CIK Receptor Kinases Maintain The Homeostasis Of Shoot Apical Meristem Through CLV Signaling

Continuous organ initiation and outgrowth in plants rely on the proliferation and differentiation of stem cells maintained by the C LAVATA(CLV)-WUSCHEL(WUS)negative feedback loop.Leucine-rich repeat receptor-like protein kinases(LRR-RLKs),including C LV1 and its homologs BARELY ANY MERISTEM(BAMs),and REC EPTO R-LIKE PROTEIN KINASE 2(RPK2),a receptor-like protein CLV2 and a pseudokinase CORYN E(CRN),are involved in the perceptio n of CLV3 signal to repress WUS expression.WUS,a homeodomain transcription factor,in turn directly binds to the promoter of CLV3 to activate its expression and promote stem cell activity in the shoot apical meristem(SAM).However,the signaling mechanism immediately following the initial perception of C LV3 by its receptors is poorly understood.Here,we show that a group of LRR-RLKs,designated as CLAVATA3 INSENSITIVE REC EPTOR KINASE 1(CIK1)to CIK4,play essential roles in regulating CLV3-mediated stem cell homeostasis.The cik1 2 3 4 quadruple mutant exhibits significantly enlarged SAM and extra rosette leaves,flowers and floral organs,resembling clv mutants.RNA in situ hybridization results show that the expression of CLV3 and WUS is dramatically expanded and enhanced.The extremely changed expression patterns of CLV3 and WUS in cik1 2 3 4 quadruple mutant suggest that the C LV-WUS pathway is disrupted.clv1-20 cik1 2 3 4,clv2-101 cik1 2 3 4 and rpk2 cik1 2 3 4 quintuple mutants were further generated for genetic analyses,but none of clv1,clv2 or rpk2 shows additive phenotype to the cik1 2 3 4 quadruple mutant,suggesting that CIKs function in all three receptor-mediated C LV signaling pathways.At meanwhile,we found that wus-101 cik1 2 3 4 shows terminated SAM as wus,indicating that CIKs act upstream of WUS.Biochemical assays showed that CIKs interact with CLV1,CRN and RPK2 both in vivo and in vitro,and CLV1 can directly phosphorylate CIKs in vitro.Furthermore,the phosphorylation level of CIKs is quickly elevated in response to exogenous application of C LV3 in wild-type plants,but the response is lost in the clv1-20 bam1 bam2 triple mutants.These data suggest that CIKs function as co-receptors of C LV1,CLV2/CRN and RPK2 to mediate C LV3 signaling through a phosphorylation manner.We further identified two groups of kinase interacting with CIKs through mass spectrum.O verexpression of these kinases can partially restore the phenotypes of cik1 2 4 weak mutant,suggesting that these two groups of kinase may be involved in CLV signaling.Based on a phylogenetic analysis,we found that CIKs exist generally in higher plants.Homologous CIK genes of maize were cloned and overexpressed in Arabidopsis cik mutants.overexpression of these maize CIK genes can greatly rescue the enlarged meristem phenotype of cik1 2 4 triple mutants,suggesting a conserved function of CIKs in regulating SAM homeostasis in different plant species.Interestingly,cik 2 3 4 mutants showed resistance to all of those CLE peptides that can inhibit root growth of wild-type plants,indicating that CIKs are likely to mediate multiple CLE signals.Taken together,our findings not only widen the understanding of the underlying mechanism of C LV3 signal transduction in regulating stem cell fate,but also reveal a novel group of RLKs functioning as co-receptors to possibly mediate multiple extrinsic and intrinsic signals during plant growth and development.