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Signaling of Cell Fate Decisions by CLAVATA3 in Arabidopsis Shoot Meristems

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Science  19 Mar 1999:
Vol. 283, Issue 5409, pp. 1911-1914
DOI: 10.1126/science.283.5409.1911

Abstract

In higher plants, organogenesis occurs continuously from self-renewing apical meristems. Arabidopsis thaliana plants with loss-of-function mutations in the CLAVATA (CLV1,2, and 3) genes have enlarged meristems and generate extra floral organs. Genetic analysis indicates that CLV1, which encodes a receptor kinase, acts with CLV3 to control the balance between meristem cell proliferation and differentiation.CLV3 encodes a small, predicted extracellular protein.CLV3 acts nonautonomously in meristems and is expressed at the meristem surface overlying the CLV1 domain. These proteins may act as a ligand-receptor pair in a signal transduction pathway, coordinating growth between adjacent meristematic regions.

The shoot apical meristem (SAM) is the source of all the aerial parts of the plant. Cells at the SAM summit serve as stem cells that divide slowly to continuously displace daughter cells to the surrounding peripheral region, where they are incorporated into differentiating leaf or flower primordia (1). A balance between creation of new meristematic cells by division and departure of cells from the meristem by differentiation is required to maintain a functional SAM. The CLV3 andCLV1 genes play critical roles in maintaining this balance, because loss-of-function mutations in either gene cause progressive SAM enlargement and floral meristem overgrowth (2–6). The phenotypes of representative wild-type and clv3 mutant plants (7) are shown inFig. 1. CLV1 encodes a leucine-rich repeat (LRR) transmembrane receptor serine-threonine kinase (8). LRRs are a common motif of protein-binding domains (9), suggesting that CLV1 may bind an extracellular protein or peptide ligand. clv1 clv3 double-mutant analysis shows that the genes are mutually epistatic (5), suggesting that the two gene products act in the same pathway. Doubly heterozygous (clv1/+; clv3/+) plants have a clvmutant phenotype (5), implying that the gene products have a quantitative interdependence, as if they acted together in a complex or in closely associated steps of a pathway. Thus, it appears that CLV3 protein acts either in the intracellular pathway leading from CLV1 activation to cellular activity, or in the production of, or as, the CLV1 ligand.

Figure 1

clv3 shoot and flower phenotypes. (A) Wild-type inflorescence meristem. (B)clv3-2 inflorescence meristems undergo fasciation, growing as a ring or line rather than a point. (C) clv3-2mutant flowers contain extra organs of all types, particularly stamens and carpels. Bars, 1 mm.

To distinguish which hypothesis is correct, we cloned theCLV3 gene using two tagged alleles, clv3-3 andclv3-7. The clv3-3 allele is caused by transferred DNA (T-DNA) integration (10) and confers a weakclv3 phenotype, whereas the clv3-7 allele is caused by integration of the maize transposable element En-1(11) and confers a strong phenotype. DNA sequence analysis of genomic clones flanking both insertion sites revealed the presence of three small, overlapping open reading frames (12). Comparison of the genomic clones with cDNA RACE products revealed a gene consisting of three exons and two small introns (Fig. 2).

Figure 2

CLV3 genomic region and peptide sequence. (A) The CLV3 genomic region. The translation start site is denoted by the arrow and the exons by boxes. The relative positions of the clv3 mutations are shown. Restriction sites: M, Mfe I; D, Dra I. The genomic DNA sequence is available through GenBank under accession number AF126009. (B) The CLV3 predicted amino acid sequence. Intron positions are indicated by arrows, the predicted signal sequence is underlined, and the amino acid altered in the clv3-1 andclv3-5 alleles is in bold type. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and W, Trp.

The nucleotide and deduced amino acid sequence of CLV3 is shown in Fig. 2. The clv3-3 T-DNA insertion site lies 175 base pairs (bp) downstream of the polyadenylate [poly(A)] addition site (Fig. 2A), potentially disrupting an enhancer element. TheEn-1 element in clv3-7 inserted in the second intron, close to the intron–exon 3 boundary. The independently derivedclv3-1 and clv3-5 ethylmethane sulfonate (EMS)–induced alleles have intermediate phenotypes and contain a G to A transition at position +266 relative to the translation initiation site (Fig. 2A) (13). The strong clv3-2 γ ray–induced allele and the clv3-4 x-ray–induced allele both contain breakpoints occurring between the Mfe I and Dra I restriction sites flanking the third exon (14), whereas the strong clv3-6 EMS-induced allele alters the third exon splice acceptor site from AG to AA (Fig. 2A). Reversion of unstableclv3-7 mutants to the wild-type phenotype was accompanied by loss of the En-1 element, verifying that the gene cloned corresponds to CLV3.

