Early responses to dehydration in contrasting wild <i>Arachis</i> species

<div><p>Wild peanut relatives (<i>Arachis</i> spp.) are genetically diverse and were selected throughout evolution to a range of environments constituting, therefore, an important source of allelic diversity for abiotic stress tolerance. In particular, <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i>, the parents of the reference <i>Arachis</i> A-genome genetic map, show contrasting transpiration behavior under limited water conditions. This study aimed to build a comprehensive gene expression profile of these two wild species under dehydration stress caused by the withdrawal of hydroponic nutrient solution. For this purpose, roots of both genotypes were collected at seven time-points during the early stages of dehydration and used to construct cDNA paired-end libraries. Physiological analyses indicated initial differences in gas exchange parameters between the drought-tolerant genotype of <i>A</i>. <i>duranensis</i> and the drought-sensitive genotype of <i>A</i>. <i>stenosperma</i>. High-quality Illumina reads were mapped against the <i>A</i>. <i>duranensis</i> reference genome and resulted in the identification of 1,235 and 799 Differentially Expressed Genes (DEGs) that responded to the stress treatment in roots of <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i>, respectively. Further analysis, including functional annotation and identification of biological pathways represented by these DEGs confirmed the distinct gene expression behavior of the two contrasting <i>Arachis</i> species genotypes under dehydration stress. Some species-exclusive and common DEGs were then selected for qRT-PCR analysis, which corroborated the <i>in silico</i> expression profiling. These included genes coding for regulators and effectors involved in drought tolerance responses, such as activation of osmosensing molecular cascades, control of hormone and osmolyte content, and protection of macromolecules. This dataset of transcripts induced during the dehydration process in two wild <i>Arachis</i> genotypes constitute new tools for the understanding of the distinct gene regulation processes in these closely related species but with contrasting drought responsiveness. In addition, our findings provide insights into the nature of drought tolerance in wild germoplasm, which might be explored as novel sources of diversity and useful wild alleles to develop climate-resilient crop varieties.</p></div

Clusters of Ortholog Groups (OGs) from four legume species.

<p>Venn diagram indicating the number of Ortholog Groups (OGs) among <i>Arachis duranensis; Arachis ipaënsis; Glycine max; and Phaseolus vulgaris</i>. The number of DEGs in the different groups is in parentheses.</p

Expression of DEGs as determined by qRT-PCR.

<p>Relative quantification of mRNA levels of 15 candidate genes in <i>A</i>. <i>duranensis</i> roots during the dehydration treatment and collected after 25 (T25); 50 (T50); 75 (T75); 100 (T100); 125 (T125) and 150 (T150) min, relative to control (T0). Bars represent the standard deviation of three biological replicates. Significantly (P < 0.05) up- or downregulated genes are indicated by asterisks.</p

Circos plot detailing chromosome distribution of DEGs.

<p>Distribution of the 1,602 DEGs in the ten chromosomes of <i>A</i>. <i>duranensis</i> (A01 to A10). The outer lines represent exclusive <i>A</i>. <i>duranensis</i> (red) and <i>A</i>. <i>stenosperma</i> (blue) and common (green) DEGs. The inner dots represent the distribution of Log2FC values for each up- and downregulated DEG in <i>A</i>. <i>duranensis</i> (red) and <i>A</i>. <i>stenosperma</i> (blue), with the line indicating Log2FC = 0.</p

Summary of sequencing data.

<p>Illumina HiSeq2000 sequencing data of <i>Arachis</i> spp. transcriptome and mapping of reads to the reference <i>A</i>. <i>duranensis</i> genome.</p

Overview of DEGs expression patterns.

<p>MapMan analysis showing molecular functional categories of DEGs expression patterns in roots of dehydration-stressed plants of <i>A</i>. <i>duranensis</i> (DD) and <i>A</i>. <i>stenosperma</i> (SD) relative to control. Squares show the different genes encoding proteins related to drought perception and oxidative responses (A) and carbohydrate and amino acid metabolism steps (B). Upregulated genes are indicated by red squares and downregulated by blue squares.</p

Physiological analysis of <i>Arachis</i> spp. plants during the early stages of dehydration.

<p>A) Photosynthetic rate; B) Stomatal conductance; C) Transpiration rate; D) Leaf temperature; E) Leaf vapor pressure deficit; and F) Internal CO₂ concentration of <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i> plants subjected to dehydration. Data represent averages calculated over the early stages (0, 25 and 50 min) of the drought imposition and standard error. Statistically significant differences (n = 3, Student’s t-test, p<0.05) are indicated by asterisk.</p

Expression patterns of <i>Arachis</i> spp. transcripts in response to dehydration.

<p>Venn diagrams showing the number of common and exclusive Differentially Expressed Genes (DEGs) in STR and CTR libraries of <i>A</i>. <i>duranensis</i> (A); <i>A</i>. <i>stenosperma</i> (B); and in both species (C). STR indicates the library from stressed (T25 to T150) samples and CTR the library from control (T0) samples. Red arrows indicate DEGs significantly upregulated during dehydration and green the downregulated DEGs. Volcano plots showing the comparison of gene expression profiles between STR and CTR samples in <i>A</i>. <i>duranensis</i> (D) and <i>A</i>. <i>stenosperma</i> (E), with each gene represented by one dot (DEGs in red and not significant in black).</p

Heatmap of the relative expression of the 20 candidate genes in <i>Arachis duranensis</i> and <i>A</i>. <i>stenosperma</i> after <i>in silico</i> (RNA-Seq data) and <i>in vitro</i> (qRT-PCR) analyses.

<p>Normalized log2FC values are shown in a red–blue scale, with darker red the most upregulated and the darker blue the most downregulated DEGs.</p

Gene ontology enrichment of <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i> DEGs during dehydration.

<p>Gene ontology enrichment of <i>A</i>. <i>duranensis</i> and <i>A</i>. <i>stenosperma</i> DEGs during dehydration.</p