Nebraska 68588-0664 (G.S., R.C.)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 4 process, with light-induced dephosphorylation occurring rapidly (Յ15 min) and dark-induced phosphorylation occurring much more slowly (Ն30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C 3 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.Pyruvate,orthophosphate (Pi) dikinase (PPDK; EC 2.7.9.1) is a well-known enzyme of the C 4 photosynthetic pathway where it catalyzes the ATP-and Pidependent formation of phosphoenolpyruvate (PEP), the primary CO 2 acceptor molecule, from pyruvate:Consistent with its being a rate-limiting enzyme in the C 4 cycle, PPDK activity is regulated in a reversible, light-dependent manner so the overall pathway can function optimally in net CO 2 assimilation (Hatch, 1987;Furbank et al., 1997). This posttranslational regulation is conferred by reversible phosphorylation of a "target" Thr residue (Thr-456 in maize [Zea mays] C 4 PPDK) proximal to a catalytically essential (phospho) His (His-458 in maize), with the enzyme being inactive in its threonylphosphorylated state. A single, bifunctional protein kinase/phosphatase, named the PPDK regulatory protein (RP), catalyzes this regulatory phosphorylation/dephosphorylation cycle ( Fig. 1; Burnell and Hatch, 1984, 1985Roeske and Chollet, 1987; Ashton et al., 1990). Along with its target enzyme, RP is specifically localized in the chloroplast stroma of the C 4 mesophyll cell. It is a highly unusual and unique RP in at least three important respects. First, it is bifunctional in that it catalyzes both PPDK in...