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

RUI：C3 植物中磷酸调节质体丙酮酸、正磷酸二激酶的意义

• 批准号: 0318568
• 负责人: Chris Chastain
• 金额: $ 12.43万
• 依托单位: Minnesota State University Moorhead
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0318568&HistoricalAwards=false
• 关键词: RUI; Significance; Phospho; Regulated; Plastidic

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.

从历史上看，植物丙酮酸正磷酸二激酶（PPDK）最初是在具有C4光合作用途径的植物叶子中发现的，它是一种丰富的酶。 C4 植物中的 PPDK 催化主要 CO2 受体磷酸烯醇丙酮酸 (PEP) 的再生。由于 C4 光合作用途径中的这一作用，主要重点放在了解其通过可逆磷酸化进行的光/暗活性调节（光、活性；暗、非活性）。这种形式的调节允许 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“昵称为”PPDK 调节蛋白或“RP”，催化这种光/暗介导的可逆磷酸化。 RP 是一种非常独特的调节酶，其催化属性仅与少数其他已知酶相同。然而，由于 RP 的基因或 cDNA 尚未被克隆，该酶也没有被可重复地纯化至同质性，因此对这种不寻常酶的结构和功能特性的离散了解仍然难以捉摸，后者妨碍了通过常规方法对其进行克隆。在C3植物（仅具有C3光合作用途径的植物）中，PPDK也存在于质体中，但不直接在光合作用中发挥作用。它显然普遍存在（例如，在根、茎、叶、种子中）并且通常是低丰度酶。尽管它不进行光合作用，但其在 C3 植物中的功能仍有待阐明。最近，有文献报道，C3 植物的叶子和叶绿体中的 PPDK 以类似于 C4 二激酶的方式经历光/暗诱导的激活状态变化。经确定，这种光/暗调节是由与 C4 植物叶绿体中发现的 PPDK 调节蛋白 (RP) 相同的酶活性介导的。该项目将基于这些最近的观察结果，最终阐明位于 C3 植物质体中的 PPDK 的具体作用。叶绿体定位的 PPDK 在 C3 植物中的几个假定作用有待解决，包括 (i) 增强质体中 PEP 供应以进行下游生物合成，(ii) 保卫细胞中的渗透调节，以及 (iii) 种子发育。一个中心策略是评估具有失活质体 PPDK 基因的拟南芥突变体的形态和生化表型。对 C3 质体 PPDK 功能的研究的补充将是获得推定的 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。该序列将用于对这种独特且潜在的“独一无二”酶进行深入的分子表达研究。