Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa.

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.

First evidence for the presence of weddellite crystallites in Opuntia ficus indica parenchyma.

Calcium oxalate crystallites occur very often in the plants tissues and their role is still poorly known. We report here the experimental protocol leading to the isolation of two forms of calcium oxalate crystallites differing in their hydration level in the parenchymal tissues of Opuntia ficus indica (Miller). Whereas the whewellite crystallites are habitual in all Opuntia species, the weddellite form has never been isolated from these species before, which is probably due to their small size (about 1 microm). We have identified these forms using X-ray diffraction and scanning electron microscopy.

Morphological and structural aspects of the giant starch granules from Phajus grandifolius.

The morphology and structure of giant starch granules from the pseudo-bulbs of Phajus grandifolius were investigated, using a number of microscopy techniques together with synchrotron radiation microdiffration analysis. Most of the granules, which had sizes between 100 and 200 microm, occurred as ogival particles with the hilum or proximal end located at the apex of the granules. A small percentage of granules held a protuberance extending orthogonally to the underlying parent granule. Growth rings were observed in all granules: strongly curved close to the hilum, but planar toward the distal end of the granules or in the protuberances. Specific mechanical disruption followed by enzymatic digestion revealed the susceptibility of the disorganized parts of the growth rings, which were preferentially carved away during the digestion, leaving behind the better-organized domains. Microdiffraction analysis achieved with synchrotron radiation revealed the crystalline features of the granules and provided orientation maps of the amylopectin molecules in the various parts of the granules. In simple ogival granules the amylopectin molecules were uniformly oriented with their axes running from the hilum toward the distal end of the granule. In granules with a protuberance, the axes of the amylopectin molecules kept their direction in the parent granule, but took an orthogonal direction in the protuberance. The occurrence of these morphological and structural features is tentatively correlated with the mode of growth of these granules.

Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii.

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.

Amylose synthesized in vitro by amylosucrase: morphology, structure, and properties.

The recombinant amylosucrase from Neisseria polysaccharea was used to synthesize in vitro amylose from sucrose as unique substrate. The morphology and structure of the insoluble residue were shown to depend only on the initial sucrose concentration (100, 300, or 600 mM), which controlled both the chain length and concentration at the precipitation stage. The average degree of polymerization (DP) in the precipitated product varied from 58 for the lowest initial sucrose concentration (100 mM) to 45 and 35 for higher sucrose concentrations (300 and 600 mM, respectively). The shorter chains (DP 35 and 45), produced in high yields (54 and 24 g/L respectively), precipitated as polycrystalline aggregates with exceptional crystallinity, without optimization of the reaction medium for crystallization. The longer chains (DP 58), produced in lower amount (2.9 g/L), formed networks similar to those observed for amylose gels. All synthesized products displayed a B-type crystal structure. Their melting behavior was also studied, the thermostability being higher for the precipitate containing the longer chains. Further thermal treatments were shown to still improve the crystallinity and yield substrates usable as new standards for the determination of the relative crystallinity of starchy products. The kinetics of chain elongation and aggregation were thoroughly investigated in order to explain how the action of amylosucrase resulted in such different amylose structures. These results emphasize the potentiality of amylosucrase in the design of amylodextrins with controlled morphology, structure, and physicochemical properties.