Environmental Stress Adaptation Strategies

A. How Many Strategies Are There?

It would be impossible to list all reported data sets on stress adaptations, each of which provides a glimpse of how plants maintain growth during stressful times or at least how they survive, but there are now sufficient data to identify probable protective mechanisms in a general sense. In broad categories, one can identify three levels of response: (1) the immediate reactions, (2) adjustments to reach a new equilibrium, and (3) long-term developmental changes. Most is known about the "downstream" reactions, the induction of new pathways or the repression of enzymes and biochemical pathways, which have been established during normal growth and which functioned prior to stress (Fig. 1). Immediate stress reactions, which represent the first line of defense, are mediated by the cellular defense mechanisms that are evolutionarily well conserved. A second category, including sensing and signaling circuits, initiates the immediate response, establishes a new equilibrium for further growth, and prepares for long-term changes. Responding to stress signaling and the concomitantly altered hormonal state are developmental changes that may provide the best answers to long-term stress.

leaf/root ratio, emergence seedling growth, leaf size, branching, flowering, seed set Development & Timing

DnaJ, HSP, chaperon ines, pept idyl-prolyl cis-trans isomerases Chaperone Action proteoses, ubiquitin, protease inhibitors Proteome Remodeling

Stress Proteins interaction & stab iiizat ion, LEA s

Surface Properties cutietilar wox, trie homes, bladder cells, glands ceil wall changes, lignification

Dehydration

Sensing & Signaling

Abiotic Environmental Stress

Sensing ~ Sensing 4

Signaling Signaling

Salinity

'Sensing & Signaling

Cold carotenoids, pigments, thioredoxins, ascorbate-glutathione cycle SOD, ASX. cotatase Radical Scavenging

Pathway Adjustment carbohydrate ffux, photosynthate partitioning C/N-adjustments, redox status, growth regulator synthesis

Osmolyte Synthesis Heat )—► A Turnover glycine be tame, polyols, proline, ectoine, trehalose, fructans, raffinose

'Calcirveurin-type

Ion Homeostasis K*-channels/transporters, Na" /H* - antiporters, Na'/inositol symport, water channels, general cation/onion transporters, ABC-transporters

Figure 1 Strategies for stress tolerance. The scheme symbolizes sensing and signaling pathways for dehydration, cold, heat, and salinity stress recognition that are largely unknown. A Hog1-like pathway leading to osmolyte synthesis and a calcineurin-type pathway for ion homeostasis, both similar to yeast signal transduction pathways, are indicated. Included are boxes that summarize biochemical, metabolic, molecular, and developmental elements that contribute to stress tolerance.

B. Protection of Downstream Reactions

Several mechanisms are known for which the evidence for a protective role seems clear: (1) scavenging of radical oxygen species (18,19), (2) controlled ion and water uptake (11,20-24), and (3) management of accumulating reducing power through adjustments in carbon-nitrogen allocation and the accumulation of compatible solutes (25-29). Responses on this level of defense are immediate, taken in an enhanced form from the repertoire of daily plant life. They encompass, for example, the down-regulation of the light-harvesting and water-splitting complexes, enhancement of the water-water cycle, increased synthesis and action of radical-scavenging enzymes and nonenzymatic scavenging molecules, the storage of reducing power in a reusable form (e.g., as proline, polyols, or fructans), and the engagement of proton pumping and ion and water transport systems. Also, the stabilization of proteins and membranes through enhanced chaperone action belongs into this category of reactions (30,31). Such mechanisms accomplish ordinary adjustments, although reaction bandwidth, speed, and magnitude seem to delineate species-specific boundaries. Several of these emergency measures have been enhanced in transgenic models with limited, albeit recognizable, success (28,32-34), but prolonged stress requires additional mechanisms. This view then assumes a common set of genes for stress responses, similar or identical for glycophytes, halophytes, and xerophytes, as well as low-temperature tolerant species. The differences between species lie in how, how fast, or how resolutely the response can be initiated, elaborated, or sustained.

