No abstract

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, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multi-gene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e., regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

26A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-27 anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE 28 (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and 29 regulate growth, development, reproduction, and immunity in Arabidopsis thaliana. 30 Duplications in each component, which potentially could generate thousands of 31 combinations of this signaling complex, are also evident in other angiosperms. 32 Widespread duplication in angiosperms raises the question what evolutionary 33 mechanisms underlie the expansion and retention of these gene families, as duplicated 34 genes are typically rendered non-functional. As genetic and genomic resources make it 35 a tractable model system, here we investigated this question using LLG gene family 36 evolution and function in Brassicaceae. We first established that the LLG homologs in the 37 Brassicaceae resulted from duplication events that pre-date the divergence of species in 38 this family. Complementation of vegetative phenotypes in llg1 by LRE, LLG2, and LLG3 39 showed that the molecular functions of LLG homologs in A. thaliana are conserved. We 40 next tested the possibility that differences in gene expression (regulatory 41 subfunctionalization), rather than functional divergence, played a role in retention of these 42 duplicated genes. For this, we examined the function and expression of LRE and LLG1 43 in A. thaliana and their single copy ortholog in Cleome violacea (Clevi LRE/LLG1), a 44 representative species outside the Brassicaceae, but from the same order (Brassicales). 45 We showed that expression of LLG1 and LRE did not overlap in A. thaliana and that Clevi-46 LRE/LLG1 expression in C. violacea encompassed all the expression domains of A. 47 thaliana LRE + LLG1. Still, complementation experiments showed that LLG1 rescued 48 reproductive phenotypes in lre and that Clevi LRE/LLG1 rescued both vegetative and 49 reproductive phenotypes in llg1 and lre. Additionally, we found that expression of LLG2 50 and LLG3 in A. thaliana have also diverged from the expression of their corresponding 51 single copy ortholog (Clevi LLG2/LLG3) in C. violacea. Our findings demonstrated how 52 regulatory subfunctionalization, rather than functional divergence, underlies the retention 53 of the LLG gene family in Brassicaceae. Our findings on the regulatory divergence and 54 functional conservation provide an experimental framework to characterize the 55 combinatorial assembly and function of this critical plant cell signaling complex. 56 57pathogen-associated molecular patterns (PAMPs) flagellin and elongation factor thermo 85 unstable (EF-TU) via the immunogenic epitopes flg22 and elf18, respectively. In this 86 context, RALF23 binds to FER-LLG1 to suppress the scaffolding function of FER, thereby 87 inhibiting ROS production and immunity [16, 17]. 88In A. thaliana, the GPI-anchored protein (GPI-AP), receptor kinase, and small CRP 89 components of the trimeric CrRLK1L-LLG-RALF signaling complex are ...