Biochemical and Proteomic Profiling of Maize Endosperm Texture and Protein Quality

The research described herein, focuses on the biochemical and proteomic analysis of the maize endosperm and what influences kernel texture. Quality Protein Maize (QPM) is a hard endosperm version of the high-lysine opaque2 (o2) mutant but the genes involved in modifying the soft o2 endosperm are unknown. Pyrophosphate (PPi)-dependent fructose 6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose 6-phosphate to fructose 1, 6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the alpha regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays show a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to wild type and o2. Furthermore, the O2 regulated pyruvate Pi dikinase (PPDK) gene is reduced in expression and activity in o2. Normal vitreous endosperm in QPM may occur due to modulation of glycolytic flux attributable to increased enzyme activity at two regulatory enzymes of glycolysis, PFP and PPDK. Opaque endosperm is most often attributed to either quantitative or qualitative changes in zein accumulation. However, some opaque endosperm mutants have normal zein accumulation. In order to identify factors involved in vitreous endosperm formation or its disruption (opacity) we conducted shotgun proteomic analysis of the nearly isogenic lines of six opaque endosperm mutant non-zein fractions. Our proteomic data indicates that there is not one specific cause of endosperm opacity apart from the non-ubiquitous reduction of zeins. We suggest that mis-regulation of zein deposition on the ER membrane or improper trafficking either upon loading or unloading from the ER membrane causes cellular stress which could invoke opacity. A mutagenized B73 population (MB73) was also created as an additional method to identify genomic regions and ultimately, genes involved in vitreous and/or opaque endosperm texture as well as create variants with proteomes rebalanced towards improved endosperm protein quality. Advisor: David R. Holdin