D-pinitol is an extremely stable end-product. The activities of IMT and OEP arenot found in tobacco and Arabidopsis (Vernon et al., 1993; Ishitani et al., 1996;Sheveleva et al., 1997); in fact, genes for these enzymes seem to be missing inthese species.

4. Functions of compatible solutes

Compatible solutes do not interfere with protein structure and function, andthey alleviate inhibitory effects of high ion concentrations on enzyme activity. Itis an osmoregulatory function as osmolytes that is typically assigned to themultitude of compatible solutes which accumulate in response to osmotic stress.


Some solutes, such as trehalose, do not respond to osmotic stress by accumulatingto high amounts but are protective even at low concentrations (Mackenzie et al.,1988; HolmstroÈm et al., 1996). When present at low, osmotically insignificant,concentration such solutes may function as osmoprotectants, i.e. active in amechanism that is non-osmotic as for example in radical oxygen scavenging. Wehave, for example, recently shown that mannitol at concentrations of less than100 mM in chloroplasts specifically reduces damage by hydroxyl radicals generatedthrough the Fenton reaction (Shen et al., 1997a, b). While the net increase of soluteslowers the osmotic potential of the cell which supports the maintenance of waterbalance under osmotic stress, the net lowering of the solute potential may notbe the only, or not even the essential function of a compatible solute.

The main function of a compatible solute may be the stabilization of proteins,protein complexes or membranes under environmental stress. In in vitroexperiments, compatible solutes at high concentrations have been found toreduce the inhibitory effects of ions on enzyme activity (Pollard and Wyn Jones,1979; Yancey et al., 1982; Brown, 1990; Solomon et al., 1994). The addition ofcompatible solutes increased the thermal stability of enzymes (Back et al., 1979;Paleg et al., 1981; Galinski, 1993), and prevented dissociation of the oxygen-evolving complex of photosystem II (Papageorgiou and Murata, 1995). Oneargument often raised against these studies is that the effective concentration ofcompatible solute necessary for protection in vitro is very high, approximately500 mM. Such high concentrations are rarely found in vivo. However, when weconsider the high concentration of proteins in cells, the concentration ofcompatible solute necessary for protection can, we think, be much lower than thatrequired for protection in in vitro assays. In addition, it may not be theconcentration of compatible solute in solution that is important. Glycine betaine(which may be present in high or low amounts), for example, protects thylakoidmembranes and plasma membranes against freezing damage or heat destabiliza-tion (Coughlan and Heber, 1982; Jolivet et al., 1982; Zhao et al., 1992), indicatingthat the local concentration on membranes or protein surfaces may be moreimportant than the absolute concentration.

Two theoretical models have been proposed to explain protective or stabilizingeffects of compatible solutes on protein structure and function. The first is termedthe `preferential exclusion model' (Arakawa and Timasheff, 1985) according towhich compatible solutes are largely excluded from the hydration shell ofproteins, which stabilizes protein structure or promotes or maintains protein/protein interactions. Compatible solutes in this model would not disturb thenative hydration water of proteins, but they would interact with the bulk waterphase in the cytosol. The second model, the `preferential interaction model', incontrast, emphasizes interactions between compatible solutes and proteins(Schobert, 1977). The protein's hydration shell is crucial for structural stability.During water deficit, compatible solutes may interact directly with the


hydrophobic domains of proteins and prevent their destabilization, or they maysubstitute for water molecules in the vicinity of such regions. While the twomodels seem to be mutually exclusive at first sight, the actual function ofcompatible solutes may in fact be explained by both models. The structures ofdifferent compatible solutes could accommodate hydrophobic, van-der-Waalsinteractions, and charged interactions, but further experiments will be necessaryto gain a better insight into the stabilizing effects of compatible solutes that havebeen documented in in vitro experiments.

