Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Abstract

Maize <i>opaque2</i> (<i>o2</i>) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by <i>o2</i> include <i>pdk1</i> and <i>pdk2</i> that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. <i>pdk1</i> and <i>pdk2</i> were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. <i>pdk2</i> accounts for the large majority of endosperm PPDK, whereas <i>pdk1</i> specifies the abundant mesophyll form. The <i>pdk1-</i> mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by <i>o2</i>-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that <i>o2</i>- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.