Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP ≤13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The α-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B2-3 chains of amylopectin in the PUL mutant was ∼3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP ≤7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities

Production and characterization of novel glycogen-based polymer. Joint research by Public University of Navarra and Niigata University

Trabajo presentado en el KAAB International Symposium (Frontiers in Plant Science and Biotechnology), celebrado en Niigata (Japón) el 29 de septiembre de 2015.Natural storage polysaccharides, like as amylopectin and glycogen, vary in form and molecular size. Glycogen molecule is smaller in size and higher in branching degree than that of amylopectin. To develop a novel type of glycogen containing a long α-1, 4 glucosyl chain, we introduced rice granule-bound starch synthase I (GBSSI) gene in Escherichia coli and analyzed its expression and effect on glycogen synthesis, Rice GBSSI synthesizes not only amylose but also amylopectin, especially synthesizes extra-long chain, whose degrees of polymerization is over one hundred (1). We constructed the expression vector, pHis:SUMO:GBSSI, and confirmed that the expressed His:SUMO:GBSSI can synthesize extra-long chain in amylopectin in vitro. We transformed the pHis:SUMO:GBSSI into various E. coli strains, BW25113 and its mutants deficient in genes related to glycogen accumulation (Keio Collection)(2,3), and then examined effect of GBSSI expression on changes in nature and the amount of glycogen. Genes involved in Glycogen metabolism in E. coli are clustered in two apparently independent transcriptional units, glgBX (encoding branching enzyme GlgB and debranching enzyme GlgX) and glgCAP (encoding ADPG pyrophosphorylase GlgC, glycogen synthase GlgA and glycogen phosphorylase GlgP),. Both of ΔglgX andΔglgP mutants show increase in glycogen accumulation than BW25113, whileΔglgA mutant accumulates no glycogen at all. We use these three mutants,ΔglgX, ΔglgP, and ΔglgA, for this study. GBSSI expressing ΔglgX,ΔglgP, and BW25113 produced much more glycogen than non-expressing E. coli mutants, whereas the GBSSI expressing mutant ΔglgA did not produce the glycogen at all. The results indicate that the rice GBSSI does not complement the glycogen synthase deficiency by ΔglgA in E. coli. Our research also indicates that GBSSI can function not only in rice, but also in E. coli and affect its glycogen accumulation.Funding acknowledgements: This work was supported by KAAB project and Grant-in-Aid for Scientific Research, No. 15K00779, JSPS.Peer Reviewe