Clv Mutants Research Articles

Transcriptome Analysis Identifies Downstream Genes of CLAVATA3 in Tomato

Fruit locule number is an important agronomic trait that affects fruit appearance, quality, and yield. CLAVATA3 (SlCLV3) is a candidate gene of the fasciated (fas) locus that plays a role in controlling the number of flower organs and fruit locules in tomato. The SlCLV3 encoding signal peptide mainly acts by inhibiting the expression of WUSCHEL (WUS) but there is little research about how the receptor transmits the CLV3 signal to WUS and inhibits its expression. The CRISPR/Cas9 method was employed to edit the first exon of tomato SlCLV3 in this study, leading to the functional deletion of SlCLV3. As a result, flowers with a high number of organs and fruits with a high number of locules were produced. We screened six candidate genes using the transcriptome of clv3 mutants, analyzed expression variations in these genes between the cultivated allele and wild-type allele of fas, and showed that only SlLET6 and SlGIF1 (GRF1-interacting factor 1) were influenced by the fas locus. SlLET6 overexpression resulted in an increase in flower carpels and fruit locules. These results suggest that SlLET6 may be the downstream gene of SlCLV3 regulating the number of carpels and fruit locules in tomato.

Knox homologs shoot meristemless (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during shoot meristem development in Arabidopsis thaliana.

InArabidopsis,the genes SHOOT MERISTEMLESS(STM) andCLAVATA3 (CLV3) antagonistically regulate shoot meristem development.STMis essential for both development and maintenance of the meristem, asstmmutants fail to develop a shoot meristem.CLV3, on the other hand, negatively regulates meristem proliferation, andclv3mutants possess an enlarged shoot meristem. Genetic interaction studies revealed thatstmandclv3 dominantly suppress each other's phenotypes. STM works in conjunction withits closely related homologue KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6)to promote meristem development and organ separation, asstmknat6double mutants fail to form shoot meristem and produce a fused cotyledon. In this study, we show thatclv3fails topromoteshoot meristem formation instm-1background if we also remove KNAT6.stm-1knat6 clv3triple mutants result in shoot meristem termination and produce fused cotyledons similar tostmknat6double mutant. Notably, the stm-1 knat6 and stm-1 knat6 clv3 alleles lack tissue in the presumed region of SAM that is a novel phenotype reported in Arabidopsis mutants.stm-1knat6 clv3also showed reduced inflorescence size as compared toclv3single orstmclv3double mutants. In contrast to previously published data, these data suggest thatSTM and KNAT6 are redundantly required for the vegetative SAM, but insufficient for the inflorescence meristem.

A group of receptor kinases are essential for CLAVATA signalling to maintain stem cell homeostasis.

Continuous organ initiation and outgrowth in plants relies on the proliferation and differentiation of stem cells maintained by the CLAVATA (CLV)-WUSCHEL (WUS) negative-feedback loop1-3. Leucine-rich repeat receptor-like protein kinases (LRR-RLKs), including CLV1, BARELY ANY MERISTEMS and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), a receptor-like protein CLV2 and a pseudokinase CORYNE (CRN) are involved in the perception of the CLV3 signal to repress WUS expression4-10. WUS, a homeodomain transcription factor, in turn directly activates CLV3 expression and promotes stem cell activity in the shoot apical meristem11,12. However, the signalling mechanism immediately following the perception of CLV3 by its receptors is poorly understood. Here, we show that a group of LRR-RLKs, designated as CLAVATA3 INSENSITIVE RECEPTOR KINASES (CIKs), have essential roles in regulating CLV3-mediated stem cell homeostasis. The cik1 2 3 4 quadruple mutant exhibits a significantly enlarged SAM, resembling clv mutants. Genetic analyses and biochemical assays demonstrated that CIKs function as co-receptors of CLV1, CLV2/CRN and RPK2 to mediate CLV3 signalling through phosphorylation. Our findings not only widen the understanding of the underlying mechanism of CLV3 signal transduction in regulating stem cell fate but also reveal a novel group of RLKs that function as co-receptors to possibly mediate multiple extrinsic and intrinsic signals during plant growth and development.

Dynamic expression reveals a two-step patterning of WUS and CLV3 during axillary shoot meristem formation in Arabidopsis

Seed plants have a remarkable capability to produce axillary meristems (AM) in the leaf axils, however, the dynamic establishment of a stem cell niche in AM is largely uncharacterized. We comprehensively examined the dynamic patterning of WUSCHEL (WUS) and CLAVATA3 (CLV3), the two key marker genes defining the shoot stem cell niches, during AM formation in Arabidopsis, and we found that a two-step patterning of WUS and CLV3 occurred during AM stem cell niche establishment. Our further work on the wus and clv3 mutants implicates that such two-step patterning is likely critical for the maintenance of AM progenitor cells and the specification of AM stem cell niche. These data provide a cytological frame for how a stem cell niche is established during AM formation.