Compatible solutes may also function as oxygen radical scavengers. Evidencefor such a function comes from studies on fungal pathogen interactions where thepathogens are protected by the synthesis and secretion of mannitol. Plants andanimals produce oxygen radicals in response to pathogen attack. The rapidproduction and local accumulation of reactive oxygen species leads to localizedcell death in the host which then may limit spread of the pathogen (Tenhakenet al., 1995). In response, some pathogens seem to have evolved mechanismswhich detoxify the reactive oxygen species produced by the host. Cryptococcusneoformans, a yeast which opportunistically infects humans with a compromisedimmune system, produces and secretes mannitol. A mutant strain that does notproduce mannitol is less virulent (Niehaus and Flynn, 1994). Similarly, thetomato pathogen, Cladosporium fulvum, produces mannitol during the infectionprocess, which seems to protect the fungus from damage by reactive oxygenspecies produced by the plants (Joosten et al., 1990).

Mannitol has been shown in vitro to function as a scavenger of reactive oxygenspecies, ROS (Elstner, 1987; Halliwell et al., 1988). ROS is a generic term whichis used to include not only free radicals, such as superoxide and hydroxyl radicalsbut also singlet oxygen and H2O2. Smirnoff and Cumbes (1989) designedexperiments that compared the radical scavenging capabilities of differentcompatible solutes. They reported that mannitol, sorbitol, glycerol, proline, ononitoland pinitol were active scavengers, although at different concentrations in vitro,whileglycine betainewas not able to scavenge radicals (Smirnoff and Cumbes, 1989;Orthen et al., 1994). The relative radical scavenging efficiency of these compoundsseemed dependent on their rate constants for reactions with hydroxyl radicals.For example, the rate constant of mannitol is four-fold higher than that of proline(Buxton et al., 1988), and thus mannitol was more effective than proline as ahydroxyl radical scavenger. Under water deficit conditions, radical productionincreases in plants (Moran et al., 1994), and it may be that the accumulation ofpolyols provides some protective effect against oxidative damage of proteins.

Recently, results have been reported which shed light on the radical scavengingcapacity of mannitol in in vivo experiments (Shen et al., 1997a, b). Mannitol1-phosphate dehydrogenase was modified such that the protein was imported intochloroplasts, and the gene construct was expressed in transgenic tobacco. Weargued that a potential function in radical scavenging in vivo might best be


demonstrated with chloroplasts which abundantly produce a variety of reactiveoxygen species when stressed by water deficit (for a recent review, see Noctorand Foyer, 1998). Mannitol was present in concentrations of approximately100 mM in the chloroplasts (Shen et al., 1997a). Using different conditions, suchas illumination with high light, paraquat treatment, enhanced H2O2 generation,and DMSO infiltration, it could be shown that plants containing mannitol in theirchloroplasts were better able to maintain high carbon fixation rates and showedless chlorophyll bleaching (Shen et al., 1997a) than plants without mannitol.Further experiments indicated that mannitol was active specifically againsthydroxyl radicals and not against hydrogen peroxide or radical oxygen. This isimportant information, considering that chloroplast detoxification systems existthat can deal with H2O2 and radical oxygen, while there is no enzyme systemdescribed that could deal with the extremely short-lived and highly reactivehydroxyl radicals.

Further experiments (Shen et al., 1997b) indicated that even under high lightconditions the major effect of increased hydroxyl radical production was largelyrestricted to the dark reactions of photosynthesis, while the photosystemsthemselves functioned normally. It could be demonstrated that some enzymes ofthe Calvin-cycle were predominantly affected by hydroxyl radicals. Phosphori-bulokinase, PRK, and likely other SH-enzymes of the Calvin-cycle, showedsensitivity to hydroxyl radicals, and the activity of PRK was protected by thepresence of mannitol (Shen et al., 1997b).

