Scientia Horticulturae 78 (1999) 237±260
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
* 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
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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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The success of plants in dealing with fluctuating, (seasonally) increasing, or
permanently high salinity is largely determined by their ability to carry out three
reactions: handling sodium exclusion or partitioning; taking up water that may be
either plentiful (e.g. in salt marshes) or scarce (e.g. in drought-stricken areas)
against an osmotic gradient; and maintaining homeostasis with respect to
essential ions. The generally accepted view is that osmotic adjustment and
compartmentation are central to accomplishing these tasks: the accumulation of
compatible solutes is often an essential component of this process. Many essential
reactions, including those that attempt to control ion influx to a species-specific
acceptable value, are not salt stress-specific, but apply similarly to drought
conditions: this allows us to deal with both salinity and drought stress
simultaneously (Bohnert et al., 1995; Bray, 1997; Jain and Selvaraj, 1997),
especially when discussing a prevalent reaction to both stress conditions, the
uptake or synthesis of specific solutes.
Most organisms increase the cellular concentration of osmotically active
compounds, termed compatible solutes, when in danger of becoming desiccated
by either drought or external lowering of the osmotic pressure accompanying, for
example, increases in soil salinity (Yancey et al., 1982; Le Rudulier et al., 1984;
McCue and Hanson, 1990; Delauney and Verma, 1993). The accumulating
compounds are `compatible' with normal cellular metabolism at high concentrations (Brown and Simpson, 1972). Typically, compatible solutes are hydrophilic
giving 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 a
physiological meaning which does not explain the function(s) such solutes carry
out. The biochemical mechanisms through which compatible solutes protect are
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239
still unknown, but this does not necessarily preclude working on the generation of
transgenic plants in which accumulation of a metabolite is enhanced. Rather than
focusing on such aspects, our concern is more with mechanisms of compatible
solute action and how compatible solutes are integrated into a whole plant stress
response 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 function
for a particular solute (see, Shen et al., 1997a, b) and, based on results from in
vitro 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 `osmotic
adjustment', had been recognized long ago (e.g. Brown and Simpson, 1972;
Borowitzka and Brown, 1974; Levitt, 1980). A correlation between compatible
solute amount and tolerance has been documented (e.g. Storey and Wyn, 1977;
Flowers and Hall, 1978; Bohnert et al., 1995 and references therein). Plant
transformation had to be developed before experiments could be designed to
replace correlative relationships by proofs. The logical next step has been the
engineering of plants to express enzymes that lead to the synthesis of compatible
solute and subsequent physiological analysis of these plants. A discussion of what
we have learned from such experiments will be presented.
Another discussion topic addresses recent experiments which replaced the
specific compatible solute found in one organism by a different compatible solute
not normally present in that organism. Preliminary results indicate that such
simple replacement might not yield similar protection (Shen et al., 1998).
Formulated as a hypothesis, it could be that a particular compatible solute and the
cell or tissue in which this solute is found must be `compatible' in a different
sense (see below), namely that the proteins/structures that need to be protected
under stress have evolved to `fit' a particular compatible solute. While this
hypothesis might be falsifiable, the important issue is the question about tissue- or
organ-specificity of the presence of compatible solute. Are there requirements for
protection of, for example, root tissue that are different from the requirements
protecting leaves or flower structures?
Compatible solute specificity and the elements that constitute stress tolerance
may be compared to interdigitated chainlinks. If the two parents in a cross
showed enhanced resistance to two different aspects of the stress, and sensitivity
in other aspects, their progeny might exhibit a weakest link that is different from
those 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 and
additively (Hickok et al., 1991; Dvorak et al., 1994). Flowers and Yeo (1995)
suggested pyramiding of physiological traits as a practical means of generating
salt-resistance at the present time. They considered a task for the distant future
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the suggestion that pyramiding might be accomplished by gene transformation
(Bohnert and Jensen, 1996a). We include a recommendation for multigene
transfer, which is possible with present genetic engineering technology, in which
several different putative stress resistance mechanisms can be targeted
simultaneously. The problems that stand in the way of technically feasible
multigene transfers are, however, not to be underestimated. A significant problem
is that our understanding of salinity tolerance has increased only with respect to
cellular aspects. In contrast, our knowledge is insufficient about the integration of
whole plant responses in a developmental context. Also, we are still largely
ignorant about the choice of appropriate gene control elements for the
engineering of stress tolerance.
