DUF581-9 (At2g44670; FLZ3) negatively regulates SnRK1 activity by interference with T-loop phosphorylation

In plants, SUCROSE NON-FERMENTING RELATED KINASE 1 (SnRK1) is a key energy-sensor that orchestrates large-scale transcriptional reprogramming to maintain cellular homeostasis under energy deficit. SnRK1 activity is under tight negative control, although the exact mechanisms leading to its activation are far from being understood. We show that the Arabidopsis DOMAIN OF UNKNOWN FUNCTION (DUF581) protein DUF581-9/FLZ3 binds to the catalytic SnRK1 subunit KIN10 to inhibit its activation by GRIK-dependent T-loop phosphorylation. Overexpression of DUF581-9 in Arabidopsis dampens SnRK1 signalling and interferes with adaptation to dark-induced starvation. The presence of DUF581-9 significantly reduced SnRK1 activity in protoplasts and in vitro. This was accompanied by a reduction in T175 T-loop phosphorylation and also diminished KIN10 auto-phosphorylation. Furthermore, DUF581-9 reduced binding of the up-stream activating kinase GRIK2 to KIN10, explaining the reduced KIN10 T-loop phosphorylation. Ectopically expressed DUF581-9 protein was rapidly turned over by the proteasome when Arabidopsis plants were subjected to starvation treatment, likely releasing its inhibitory activity on the SnRK1 complex. Taken together, our results support a model in which DUF581-9 negatively regulates SnRK1 activity under energy sufficient conditions. Turnover over of the protein provides a rapid way for SnRK1 activation under energy deficit without the need of de-novo protein synthesis.