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.

从历史上讲，植物丙酮酸正磷酸二激酶（PPDK）最初是在具有C4光合途径的植物叶片中发现的，在那里它是一种丰富的酶。C4植物中的PPDK催化初级CO2受体磷酸烯醇丙酮酸（PEP）的再生。由于C4-光合途径中的这种作用，主要重点是通过可逆磷酸化来了解其现在已充分阐明的光/暗调节活性（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，具有仅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的理解仍然难以捉摸，因为RP的基因或cDNA尚未被克隆，也没有酶被可重复地纯化到同质性，后者排除了它的克隆通过常规手段。在C3植物（仅具有C3光合途径的植物）中，PPDK也存在于质体中，但不直接在光合作用中起作用。它显然是普遍存在的（例如，在根、茎、叶、种子中）并且通常是低丰度酶。它在C3植物中的功能，虽然非光合作用，仍有待阐明。最近，据报道，PPDK在叶片和叶绿体的C3植物经历光/暗诱导的变化，在激活状态的方式类似于C4双激酶。据确定，这种光/暗调节介导的酶活性相同的PPDK调节蛋白（RP）中发现的叶绿体的C4植物。本项目将建立在这些最近的观察，最终阐明的具体作用（S）的PPDK定位在质体的C3植物。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）增加下游生物合成质体中PEP供应，（ii）保卫细胞中的PEP调节，和（iii）种子发育。一个中心的战略将是评估形态和生化表型的拟南芥突变体与失活的质体PPDK基因。补充C3质体PPDK功能的调查将获得一个cDNA克隆的推定C3（拟南芥）RP基因，可能介导可逆磷酸化的PPDK在C3叶绿体。该序列将用于深入研究这种独特的和潜在的“独一无二”的酶的分子表达。