2. Engineering regulatory circuits or engineering metabolic functions?
Two schools of thought approach crop stress tolerance engineering with
fundamentally different concepts. One strategy focuses on biochemical aspects,
i.e., engineering downstream reactions that are at the end of signal transduction
chains. In this view, what should be engineered are pathways or end-points of biochemical pathways and, also, pathways that are absent in the target crop should
be introduced. Modifying a species through either overexpression or antisense
suppression of individual proteins, typically enzymes for a desired biochemical
reaction, are frequently used schemes which have become routine; most
transgenic experiments through which stress tolerance has been analyzed utilized
this 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 stress
perception and signaling. Ideally, this would utilize components of the
endogenous stress-relieving mechanisms in the target species. Constitutive or
inducible elicitation of a water-stress perceiving and responding signaling system
could be accomplished that would result in the enhancement or induction of
stress-relieving functions. Variations of this strategy can be imagined, e.g., for
stress-sensitive species which lack the appropriate connection between signal and
response modules. The stress-response itself could also be engineered to be
hyper-inducible by the stress. All plants include a genetic makeup for stress
responses, and these responses are, likely, coordinately regulated following the
recognition of stress. In this scenario, the overexpression of a single signal
transduction pathway intermediate would be sufficient to induce global stress
responses. In higher plants, a (water) stress-related phosphorylation cascade
analogous to the HOG (`high osmolarity glycerol') pathway for osmotic stress
signaling in yeast seems to act in a very similar fashion (Jonak et al., 1994; Ruis
and Schuller, 1995; Shinozaki and Yamaguchi-Shinozaki, 1997). Overexpression
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241
of a stress-related protein kinase or protein phosphatase of this MAP (`mitogenactivated protein')-HOG kinase cascade, for example, would lead to the
activation of the pathway or pathways controlled by this particular stresssignaling intermediate.
A test permitting judgment about the validity of the second concept has not yet
been reported. However, the gene encoding a subunit of the yeast protein
phosphatase, calcineurin (Di Como et al., 1995; Marquez and Serrano, 1996), has
recently been expressed in transgenic tobacco (R.A. Bressan and P.M. Hasegawa,
personal communication). In yeast, calcineurin has been shown to be involved in
the regulation of several pathways, foremost in the regulation of ion homeostasis
(Cyert et al., 1991; Ferrando et al., 1995; Guerini, 1997). According to the
preliminary data, a calcineurin-regulated plant pathway exists which affects ion
homeostasis in transgenic tobacco, and overexpression of the yeast calcineurin
increases potassium uptake and sodium exclusion. Thus, a global enhancement of
existing plant signal transduction chains for dealing with salt stress could provide
valuable information which may lead to applicable protection strategies.
One concern must be raised. If a global increase in tolerance could be
accomplished by the mutation of a single gene or very few genes in a stressrelevant signaling pathway, such a mutation should have been hit upon in various
breeding programs. Apparently, this has not happened. This might mean that
increases in signaling-mediated tolerance must occur in more than one signal transduction pathway for any increase in salt tolerance to be exhibited. Alternatively,
constitutive increases in stress signaling affecting many downstream reactions
might have other detrimental effects, e.g. on productivity, so that the mutants were
not recognized and then eliminated during screening in classical breeding programs.
Yet another possibility is that the permanent enhancement of stress-relieving
pathways 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 can
function as compatible solutes. Potassium, if available, serves this function in
many unicellular organisms (Serrano, 1996) and sufficient potassium in the soil
leads to more efficient exclusion of sodium in higher plants (Niu et al., 1995, and
references therein). Also amino acids and some amino acid derivatives, sugars,
acyclic and cyclic polyols, fructans, and quaternary amino and sulfonium
compounds frequently act as compatible solutes (Levitt, 1980; McCue and
Hanson, 1990; Delauney and Verma, 1993; Bartels and Nelson, 1994; Bohnert
and Jensen, 1996b). Recently, genes have been characterized leading to ectoine
(1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), a zwitterionic compatible solute found in a number of halobacteria which shows exceptional
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protection of protein function in in vitro assays (Galinski, 1993; Louis and
Galinski, 1997). Typically, pathways leading to their synthesis are connected to
pathways 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 pathway
leading to the methylated inositol, D-pinitol (Vernon and Bohnert, 1992; Ishitani
et al., 1996; Bohnert and Jensen, 1996a, b). We present a description of pinitol
biosynthesis, highlighting essential features that seem to characterize what is
required of compatible solutes.
