Reevaluation of the CLV3-receptor interaction in the shoot apical meristem: dissection of the CLV3 signaling pathway from a direct ligand-binding point of view

The CLAVATA signaling pathway is a key component of the network that controls stem cell renewal and differentiation in Arabidopsis thaliana. CLAVATA3 (CLV3) is a post-translationally arabinosylated secreted peptide signal that regulates WUSHEL (WUS) transcription to affect the balance of stem cell differentiation and proliferation in the shoot apical meristem (SAM). Known membrane-localized receptors involved in the perception of CLV3 signaling include CLV1, the CLV2/CORYNE (CRN) complex and RPK2. The CLV3 peptide can directly bind to CLV1; however, it is unclear whether the CLV3 peptide directly binds to CLV2 or RPK2. In this study, we re-evaluated the direct interaction between CLV3 and its receptors by photoaffinity labeling with photoactivatable arabinosylated CLV3. We showed that CLV2 and RPK2 exhibited no direct binding to the CLV3 peptide. Further analysis showed that the receptor kinase BAM1 directly binds the CLV3 peptide. A loss-of-function clv1 bam1 double mutant exhibited a large number of stem cells that accumulated in the SAM and was insensitive to exogenous treatment with the arabinosylated CLV3 peptide. WUS gene transcripts were up-regulated, and the region of WUS expression was enlarged at the SAM in the clv1 bam1 double mutant. These results indicate that CLV1 and BAM1 are direct receptors that are sufficient to affect the regulatory network controlling stem cell number in the SAM. In contrast, the CLV2/CRN complex and RPK2 are not involved in direct ligand interactions but may act as co-receptors. Significance Statement We re-evaluated the direct interaction between CLV3 and its receptors by photoaffinity labeling. The receptor kinase BAM1 could directly bind CLV3 peptide as well as CLV1. A loss-of-function clv1 bam1 double mutant exhibited a large number of stem cells that accumulated in the SAM and was insensitive to arabinosylated CLV3. These results indicate that CLV1 and BAM1 are direct receptors that are sufficient to affect the regulatory network controlling stem cell number in the SAM.