5. Compatible solutes and water movement

Little is known about the relationships between compatible solute synthesis,water transport and ion uptake (or sodium exclusion). Loss of turgor followingwater deficit caused either by lowering of water uptake through roots orcontinued evapotranspiration through stomata is very likely a signal forcompatible solute synthesis, possibly through a pathway that is similar to theyeast high osmolarity glycerol osmotic signaling pathway (Shinozaki andYamaguchi-Shinozaki, 1997). That water movement is not regulated, but simplyfollows osmotic gradients, must be questioned.

Recently, water channel proteins (aquaporins), which have been detected in allorganisms from bacteria to humans, have been implicated as the major facilitatorsfor the movement of water across membranes (Chrispeels and Agre, 1994). Forsome of these proteins it has been demonstrated that they function as aquaporinsby a swelling test of Xenopus oocytes after the RNA encoding the putativeaquaporin has been injected into the oocytes. Most of these proteins seem tofacilitate water flux along an existing osmotic gradient, while others transportother metabolites, such as glycerol (Luyten et al., 1995; Yang and Verkman,


1997). In plants, two subfamilies of water channel genes have been distinguished.In Arabidopsis thaliana, for example, each subfamily of genes encompasses 10±12 members (Weig et al., 1997) which are expressed in a organ- anddevelopmental stage-specific mode. The protein products from each subfamilyare targeted to either the plasma membrane or the tonoplast, respectively(Kammerloher et al., 1994; Yamada et al., 1995; Weig et al., 1997). Several iceplant water channel mRNAs and proteins have been found to decline during saltstress (Yamada et al., 1995; Kirch, H.H. and Bohnert, H.J., unpublished). Adynamic way of regulating water movement seems to involve aquaporin turnoverand/or post-translational modifications. A decrease in water channel proteins intransgenic Arabidopsis by an antisense strategy lowered the water permeability ofthe plasma membrane of protoplasts in comparison to wild type plants andreduced bursting of protoplasts in hypo-osmotic media (Kaldenhoff et al., 1995).The whole-plant phenotype of Arabidopsis containing less aquaporin proteinsseems to be that the root to shoot ratio is drastically increased (Kaldenhoff, R.,personal communication). Regulation of water permeability may also be throughregulated protein modifications (Maurel et al., 1995; Johanson et al., 1996). Aputative plasma membrane aquaporin from spinach is reversibly phosphorylatedat multiple sites in the protein in response to changes in calcium and a lowering ofthe apoplastic water potential (Johanson et al., 1996). The function of waterchannels in maintaining water balance across membranes under osmotic stress isstill debated. Water channels could facilitate water uptake in roots if theirpresence in the plasma membrane were synchronized with the synthesis andaccumulation of metabolites that lead to osmotic adjustment; their removal fromthe membrane might restrict the loss of water. Similarly, the amount or regulationof tonoplast-located water channel proteins might be involved in determining ionpartitioning to the vacuole. As one example, we have observed using peptide-specific antibodies that tonoplast-located water channel proteins decline in theroot of the ice plant within hours following salt stress (Golldack, D., Kirch, H.H.,Bohnert, H.J., unpublished) which will alter water and ion flux into thevasculature. Additional experiments are needed to investigate whether and howmuch water channel proteins are involved in the regulation of water movement inwhole plants under stress conditions.

6. Replacement of a compatible solute in yeast

When yeast, S. cerevisiae, is stressed by high salinity, a signal transductionrelay of phosphorylation events (high osmolarity glycerol-pathway; Albertynet al., 1994; Posas et al., 1996) leads to induction of several genes. Among these,one of two GPD genes, encoding a glycerol 3-phosphate dehydrogenase, isinduced resulting in increased production of glycerol (Luyten et al., 1995). The


