Pyruvate,orthophosphate dikinase in leaves and chloroplasts of C3 plants undergoes light-/dark-induced reversible phosphorylation

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C-4 cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C-3 plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C-3 PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C-4 dikinase. In addition, the kinetics of this process closely resemble the reversible C, process, with light-induced dephosphorylation occurring rapidly (less than or equal to15 min) and dark-induced phosphorylation occurring much more slowly (greater than or equal to30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C3 PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C-3-PPDK. Last we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C-3 plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C-3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C-4 pathway regulatory "converter" enzyme.