Genome Duplication and Gene Loss Affect the Evolution of Heat Shock Transcription Factor Genes in Legumes

<div><p>Whole-genome duplication events (polyploidy events) and gene loss events have played important roles in the evolution of legumes. Here we show that the vast majority of Hsf gene duplications resulted from whole genome duplication events rather than tandem duplication, and significant differences in gene retention exist between species. By searching for intraspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found that genome duplications accounted for 42 of 46 Hsf-containing segments in <i>Glycine max</i>, while paired segments were rarely identified in <i>Lotus japonicas</i>, <i>Medicago truncatula</i> and <i>Cajanus cajan</i>. However, by comparing interspecies microsynteny, we determined that the great majority of Hsf-containing segments in <i>Lotus japonicas</i>, <i>Medicago truncatula</i> and <i>Cajanus cajan</i> show extensive conservation with the duplicated regions of <i>Glycine max</i>. These segments formed 17 groups of orthologous segments. These results suggest that these regions shared ancient genome duplication with Hsf genes in <i>Glycine max</i>, but more than half of the copies of these genes were lost. On the other hand, the <i>Glycine max</i> Hsf gene family retained approximately 75% and 84% of duplicated genes produced from the ancient genome duplication and recent <i>Glycine</i>-specific genome duplication, respectively. Continuous purifying selection has played a key role in the maintenance of Hsf genes in <i>Glycine max</i>. Expression analysis of the Hsf genes in <i>Lotus japonicus</i> revealed their putative involvement in multiple tissue-/developmental stages and responses to various abiotic stimuli. This study traces the evolution of Hsf genes in legume species and demonstrates that the rates of gene gain and loss are far from equilibrium in different species.</p></div

Expression of <i>L. japonicus</i> Hsf genes in response to abiotic stress measured by quantitative real-time PCR.

<p>The mRNA level of each gene in <i>L. japonicus</i> seedlings given heat (HS: 42°C), cold (4°C) and oxidative (OS: 10 mM H₂O₂) stress for 1 h was plotted relative to the value obtained for the unstressed contral. Error bars represent standard errors.</p

Sliding window plots of representative duplicated Hsf genes in <i>G. max</i>.

<p>As shown in the key, the gray blocks, from dark to light, indicate the positions of the DBD domain, HR-A/B region, NLS, NES and AHA motifs, respectively. The window size is 150 bp, and the step size is 9 bp. The data for all pairs of duplicated Hsf genes of soybean are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102825#pone.0102825.s006" target="_blank">Figure S6</a>.</p

Phylogenetic tree of 140<i>L. japonicus</i>, <i>M. truncatula</i>, <i>C. arietinum</i>, <i>G. max</i>, <i>C. cajan</i> and <i>P. vulgaris</i>.

<p>This tree was constructed based on amino acid sequence comparison of the conserved N-terminal regions of Hsfs including the DNA-binding domain, the HR-A/B region and parts of the linker between them, using the neighbor-joining method with 1,000 bootstrap replicates. The Hsf of <i>Saccharomyces cerevisiae</i> (ScHsf1) and the Hsfs of <i>C. reinhardtii</i>, <i>S. moellendorffii</i> and <i>P. patens</i> were used as the outgroup. The colors indicate the species background of the Hsfs. The tree was divided into 18 shared clades (Clades 1–18) according to evolutionary distances. The bootstrap values of both neighbor-joining (NJ) tree (first number; 1000 replicates) and maximum likelihood (ML) tree (second number; 1000 replicates) were shown on the branches leading to each of the clades. The clades were supported by high bootstrap values in neighbor-joining and maximum likelihood analyses. Different subclasses of Hsfs are indicated in brackets. Gene names are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102825#pone.0102825.s008" target="_blank">Table S2</a>. The scale bar represents 0.1 amino acid changes per site.</p

Comparative maps of representative Hsf genes and their flanking genes within syntenic chromosomal intervals across selected legume species.

