Expression Profiles and Post-Translational Modifications of Phosphoenolpyruvate Carboxylase Isozymes of Bienertia sinuspersici during Leaf Development

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is the primary acceptor of carbon dioxide in C₄ photosynthesis. Three isozymes of PEPC: PEPC1, PEPC2, and PEPC3, in the single cell, C₄ plant Bienertiasinuspersici Akhani were determined in this study. Differential expressions of these isoforms were observed at different stages of leaf development. Amino acid sequence alignment revealed that PEPC1, PEPC2, and PEPC3 had characteristics of C₃-C₄ PEPC type, C₄ PEPC, and C₃ PEPC, respectively. Gene expression profiles showed that PEPC3 was highly expressed in young leaves while PEPC1 and PEPC2 were more expressed in intermediate and mature leaves, respectively. Using immunoblotting, we also assessed tissue-specific expression and post-translational modifications (PTMs) of PEPC in B. sinuspersici. During leaf maturation, phosphorylation at Ser 11 and monoubiquitination at Lys 629 were highly increased. The phosphorylation pattern on PEPC was diurnally regulated but monoubiquitination was not. Moreover, dimorphic chloroplasts from B. sinuspersici chlorenchyma cell of mature leaf were isolated to study spatial localization of PEPC by PTM. Monoubiquitinated PEPC were specifically interacting with peripheral chloroplasts. These results indicated that phosphorylation and monoubiquitination on PEPC were mutually exclusive as well as affecting spatial localization in single cell C₄ plants.