Plant F-Box Protein Evolution Is Determined by Lineage-Specific Timing of Major Gene Family Expansion Waves

<div><p>F-box proteins (FBPs) represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in <i>Arabidopsis thaliana</i>, which carries a C-terminal F-box associated domain (FBA proteins) shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we <i>de novo</i> identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.</p></div

Number of unstable, stable and singleton FBA proteins in <i>A. thaliana</i>, <i>V. vinifera</i>, <i>P. trichocarpa</i>, <i>O. sativa</i>, <i>S. bicolor</i>, <i>S. moellendorffii</i>, <i>P. patens, and C.</i> sp. C-169.

<p>Number of unstable, stable and singleton FBA proteins in <i>A. thaliana</i>, <i>V. vinifera</i>, <i>P. trichocarpa</i>, <i>O. sativa</i>, <i>S. bicolor</i>, <i>S. moellendorffii</i>, <i>P. patens, and C.</i> sp. C-169.</p

Number of FBA proteins in non-land plant model species.

<p>FBA proteins in non-land plant species were identified by screening the Interpro database from the EMBL-EBI website (<a href="http://www.ebi.ac.uk/interpro/" target="_blank">http://www.ebi.ac.uk/interpro/</a>) for proteins with the following domains: IPR007397 (F-box associated domain), IPR006527 (F-box associated domain_type1), IPR012885 (F-box associated domain_type2) and IPR013187 (F-box associated domain_type3). Identified proteins were verified for the presence of F-box and FBA domain using Pfam 26.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068672#pone.0068672-Punta1" target="_blank">[27]</a>.</p

Patterns of selection in <i>FBA</i> genes.

<p>A, Average <i>K_a/K_s</i> [ω by Yang <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068672#pone.0068672-Yang3" target="_blank">[40]</a> and <i>g</i> by Comeron <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068672#pone.0068672-Comeron1" target="_blank">[41]</a>] ratios calculated over the complete coding sequence of all stable and unstable genes in <i>A. thaliana</i> by comparison to <i>A. lyrata</i> orthologs. Error bars represent SE. The difference is significant according to Kruskal-Wallis test (P<0.01). B, Sliding window plots for the four stable genes. C, Sliding window plots for randomly chosen unstable genes. For sliding window analysis, nucleotide sequences of <i>A. thaliana</i> (indicated by Arabidopsis Genome Initiative identifier) and orthologous nucleotide sequences of <i>A. lyrata</i> (indicated by protein identifier according to the Joint Genome Institute) were used. Window size was 150 bp, and step size was 9 bp. Light gray boxes highlight the position of the F-box domain, and dark gray boxes highlight the FBA-D position.</p

Evolutionary change in the number of FBA proteins in land plants.

<p>The numbers in rectangles and circles represent the maximum number of genes in ancestral and extant species, respectively. The numbers with plus and minus signs indicate gene gains and losses, respectively, for each branch. Bold lines represent branches with high gene expansion rate. N0: Chloroplastida ancestor, N1: land plant ancestor, N2: angiosperm ancestor, N3: monocot ancestor, N4: eudicot ancestor. Branch lengths are not in proportion to evolutionary time.</p

Phylogeny of F-box proteins with C-terminal FBA domains in land plant species.

<p>A, Phylogenetic tree of <i>A. thaliana, V. vinifera, P. trichocarpa, O. sativa, S. bicolor, S. moellendorffii</i> and <i>P. patens</i> FBA proteins. Multiple sequence alignments of the full-length FBA protein sequences were performed using MUSCLE. The phylogenetic tree was generated using ML methods in GARLI. The tree was rooted with the FBA protein sequence of <i>Coccomyxa</i> sp. C-169. The color code corresponds to the different species. B, Ratios of unstable, stable and singleton FBA proteins in the seven analyzed land plant species.</p

Differential expression profiles of phylogenetically closely related <i>FBA</i> genes.

<p>A, Mean expression values for unstable and stable <i>A. thaliana FBA</i> genes extracted from AtGenExpress_Plus-extended_tissue_series <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068672#pone.0068672-Toufighi1" target="_blank">[42]</a>. Error bars represent SE. Statistical significance was assessed using Student´s t-test (***P<0.001). B, NJ tree of 211 <i>A. thaliana</i> FBA proteins based on amino acid sequence homology. C, Clustering of 102 <i>A. thaliana</i> FBA proteins based on co-expression data from the AtGenExpress_Plus-extended_tissue_series. Discrepancy in the number of FBA genes/proteins between the trees in A and B results from 109 missing <i>FBA</i> genes on the ATH1 microarray. Two clades were selected for which all genes had representatives in the ATH1-Chip for illustration purposes (highlighted in green and red font). All other genes were collapsed and labeled according to the number of <i>FBA</i> genes within the respective branches.</p

Phylogeny of species included in this analysis and number of FBA proteins identified.

<p>The tree represents the phylogenetic relationships between the species analyzed. Branch lengths are not in proportion to evolutionary time. The number of FBA proteins identified per species is indicated next to the species name. Species included in the phylogenetic reconstruction of FBA protein evolution are indicated.</p