Much of what we know of the cellular protection mechanisms involved in desiccation tolerance in plants comes from studies of orthodox seeds (Bewley and Black, 1994; Chapter 5) and, to a slightly lesser extent, pollen (Crowe et al., 1992; Hoekstra et al., 1992). The ability of seeds to withstand desiccation is acquired during their development. This acquisition is usually substantially earlier than the culmination of the drying event itself, which is the terminal event in orthodox seed maturation. Seeds of some species can withstand premature desiccation well before the midpoint of their development (Bewley and Black, 1994; Chapter 5). Among the metabolic changes that take place just prior to or during drying is the synthesis of proteins and sugars, which have long been postulated to form the basis of a series of overlapping protective mechanisms that limit damage to cellular constituents (Bewley, 1979; Leprince et al., 1993; Oliver and Bewley, 1997). These two components have since been widely implicated as being critical for desiccation tolerance in all plant cells including vegetative cells (Ingram and Bartels, 1996; Oliver and Bewley, 1997; Scott, 2000). Over the years it has also become clear that the synthesis of antioxi-dants and enzymes involved in oxidative metabolism also play a critical role in cellular protection and desiccation tolerance (Chapter 10). However, this aspect of protection will not be addressed here.
Only one subset of proteins that accumulate at the time of the acquisition of desiccation tolerance has been extensively investigated, the late embryogenesis abundant (LEA) proteins, first described in cotton (Galau and Hughes, 1987; Galau et al., 1987, 1991; Chapter 5). The genes that encode LEA proteins in developing cottonseeds are comprised of two distinct classes whose regulation is coordinated. One class contains six different lea transcripts, which appear relatively early in development and reach a maximum about three days before the seed begins to desiccate (Galau and Hughes, 1987; Galau et al., 1987). The other class contains 12 transcripts, which appear late in maturation and achieve maximum expression just before and during desiccation. LEA proteins make up 30% of the non-storage protein and 2% of the total soluble protein in the mature cotton embryos and are uniformly localized throughout the cytoplasm (Roberts et al., 1993). LEA proteins and the acquisition of desiccation tolerance during seed maturation have been linked in other dicots (e.g. soybean: Blackman et al., 1995) and in monocots (e.g. maize: Mao et al., 1995; Wolkers et al., 1998).
A set of LEA proteins arises in developing barley and maize embryos at the time that tolerance of desiccation is acquired. A small subset of these proteins is induced when barley embryos at the intolerant stage are cultured in abscisic acid (ABA) (Bartels et al., 1988; Bochicchio et al., 1991), and a causal relationship between ABA and lea gene expression has been suggested. Evidence for, and against, this relationship exists in the literature. In cotton embryos, high expression of the first class of lea genes occurs as ABA content increases. High expression of the second set of lea genes, however, occurs at the start of, and during, maturation drying, when the endogenous ABA content is low. There are explanations for this lack of correlation, e.g. there is an early-regulated, ABA-controlled mechanism, which operates only later when drying commences. On the other hand, an ABA-independent pathway may be involved in the synthesis of the second group of LEA proteins.
LEA proteins have been identified in the vegetative tissues of all desiccation-tolerant plants studied so far (Ingram and Bartels, 1996; Oliver and Bewley, 1997; Blomstedt et al., 1998) and proteins related to some of the LEA proteins, e.g. dehydrins (see below), have been associated with the response of non-tolerant plants to water stress (Skriver and Mundy, 1990; Bray, 1997). In nearly all instances, the induction of LEA protein synthesis in vegetative tis sues can be elicited by exogenous ABA application (Ingram and Bartels, 1996; Campalans et al., 1999).
LEA proteins fall into five groups by virtue of sequence similarities (Dure et al., 1989; Ingram and Bartels, 1996; Cuming, 1999). All are highly hydrophilic and all are very stable, as evidenced by their resistance to the denaturing effects of boiling (with the exception of Group 5 LEA proteins). Group
1 LEA proteins are characterized by a 20-amino acid motif and are represented by the wheat Em protein, the first LEA protein identified (Cuming and Lane, 1979). Group
2 LEA proteins are characterized by a 15-amino acid motif, the K-segment, a stretch of serine residues and a conserved motif near the N-terminus of the protein (Close, 1997). This group of proteins is also called the dehydrins and these are the most widespread and most studied of the LEA proteins. Group 3 LEA proteins share a characteristic 11-amino acid repeat motif (Dure et al., 1989), which is predicted to form an amphipathic a-helix. These amphi-pathic helices are postulated to form intra-and intermolecular interactions that may have important consequences for their function (Baker et al., 1988; Dure, 1993a). The least studied of the LEA proteins are those in Groups 4 and 5, which are somewhat atypical (Dure, 1993b; Galau et al., 1993). Group 5 LEA proteins are more hydrophobic than other LEA proteins and are not resistant to high temperature. Most of the LEA protein groups have been identified in many different plants. All groups are thought to play a role in desiccation tolerance, and the evidence for this viewpoint is growing.