Biosynthesis of D-pinitol in the halophyte Mesembryantheumum crystallinum
(ice plant) requires increased flux of carbon from glucose 6-phosphate to myoinositol 1-phosphate and then myo-inositol (Ishitani et al., 1996). The first gene in
the pathway, encoding inositol-1P synthase is transcriptionally upregulated, and
increased protein amounts can be detected (Ishitani et al., 1996; Nelson et al.,
1998b). The second enzyme, inositol monophosphatase, is not regulated under
stress conditions in the ice plant (Mesembryantheumum crystallinum) (Nelson
DE, personal communication). Utilizing increased amounts of inositol following
stress, the enzyme myo-inositol O-methyltransferase (IMT), generates D-ononitol
(Vernon and Bohnert, 1992). In the ice plant, IMT is only expressed following salt
stress, i.e. the protein is virtually absent in unstressed plants and increases
dramatically within one to two days of stress (Vernon and Bohnert, 1992; Nelson
et al., 1998b). Finally, D-ononitol is converted into D-pinitol by an epimerization
reaction which may include more than one enzyme. This activity, which we term
OEP, has not yet been characterized biochemically or genetically. There are two
signature features of this pathway. First, the pathway is connected to inositol
synthesis and phospholipid biosynthesis, pathways which are tightly controlled in
organisms in which they have been studied (Nikoloff and Henry, 1991), and
beyond that the synthesis of the methylated inositols is connected to the major
flux of carbon in photosynthetic cells. The second feature is that the pathway
includes additional enzymes which remove the product from general metabolism.
D-pinitol is an extremely stable end-product. The activities of IMT and OEP are
not 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 in
these species.
4. Functions of compatible solutes
Compatible solutes do not interfere with protein structure and function, and
they alleviate inhibitory effects of high ion concentrations on enzyme activity. It
is an osmoregulatory function as osmolytes that is typically assigned to the
multitude of compatible solutes which accumulate in response to osmotic stress.
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Some solutes, such as trehalose, do not respond to osmotic stress by accumulating
to 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 a
mechanism that is non-osmotic as for example in radical oxygen scavenging. We
have, for example, recently shown that mannitol at concentrations of less than
100 mM in chloroplasts specifically reduces damage by hydroxyl radicals generated
through the Fenton reaction (Shen et al., 1997a, b). While the net increase of solutes
lowers the osmotic potential of the cell which supports the maintenance of water
balance under osmotic stress, the net lowering of the solute potential may not
be 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 vitro
experiments, compatible solutes at high concentrations have been found to
reduce 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 of
compatible solutes increased the thermal stability of enzymes (Back et al., 1979;
Paleg et al., 1981; Galinski, 1993), and prevented dissociation of the oxygenevolving complex of photosystem II (Papageorgiou and Murata, 1995). One
argument often raised against these studies is that the effective concentration of
compatible solute necessary for protection in vitro is very high, approximately
500 mM. Such high concentrations are rarely found in vivo. However, when we
consider the high concentration of proteins in cells, the concentration of
compatible solute necessary for protection can, we think, be much lower than that
required for protection in in vitro assays. In addition, it may not be the
concentration of compatible solute in solution that is important. Glycine betaine
(which may be present in high or low amounts), for example, protects thylakoid
membranes and plasma membranes against freezing damage or heat destabilization (Coughlan and Heber, 1982; Jolivet et al., 1982; Zhao et al., 1992), indicating
that the local concentration on membranes or protein surfaces may be more
important than the absolute concentration.
Two theoretical models have been proposed to explain protective or stabilizing
effects of compatible solutes on protein structure and function. The first is termed
the `preferential exclusion model' (Arakawa and Timasheff, 1985) according to
which compatible solutes are largely excluded from the hydration shell of
proteins, which stabilizes protein structure or promotes or maintains protein/
protein interactions. Compatible solutes in this model would not disturb the
native hydration water of proteins, but they would interact with the bulk water
phase in the cytosol. The second model, the `preferential interaction model', in
contrast, 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
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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 sight, the actual function of
compatible solutes may in fact be explained by both models. The structures of
different compatible solutes could accommodate hydrophobic, van-der-Waals
interactions, and charged interactions, but further experiments will be necessary
to gain a better insight into the stabilizing effects of compatible solutes that have
been documented in in vitro experiments.
Compatible solutes may also function as oxygen radical scavengers. Evidence
for such a function comes from studies on fungal pathogen interactions where the
pathogens are protected by the synthesis and secretion of mannitol. Plants and
animals produce oxygen radicals in response to pathogen attack. The rapid
production and local accumulation of reactive oxygen species leads to localized
cell death in the host which then may limit spread of the pathogen (Tenhaken
et al., 1995). In response, some pathogens seem to have evolved mechanisms
which detoxify the reactive oxygen species produced by the host. Cryptococcus
neoformans, a yeast which opportunistically infects humans with a compromised
immune system, produces and secretes mannitol. A mutant strain that does not
produce mannitol is less virulent (Niehaus and Flynn, 1994). Similarly, the
tomato pathogen, Cladosporium fulvum, produces mannitol during the infection
process, which seems to protect the fungus from damage by reactive oxygen
species produced by the plants (Joosten et al., 1990).
