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Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C4 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 C3 plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C3 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 C4 dikinase. In addition, the kinetics of this process closely resemble the reversible C4 process, with light-induced dephosphorylation occurring rapidly ( 15 min) and darkinduced phosphorylation occurring much more slowly ( 30-60 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 C3-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C3 plants Vicia faba and rice (Oryza sativa). We conclude that an RPlike activity mediates the light/dark modulation of PPDK phosphorylation state in C3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C4 pathway regulatory "converter" enzyme.