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During the last couple of decades, rapid progress has been made in increasing our understanding of the specific contributions of the most important enzymes to starch biosynthesis in cereal endosperms, particularly in rice endosperm. Now, we can basically predict how and to what extent the starch-related phenotypes can be modified in the endosperm in accordance with alterations of activities of single or simultaneously multiple starch biosynthetic isozymes. Thus, starch bioengineering in planta is now a most particularly promising field of plant biotechnology. Here, an overview of the present status of biochemical, molecular, and genetic research is presented, regarding starch biosynthesis and its engineering for the generation of new cultivars with improved cooking or novel functional properties of starch in rice endosperm. A huge number and variety of cultivars have been developed and cultivated mainly in paddy fields of Asian countries because rice is the most important staple food grain there. The availability of such invaluable genetic resources, as well as databases for various types of mutants, complete genome sequences, and omics tools should ensure further progress in the rice starch biotechnology research field. [ABSTRACT FROM AUTHOR]

In maturing endosperms of a variety of sugary mutants of rice, phytoglycogen-like polysaccharides with highly branched α-glucans were accumulated instead of amylopectin, while the amylose content greatly decreased. Measurement of activities per endosperm of the 10 major enzymes involved in starch and sucrose metabolism revealed that the activity of starch debranching enzyme (R-enzyme) was specifically reduced in the sugary mutants. The activity of starch branching enzyme I (Q-enzyme 1) was also significantly decreased, but less so than the R-enzyme, in the mutants, suggesting some coordination of the expression of the genes coding for R-enzyme and Q-enzyme I. Western blot analysis showed that the sugary mutations of rice resulted in a decrease in the amount of R-enzyme protein, but not in major modification of the enzyme. The findings strongly suggest that R-enzyme plays a critical role in determining the amylopectin fine structure, since at the extremely low level of R-enzyme activity as compared with Q-enzyme activity, as found in sugary mutants, the rice endosperm produced phytoglycogen. We hypothesize that balance of activities or interaction between Q-enzyme and R-enzyme may be responsible for the fine structure of α-polyglucans in plant tissues. [ABSTRACT FROM AUTHOR]

The pattern of isoforms of starch branching enzyme II or Q‐enzyme II in the tuberous root of sweet potato was distinct from those of other organs; altogether 7 isoforms of QEII were contained in the sweet potato plant. The QEIIf isoform, one of the two major QEII isoforms in the tuberous root, was purified to homogeneity by using a variety of HPLC columns. The purified QEIIf was a monomeric protein with a molecular mass of about 85 kDa. Western blot analysis showed that the polyclonal antibodies raised against the purified QEIIf was significantly reactive to the rice endosperm QEI, but not to the rice endosperm QEIIa. Furthermore, the sweet potato QEIIf reacted with the antiserum raised against the rice endosperm QEI, but not with that against the rice endosperm QEIIa. The results suggest that the sweet potato QEIIf is more similar to the rice endosperm QEI than to the rice endosperm QEIIa. [ABSTRACT FROM AUTHOR]

ADP glucose pyrophosphorylase from developing rice (Oryza saliva) endosperm was purified. The final preparation yielded 6 major protein spots as separated by two-dimensional polyacrylamide electrophoresis. All 6 polypeptides had similar molecular weights of ca 50 kDa and cross-reacted with polyclonal antibodies raised against two main protein bands among them. The results suggest that the rice endosperm ADPglucose pyrophorsphorylase is tetrameric and composed of multiple subunits with similar amino acid structure. [ABSTRACT FROM AUTHOR]

Two isoforms of starch branching enzyme (Q-enzyme), QEI and QEII, have been purified to homogeneity from developing rice endosperm. QEI and QEII, with molecular weights of about 80 and 85 kDa, respectively, could be fully separated by anion-exchange or hydrophobic chromatography. The peptide maps obtained after V8 proteinase digestion were quite different between the two enzymes. Antibodies prepared against QEI showed no immunological cross-reaction with the QEII protein in Western blot experiments, and anti-QEII serum did not react with the QEI protein. The data indicate that QEI and QEII are distinct proteins encoded by different genes in rice plants. [ABSTRACT FROM AUTHOR]

The effects of a previously reported shear and heat milling machine (SHMM) on the crystallinity and molecular architecture of starch are examined. The SHMM produces amorphous starch flour from rice grains containing crystalline starch. A high temperature of 120 °C and a distance of 10 μm between milling mortars were essential for the complete conversion of crystalline starch to amorphous starch. The effects of increasing the gap between mortars to 100 μm and decreasing the temperature to 15 °C were tested. The use of highly dried rice flour containing only 1% water (w/w) was also examined. The results suggest that the transition from crystalline starch to amorphous starch produced by the SHMM is a gelatinization process because a significant amount of water in the rice grains contributes to the formation of amorphous starch. It was also found that high temperature milling specifically, but only partially, cleaved the cluster-interconnecting chains of amylopectin, resulting in the formation of glucans smaller than normal amylopectin. Novel finding from these results is that the coordinated action of high temperature milling, a small gap, and endogenous water in amylopectin induces gelatinization, accompanied by a reduction in the molecular weight of amylopectin cut at the B2-3 branches. [ABSTRACT FROM AUTHOR]

Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700 kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400 kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. [ABSTRACT FROM AUTHOR]

In monocots, starch branching enzyme II (BEII) was functionally differentiated into BEIIa and BEIIb after separation from the dicots, and in cereals BEIIb plays a distinct role in amylopectin biosynthesis in the endosperm. The present study was conducted to examine to what extent a green algal BEII has an overlapping function with BEIIb in starch biosynthesis by introducing the Chlorella BEII gene into an amylose-extender (ae) mutant of rice. Chlorella BEII was found to complement the contribution of the rice endosperm BEIIb to the structures of amylopectin and starch granules because these mutated phenotypes were recovered almost completely to those of the wild type by the expression of Chlorella BEII. When the recombinant BE enzymes were incubated with the rice ae amylopectin, the branching pattern of Chlorella BEII was much more similar to that of rice BEIIb rather than rice BEIIa. Detailed analyses of BE reaction products suggests that BEIIb and Chlorella BEII only transfer chains with a degree of polymerization (DP) of 6 and 7, whereas BEIIa preferably transfers short chains with a DP of about 6–11. These results show that the Chlorella BEII is functionally similar to rice BEIIb rather than BEIIa. [ABSTRACT FROM AUTHOR]