A Comprehensive Analysis of RALF Proteins in Green Plants Suggests There Are Two Distinct Functional Groups

Abstract

Rapid Alkalinization Factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth. Although RALF peptides have been identified within many species, a single wide-ranging phylogenetic analysis of the family across the plant kingdom has not yet been undertaken. Here, we identified RALF proteins from 51 plant species that represent a variety of land plant lineages. The inferred evolutionary history of the 795 identified RALFs suggests that the family has diverged into four major clades. We found that much of the variation across the family exists within the mature peptide region, suggesting clade-specific functional diversification. Clades I, II, and III contain the features that have been identified as important for RALF activity, including the RRXL cleavage site and the YISY motif required for receptor binding. In contrast, members of clades IV that represent a third of the total dataset, is highly diverged and lacks these features that are typical of RALFs. Members of clade IV also exhibit distinct expression patterns and physico-chemical properties. These differences suggest a functional divergence of clades and consequently, we propose that the peptides within clade IV are not true RALFs, but are more accurately described as RALF-related peptides. Expansion of this RALF-related clade in the Brassicaceae is responsible for the large number of RALF genes that have been previously described in Arabidopsis thaliana. Future experimental work will help to establish the nature of the relationship between the true RALFs and the RALF-related peptides, and whether they function in a similar manner.

Keywords: RALF; development; evolution; growth; peptide; phylogeny.

Figure 1

The relationship between the total number of genes within the genome and the number of identified RALFs for the monocots (red) and eudicots (blue). Species with unusually high numbers of RALFs based upon their genome content are circled.

Figure 2

An unrooted approximately-maximum likelihood phylogenetic tree of the 795 aligned RALF proteins from 51 plant species. (A) A simplified cladogram representing the high-level splits of the tree, with the four major clades denoted by a number and sub-clades denoted by a letter. Local support values of these splits are provided, as calculated by the Shimodaira-Hasegawa test. (B) The full tree inferred from the 795 proteins. Pink, red, green and blue colors indicate clades I, II, III, and IV respectively and sub-clades are shaded appropriately.

Figure 3

A CLANS analysis of RALF protein similarity. Each of the 795 full-length protein sequences is represented by a colored dot that relates to the placement of that protein within the phylogenetic tree clades and subclades. Proteins that are closer within the 2D space are considered to have more sequence similarity.

Figure 4

Divergence of the mature peptide region. (A) A CLANS sequence similarity analysis of the region downstream of the YISY motif from 795 RALF proteins. The colored dots represent the placement of each peptide within the full-length preproprotein tree (Figure 2), as denoted by the key. (B) An approximate-maximum likelihood tree of the mature RALF peptides placed within the full-length preproprotein clades I, II, and III. As a measure of the correspondence between the two trees, the numbers to the side of each clade show the percentage of RALFs that are placed within clade I/clade II/clade III on the full-length tree, respectively.

Figure 5

Divergence of RALF protein sequences across the four major phylogenetic clades. (A) WebLogo3 plots to demonstrate residue conservation within the mature peptide region of the four major clades. Clade I and II (A) are shown together as the mature peptides of these sub-clades are very similar. (B) A schematic representation of the motif structure of RALF proteins from clades I, II and III in comparison to the shorter RALFs of clade IV. Proteins from both clades contain an N-terminal signal peptide, but the variable and acidic region downstream of this is much shorter and frequently absent within the clade IV RALFs. The di-basic mature peptide cleavage site is absent within clade IV, suggesting an alternate cleavage mechanism. Additionally, the YISY motif thought to be required for receptor binding is highly variable within clade IV RALFs and many proteins in this clade do not contain the second of the four typically conserved cysteine residues.

Figure 6

Clustered mRNA expression values of 37 Arabidopsis thaliana RALF genes across a variety of tissues. Each gene is colored according to its phylogenetic clade (see key). Asterisks indicate the five genes belonging to the sub-clade III(B).