A Bistable Switch Mechanism for Stem Cell Domain Nucleation in the Shoot Apical Meristem.

In plants, the stem cells residing in shoot apical meristems (SAM) give rise to above-ground tissues (Aichinger et al., 2012). Hence, the maintenance of stem cell niches is of central importance to a plant's continued growth and development (Fletcher and Meyerowitz, 2000; Gordon et al., 2009). For the flowering plant Arabidopsis thaliana, the genetic determinants of stem cell growth, division, and localization have been identified, and negative feedback between a homeodomain transcription factor, WUSCHEL (WUS), and a receptor kinase, CLAVATA (CLV), is known to play a crucial role in controlling the reservoir of stem cells in the central domain of a SAM. The morphology of plant stems and floral organs is controlled in large part by the size and stability of SAMs, which is controlled, in turn, by spatiotemporal patterns of WUS and CLV expression in meristems. For example, loss of restrictive signals in clv mutants of Arabidopsis leads to enlargement of shoot and floral meristems, resulting in extra floral organs and club-shaped siliques (Jonsson et al., 2005). The size, localization and stability of stem cell domains should be determined, in principle, by the interactions of WUS and CLV proteins, especially by their propensities to diffuse through the domain and by the rates of the molecular reactions that control their activities. Within this paradigm, reaction-diffusion (RD) models of WUS-CLV interactions have been popular mathematical models of SAM dynamics (Jonsson et al., 2005; Hohm et al., 2010; Fujita et al., 2011). In RD models, the spontaneous generation of inhomogeneous distributions of WUS and CLV in SAM domains is usually attributed to mechanisms based on a “Turing” instability (Turing, 1952; Segel and Jackson, 1972). The generic RD equations for spatiotemporal changes in the concentrations, u(x,t) and v(x,t), of two interacting proteins are ∂u∂t=f(u,v)+Du∂2u∂x2, ∂v∂t=g(u,v)+Dv∂2v∂x2, where f(u,v) and g(u,v) are nonlinear functions describing their local chemical interactions. A unique, uniform, steady-state solution, u(x,t) = u0 = constant and v(x,t) = v0, of these equations can become unstable with respect to small, non-uniform perturbations, u(x,t) = u0 + eλt·δu·cos(qx) and v(x,t) = v0 + eλt·δv·cos(qx), δu > Du, generating standing waves of wavelength l ≈ 2π/qcrit in the simulations of the RD system (Gierer and Meinhardt, 1972; Murray, 2003). At present, the diffusive lengths of CLV and WUS in SAMs have not been determined, and there is no evidence to suggest that the Turing condition (diffusivity of CLV >> diffusivity of WUS) is satisfied in the central zone of a SAM.

Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute toRalstonia solanacearum pathogenicity through a miR169-dependent pathway.

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R.solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module.

Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases.

The CLAVATA3 (CLV3)-CLAVATA1 (CLV1) ligand-receptor kinase pair negatively regulates shoot stem cell proliferation in plants. clv1 null mutants are weaker in phenotype than clv3 mutants, but the clv1 null phenotype is enhanced by mutations in the related receptor kinases BARELY ANY MERISTEM 1, 2 and 3 (BAM1, 2 and 3). The basis of this genetic redundancy is unknown. Here, we demonstrate that the apparent redundancy in the CLV1 clade is in fact due to the transcriptional repression of BAM genes by CLV1 signaling. CLV1 signaling in the rib meristem (RM) of the shoot apical meristem is necessary and sufficient for stem cell regulation. CLV3-CLV1 signaling in the RM represses BAM expression in wild-type Arabidopsis plants. In clv1 mutants, ectopic BAM expression in the RM partially complements the loss of CLV1. BAM regulation by CLV1 is distinct from CLV1 regulation of WUSCHEL, a proposed CLV1 target gene. In addition, quadruple receptor mutants are stronger in phenotype than clv3, pointing to the existence of additional CLV1/BAM ligands. These data provide an explanation for the genetic redundancy seen in the CLV1 clade and reveal a novel feedback operating in the control of plant stem cells.