1. Osmotic Adjustment

This term was coined to explain functions of accumulating metabolites. As the external osmotic potential decreases, internal accumulations restore the differential necessary for the plant to take up water. Potassium, if available, can serve in this function. Also, amino acids and some amino acid derivatives, sugars, acyclic and cyclic polyols, fructans, and quaternary amino and sulfonium compounds accumulate (28,35-38). Typically, pathways leading to their synthesis are connected to pathways in general metabolism with high flux rates. Examples are the proline biosynthetic pathway (36), glycine betaine synthesis (35,39), sulfonium compounds (40), and the pathway leading to the methylated inositol, D-pinitol (41-43). In the past, it had been assumed that the enzymatic activities that lead to accumulation of one or the other metabolite indicated differences in gene complement between species. This seems not to be the case; rather, different plant species activate different sets of genes (40). The osmotic effect of high accumulation is documented by the ice plant (Mesembryanthemum crystallinum), although this may be an extreme case. This plant, a salt accumulator, contains sodium in vacuoles of epidermal bladder cells at a concentration exceeding 1 M and a methylated inositol, D-pinitol, in the cytosol at a concentration that may be as high as 600-800 mM (44-46).

Whether the function of these accumulators is simply or only in mass action is questionable. In experiments with transgenic plants (28,32,47), low to moderate accumulation of mannitol, glycine betaine, D-ononitol, or sorbitol has had a measurable protective effect. Experiments indicate that these osmolytes at low concentration within cells also have a protective function by preventing the formation of hydroxyl radicals (48-50). Another possibility, yet to be rigorously tested, is that transiently accumulating metabolites could defuse reductive power during stress (26,51). As the stress-induced decline in growth increases the ratio of [NAD(P)H + H+] to [NAD(P)+], the accumulation of an osmolyte could lead to adjustment of the cellular redox state. Thus, current understanding places less emphasis on the previously assumed function of osmolytes, the stabilization of proteins, protein complexes, or membranes by mass action, or either protection or replacement of the water shell around proteins (52,53).

2. Radical Scavenging Capacity

The production of reactive oxygen species (ROS) is unavoidable in chlo-roplasts, but ROS are also produced in mitochondria, peroxisomes, and cytosol in all cells. The ROS, including singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals, react with and can damage proteins, membrane lipids, and other cellular components (19,54). The ROS also serve as signaling molecules, for example, in the recognition of attack by pathogens

(55). Under water deficit conditions, radical production increases in plants (18,19). ROS toxicity is clearly evident during drought (18), chilling stress

(56), and high salinity (50) (see Ref. 28 for a review). Superoxide and H,0, concentrations increase during drought and at low temperature (57). Enhanced production of ROS results in an increase in lipid peroxidation, as documented by a more than fivefold increase of malonaldehyde production in wheat (58). The concentration of nonbound iron increases under drought stress (59), which stimulates the production of hydroxyl radicals in the presence of H202 in a Fenton reaction. Compared with superoxide and H202, hydroxyl radicals oxidize a variety of molecules at nearly diffusion-controlled rates. Finally, levels of nonenzymatic radical scavengers, such as ascorbate, carotenoids, flavonoids, sugar polyols, and proline, increase and can complement the existing enzyme-based protection systems, such as superoxide dismutase, ascorbate peroxidase, or catalase. Together, the scavenging enzymes and nonenzymatic antioxidants provide sufficient protection under normal growth conditions and can handle moderate increases of ROS, unless long-term stress exceeds the detoxification capacity (6,18).

Evidence for a protective effect of enhanced ROS scavenging systems has been provided by the overexpression of an enzyme with the combined activities of glutathione ^-transferase (GST), and glutathione peroxidase (GPX) (60). With doubling of the GST-GSX activity in transgenic tobacco, the seedlings and plants showed significantly faster growth than the wild type during chilling and salt stress episodes. The increased enzyme activities resulted in higher amounts of oxidized glutathione (GSSG) in the stressed plants, indicating that the oxidized form could provide an increased sink for reducing power. Even more striking is the detection of a gain-of-function mutation, improved growth under salt stress, in Arabidopsis (61). The recessive mutation is characterized by constitutively increased activities for ROS-scavenging enzymes. In a different strategy, the transgenic increase of a catalase in tobacco resulted in protection of leaves against paraquat-induced bleaching (62). The experiment indicated as a major stress factor the depletion of chloroplastic ASA affecting ascorbate peroxidase amounts.

