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

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day

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

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

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

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

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

Information on reference and target genes.

<p>^a New reference candidate genes</p><p>^b Commonly used reference genes</p><p>^c Target gene</p><p>Information on reference and target genes.</p

Commonly used RT-qPCR reference genes from soybean, according to Hu and colleagues (2009).

<p>Commonly used RT-qPCR reference genes from soybean, according to Hu and colleagues (2009).</p

Intra- and intergroup variation of gene expression.

<p>The intragroup variation within the <b>(A)</b> control (non-stressed plants) and <b>(B)</b> stressed (plants under drought stress) and <b>(C)</b> the intergroup variation are presented.</p