Plastidial Starch Phosphorylase in Sweet Potato Roots Is Proteolytically Modified by Protein-Protein Interaction with the 20S Proteasome

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions

Établissement de l'implication des a- et b-amylases et des a-glucanes phosphorylases au cours de la dégradation de l'amidon dans la feuille d'Arabidopsis thaliana

Différentes classes d enzymes hydrolytiques (a- et b-amylases) et phosphorolytiques (amidon-phosphorylases) des a-glucanes sont impliquées dans le métabolisme de l amidon dans la feuille d Arabidopsis thaliana. Pas moins de 9 b-amylases, 3 a-amylases et 2 glucane-phosphorylases ont été répertoriées dans le génome de cette plante. Afin de leur attribuer une fonction précise, une étude phénotypique complète de lignées mutantes pour les formes plastidiales de ces enzymes a été menée au cours de ce travail. Chaque isoforme d amylase a également été croisée avec un double mutant d enzymes de branchement, be2- be3-, qui est totalement dépourvu d amidon et accumule du maltose de manière inhabituelle. Afin de préciser l implication de chaque forme d amylase pour la production de ce maltose, le contenu en maltose des triples mutants a été analysé. Nos résultats nous permettent de conforter l hypothèse d une fonction de régulation de BAM4. Les perturbations observées dans les mutants bam1- et bam8- montrent l importance de ces enzymes dans le métabolisme de l amidon. Enfin, les résultats obtenus avec le mutant aam3- ne nous révèlent rien de sa fonction. L analyse de l a-glucane phosphorylase plastidiale PHS1 a été couplée à celle du mutant ss4- (une forme d amidon synthétase soluble) et du double mutant ss4- phs1-. En effet, on observe au sein de ce double mutant des grains d une taille 4 à 5 fois supérieure à ceux de la souche sauvage ainsi qu une très forte augmentation de la quantité d amidon. Les résultats nous indiquent que PHS1 est impliquée dans la dégradation de l amidon, n attaquant pas le grain natif, et qu elle est similaire à ses homologues des plantes supérieures.Different classes of a-glucans hydrolytic (a- and b-amylases) and phosphorolytic enzymes are implicated in starch metabolism in Arabidopsis thaliana leaves. At least, 9 b-amylases, 3 a-amylases and 2 a-glucan phosphorylases are listed in this plant genome. In order to allocate them a precise function, a complete phenotypic study of mutant lines for plastidial forms of these enzymes was investigated. Each isoform of amylase studied was crossed with a double mutant of branching enzymes, be2- be3-, wich is free of starch and accumulating maltose which is unusual in this plant. In order to specify the implication of each form of amylase for the production of maltose in the be2- be3- mutant, maltose content of the triple mutants was analyzed. Our results allow us to strengthen the hypothesis of a regulating function of BAM4. Alterations observed in mutants bam1- and bam8- show us importance of these enzymes in starch metabolism. Finally, results obtained from analysis of aam3- mutant don t reveal anything about its function. Analysis of plastidial a-glucan phosphorylase PHS1 was coupled with that of the mutant ss4- (a form of soluble starch synthase) and the double mutant ss4- phs1-. In fact, in this double mutant we observed starch granules 4 to 5 fold bigger than those of the wild-type strain and a strong increase of starch content. Results obtained show that PHS1 is implicated in starch degradation, don t breaking native granule directly, and that it is similar to phosphorylases of land plants.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains ▿

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium

Biochemical characterization of the chlamydomonas reinhardtii alpha-1,4 glucanotransferase supports a direct function in amylopectin biosynthesis

Plant alpha-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the alpha-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with the sta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure

Genetic and biochemical evidence for the involvement of alpha-1,4 glucanotransferases in amylopectin synthesis

We describe a novel mutation in the Chlamydomonas reinhardtii STA11 gene, which results in significantly reduced granular starch deposition and major modifications in amylopectin structure and granule shape. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides. The sta11-1 mutation causes the absence of an alpha-1,4 glucanotransferase known as disproportionating enzyme (D-enzyme). D-enzyme activity was found to be correlated with the amount of wild-type allele doses in gene dosage experiments. All other enzymes involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, debranching enzymes, soluble and granule-bound starch synthases, branching enzymes, phosphorylases, alpha-glucosidases (maltases), and amylases, were unaffected by the mutation. These data indicate that the D-enzyme is required for normal starch granule biogenesis in the monocellular alga C. reinhardtii

Red light induces starch accumulation in Chlorella vulgaris without affecting photosynthesis efficiency, unlike abiotic stress

International audienceMicroalgae show great promise as sources of starch, one of the most widely consumed macromolecules. In this study, we evaluated the impact of three starch-inducing factors, namely nitrogen deprivation, supra-optimal temperature, and red light, on the physiology and starch accumulation capacity of Chlorella vulgaris. This starch accumulation was monitored by measuring the total carbohydrate content and transmission electron microscopy (TEM) imaging. Nitrogen deprivation and a supra-optimal temperature of 39 °C resulted in carbohydrate contents of 69.7 and 64.3 % of dry weight (DW) respectively. This constituted a 5.3- and 3.3-fold increase in carbohydrate productivity compared to the control, after 4 days of cultivation. During this period, carbohydrates represented over 80 % of the produced material (DW basis). However, nitrogen deprivation and supra-optimal temperature were accompanied by extensive stress, leading to lower cell division rates and damage to the photosynthetic apparatus. Red light illumination resulted in a more moderate production of carbohydrates. After 4 days of cultivation, the carbohydrate content reached 46.8 %, representing a 3.0-fold increase in productivity compared to control. The composition of the starch formed under red light was surprisingly poor in amylose, similar to transitory-type starch rather than storage starch. Most notably, the starch accumulation under red light was sustained over 7 days without affecting the rate of cell division and quantum yield efficiency. To the best of our knowledge, red light is the only factor reported so far to induce a significant starch accumulation without hindering cell division and photosynthesis efficiency, even after long-term exposure (7 days). Furthermore, all three conditions induced a cell wall thickening, albeit without affecting the recovery of accumulated starch by high-pressure homogenization. These results highlight the potential of red light as a starch inducer in Chlorella vulgaris and open up perspectives for the production of starch-based bioplastics from microalgae