CLE Signaling Systems During Plant Development and Nematode Infection

Plants contain numerous CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR) (CLE) genes encoding small secreted peptide hormones that function in a variety of developmental and physiological processes. The first known Arabidopsis CLE gene was originally discovered through the analysis of clv3 mutants, which exhibit fasciated stems and an increased number of floral organs. In total, 32 CLE genes have been identified in Arabidopsis. Amongst these are CLV3 and CLE40, which repress the expression of homeobox-containing genes WUSCHEL (WUS) and WUSCHEL-related homeobox 5 (WOX5) to control shoot apical meristem (SAM) and root columella initial cell activity, respectively. Interestingly, the CLE signaling pathway appears to be conserved amongst plants. In this review, we discuss the latest research uncovering the diverse functions and activities of CLE peptides in plants; especially during shoot, root and vascular development. In addition, we discuss the important role of CLE peptides during infection by phytoparasitic nematodes. Understanding the molecular properties of CLE peptides and their modes of action will provide further insight into plant cell-cell communication, which could also be applied to manipulate plant-nematode interactions.

Reaction-diffusion pattern in shoot apical meristem of plants.

A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.

Identification of novel markers for stem-cell niche of Arabidopsis shoot apex

In Arabidopsis, shoot stem-cells give rise to all the aerial parts of plant body and are localized at the tip of the shoot apex. Feedback signaling mediated by CLAVATA3 (CLV3) and WUSCHEL (WUS), specify and maintain stem-cells in the shoot apical meristem (SAM). Although CLV3 and WUS have been used extensively as stem-cell markers, their expression levels are mutually influenced by each other. Thus, CLV3 and WUS are not ideal markers for analyzing genes that function in this feedback regulation. Here, we present the expression profiles of genes that can be used as markers for cells of the stem-cell niche based on the following criteria: (1) they are expressed in cells of the central zone (CZ) or the rib-meristem (RM)/organizing center (OC), (2) their expression levels are not influenced by alterations in CLV3 or WUS levels, and (3) their expression is initiated during embryonic stages and is maintained in post-embryonic development. By RNA in- situ hybridization, we also found that the expression of these genes follow two distinct patterns: (1) CLV3-like and (2) WUS-like. Moreover, the expression domains of these genes expand in SAMs of clv3 mutants, which accumulate higher numbers of stem-cells. A comprehensive expression patterns provided here suggests that they are ideal markers for stem-cell niche of Arabidopsis shoot apex.

BAM receptors regulate stem cell specification and organ development through complex interactions with CLAVATA signaling.

The CLAVATA1 (CLV1) receptor kinase regulates stem cell specification at shoot and flower meristems of Arabidopsis. Most clv1 alleles are dominant negative, and clv1 null alleles are weak in phenotype, suggesting additional receptors functioning in parallel. We have identified two such parallel receptors, BAM1 and BAM2. We show that the weak nature of the phenotype of clv1 null alleles is dependent on BAM activity, with bam clv mutants exhibiting severe defects in stem cell specification. Furthermore, BAM activity in the meristem depends on CLV2, which is required in part for CLV1 function. In addition, clv1 mutants enhance many of the Bam(-) organ phenotypes, indicating that, contrary to current understanding, CLV1 function is not specific to the meristem. CLV3 encodes a small, secreted peptide that acts as the ligand for CLV1. Mutations in clv3 lead to increased stem cell accumulation. Surprisingly, bam1 and bam2 mutants suppress the phenotype of clv3 mutants. We speculate that in addition to redundant function in the meristem center, BAM1 and BAM2 act to sequester CLV3-like ligands in the meristem flanks.

The Receptor Kinase CORYNE ofArabidopsisTransmits the Stem Cell–Limiting Signal CLAVATA3 Independently of CLAVATA1

Stem cells in shoot and floral meristems of Arabidopsis thaliana secrete the signaling peptide CLAVATA3 (CLV3) that restricts stem cell proliferation and promotes differentiation. The CLV3 signaling pathway is proposed to comprise the receptor kinase CLV1 and the receptor-like protein CLV2. We show here that the novel receptor kinase CORYNE (CRN) and CLV2 act together, and in parallel with CLV1, to perceive the CLV3 signal. Mutations in CRN cause stem cell proliferation, similar to clv1, clv2, and clv3 mutants. CRN has additional functions during plant development, including floral organ development, that are shared with CLV2. The CRN protein lacks a distinct extracellular domain, and we propose that CRN and CLV2 interact via their transmembrane domains to establish a functional receptor.

