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in maize plants (Rowland and Chourey, 1990). The Sh and Sws-encoded protein
subunits, SSI and SS2, polymerize randomly in seedling roots and shoots to
produce the five different sucrose synthase isozymes, two homotetramers (S1S1S1S1 and S2S2S2S2) and three heterotetramers (S1S1S1S2, S1S1S2S2 and S1S2S2S2) (Chourey, 1988; Rowland and Chourey, 1990). Developing endosperm cells contain the two homotetramers, whereas in young roots and shoots, the three heterotetramers are also present (Chourey et al., 1986). Similarly, five isozymes have been detected in sorghum but, in contrast to maize, both sucrose synthase genes are expressed simultaneously in the endosperm, leading to the additional presence of the heterotetramers in this tissue (Chourey et al., 1991).
A cDNA encoding sucrose synthase in potato has been cloned and sequenced and a comparison made with the maize sucrose synthase cDNA (Salanoubat and Belliard, 1987; Werr et al., 1985). The nucleotide sequence and deduced amino acid sequence for the two clones show 70% and 75% identity, respectively. Three amino acid regions are about 90% homologous and two are considered important regions for enzyme function due to strong constraints at the amino acid sequence level.
A sucrose synthase cDNA was also cloned and sequenced from mung bean seedlings (Arai et al., 1992a). The extent of the similarity between mung bean sucrose synthase and the enzymes from maize and potato was 71% and 72%, respectively, at the nucleotide level, and 76% and 81% at the deduced amino acid level. The deduced amino acid sequence from a cDNA sucrose synthase clone from Vida faba cotyledons (Heim et al., 1993) was closely related to both the mung-bean and potato sucrose synthase (95% homology) and to a lesser extent the maize sucrose synthase (approx. 75% homology). These molecular studies indicate a high degree of homology between the sucrose synthase genes from different plant species.
Sucrose synthase appears to be highly regulated both at the level of fine and coarse control. Discussion of possible effectors of sucrose synthase activity will be limited to those involved in sucrose cleavage only. The products of the reaction, UDP-glucose and fructose, both inhibit the enzyme. Pinyon (Pinus
edulis) sucrose synthase was non-competitively inhibited by UDP-glucose (K- - 0.011 mM) when tested over a range of UDP concentrations (Hammer and Murphy, 1993); whereas sucrose synthase from Jerusalem artichoke (Helianthus
tuberosus) was competitively inhibited by UDP-glucose (Wolosuik and Pontis, 1974). Fructose acts as a competitive inhibitor with respect to sucrose but as an uncompetitive inhibitor with respect to UDP (Wolosuik and Pontis, 1974). Fructose and glucose both uncompetitively inhibited maize endosperm sucrose synthase by binding to the enzyme-UDP complex (Doehlert, 1987). Soybean nodule sucrose synthase was inhibited 25% and 50% by glucose concentrations of 2 and 5 mM, respectively (Morell and Copeland, 1985). It is of physiological significance that the enzyme shows inhibition to the products of sucrose hydrolysis by invertase. By contrast, none of the following metabolites (at a final concentration of 5 mM) had any effect on soybean nodule sucrose synthase: galactose, mannose, maltose, raffinose, glucose-l-P, glucose-6-P, fructose-6-P, fructose-1,6-P2, 3-P-glycerate, P-enolpyruvate, ethanol, succinate, 2-oxoglutarate, glutamate, glutamine, NAD, AMP, and PPi (Morell and Copeland, 1985).
A cDNA sequence encoding a nodule-specific protein, nodulin-100, from soybean nodules was identified as a subunit of sucrose synthase (Thummler and Verma, 1987). Nodule sucrose synthase protein was shown to dissociate into its monomers in the presence of heme and it has been suggested that the availability of free heme may regulate the activity of the nodule sucrose synthase.
At the gene level, sucrose synthase expression is not only spatially controlled within the plant (Heinlein and Starlinger, 1989; Chourey et al., 1991) but is also regulated by tissue carbohydrate levels (Koch et al., 1992; Salanoubat and Belliard, 1989; Sowokinos and Varns, 1992) and by anaerobic conditions (Salanoubat and Belliard, 1989; Taliercio and Chourey, 1989; Chourey et al., 1991). The results of Koch and coworkers (1992) demonstrate for the first time that there is differential regulation of the sucrose synthase isozyme genes within a plant organ. Metabolisable sugars had both positive and negative effects on the sucrose synthase gene system with Shi transcript levels decreasing in the presence of 4% glucose while Susl levels increased. Sucrose was shown to down regulate the Shi message to an equal or greater extent than did glucose but had little effect on Susl. Fructose affected transcript levels of both Shi and Susl in a similar manner to glucose although the negative and positive changes in the expression of the two genes with fructose was not so great as with glucose.
In potato leaf petioles supplied with a range of sucrose solutions (50, 100, 150, 200 and 250 mM), an almost linear increase in sucrose synthase transcript levels was observed by Northern blotting indicating that sucrose is involved in the coarse control of the enzyme (Salanoubat and Belliard, 1989). Additional evidence on sucrose induction of sucrose synthase was demonstrated with cultured potato cells (Sowokinos and Varns, 1992). Anaerobic incubation conditions had a stimulating effect on the level of sucrose synthase mRNA in potato tubers (Salanoubat and Belliard, 1989) and on the level of both sucrose synthase genes in sorghum (Chourey et al., 1991).
The importance of a high degree of metabolic regulation will be evident when considering the involvement of sucrose synthase activity in carbohydrate metabolism.
1.4 The roles of the sucrose-cleaving enzymes in carbohydrate metabolism
1.4.1 Cell wall acid invertase
Evidence presented in section 1.3.1.1 clearly identifies a distinction between cell wall and vacuolar acid invertases based on their properties and structure. Studies on a number of the cell wall invertases, whose properties are listed in Table 1.1, have helped to elucidate the function of these enzymes in planta. In maize kernels, 60 to 80% of acid invertase activity was found to result from insoluble acid invertase which was apparently bound to cell walls by ionic interactions (Doehlert and Felker, 1987). In addition, most of the activity was restricted to the pedicel tissues and the basal region of the endosperm (Felker, 1986), suggesting a role for the cell wall invertase in the unloading of sucrose from the phloem and subsequent uptake into the basal endosperm. This possibility has been further investigated using the maize invertase-deficient
miniature-1 seed mutation which has abnormal pedicel and endosperm development (Miller and Chourey, 1992). Biochemical and histochemical analysis of the mutant maize kernels showed that extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities were present. The data is thought to provide strong evidence that invertase in basal endosperm cells is required for the cleavage of sucrose to allow its subsequent mobilisation to the upper parts of the maize endosperm. In its absence, the normal movement of photoassimilate between the pedicel and endosperm is disturbed resulting in cell degeneration and the characteristic small seed size of the miniature-1 mutants.
Evidence for the involvement of cell wall acid invertase in the cleavage of apoplastic sucrose prior to uptake into the parenchyma cells in sugar beet taproots, based on protoplast studies, is provided by the preferential uptake of glucose (Fieuw and Willenbrink, 1990). The glucose influx, mediated by a