Suppression of starch synthase I expression affects the granule morphology and granule size and fine structure of starch in wheat endosperm.

Studies in Arabidopsis and rice suggest that manipulation of starch synthase I (SSI) expression in wheat may lead to the production of wheat grains with novel starch structure and properties. This work describes the suppression of SSI expression in wheat grains using RNAi technology, which leads to a low level of enzymatic activity for SSI in the developing endosperm, and a low abundance of SSI protein inside the starch granules of mature grains. The amylopectin fraction of starch from the SSI suppressed lines showed an increased frequency of very short chains (degree of polymerization, dp 6 and 7), a lower proportion of short chains (dp 8-12), and more intermediate chains (dp 13-20) than in the grain from their negative segregant lines. In the most severely affected line, amylose content was significantly increased, the morphology of starch granules was changed, and the proportion of B starch granules was significantly reduced. The change of the fine structure of the starch in the SSI-RNAi suppression lines alters the gelatinization temperature, swelling power, and viscosity of the starch. This work demonstrates that the roles of SSI in the determination of starch structure and properties are similar among different cereals and Arabidopsis.

Real-time quantitative RT-PCR assay of gene expression in plant roots during fungal pathogenesis.

Real-time quantitative RT-PCR is becoming the preferred method for high-sensitivity, rapid-throughput RNA transcript quantification. However, due to the significant developmental costs of dedicated fluorogenic probes, a real-time assay that is simple to establish, comparatively inexpensive, and readily adaptable would be advantageous for the detailed analysis of large sets of expressed sequences. We have devised a flexible real-time quantitative RT-PCR assay that employs a nonspecific DNA binding dye for product detection and uses a relative quantification formula to account for differences in PCR amplification efficiency between the target and reference products. The latter permits the use of an exogenous reference transcript and therefore avoids the normal requirement for the construction of a recombinant RNA reference transcript or extensive characterization of housekeeping gene expression. In an investigation of class II chitinase expression in two varieties of Bermuda grass (Cynodon spp.), following infection with the fungal root pathogen Ophiosphaerella narmari, this assay identified 16- and 28-fold peaks in gene expression at 24 and 96 h after inoculation, respectively.