Identification of ABA-dependent phosphorylated bHLH transcription factors in guard cells of Vicia faba by mass spectrometry

A previous investigation showed that an unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase (AAPK) in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard cell protoplasts (GCPs) from Vicia faba. Later, ABA-dependent phosphorylated proteins were identified as basic helix-loop-helix (bHLH) transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, it is still unknown whether the 61 kDa in Vicia GCPs is AKS or not. In this study, we performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in Vicia faba (VfAKSs) by mass spectrometry. Moreover, we found that the 61 kDa protein is VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated on Ser residues, which are important for 14-3-3 protein binding and monomerization, in response to ABA in GCPs. In addition, orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain, were also co-immunoprecipitated with 14-3-3 protein in guard cells.