Mannitol has been shown in vitro to function as a scavenger of reactive oxygen
species, ROS (Elstner, 1987; Halliwell et al., 1988). ROS is a generic term which
is used to include not only free radicals, such as superoxide and hydroxyl radicals
but also singlet oxygen and H2O2. Smirnoff and Cumbes (1989) designed
experiments that compared the radical scavenging capabilities of different
compatible solutes. They reported that mannitol, sorbitol, glycerol, proline, ononitol
and pinitol were active scavengers, although at different concentrations in vitro,
while glycine betaine was not able to scavenge radicals (Smirnoff and Cumbes, 1989;
Orthen et al., 1994). The relative radical scavenging efficiency of these compounds
seemed 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 a
hydroxyl radical scavenger. Under water deficit conditions, radical production
increases in plants (Moran et al., 1994), and it may be that the accumulation of
polyols provides some protective effect against oxidative damage of proteins.
Recently, results have been reported which shed light on the radical scavenging
capacity of mannitol in in vivo experiments (Shen et al., 1997a, b). Mannitol
1-phosphate dehydrogenase was modified such that the protein was imported into
chloroplasts, and the gene construct was expressed in transgenic tobacco. We
argued that a potential function in radical scavenging in vivo might best be
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245
demonstrated with chloroplasts which abundantly produce a variety of reactive
oxygen species when stressed by water deficit (for a recent review, see Noctor
and Foyer, 1998). Mannitol was present in concentrations of approximately
100 mM in the chloroplasts (Shen et al., 1997a). Using different conditions, such
as illumination with high light, paraquat treatment, enhanced H2O2 generation,
and DMSO infiltration, it could be shown that plants containing mannitol in their
chloroplasts were better able to maintain high carbon fixation rates and showed
less chlorophyll bleaching (Shen et al., 1997a) than plants without mannitol.
Further experiments indicated that mannitol was active specifically against
hydroxyl radicals and not against hydrogen peroxide or radical oxygen. This is
important information, considering that chloroplast detoxification systems exist
that can deal with H2O2 and radical oxygen, while there is no enzyme system
described that could deal with the extremely short-lived and highly reactive
hydroxyl radicals.
Further experiments (Shen et al., 1997b) indicated that even under high light
conditions the major effect of increased hydroxyl radical production was largely
restricted to the dark reactions of photosynthesis, while the photosystems
themselves functioned normally. It could be demonstrated that some enzymes of
the Calvin-cycle were predominantly affected by hydroxyl radicals. Phosphoribulokinase, PRK, and likely other SH-enzymes of the Calvin-cycle, showed
sensitivity to hydroxyl radicals, and the activity of PRK was protected by the
presence 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 following
water deficit caused either by lowering of water uptake through roots or
continued evapotranspiration through stomata is very likely a signal for
compatible solute synthesis, possibly through a pathway that is similar to the
yeast high osmolarity glycerol osmotic signaling pathway (Shinozaki and
Yamaguchi-Shinozaki, 1997). That water movement is not regulated, but simply
follows osmotic gradients, must be questioned.
Recently, water channel proteins (aquaporins), which have been detected in all
organisms from bacteria to humans, have been implicated as the major facilitators
for the movement of water across membranes (Chrispeels and Agre, 1994). For
some of these proteins it has been demonstrated that they function as aquaporins
by a swelling test of Xenopus oocytes after the RNA encoding the putative
aquaporin has been injected into the oocytes. Most of these proteins seem to
facilitate water flux along an existing osmotic gradient, while others transport
other metabolites, such as glycerol (Luyten et al., 1995; Yang and Verkman,
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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- and
developmental stage-specific mode. The protein products from each subfamily
are targeted to either the plasma membrane or the tonoplast, respectively
(Kammerloher et al., 1994; Yamada et al., 1995; Weig et al., 1997). Several ice
plant water channel mRNAs and proteins have been found to decline during salt
stress (Yamada et al., 1995; Kirch, H.H. and Bohnert, H.J., unpublished). A
dynamic way of regulating water movement seems to involve aquaporin turnover
and/or post-translational modifications. A decrease in water channel proteins in
transgenic Arabidopsis by an antisense strategy lowered the water permeability of
the plasma membrane of protoplasts in comparison to wild type plants and
reduced bursting of protoplasts in hypo-osmotic media (Kaldenhoff et al., 1995).
