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Starch phosphorylase (Pho), especially the plastidial type, plays an important role in starch biosynthesis. The availability of a complete cassava (Manihot esculenta Crantz) genome database allowed the identification of five Pho family members, which could be grouped into the plastidial type (MePho1a, MePho1b, and MePho1c) and cytosolic type (MePho2 and MePho3) on the basis of phylogenetic analysis. Spatial gene expression analysis of various cassava tissues revealed that plastidial MePho1b had the highest transcriptional activity in root stele, whereas the cytosolic MePho2 was expressed with higher levels in leaves and petiole. Spatial expression analysis showed that MePho1b was strongly expressed in the tuber during root development process, especially at 70 and 180 d after planting, whereas MePho1a had highest expression in leaves at 180 d after planting. Expression of MePho1b and MePho2 could be induced by salt, drought, exogenous abscisic acid, methyl jasmonate, and salicylic acid signal in the leaves of abiotic stress-treated plantlets. In addition, the expression profiles of MePho1b and starch branching enzyme gene member MeSBE2.2 exhibited significant correlation under diverse stress environments, indicating that they may have coordinated functions. [ABSTRACT FROM AUTHOR]

Background Starch is synthesized in both leaves and storage tissues of plants. The role of starch syntheses and branching enzymes is well understood; however, the role of starch phosphorylase is not clear. Results A gene encoding Pho1 from barley was characterized and starch phosphorylases from both developing and germinating grain were characterized and purified. Two activities were detected: one with a molecular mass of 110 kDa and the other of 95 kDa. It was demonstrated through the use of antisera that the 110 kDa activity was located in the amyloplast and could correspond to the polypeptide encoded by the Pho1 gene cloned. The 95 kDa activity was localized to the cytoplasm, most strongly expressed in germinating grain, and was classified as a Pho2-type sequence. Using RNAi technology to reduce the content of Pho1 in the grain to less than 30% of wild type did not lead to any visible phenotype, and no dramatic alterations in the structure of the starch were observed. Conclusion Two starch phosphorylase activities were identified and characterized in barley grains, and shown to be present during starch synthesis. However, their role in starch synthesis still remains to be elucidated.© 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5′-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose ( kcat P = 0.033 s−1) and substrate binding affinity in the ternary complex with enzyme bound to phosphate ( Km = 280 mm), respectively. The 31P chemical shift of pyridoxal 5′-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334→Gly. Analysis of interactions of α-d-glucose 1-phosphate and α-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme–substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5–7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced. [ABSTRACT FROM AUTHOR]

The transfer of potato (Solanum tuberosum) tubers from 10 to 1°C was associated with an initial decline in the rate of CO2 output followed by a rapid increase reaching, within some 12 days, a peak which was about 3-fold higher than at 10°C. Thereafter the rate of CO2 evolution declined gradually for the duration of the experiment. The specific rate of mitochondrial O2 uptake decreased initially, followed by a rise to a level similar to that of mitochondria prepared from tubers stored at 10°C. Low temperature decreased by 30% the capacity of the cytochrome pathway while it sharply increased the capacity of the alternative pathway. Sucrose was the first sugar to accumulate at 1°C, followed after a delay of 6-7 days by glucose and fructose. Low temperature induced within 4-5 days a rise in amylase activity which increased by 10-fold after 30 days. The increase was reflected in only two out of four existing isoforms. In addition a novel isoform of amylase was detected later in storage. The induction and the accumulation of invertase mRNA and extractable activity followed the increase in sucrose but preceded that of hexoses. The activity of starch phosphorylase isoforms was not affected by temperature. There was a 3-fold increase in chlorogenic acid at 1°C. Hypoxia strongly inhibited the accumulation of sugars and chlorogenic acid, the increase in the amylase activity, and the appearance of the novel isoform. Low O2 totally suppressed the induction of invertase mRNA and increased the capacity of the alternative oxidase. It did not, however, prevent the decrease in cytochrome capacity; neither did it affect the activity of starch phosphorylase isoforms. [ABSTRACT FROM AUTHOR]

Starch and sucrose metabolism of one- and two-year-old needles of Norway spruce (Picea abies [L.] Karst., about 30 years old) was investigated from three months before until three months after bud break at a natural site. We distinguish different metabolic states according to the extractable activities of enzymes (α-amylase [EC 3.2.1.1 ], ADP-glucose pyrophosphorylase [AGP, EC 2.7.7.27], D-enzyme [EC 2.4.1.25], starch phosphorylase [STP, EC 2.4.1.1]), sucrose phosphate synthase [SPS, EC 2.4.1.14], sucrose synthase [SS, EC 2.4.1.13], acid invertase [AI, EC 3.2.1.26]) and pool sizes of related metabolites (starch, glucose, fructose, sucrose, raffinose, stachyose, fructose 6-phosphate [F6P], glucose 6-phosphate [G6P], fructose 2,6-bisphosphate [F26BP], and inorganic phosphate [Pi]). The period ending with bud break was characterized by high rates of net photosynthesis, a pronounced decrease in the amount of soluble sugars, and a steep rise in starch (from the detection limit to approximately 600 nmol glycosyl units [mg dry weight]-1). In parallel, the extractable activity of AGP increased, while D-enzyme was on a relative high level when compared with the period after bud break. With respect to sucrose metabolism, F26BP, an inhibitor of sucrose synthesis, decreased from 1 to 0.4 pmol (mg dry weight)-1. This was complemented by SPS activity, which was due to both increased protein levels shown by immunoblotting and activation under metabolite control (high levels of G6P and a low Pi/G6P ratio). This indicates a high capacity of synthesis of starch and sucrose in the period before bud break. These observations are in accordance with estimates of photosynthetic carbon gain, which indicate that in early spring large amounts of carbon from current photosynthesis are exported out of the needles. In addition, the content of nonstructural carbohydrates (expressed as hexoses) increased in the bark of the stem. This could also be a consequence of an enhanced carbon export from the needles. After the onset of bud break, starch concentration decreased in all tissues under investigation. In contrast, the level of total nonstructural carbohydrates in the outermost sapwood nearly doubled from bud break until the end of sampling. In the needles, net photosynthesis was reduced by about 75% and a decrease in SPS activity and protein level were found together with lower G6P concentration, and an increased Pi/G6P ratio. These results suggest that during that period sucrose synthesis was reduced in the older needles. In addition, under conditions of reduced photosynthesis, carbon demand of current year needles was in part ensured by the mobilization of starch in the older needles. Taken together our data show that before bud break carbon metabolism of mature leaves is related with the sink demands of storage organs. After bud break the accumulated assimilate pools in needles and stem, mainly the bark, are mobilized and support carbon supply to new tissues. [ABSTRACT FROM AUTHOR]

Changes in the contents of starch, protein, DNA, RNA, total phosphorus., acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes. of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis. Starch content in Cuscuta seeds showed: .a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination aid then decreased, Total phosphorus in the seeds remained almost unchanged during the period of study, Both trichioroacetic add soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and ,deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed .a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds. and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed. [ABSTRACT FROM AUTHOR]