second of the GPD genes, which is not induced by osmotic stress but by anaerobiosis,contributes less enzyme (Ansell et al., 1997). Simultaneously, regulation of theglycerol facilitator protein, belonging to the water channel gene family, retardsthe cell's loss of glycerol which then accumulates to approximately 400 mMinside cells. When both GPD genes are inactivated, the cells become extremelysalt-sensitive (Albertyn et al., 1994; Ansell et al., 1997), which provides furthercredence to the view of glycerol as a compatible solute. We have used a mutantlacking both GPD genes, and replaced their function by enzymes leading tomannitol and sorbitol synthesis, respectively (Shen et al., 1998). The expressionof a sorbitol dehydrogenase and a mannitol dehydrogenase led to sorbitolamounts, 375 mM, which were as high as the amounts of glycerol in wild type;mannitol accumulated to a slightly lower amount, 213 mM. In all lines, trehaloseaccumulation was comparable. Surprisingly, the degree of salt tolerance acquiredby the sorbitol- and mannitol-accumulating lines was not comparable to thetolerance generated by 400 mM glycerol. The I50, the concentration of NaCl thatinhibited growth by 50%, was 1 M for the mutant line into which the GPD genewas re-introduced and 0.55 M for sorbitol- and mannitol-producing plants, whichwas not much different from the I50 of 0.4 M for the mutant line lacking GPDenzymes (Shen et al., 1998). The result seems to indicate that the osmoticadjustment component of compatible solutes must be considered marginal.

This result argues against compatible solute having an unspecific function.They seem to be more than simple osmolytes. It could be that the biochemicalpathway through which glycerol is synthesized in yeast is more important thanthe amount that is being made. In favour of this argument, yeast does not retainglycerol very well once synthesized; more than 90% of the glycerol is found inthe medium over a period of approximately three generations (Shen et al., 1998).The pathway leading to sorbitol or mannitol, respectively, may not be compatiblewith the function that is provided by increased glycerol synthesis. Since the entirepathway leading to glycerol biosynthesis consumes more NADH than mannitolbiosynthesis, another possible explanation is that compatible solute biosynthesisserves the purpose of reducing cellular NADH levels. Alternatively, glycerolmight interact with yeast proteins better than sorbitol or mannitol. Thisdisconcerting notion might mean that protein structure and the type of compatiblesolute found in a particular species have adapted evolutionarily, clearly a dauntingprospect for stress tolerance engineering.

7. Analysis of transgenic plants

Plant transformation/regeneration has become routine in many species, even inthose that were considered recalcitrant, such as rice or corn (Komari et al., 1996;Heath et al., 1997). Equally, the necessary manipulations and sequence


modifications for gene constructions are commonplace techniques by now andmay in the near future become contract services. Genes encoding enzymes whichenhanced the synthesis of putatively protective osmolytes have been transferredby several groups, and the transgenic plants were analyzed for their performanceduring chilling, drought, salinity stress and an increase in reactive oxygen species,for example, by exposure to methyl viologen (Table 1). However, plant growthand stress treatments were invariably carried out under controlled conditions ingrowth chambers, not under field conditions, and small populations of seedlingsor young plants have been used in these experiments (Table 1).

In all experiments, the transgenic plants showed differences compared to wildtype, but these differences were small, or were restricted to narrow developmentalstages, or they were followed for short time periods, never for an entire growingseason. Most clearly documented is a protective effect of enzymes that act inH2O2 detoxification, superoxide dismutases and enzymes of the ascorbate±glutathione cycle (Bowler et al., 1991; McKersie et al., 1993; Van Camp et al.,1996; Roxas et al., 1997). In addition, mannitol, possibly not only in chloroplasts,seems to act in hydroxyl radical scavenging (Shen et al., 1997a, b). Unknown isthe function of over-expression of a barley LEA (late embryogenesis-abundant)protein in rice, which leads to a higher than wild type growth rate of the plantsunder stress (Xu et al., 1996). The expression of a bacterial levansucrase led tofructan accumulation (Pilon-Smits et al., 1995), and expression of a subunit of theyeast trehalose synthase resulted in low amounts of trehalose (HolmstroÈm et al.,1996). In both cases, and in the case of mannitol accumulation in the cytosol(Tarczynski et al., 1993; Thomas et al., 1995), the protective effects cannot beexplained by osmotic adjustment because of the low amounts of accumulatingmetabolites. In contrast, the overexpression of a D

1-pyrroline-5-carboxylatesynthetase, leading to increased proline biosynthesis (Kishor et al., 1995),generated proline in an osmotically significant high amount. Similarly, themethylated inositol, D-ononitol, produced by the action of a methyltransferase(Sheveleva et al., 1997) accumulated under drought conditions to approximately600 mM with a small protective effect.