The whole-plant phenotype of Arabidopsis containing less aquaporin proteins
seems to be that the root to shoot ratio is drastically increased (Kaldenhoff, R.,
personal communication). Regulation of water permeability may also be through
regulated protein modifications (Maurel et al., 1995; Johanson et al., 1996). A
putative plasma membrane aquaporin from spinach is reversibly phosphorylated
at multiple sites in the protein in response to changes in calcium and a lowering of
the apoplastic water potential (Johanson et al., 1996). The function of water
channels in maintaining water balance across membranes under osmotic stress is
still debated. Water channels could facilitate water uptake in roots if their
presence in the plasma membrane were synchronized with the synthesis and
accumulation of metabolites that lead to osmotic adjustment; their removal from
the membrane might restrict the loss of water. Similarly, the amount or regulation
of tonoplast-located water channel proteins might be involved in determining ion
partitioning to the vacuole. As one example, we have observed using peptidespecific antibodies that tonoplast-located water channel proteins decline in the
root 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 the
vasculature. Additional experiments are needed to investigate whether and how
much water channel proteins are involved in the regulation of water movement in
whole plants under stress conditions.
6. Replacement of a compatible solute in yeast
When yeast, S. cerevisiae, is stressed by high salinity, a signal transduction
relay of phosphorylation events (high osmolarity glycerol-pathway; Albertyn
et 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, is
induced resulting in increased production of glycerol (Luyten et al., 1995). The
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247
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 the
glycerol facilitator protein, belonging to the water channel gene family, retards
the cell's loss of glycerol which then accumulates to approximately 400 mM
inside cells. When both GPD genes are inactivated, the cells become extremely
salt-sensitive (Albertyn et al., 1994; Ansell et al., 1997), which provides further
credence to the view of glycerol as a compatible solute. We have used a mutant
lacking both GPD genes, and replaced their function by enzymes leading to
mannitol and sorbitol synthesis, respectively (Shen et al., 1998). The expression
of a sorbitol dehydrogenase and a mannitol dehydrogenase led to sorbitol
amounts, 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, trehalose
accumulation was comparable. Surprisingly, the degree of salt tolerance acquired
by the sorbitol- and mannitol-accumulating lines was not comparable to the
tolerance generated by 400 mM glycerol. The I50, the concentration of NaCl that
inhibited growth by 50%, was 1 M for the mutant line into which the GPD gene
was re-introduced and 0.55 M for sorbitol- and mannitol-producing plants, which
was not much different from the I50 of 0.4 M for the mutant line lacking GPD
enzymes (Shen et al., 1998). The result seems to indicate that the osmotic
adjustment 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 biochemical
pathway through which glycerol is synthesized in yeast is more important than
the amount that is being made. In favour of this argument, yeast does not retain
glycerol very well once synthesized; more than 90% of the glycerol is found in
the medium over a period of approximately three generations (Shen et al., 1998).
The pathway leading to sorbitol or mannitol, respectively, may not be compatible
with the function that is provided by increased glycerol synthesis. Since the entire
pathway leading to glycerol biosynthesis consumes more NADH than mannitol
biosynthesis, another possible explanation is that compatible solute biosynthesis
serves the purpose of reducing cellular NADH levels. Alternatively, glycerol
might interact with yeast proteins better than sorbitol or mannitol. This
disconcerting notion might mean that protein structure and the type of compatible
solute found in a particular species have adapted evolutionarily, clearly a daunting
prospect for stress tolerance engineering.
7. Analysis of transgenic plants
Plant transformation/regeneration has become routine in many species, even in
those that were considered recalcitrant, such as rice or corn (Komari et al., 1996;
Heath et al., 1997). Equally, the necessary manipulations and sequence
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modifications for gene constructions are commonplace techniques by now and
may in the near future become contract services. Genes encoding enzymes which
enhanced the synthesis of putatively protective osmolytes have been transferred
by several groups, and the transgenic plants were analyzed for their performance
during chilling, drought, salinity stress and an increase in reactive oxygen species,
for example, by exposure to methyl viologen (Table 1). However, plant growth
and stress treatments were invariably carried out under controlled conditions in
growth chambers, not under field conditions, and small populations of seedlings
or young plants have been used in these experiments (Table 1).
