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

Minnesota State University Moorhead, Moorhead MN

Investigators

Abstract

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.