Osmotic Adjustments and Controlling Factors

Among the accumulators are amino acids, predominantly proline, sugars and sugar alcohols, such as sucrose or mannitol, trehalose, mannitol/sorbitol, or inositol derivatives, and more complex carbohydrates, such as fructans and raffinose-related compounds. This list is likely to expand in the future as other models are investigated. In a drought-adapted watermelon, for example, citrulline, an intermediate in the urea cycle, has been detected as a drastically accumulating metabolite during drought stress. Citrulline function may be in radical oxygen scavenging (Akashi et al., 2001, 2004). A number of experiments have been reported that attempted to engineer osmolyte accumulation into glycophytic plants that show only marginal accumulation of metabolites with the intention to improve tolerance. Table 12.2 provides a selection of such studies, also including experiments to transgenically engineer ionic stress tolerance and to engineer regulatory circuits. The range included exemplifies the major categories of genes or cDNAs that have been used in engineering: osmolytes and other protectants [chaperones, late-embryogenesis-abundant (LEA) and heat-shock proteins (HSPs)], transporters and pumps, scavengers of radicals, adjustments in hormone biosynthesis, and regulatory genes, outlined in detail at: www.plantstress.com/Files% 5CAbiotic-stress_gene.htm. Under strictly controlled growth conditions, it has been shown in many of these experiments that the plants exhibited showed increased osmotic, ionic, or temperature tolerance (Table 12.2). Often the actual increase or accumulation did not amount to concentrations found in the natural models gave rise to the ‘‘compatible osmolyte’’ concept.

These experiments demonstrated several other aspects as well. First, osmotic/ ionic abiotic stress tolerance seems to be controlled by different mechanisms depending on age or developmental stage, that is, seedling, vegetative, and reproductive stages, each seem to require stage-specific regulation of tolerance and protective determinants (Rontein et al., 2002).

FIGURE 12.2 Biochemical and metabolic determinants of salinity stress tolerance. (A) A schematic representation of cellular mechanisms depicted as functional categories (e.g., maintenance of membrane potential, ROS-scavenging, altered membrane traffic, or protein turnover), and identification of major metabolites and protein families that constitute cellular defenses against ionic stress (modified after Hasegawa et al., 2000b). Included are functions such as protein turnover, membrane structure, and vesicular traffic reorientations. Not included are molecular functions that also play important roles: chromatin remodeling, transcription/splicing, RNA transport, or regulation of translation. (B) Metabolic reactions leading to trehalose synthesis and reutilization in S. cerevisiae stressed by addition of 1 M NaCl (90 min). Upregulated transcripts are indicated by filled circles, and the presence of a stress response element (STRE) in promoters of individual genes is indicated (*). Although trehalose accumulates long term, the trehalose- cleaving enzyme, trehalase, is also upregulated (Yale and Bohnert, 2001).

In one of the first attempts at engineering, for example, salinity stress tolerance (mannitol accumulation) was observed only when the transgenic tobacco plants received stress during early vegetative growth (Tarczynski et al., 1993). Second, the common use of strong, constitutively expressed regulatory elements, while potentially leading to high (enzyme) expression, product accumulation, and vegetative tolerance, is or can be nonphysiological. This was demonstrated by the high accumulation of D-ononitol and mannitol in transgenic tobacco that protected the plants at vegetative growth stages (salinity and drought), but prevented normal seed formation due to the interference of the accumulating metabolites, both nonutilizable metabolic endproducts in tobacco, with sucrose unloading in the developing seeds (Sheveleva et al., 2000). Figure 12.2A gives a schematic rendition of biochemical and physiological mechanisms, structures, and determinants that have recognized as stress relevant from experiments conducted during the last decade.

Third, synthesis leading to accumulation is not necessarily the major purpose of a purported osmolyte, while the accumulation may be a pathological side effect. One example is yeast that increases the pathway leading to increased synthesis of trehalose (Hohmann, 2002). However, yeast also increases levels of enzymes that degrade trehalose, indicating that flux through the pathway (consuming reducing power) may be more important than simply making more (Fig. 12.2B). This behavior has been shown in several studies (Yale and Bohnert, 2001). Only at very high salt concentrations, which arrests or slows growth, will trehalose accumulate drastically in yeast. Such a view might then lead to a different interpretation for the compatible solutes. They might be seen as metabolic valves that adjust or lower the redox potential of cells in order to prevent or minimize production of radical oxygen species (ROS) in mitochondria and chloroplasts. Finally, attempts at engineering salinity stress tolerance have only begun to pay attention to compartment-specific strategies and the appreciation of stress tolerance as a multigenic network has not been tackled, for example, by the accumulation of different stress alleviating determinants.