Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2

Expression profiles of 12 callose synthases in different sporophytic tissues and during male gametophyte development of <i>Arabidopsis thaliana</i>.

<p>SUS, suspension cultures; SDL, seedling; LF, leaf; RT, root; FWB, flower buds; FLW, flower; SD, seed; UNM, uninuclear microspore; BCP, bicellular pollen; TCP, tricellular pollen; MPG, mature pollen grain; PT4, 4h pollen tubes; SPC, sperm cells.</p

Phylogenetic tree of 12 <i>Arabidopsis thaliana</i> callose synthases.

<p>The evolutionary history was inferred using maximum likelihood. The bootstrap support values are shown next to the branches. Accessions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187331#pone.0187331.t001" target="_blank">Table 1</a>.</p

Evolutionary history of callose synthases in terrestrial plants with emphasis on proteins involved in male gametophyte development

<div><p>Callose is a plant-specific polysaccharide (β-1,3-glucan) playing an important role in angiosperms in many developmental processes and responses to biotic and abiotic stresses. Callose is synthesised at the plasma membrane of plant cells by callose synthase (CalS) and, among others, represents the main polysaccharide in the callose wall surrounding the tetrads of developing microspores and in the growing pollen tube wall. CalS proteins involvement in spore development is a plesiomorphic feature of terrestrial plants, but very little is known about their evolutionary origin and relationships amongst the members of this protein family. We performed thorough comparative analyses of callose synthase family proteins from major plant lineages to determine their evolutionary history across the plant kingdom. A total of 1211 candidate CalS sequences were identified and compared amongst diverse taxonomic groups of plants, from bryophytes to angiosperms. Phylogenetic analyses identified six main clades of CalS proteins and suggested duplications during the evolution of specialised functions. Twelve family members had previously been identified in <i>Arabidopsis thaliana</i>. We focused on five CalS subfamilies directly linked to pollen function and found that proteins expressed in pollen evolved twice. CalS9/10 and CalS11/12 formed well-defined clades, whereas pollen-specific CalS5 was found within subfamilies that mostly did not express in mature pollen vegetative cell, although were found in sperm cells. Expression of five out of seven mature pollen-expressed CalS genes was affected by mutations in <i>bzip</i> transcription factors. Only three subfamilies, CalS5, CalS10, and CalS11, however, formed monophyletic, mostly conserved clades. The pairs CalS9/CalS10, CalS11/CalS12 and CalS3 may have diverged after angiosperms diversified from lycophytes and bryophytes. Our analysis of fully sequenced plant proteins identified new evolutionary lineages of callose synthase subfamilies and has established a basis for understanding their functional evolution in terrestrial plants.</p></div

Callose synthase expression profiles in <i>Nicotiana tabacum</i> male gametophyte.

<p>A) Expression profile of five male gametophyte-expressed CalS mRNAs during pollen development and progamic phase, B) Distribution of transcripts encoding three most abundant tobacco pollen CalS mRNAs (CalS5, CalS10, CalS12) between actively translated polysomal and storage EPP fractions. UNM, uninuclear microspore; early BCP, early bicellular pollen; late BCP, late bicellular pollen; MPG, mature pollen grain; PT4, 4h pollen tubes; PT24, 24h pollen tubes; EPP, mRNA storage EPP complexes; POL, polysomal fraction.</p

Summary of callose synthase proteins their function, and location in the <i>Arabidopsis thaliana</i>.

<p>Proteins active in pollen are underlined.</p

Callose synthase phylogeny in terrestrial plants inferred using maximum parsimony.

<p><b>The tree is rooted by green alga <i>Auxenochlorella protothecoides</i> representing one of possible outgroup for terrestrial plants.</b> The most parsimonious tree with length 90012, CI = 0,223978, RI = 0,828990 is shown. The data contains 655 protein sequences and 4970 positions in the final dataset. Bootstrap support values ≥50% are shown next to the branches. Numbers in circles indicate main clades described in the text.</p

Unrooted phylogenetic tree of 655 proteins sequences of the callose synthase.

<p>The evolutionary history was inferred using maximum likelihood. Bootstrap support values ≥50% are shown on the branch above. The ML log likelihood -482640.553247. The analysis included 655 amino acid sequences and 3435 positions in the final data set. Numbers in circles indicate main clades described in the text.</p

Callose synthase expression in <i>Arabidopsis thaliana</i> pollen.

<p>A) Abundance of expressed CalS transcripts in mature pollen and sperm cells. B) Expression profiles of pollen-expressed CalS transcripts in wild type Col-0 and <i>bzip18/-</i> and <i>bzip34/-</i> mutant <i>A</i>. <i>thaliana</i> mature pollen.</p

Consensus domains in the callose synthase proteins.

<p>A) Three main domains from the Vta1 superfamily, 1,3-beta-glucan synthase subunit FKS1, and the 1,3-beta-glucan synthase superfamily. B) Specific domains for each callose synthase. Domains were predicted by Pfam, and the domain in the boxes was predicted by DART. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187331#pone.0187331.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187331#pone.0187331.s002" target="_blank">S2</a> Tables for details.</p