Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines.

BACKGROUND: Starch is the most important carbohydrate in plant storage tissues. Multiple isozymes in at least four enzyme classes are involved in starch biosynthesis. Some of these isozymes are thought to interact and form complexes for efficient starch biosynthesis. Of these enzyme classes, starch synthases (SSs) and branching enzymes (BEs) play particularly central roles. RESULTS: We generated double mutant lines (ss1/be1 and ss1L/be2b) between SSI (the largest component of total soluble SS activity) and BEI or BEIIb (major BEs in developing rice endosperm) to explore the relationships among these isozymes. The seed weight of ss1/be1 was comparable to that of wild type, although most ss1/be2b seeds were sterile and no double recessive plants were obtained. The seed weight of the double recessive mutant line ss1L/be2b, derived from the leaky ss1 mutant (ss1L) and be2b, was higher than that of the single be2b mutant. Analyses of the chain-length distribution of amylopectin in ss1/be1 endosperm revealed additive effects of SSI and BEI on amylopectin structure. Chain-length analysis indicated that the BEIIb deficiency significantly reduced the ratio of short chains in amylopectin of ss1L/be2b. The amylose content of endosperm starch of ss1/be1 and ss1L/be2b was almost the same as that of wild type, whereas the endosperm starch of be2b contained more amylose than did that of wild type. SSI, BEI, and BEIIb deficiency also affected the extent of binding of other isozymes to starch granules. CONCLUSIONS: Analysis of the chain-length distribution in amylopectin of the double mutant lines showed that SSI and BEI or BEIIb primarily function independently, and branching by BEIIb is followed by SSI chain elongation. The increased amylose content in be2b was because of reduced amylopectin biosynthesis; however, the lower SSI activity in this background may have enhanced amylopectin biosynthesis as a result of a correction of imbalance between the branching and elongation found in the single mutant. The fact that a deficiency of SSI, BEI, or BEIIb affected the affinity of other starch biosynthetic isozymes for the starch granule implies that there is a close interaction among SSI, BEI and BEIIb during amylopectin biosynthesis in rice endosperm.

Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage α-glucan.

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the ß-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).

Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa.

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ∼90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.

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 < or = 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 alpha-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 B(2-3) chains of amylopectin in the PUL mutant was approximately 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 < or = 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.

Lack of starch synthase IIIa and high expression of granule-bound starch synthase I synergistically increase the apparent amylose content in rice endosperm.

Rice endosperm starch is composed of 0-30% linear amylose, which is entirely synthesized by granule-bound starch synthase I (GBSSI: encoded by Waxy, Wx). The remainder consists of branched amylopectin and is elongated by multiple starch synthases (SS) including SSI, IIa and IIIa. Typical japonica rice lacks active SSIIa and contains a low expressing Wx(b) causing a low amylose content (ca. 20%). WAB2-3 (SS3a/Wx(a)) lines generated by the introduction of a dominant indica Wx(a) into a japonica waxy mutant (SS3a/wx) exhibit elevated GBSSI and amylose content (ca. 25%). The japonica ss3a mutant (ss3a/Wx(b)) shows a high amylose content (ca. 30%), decreased long chains of amylopectin and increased GBSSI levels. To investigate the functional relationship between the ss3a and Wx(a) genes, the ss3a/Wx(a) line was generated by crossing ss3a/Wx(b) with SS3a/Wx(a), and the starch properties of this line were examined. The results show that the apparent amylose content of the ss3a/Wx(a) line was increased (41.3%) compared to the parental lines. However, the GBSSI quantity did not increase compared to the SS3a/Wx(a) line. The amylopectin branch structures were similar to the ss3a/Wx(b) mutant. Therefore, Wx(a) and ss3a synergistically increase the apparent amylose content in rice endosperm, and the possible reasons for this increase are discussed.

Preparation of carbocycles via base-catalyzed endo-mode cyclization of allenes.

A new and reliable procedure for constructing five- to seven-membered carbocycles via an endo-mode ring-closing reaction of 1-phenylsulfonylallenes with a substituent that has a terminal active methine moiety at the C1-position has been developed. Trisubstituted 1-phenylsulfonylallenes underwent a similar endo-mode ring-closing reaction to produce the corresponding five- to seven-membered carbocycles, while the formation of six- and seven-membered carbocycles from the corresponding tetrasubstituted allene was not realized. In addition, the introduction of an aromatic ring to the alkyl side chain of the starting allenes made possible the construction not only of normal-sized carbocycles but also an eight-membered framework.