Novel starch-related enzymes and carbohydrates.

In chloroplasts, both biosynthesis and degradation of starch are strictly regulated but the mechanisms involved are still incompletely understood. Recent studies revealed two novel and regulatory relevant aspects in the biochemistry of starch: the phosphorylation of starch and the starch-related metabolism of cytosolic heteroglycans. Starch phosphorylation occurs by a sequential action of two plastidial enzymes, the glucan, water dikinase (GWD; EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD; EC 2.7.9.5). Both enzymes utilize ATP as dual phosphate donor and transfer the terminal phosphate group to water whereas the beta-phosphate is used for esterification of glucosyl moieties. The metabolism of starch-derived degradation products is closely linked to recently discovered cytosolic heteroglycans that possess, as prominent constituents, arabinose, galactose, glucose and fucose. The pattern of glycosidic linkages is highly complex comprising more than 25 different bonds. During the dark period the size distribution or the amount of the cytosolic heteroglycans increases depending on the plant species. As revealed by in vitro 14C labeling assays, the heteroglycans act as both glucosyl acceptors and donors for two cytosolic glucosyl transferases, the phosphorylase (EC 2.4.1.1) and the transglucosidase (EC 2.4.1.25) and, at least in part, both enzymes utilize the same glucosyl acceptor and donor sites. In mutants of Arabidopsis thaliana L. that are deficient in the cytosolic transglucosidase both the structure and (bio)chemical properties of the heteroglycans are altered.

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter.

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

Reversible binding of the starch-related R1 protein to the surface of transitory starch granules.

Intact starch granules were isolated from leaves of Solanum tuberosum L. (and from Pisum sativum L.), and the patterns of starch-associated proteins were determined by SDS-PAGE. Depending on the pretreatment of the leaves the protein patterns varied: a 160 kDa compound was present in the starch-associated protein fraction when the leaves were darkened and performed net starch degradation. However, following illumination (i.e. during net starch biosynthesis) the 160 kDa protein was recovered almost exclusively in a soluble state. The 160 kDa protein was identified to be the recently described starch-related R1 protein. In in vitro assays recombinant R1 did bind to starch granules isolated from either illuminated or darkened leaves. However, binding to the latter was more effective. It is concluded that, depending upon the metabolic state of the cells, the starch granule surface changes and thereby affects binding of the R1 protein.

Rates of sugar uptake by guard cell protoplasts of pisum sativum L. Related To the solute requirement for stomatal opening

We wished to determine whether the capacity of the sugar uptake mechanisms of guard cells of the Argenteum mutant of pea (Pisum sativum L.) sufficed to support a concurrent stomatal opening movement. Sugar uptake by guard cell protoplasts was determined by silicone-oil-filtering centrifugation. The protoplasts took up [(14)C]glucose, [(14)C]fructose, and [(14)C]sucrose (Suc), apparently in symport with protons. Mannose, galactose, and fructose competed with Glc for transport by a presumed hexose carrier. The uptake of Glc saturated with a K(m) of 0.12 mM and a V(max) of 19 fmol cell(-1) h(-1). At external concentrations <1 mM, the uptake of Suc was slower than that of Glc. It exhibited a saturating component with a K(m) varying between 0.25 and 0.8 mM and a V(max) between 1 and 10 fmol cell(-1) h(-1), and at external concentrations >1 mM, a non-saturating component. At apoplastic sugar concentrations below 4 mM, sugar import was estimated to be mainly in the form of hexoses and too slow to support a simultaneous stomatal opening movement. If, however, during times of high photosynthesis and transpiration, the apoplastic Suc concentration rose and entered the range of non-saturating import, absorbed Suc could replace potassium malate as the osmoticum for the maintenance of stomatal opening.

Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening.

We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules. Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch. The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen. It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level. The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.