Regulation Of NADPH Oxidase RBOHF By Protein Kinases In Arabidopsis Thaliana

During the process of evolution the simple ion Ca2+ has become one of the most important second messengers in eukaryotic species.The specificity and information content of a Ca2+signal is defined by its distinct stimulus specific temporal and spatial changes in concentration in the cytoplasm of cells.Plants are equipped with an elaborate repertoire of Ca2+ sensors and responders allowing them to appropriately perceive and respond to specific Ca2+ signals.Among these,Arabidopsis CBL/CIPK complexes represent a sophisticated Ca2+ decoding machinery,which regulates a wide range of targets and plant physiological processes.Recently,reactive oxygen species(ROS)were recognized as another important second messenger,which fulfills crucial roles in plant immune responses and long distance signaling.Recent studies suggested that there appears to be an interconnection between Ca2+ and ROS signaling.Our study suggests the Arabidopsis NADPH oxidase RBOHF is a key convergence point interconnecting Ca2+ and ROS signaling.The regulation of RBOHF protein activity by CBL/CIPK complexes was investigated in this study by different approaches,including pathway reconstitution in heterologous HEK293T cell culture system.We found that in addition to CIPK26,CIPK11 with CBL1/9 could also activate RBOHF.Further investigation showed CIPK11 and CIPK26 did not provide synergistic activation of RBOHF.This finding suggests that both kinases might function in activation RBOHF in response to different stimuli or they may have overlapping functions.Biochemical results indicated that both CIPK11 and CIPK26 could phosphorylate RBOHF-N in vitro and that Ca2+ binding to RBOHF enhanced this phosphorylation.In HEK293T cells,further analyses showed the activation of RBOHF is dependent on the kinase activity of CIPK26 and the plasma membrane localization of CBL1/CIPK26 complexes.Moreover,the further enhanced activity of RBOHF is also Ca2+dependent.Therefore,both phosphorylation and Ca2+ binding are essential for the activation of RBOHF and appear to mutually enhance each other in activating this NADPH oxidase.In this study,another well-known protein kinase OST(SnRK2.6)was found to activate RBOHF in HEK293T cells as well.Not wild-type OST1 but OST1 fused with a plasma membrane targeting domain(designated as PM-OST1)could strongly activate RBOHF.This observation suggests,that for activity of OST1 towards plasma membrane targets some currently unknown mechanism(like protein-protein interaction or protein modification)appears to be required to convey a localization of this kinase at the plasma membrane.Importantly,this study uncovered synergistic activation of RBOHF by combined Ca2+-dependent and ABA-mediated phosphorylation through CBL1/CIPK26 and OST1.Mass spectrometric analyses revealed an overlapping phosphorylation pattern in which both kinases targeted the same amino acid residues but also each phosphorylated distinct sites.Further functional analyses of these phosphorylation when mutated in HEK293T cells provided novel important insights into the regulatory mechanism of RBOHF.Lastly,we found ABI1,one member of protein phosphatase 2C family,could abolish the activation of RBOHF by CBL/CIPK26,PM-OST1 or their combination to convey negative regulation of this pathway.Above all,our research revealed that the ROS generation could be quickly enhanced after the phosphorylation and Ca2+ binding dependent activation of RBOHF.We also found the activation of RBOHF was not only regulated by the Ca2+ dependent kinases,but also regulated by Ca2+ independent kinase which made the regulatory mechanism of RBOHF more complex.Collectively,the results of this study support a novel model of RBOHF regulation:Upon a stress or developmental stimulus a rapid Ca2+ signal can activate RBOHF by Ca2+ binding and Ca2+ dependent phosphorylation very rapidly within seconds or minutes.More long-term synthesis of ABA can activate OST1 to enhance and sustain ROS production by RBOHF.Decline of the stress would result in a decrease of ABA concentration and favor activation of ABI1 to dephosphorylate RBOHF.In this way the balance of RBOHF phosphorylation and dephosphorylation can fine tune the ROS production in plants.These results can inform future studies to establish the biological significances of this complex mechanism and to elucidate its general importance for the regulation of other NADPH oxidases.