Jean-Philippe Ral

Circadian Clock Regulation of Starch Metabolism Establishes GBSSI as a Major Contributor to Amylopectin Synthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii displays a diurnal rhythm of starch content that peaks in the middle of the night phase if the algae are provided with acetate and CO2 as a carbon source. We show that this rhythm is controlled by the circadian clock and is tightly correlated to ADP-glucose pyrophosphorylase activity. Persistence of this rhythm depends on the presence of either soluble starch synthase III or granule-bound starch synthase I (GBSSI). We show that both enzymes play a similar function in synthesizing the long glucan fraction that interconnects the amylopectin clusters. We demonstrate that in log phase-oscillating cultures, GBSSI is required to obtain maximal polysaccharide content and fully compensates for the loss of soluble starch synthase III. A point mutation in the GBSSI gene that prevents extension of amylopectin chains, but retains the enzymes normal ability to extend maltooligosaccharides, abolishes the function of GBSSI both in amylopectin and amylose synthesis and leads to a decrease in starch content in oscillating cultures. We propose that GBSSI has evolved as a major enzyme of amylopectin synthesis and that amylose synthesis comes as a secondary consequence of prolonged synthesis by GBSSI in arrhythmic systems. Maintenance in higher plant leaves of circadian clock control of GBSSI transcription is discussed.

Starch Division and Partitioning. A Mechanism for Granule Propagation and Maintenance in the Picophytoplanktonic Green Alga Ostreococcus tauri

Whereas Glc is stored in small-sized hydrosoluble glycogen particles in archaea, eubacteria, fungi, and animal cells, photosynthetic eukaryotes have resorted to building starch, which is composed of several distinct polysaccharide fractions packed into a highly organized semicrystalline granule. In plants, both the initiation of polysaccharide synthesis and the nucleation mechanism leading to formation of new starch granules are currently not understood. Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, defines the tiniest eukaryote with one of the smallest genomes. We show that it accumulates a single starch granule at the chloroplast center by using the same pathway as higher plants. At the time of plastid division, we observe elongation of the starch and division into two daughter structures that are partitioned in each newly formed chloroplast. These observations suggest that in this system the information required to initiate crystalline polysaccharide growth of a new granule is contained within the preexisting polysaccharide structure and the design of the plastid division machinery.

Pathway of Cytosolic Starch Synthesis in the Model Glaucophyte Cyanophora paradoxa

ABSTRACT The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.

STA11, a Chlamydomonas reinhardtii Locus Required for Normal Starch Granule Biogenesis, Encodes Disproportionating Enzyme. Further Evidence for a Function of α-1,4 Glucanotransferases during Starch Granule Biosynthesis in Green Algae

In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS). The mutants ofSTA11 were showed to lack d-enzyme, a plant α-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase. We have cloned and characterized both the cDNA and gDNA corresponding to the C.reinhardtii d-enzyme. We now report allele-specific modifications of the d-enzyme gene in the mutants of STA11. These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodesd-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutantC. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.

Genetic dissection of floridean starch synthesis in the cytosol of the model dinoflagellate Crypthecodinium cohnii

Starch defines an insoluble semicrystalline form of storage polysaccharides restricted to Archaeplastida (red and green algae, land plants, and glaucophytes) and some secondary endosymbiosis derivatives of the latter. While green algae and land-plants store starch in plastids by using an ADP-glucose-based pathway related to that of cyanobacteria, red algae, glaucophytes, cryptophytes, dinoflagellates, and apicomplexa parasites store a similar type of polysaccharide named floridean starch in their cytosol or periplast. These organisms are suspected to store their floridean starch from UDP-glucose in a fashion similar to heterotrophic eukaryotes. However, experimental proof of this suspicion has never been produced. Dinoflagellates define an important group of both photoautotrophic and heterotrophic protists. We now report the selection and characterization of a low starch mutant of the heterotrophic dinoflagellate Crypthecodinium cohnii. We show that the sta1-1 mutation of C. cohnii leads to a modification of the UDP-glucose-specific soluble starch synthase activity that correlates with a decrease in starch content and an alteration of amylopectin structure. These experimental results validate the UDP-glucose-based pathway proposed for floridean starch synthesis.

Suppression of glucan, water dikinase in the endosperm alters wheat grain properties, germination and coleoptile growth

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.

New insight in cereal starch degradation: identification and structural characterization of four α-amylases in bread wheat

Abstract Grain α-amylase presents an apparent paradox for the wheat community. Despite the necessity of α-amylase for the seed germination process, high levels of amylase activity in the grain are considered detrimental for grain quality. Wheat α-amylases (EC 3.2.1.1) are endohydrolases belonging to the GH13_6 subfamily, one of the most studied subclasses of glycoside hydrolase (GH) family GH13. However, no comprehensive study had been done so far to describe and catalogue all the wheat α-amylase isoforms, despite compelling information on the involvement of two α-amylases on economically important issues for the international cereal community, namely pre-harvest sprouting and late maturity α-amylase. This study describes for the first time the genomic localization, nucleotide and amino acid sequences, phylogeny and expression profile of all known α-amylases in wheat, including a hitherto unknown fourth isoform here designated as TaAMY4. Isoform profiling strongly suggested α-amylases to be working in partnership to achieve complete degradation of a starch granule, whereas expression profiling revealed a potential involvement of TaAMY4 in the late maturity α-amylase problem.