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Starch is an important component of a wheat grain, comprising 50-70% of its dry weight. Its biosynthesis involves a complex pathway mediated by several enzymes, each of which is encoded by genes that have more than one family member. To better understand starch synthesis in wheat grains, this study characterized the sucrose-starch metabolic pathway using physiological, molecular, biochemical and metabolic approaches. These analyses led to the identification of genes that appear to have predominant expression during grain development in wheat including, TaSUT1, TaSuSy2, AGPL1, SSI, SSIIIa and SBEIIa, suggesting that these genes play a regulatory role in starch accumulation. This was further confirmed by comparative analyses of starch synthesis between cultivars with contrasting thousand kernel weights, which revealed a closer association of the expression of the same set of genes with starch accumulation in developing wheat grains. The effect on starch yield of one of the candidate genes identified, AGPase, was examined through a transgenic approach, which involved expression of a gene encoding modified version of maize AGPase large subunit, designated as Sh2r6hs, in wheat under the control of maize’s constitutive Ubiquitin1 promoter. This manipulation of the wheat AGPase activity produced wheat lines with increased AGPase activity, grain weight and grain starch level, suggesting that the wheat grain size can be enhanced through increasing the capacity of starch synthesis both in the source and sink tissues. The study also identified and characterized a partial fragment of wheat rbcS promoter, and indicated that the promoter fragment can potentially be used as a tool for targeting the expression of genes of interest in photosynthetic source tissues. / October 2014

Doctor of Philosophy / Department of Agronomy / Allan K. Fritz / Heat stress is one of the major environmental constraints for wheat production worldwide. High temperature during grain filling in wheat leads to a significant reduction in yield. In this research, three different projects were completed. The first project was to study cytoplasmic effects on heat tolerance in wheat, where ten different alloplasmic lines of wheat were backcrossed with four different wheat varieties: „Karl 92‟, „Ventnor‟, „U1275‟ and „Jagger‟. The nuclear genome of the alloplasmic lines was substituted by backcrossing six times using the recurrent parents as males. During the fifth and/or fourth backcross, reciprocal crosses were made to develop NILs (Near Isogenic Lines) for cytoplasm. Sixty-eight NILs and their parents were evaluated in growth chambers for post-anthesis heat tolerance. Plants were grown in the greenhouse and placed under heat stress for 14 days starting at 10 days after anthesis. Growth chambers were maintained at 35°/30°C for heat stress and the greenhouse was maintained at 20°/15°C as the optimum temperature. Effects of high temperature on chlorophyll content and Fv/Fm (a chlorophyll fluorescence measuring parameter) were found to be significant. Cytoplasms 1, 4, 5, 8, 9 and10 provided greater tolerance in one or more nuclear backgrounds. These results indicated that cytoplasmic effects can contribute to heat tolerance of wheat. The second project focused on identification of quantitative trait loci (QTL) for thylakoid membrane damage (TMD), SPAD chlorophyll content (SCC) and plasma membrane damage (PMD), as these traits are found to be associated with resistance to heat stress and contributes to relatively stable yield under high temperature. A RIL (Recombinant Inbred line) population of a cross between winter wheat cultivars „Ventnor‟ and „Karl 92‟ was evaluated using two different temperature regimes (20°/15°C, 36°/30°C) imposed at ten days after anthesis. The aforementioned traits were evaluated and associated with various molecular markers (SSR, AFLP and SNP). The putative QTL associated are localized on chromosomes 6A, 7A, 1B, 2B and 1D and have the potential to be used in marker assisted selection for improving heat tolerance in wheat. In the third project, a transgenic approach to increase grain fill during high temperatures was investigated. Grain fill is reduced at temperatures above 25°C in wheat partly due to the inactivity of soluble starch synthase. We isolated a soluble starch synthase gene from rice that has the potential to overcome this deficiency during high temperatures and placed it behind both a constitutive promoter and an endosperm-specific promoter. Transgene expression and the effects of the transgene expression on grain yield-related traits for four generations (T0, T1, T2 and T3) were monitored. The results demonstrated that even after four generations, the transgene was still expressed at high levels, and transgenic plants produced grains of greater seed weight than Bobwhite control plants under the same environmental conditions. Thousand-seed weight under high temperatures increased 21-34% in T2 and T3 transgenic plants when compared to the non-transgenic control plants. In addition, the duration of photosynthesis was longer in transgenic wheat than in non-transgenic controls. Our study demonstrated that expression of rice soluble starch synthase gene in wheat can improve wheat yield under heat stress conditions.

Storage starch is synthesized in sub-cellular organelles called amyloplasts in higher plants. The synthesis of the starch granule is a result of the coordinated activity of several groups of starch biosynthetic enzymes. There are four major groups of these enzymes, ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDE). Starch phosphorylase (SP) exists as both dimeric and tetrameric forms in plastids in developing cereal endosperm and catalyses the reversible transfer of glucosyl units from glucose-1-phosphate to the non-reducing end of α-1-4 linked glucan chains, although the precise role in the pathway remains unclear. The present study was conducted to investigate the role and regulation of SP and SSIV in starch biosynthesis in developing maize endosperm. The results of this study showed that the tetrameric form of SP accounts for the majority of measurable catalytic activity, with the dimeric form being barely active and the monomer catalytically inactive. A catalytically active recombinant maize SP was heterologously expressed and used as an affinity ligand with amyloplast lysates to test protein-protein interactions in vitro. Results showed that the different multimeric status of SP influenced interactions with other enzymes of starch synthesis. Tetrameric SP interacted with SBEI and SSIIa, whilst the dimeric form of the enzyme interacted with SBEI, SBEIIb. All of these interactions were enhanced when amyloplasts were pre-treated with ATP, and broken following treatment with alkaline phosphatase (APase), indicating these interactions are regulated by protein phosphorylation. In addition, the catalytic activity of SSIV was reduced following treatment with APase, indicating a role for protein phosphorylation in the regulation of SSIV activity. Protein-protein interaction experiments also suggested a weak interaction between SSIV and SP. Multimeric forms of SP regulated by protein-protein interactions and protein phosphorylation suggested a role for SP in starch biosynthesis in maize endosperm.