With the exception of the "resurrection plants", Selaginella lepidophylla and Myrothamnus flabellifolius, which utilize trehalose to survive extreme desiccation, the disaccharide was thought to be largely absent in plants (79). At the same time, however, extending the protective effects of trehalose to plants had been a sought after goal of work in stress tolerance (80,81). A number of exciting recent discoveries have dispelled the notion that trehalose is rare in plants. Equally striking is the discovery that the same basic parameters which characterize trehalose in S. cerevisiae are also seen in plants, and now in Drosophila as well: trehalose enhances resistance to environmental stress; accumulation of trehalose can be detrimental during growth; and finally, components and intermediates of the trehalose synthesis process are intimately connected to metabolism and gene expression.
The finding that trehalose is widespread in plants was made serendipitously: in the course of efforts to engineer trehalose production in tobacco and potato, the control plants were found to contain trace levels of the sugar (81). Other work demonstrated that treatment of Arabidopsis with the trehalase inhibitor validomycin A resulted in significant accumulation of trehalose (79,82). TPS and TPP homologs have since been identified in rice, soybean, and tomato, and more recently, a host of related genes, as well as a putative trehalose, have been found in Arabidopsis (71).
The Arabidopsis sequences cluster into two groups (71). The first subfamily, members of which most resemble Tps1, contains four genes which are 63-80% identical in sequence. One gene, designated AtTps1, restores the ability of S. cerevisiae tps1 mutants to produce trehalose (67). The AtTps1 sequence includes a 100 amino acid N-terminal region not present in yeast Tps1 or the other plant Tps1 homologs. This sequence is homologous to segments of the yeast regulator protein Tsl1 and likewise appears to have a regulatory function: when deleted, activity of the enzyme increases (83). Members of the second subfamily are most similar to Tps2. This group shares 54-83% sequence identity, and all members contain a C-terminal sequence common to phosphatases (71).
Overexpression of yeast and E. coli trehalose synthesis enzymes in transgenic plants indeed enhanced resistance to stress (82,84,85). As with S. cerevisiae nth1 mutants, however, this benefit was not without cost: the transgenic plants displayed altered metabolism and morphological growth defects, such as stunted growth and lancet shaped leaves (82,86).
Studies in Arabidopsis revealed that AtTps1 is expressed at low levels in all tissues examined, and is strongly upregulated during embryo maturation (seed development) (87). Perhaps the most dramatic parallels to yeast were seen in a key study in which the gene was mutated. Although the thermotolerance properties of the mutant have not yet been reported, the effects on development were manifest, resulting in recessive embryonic lethality (87). Development in higher plants such as Arabidopsis proceeds in three overlapping stages. Cells first divide and differentiate, establishing the pattern of the embryo. Next, cells expand in size and accumulate storage reserves. Finally, the embryo desiccates and growth halts. The AtTps1 mutant plants were unable to proceed past the torpedo stage, at the transition between pattern formation and growth (87).
Under normal conditions, sucrose levels rise dramatically in Arabidopsis embryos at this juncture, as does expression of AtTps1. The increase is thought to be required both for producing storage reserves and for influencing gene expression to control this developmental transition. The latter suggestion is supported by abnormalities in expression of the seed maturation marker genes At2S2 and AtOLEOSN2 seen in the mutant.
As mentioned earlier, S. cerevisiae tps1 mutants die in the presence of glucose. The sugar enters the cells and is phosphorylated in an unrestricted manner, depleting cellular phosphate and ATP stores. Moreover, Tps1 is also required for carbon catabolites to control gene expression (49). These cells are, however, able to grow on carbon sources such as galactose. As discussed earlier, growth on glucose can be restored by mutating the gene encoding Hxk2, the enzyme primarily responsible for phosphorylating glucose for entry into glycolysis (65). Just as S. cerevisiae tps1 mutants can be rescued by restricting entry of glucose into glycolysis, AtTps1 mutants similarly are able to proceed with development in vitro if sucrose levels are reduced. Unlike in yeast, growth could not be restored in these cells by targeting Hxk2 (here by antisense methods or treatment with the hexokinase inhibitor glucosamine), nor is Arabidopsis Hxk2 inhibited by trehalose-6-phosphate (87). The reason for this failure is not clear, but several explanations have been proposed. The HXK gene family in Arabidopsis is not well characterized, so another isoform (or perhaps an entirely distinct kinase) may be the critical target. Alternatively, sucrose can enter gly-colysis through more than one route in plants, so its catabolism at this developmental stage may proceed by a different pathway, such as that utilizing sucrose synthase, which does not require phosphorylation of glucose (87,88). Finally, it is also possible that AtTps1 acts directly or via trehalose to influence development and gene expression. Based on their finding that the E. coli trehalose synthase gene can complement the AtTps1 mutation, Schluepmann et al. favor the view that the mutant phenotype results from the absence of activity of AtTps1, rather than a regulatory property of the protein (89).
The likelihood that the protean roles of the trehalose pathway are broadly conserved was underscored by exciting work in Drosophila paralleling these findings. Drosophila is unusually resistant to anoxic injury, a property of considerable medical interest because of its potential to help limit damage to the brain in patients following stroke or cardiac arrest. By overexpressing the Drosophila Tps1 gene, Chen et al. succeeded in making that organism even more resistant. Strikingly, deletion of the gene or its overexpression under certain conditions led to developmental abnormalities or death of the developing flies (4).
