Funktionale Analyse des Phytosulfokin Rezeptors 1 aus Arabidopsis thaliana
In Arabidopsis thaliana, the peptide growth factor phytosulfokine (PSK) is perceived by two plasma membrane-localized receptors termed PSKR1 and PSKR2 that belong to the leucine-rich repeat class of receptor kinases. In this study functional analysis in particular of PSKR1 from Arabidopsis was performed. Overexpression of PSKR1 and PSKR2 in the wild type resulted in promotion of root and hypocotyl growth showing that abundance of PSK receptors limits growth. Epidermis-specific expression of PSKR1 in the pskr1-3 pskr2-1 (r1r2) background driven by the CER6 promoter showed that perception of PSK in the epidermis is sufficient to stimulate growth not only of the outer but also of the inner cell layers. Furthermore, expression of PSKR1 driven by the atrichoblast-specific promoter GL2 in r1r2 promoted growth not only of atrichoblast cells but also of root hair-forming trichoblasts. Hence, expression of PSKR1 in one cell type of the epidermis is sufficient to stimulate whole-organ growth indicating that PSK acts as a non-cell autonomous growth factor in planta. Bioinformatic analysis identified a calmodulin (CaM)-binding site in PSKR1. Replacement of the conserved tryptophan W831 in the CaM binding site by a hydrophilic serine resulted in loss of CaM binding. Expression of the CaM binding site mutated receptor variant in r1r2 enhanced the r1r2 phenotype with reduced root and shoot growth.Expression of the kinase-inactive PSKR1(K762E) in r1r2 resulted in reduced root and shoot growth. Hence, both, PSKR1 kinase activity and CaM binding to the receptor are essential for the growth promoting effect of PSK in planta. To study the regulation of the receptor by phosphorylation, a phosphosite mapping of PSKR1 was performed in collaboration. Subsequent functional analysis of identified phosphorylation sites by site-directed mutagenesis demonstrated that three conserved threonines and one serine (T890, S893, T894 and T899) located in the activation loop of PSKR1 are required for in vitro PSKR1 kinase activity and for receptor function in planta.