Another set of experiments shed light on the relationships between ROS and the accumulation of polyols. When a bacterial gene (mrtD) encoding mannitol-1-phosphate dehydrogenase was modified so that the enzyme was expressed in chloroplasts, transgenic tobacco contained 60-100 mM mannitol in the plastids. Using transgenic plants, freshly prepared cells, or a thylakoid in vitro system, the protective effect exerted by mannitol on photosynthesis characteristics could be shown (50,63). The presence of mannitol resulted in increased resistance to oxidative stress generated by methyl-viologen, and cells exhibited significantly higher C02 fixation rates than controls during stress. After impregnation of tissues, isolated cells, or a reconstituted thylakoid system with dimethyl sulfoxide, a hydroxyl radical generator, mannitol-containing cells showed a lower rate of methanesulfinic acid production than the wild type, indicating that mannitol acted specifically as a hydroxyl radical scavenger. Calvin cycle enzymes sustained primary damage rather than components of the light-harvesting and electron transfer systems. Phosphoribulokinase (PRK) and probably other SH enzymes of the Calvin cycle showed sensitivity to hydroxyl radicals, and the activity of PRK was protected by the presence of mannitol (63,64). At present, a possible interpretation is that mannitol reduces the amount of hydroxyl radicals produced in Fenton reactions, possibly by complexing free iron (Fe2+) (G. Bongi, personal communication).

3. Water and Ion Relations

Abiotic stresses affect plant water and ion relations, and both are intricately connected. The most crucial aspects during drought and salinity stress are continued uptake of water and potassium and the exclusion or internal sequestration of sodium ions. Lowering the plant's internal osmotic potential to match or exceed that of the medium is the generally assumed function of potassium and the accumulating osmolytes. In reality, little is known about the relationships between the adaptive responses that initiate solute production and accumulation, water transport (either loss, acquisition, or movement), and ion uptake under water deficit (or sodium uptake or exclusion in high salinity). Loss of turgor following water deficit caused either by lowering of water uptake through roots or continued évapotranspiration through stomata very likely constitutes a signal, possibly transduced through phosphorylation cascades similar to the yeast high osmolarity glycerol (HOG) and calcineurin osmotic and ionic signaling pathways (10,65).

a. Water Channels. Such channels (aquaporins) enhance membrane permeability to water in both directions, depending on osmotic pressure differences across a membrane. They are present in all organisms as facilitators for the movement of water and metabolites, such as urea or glycerol (65-67). In plants, two subfamilies can be distinguished: aquaporins targeted to the plasma or vacuolar membrane, termed PIP and TIP, respectively. As in animal cells (68), plant aquaporins seem to be controlled by posttransla-tional protein modification and cycling through the endomembrane system (69-72). For example, a putative plasma membrane aquaporin from spinach is reversibly phosphorylated at multiple sites in response to changes in calcium and a lowering of the apoplastic water potential (69). The function of water channels in maintaining water balance under osmotic stress is still debated. Their presence increases water movement across a membrane by a certain factor, which is different for each channel, but water movement through lipid membranes alone is still substantial. The significance of these channels seems to lie in determining the flux rate of water and solutes through tissues rather than on their maintaining water relations of individual cells. Important to the stress aspect is that the protein amount and the spatial expression patterns of water channels are regulated. There is evidence that location may vary with changes in physiological state (67). Plasma membrane-located proteins appear in membrane fractions that are cell internal under salt stress in Mesembryanthemum (72) (H. J. Bohnert, unpublished data). High salinity leads to the disappearance of tonoplast-localized channels, whereas drought causes the decline of plasma membrane-localized water channel proteins (72) (R. Vera-Estrella, B. Barkla, H. J. Bohnert, unpublished results).

