Motif analysis.

<p>(A) Consensus distribution of motifs in all Viridiplantae sequences. (B) Distribution of motifs across 67 representative Viridiplantae CLO/PXG proteins grouped in eight taxonomic clades as follows. (i) Trebouxiophyceae, (ii) Chlorophyceae, (iii) Charophyta, (iv) Non-seed plants, (v) Gymnosperms, (vi) basal Angiosperms, (vii) Monocots and (viii) Dicots.(C) Sequences of the 7 major motifs found in Viridiplantae CLO/PXG proteins.</p

Sequence alignments of CLO/PXG protein families from three representative Charophyte species plus 67 sequences from 34 species across the Viridiplantae.

<p>(A) <i>Klebsormidium nitens</i> alignment. (B) <i>Oryza sativa</i> alignment. (C) <i>Arabidopsis thaliana</i> alignment. (D) 67 protein sequences alignment from 34 Viridiplantae species. The five major structural domains are shown respectively as the N-terminal H-caleosin, Ca 2⁺ binding EF Hand, Lipid-binding, Heme binding and kinase phosphorylation and C-terminal kinase phosphorylation domains. The proline knot region and two conserved Histidines are also shown.</p

Evolutionary and genomic analysis of the caleosin/peroxygenase (CLO/PXG) gene/protein families in the Viridiplantae

<div><p>Bioinformatics analyses of caleosin/peroxygenases (<i>CLO/PXG</i>) demonstrated that these genes are present in the vast majority of Viridiplantae taxa for which sequence data are available. Functionally active CLO/PXG proteins with roles in abiotic stress tolerance and lipid droplet storage are present in some Trebouxiophycean and Chlorophycean green algae but are absent from the small number of sequenced Prasinophyceaen genomes. <i>CLO/PXG</i>-like genes are expressed during dehydration stress in Charophyte algae, a sister clade of the land plants (Embryophyta). <i>CLO/PXG</i>-like sequences are also present in all of the >300 sequenced Embryophyte genomes, where some species contain as many as 10–12 genes that have arisen via selective gene duplication. Angiosperm genomes harbour at least one copy each of two distinct CLO/PX isoforms, termed H (high) and L (low), where H-forms contain an additional C-terminal motif of about 30–50 residues that is absent from L-forms. In contrast, species in other Viridiplantae taxa, including green algae, non-vascular plants, ferns and gymnosperms, contain only one (or occasionally both) of these isoforms per genome. Transcriptome and biochemical data show that <i>CLO/PXG</i>-like genes have complex patterns of developmental and tissue-specific expression. CLO/PXG proteins can associate with cytosolic lipid droplets and/or bilayer membranes. Many of the analysed isoforms also have peroxygenase activity and are involved in oxylipin metabolism. The distribution of <i>CLO/PXG</i>-like genes is consistent with an origin >1 billion years ago in at least two of the earliest diverging groups of the Viridiplantae, namely the Chlorophyta and the Streptophyta, after the Viridiplantae had already diverged from other Archaeplastidal groups such as the Rhodophyta and Glaucophyta. While algal CLO/PXGs have roles in lipid packaging and stress responses, the Embryophyte proteins have a much wider spectrum of roles and may have been instrumental in the colonisation of terrestrial habitats and the subsequent diversification as the major land flora.</p></div

Transcriptome analysis of <i>CLO/PXG</i> gene expression in date palm and oil palm tissues.

<p>(A) The oil palm genome P5-build was used to read map the RNA seq data from Roche 454 reads (a full dataset is available from NCBI BioProject PRJNA201497). Reads from Roche/454-derived libraries were assembled into isotigs, which were blasted onto Arabidopsis thaliana gene models with a threshold of E-value < 10−5. The best-hit <i>A</i>. <i>thaliana</i> gene model was assigned to the homologue of the query isotig. To estimate expression levels of genes in mesocarp and kernel tissues, Illumina HiSeq 2000 reads from each library were mapped to assembled isotigs from all <i>Elaeis guineensis</i> reads by using the Burrows–Wheeler Aligner. Gene group expression levels were calculated as the number of mapped reads on each isotig divided by the total number of isotigs, multiplied by 100,000, and scaled by the number of genes in each gene group. Both copy number and read coverage were the mean of measures from two biological replicates. Data were analysed as described above for Roche/454 data, except that expression levels were calculated as transcripts per million tags. Identification of expression caleosin in oil palm was done using open source Tuxedo suite software[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196669#pone.0196669.ref050" target="_blank">50</a>]. (B) Transcriptional analysis of <i>CLO/PXG</i> gene expression in date palm tissues and treatments as follows: a) exposure to 0, 10 and 100 ng.L^-1 of the dioxin, TCDD; b) drought for 2, 4 and 6 days; c) exposure to 0, 150 and 300 ng.L^-1 NaCl. Seedlings with a radicle length of 0.5 or 2 or 4.5 cm were referred as stage I, II and III respectively.</p

Alignments of anomalous CLO/PXG sequences from two non-Viridiplantae species.

<p>(A) Alignments of seven CLO/PXG sequences from <i>S</i>. <i>cellulosum</i>. (B) Alignments of two CLO/PXG sequences from <i>C</i>. <i>owczarzaki</i> with a range of green algal sequences. <b>(</b>C) Alignments of five putative CLO/PXG sequences from the Metazoan nematode genus, <i>Panagrolaimus spp</i>, with sequences from <i>A</i>. <i>thaliana</i>.</p

Phylogenetic analysis of 67 CLO/PXG sequences.

<p>(A) 67 CLO/PXG sequences from 34 species across the Viridiplantae. (B) 67 CLO/PXG sequences from 34 species across the Viridiplantae plus six selected fungal species.</p