RGF1-RGI1,a Peptide-Receptor Complex,Regulates 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 FACTOR 1(RGF1)is a critical peptide hormone regulating root stem cell niche maintenance.Previous studies discovered that five closely related leucine-rich repeat receptor-like protein kinases(LRRRLKs),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.Here,we report that the mitogen-activated protein(MAP)kinase kinase 4(MKK4)and MAP kinase 3(MPK3)were coimmunoprecipitated with RGI1-FLAG after Arabidopsis seedlings were treated with RGF1.Genetic and biochemical assays confirmed that MKK4 and MKK5,and their downstream targets MPK3 and MPK6,are essential RG卜dependent regulators of root meristem development.In addition,we found that the MKK4/MKK5-MPK3/MPK6 module functions downstream of YDA,a MAPKKK.Our results demonstrate that RGF1-RGI1 regulate the expression of PLT1/PLT2 via a YDA-MKK4/MKK5-MPK3/MPK6 signaling cascade.

The YDA-MKK4/MKK5-MPK3/MPK6 Cascade Functions Downstream of the RGF1-RGI Ligand-Receptor Pair in Regulating Mitotic Activity in Root Apical Meristem

The mitotic activity of root apical meristem(RAM)is critical to primary root growth and development.Previous studies have identified the roles of ROOT GROWTH FACTOR 1(RGF1),a peptide ligand,and its receptors,RGF1 INSENSITIVEs(RGIs),a clade of five leucine-rich-repeat receptor-like kinases,in promoting cell division in the RAM,which determines the primary root length.However,the downstream signaling components remain elusive.In this study,we identify a complete mitogen-activated protein kinase(MAPK or MPK)cascade,composed of YDA,MKK4/MKK5,and MPK3/MPK6,that functions downstream of the RGF1-RGI ligand-receptor pair.Similar to the rgi1/2/3/4/5 quintuple mutant,loss-of-function mutants of MPK3 and MPK6,MKK4 and MKK5,or YDA show a short-root phenotype,which is associated with reduced mitotic activity and lower expression of PLETHORA 1(PLT1)/PLT2 in the RAM.Furthermore,MPK3/MPK6 activation in response to exogenous RGF1 treatment is impaired in the rgi1/2/3/4/5 quintuple,yda single,and mkk4 m kk5 double mutants.Epistatic analyses demonstrated that the expression of constitutively active MKK4,MKK5,or YDA driven by the RGI2 promoter can rescue the short-root phenotype of the rgi1/2/3/4/5 mutant.Taken together,these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the RGF1-RGI ligand-receptor pair and upstream of PLT1/PLT2 to modulate the stem cell population and primary root growth in Arabidopsis.