Elucidating the controlling role of the pyruvate phosphate dikinase regulatory protein on C4 photosynthesis

Pyruvate phosphate dikinase (PPDK) is a C 4 photosynthetic pathway enzyme that converts Pyr to PEP, the substrate for CO 2 fixation in C 4 leaves. PPDK activity is regulated in response to light intensity to ensure the PEP synthesis rate is in balance with the amount of light energy incident on the leaf. Light regulation of PPDK occurs by reversible phosphorylation of an active‐site Thr. A bifunctional protein kinase/phosphotransferase, the PPDK regulatory protein (PDRP), catalyzes this reversible phosphorylation. Although the biochemical properties of PDRP are well known, its role in overall C 4 cycle regulation in leaves is unclear. Our approach for resolving this question entails creating PDRP‐RNAi transgenic knockdown maize lines for assessing the interdependence of C 4 PS and PDRP>;PPDK regulation. The first phase of the study involved isolation of homozygous PDRP knockdown T 2 maize lines. This was accomplished using PCR and qPCR based screen. A total of 22/134 T 2 plants were found to be homozygous for the PDRP‐RNAi transgene. A subset of these T 2 plants was analyzed for leaf PDRP transcript abundance via Taqman based qPCR assay. This revealed a 5–50% suppression of PDRP transcript vs. Wt. Western blot analysis of leaf extracts showed a corresponding decrease in PDRP protein. Lines showing severe PDRP suppression developed chlorotic leaves and failed to reach maturity, suggesting PDRP is required for optimal C 4 PS.