In all experiments, the transgenic plants showed differences compared to wild
type, but these differences were small, or were restricted to narrow developmental
stages, or they were followed for short time periods, never for an entire growing
season. Most clearly documented is a protective effect of enzymes that act in
H2O2 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 is
the 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 plants
under stress (Xu et al., 1996). The expression of a bacterial levansucrase led to
fructan accumulation (Pilon-Smits et al., 1995), and expression of a subunit of the
yeast 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 be
explained by osmotic adjustment because of the low amounts of accumulating
metabolites. In contrast, the overexpression of a D1-pyrroline-5-carboxylate
synthetase, leading to increased proline biosynthesis (Kishor et al., 1995),
generated proline in an osmotically significant high amount. Similarly, the
methylated inositol, D-ononitol, produced by the action of a methyltransferase
(Sheveleva et al., 1997) accumulated under drought conditions to approximately
600 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 the
absence of stress (Karakas et al., 1997). Consequently, the purported stress
protection has been interpreted as an effect of slower growth leading to less
sodium uptake under stress. Growth retardation has indeed been observed in some
lines which are characterized by very high mannitol or sorbitol accumulation
(Sheveleva et al., 1998), but growth is not significantly lower in lines that contain
less than approximately 50 mM of mannitol in total cell water, while a slight
protective effect can still be demonstrated.
Other experiments cast a more significant shadow on the overexpression
schemes 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
Enzyme
Host species
Notes
Reference
MnSOD
(N. plumbaginifolia)
Mn-SOD
N. tabacum
Organelle targeted expression leading
to reduced damage by ROSa
Bowler et al., 1991
MtlD (E. coli)
Mannitol 1-P DH
Hva1 (H. vulgare)
HVA1-LEA
O. sativa
Imt1 (ice plant)
N. tabacum
SacB (B. subtilis)
myo-inositol
O-methyltransferase
Levansucrase
N. tabacum
Tps1 (S. cerevisiae)
Trehalose synthase
N. tabacum
CodA (A. globiformis)
Choline oxidase
A. thaliana
P5CS (V. aconitifolia)
P5CS
N. tabacum
FeSOD (A. thaliana)
Fe-SOD
N. tabacum
Gst/Gpx (N. tabacum)
GST/GPX
N. tabacum
M. sativa
N. tabacum
A. thaliana
N. tabacum
Sodium tolerance at early growth
Enhanced seed germination in NaCl
Chloroplast location, ROS scavenging;
protection of calvin-cycle
Maintenance of higher growth rate by
stressed plants
Stress-induced accumulation of D-ononitol
Fructan accumulation; higher growth rate
during drought stress
Low conc. trehalose increased drought
tolerance
Glycine betaine accumulation enhanced
tolerance
Proline accumulation leading to lowering
of osmotic potential
PSII/plasma membrane protection/methyl
viologen
Increase of oxidized glutathione (GSSG)
enhanced seedling growth
McKersie et al., 1993, 1996
Tarczynski et al., 1992, 1993
Thomas et al., 1995
Shen et al., 1997a, b
Xu et al., 1996
Sheveleva et al., 1997
Pilon-Smits et al., 1995
HolmstroÈm et al., 1996
Hayashi et al., 1997
Kishor et al., 1995
Van Camp et al., 1996
Roxas et al., 1997
H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260
Gene (species)
a
249
Reactive 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.
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H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260
lines obtained by classical breeding strategies and differing only in glycine
betaine content, responses to salinity stress were monitored. A correlation was
established between the amount of glycine betaine and tolerance (Saneoka et al.,
1995). Clearly, the lines containing glycine betaine showed higher carbon
assimilation and maintained a higher leaf relative water content than the glycine
betaine-deficient lines. In field trials, however, the glycine betaine accumulators
were more prone to fungal diseases and lodging, which negated the effect that
might be provided by a high osmolyte content under drought conditions as they
might 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 but
experimental results have not yet become available. We will discuss requirements that seem to emerge from published attempts for the purpose of increasing
water deficit/salinity stress tolerance. First, the low amounts of fructan, mannitol,
or trehalose that show some protection seem to indicate that the compatible
solutes have a specific protective role, rather than acting as osmolytes.
Accumulation might not be necessary. Also, different compatible solutes seem
to have different functions. Clearly, glycine betaine does not act as a hydroxyl
radical scavenger in the way mannitol does (Shen et al., 1997b), but glycine
betaine does confer some protection against sodium effects when present in
transgenic plants (Hayashi et al., 1997). Thus, one suggestion is that we should
transfer genes leading to the synthesis of different compatible solutes at low or
moderate amounts.
