Starch-branching enzymes (SBE) break the alpha-1,4 linkage of starch, re-attaching the chain to a glucan chain by an alpha-1,6 bond, altering starch structure. SBEs also facilitate starch accumulation by increasing the number of non-reducing ends on the growing chain. In maize (Zea mays), three isoforms of SBE have been identified. To examine the function of the SBEIIa isoform, a reverse genetics polymerase chain reaction-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe2a. To locate the insertion within the second exon of Sbe2a, the genomic sequence of Sbe2a containing the promoter and 5' end was isolated and sequenced. Plants homozygous for sbe2a::Mu have undetectable levels of Sbe2a transcripts and SBEIIa in their leaves. Characterization of leaf starch from sbe2a::Mu mutants shows reduced branching similar to yet more extreme than that seen in kernels lacking SBEIIb activity. Characterization of endosperm starch from sbe2a::Mu mutants shows branching that is indistinguishable from wild-type controls. These mutant plants have a visible phenotype resembling accelerated senescence, which was correlated with the Mutator insertion within Sbe2a. This correlation suggests a specific role for SBEIIa in leaves, which may be necessary for normal plant development.

Cereal Chem. 75(6):887-896Three high-amylose maize starches (HAS) and a common corn starch (CCS) were subjected to differential alcohol precipitation using isoamyl alcohol and 1-butanol to obtain fractions designated as amylose (AM), amylopectin (AP), and intermediate material (IM). For each starch, IM had a blue value and an iodine binding wavelength maximum (λ max ) between the λ max of the respective AM and AP. Size-exclusion chromatography (SEC) showed similarities in the AM from CCS and HAS. HAS AP had higher blue values and iodine binding λ max values than CCS AP. SEC of the intact HAS AP and IM both showed large proportions of material eluting after the void volume (45-85%) when compared to CCS AP and IM. Chain length (CL) distributions of debranched AP and IM indicated that these fractions from each starch were highly branched, and that AP had a shorter average chain length than IM. Consequently, the differential precipitation behavior of the HAS AP and IM appears dependent on general branching structure rather than size. We conclude that in both CCS and HAS, AP and IM are subsets of the branched molecules with AP as the predominant fraction. For HAS, AP and IM include molecules of a size typical for AM and contain a higher proportion of chains that are longer than those of CCS AP. Differential alcohol precipitation is a useful method of separating amylose, amylopectin, and intermediate material from HAS.

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Several established methods of phytic acid determination in soybeans were evaluated. Iron analysis methods, which rely on a 4:6 molar ratio of Fe:P, were eliminated because this ratio was not dependable. Three assay methods relying on phosphorus analysis were then compared. The anion-exchange method was considered most accurate but not convenient for routine analysis. Analysis of the ferric phytate precipitate and a new method, analysis of the supernatant before and after ferric chloride precipitation, were judged against the anion-exchange method under different extraction conditions. Based upon good agreement with anion-exchange column results and acceptable reproducibility, the best methods were (1) precipitate analysis of phosphorus after extraction with 3% TCA + 10% sodium sulfate, or (2) the supernatant difference method after extraction with 1.2% HCl.

Previous studies indicated that the deficiency of starch-branching enzyme (SBE) Ia in the single mutant sbe1a::Mu (sbe1a) has no impact on endosperm starch structure, whereas the deficiency of SBEIIb in the ae mutant is well known to reduce the branching of starch. We hypothesized that in maize (Zea mays) endosperm, the function of SBEIIb is predominant to that of SBEIa, and SBEIa would have an observable effect only on amylopectin structure in the absence of SBEIIb. To test this hypothesis, the mutant sbe1a was introgressed into lines containing either wx (lacking the granule-bound starch synthase GBSSI) or ae wx (lacking both SBEIIb and GBSSI) in the W64A background. Both western blotting and zymogram analysis confirmed the SBEIa deficiency in sbe1a wx and sbe1a ae wx, and the SBEIIb deficiency in ae wx and sbe1a ae wx. Using zymogram analysis, no pleiotropic effects of sbe1a genes on SBEIIa, starch synthase, or starch-debranching enzyme isoforms were observed. Highperformance size exclusion chromatography analysis shows that the chain-length profiles of amylopectin as well as b-limit dextrin were indistinguishable between wx and sbe1a wx, whereas significant differences for both were observed between ae wx and sbe1a ae wx, suggesting an effect of SBEIa on amylopectin biosynthesis that is observable only in the absence of SBEIIb. The amylopectin branch density and the average number of branches per cluster were both higher in endosperm starch from sbe1a ae wx than from ae wx. These results indicate possible functional interactions between SBE isoforms that may involve enzymatic inhibition. Both the cluster repeat distance and the distance between branch points on the short intracluster chains were similar for all genotypes however, suggesting a similar pattern of individual SBE isoforms in cluster initiation and the determination of branch point location.Starch biosynthesis involves the activities of four enzymes: ADP-Glc pyrophosphorylase, starch synthase (SS), starch-branching enzyme (SBE), and starch-debranching enzyme (DBE;Smith et al., 1997). The coordinated functions of these enzymes result in two forms of starch molecules: amylose, an essentially linear glucan chain consisting of a-1,4 linkages, and amylopectin, a highly branched glucan chain with multiple branch points formed by a-1,6 linkages.SBE catalyzes the formation of branch points within glucan chains by cleaving an a-1,4 linkage and reattaching the chain to a glucan chain via a-1,6 bond. Three isoforms of SBE (SBEIa, SBEIIa, and SBEIIb) have been identified in maize (Zea mays; Boyer and Preiss, 1978;Dang and Boyer, 1988), and their expression patterns differ considerably (Gao et al., 1996). While the in vitro properties of SBEIa differ from those of SBEIIa and SBEIIb (Guan and Preiss, 1993;Takeda et al., 1993), the sbe1a or sbe2a single mutants obtained using Mu-mediated transposon mutagenesis indicated that the chain-length (CL) profiles of starch produced in endosperm in vivo were not affected by the deficiency of either SBEIa or SBEIIa (Blau...