CLAVATA2 forms a distinct CLE‐binding receptor complex regulating Arabidopsis stem cell specification

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3‐derived CLE ligand. The biochemical role of the receptor‐like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2‐CRN heteromultimeric and CLV1‐BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane‐localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3‐derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand‐binding receptor complexes controlling stem cell specification in Arabidopsis.     

Strategy for shoot meristem proliferation in plants

Shoot apical meristem (SAM) of plants harbors stem cells capable of generating the aerial tissues including reproductive organs. Therefore, it is very important for plants to control SAM proliferation and its density as a survival strategy. The SAM is regulated by the dynamics of a specific gene network, such as the WUS-CLV interaction of A. thaliana. By using a mathematical model, we previously proposed six possible SAM patterns in terms of the manner and frequency of stem cell proliferation. Two of these SAM patterns are predicted to generate either dichotomous or axillary shoot branch. Dichotomous shoot branches caused by this mechanism are characteristic of the earliest vascular plants, such as Cooksonia and Rhynia, but are observed in only a small minority of plant species of the present day. On the other hand, axillary branches are observed in the majority of plant species and are induced by a different dynamics of the feedback regulation between auxin and the asymmetric distribution of PIN auxin efflux carriers. During evolution, some plants may have adopted this auxin-PIN system to more strictly control SAM proliferation.     

Induction of Multichotomous Branching by CLAVATA Peptide in Marchantia polymorpha

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Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development

Multilocular silique is a desirable agricultural trait with great potential for the development of high‐yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T₀ with different single‐guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild‐type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

The CLAVATA1-related BAM1, BAM2 and BAM3 receptor kinase-like proteins are required for meristem function in Arabidopsis

Organ formation at shoot and flower meristems in plants requires the maintenance of a population of centrally located stem cells and the differentiation of peripherally located daughter cells. The CLAVATA (CLV) gene products in Arabidopsis, including the CLV1 receptor-kinase, regulate this process by promoting the differentiation of stem cells on the meristem flanks. Here, we have analyzed the developmental roles of the CLV1-related BAM1 (derived from barely any meristem 1), BAM2 and BAM3 receptor-like kinases. Loss-of-function alleles of these receptors lead to phenotypes consistent with the loss of stem cells at the shoot and flower meristem, suggesting that their developmental role is opposite to that of CLV1. These closely related receptors are further distinguished from CLV1, whose expression and function is highly specific, by having broad expression patterns and multiple developmental roles. These include a requirement for BAM1, BAM2 and BAM3 in the development of high-ordered vascular strands within the leaf and a correlated control of leaf shape, size and symmetry. In addition, BAM1, BAM2 and BAM3 are required for male gametophyte development, as well as ovule specification and function. Significantly, the differing roles of CLV1 and BAM receptors in meristem and organ development are largely driven by differences in expression patterns.     

The systemic nodule number regulation kinase SUNN in Medicago truncatula interacts with MtCLV2 and MtCRN

Autoregulation of nodulation (AON), a systemic signaling pathway in legumes, limits the number of nodules formed by the legume in its symbiosis with rhizobia. Recent research suggests a model for the systemic regulation in Medicago truncatula in which root signaling peptides are translocated to the shoot where they bind to a shoot receptor complex containing the leucine‐rich repeat receptor‐like kinase SUNN, triggering signal transduction which terminates nodule formation in roots. Here we show that a tagged SUNN protein capable of rescuing the sunn‐4 phenotype is localized to the plasma membrane and is associated with the plasmodesmata. Using bimolecular fluorescence complementation analysis we show that, like its sequence ortholog Arabidopsis CLV1, SUNN interacts with homologous CLV1‐interacting proteins MtCLAVATA2 and MtCORYNE. All three proteins were also able to form homomers and MtCRN and MtCLV2 also interact with each other. A crn Tnt1 insertion mutant of M. truncatula displayed a shoot controlled increased nodulation phenotype, similar to the clv2 mutants of pea and Lotus japonicus. Together these data suggest that legume AON signaling could occur through a multi‐protein complex and that both MtCRN and MtCLV2 may play roles in AON together with SUNN.     

Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs

Leucine-rich repeat receptor kinases (LRR-RKs) are the largest sub-family of transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an 'island domain', which intercepts the extracellular tandem LRRs at a position near the transmembrane domain, constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain recognizes the ligand in non-island domain LRR-RKs. Here, we used photoaffinity labeling followed by chemical and enzymatic digestion to show that BAM1, a CLV1/BAM-family LRR-RK whose extracellular domain comprises 22 consecutive LRRs, directly interacts with the small peptide ligand CLE9 at the LRR6-LRR8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-LRR8 of CLV1/BAM-family LRR-RKs deviates slightly from the LRR consensus. In support of our findings, the clv1-4 mutant carries a missense mutation at the inner concave side of LRR6 of CLV1, and introduction of the corresponding mutation in BAM1 resulted in complete loss of ligand binding activity. Our results indicate that the ligand recognition mechanisms of plant LRR-RKs are more complex and diverse than anticipated.     

Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co‐immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2–CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two‐parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.     

New aspects of CLAVATA2, a versatile gene in the regulation of Arabidopsis development

In order to unravel the functions of receptor-like proteins in Arabidopsis (AtRLPs), including the CLAVATA2 (CLV2) receptor, we employed a reverse genetics approach. In previous studies, we assembled a genome-wide collection of AtRLP gene T-DNA insertion mutants and reported on the biological roles of a few AtRLPs closely related to CLV2. In this study, we showed that over-expression of CLV2 in wild-type plants unexpectedly resulted in a multi-carpel phenotype, mimicking the clv2 mutant, which suggests a co-suppression effect on the endogenous CLV2. We also found evidence that the phenotypes of various clv2 alleles are significantly affected by the genetic background of Arabidopsis ecotypes, which, however, did not depend on the ER locus.