These analyses are not without criticism. Mannitol accumulation, for example,has been reported to lead to slower growth of the transgenic plants even in theabsence of stress (Karakas et al., 1997). Consequently, the purported stressprotection has been interpreted as an effect of slower growth leading to lesssodium uptake under stress. Growth retardation has indeed been observed in somelines which are characterized by very high mannitol or sorbitol accumulation(Sheveleva et al., 1998), but growth is not significantly lower in lines that containless than approximately 50 mM of mannitol in total cell water, while a slightprotective effect can still be demonstrated.

Other experiments cast a more significant shadow on the overexpressionschemes that have been employed (Table 1). In a series of near-isogenic maize


Table 1

Transgenically expressed proteins with an effect in water deficit, salinity stress, or oxygen radical protection

Gene (species) Enzyme Host species Notes Reference

MnSOD

(N. plumbaginifolia)

Mn-SOD N. tabacum Organelle targeted expression leading

to reduced damage by ROSaBowler et al., 1991

M. sativa McKersie et al., 1993, 1996

MtlD (E. coli) Mannitol 1-P DH N. tabacum Sodium tolerance at early growth Tarczynski et al., 1992, 1993

A. thaliana Enhanced seed germination in NaCl Thomas et al., 1995

N. tabacum Chloroplast location, ROS scavenging;

protection of calvin-cycle

Shen et al., 1997a, b

Hva1 (H. vulgare) HVA1-LEA O. sativa Maintenance of higher growth rate by

stressed plants

Xu et al., 1996

Imt1 (ice plant) myo-inositol

O-methyltransferase

N. tabacum Stress-induced accumulation of D-ononitol Sheveleva et al., 1997

SacB (B. subtilis) Levansucrase N. tabacum Fructan accumulation; higher growth rate

during drought stress

Pilon-Smits et al., 1995

Tps1 (S. cerevisiae) Trehalose synthase N. tabacum Low conc. trehalose increased drought

tolerance

HolmstroÈm et al., 1996

CodA (A. globiformis) Choline oxidase A. thaliana Glycine betaine accumulation enhanced

tolerance

Hayashi et al., 1997

P5CS (V. aconitifolia) P5CS N. tabacum Proline accumulation leading to lowering

of osmotic potential

Kishor et al., 1995

FeSOD (A. thaliana) Fe-SOD N. tabacum PSII/plasma membrane protection/methyl

viologen

Van Camp et al., 1996

Gst/Gpx (N. tabacum) GST/GPX N. tabacum Increase of oxidized glutathione (GSSG)

enhanced seedling growth

Roxas et al., 1997

aReactive oxygen species.

While the effects of overexpression indicate protection, the mechanisms leading to enhanced tolerance under controlled growth conditions are not

understood. A note of caution has recently been voiced (Karakas et al., 1997). Accumulation of mannitol transgenic tobacco line was shown to reduce

growth by up to 40%. Such reduction in growth might lead to less sodium uptake which might be misinterpreted as an increase in tolerance.

H.J.

Bo

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ert,B

.S

hen

/Scien

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orticu

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e78

(1999)

237±260

249

lines obtained by classical breeding strategies and differing only in glycinebetaine content, responses to salinity stress were monitored. A correlation wasestablished between the amount of glycine betaine and tolerance (Saneoka et al.,1995). Clearly, the lines containing glycine betaine showed higher carbonassimilation and maintained a higher leaf relative water content than the glycinebetaine-deficient lines. In field trials, however, the glycine betaine accumulatorswere more prone to fungal diseases and lodging, which negated the effect thatmight be provided by a high osmolyte content under drought conditions as theymight occur in the field (Rhodes, D., personal communication).

