Functions of maize genes encoding pyruvate orthophosphate dikinase in developing maize endosperm and their enzymatic properties

The research described herein, focuses on investigating the role of pyruvate phosphate dikinase (PPDK) and factors influencing its activity in maize starchy endosperm (SE) tissue. PPDK reversibly converts phosphoenolpyruvate (PEP), pyrophosphate (PPi), and AMP, to pyruvate, phosphate (Pi), and ATP. PPDK is found in microorganisms and plant tissues with diverse functions. PPDK is abundant in maize SE during grain fill, but little is known about its role. Hypotheses were tested using transposon-insertion alleles affecting individual genes and by eliminating all PPDK expression using RNAi. PPDK\u27s C4 metabolism function in mesophyll was proven to be essential in maize. RNAi transgenes completely eliminated PPDK protein and enzyme activity from developing SE. An opaque kernel phenotype resulted, indicative of changes in storage compound organization. Metabolite analyses indicated PPDK acts in the pyruvate-forming direction but is not necessary for synthesis of sufficient ATP to generate normal storage compound abundance. PPDK activity affects energy charge, i.e., ATP/(ADP + AMP) ratio, and through this is proposed to regulate central metabolic fluxes. PPDK activity in SE decreases during prolonged growth in high temperature, coincident with kernel weight decrease compared to normal. Recombinant PPDK was used to test whether this effect is inherent to the enzyme. When incubated without substrates, PPDK maintained full activity up to 35oC, but was completely inactivated within 5 min at 45oC. Pre-incubation with AMP prevented PPDK from inactivation at 45oC. The data suggest PPDK activity in vivo can be affected by availability of AMP, and confirm that PPDK is a particularly temperature-labile enzyme that could contribute to reduced plant performance at elevated temperatures. Recombinant PPDK isoforms were also used in protein-protein interaction tests that demonstrated formation of hetermultimeric forms including both the PPDK1 and PPDK2 polypeptides in addition to homomultimers of each isoform individually. Technical capacities developed to perform these experiments were distributed to the research community. These include a new series of vectors designed for SE-specific, high-level expression of transgenes through the course of grain fill, which were proven highly effective for plant transformation and transgene expression. New targeted metabolomics methods were developed to detect PEP, pyruvate, and PPi in total SE extracts using GC-MS