Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin, Expansion and Divergence.

Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes

Analysis of evolutionary pressure on <i>FLZ</i> genes (A) Ka/Ks ratio of gene pair of <i>P</i>. <i>patens</i> and <i>S</i>. <i>moellendorffii</i>, <i>P</i>. <i>abies</i> and duplicated gene pairs of <i>A</i>. <i>trichopoda</i>, <i>A</i>. <i>thaliana</i> and <i>O</i>. <i>sativa</i>. The Ka/Ks ratio of whole gene and FLZ domain region is individually calculated. (B) averaged Ka/Ks ratio of whole gene and FLZ domain region of selected species studied. (C) and (D) averaged Ka/Ks ratio between <i>A</i>. <i>thaliana</i> (C) and <i>O</i>. <i>sativa</i> (D) genes and their orthologous genes from selected species.

<p>The Ka/Ks ratio of each pair is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134328#pone.0134328.s010" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134328#pone.0134328.s011" target="_blank">S3</a> Tables.</p

Analysis of phylogenetic origin of FLZ proteins.

<p>Phylogram of <i>P</i>. <i>patens</i>, <i>S</i>. <i>moellendorffii</i>, <i>P</i>. <i>abies</i> and <i>A</i>. <i>trichopoda</i> FLZ proteins was inferred by BI method based on JTT model. The numbers adjacent to the branches represent posteriori probability values. The tree is drawn to scale, with branch lengths measured in the number of amino acid substitutions per site.</p

Expression analysis of <i>Arabidopsis thaliana FLZ</i> gene family during phase transition and bolting using real-time PCR.

<p><i>UBQ10</i> is used as endogenous control. The absolute expression is calculated from ΔCT value. Graphs represent average and error bars represent mean ± SD of two biological replicates. X axis represent samples and Y axis represent absolute expression value. Samples are abbreviated in graph is as follows, 22d, rosette 22 day old; 26d, rosette 26 day old; 28d, rosette 28 day old; 32d, bolted rosette 32d day old.</p

Duplication history of <i>FLZ</i> gene family.

<p>TNG: Total number of genes, BD: Block duplicates, BDN: Block duplication, TD: Tandem duplicates, TDN: Tandem duplication, TD&BD: Tandem & block duplicates, TDN&BDN: Tandem & block duplication, ND: Non-duplicates, NDN: Non-duplication</p><p>Duplication history of <i>FLZ</i> gene family.</p

Chromosomal location and evolutionary history of <i>Oryza sativa FLZ</i> gene family.

<p>The gene duplication analysis is done using PLAZA v2.5and verified by the position of duplicates in phylogram. Solid line connects the block duplicates and dotted line connects the tandem duplicates. The numbers indicated at the top represents the chromosome number.</p

Expression analysis of <i>Arabidopsis thaliana FLZ</i> gene family in different developmental stages and tissues using real-time PCR.

<p><i>UBQ10</i> is used as endogenous control. The absolute expression is calculated from ΔCT value. Graphs represent average and error bars represent mean ± SD of two biological replicates with three technical replicates each. X axis represent samples and Y axis represent absolute expression value. Samples are abbreviated in graph is as follows, RS, radicle emerged; CS, cotyledon stage seedling; LS, 2 leaves stage seedling; MR, mature plant root; ML, mature bolts rosette leaf; FB, flower bud; FO, flower open; SS, silique small; SM, silique mature.</p