Plant Physiol, April 2002, Vol. 128, pp. 1368-1378

Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of C₃ Plants Undergoes Light-/Dark-Induced Reversible Phosphorylation¹

Department of Biology, Minnesota State University, Moorhead, Minnesota 56563 (C.J.C., J.P.F., J.A.V., C.L.R., A.P.V., S.K.D., E.E.W., L.J.F., S.A.W., K.C.M.); and Department of Biochemistry, George W. Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664 (G.S., R.C.)

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 dark-induced 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 RP-like 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.