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.

Arabidopsis cytosolic alpha-glycan phosphorylase, PHS2, is important during carbohydrate imbalanced conditions.

Arabidopsis thaliana has two isoforms of alpha-glycan phosphorylase (EC 2.4.1.1), one residing in the plastid and the other in the cytosol. The cytosolic phosphorylase, PHS2, acts on soluble heteroglycans that constitute a part of the carbohydrate pool in a plant. This study aimed to define a physiological role for PHS2. Under standard growth conditions phs2 knock-out mutants do not show any clear growth phenotype, and we hypothesised that during low-light conditions where carbohydrate imbalance is perturbed, this enzyme is important. Soil-grown phs2 mutant plants developed leaf lesions when placed in very low light. Analysis of soluble heteroglycan (SHG) levels showed that the amount of glucose residues in SHG was higher in the phs2 mutant compared to wild-type plants. Furthermore, a standard senescence assay from soil-grown phs2 mutant plants showed that leaves senesced significantly faster in darkness than the wild-type leaves. We also found decreased hypocotyl extension in in vitro-grown phs2 mutant seedlings when grown for long time in darkness at 6 °C. We conclude that PHS2 activity is important in the adult stage during low-light conditions and senescence, as well as during prolonged seedling development when carbohydrate levels are unbalanced.

The role of plastidial glucose-6-phosphate/phosphate translocators in vegetative tissues of Arabidopsis thaliana mutants impaired in starch biosynthesis.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.