Millie Smith, MSc Candidate, Plaxton Lab Phosphorylation of the cytosolic glucose-6-phosphate dehydrogenase isozyme AtG6PD6 in response to phosphate nutrition of the model plant Arabidopsis thaliana Glucose-6-phosphate dehydrogenase (G6PD) is a tightly regulated enzyme that catalyzes the first committed step of the oxidative pentose phosphate pathway (OPPP). The OPPP plays a pivotal role in generating reducing power in the form of NADPH, and carbon skeletons (e.g., ribose-5-P) needed for anabolism and cell growth. Our recent phosphoproteomics study discovered that the cytosolic G6PD isozyme, AtG6PD6, became hyperphosphorylated at multiple N-terminal residues 48 h following resupply of 2 mM phosphate (Pi) to Pi-starved (–Pi) Arabidopsis thaliana cell cultures. Although other phosphoproteomic studies have documented in vivo N-terminal phosphorylation of AtG6PD6 and its orthologs from other plant species, the functions and mechanisms of this phosphorylation event remain unknown. My research seeks to test the hypothesis that phosphorylation activates AtG6PD6 to enhance OPPP flux during the rapid resumption of cell growth that follows Pi resupply to –Pi Arabidopsis. Immunoblotting with a phosphosite-specific antibody confirmed that AtG6PD6 from Pi-resupplied, but not –Pi, Arabidopsis suspension cells and roots was phosphorylated at Ser18. This correlated with an increase in extractable G6PD activity following Pi-resupply to –Pi Arabidopsis seedlings (i.e., root extracts). Although the native enzyme is highly unstable in vitro, a partial purification of phosphorylated AtG6PD6 was achieved from Pi-replete suspension cells. The purified preparation was rapidly dephosphorylated by incubating with exogenous λ-phosphatase, and the kinetic properties were investigated. Assessing the interplay between Pi nutrition and cytosolic G6PD phosphorylation may contribute to our understanding of post-translational OPPP control in plants, while identifying targets for developing Pi-efficient crop varieties. Such cultivars are urgently needed to alleviate agriculture’s over-reliance on massive applications of non-renewable and polluting Pi fertilizers.
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