The CLV3 gene encodes a protein of 96 amino acids (Fig. 2B) that shows no appreciable similarity to other sequences or sequence motifs of known functional domains. An 18–amino acid NH2-terminal hydrophobic region presumably acts as a signal peptide to direct the protein into the secretory pathway (15). No signals that would cause retention of the protein along the secretory pathway (16) were detected, indicating that the CLV3 protein may be extracellular and could therefore act as the ligand for the CLV1 receptor kinase. Alternatively, CLV3 could act to produce the ligand, function as a coligand with another small molecule, or assist CLV1 in ligand binding.

We investigated the cell autonomy of CLV3 function using periclinal chimeras derived from the unstable En-1–inducedclv3-7 mutants. Secondary shoots on clv3-7 plants that displayed a wild-type phenotype were isolated and allowed to self-pollinate. The progeny of these somatic revertants would segregate a wild-type (revertant) clv3 allele if the reversion occurred in the L2 meristem cell layer that gives rise to the gametes (17), resulting in 75% wild-type progeny plants. Reversions occurring in the L1 or L3 layer would not be transmitted to the next generation, although secondary germinal reversion events (18) contribute to the appearance of up to 30% wild-type phenotypes among the otherwise clv3 mutant progeny of L1 or L3 chimeras. Of 24 analyzed sectors, 11 segregated 0 to 30% wild-type plants, indicating that CLV3 function was restored somatically in the L1 or L3 cell layer of the revertants, but not in the L2 (19). CLV3 activity in one cell layer is therefore sufficient to control proliferation and differentiation across the entire meristem, possibly by communicating information across cell layers in a non–cell-autonomous manner.

The expression pattern of the CLV3 gene during wild-type development was analyzed by RNA in situ hybridization (20).CLV3 mRNA expression is first detected in heart stage embryos, in a patch of cells between the developing cotyledons (Fig. 3A) predicted to give rise to the SAM (21). In vegetative and inflorescence meristems,CLV3 mRNA accumulates in a small zone of cells at the meristem apex (Fig. 3, B and C). It is expressed in the L1 and L2 cell layers, and in a few underlying L3 cells. CLV3signal is not detected on the shoot meristem flanks in cells of presumptive leaf and flower anlagen, but reappears in floral meristem apices at stage 2 (22). At stages 3 to 4, CLV3mRNA expression continues in the floral meristem central region (Fig. 3D) and persists there through stage 6, when the sepal primordia completely enclose the bud (19). Throughout development the RNA signal is always restricted to the most central, nondifferentiating meristem cells, the putative stem cells. The CLV3 andCLV1 (6, 23) temporal expression patterns are very similar, but there are important spatial differences.CLV1 mRNA, unlike CLV3 mRNA, is not detected in the L1 cell layer of shoot or floral meristems, and CLV1 is expressed more deeply in the L3 region than CLV3 (compareFig. 3E with Fig. 3D).

Figure 3

CLV3 and CLV1 mRNA expression patterns in wild-type (Landsberg erecta) tissues. (A and B) In situ hybridization withCLV3 radiolabeled antisense probe. Each longitudinal section was photographed with brightfield–darkfield double exposure, using a red filter for the darkfield exposure. (C andD) In situ hybridization with CLV3digoxygenin-labeled antisense probe. (E) In situ hybridization with CLV1 digoxygenin-labeled antisense probe. (A) Heart stage embryo. (B) Ten-day-old seedling. (C) Inflorescence meristem. (D) Stage 3 flower. (E) Stage 3 flower. Bars, 50 μm. Control experiments with the sense strand of CLV3 orCLV1 detected no signal in tissue.