<p>The relative positions of all flanking protein-coding genes were defined by the anchored Hsf genes, highlighted in red. The chromosome segments are shown as gray horizontal lines, with arrows corresponding to individual genes and their transcriptional orientations. All genes are numbered from left to right, in order, for each segment. Where several duplicated genes were present within a region, these genes were given the same number, with the letters a, b, c… appended in order. Conserved gene pairs among the segments are connected with lines. (A) The syntenic chromosomal intervals containing <i>MtHsf-17</i>, <i>LjHsf-07</i>, <i>CcHsf-09</i>, <i>GmHsf-13</i> and <i>GmHsf-15</i> across <i>M. truncatula</i>, <i>L. japonicus</i>, <i>C. cajan</i> and <i>G. max</i>. (B) The syntenic chromosomal intervals containing <i>MtHsf-03, MtHsf-16</i>, <i>LjHsf-06</i>, <i>LjHsf-11</i>, <i>GmHsf-12</i>, <i>GmHsf-17</i>, <i>GmHsf-32</i> and <i>GmHsf-36</i> across <i>M. truncatula</i>, <i>L. japonicus</i>, and <i>G. max</i>. (C) The syntenic chromosomal intervals containing <i>MtHsf-12</i>, <i>GmHsf-01</i> and <i>GmHsf-20</i> across <i>M. truncatula</i> and <i>G. max</i>. The full microsynteny maps of the regions containing Hsf genes within <i>M. truncatula</i>, <i>L. japonicus</i>, <i>C. cajan</i> and <i>G. max</i> are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102825#pone.0102825.s005" target="_blank">Figure S5</a>.</p

Extensive microsynteny of Hsf regions across <i>L</i>. <i>japonicus</i>, <i>M</i>. <i>truncatula</i>, <i>G. max</i> and <i>C. cajan</i>.

<p><i>G. max</i> chromosomes, labeled Gm, are indicated by red boxes. The <i>L</i>. <i>japonicus</i>, <i>M</i>. <i>truncatula</i> and <i>C. cajan</i> chromosomes, shown in different colors, are labeled Lj, Mt and Cc, respectively. Numbers along each chromosome box indicate sequence lengths in megabases. The whole chromosomes of these four legumes, harboring Hsf regions, are shown in a circle. Black lines represent the syntenic relationships between Hsf regions.</p

Estimates of the dates for the large scale duplication events in legume species.

<p>The Hsf gene pairs from flexible sets were not used for calculation.</p

Estimates of Ks and Ka/Ks ratios in pairwise comparisons.

<p>(A) Distribution of synonymous distances (Ks) between paralogous genes flanking duplicated Hsf genes in <i>G. max</i>. The histogram shows the number of duplicate gene pairs (y-axis) versus synonymous distance between pairs (x-axis). The Ka/Ks ratios of the duplicated Hsf genes (B) and their flanking paralogs (C) in <i>G. max</i> are shown in the scatter plots; the y and x axes denote the Ka/Ks ratio and synonymous distance for each pair, respectively.</p

<i>L. japonicus</i> Hsf genes expression in various plant tissues.

<p>The type of tissue (nodule, root, stem, leaf and flower) and the gene name are shown on the y-axis and x-axis, respectively. Hierarchical clustering based on average log signal values in various tissues grouped 10 of the <i>L. japonicus</i> Hsf genes into four types (A–D).</p

Schematic presentation of a mechanism for salt tolerance in <i>K. candel</i>.

<p>Most differentially expressed proteins were integrated, and were indicated in red (up-regulated at least under 450 mM NaCl treatment) or blue (down-regulated), respectively. Abbreviations: ADP, adenosine diphosphate; AKG, oxoglutarate; BPG, 1,3-bisphosphoglycerate; cytb6f, cytochrome b6f; DHA, dehydroascrobate; DHAP, dihydroxyacetone phosphate; EA, enolase; eIF, eukaryotic translation initiation factor; F6P, fructose-6-phosphate; FADH₂, reduced flavin adenine dinucleotide; FtsH, Cell division protein ftsH; G3P, glyceraldehydes-3-phosphate; G6P, glucose-6-phosphate; GS, glutamine synthetase; GSH, reduced glutathione; GSSH, oxidized glutathione; IMD, isopropylmalate dehydratase; MDHA, monodehydroascorbate; MDHAR, MDHA reductase; NADP⁺/NADPH, nicotinamide adenine dinucleotide phosphate; OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; PG, phosphoglycolate; PGD, phosphoglycerate dehydrogenase; PPIase, peptidyl-prolyl cis-trans isomerase; PRS, proteasome; Q, quinone; R5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; RIM, reductoisomerase; TPI, triosephosphate isomerase.</p