RUI: Significance of Phospho-Regulated Plastidic Pyruvate,Orthophosphate Dikinase in C3 Plants

历史上，植物丙酮酸正磷酸二激酶（plant pyruvate,orthophosphate dikinase， PPDK）最初是在植物C4光合途径的叶片中发现的，它是一种丰富的酶。在C4植物中，PPDK可以催化二氧化碳主要受体磷酸烯醇丙酮酸酯（PEP）的再生。由于这在c4 -光合途径中的作用，主要的重点放在理解其通过可逆磷酸化对活性（光，活跃；暗，不活跃）的光/暗调节上。这种调节形式允许PPDK活性与光可用性的有效同步，以实现C4循环的最佳功能。另一种酶“昵称”PPDK调节蛋白或“RP”催化这种光/暗介导的可逆磷酸化。RP是一种高度独特的调节酶，具有与少数其他已知酶共享的催化属性。然而，这种不寻常的酶的结构和功能特性的离散理解仍然难以捉摸，因为RP的基因或cDNA尚未被克隆，也没有酶可重复纯化到同质性，后者排除了用传统方法克隆它。在C3植物（仅具有C3光合途径的植物）中，PPDK也存在于质体中，但不直接参与光合作用。它显然无处不在（例如，在根、茎、叶、种子中），通常是一种低丰度的酶。它在C3植物中的作用，虽然不进行光合作用，仍有待阐明。最近有研究表明，C3植物叶片和叶绿体中的PPDK在光/暗诱导下的激活状态变化与C4二激酶相似。结果表明，这种光/暗调节是由一种酶活性介导的，这种酶活性与C4植物叶绿体中发现的PPDK调节蛋白（RP）相同。本项目将以这些最近的观察结果为基础，最终阐明PPDK在C3植物质体中的具体作用。在C3植物中，叶绿体定位PPDK的几个假定作用有待解决，包括(i)增加质体中用于下游生物合成的PEP供应，（ii）保护细胞的渗透调节，以及（iii）种子发育。一个中心策略将是评估具有失活塑性PPDK基因的拟南芥突变体的形态和生化表型。为了补充对C3质体PPDK功能的研究，我们将获得C3（拟南芥）RP基因的cDNA克隆，该基因可能介导C3叶绿体中PPDK的可逆磷酸化。该序列将用于深入的分子表达研究这种独特的和潜在的“独一无二”的酶。

Historically speaking, plant pyruvate,orthophosphate dikinase (PPDK) was initially discovered in leaves of plants possessing the C4 photosynthetic pathway where it is an abundant enzyme. PPDK in C4 plants catalyzes the regeneration of the primary CO2 acceptor phophoenolpyruvate (PEP). Because this role in the C4-photosynthetic pathway, major emphasis was placed on gaining an understanding of its now well-elucidated light/dark regulation of activity (light, active; dark, inactive) via reversible phosphorylation. This form of regulation allows the efficient synchronization of PPDK activity with light availability for optimal functioning of the C4 cycle. Another enzyme "nicknamed" the PPDK regulatory protein or "RP" catalyzes this light/dark mediated reversible phosphorylation. RP is an highly unique regulatory enzyme with catalytic attributes shared with only a handful of other known enzymes. However, a discrete understanding of the structural and functional properties of this unusual enzyme has remained elusive since the gene or cDNA for RP has not yet been cloned, nor has the enzyme been reproducibly purified to homogeneity, the latter precluding its cloning by conventional means. In C3 plants (plants possessing only the C3 photosynthetic pathway), PPDK is also present in plastids but does not function directly in photosynthesis. It apparently occurs with ubiquity (e.g., in roots, stems, leaves, seeds) and is usually a low abundance enzyme. Its function(s) in C3 plants, although nonphotosynthetic, remain to be elucidated. Recently, it was documented that PPDK in leaves and chloroplasts of C3 plants undergoes light/dark-induced changes in activation state in a manner similar to C4 dikinase. It was determined that this light/dark regulation was mediated by an enzyme activity identical to that of the PPDK regulatory protein (RP) found in chloroplasts of C4 plants. This project will build upon these recent observations for ultimately elucidating the specific role(s) of PPDK localized in plastids of C3 plants. The several putative roles of chloroplast localized PPDK in C3 plants to be resolved include roles in (i) augmenting PEP supply in plastids for downstream biosynthesis, (ii) osmoregulation in guard cells, and (iii) seed development. A central strategy will be to assess morphological and biochemical phenotypes of Arabidopsis mutants with an inactivated plastidic PPDK gene. Complementing the investigation into C3 plastid PPDK function will be to obtain a cDNA clone for the putative C3 (Arabidopsis) RP gene that likely mediates reversible phosphorylation of PPDK in C3 chloroplasts. This sequence will be utilized for in-depth molecular expression studies of this unique and potentially "one-of-a-kind' enzyme.