It has been postulated that the SAM enlargement observed inclv1, clv2, and clv3 mutants (3, 5, 6) is due to central-zone expansion. Because CLV3 mRNA coincides with the central zone, we used in situ hybridization to test this hypothesis. Inflorescence meristems of homozygous clv1-4 plants (Fig. 4A) were hybridized to a CLV3probe, which showed that the CLV3 expression domain is markedly expanded relative to the wild type (Fig. 4B). CLV3signal is detected throughout the enlarged shoot meristem, except for a narrow strip of cells closest to newly initiating floral primordia. A broadened CLV3 expression domain is also observed in youngclv1-4 floral meristems (Fig. 4C). Although CLV3mRNA is undetectable in wild-type flowers after carpel initiation, it is present in more mature clv1-4 flowers in a domain between the developing carpels (Fig. 4D). This domain gives rise to a nested fifth whorl ovary, containing cells that continue to proliferate and express CLV3 even after the formation of ovules in the fourth whorl (Fig. 4E). Comparable enlargement of the CLV3expression domain is observed in clv2-1 andclv3-2 mutants, indicating that CLV1,CLV2, and CLV3 all act to limit the number of cells in the CLV3 expression domain. In situ hybridization also shows that the CLV1 domain is enlarged inclv1, clv2, and clv3 meristems proportionately to the enlargement of the CLV3-expressing domain (6, 24).

Figure 4

CLV3 mRNA expression patterns inclv1 mutant tissues. (A) clv1-4inflorescence meristem and flowers. The line denotes the approximate orientation of the section taken in (B). (B to E) In situ hybridization with CLV3 radiolabeled antisense probe. Each section was photographed with brightfield-darkfield double exposure, using a red filter for the darkfield exposure. (B) Abnormally enlarged clv1 inflorescence meristem. CLV3 is expressed throughout the enlarged meristem except at the margins (arrows). (C) Stage 5 clv1 flower. (D) Stage 8clv1 flower. (E) Gynoecium of mature clv1 flower. Abbreviations: st, stamen; ca, carpel; 5W, fifth whorl. Bars, 50 μm.

Loss of CLV1, CLV2, or CLV3activity causes accumulation of undifferentiated cells in the shoot apex, indicating that the CLV genes together promote the timely transition of stem cells into differentiation pathways, or repress stem cell division, or both. Loss of CLV1 orCLV3 activity also results in enlargement of the underlyingCLV1 domain. One hypothesis that fits these observations is that CLV3 encodes a protein secreted from superficial cell layers in the central region, which acts as a ligand to activate theCLV1 receptor kinase in underlying cells. The normal action of CLV3 would be to repress enlargement of theCLV1 domain. Because loss of CLV1 orCLV3 activity results in enlargement of the CLV3domain as well as the CLV1 domain, one must infer that in addition to the size-repressing CLV3/CLV1 pathway there is a size-enhancing, CLV-independent positive pathway from the underlying cells to the overlying ones that coordinates the size of the CLV3 domain to match that of the CLV1 domain. The net effect of the two pathways is to keep the relative sizes of theCLV3 and CLV1 domains fixed, allowing for meristem maintenance throughout the life of the plant. Disruption of the negative pathway, by mutating either CLV3 orCLV1, results in enlargement of the CLV1 domain, which in turn causes enlargement of the CLV3 domain and progressive SAM enlargement.

CLV1 is a member of a plant-specific family of receptor protein kinases (25) that span the plasma membrane and allow cells to recognize and respond to their extracellular environment. CLV3 appears to be a ligand, or a molecule involved in ligand synthesis or binding, that is produced in one SAM region and acts on a receptor in another region, allowing for coordinated growth between them. The cloning of CLV3 thus allows a view of meristems as collections of intercommunicating cells, each synthesizing and secreting its own set of protein ligands and responding to its neighbors through action of its own complement of transmembrane receptor kinases. Such kinases are also common components of animal signal transduction pathways, although most developmentally important receptor protein kinases in animals are tyrosine kinases. Thus, plants and animals seem to have converged on independent but parallel mechanisms for sending similar sorts of signals, which can have profound effects on the development of the organisms.

  • * Present address: University of California Berkeley, U.S. Department of Agriculture–Agricultural Research Service Plant Gene Expression Center, 800 Buchanan Street, Albany, CA 94710, USA.

  • To whom correspondence should be addressed at Division of Biology 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. E-mail: meyerow{at}cco.caltech.edu

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