8. Transfer of multiple genes into model species

Transfer of multiple genes has been contemplated by several groups butexperimental results have not yet become available. We will discuss require-ments that seem to emerge from published attempts for the purpose of increasingwater deficit/salinity stress tolerance. First, the low amounts of fructan, mannitol,or trehalose that show some protection seem to indicate that the compatiblesolutes have a specific protective role, rather than acting as osmolytes.Accumulation might not be necessary. Also, different compatible solutes seemto have different functions. Clearly, glycine betaine does not act as a hydroxylradical scavenger in the way mannitol does (Shen et al., 1997b), but glycinebetaine does confer some protection against sodium effects when present intransgenic plants (Hayashi et al., 1997). Thus, one suggestion is that we shouldtransfer genes leading to the synthesis of different compatible solutes at low ormoderate amounts.

Engineering of compatible solutes should be supported by engineering ofscavenging systems for reactive oxygen species. This is not only indicated by theslight protective effects that have been observed after engineering of reactiveoxygen species scavenging enzymes into plants (e.g. Bowler et al., 1991;McKersie et al., 1996; Noctor and Foyer, 1998), but also from the results obtainedwith plants containing engineered mannitol in the chloroplast compartment (Shenet al., 1997a, b). In a model study, we attempted to measure the production ofhydroxyl radicals via the product, MSA (methane sulfinic acid), of hydroxylradicals with DMSO (dimethyl sulfoxide). In these experiments, mannitol in thechloroplasts clearly competed with DMSO for hydroxyl radicals because lessMSA was formed. It was found that cells could tolerate a stress that nearlydoubled the amount the hydroxyl radicals that are found under non-stressconditions, and that mannitol at 100 mM in the plastids again approximatelydoubled the hydroxyl radical scavenging capacity of these cells (Shen, 1997).These results are compatible with the measurements and estimations of hydroxylradical production in cells in vitro and with the estimations about the contribution


of endogenous reactive oxygen species scavenging systems (Smirnoff, 1993;Asada, 1994; Allen, 1995; Noctor and Foyer, 1998).

A total of seven genes are being used for our first attempts at multigenetransfer, again using tobacco as the model species (Nelson, D.E., Zhu, G.,Michalowski, C.B. and Bohnert, H.J., unpublished). Included in the geneconstructions are three genes which enhance the biosynthesis of myo-inositol andD-ononitol in the cytosol; one gene leads to moderate mannitol accumulation inplastids; trehalose synthesis is enhanced in the cytosol; and two genes, Fe-SODand a cytosolic ascorbate peroxidase, respectively, are included for the provisionof enhanced reactive oxygen species scavenging. Most of the genes are derivedfrom the halophytic ice plant which has become the most intensely studied modelfor natural salinity stress tolerance (Adams et al., 1998). The genes which weutilized for the transfer into tobacco show up-regulated mRNA amounts undersalt stress in the ice plant (Michalowski, C.B. and Bohnert, H.J., unpublished).Many of these upregulated transcripts are also regulated under salinity stress inyeast which must be considered the best model for cellular stress tolerance(Serrano, 1996; Nelson et al., 1998a).