POL and related phosphatases are dosage-sensitive regulators of meristem and organ development in Arabidopsis

CLAVATA1 ( CLV1) regulates stem cell accumulation at Arabidopsis shoot and flower meristems. CLV1 encodes a receptor-like kinase, but very little is known about downstream signaling components of receptor-kinase signaling in plants. poltergeist ( pol) mutants suppress the accumulation of stem cells that occur in clv mutants, and POL has been hypothesized to modulate CLV1 signaling. The POL gene, which encodes a functional protein phosphatase type 2C, is a member of a six-gene family in Arabidopsis. We have isolated loss-of-function alleles for each of the five POL-like genes ( PLL1– PLL5). All gene family members, with the exception of PLL3, are expressed broadly within the plant, albeit at differing levels. We show that PLL1 regulates meristem development in parallel with POL. We observe a strong dosage sensitivity at the meristem for POL and PLL1 function in both loss- and gain-of-function analyses, suggesting that these proteins are rate-limiting modulators of stem cell specification. PLL genes also function outside of the meristem: POL and PLL1 regulate pedicel length in interaction with ERECTA, while PLL4 and PLL5 regulate leaf development. We observed no developmental role for either PLL2 or PLL3 based on single and double mutant analysis.

The 14–Amino Acid CLV3, CLE19, and CLE40 Peptides Trigger Consumption of the Root Meristem inArabidopsisthrough aCLAVATA2-Dependent Pathway

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.

Photoperiod regulates flower meristem development in Arabidopsis thaliana.

Photoperiod has been known to regulate flowering time in many plant species. In Arabidopsis, genes in the long day (LD) pathway detect photoperiod and promote flowering under LD. It was previously reported that clavata2 (clv2) mutants grown under short day (SD) conditions showed suppression of the flower meristem defects, namely the accumulation of stem cells and the resulting production of extra floral organs. Detailed analysis of this phenomenon presented here demonstrates that the suppression is a true photoperiodic response mediated by the inactivation of the LD pathway under SD. Inactivation of the LD pathway was sufficient to suppress the clv2 defects under LD, and activation of the LD pathway under SD conditions restored clv2 phenotypes. These results reveal a novel role of photoperiod in flower meristem development in Arabidopsis. Flower meristem defects of clv1 and clv3 mutants are also suppressed under SD, and 35S:CO enhanced the defects of clv3, indicating that the LD pathway works independently from the CLV genes. A model is proposed to explain the interactions between photoperiod and the CLV genes.

HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis.

We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM) development in Arabidopsis thaliana. Mutants have fused sepals and reduced organ numbers in all four whorls, especially in the 2nd (petal) and 3rd (stamen) whorls. han meristems can become flatter or smaller than in the wild type. HAN encodes a GATA-3-like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the meristem and its newly initiated organ primordia and at the boundaries between different floral whorls. It is also expressed in vascular tissues, developing ovules and stamens, and in the embryo. han interacts strongly with clavata (clv) mutations (clv1, clv2, and clv3), resulting in highly fasciated SAMs, and we find that WUS expression is altered in han mutants from early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is normally required for establishing organ boundaries in shoots and flowers and for controlling the number and position of WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of meristem activity, suggesting that HAN is involved in controlling cell proliferation and differentiation.

CLV1 and CLV3: negative regulators of SAM stem cell accumulation

Unlike most animals, higher plants continue to generate new organs post-embryonically from stem cells located within shoot, root and floral meristems. These stem cells are not permanent initials, but instead are positionally determined. Therefore, meristem maintenance depends upon a balance between stem cell division within the meristem center and differentiation toward the periphery. In the shoot apical meristem (SAM) of Arabidopsis thaliana, this balance is partly regulated by the signaling molecules CLAVATA1 (CLV1), a receptor protein kinase, and CLV3, a putative extracellular protein. Loss-of-function CLV1 and CLV3 mutants accumulate stem cells, resulting in meristem enlargement. Although genetic data suggests CLV1 and CLV3 act in the same signaling pathway, expression studies reveal that the two genes are expressed in different cell layers within the SAM. One model for their activity is that secreted CLV3 binds to the plasma membrane-associated CLV1 complex to limit stem cell accumulation either by decreasing stem cell division or increasing differentiation. Two recent papers1,2 support this role for CLV1 and CLV3 as a receptor and ligand pair negatively regulating SAM stem cell proliferation.

POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci.

Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.

The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein.

The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) genes are required to maintain the balance between cell proliferation and organ formation at the Arabidopsis shoot and flower meristems. CLV1 encodes a receptor-like protein kinase. We have found that CLV1 is present in two protein complexes in vivo. One is approximately 185 kD, and the other is approximately 450 kD. In each complex, CLV1 is part of a disulfide-linked multimer of approximately 185 kD. The 450-kD complex contains the protein phosphatase KAPP, which is a negative regulator of CLV1 signaling, and a Rho GTPase-related protein. In clv1 and clv3 mutants, CLV1 is found primarily in the 185-kD complex. We propose that CLV1 is present as an inactive disulfide-linked heterodimer and that CLV3 functions to promote the assembly of the active 450-kD complex, which then relays signal transduction through a Rho GTPase.