posted on 2016-02-20, 05:42authored byVito M. Butardo, Venea Dara Daygon, Michelle L. Colgrave, Peter M. Campbell, Adoracion Resurreccion, Rosa Paula Cuevas, Stephen
A. Jobling, Ian Tetlow, Sadequr Rahman, Matthew Morell, Melissa Fitzgerald
Elevated proportions of amylose in cereals are commonly
associated
with either the loss of starch branching or starch synthase activity.
Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo
carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound
proteins revealed that SSI and SSIIa accumulate inside the mature
starch granules of Goami 2, which is similar to the amylose
extender mutant IR36ae. However, unlike the amylose
extender mutants, SBEIIb was still detectable inside the
starch granules of Goami 2. Detection of SBEIIb after protein fractionation
revealed that most of the SBEIIb in Goami 2 accumulates inside the
starch granules, whereas most of it accumulates at the granule surface
in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound
proteins failed to detect any peptide sequence mutation or major post-translational
modifications in Goami 2. Moreover, the signal peptide was found to
be cleaved normally from the precursor protein, and there is no apparent
N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with
Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between
exon and intron 11, which could potentially introduce a premature
stop codon and produce a truncated form of SBEIIb. It is suggested
that the mutation responsible for producing high amylose in Goami
2 is not due to a defect in SBEIIb gene as was observed
in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic
basis of this mutation will be important in identifying novel targets
for increasing amylose and resistant starch contents in rice and other
cereals.