9. Models for cellular stress tolerance

The most widely used organism for transgenic analyses of environmental stresstolerance is Nicotiana tabacum L. It has been pointed out that tobacco, which isnot particularly salt sensitive, may be less than ideal as a salt stress model(Murthy and Tester, 1996), because the plant reacts predominantly to the decreasein water potential that accompanies salt stress and not to sodium toxicity. Whilethis is indeed the case, the advantages of using tobacco as a transgenicbiochemical model outweigh this problem. In fact, we consider the osmoticaspects of salinity stress to have a far greater effect on plants than the toxic effectsof sodium (see also, Cheeseman, 1988). In other models used, Arabidopsisthaliana, Medicago sativa, and Oryza sativa, equally successful, and equallylimited, improvements of abiotic stress tolerance have been reported (Thomaset al., 1995; Xu et al., 1996; McKersie et al., 1996). Another model species,Saccharomyces cerevisiae, currently may be the best model for understandingsalinity tolerance mechanisms (Serrano, 1996; Nelson et al., 1998a). First, yeastis salt tolerant, and mutants which are salt sensitive are readily identifiable. Notonly do these mutants allow identification of important salinity tolerance genes,but also by complementation, they allow the identification of homologs fromother species as well as providing useful salt-sensitive strains for a variety ofphysiological and transgenic experiments. Second, the entire genomic andmitochondrial sequences are available. Analysis of the yeast genes has broken thebarrier of gene availability, and with the plant genes identified by complementa-


tion we now have a large repertoire of coding sequences available ± a prerequisitefor multigene transfer for pyramiding desirable traits. Finally, the completion ofthe yeast genome sequence now allows analysis of all proteins (`proteome';Oliver, 1996) in their location during the life-cycle through which additional,important aspects of gene expression will be identified, and the function of allreading frames will become known.

The yeast genome includes approximately 5800 translated reading frames(Dujon, 1996). Based on several analyses and the following considerations,approximately 100 may be basic to salinity stress tolerance. When the genePBS2, encoding MAP kinase of the high osmolarity glycerol osmosignalingpathway, was deleted, proteins controlled by this pathway could be documentedby their disappearance from 2D gels (Akhtar et al., 1997). The authors reported29 protein spots affected by this deletion. Assuming that maybe one third of theyeast proteins are of sufficiently high abundance to be visible on 2D gels, byextrapolation a number of 100 closely stress-associated genes seems a reasonableestimate. Also, the number of genes that are essential to tolerance as determinedby gene deletion is approximately 10±20. That a significantly larger numbershould aid in tolerance but not be absolutely necessary is reasonable, and thisconsideration, again, puts the estimate in the 10±100 genes. A similar estimate,on the order of 100 up-regulated transcripts under salt stress, has been obtainedafter screening lambda phage clones representing 3.2 Mb, or approximately 1%,of the ice plant genome (Meyer et al., 1990). Since the processes controlled bythe known yeast genes are similar to those found or suggested as essentialreactions of plants under salt/drought stress, we suggest that large scale metabolicengineering of crop plants should start by utilizing the plant genes that arefunctionally equivalent to these yeast genes.

10. Conclusions

Yeast as a model for cellular osmotic stress tolerance in plants is studied byseveral groups at present. The integration of the cellular responses with thoseexhibited by the whole plant will be the next challenge. In higher plants it is stilldifficult to connect putative mechanisms with the phenotypes that characterizeosmotic and ionic stress tolerance, and it has been impossible to identify all geneswhose products generate the mechanisms. Even in the cases where sufficientcorrelative evidence exists, metabolic engineering remains risky without knowingthese genes and their functions. Yeast can provide many of the genes, and theirhomologs in higher plants can be studied. At present, attempts at engineeringsalinity tolerance in whole plants can be compared to following single pages of aninstruction manual without page numbers. Work with yeast has providedindividual chapters, but we still have to guess how the sections fit together for


understanding the cellular salinity stress tolerance manual in higher plants. Forother sections, covering developmental aspects of stress tolerance, for example,tissue and organ interactions, yeast will be a less suitable model.

Apart from knowing about the genes, it is also important to know how they areexpressed. The technical capability exists for analyzing and quantifying order,magnitude, and complexity of the expression of all genes during a growth cycleby microarray analysis (Schena et al., 1996; Shalon et al., 1996). Then, byobserving changes in the order and amount of expression following the additionof NaCl, we will gain information about the dynamic progression of gene activitychanges, about which gene products may be rate-limiting, and which arenecessary for maintenance of a new steady-state. This analysis is already feasiblefor yeast. In higher plants, similar analyses will soon be possible with thecollections of cloned expressed sequence tags from several model organisms,mainly from Arabidopsis thaliana, rice and corn (several addresses can be foundon the world-wide-web, although most of the corn EST [expressed sequence tags]are not available to the public).