Engineering of compatible solutes should be supported by engineering of
scavenging systems for reactive oxygen species. This is not only indicated by the
slight protective effects that have been observed after engineering of reactive
oxygen species scavenging enzymes into plants (e.g. Bowler et al., 1991;
McKersie et al., 1996; Noctor and Foyer, 1998), but also from the results obtained
with plants containing engineered mannitol in the chloroplast compartment (Shen
et al., 1997a, b). In a model study, we attempted to measure the production of
hydroxyl radicals via the product, MSA (methane sulfinic acid), of hydroxyl
radicals with DMSO (dimethyl sulfoxide). In these experiments, mannitol in the
chloroplasts clearly competed with DMSO for hydroxyl radicals because less
MSA was formed. It was found that cells could tolerate a stress that nearly
doubled the amount the hydroxyl radicals that are found under non-stress
conditions, and that mannitol at 100 mM in the plastids again approximately
doubled the hydroxyl radical scavenging capacity of these cells (Shen, 1997).
These results are compatible with the measurements and estimations of hydroxyl
radical production in cells in vitro and with the estimations about the contribution
H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260
251
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 multigene
transfer, again using tobacco as the model species (Nelson, D.E., Zhu, G.,
Michalowski, C.B. and Bohnert, H.J., unpublished). Included in the gene
constructions are three genes which enhance the biosynthesis of myo-inositol and
D-ononitol in the cytosol; one gene leads to moderate mannitol accumulation in
plastids; trehalose synthesis is enhanced in the cytosol; and two genes, Fe-SOD
and a cytosolic ascorbate peroxidase, respectively, are included for the provision
of enhanced reactive oxygen species scavenging. Most of the genes are derived
from the halophytic ice plant which has become the most intensely studied model
for natural salinity stress tolerance (Adams et al., 1998). The genes which we
utilized for the transfer into tobacco show up-regulated mRNA amounts under
salt stress in the ice plant (Michalowski, C.B. and Bohnert, H.J., unpublished).
Many of these upregulated transcripts are also regulated under salinity stress in
yeast 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 stress
tolerance is Nicotiana tabacum L. It has been pointed out that tobacco, which is
not particularly salt sensitive, may be less than ideal as a salt stress model
(Murthy and Tester, 1996), because the plant reacts predominantly to the decrease
in water potential that accompanies salt stress and not to sodium toxicity. While
this is indeed the case, the advantages of using tobacco as a transgenic
biochemical model outweigh this problem. In fact, we consider the osmotic
aspects of salinity stress to have a far greater effect on plants than the toxic effects
of sodium (see also, Cheeseman, 1988). In other models used, Arabidopsis
thaliana, Medicago sativa, and Oryza sativa, equally successful, and equally
limited, improvements of abiotic stress tolerance have been reported (Thomas
et al., 1995; Xu et al., 1996; McKersie et al., 1996). Another model species,
Saccharomyces cerevisiae, currently may be the best model for understanding
salinity tolerance mechanisms (Serrano, 1996; Nelson et al., 1998a). First, yeast
is salt tolerant, and mutants which are salt sensitive are readily identifiable. Not
only do these mutants allow identification of important salinity tolerance genes,
but also by complementation, they allow the identification of homologs from
other species as well as providing useful salt-sensitive strains for a variety of
physiological and transgenic experiments. Second, the entire genomic and
mitochondrial sequences are available. Analysis of the yeast genes has broken the
barrier of gene availability, and with the plant genes identified by complementa-
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tion we now have a large repertoire of coding sequences available ± a prerequisite
for multigene transfer for pyramiding desirable traits. Finally, the completion of
the 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 all
reading 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 gene
PBS2, encoding MAP kinase of the high osmolarity glycerol osmosignaling
pathway, was deleted, proteins controlled by this pathway could be documented
by their disappearance from 2D gels (Akhtar et al., 1997). The authors reported
29 protein spots affected by this deletion. Assuming that maybe one third of the
yeast proteins are of sufficiently high abundance to be visible on 2D gels, by
extrapolation a number of 100 closely stress-associated genes seems a reasonable
estimate. Also, the number of genes that are essential to tolerance as determined
by gene deletion is approximately 10±20. That a significantly larger number
should aid in tolerance but not be absolutely necessary is reasonable, and this
consideration, 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 obtained
after screening lambda phage clones representing 3.2 Mb, or approximately 1%,
of the ice plant genome (Meyer et al., 1990). Since the processes controlled by
the known yeast genes are similar to those found or suggested as essential
reactions of plants under salt/drought stress, we suggest that large scale metabolic
engineering of crop plants should start by utilizing the plant genes that are
functionally equivalent to these yeast genes.
