Expression Patterns of GmAP2/EREB-Like Transcription Factors Involved in Soybean Responses to Water Deficit

<div><p>Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a <i>GmDRIP</i>-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 <i>GmAP2/EREB</i>-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the <i>GmDREB1F</i>-like gene, for example, was four times higher in roots than in leaves). The <i>GmDRIP-like</i> gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several <i>GmDREB</i>-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.</p></div

Selected DRIP and ERF superfamily target genes.

<p>The BLAST description and Gene Ontology are presented for each gene, and the sequences with greater similarity were used (GenBank access #). The BLAST results are from Aug. 2012 and the GO terms for Biological Process and Molecular Function are listed in the Gene Ontology annotation.</p><p>The BLAST description and Gene Ontology are presented for each gene; the sequences with greater similarity were used (GenBank accession #). The BLAST results are from Aug. 2012, and the GO terms for Biological Processes and Molecular Function are listed in the Gene Ontology annotation.</p

Stability analyses of endogenous genes.

<p>In total, six candidate genes were evaluated using the NormFinder and GeNorm programs to select the most stable genes. The <i>Y</i> axis represents the <i>Expression Stability Measure (M)</i> from the GeNorm program and the Stability value from the NormFinder program. Genes are ranked from less stable (higher values) to most stable (genes with lower values).</p

Amino-acid sequence alignment of the AP2 domains.

<p>Regions of amino-acid conservation are shown. Letters represent the amino acids of the protein sequences, and dashes delimit the specific 14^th and 19^th positions for each DREB or ERF subfamily member.</p

Phylogenetic tree. Proteins encoded by the candidate genes and the DREB/ERF protein that was described in the NCBI database were used to construct the tree using the ClustalW algorithm with the MEGA 5 program.

<p>The Neighbor-Joining (NJ) method was used with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates; random seed). Candidate genes are represented by the GeneModels, and the homologous DREB/ERF sequences from Fabaceae (<i>Glycine max, Medicago truncatula, Cypripedium arietinum, Trifolium repens, Glycine soja, Caragana korshinskii, Pisum sativum,</i> and <i>Galega orientalis</i>) are represented by GI.</p

Quantitative PCR of the <i>AP2/EREB</i> genes.

<p>Gene expression was measured in root and leaf tissues of BR 16 and Embrapa 48 soybean cultivars that were subjected to different periods of water deficit (25 to 150 min). The raw data were normalized to the expression of the <i>ELF1-β</i> and the <i>β-actin</i> endogenous genes, and the relative expression was determined and compared with the control sample (T0 min).</p

Early Transcriptional Response of Soybean Contrasting Accessions to Root Dehydration

<div><p>Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration) for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO) categories for the tolerant accession revealed the expression “protein binding” as the most represented for “Molecular Function”, whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to “hormone response” (LOX, ERF1b, XET), “water response” (PUB, BMY), “salt stress response” (WRKY, MYB) and “oxidative stress response” (PER) figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY) validated by RT-qPCR (four different time points) confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important differences between both accessions. </p> </div

Hierarchical clusterization¹ regarding GO categories [(A) Response to hormones; (B) Response to water], and several contrasts²

<p>¹Gray spots: no expressed unitags; Red: up-regulated unitags; green: down-regulated unitags; black: constitutive expression ². Tolerant accession under stress vs. respective control (I); Sensitive accession under stress vs. respective control (II); and Tolerant accession vs. Sensitive accession, both after root dehydration stress (III). Arrows indicate transcripts mentioned in the discussion.</p

RT-qPCR of the unitags measured at the appropriate sample time using REST2009 software.

<p>RT-qPCR of the unitags measured at the appropriate sample time using REST2009 software.</p

Gene Ontology categorization of the tolerant accession transcripts based on the UR^* and DR^* soybean DeepSuperSAGE unitags.

<p>UR: up-regulated; DR: down-regulated ; *Unitags from tolerant accession under stress versus respective control.</p