The Arabidopsis thaliana Histidine Kinase 5 - Towards Specificity in H2O2 and MSP Signaling

Abstract

The ARABIDOPSIS HISTIDINE KINASE 5 (AHK5) is thought to act as a cytosolic ROS sensor, mediating physiological responses to various ROS and ROS- inducing stimuli via a multistep phosphorelay (MSP) (Iwama et al., 2007; Desikan et al., 2008; Pham et al., 2012; Heunemann, 2016). To better understand the mechanism of perception by AHK5 and the biological significance of downstream posttranslational and transcriptional changes, in vitro protein assays, in vivo interaction studies and analysis of the phosphoproteome and transcriptome of ahk5-1 plants in dependence on exogenous H2O2 (eH2O2) were performed. In in vitro experiments, the glutathione redox midpoint potential of the AHK5 input domain was determined. It could further be shown that the recombinant AHK5 output domain not only phosphorylates ARABIDOPSIS HISTIDINE CONTAINING PHOSPHOTRANSFER PROTEIN 1 (AHP1), but also AHP2, and that AHK1 may have a function as a His or Asp phosphatase. Furthermore, the existence of a high molecular weight complex consisting of AHK5, RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), RBOHF, and possibly FLAGELLIN-SENSITIVE 2 (FLS2) is postulated. Preliminary in vitro phosphorylation experiments with recombinant AHK5 from insect cells indicate constitutive autoactivity independent of the dimerization status of the input domain and inability to transfer phosphate to AHP1. In presence and absence of eH2O2, a rapid and extensive transition of the AHK5 mediated MSP to classical Ser/Thr phosphorylation was observed. The transcriptome data show strikingly early peaking of AHK5 dependent changes in gene expression in response to eH2O2. The importance of posttranslational and transcriptional AHK5 mediated changes are discussed and related to other AHK-dependent phosphoproteins, in an attempt to advance understanding of MSP signaling specificity. The generated stable lines of Arabidopsis thaliana, which carry wild-type or mutant full-length genomic AHK5 under the control of the endogenous promoter in the ahk5-1 background, could shed further light on the AHK5 dependent MSP signaling network.