Plant genomes contain more than 23 genes for water (and/or solute) channels (73,74). Studies indicate that water channel transcripts are expressed with developmental and spatial complexity in diverse tissues

(73,75,76). An analysis of more than 3000 transcripts comparing control and salt-stressed maize ESTs documented that water channels are highly abundant in roots and roots contain a greater diversity of different transcripts than leaves with a more than 10-fold decline in transcript number following salinity stress (H. Wang, G. Zepeda, H. J. Bohnert, unpublished results). These dynamic changes indicate that control over water channel distribution or activity is important under stress conditions, but additional data are required to verify this. At present, we know only that transcriptional and post-transcriptional controls exist, which seem to involve synthesis, membrane trafficking, and possibly reversible insertion into or removal from membranes and aquaporin half-life.

b. Proton-ATPases and Vacuolar Pyrophosphatase. Plasma membrane and vacuolar proton transporters play essential roles in plant salinity stress tolerance by maintaining the transmembrane proton gradient that ensures control over ion fluxes and pH regulation (77-79). Three proteins or protein complexes exist for this purpose: the plasma membrane (H+)-aden-osine triphosphatase (ATPase) (P-ATPase) and two vacuolar transport systems, an (H+)-ATPase (V-ATPase) and a pyrophosphatase (PP;ase).

The plant P-ATPase (100 kDa) is represented by a family of more than 10 genes with homology to the yeast PMAs (80,81). As the main proton pump in the plasma membrane, this ATPase shows increased activity accompanying salt stress. Halophytic plants have been shown to increase pump activity under salt stress conditions more drastically than glycophytes (82,83), but we know little about regulatory circuits that lead to increased activity during salt stress. The V-ATPase, a multisubunit complex homologous to organellar, yeast (VMA), and bacterial FoFj-ATPases, has already been shown to be important in plant salinity tolerance. Electrophysiological studies revealed increased activity of this ATPase when cells or tissues from stressed plants were analyzed (84). Transcripts for subunits of the V-ATPase are up-regulated following salt shock (85-87). In Mesembryanthemum, V-ATPase activity increases severalfold following stress without affecting protein amount, although some of the subunits are expressed in increased abundance in complementary DNA (cDNA) libraries from the stressed plant (88-90). The response is specific for NaCl and is not elicited by mannitol-induced osmotic stress. Little is known about the function of the PPiase enzyme in stressed plants. PPase activity declined under salt stress in some species but increased in others (91-93). Overexpression of the Na+/H+ antiporter from Arabidopsis has been shown to confer salt tolerance to the saltsensitive enal mutant of yeast; however, this phenotype required the expression of both a chloride ion transporter and an Na+/H+-antiporter (94).

c. Potassium Transporters and Channels. One possible passage for sodium across the plasma membrane is through transport systems for other monovalent cations (11,22-24). Among those, the most significant is the uptake system for potassium, the most abundant cation in the cytosol with important roles in plant nutrition, development, and physiological regulation. Physiological observations indicating a biphasic uptake of K+ into roots (95) gave rise to the assumption that two uptake entities should be involved, a high-affinity system functioning at pM concentrations of external K+ and a low-affinity system active in the mM range of potassium. In reality, a complex assortment of different proteins participate in potassium uptake. Analysis of transcripts, proteins, and electrophysiological studies indicated not only cell-, tissue-, and stress-specific expression differences but also regulation that allows some transport systems to function in both high- and low-affinity mode, depending on external potassium concentrations (96,97). In contrast to earlier assumptions, K+ channels are highly selective against Na+ (98) and thus play an insignificant role in inadvertent sodium uptake (22).

