Characterizing TsBAHD in Turnera & cloning wheat TaAMY genes for protein expression

Courtney Marie Matzke

doi:10.7273/000004101

TsBAHD in Turnera: In heterostyly, individual flowering plants bear one of two floral morphs between which male and female organs are reciprocally positioned to promote outcrossing. These features are controlled by the single multigene S-locus. In Turnera, the S-locus contains tightly linked genes, which were identified recently and have yet to be characterized biochemically. These genes are only present in the hemizygous S-haplotype, which has a shortened style. TsBAHD is the only S-locus gene expressed in pistils and necessarily controls style length. Based on homology, TsBAHD is predicted to be a brassinosteroid (BR) degrading acyltransferase, but such predictions are notoriously unreliable for acyltransferases. In this study we test the hypothesis that TsBAHD possesses brassinosteroid degrading activity. Constitutive overexpression of TsBAHD in Arabidopsis caused phenotypes (including dwarfism and inhibition of hypocotyl extension) characteristic of BR-deficient mutants. Hypocotyl extension increased with application of exogenous BL in light grown seedlings but not in dark grown seedlings, as has been previously reported in Arabidopsis BR mutants. Further, expression of BR biosynthesis genes was upregulated and BR degrading genes was downregulated in these plants. These results provide empirical support for the hypothesis that TsBAHD is a BAHD acyltransferase that degrades BRs, inhibiting cell expansion and shortening style length. TaAMYs in wheat: [alpha]-amylase is an enzyme in bread wheat (Triticum aestivum) that breaks down starch to fuel embryo growth after germination, but may have detrimental effects when present in harvested grain. Two genetic traits in wheat, Late Maturity Amylase (LMA) and preharvest sprouting (PHS), generate significant problems that severely reduce the commercial value of grain. There are four genetic isoforms of [alpha]-amylase in wheat. TaAMY1 and TaAMY2 are most commonly connected to lowered market value. Although TaAMY1 and TaAMY2 have been most extensively studied, further analyses are needed to determine the specific functions of all four isoforms and their potential involvement in LMA and PHS. In this study, the Chinese Spring cultivar was used to clone and sequence each TaAMY gene. TaAMY1 was then expressed to generate protein which will be essential in developing immunoassays capable of detecting the presence of each TaAMY isoform.

Characterizing TsBAHD in Turnera & cloning wheat TaAMY genes for protein expression

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