Peptide RGF1 And Its Receptor RGI1 Regulate Arabidopsis Root Meristem Development Via A MAPK Signaling Cascade

Root growth is maintained by the continuous division of cells in the apical meristem.ROOT MERISTEM GROWTH FACTORs?RGFs?are a group of critical peptide hormones regulating root stem cell niche maintenance.Previous studies from our lab and others discovered that five closely related leucine-rich repeat receptor-like protein kinases?LRR-RLKs?,named RGF1 INSENSITIVES?RGIs?or RGF1 RECEPTORS?RGFRs?,are able to perceive the RGF1 signal and redundantly control root stem cell niche maintenance.RGF1 regulates root meristem activity mainly via two downstream transcription factors,PLETHORA 1?PLT1?and PLT2.Regulatory proteins connecting cell surface RGF1-RGI1 and nuclear PLTs,however,were not identified.In order to identify novel regulatory components in RGIs-mediated signaling pathway,we established 35S::RGI1-FLAG transgenic plants and tried to identify all possible interacting proteins in RGI1 complex utilizing immunoprecipitation and proteomic approaches.Interestingly we found that a mitogen-activated protein kinase kinase 4?MKK4?and MAP kinase 3?MPK3?were co-immunoprecipitated with RGI1-FLAG only after the Arabidopsis seedlings were treated with RGF1.Previous studies indicated that a number of LRR-RLK-mediated signaling pathways use MAPK pathway to transduce their signals to downstream transcription factors.We therefore tested whether MAPK cascade is involved in RGIs-mediated signaling pathway.We found expression of constitutively active forms of MKK4 and MKK5,MKK4^DD and MKK5^DD,driven by an RGI1 promoter were able to partially rescue the root defective phenotypes of the rgi1/2/3/4 and rgi1/2/3/4/5 mutants.Loss-of-function mutations of MKK4/MKK5 or MPK3/MPK6 resulted in rgi1/2/3/4-like or rgi1/2/3/4/5-like short root phenotypes,respectively.Our gain-of-function and loss-of-function genetic data support YDA is an upstream MAPKKK of MKK4/MKK5 in the RGF1-RGI1 signaling pathway.Our biochemical data indicated that RGF1 treatment could induce the phosphorylation of MPK3 and MPK6.The activation of MPK3 and MPK6 by RGF1 relies on the functional RGIs as well as their downstream MKK4/MKK5.Finally,the expression of downstream transcription factors,PLT1 and PLT2,are largely controlled by RGF1-RGI1-mediated signaling pathway.In mkk4 mkk5,the expression of PLT1 and PLT2 are greatly reduced.From all the results presented in this and other studies,we put forward a signaling pathway that controls root meristem development in Arabidopsis from RGF1,to RGI1,to YDA-MKK4/MKK5-MPK3/MPK6 cascade,and finally to regulate the expression of transcription factors,such as PLT1 and PLT2.