Stem cell signalling in Arabidopsis requires CRN to localize CLV 2 to the plasma membrane

Stem cell number in shoot and floral meristems of Arabidopsis is regulated by the CLAVATA3 (CLV3) signalling pathway. Perception of the CLV3 peptide requires the receptor kinase CLAVATA1 (CLV1), the receptor-like protein CLAVATA2 (CLV2) and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together, and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags, and their capacities for interaction via efficiency of fluorescence resonance energy transfer (EFRET). We found that CLV2 and CRN require each other for export from the endoplasmatic reticulum (ER), and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN. 2 www.plantphysiol. org on October 1, 2017 Published by Downloaded from Copyright © 2009 American Society of Plant Biologists. All rights reserved. Bleckmann et al., CRN localizes CLV2 to the PM Introduction: In Arabidopsis, the stem cell number in the shoot apical meristem (SAM) is regulated by negative feedback regulation. Stem cell induction and maintenance is controlled by the homeodomain protein WUSCHEL (WUS), and WUS expression is in turn repressed by CLAVATA3 (CLV3) (Brand et al., 2000; Schoof et al., 2000), which encodes a 13 amino acid arabinosylated glycopeptide that is secreted from stem cells (Ohyama et al., 2009). Three genes have been identified that encode receptors for CLV3 signalling. Mutations in CLAVATA1 (CLV1) (Clark et al., 1997), encoding a leucine rich repeat (LRR) receptor kinase, CLAVATA2 (CLV2), encoding a LRR receptor-like protein (Jeong et al., 1999), or CORYNE (CRN), encoding a receptorlike kinase, disrupt CLV3 signalling and allow the stem cell domain to expand (Sablowski, 2007; Müller et al., 2008). Binding of CLV3 to the LRR domains of CLV1 was recently shown (Ogawa et al., 2008). A simple readout for CLV3 signalling is carpel number. Stem cells of floral meristems are normally consumed with the production of two central carpels. Any reduction in CLV3 signalling, which results in increased WUS expression and production of more stem cells, causes an increase in carpel number. Mutations in CLV1, CLV2 or CRN showed an intermediate carpel number phenotype and reduced CLV3 signalling (Müller et al., 2008). Double mutants of clv2 with crn were epistatic, but double mutants of clv1 with clv2 or crn were synergistic and abolished CLV3 signalling. This indicated that CLV1 acts independently from, and in parallel with, CLV2 and CRN to transmit the CLV3 signal. Furthermore, clv2 and crn mutants showed additional phenotypes, such as elongated pedicels and defects in stamen development, suggesting that CLV2 and CRN act in a common pathway (Müller et al., 2008). Both CRN and CLV2 were proposed to be membrane localized, and may physically 3 www.plantphysiol. org on October 1, 2017 Published by Downloaded from Copyright © 2009 American Society of Plant Biologists. All rights reserved. Bleckmann et al., CRN localizes CLV2 to the PM interact via their transmembrane domains, or immediately adjacent sequences (Fig. 1A). A loss-of-function mutation of CRN, crn-1, is caused by an amino acid exchange within the predicted transmembrane domain, suggesting that membrane localization, interaction with a partner protein, or both is essential for CRN function. We have here investigated the intracellular localization of CLV1, CLV2 and CRN in plant cells, and their tendencies for protein-protein interactions. Using fluorescent protein tags, we show that CLV1 resides at the plasma membrane. We found that CLV2 and CRN require each other for transport from the ER to the plasma membrane. Via FRET, we show that CLV2 and CRN form complexes at the ER that then relocalize to the plasma membrane, and we identified the protein domains required for this interaction. Futhermore, we found that CLV1 homomerizes, but can also interact with CRN and CLV2, suggesting a mechanism for crosstalk between the two receptor complexes for CLV3 signalling. 4 www.plantphysiol. org on October 1, 2017 Published by Downloaded from Copyright © 2009 American Society of Plant Biologists. All rights reserved. Bleckmann et al., CRN localizes CLV2 to the PM