The evidence for the involvement of LEA proteins in desiccation tolerance is circumstantial but compelling. LEA protein synthesis in seeds, as mentioned above, is associated with both the acquisition of desiccation tolerance and the final stage of seed maturation just prior to desiccation. In addition, ABA-deficient (aba) and ABA-insensitive (abi3) double-mutants of Arabidopsis seeds do not dry on the parent plant, do not tolerate desiccation and lack several LEA proteins (Koorneef et al., 1989; Meurs et al., 1992).
LEA protein synthesis is also highly induced in the vegetative tissues of desiccation-tolerant angiosperms during drying (Bartels et al., 1993; Blomstedt et al., 1998; Bartels, 1999). Callus derived from vegetative tissue of the desiccation-tolerant plant Craterostigma plantagineum is not inherently tolerant but can be made so by the application of ABA (Bartels et al., 1990). The application of ABA to this tissue results in the synthesis of novel proteins, some of which are LEA proteins including the Group 2 LEA proteins, the dehydrins (Bartels et al., 1993). The desiccation-tolerant moss T. ruralis utilizes a more primitive mechanism of desiccation tolerance (Oliver et al., 2000), which involves a constitutive cellular protection strategy, and in this plant, unlike others, dehydrins are not induced by dehydration or by ABA but are constitutively expressed (Bewley et al., 1993). Dessication-sensitive species exposed to sub-lethal dehydration stress also respond by synthesizing LEA proteins and LEA-like proteins, in particular dehy-drins (Close, 1997). These examples and many more all point to the importance of LEA proteins in dehydration responses and desiccation tolerance.
The most convincing pieces of evidence to suggest that LEA proteins have an important role in cellular protection come from transgenic studies using a barley Group 3 lea gene, HVA1. This gene, when expressed in a constitutive fashion in transgenic rice, increased its tolerance to water and salt stress (Xu et al., 1996). HVA1 overexpression in wheat, driven by a maize ubiquitin promoter, resulted in transgenic lines that performed in a superior fashion under soil-water deficits (Sivamani et al., 2000).
There are a variety of suggested mechanisms by which LEA proteins might protect cellular components. Many LEA proteins have extensive regions of random coiling, which has been postulated to promote the binding of water, helping to maintain a minimum water requirement (Ingram and Bartels, 1996). For instance, the Em protein of wheat is considerably more hydrated than most common proteins, and over 70% of the Em protein is configured as random coils (McCubbin et al., 1985). Baker et al. (1988) suggested that the random coil nature of some LEA proteins may allow them to conform to the shape of cellular constituents and thus, by virtue of their hydroxyl groups, help to maintain their solvation state when water is removed. These authors also suggested that the Group 2 LEA proteins (dehydrins), by virtue of their amphipathic helical repeats, provide surfaces when bundled together that would sequester ions. This may be crucial as the increasing ionic strength during drying could cause irreversible damage to cellular proteins and structural components. Recently, Velten and Oliver (2001) described an LEA-like protein from T. ruralis that contains 15 15-amino-acid repeats predicted to form amphipathic helices. This protein appears to be synthesized during the rehydration event and may serve to trap valuable ions that would otherwise be lost. Studies using individual LEA proteins in in vitro assays also add to the possible mechanisms by which these proteins exert protection of cellular components. Wolkers (1998) suggested from data obtained from the study of a pollen Group 3 LEA protein and its effect on sucrose glass formation that LEA proteins may act as anchors in a structural network that stabilizes cytoplasmic components during drying and in the dried state.
At this point it seems likely that each individual group of LEA proteins may have different, complementary effects. Most desiccation-tolerant tissues contain a representative of most, if not all, of the different groups of LEA proteins, and it is also likely that all are needed to achieve the highest degree of desiccation tolerance.
There is mounting evidence that another class of proteins, the small heat-shock proteins (HSPs), may play a role in cellular protection during desiccation. Small HSPs accumulate in maturing seeds of many plant species (Vierling, 1991; Wehmeyer et al., 1996) prior to desiccation. Alamillo et al. (1995) reported that small HSPs are expressed constitutively in the vegetative tissues of C. plantagineum and increased in accumulation during desiccation. Constitutive expression of HSPs is unusual in vegetative tissues and resembles the expression pattern of these proteins in seeds. In addition, exogenous ABA induced both the expression of HSPs and the acquisition of desiccation tolerance in C. plantagineum callus tissues (Alamillo et al., 1995). Finally, a LEA-like HSP, HSP-12, from yeast was shown to be capable of protecting liposomal membranes from the damaging effects of desiccation in a way similar to that seen with the sugar tre-halose (Sales et al., 2000). Thus it appears that small HSPs may also play a role in cellular protection during desiccation: perhaps this capability is related to their chaperonin-like activities, which may help maintain protein structure under denaturing conditions. Other proteins whose transcripts accumulate during the dehydration phases of vegetative desiccation-tolerant angiosperms have been identified but little has been done to confirm their roles in desiccation tolerance (Kuang et al., 1995; Ingram and Bartels, 1996; Blomstedt et al., 1998; Bockel et al., 1998; Neale et al., 2000). See Chapters 5 and 11 for further discussion of all these proteins.
