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Arabidopsis CLV3 Peptide Directly Binds CLV1 Ectodomain

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Science  18 Jan 2008:
Vol. 319, Issue 5861, pp. 294
DOI: 10.1126/science.1150083

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Abstract

CLV1, which encodes a leucine-rich repeat receptor kinase, and CLV3, which encodes a secreted peptide, function in the same genetic pathway to maintain stem cell populations in Arabidopsis shoot apical meristem. Here, we show biochemical evidence, by ligand binding assay and photoaffinity labeling, that the CLV3 peptide directly binds the CLV1 ectodomain with a dissociation constant of 17.5 nM. The CLV1 ectodomain also interacts with the structurally related CLE peptides, with distinct affinities depending on the specific amino acid sequence. Our results provide direct evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance.

Plants continuously produce organs from self-renewing shoot apical meristem (SAM). A feedback signaling loop between stem cells and the underlying organizing center balances stem cell renewal and differentiation (1). CLAVATA1 (CLV1) and CLAVATA3 (CLV3), which are expressed in adjacent regions in SAM and function in the same genetic pathway, are key components of the regulatory network controlling this balance in Arabidopsis. Mutations in the CLV genes cause accumulation of stem cells and enlargement of the SAM. In contrast, overexpression of CLV3 causes loss of stem cells and, in turn, developmental arrest. CLV1 encodes a leucine-rich repeat receptor kinase (LRR-RK) (2), and CLV3 encodes a secreted polypeptide that is converted into a 12–amino acid peptide by proteolytic processing (3, 4). Although these molecules are thought to act as a ligand-receptor pair, biochemical evidence remains lacking.

The ligand-binding characteristics of CLV1 are difficult to assess because it acts as a negative regulator of cell growth and therefore cannot be overexpressed in plant cells in sufficient quantities for ligand-binding assays. Deletion of the cytoplasmic kinase domain improves expression of LRR-RK proteins without affecting ligand-binding affinity of the extracellular domain (5). We therefore overexpressed a CLV1 in which the kinase domain was replaced with HaloTag (Promega, Madison, WI) (CLV1-ΔKD-HT) in tobacco BY-2 cells (Fig. 1, A and B).

Fig. 1.

(A) Schematic structures of CLV1 and its HaloTag fusion protein (CLV1-ΔKD-HT). CLV1 is a 980–amino acid LRR-RK containing a signal peptide (SP), 22 tandem LRRs, a transmembrane domain (TM), and a cytoplasmic kinase domain (KD). (B) Visualization of CLV1-ΔKD-HT in wild-type (WT) BY-2 cells and in transgenic BY-2 cells overexpressing CLV1-ΔKD-HT (OX) by specific incorporation of the fluorescent dye HaloTag TMR (Promega, Madison, WI). (C) [3H]CLV3 binding assay of microsomal fractions derived from WT cells and transgenic cells overexpressing CLV1-ΔKD-HT (OX) in the absence or presence of excess unlabeled CLV3 as a competitor (mean ± SD, n = 3). (D) Photoaffinity labeling of CLV1-ΔKD-HT by [125I]ASA-CLV3 in the absence or presence of excess unlabeled CLV3. (E) Scatchard plot of the [3H]CLV3 binding to CLV1-ΔKD-HT. (F) Competitive replacement of [3H]CLV3 (32 nM) binding to CLV1-ΔKD-HT by CLV3, [Ala6]CLV3, CLE2, CLE9, CLE44, and [1-8]CLV3 peptides (mean ± SD, n = 3). Aminoacid sequence of each peptide is given in fig. S1D.

To test whether CLV1-ΔKD-HT specifically interacts with CLV3, we incubated membrane fractions derived from wild-type and transgenic BY-2 cells with [3H]CLV3. Membrane fractions derived from transgenic BY-2 cells overexpressing CLV1-ΔKD-HT had significantly higher specific [3H]CLV3 binding activity than fractions derived from wild-type BY-2 cells (Fig. 1C). The specific [3H]CLV3 binding to membranes was rapid, saturable, and reversible (fig. S1A). Incubation of the membrane fractions derived from transgenic BY-2 cells with a photoactivable [125I]ASA-CLV3 (fig. S1, B and C) followed by cross-linking by ultraviolet (UV) irradiation resulted in specific labeling of the 130-kD band that corresponds to CLV1-ΔKD-HT (Fig. 1D). These results indicate that CLV3 directly binds the CLV1 ectodomain. A broad 90-kD labeled band probably represents C-terminal–truncated CLV1-ΔKD-HT in which the HT domain was lost. Truncated receptor kinases are often detected at considerable amounts in membrane fractions (5). Scatchard analysis revealed that CLV1-ΔKD-HT interacts with [3H]CLV3 with a dissociation constant (Kd) of 17.5 nM (Fig. 1E). The interaction was only partially inhibited by excess [Ala6]CLV3, a less-active CLV3 analog corresponding to clv3-1 and clv3-5 alleles (3), indicating that the observed binding is specific (Fig. 1F).

Several CLE peptides, structurally related to CLV3, play pleiotropic roles in plant growth and development (6). Of the total of 31 CLE genes in Arabidopsis, 16 can be grouped into three phylogenetic clades: CLE1 to CLE7, CLE9 to CLE13, and CLE41 to CLE44 (7). Overexpression of the first group of CLE genes caused phenotypes similar to, but milder than, those caused by CLV3 overexpression (7). Overexpression of the second CLE group induced termination of SAM with high mortality, similar to the SAM of CLV3-overexpressing plants. Overexpression of the third group of CLE genes did not affect the SAM. [3H]CLV3 binding to CLV1-ΔKD-HT was inhibited similarly by either CLE9 or CLV3 (Fig. 1F). CLE2 also inhibited [3H]CLV3 binding to a considerable degree, whereas CLE44 only partially inhibited binding (Fig. 1F). Thus, CLV1 interacts both with CLV3 and also CLE peptides, each with unique affinities. The relative binding affinities of the CLV and CLE peptides for the ectodomain of CLV3 and related receptors may, together with the location and timing of expression of these components, form the basis for the physiological importance of these signals in the SAM and other tissues.

Our results provide direct evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance. Our results also indicate that the CLV1 ectodomain alone is sufficient for CLV3 binding. This suggests that arrays of ectodomains derived from LRR-RKs phylogenetically related to CLV1 may be able to bind CLE peptides in a sequence-specific manner in vitro. Such a biochemical approach could help clarify highly redundant and pleiotropic CLE signaling by identifying the individual ligand-receptor interactions.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5861/294/DC1

Materials and Methods

Fig. S1

References

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