Conserved Molecular Function and Regulatory Subfunctionalization of the LORELEI Gene Family in Brassicaceae

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, development, reproduction, and immunity in Arabidopsis thaliana. Duplications in each component, which potentially could generate thousands of combinations of this signaling complex, are also evident in other angiosperms. Widespread duplication in angiosperms raises the question what evolutionary mechanisms underlie the expansion and retention of these gene families, as duplicated genes are typically rendered non-functional. As genetic and genomic resources make it a tractable model system, here we investigated this question using LLG gene family evolution and function in Brassicaceae. We first established that the LLG homologs in the Brassicaceae resulted from duplication events that pre-date the divergence of species in this family. Complementation of vegetative phenotypes in llg1 by LRE, LLG2, and LLG3 showed that the molecular functions of LLG homologs in A. thaliana are conserved. We next tested the possibility that differences in gene expression (regulatory subfunctionalization), rather than functional divergence, played a role in retention of these duplicated genes. For this, we examined the function and expression of LRE and LLG1 in A. thaliana and their single copy ortholog in Cleome violacea (Clevi LRE/LLG1), a representative species outside the Brassicaceae, but from the same order (Brassicales). We showed that expression of LLG1 and LRE did not overlap in A. thaliana and that Clevi-LRE/LLG1 expression in C. violacea encompassed all the expression domains of A. thaliana LRE + LLG1. Still, complementation experiments showed that LLG1 rescued reproductive phenotypes in lre and that Clevi LRE/LLG1 rescued both vegetative and reproductive phenotypes in llg1 and lre. Additionally, we found that expression of LLG2 and LLG3 in A. thaliana have also diverged from the expression of their corresponding single copy ortholog (Clevi LLG2/LLG3) in C. violacea. Our findings demonstrated how regulatory subfunctionalization, rather than functional divergence, underlies the retention of the LLG gene family in Brassicaceae. Our findings on the regulatory divergence and functional conservation provide an experimental framework to characterize the combinatorial assembly and function of this critical plant cell signaling complex.