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CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development - PubMed

CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development

Anne Diévart et al. Plant Cell. 2003 May.

Abstract

The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

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Figures

Figure 1.
Figure 1.

Alleles of clv1. (A) Scheme of known alleles of clv1 and locations of the DsE insertion (clv1-11) and the T-DNA insertions (clv1-12 and clv1-13) of three novel null alleles of clv1. The amino acid sequences surrounding the integration sites are indicated. The LRRs (black boxes), transmembrane domain (gray box), and kinase domains (white box) are represented. (B) Ethidium bromide agarose gels from the RT-PCR performed on Col (lane 1), clv1-12 (lane 2), clv1-13 (lane 3), Ler (lane 4), and clv1-11 (lane 5) samples. mRNA isolated from inflorescences was analyzed by RT-PCR to monitor the transcript accumulation of CLV1. Each PCR amplification of the cDNA was generated in parallel with specific primers for CLV1 (top gel) and control (βATPase; bottom gel). RT-PCR products from Col and Ler plants were examined as controls. (C) For each allele, the phenotypic severity, the amino acid mutated, and the domain affected are indicated. (D) Relative size of the fifth whorl compared with the number of carpels in clv1-1, clv1-6, clv1-11, clv1-4, and Ler. Ten flowers from at least 20 primary inflorescences of 30-day-old plants were scored for the number of carpels (means ±

se

). The size of the silique represents the length between the attachment site for the sepals, petals, and stamens and the top of the gynoecium. The same references were used for the measurement of fifth-whorl length. The measured size of the fifth whorl is represented as a percentage of total silique length. The vertical and horizontal bars represent standard errors for the relative fifth-whorl size and the number of carpels, respectively. Note that there is no fifth whorl in Ler and that the number of carpels does not vary from two, so the standard error is 0. (E) Comparison of siliques from Ler plants. The shape of the siliques from Ler, clv1-11, clv1-1, and clv1-4 varies with the severity of the Clv1− phenotype. Note the club shape of the intermediate clv1-1 and strong clv1-4 alleles compared with the weak clv1-11 silique shape. At right is shown a dry clv1-1 silique revealing the fifth whorl of organ growing inside the gynoecium and representing ∼70% of the size of the silique. Bar = 2 mm.

Figure 2.
Figure 2.

Schemes (Not to Scale) of the CLV1 cDNA (ER:CLV1), ER:CB1, ER:CB2, ER:CB2m, ER:CB3, and ER:CB4 Chimeric Receptor Kinase, and Control Transgenes. The CLV1 and BRI1 protein structures are shown as white and black boxes, respectively. The junction between CLV1 and BRI1 in each chimeric construct did not alter the amino acid sequence of CLV1 or BRI1. The mutation introduced in the BRI1 kinase domain to produce the ER:CB2m transgene (asterisk) is located in domain IX and is the same as the bri1-101 mutation, which results in a loss of kinase activity (Friedrichsen et al., 2000). These constructs are driven by the ERECTA promoter (black line; 1678 bp of sequence upstream of ER), contain the E9 terminator (hatched box), and are cloned into pCB302 (Xiang et al., 1999) (see Methods). CT, C-terminal tail; ER prom., ER promoter; JM, juxtamembrane domain; KD, kinase domain; E9 term., E9 terminator; TM, transmembrane domain.

Figure 3.
Figure 3.

CLV1/BRI1 (CB) Chimeric Receptors Rescue the clv1-1 Mutant Phenotype. (A) Relative size of the fifth whorl compared with the number of carpels in Ler, clv1-1, and clv1-1 transformed with the ER:CLV1, ER:CB1, ER:CB2, ER:CB2m, ER:CB3, ER:CB4, empty vector, and control transgenes. The number of independent transgenic lines and the number of T3 plants analyzed are shown in the table at bottom. Between 10 and 20 fully expanded siliques of 30-day-old plants were counted for the number of carpels and dissected for the size of the fifth whorl before desiccation. The size of the fifth whorl growing inside the gynoecium was measured and compared with the size of the silique to give its relative size (% ±

se

) (cf. Figure 1D). The vertical and horizontal bars represent standard errors for the fifth whorl size and the number of carpels, respectively. (B) Scanning electron micrographs of the shoot apical meristems of 30-day-old plants. Note that the shape of the meristems of clv1-1 ER:CLV1 and clv1-1 ER:CB2 transgenic plants is closer to that of Ler than to the fasciated clv1-1 meristem. Bars = 500 μm.

