Molecular Mechanisms Of Two Groups Of LRR-RLKs In Regulating Arabidopsis Root Growth And Development

During adaptation to terrestrial environment,plants evolved an important vegetative organ,root,to achieve immobilization,absorption of water and nutrients,biosynthesis,transportation and other physiological functions.Stem cell niche in root apical meristem,including quiescent center(QC)cells and their surrounding stem cells,is critical for normal root growth and development.During the maintenance of stem cell niche,frequent signal exchange among different cell types is critical for cell fate determination.RGFs are mainly produced from the QC cells and secreted to surrounding extracellular space,up-regulate the expression of PLT1 and PLT2 to promote rapid division of stem cells and maintain stem cell niche activity.Yet,the receptor of RGFs remained a mystery.Serial studies from our group demonstrated that BAK1 not only acts as an essential co-receptor regulating brassinosteroid(BR)signaling pathway,but also is involved in a root developmental pathway independent of BR signaling.We screened all 161 available LRR-RLKs for their possible interactions with BAK1 using a yeast two-hybrid system,and identified 83 LRR-RLK candidates.We functionally characterized two groups of LRR-RLKs by genetic approaches.A clade of LRR-RLKs including five members was designated as RGF1 INSENSITIVE 1(RGI1)to RGI5.Single T-DNA insertion mutants were isolated and none of them showed any obvious defective phenotypes,we generated a series of double,triple,quadruple,and quintuple mutants and found that these mutants showed more or less root defective phenotypes.The quintuple mutant showed an extremely short root phenotype with minimal meristem size.We further generated the second quintuple mutant by using five independent single T-DNA insertion mutants and the resulting second quintuple mutant showed root growth defects identical to the first quintuple mutant.The expression levels of PLT1 and PLT2 are almost undetectable in the quintuple mutant.Ectopic expression of PLT2 driven by an RGI2 promoter in the quintuple mutant greatly rescued its root meristem defects.The quintuple mutant is completely insensitive to synthesized sulfonated RGF1.In vitro dot-blotting and pull-down analysis indicated that RGI1 can physically interact with RGF1.Exogenous application of RGF1 can quickly induce the phosphorylation of RGI1 and RGI2,indicating that RGIs can perceive and transduce the RGF1 peptide signal.RGF1 can rapidly induce the poly-ubiquitination and the degradation of RGI1.RGF1 can also quickly induce the phosphorylation of BAK1.These results demonstrated that RGIs,acting as the receptors of RGF1,play essential roles in RGF1-PLT-mediated root meristem development in Arabidopsis thaliana.The second group of LRR-RLKs contains three members,which are expressed in root to a certain extent.Overexpression of RLK101 or RLK219 resulted in a short root phenotype.Triple mutant showed accelerated germination and root growth with upregulation of several ARRs.In summary,we carried out an in-depth study on the first group of LRR-RLKs,RGIs.Using genetic,physiological,biochemical,cellular biology and other strategies,we proved that RGIs,as the receptors of RGF1,are responsible for the maintenance of stem cell niche in Arabidopsis thaliana.We have also made a preliminary study on the second group of LRR-RLKs,and demonstrated their negative roles in the root growth and development in Arabidopsis thaliana.