The Wheat NAC Transcription Factor TaNAC2L Is Regulated at the Transcriptional and Post-Translational Levels and Promotes Heat Stress Tolerance in Transgenic Arabidopsis

<div><p>Heat stress poses a serious threat to global crop production. In efforts that aim to mitigate the adverse effects of heat stress on crops, a variety of genetic tools are being used to develop plants with improved thermotolerance. The characterization of important regulators of heat stress tolerance provides essential information for this aim. In this study, we examine the wheat (<i>Triticum aestivum</i>) NAC transcription factor gene <i>TaNAC2L</i>. High temperature induced <i>TaNAC2L</i> expression in wheat and overexpression of <i>TaNAC2L</i> in <i>Arabidopsis thaliana</i> enhanced acquired heat tolerance without causing obvious alterations in phenotype compared with wild type under normal conditions. <i>TaNAC2L</i> overexpression also activated the expression of heat-related genes in the transgenic Arabidopsis plants, suggesting that TaNAC2L may improve heat tolerance by regulating the expression of stress-responsive genes. Notably, TaNAC2L is also regulated at the post-translational level and might be degraded via a proteasome-mediated pathway. Thus, this wheat transcription factor may have potential uses in enhancing thermotolerance in crops.</p></div

Expression of six stress marker genes in <i>TaNAC2L</i> transgenic Arabidopsis plants and wild type (WT) under normal and heat stress conditions.

<p>The transcript levels of <i>DREB2A</i>, <i>DREB2B</i>, <i>AtCYP18-1</i>, <i>RD17</i>, <i>HSP26</i>.<i>5</i>, <i>LEA</i>, <i>AtGolS1</i>, <i>HSP70</i>, <i>AtHsfA3</i>, and <i>RD29A</i> were determined by quantitative RT-PCR. Values represent the mean ± SD of three independent experiments and were normalized to <i>ACTIN</i>.</p

Hypocotyl elongation of <i>TaNAC2L</i>-overexpressing transgenic Arabidopsis plants.

<p>A. Levels of the <i>TaNAC2L</i> transcript in the wild type (WT) and 20 transgenic Arabidopsis lines according to real-time quantitative PCR analyses. B. Hypocotyl elongation of WT and transgenic plants (#13) after growth for 2.5 days in the dark at 22°C. Seedling treatments included: 1) maintained at 22°C; 2) treated at 38°C for 90 min; 3) treated at 45°C for 2 h; 4) first treated at 38°C for 90 min followed by 2 h at 22°C and then 2 h at 45°C. For all treatments, seedlings were returned to 22°C for 2.5 days and then photographed. The quantitative analysis of the hypocotyl length of the wild-type and transgenic plants is presented below the photographs.</p

Transcript and protein levels of TaNAC2L in the <i>drip1 drip2</i> double mutant background.

<p><i>TaNAC2L</i> was highly transcribed in three independent siblings of <i>TaNAC2L</i>-overexpressed lines crossed with the <i>drip1 drip2</i> double mutant; the absence of DRIP1 and DRIP2 failed to block the degradation of the TaNAC2L protein.</p

Protein levels of TaNAC2L in <i>TaNAC2L-OX</i> transgenic lines.

<p>A. TaNAC2L protein and transcript levels in <i>TaNAC2L</i>-overexpressing lines (#2, #9 and #13) under normal conditions and 38°C heat stress. Western blot analyses were performed with the anti-HA monoclonal antibody. B. Seven-day-old wild-type (WT) and <i>TaNAC2L-OX</i>-#13 seedlings were treated at 38°C for 2 h. After heat treatment, seedlings were transferred to dimethylsulfoxide (DMSO) for 0.5, 1, 2, 3, 4, 6, or 8 h; or DMSO with 50 μM MG132 for 1, 3, 4, 6, 8, 12, or 24 h before harvesting. The HA antibody was used for the immunoblot analyses.</p

Overexpression of Three <i>TaEXPA1</i> Homoeologous Genes with Distinct Expression Divergence in Hexaploid Wheat Exhibit Functional Retention in <i>Arabidopsis</i>

<div><p>Common wheat is a hexaploid species with most of the genes present as triplicate homoeologs. Expression divergences of homoeologs are frequently observed in wheat as well as in other polyploid plants. However, little is known about functional variances among homologous genes arising from polyploidy. Expansins play diverse roles in plant developmental processes related to the action of cell wall loosening. Expression of the three <i>TaEXPA1</i> homoeologs varied dynamically at different stages and organs, and epigenetic modifications contribute to the expression divergence of three <i>TaEXPA1</i> homoeologs during wheat development. Nevertheless, their functions remain to be clarified. We found that over expression of <i>TaEXPA1-A</i>, <i>-B</i> and <i>-D</i> produced similar morphological changes in transgenic <i>Arabidopsis</i> plants, including increased germination and growth rate during seedling and adult stages, indicating that the proteins encoded by these three wheat <i>TaEXPA1</i> homoeologs have similar (or conserved) functions in <i>Arabidopsis</i>. Collectively, our present study provided an example of a set of homoeologous genes expression divergence in different developmental stages and organs in hexaploid wheat but functional retention in transgenic <i>Arabidopsis</i> plants.</p></div

Additional file 1: Table S1. of Characterization of wheat MYB genes responsive to high temperatures

Primer sequences used in this study (DOCX 24 kb

Supplemental Dataset

Supplemental Datase

Overexpression of <i>ZmARF25</i> in <i>Arabidopsis</i>.

<p>(A) The genomic PCR results for four homozygous T₃ transgenic plants (35S::<i>ZmARF25</i> #1–4) using <i>Bar</i> gene specific primer. PCR products were determined visually by running on 1% agarose gel and stained by Ethidium bromide. Col represents wild-type and #1–4 represent 35S::<i>ZmARF25</i> transgenic lines, which is the same in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094830#pone-0094830-g003" target="_blank">Figure 3B</a>; “+” represents the recombinant plasmid. (B) Relative expression levels of <i>ZmARF25</i> gene in four homozygous T₃ transgenic lines. Total RNA was isolated from the 30-day-old aerial of <i>Arabidopsis</i>. Data are represented as the mean ± SE (n = 3). <i>ACTIN2</i> was used as an endogenous control.</p

Morphology of 35S::<i>ZmARF25</i> and wild-type plants.

<p>Morphology of (A) leaf size, (B) Inflorescences, flowers and siliques and (C) plant height. Statistics of (D) fully expended fifth leaves area, (E) siliques and (F) plant height. Col represents the wild-type; #1–4 represent the four 35S::<i>ZmARF25</i> transgenic lines; data are represented as the mean ± SE (n = 10). <i>t</i>-test compared with the wild-type control: **refers to significant at <i>p</i><0.01.</p