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.
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