K+ transporters, first isolated from wheat (HKT1) (99), operate at low external potassium and may mediate entry of sodium in saline soil. The high-affinity HKT1 was indicated as a K 7Na" symporter with high-affinity binding sites for both Kf and Na+ (99) (see Ref. 23). Another line of evidence for the involvement of the high-affinity K+ uptake system in salt tolerance came from the study of salt-sensitive mutants. The sosl mutant of Arabidopsis thaliana is hypersensitive to Na+ and Li' and unable to grow on low potassium (100). The Sosl gene encodes a sodium/proton antiporter (J. K. Zhu, personal communication) different from the proteins detected by other groups (94,101). Other transporters in a different protein family complicate the picture even further; these take up potassium with dual affinity (102,103).

d. Sodium Transport Systems. Sodium is thought to enter plant cells by two different routes: through the apoplast or through the symplast. Multiple cellular uptake mechanisms through the plasma membrane have been documented that display varying amounts of permeability to Na+ (23). In particular, voltage-insensitive monovalent cation (VIC) channels have been argued to play important roles in allowing the bulk of Na+ influx (22,24,104). Therefore, genetic manipulation of VIC channels to improve their selectivity against Na+ influx or alter their expression and regulation represents possible strategies for reducing Na+ uptake (24).

The existence of an antiport system, connected to transmembrane proton gradients, had been observed before (105). Increased sodium/proton antiport activity during salt stress has been measured in several model systems, tissues, cells, and isolated vacuoles; it parallels the increase in proton-pump ing V-ATPase activity (46,90,106,107). It was shown that sodium transport from cytosol to vacuoles is accomplished by a sodium/proton antiporter (34,53,101). These Arabidopsis antiporters seem to be associated with vacuolar or prevacuolar membranes, suggesting that sodium would either be deposited into the central vacuole or partitioned into an exocytotic pathway (108). The overexpression of this antiporter, NHE1, in Arabidopsis had a remarkably protective effect under salt stress conditions (34). Ultimately, however, the capacity to sequester Na+ into the vacuole via Na+/H+ antiport activity will depend on H+-ATPase and/or H+-PPiase proton-pumping capacity. Thus, engineering strategies exploiting Na+/H+ antiport overexpression as a means of improving Na+ compartmentation must also increase proton-pumping activities, as was demonstrated by the expression of a plant vacuolar H+-PPiase in yeast (94). Furthermore, the inhibition of Na'/Hr antiport activity by high salinity can be influenced by the degree of membrane desaturation in membrane lipids. Synechocystis mutants deficient in desaturase genes and thus lacking polyunsaturated fatty acids were found to be more sensitive to salinity-mediated inhibition of photosystem II (PSII) activity (109).

Yet another pathway for sodium transport may exist that functions in concert with the antiporters. In Mesembryanthemum, the synthesis of myoinositol and its transport through phloem to the roots are correlated with sodium and rayo-inositol transport from roots to the leaves (43,110). Only in the leaves, sodium accumulates to high amounts in vacuoles and methylated inositols accumulate in the cytosol of mesophyll cells. Stress-induced sodium/wyo-inositol symporters (111), together with Na+/H+-antiporters (34), could account for long-distance transport of sodium. Also, an Na+/ myoinositol symport mechanism provides an attractive hypothesis considering that the stress-enhanced passage of myo-inositol through the ice plant vasculature connects leaf photosynthesis to root sodium uptake and its sequestration into vacuoles (110).

e. The Essentiality of Calcium. Increasing calcium improves the salinity tolerance of plants. The effect is mediated through an increase of intracellular calcium, changes in vacuolar pH, and activation of the vacuolar Na+/H+-antiporter (112-114). The strict control over calcium concentrations in the cytosol and calcium storage in a number of locations (vacuole, mitochondria, endoplasmic reticulum) demonstrates the crucial role of calcium in plant salinity stress responses. An Arabidopsis mutant, sos3, with hypersensitivity to NaCl has been characterized (115). The mutant phenotype can be masked by the external addition of calcium, and it reveals the link between calcium and salinity stress tolerance. Sos3 encodes a subunit of a calcineurin-related signal transduction chain (115) involved in altering K+/ Na+ selectivity.

