The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.
The β-, γ- and δ-kafirin genes were sequenced from 35 Sorghum genotypes to investigate the allelic diversity of seed storage proteins. A range of grain sorghums, including inbred parents from internationally diverse breeding programs and landraces, and three wild Sorghum relatives were selected to encompass an extensive array of improved and unimproved germplasm in the Eusorghum. A single locus exists for each of the expressed kafirin-encoding genes, unlike the multigenic α-kafirins. Significant diversity was found for each locus, with the cysteine-rich β-kafirin having four alleles, including the first natural null mutant reported for this prolamin subfamily. This allele contains a frame shift insertion at +206 resulting in a premature stop codon. SDS-PAGE revealed that lines with this allele do not produce β-kafirin. An analysis of flour viscosity reveals that these β-kafirin null lines have a difference in grain quality, with significantly lower viscosity observed over the entire Rapid ViscoAnalyser time course. There was less diversity at the protein level within the cysteine-rich γ-kafirin, with only two alleles in the cultivated sorghums. There were only two alleles for the δ-kafirin locus among the S. bicolor germplasm, with one allele encoding ten extra amino acids, of which five were methionine residues, with an additional methionine resulting from a nucleotide substitution. This longer allele encodes a protein with 19.1% methionine. The Asian species, S. propinquum, had distinct alleles for all three kafirin genes. We found no evidence for selection on the three kafirin genes during sorghum domestication even though the δ-kafirin locus displayed comparatively low genetic variation. This study has identified genetic diversity in all single copy seed storage protein genes, including a null mutant for β-kafirin in Sorghum.
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