PRE-HARVEST SPROUTING AND LATE MATURITY ALPHA-AMYLASE IN TRITICUM AESTIVUM (COMMON WHEAT), A PROTEOMICS PERSPECTIVE

John Holden Kelly

Pre-harvest sprouting (PHS) and late maturity alpha-amylase (LMA) are two weather related events that can occur in wheat prior to harvest and lead to the over-expression of the alpha-amylase enzyme. Alpha-amylase degrades starches in the wheat endosperm, reducing wheat soundness, lowering end-use quality, and causing substantial financial losses for growers. Increases in the amount and severity of PHS since the early 90s, and the discovery of LMA in the PNW in 2013, have necessitated a proportional increase in efforts to better understand the PHS and LMA problem and develop better management strategies and tools to mitigate the financial burden for growers. Chapter one summarizes what is currently known about the PHS and LMA proteomes of wheat seeds, with a particular focus on the endosperm, as it is critical to wheat product quality. Specific proteins and their significance to PHS and LMA were explored, and the impact that PHS and LMA have on end-use quality was reviewed based on the current literature. Possible directions for future research were discussed, including further exploration of the germination proteome Chapter two explores expression of the alpha-amylase enzyme during GA treatment of aleurones and germination, and validates antibodies created for this purpose. Two antibodies were developed: anti-TaAMY1-D, a generalist which can detect all four alpha-amylase isoforms, and anti-TaAMY2-B, which can specifically detect TaAMY2. Functionality of both antibodies was validated for purified proteins, for endogenous wheat proteins expressed from isolated aleurones, and endogenous proteins expressed in germination. Targeted proteomics was performed to quantify alpha-amylase changes throughout germination and identify homeoforms and subgenomes of origin for each return. Different patterns of expression were observed across alpha-amylase isoforms, a rapid and substantial regulation of TaAMY1, a delayed and less substantial upregulation of TaAMY2, an absence of TaAMY3, and low and delayed upregulation of TaAMY4. We recommend further examination of alpha-amylase expression in other varieties to examine if TaAMY expression patterns are reproducible. In chapter three, the investigation into protein expression during germination continued with a whole seed protein extraction for germinated seed modeling the PHS phenotype. Short germination time courses for the ‘Seahawk’ and ‘Jasper’ varieties were run to validate the presence of alpha-amylase, and long germination time courses were run to examine alpha-amylase expression. Whole seed proteomics analysis revealed a small subset of proteins differentially expressed between germinated and dry seeds, and an even smaller subset that were consistently differentially expressed across varieties. Of these proteins, alpha-amylase was successfully validated. The examination of alpha-amylase expression in the longer-time course revealed similar patterns of expression as to what was observed in chapter two. More work should be done to explore the role of each alpha-amylase isoform in germination and to validate the identity of homeoforms and subgenomes of origin. In chapter four, LMA physiology was explored, and the window of vulnerability for LMA induction was defined. A novel method for identifying the LMA susceptibility window known as the ‘squish test’ was developed for use by agronomists. The squish test may be an easy way to judge if wheat in the field is at the right development stage for LMA induction.

PRE-HARVEST SPROUTING AND LATE MATURITY ALPHA-AMYLASE IN TRITICUM AESTIVUM (COMMON WHEAT), A PROTEOMICS PERSPECTIVE

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