C. Ubiquitous Cellular Stress Tolerance Mechanisms— Comparison of Models

A comparison of stress-regulated transcripts detected in model organisms seems to represent a common set of transcripts for the cellular complement of genes for immediate, stress-relieving emergency reactions (11,116). Many up-regulated genes reveal stress-regulated functions found in cyanobacteria and yeasts, which are similar to those regulated in xerophytic, halophytic, or glycophytic species. Figure 2 summarizes these in terms of proteins that

Figure 2 Cellular stress tolerance elements. The schematic drawing of a cell includes the vacuole into which sodium is partitioned, a chloroplast, mito-chondrium, and peroxisome, which are prone to oxygen radical damage. Included are symbols for H+-ATPases/PPiases that maintain transmembrane proton gradients and symbols for potassium uptake, sodium transport, and controlled water flux. Also included are protective proteins (chaperone [heat shock proteins] and late embryogenesis abundant [LEA] proteins) and products (glycine betaine, polyols, proline) of biochemical pathways that aid in osmotic adjustment. (Modified from Ref. 11.)

Figure 2 Cellular stress tolerance elements. The schematic drawing of a cell includes the vacuole into which sodium is partitioned, a chloroplast, mito-chondrium, and peroxisome, which are prone to oxygen radical damage. Included are symbols for H+-ATPases/PPiases that maintain transmembrane proton gradients and symbols for potassium uptake, sodium transport, and controlled water flux. Also included are protective proteins (chaperone [heat shock proteins] and late embryogenesis abundant [LEA] proteins) and products (glycine betaine, polyols, proline) of biochemical pathways that aid in osmotic adjustment. (Modified from Ref. 11.)

represent these "downstream" biochemical mechanisms, compiled from work mainly with Arabidopsis mutants, yeast, and several halophytic and xerophytic models.

Transgenic tobacco expressing selected genes has frequently been utilized for its usefulness as a biochemical model. Effects have been studied in transgenic plants in which several mechanisms were tested: enhanced osmotic adjustment, the expression of late embryogenesis abundant (LEA) proteins, or proteins leading to radical scavenging through the overexpression of foreign proteins (33). Moderate improvements of tolerance have been documented. In other models, Arabidopsis thaliana, Medicago sativa, and Oryza sativa, equally successful minor improvements of abiotic stress tolerance have been reported (38,117-121) (for reviews see Refs. 38,120,121).

Saccharomyces cerevisiae is by far the best model for understanding salinity tolerance mechanisms at the cellular level (20,33,122). Yeast is salt tolerant, and salt-sensitive mutants are readily obtained. These mutants allow the identification of important salinity tolerance genes, and complementation allows the identification of homologues from other species as well as providing useful strains for a variety of physiological and transgenic experiments at the cellular level. The yeast genome includes approximately 6000 open reading frames (123). Based on several analyses and the following considerations, approximately 100 ORFs seem to provide the basis for salinity stress tolerance (33). For example, through the deletion of PBS2, encoding a mitogen-activated protein (MAP) kinase kinase of the HOG osmotic stress signaling pathway, proteins controlled by this pathway could be documented by their disappearance from two-dimensional gels (124). The authors found 29 proteins affected by this deletion. Assuming that only a small number of the yeast proteins are of sufficiently high abundance to be visible, by extrapolation a number of approximately 100 closely stress-associated genes seems a reasonable estimate. That a larger number should aid in tolerance but not be absolutely necessary is reasonable, and this consideration puts the estimate of stress-affected genes in yeast in the low hundreds.

An analysis by microarray of all yeast ORFs indicated that approximately 300 transcripts, or 5% of the yeast genes, are significantly increased following salt stress and approximately 200 transcripts are down-regulated to a similar degree (J. Yale, H. J. Bohnert, unpublished results). When the cells experienced oxidative stress or heat shock, similar numbers of up-regulated transcripts were observed, although the overlap between the different stresses was only approximately 25%. The processes controlled by the known yeast genes are similar to those found or suggested as essential reactions of stressed plants. The majority of these genes, including those most strongly up-regulated, encode functions in energy metabolism, ion homeostasis, cell defense, chaperone functions, and transport facilitation. Approximately one third of the up-regulated yeast transcripts encode proteins of unknown function. We think that in plants a similar number of genes, maybe up to 10% of the plant genome or roughly 3000 genes, may participate in adaptive stress reactions (116).

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