Functional Analyses of the CLAVATA2-Like Proteins and Their Domains That Contribute to CLAVATA2 Specificity

Abstract

The Arabidopsis (Arabidopsis thaliana) CLAVATA2 (CLV2) gene encodes a leucine-rich repeat receptor-like protein (RLP) that is involved in controlling the stem cell population size in the shoot apical meristem. Our previous genome-wide functional analysis of 57 AtRLP genes revealed only a few phenotypes for mutant alleles, despite screening a wide range of growth and developmental stages and assaying sensitivity to various stress responses, including susceptibility toward pathogens. To gain further insight into the biological role of AtRLPs, in particular CLV2-related AtRLP genes, we tested their ability to complement the clv2 mutant phenotype. We found that out of four close CLV2 homologs tested, AtRLP2 and AtRLP12 could functionally complement the clv2 mutant when expressed under the control of the CLV2 promoter. This indicates that the functional specificity of these three genes is determined at the level of their transcriptional regulation. Single and double mutant combinations with impaired AtRLP2 and/or AtRLP12 did not show an aberrant phenotype, suggesting that other genes are redundant with these CLV2-like genes. To understand which protein domains are essential for CLV2 function and which parts are interchangeable between related CLV2-like proteins, we performed domain-deletion and domain-swap experiments. These experiments revealed that CLV2 remains functional without the island domain, whereas the C1 and C3 regions of the leucinerich repeat domain are essential for functionality. Analysis of domain-swap constructs showed that the C3-G region of CLV2 can be replaced by that of AtRLP38, although it could not complement the clv2 mutant under control of the CLV2 promoter. This suggests that the C3-G region is conserved among related AtRLP members, whereas the C1 domain may determine the functional specificity of CLV2.

New insights into receptor-like protein functions in Arabidopsis

“…19 However, no phenotypic alterations were observed for different combinations of the T-DNA insertion mutants of AtRLP2, AtRLP12 and CLV2. 19 These results highlighted the existence of both functional redundancy and functional divergence among AtRLP proteins. Despite these achievements on the studies of AtRLP genes, only a few AtRLPs have been characterized to date.…”

“…To gain further insights into the biological functions of AtRLPs, several AtRLPs that are closely-related with CLV2 were identified. 19 Of the CLV2 homologues exmined, AtRLP2 and AtRLP12, when driven by the endogenous promoter of CLV2, could rescue the clv2 meristem defects. 19 However, no phenotypic alterations were observed for different combinations of the T-DNA insertion mutants of AtRLP2, AtRLP12 and CLV2.…”

“…19 Of the CLV2 homologues exmined, AtRLP2 and AtRLP12, when driven by the endogenous promoter of CLV2, could rescue the clv2 meristem defects. 19 However, no phenotypic alterations were observed for different combinations of the T-DNA insertion mutants of AtRLP2, AtRLP12 and CLV2. 19 These results highlighted the existence of both functional redundancy and functional divergence among AtRLP proteins.…”

“…Our previous study implied that the functional specificity of AtRLP genes, at least in part, was determined at the level of their transcriptional regulation. 19 In addition, the fact that phenotypic alterations only be visible under suitable conditions makes studies on the function of AtRLP genes even more difficult. To obtain a more precise understanding of the transcriptional regulation of AtRLP genes, in a recent study we undertook a systematic analysis of the expression patterns of AtRLP genes with respect to a broad range of conditions including abiotic/biotic stresses and hormones.…”

“…12,19 In addition, it is nearly impossible for genetically crossing individual T-DNA insertion lines to obtain high-order knockout mutants because many AtRLP genes are clustered at the same locus. 11,12,20 Also it is difficult to elucidate the function of AtRLPs due to the high diversity and extremely low abundance of their putative ligands.…”