The cell cycle machinery regulates glucose-6-phosphate dehydrogenase activity in maize

Glucose-6-phosphate dehydrogenase (G6PDH), a key regulatory enzyme of the oxidative pentose phosphate pathway, plays a central role during seed germination through the generation of NADPH and metabolic intermediates required for biosynthetic processes, including DNA synthesis. Therefore, cell proliferation must be linked to OPPP activity to ensure the supply of metabolites and reducing equivalents necessary for cell cycle initiation. Cell cycle progression is regulated by heterodimeric complexes of cyclins (Cyc) and cyclin-dependent kinases (CDKs), able to phosphorylate target proteins, thus we hypothesized that G6PDH could be an integral part of this regulatory network. To test this, we investigated the interaction of the cytosolic G6PDH from maize (ZmCy-G6PDH) with ZmCycs or ZmCDKs, its phosphorylation by Cyc/CDK complexes, and if this phosphorylation affects its activity. Pull-down assays showed that ZmCy-G6PDH interacted with CycD2; 2, CycD3; 1 or CycB2; 1 and CDKA; 1, but not with CycB1; 2 or CDKB1; 1. ZmCy-G6PDH was phosphorylated in vitro and semi-in vivo by different Cyc/CDK complexes exclusively in its homodimeric and homotetrameric conformations, leading to significant increase in ZmCy-G6PDH activity. Unexpectedly, in vitro treatment with the CDK-specific and ATP-competitive inhibitor RO-3306, also enhanced ZmCy-G6PDH activity. G6PDH activity increased as germination advanced in maize embryo axes, but a parallel treatment with RO-3306 decreased this activity, it being more evident at 24 and 36 h. Incidentally, the main reactive oxygen species (ROS) contention barriers, played by glutathione reductase and catalase, also showed reduced activity in embryo axes treated with the inhibitor. These results suggest a correlation between cell cycle machinery and the oxidative pentose phosphate pathway via ZmCy-G6PDH phosphorylation during germination, and this might become relevant to the contention of the oxidative stress.