Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochond rial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA ( AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense d own-regulation of its expression on plant growth and development. The deduc ed amino acid sequence of AtPDHK exhibits extensive similarity to other pla nt and mammalian PDHKs, containing conserved domains typical of two-compone nt histidine protein kinases. The Escherichia coli expressed AtPDHK specifi cally phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to con trol plants. Immunoblot analyses performed with a monoclonal antibody to th e E1 alpha mtPDH component (the subunit phosphorylated by PDHK) indicated t hat the increased mtPDC activity was not the result of an increase in the l evel of PDH protein. MtPDC from transgenic plants showed a reduced sensitiv ity to ATP-dependent inactivation compared to that observed in wild-type pl ants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC a ctivity observed in the antisense PDHK plants. Transgenic plants with parti ally repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter g eneration time. The potential role for AtPDHK gene manipulation in crop imp rovement is discussed.