Improvements in gene transfer technology are a third essential requisite for arational tolerance-engineering strategy. Following the identification of importantgenes and their expression during salinity stress, multiple genes will have to betransferred for the analysis of salinity tolerance in transgenic model plants. Theability now exists for the transfer of fragments of DNA, which could includehundreds of genes (Hamilton et al., 1996), to an increasing number of plants,including many important crop species (Zupan and Zambryski, 1995; Hanson andChilton, 1996; Komari et al., 1996; Heath et al., 1997). In addition, methods arenow available for removing undesirable selectable markers after transformation(Komari et al., 1996). Removing coding regions by targeted gene disruptionthrough homologous recombination, which has recently been reported forArabidopsis (Kempin et al., 1997), poses no insurmountable problem any longer.One significant limitation still exists. Currently missing is a sufficiently large andcomplex set of plant promoters with cell-specific, tissue-specific, developmentalstage-specific, and/or inducible patterns of expression. It can be expected that theongoing genome and EST sequencing projects will provide some information.Microarray analysis of gene expression in a judicious selection of species,utilizing halophyte and glycophyte models, will, we hope, eliminate this problemin the near future and will allow the assembly of a library of plant promoters. Wehave begun multigene transfer into tobacco as a model plant. Determined by theavailability of genes for functions that have been identified in previous experiments,our first attempts are designed to achieve an increase of reactive oxygen speciesscavenging, metabolite accumulation in cytosol and organelles, and increasedsynthesis of inositol which we think is essential for growth under stress.

Finally, the impact of salinity tolerance-engineering, if we assume thatsignificant improvements in plant tolerance can be achieved, must be considered.


The extent to which plants can be made tolerant to sodium in the soil is unknown.More significantly, stress tolerance and productivity must be positivelycorrelated. The concentration of NaCl that higher plants may tolerate is limitedby the ability of the plants to store NaCl in vacuoles, by the plant's capability foropening stomata under saline conditions, by the energy drain that is associatedwith increased proton pumping, and the energy expenditure for maintainingsignificant amounts of osmotically compensating metabolites. It is impossible topredict where the limits may be, but it is certain that constitutively expressedincreased tolerance will be associated with a cost that will limit productivity. Thiscost, possibly, might be minimized by utilizing stress-inducible transgenes. Also,targeting tolerance to seawater strength sodium, approximately 430 mM,approximately 33 parts per thousand (ppt) of sodium, seems unrealistic. It doesseem possible, however, that an improvement in tolerance to half seawater or to10±15 ppt of sodium will be achievable. Tolerance of this level, whilemaintaining growth and seed set, would constitute a significant improvementconsidering that most crop species are adapted to produce only at approximately5 ppt or less sodium in the soil. The path towards this goal will require thetransfer of many genes. Time will have to be devoted to testing appropriateexpression characteristics of these transgenes so that the transgenic plants canbecome material for breeding programs.

Acknowledgements

Work in the laboratory has been or is supported by the U.S. Department ofEnergy (Biological Energy Program), the National Science Foundation(Integrative Plant Biology Program), the U.S. Department of Agriculture(National Research Initiative, Plant Responses to the Environment Program)and the Arizona Agricultural Experiment Station. Part of the work has also beenfunded by NEDO, Japan, in a collaborative international program. Visitingscientists have been supported by the Rockefeller Foundation, Japan Tobacco,Inc., the Smithsonian Institution/Carnegie-Mellon Foundation, the JapaneseSociety for the Promotion of Science, and the Deutsche Forschungsgemeinschaft.

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