New insights into the regulation of the vacuolar antiporters NHX1 and NHX2 activity by pH and phosphorylation

Abstract

The tonoplast-localized K+ ,Na+ /H+ exchangers NHX1 and NHX2 of Arabidopsis are involved in the accumulati on of K+ into the vacuole of plant cells, thereby increasing the osmoti c potenti al, water uptake and the turgor pressure necessary for cell expansion and growth. These proteins are also required for vacuolar remodelling during stomatal movements. Phosphoproteomic analyses and preliminary results from our laboratory strongly support that NHX1 and NHX2 functi on is regulated by phosphorylati on of their C-terminal domain by CBL-interacti ng protein kinases (CIPKs). Functi onal and in vitro phosphorylati on assays demonstrati ng a diff erenti al regulati on of NHX1 and NHX2 by CIPKs will be presented. We have also identi fi ed by phylogeneti c analysis and computati onal modelling of the NHX1 protein various structural domains and amino acid residues putati vely involved in ion transport, cati on coordinati on, and pH sensing of NHX1. Point-mutati on alleles for these relevant residues have been generated to analyse their impact in the biochemical acti vity and pH dependence of NHX1 by functi onality tests in yeast and in vitro ion transport assays. Our results point to the fi netuning of NHX1 and/or NHX2 acti vity in response to developmental and environmental cues. In additi on, we expect to unravel the biochemical mechanisms for pH sensing and regulati on of these criti cal K+ transporters of Arabidopsis.