The accumulation of soluble sugars is also strongly correlated to the acquisition of desiccation tolerance in plants and other organisms (for reviews see Crowe et al., 1992; Leprince et al., 1993; Vertucci and Farrant, 1995; Chapters 5 and 10). Soluble sugars, especially sucrose, accumulate in seeds (Leprince et al., 1993), pollen (Hoekstra et al., 1992) and in desiccation-tolerant vegetative tissues (Bewley and Krochko, 1982; Ingram and Bartels, 1996; Oliver and Bewley, 1997). In Craterostigma plantagineum, 2-octulose stored in the hydrated leaves is converted to sucrose during drying to such an extent that in the dried state it comprises about 40% of the dry weight (Bianchi et al., 1991).
Sucrose is the only free sugar available for cellular protection in desiccation-tolerant mosses, including Tortula ruraliformis and T. ruralis (Bewley et al., 1978; Smirnoff, 1992). The amount of this sugar in gametophytic cells of T. ruralis is approximately 10% of dry mass, which is sufficient to offer membrane protection during drying, at least in vitro (Strauss and Hauser, 1986). Moreover, neither drying nor rehydration in the dark or light results in a change in sucrose concentration, suggesting that it is important for cells to maintain sufficient amounts of this sugar (Bewley et al., 1978). The lack of an increase in soluble sugars during drying appears to be a common feature of desiccation-tolerant mosses (Smirnoff, 1992).
It is thought that sugars protect the cells during desiccation by two mechanisms. First, the hydroxyl groups of sugars may substitute for water to maintain hydrophilic interactions in membranes and proteins during dehydration (Crowe et al., 1992). This has so far only been demonstrated in vivo, using liposomes and isolated proteins (Crowe et al., 1992). Secondly, sugars are a major contributing factor to vitrification, the formation of a biological glass, of the cytoplasm of dry cells (Leopold et al., 1994; Chapter 10). This mechanism has been the subject of intense research over the last 15 years.
Vertucci and Leopold (1986) suggested that desiccation tolerance in seeds had to be associated with some feature or solute combination that would avoid crystallization of the cytoplasm as dehydration progressed. Burke (1986) proposed that high concentrations of sugars lead to vitrification of the cytoplasm during desiccation and thus prevent crystallization. Glass formation has since been demonstrated in seeds (Williams and Leopold, 1989; Leopold et al., 1994; Leprince and Walters-Vertucci, 1995), pollen (Buitink et al., 1996) and in leaf tissues of C. plantagineum (Wolkers et al., 1998). Walters (1998) went as far as to say that glass formation is an intrinsic property of any complex system that can survive desiccation. However, glass formation may not be sufficient to confer desiccation tolerance since desiccation-sensitive tissues are capable of forming cytoplasmic glasses (Sun et al., 1994; Buitink et al., 1996).
Cytoplasmic glass formation has also been postulated to maintain the structural and functional integrity of macromolecules (Sun and Leopold, 1997; Crowe et al., 1998b), which has been well demonstrated with in vitro models (Roos, 1995). Intracellular glasses, by virtue of their high viscosity, drastically reduce molecular movement and impede diffusion of reactive compounds in the cell. It is by this property that glasses are thought to prolong the longevity of desiccated tissues by slowing down degradative processes during storage. Buitink et al. (1998) recently demonstrated a strong relationship between molecular mobility and storage longevity in both pollen and pea seeds. Thus, although glass formation may not be important in the initial acquisition of desiccation tolerance, it may be crucial for survival of the dried state (as suggested by Buitink, 2000; Chapter 10).
Other carbohydrates besides sucrose accumulate in desiccation-tolerant tissues, the principal ones being the oligosaccha-rides stachyose and raffinose (Horbowicz and Obendorf, 1994), and have been postulated to play a part in desiccation tolerance. The presence of these compounds has also been correlated with seed longevity (Hoekstra et al., 1994; Horbowicz and Obendorf, 1994), which has linked them to a possible role in the stabilization of intracellular glasses (Leopold et al., 1994; Bernal-Lugo and Leopold, 1995; Sun, 1997). However, Buitink et al. (2000) demonstrated that the reduction in oligosaccharides in primed seeds did not alter Tg (the glass-to-liquid transition temperature) or viscosity and thus they contended that oligosaccharides do not affect the stability of intracellular glasses. These results support the earlier studies of Black et al. (1999), which had shown a lack of a temporal correlation between the induction of desiccation tolerance by a mild dehydration treatment and the appearance of raffinose in wheat embryos. These studies cast doubt on the role of oligosaccharides in the acquisition of tolerance and the maintenance of viability in the dried state.
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