Efforts to elucidate the role of trehalose in adverse conditions and in growth and metabolism have advanced concurrently with the drive to harness its protective properties in food biotechnology. That work, which has primarily involved genetic engineering and exogenous use of the sugar, also holds outstanding promise broadly in industry and medicine.
Utilization of trehalose as an additive for food preservation or storage has recently become feasible through the development of new techniques enabling economical large scale production from starch (24,29). The sugar's mild sweetness — 45% that of sucrose (29) — and "masking effect" of bitter-tasting compounds, as well as lack of an aftertaste or laxative effect, enhance its versatility as a contributor to the texture and balance of flavors in foods (90). It is not easily degraded during processing, and its resistance to taking up moisture reduces caking in mixtures with other ingredients (29). The protective effects of trehalose prolong the shelf life of starch-containing products by preventing retrogradation, as noted; similarly, the stabilization of proteins and lipids during freezing and drying extends the shelf life of products containing eggs or meat (29).
Genetic methods have successfully been employed to improve the freeze tolerance of frozen dough (9l,92). As mentioned earlier, tobacco and potato plants modified to produce trehalose display enhanced tolerance to a diverse range of stresses, including salt, drought, and low temperatures (79). Considerable potential also holds for the use of such methods to promote proper folding and prevent aggregation of recombinant proteins synthesized in E. coli or other organisms, for production of foods or pharmaceuticals (ll). Trehalose has already been shown to help preserve vaccines and liposomes, as well as organs in preparation for transplant (30,90). The novel finding that disruption of Tps2 in Candida leads to diminished infectivity of that organism suggests that the trehalose pathway may be an attractive target for antibiotics (93,94). The same may also hold true for pesticides: diminished infectivity and pathogenicity was similarly seen for trehalose mutants of the rice blast fungus Magnaporthe grisea (95).
Finally, trehalose may be helpful in the treatment and prevention of disorders resulting from misfolding or aggregation of proteins (9,96,97). Expression of a-synuclein, a protein involved in Parkinson's disease, is toxic to yeast. In a genetic screen to identify modulators of this toxicity, Willingham et al. discovered that cells lacking Tsll were more susceptible to the deleterious effects of a-synuclein (98). Another recent study, involving a transgenic mouse model of Huntington's disease, demonstrated that mice fed trehalose dosed water developed far fewer polyglutamine aggregates (one of the hallmarks of Huntington's disease pathology) than did untreated controls (99). The trehalose group also lived longer and performed better in coordination tests, suggesting a correlation between the decreased number of aggregates and improvements in the Huntington's-like symptoms.
Overall, three general principles are emerging from work on trehalose in S. cerevisiae and its striking correlates in plants and Drosophila: (1) trehalose has a protective role in vivo as well as in vitro in stress tolerance; (2) the same properties which can protect macromol-ecules may also be detrimental as environmental circumstances change; and (3) a critical but still elusive connection exists between the trehalose pathway and cellular metabolism, gene expression, and development. It is constructive to consider these principles in the broader context of stress tolerance mechanisms, particularly other organic solutes and the heat shock proteins (HSPs).
Regarding the former, trehalose is not unique in its protective effects. Rather, similar properties are shared by a host of other small molecules, such as the amino acids proline and betaine and the sugars sucrose and sorbitol (8,28). The diversity in nature of these "compatible solutes" or "chemical chaperones" is intriguing. It may reflect propensities to stabilize different sensitive structures in different organisms, or perhaps regulatory and metabolic functions (such as the interaction of trehalose with glycolysis) that make certain osmolytes of greater utility in particular organisms. Thus, a vital connection to cell biology and organismal development may exist more generally with compatible solutes, providing another unifying characteristic and potentially powerful tool in food biotechnology.
A paradigm for such an association between fundamental cellular processes and stress tolerance already exists, with the heat shock proteins. While a subgroup are expressed only under adverse environmental conditions, many others, such as the members of the Hsp70 and Hsp40 families, have variants which play essential roles in protein folding and translocation under normal circumstances (100). Even Hsp104, an S. cerevisiae heat shock protein initially thought to function exclusively in survival of extreme stress (75,77), subsequently was found to have a key role at normal temperatures for propagation of [PSI+], [RNQ+], and [URE3] (101). Also known as yeast prions, these unconventional genetic elements represent alternative but stable conformations of a particular protein, which propagate heritable information through maintenance of a particular conformation (101-03).
This finding and other work in HSPs highlight the subtleties of cellular adaptation to environmental challenges. Drosophila utilize Hsp70 as their primary protective mechanism in response to heat shock. In tissue culture cells and embryos, that protein is sequestered into granules with astonishing speed after return to normal conditions. As with trehalose, its removal likely occurs because of interference with protein folding and progress of development (104,105). A similar explanation may underlie the puzzling finding that although AtTpsl expression has been found in all Arabidopsis tissues examined, the disaccharide itself under normal conditions is detectable only at low levels, if at all (88).
As these incongruities continue to inspire further efforts, it is clear that in the nearly two centuries since its discovery, trehalose has indeed come full circle - from a component of the toxin ergot contaminating rye, to a versatile and valuable tool - in a transcendent irony with great promise for food biotechnology.
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