Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane

Abstract

Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein 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. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum 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.

Figure 1.

Inducible transgene expression rescues the corresponding mutants. A, Speculative model for interactions of CLV3 with receptor complexes. CLV3 peptide is proposed to bind to two separate receptor systems, consisting of CLV1 and CLV2 together with CRN. CLV2 homodimers could interact with CRN via their transmembrane domains. Receptor activity restricts stem cell fate, and the separate receptor complexes may interact through their kinase domains. B, T-DNA for inducible expression of translational fusions with the FPs GFP, mCherry, or both. G10-90, Constitutive promoter; XVE, chimeric transcription factor that activates transcription from the lexA-46 35S promoter upon estradiol induction. FPs are GFP in pABindGFP, mCherry in pABindmCherry, and GFP-mCherry in pABindFRET. C, Schematic representation of CLV1-FP, CLV2-FP, and CRN-FP. D to F, Examples of partial phenotypic restoration in clv1-11, clv2-1, and crn-1 mutants upon induced expression of the corresponding FP fusion protein. Left panels show whole plants; top insets show higher magnification of the primary shoot; bottom insets show mutant siliques with four carpels and rescued siliques (asterisks) with two carpels. D, clv1-11 carrying the iCLV1-GFP transgene. Inducing iCLV1-GFP expression led to the formation of a single silique with two carpels, whereas older and younger siliques form four carpels. E, clv2-1 carrying iCLV2-GFP. iCLV2-GFP induction restored carpel number to two in four siliques. F, crn-1 carrying iCRN-GFP. iCRN-GFP induction led to the formation of three siliques with only two carpels; all older and younger siliques consisted of four carpels. Bars = 1 cm.

Figure 2.

Analysis of receptor-GFP fusion protein expression. A and B, Transient expression of iCLV1-GFP in leaf epidermis cells of N. benthamiana. Bars = 20 μm. A, Long induction (>12 h) of iCLV1-GFP causes formation of fluorescent aggregates (inset shows close-up). B, At 4 h after induction, CLV1-GFP localizes predominantly to the PM. C, Western-blot analysis of protein extracts from N. benthamiana leaf cells transiently expressing CLV1-GFP, CLV2-GFP, or CRN-GFP. An anti-GFP antibody was used for detection; sizes of protein markers are given in kD. The Ponceau S-stained protein bands of Rubisco are shown as a loading control. wt, Wild type.

Figure 3.

Intracellular localization of CLV1, CLV2, and CRN. Transient expression of FP-tagged receptor chimeras in epidermis cells of N. benthamiana. Confocal sections of epidermis cells either through the middle of a cell (A–A″, C–C″, E–E″, and G–L″) or beneath the outer cell wall (B–B″, D–D″, and F–F″) are shown. A to A″, C to C″, E to E″, J to J″, and K to K″, Receptor-GFP colocalization with FM4-64. B to B″, D to D″, and F to F″, Receptor-GFP colocalization with an mCherry ER-reporter. A to A″, CLV1-GFP colocalizes with the FM4-64 dye at the PM and in a few transport vesicles (arrow). B to B″, Weak CLV1-GFP expression in the ER. C to C″, CLV2-GFP is found next to the PM in the ER and does not colocalize with FM4-64. D to D″, Colocalization of CLV2-GFP with the ER reporter mCherry. E to E″, Like CLV2, CRN-GFP is not found at the PM. F to F″, CRN-GFP colocalization with the ER-mCherry protein. G to G″, Coexpression of CLV1-mCherry with CLV2-GFP does not affect their localization in the cell. H to H″, Coexpression of CLV1-mCherry with CRN-GFP does not affect their localization in the cell. I to I″, Coexpression of CLV2-GFP with CRN-mCherry leads to their relocation to the PM and the formation of transport vesicles (arrow). J to J″, CLV2-GFP colocalizes with FM4-64 at the PM if CRN is coexpressed. K to K″, CRN-GFP colocalizes at the PM with FM4-64 in the presence of CLV2. L to L″, CRN-GFP colocalized with CLV1-mCherry at the PM in the presence of CLV2. Bars = 20 μm.

Figure 4.

CRN localization at the PM requires the TM domain and adjacent sequences. Transient expression of receptor-FP fusions in N. benthamiana. Confocal sections through the middle of an epidermis cell are shown. First to third columns show coexpression of CRN-GFP or derivatives with CLV2-mCherry. Fourth column shows localization of CRN-GFP or derivatives in the absence of CLV2 and PM labeling with FM4-64 (red). A to A″′, CRN and CLV2 localize to the PM. B to B″′, CR(ΔSP-TM)-GFP is found in the cytoplasm and nucleus, and CLV2 is now found in the ER. C to C″′, The point mutation in crn-1 reduces PM localization. D to D″′, The TM of BAK1 is insufficient to replace the TM of CRN. E to E″′, Deleting the EC of CRN abolishes PM localization of CRN and CLV2. F to F″′, Exchanging the CRN kinase domain against the CLV1 kinase domain weakly interferes with PM localization. G to G″′, Deleting the CRN kinase domain does not affect PM localization. H to H″′, Fusion of CLV2 to the CRN kinase domain abolishes PM localization. Insets show close-ups. Scale bars = 20 μm.

Figure 5.

Receptor interaction revealed by E_FRET. A, Principle of E_FRET measurement by acceptor photobleaching. Fluorescence intensities of GFP and mCherry are recorded. E_FRET is calculated as relative increase of GFP fluorescence intensity (%) after photobleaching of the mCherry FRET acceptor. B, E_FRET control measurements. GFP background fluctuations of C1-G, C2-G, and CR-G at the PM and measurement of intramolecular FRET are shown. C, Receptor homomers. D, Capacity of CRN deletion derivatives and domain swaps to interact with CLV2. E, Formation of receptor heteromers. Gray bars show mean values, with se indicated. Asterisks mark E_FRET levels significantly different from GFP fluorescence fluctuation. Red lines at 4% indicate background fluctuation level of GFP. B1, BAK1; C, mCherry; C1, CLV1; C2, CLV2; CR, CRN; G, GFP; Ki, kinase domain; square brackets, coexpression of unlabeled protein; <>, domain exchange.

Figure 6.

Model of CLV receptor complexes. CRN dimer interacts with two CLV2 receptors via the TM domain. Juxtamembrane sequences are required to secure interaction and for localization of the complex at the PM (left). CLV1 forms homodimers (right), which can also bind the tetrameric CLV2/CRN complex (middle). This interaction is mediated by CRN. Binding of CLV3 to the three complexes may trigger different signal transduction cascades.