MiR159 directs the cleavage of <i>TaGAMYB</i> and the cleavage site.

<p>(A) 5′-RACE was performed to map the cleavage site of <i>TaGAMYB</i>. The conserved domains (R2R3, BOX1, BOX2 and BOX3) of GAMYB in three cereals and Arabidopsis are shown in black. The complementary sequences between miR159 and <i>GAMYB</i> genes are shown in grey. The arrow indicates the cleavage site, and the sequence of the mutated miR159 target site is illustrated (bottom). Numbers in italics indicate the proportion of clones analyzed that mapped to the miR159 cleavage position. (B) Expression analysis of miR159, <i>TaGAMYB1</i> and <i>mTaGAMYB1</i> in <i>N. benthamiana</i> leaves co-Agro-infiltrated with different combinations of <i>35S::TamiR159</i>, <i>35S::TaGAMYB1</i>, <i>35S::mTaGAMYB1</i> and <i>35S::GFP.</i> (C) Expression analysis of miR159, <i>TaGAMYB2</i> and <i>mTaGAMYB2</i> in <i>N. benthamiana</i> leaves co-Agro-infiltrated with different combinations of <i>35S::TamiR159</i>, <i>35S::TaGAMYB2</i>, <i>35S::mTaGAMYB2</i> and <i>35S::GFP.</i></p

<i>TaGAMYB1-A, B, D</i> chromosome locations expression patterns in various tissues and responses to heat stress.

<p>(A) Genome-specific PCR amplification for the three homeologous <i>TaGAMYB1</i> genes. Each primer was used to amplify a Chinese Spring (CS) nulli-tetrasomic set. N3AT3B indicates a nulli-3A-tetra-3B line of CS, and so on. (B) Amplification efficiency of primers for the three homeologous <i>TaGAMYB1</i> genes estimated by Q-PCR using CS DNA. (C) The expression patterns of <i>TaGAMYB1-A, TaGAMYB1-B,</i> and <i>TaGAMYB1-D</i> in leaves, roots, young spikes, anthers and developing seeds. (D) The expression pattern of <i>TaGAMYB1-A, TaGAMYB1-B,</i> and <i>TaGAMYB1-D</i> in response to heat stress. Heat-tolerant cultivar TAM107 and heat-susceptible cultivar CS seedlings were treated at 42°C for 0.5 hr, 1 hr and 2 hrs. Those seedlings treated for 2 hrs were returned to normal growth conditions for 24 hrs (R).</p

Expression of <i>TamiR159</i>, <i>TaGAMYB1</i> and <i>OsGAMYB</i> in transgenic <i>Ubi::TamiR159</i>, <i>Ubi::TaGAMYB1</i> and <i>Ubi::mTaGAMYB1</i> rice lines.

<p>(A) Real-time PCR and Northern blot analysis of leaves from <i>Ubi::TamiR159</i> transgenic plants were used to determine the relative expression of endogenous <i>OsGAMYB</i> and mature miR159 levels. (B) Real-time PCR was used to determine the relative expression of <i>TaGAMYB1</i> in leaves from <i>Ubi::TaGAMYB1</i> transgenic plants. (C) Real-time PCR was used to determine the relative expression of <i>TaGAMYB1</i> in leaves from <i>Ubi::mTaGAMYB1</i> trangenic plants.</p

Heat tolerance testing of <i>Arabidopsis</i> wild-type (WT) and <i>myb33myb65</i> double mutant plants.

<p>(A) Phenotype of WT and <i>myb33myb65</i> 2-weeks-old seedlings after heat stress for 4 hr at 44°C Identical samples was planted diagonally. WT seedlings were planted in the northwest and southeast corners, while double mutant seedlings were planted in the other two corners as indicated in the schematic. (B) Relative electrical conductivity test of WT and <i>myb33myb65</i> double mutants after heat treatment.</p

Phylogenetic tree of GAMYB proteins from <i>Triticum aestivum</i> (<i>Ta</i>), <i>Zea mays</i> (<i>Zm</i>), <i>Arabidopsis thaliana</i> (<i>At</i>), <i>Oryza sativa</i> (<i>Os</i>) and <i>Hordeum vulgare</i> (<i>Hv</i>).

<p>All GAMYB proteins were clustered using ClustalX, and the phylogenetic tree was generated by MEGA.</p

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

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

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

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