10. Conclusions
Yeast as a model for cellular osmotic stress tolerance in plants is studied by
several groups at present. The integration of the cellular responses with those
exhibited by the whole plant will be the next challenge. In higher plants it is still
difficult to connect putative mechanisms with the phenotypes that characterize
osmotic and ionic stress tolerance, and it has been impossible to identify all genes
whose products generate the mechanisms. Even in the cases where sufficient
correlative evidence exists, metabolic engineering remains risky without knowing
these genes and their functions. Yeast can provide many of the genes, and their
homologs in higher plants can be studied. At present, attempts at engineering
salinity tolerance in whole plants can be compared to following single pages of an
instruction manual without page numbers. Work with yeast has provided
individual chapters, but we still have to guess how the sections fit together for
H.J. Bohnert, B. Shen / Scientia Horticulturae 78 (1999) 237±260
253
understanding the cellular salinity stress tolerance manual in higher plants. For
other 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 are
expressed. The technical capability exists for analyzing and quantifying order,
magnitude, and complexity of the expression of all genes during a growth cycle
by microarray analysis (Schena et al., 1996; Shalon et al., 1996). Then, by
observing changes in the order and amount of expression following the addition
of NaCl, we will gain information about the dynamic progression of gene activity
changes, about which gene products may be rate-limiting, and which are
necessary for maintenance of a new steady-state. This analysis is already feasible
for yeast. In higher plants, similar analyses will soon be possible with the
collections of cloned expressed sequence tags from several model organisms,
mainly from Arabidopsis thaliana, rice and corn (several addresses can be found
on 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 a
rational tolerance-engineering strategy. Following the identification of important
genes and their expression during salinity stress, multiple genes will have to be
transferred for the analysis of salinity tolerance in transgenic model plants. The
ability now exists for the transfer of fragments of DNA, which could include
hundreds of genes (Hamilton et al., 1996), to an increasing number of plants,
including many important crop species (Zupan and Zambryski, 1995; Hanson and
Chilton, 1996; Komari et al., 1996; Heath et al., 1997). In addition, methods are
now available for removing undesirable selectable markers after transformation
(Komari et al., 1996). Removing coding regions by targeted gene disruption
through homologous recombination, which has recently been reported for
Arabidopsis (Kempin et al., 1997), poses no insurmountable problem any longer.
One significant limitation still exists. Currently missing is a sufficiently large and
complex set of plant promoters with cell-specific, tissue-specific, developmental
stage-specific, and/or inducible patterns of expression. It can be expected that the
ongoing 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 problem
in the near future and will allow the assembly of a library of plant promoters. We
have begun multigene transfer into tobacco as a model plant. Determined by the
availability of genes for functions that have been identified in previous experiments,
our first attempts are designed to achieve an increase of reactive oxygen species
scavenging, metabolite accumulation in cytosol and organelles, and increased
synthesis of inositol which we think is essential for growth under stress.
Finally, the impact of salinity tolerance-engineering, if we assume that
significant improvements in plant tolerance can be achieved, must be considered.
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The extent to which plants can be made tolerant to sodium in the soil is unknown.
More significantly, stress tolerance and productivity must be positively
correlated. The concentration of NaCl that higher plants may tolerate is limited
by the ability of the plants to store NaCl in vacuoles, by the plant's capability for
opening stomata under saline conditions, by the energy drain that is associated
with increased proton pumping, and the energy expenditure for maintaining
significant amounts of osmotically compensating metabolites. It is impossible to
predict where the limits may be, but it is certain that constitutively expressed
increased tolerance will be associated with a cost that will limit productivity. This
cost, 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 does
seem possible, however, that an improvement in tolerance to half seawater or to
10±15 ppt of sodium will be achievable. Tolerance of this level, while
maintaining growth and seed set, would constitute a significant improvement
considering that most crop species are adapted to produce only at approximately
5 ppt or less sodium in the soil. The path towards this goal will require the
transfer of many genes. Time will have to be devoted to testing appropriate
expression characteristics of these transgenes so that the transgenic plants can
become material for breeding programs.
Acknowledgements
Work in the laboratory has been or is supported by the U.S. Department of
Energy (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 been
funded by NEDO, Japan, in a collaborative international program. Visiting
scientists have been supported by the Rockefeller Foundation, Japan Tobacco,
Inc., the Smithsonian Institution/Carnegie-Mellon Foundation, the Japanese
Society for the Promotion of Science, and the Deutsche Forschungsgemeinschaft.
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