Chastain Chris J, Fries Jason P, Vogel Julie A, Randklev Christa L, Vossen Adam P, Dittmer Sharon K, Watkins Erin E, Fiedler Lucas J, Wacker Sarah A, Meinhover Katherine C, Sarath Gautam, Chollet Raymond
Department of Biology, Minnesota State University, Moorhead, MN 56563, USA.
Plant Physiol. 2002 Apr;128(4):1368-78. doi: 10.1104/pp.010806.
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 (< or =15 min) and dark-induced phosphorylation occurring much more slowly (> or =30-60 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.
丙酮酸、正磷酸（Pi）双激酶（PPDK）作为一种叶绿体C4循环酶最为人所知。作为控制该光合途径通量的关键调控位点之一，它受到光的严格且可逆的调控。这种光/暗调节是由PPDK调节蛋白（RP，一种双功能蛋白激酶/磷酸酶）对活性位点结构域中一个保守的苏氨酸残基进行可逆磷酸化介导的。PPDK也存在于C3植物中，尽管其尚无已知的光合功能。然而，在本报告中我们表明，几种被子植物叶片中的C3 PPDK以及分离的完整菠菜（Spinacia oleracea）叶绿体中的C3 PPDK，其磷酸化状态会以类似于C4双激酶的方式发生光/暗诱导变化。此外，该过程的动力学与可逆的C4过程非常相似，光诱导的去磷酸化迅速发生（≤15分钟），而暗诱导的磷酸化则慢得多（≥30 - 60分钟）。在完整的菠菜叶绿体中，C3 PPDK的光诱导去磷酸化被证明依赖于外源Pi和光系统II的活性，但不依赖于光系统I的电子传递。这些细胞器内的结果表明，Pi和腺苷酸的基质池在调节C3 - PPDK的可逆磷酸化中发挥作用。最后，我们使用体外RP测定法直接证明了在C3植物蚕豆（Vicia faba）和水稻（Oryza sativa）的脱盐叶片提取物中，PPDK的磷酸化依赖于ADP。我们得出结论，一种类似RP的活性介导了C3叶片和叶绿体中PPDK磷酸化状态的光/暗调节，并且可能代表了这种不寻常且关键的C4途径调节“转换”酶的祖先同工型。
