ADP-glucose pyrophosphorylase is posttranslational regulated by phosphorylation in wheat seeds

BALLICORA MA; FERRERO DML; IGLESIAS AA; PIATTONI CV; ASENCION DIEZ MD

Buenos Aires

Congreso; LIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR; 2017

REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS

ADP glucose pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27), a heterotetramer comprised of two small (S) and large (L) subunits, catalyzes the first and rate-limited step of starch biosynthesis pathway. In plants, the activity of ADPGlcPPases is regulated allosterically by metabolites, by redox mechanisms and recently, phosphoproteomic and bioinformatic studies suggest its regulation by phosphorylation. In comparative studies along wheat (storing starch) and castor (storing triacylglycerides) seeds development, we found that ADPGlcPPase is phosphorylated only in wheat seeds at stages with increased ADPGlcPPase activity and higher starch accumulation. To study the effect of phosphorylation in wheat ADPGlcPPase, we used recombinant co-expression strategies in Escherichia coli to produce the subunits of wheat endosperm ADPGlcPPase, separately (TaeS, TaeL) or together (TaeSL). Also, we designed and produced different TaeS and TaeL Ser mutants located in putative phosphorylation sites. In vitro phosphorylation assays with 32P-γ-ATP, wheat seed extracts (as kinases resource) or specific recombinant kinases (SnRK1, SOS2 and CDPK) showed that TaeES, TaeEL and TaeESL were phosphorylated. As well, all mutants were phosphorylated too, suggesting that TaeEADPGlcPPase is phosphorylated in other Ser/Thr residue or the occurrence of many phosphorylation sites. When we evaluated the effect of phosphorylation, the result showed a 4-fold increase of TaeSL activity. Altogether, the results point out that posttranslational regulation of TaeEADPGlcPPase by phosphorylation would increase the enzyme activity at specific stages of wheat seed development. This work describes a new posttranslational mechanism in the regulation of starch metabolism at the level of ADPGlcPPase, that will required further study to deeply comprehend the interplay with other regulation mechanism and explore possible biotechnological developments.