Figure 4.
Figure 4.

clv1-1 Is Dominant Negative. (A) Ethidium bromide agarose gels from RT-PCR performed on individual suppressed transgenic lines (see Methods). T1 inflorescence RNA samples for clv1-1 ER:CLV1, clv1-1 ER:CB2, clv1-1 ER:CB3, and clv1-1 ER:CB2m transgenic plants were analyzed by RT-PCR to monitor transcript accumulation of the endogenous clv1-1 and transgene mRNA. Each lane corresponds to one transgenic line. RT-PCR products from clv1-1 nontransgenic plants, clv1-1 control transgenic plants, and no RNA were examined as controls (c) in all cases. Each PCR amplification of the cDNA was generated in parallel with specific primers for clv1-1 (top gel), the appropriate transgene (middle gel), and the control (βATPase; bottom gel). Top panel, clv1-1 ER:CLV1 transgenic lines; second panel, clv1-1 ER:CB2; third panel, clv1-1 ER:CB3; fourth panel, clv1-1 ER:CB2m. (B) Quantification and normalization of the RT-PCR agarose gel data (see Methods). Black boxes indicate clv1-1 expression, and white boxes indicate transgene (Tg) expression.

Figure 5.
Figure 5.

The Chimeric Receptors CB2, CB2m, CB3, and CB4 Are Dominant Negative. (A) The relative size of the fifth whorl compared with the number of carpels in clv1-11 and clv1-11 transformed with ER:CLV1, ER:CB1, ER:CB2, ER:CB2m, ER:CB3, ER:CB4, empty vector, and control transgenes. The number of independent transgenic lines and the number of T3 plants analyzed are shown in the table at bottom. Ten fully expanded siliques of 30-day-old plants were counted for the number of carpels and dissected for the size of the fifth whorl before desiccation. The vertical and horizontal bars represent standard errors for the fifth whorl size and the number of carpels, respectively. Note that the almost complete rescue of the clv1-11 phenotype is observed for ER:CLV1 and ER:CB1. On the other hand, the chimeric receptors ER:CB2, ER:CB2m, ER:CB3, and ER:CB4 enhance the phenotypes of clv1-11 plants. (B) Comparison of the silique shapes of clv1-11 plants transformed with ER:CLV1, ER:CB2, ER:CB2m, ER:CB3, empty vector, and control transgenes. Note that the shape of the ER:CLV1 siliques is close to that of wild-type siliques and that the siliques of ER:CB2, ER:CB2m, and ER:CB3 transgenic plants are club shaped, reflecting a more severe phenotype than that seen in the clv1-11 and control lines. Bar = 2 mm.

Figure 6.
Figure 6.

Model of Dominant-Negative Receptor Action. A speculative model for the role of CLV1, an additional unknown receptor kinase with functional overlap (RLK), and ER in regulating meristem development. Scenarios for wild-type plants (WT), clv1 null mutants, intermediate clv1 mutants, strong clv1 mutants, and the dominant-negative chimeric receptors are indicated. The model predicts receptor interactions between CLV1 and RLK, which can lead to interference with RLK function in the presence of clv1 dominant-negative isoforms and in the presence of chimeric receptors. The thickness of the arrows suggests the relative strength of signaling, and lesions in clv1 proteins are indicated with a red X.

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References

    1. Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris 316, 1194–1199.
    1. Bechtold, N., and Pelletier, G. (1998). In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266. - PubMed
    1. Brand, U., Fletcher, J.C., Hobe, M., Meyerowitz, E.M., and Simon, R. (2000). Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science 289, 617–619. - PubMed
    1. Brand, U., Grunewald, M., Hobe, M., and Simon, R. (2002). Regulation of CLV3 expression by two homeobox genes in Arabidopsis. Plant Physiol. 129, 565–575. - PMC - PubMed
    1. Chen, R.H., Ebner, R., and Derynck, R. (1993). Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-beta activities. Science 260, 1335–1338. - PubMed

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