Table4.XLSX

<p>Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.</p

Table1.XLSX

<p>Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.</p

Table3.XLSX

<p>Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.</p

Genes identified in the inflammatory response and autoimmunity RT² Profiler Array.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 100 μM S31-201 or 25 μg ml^-1 p37 and 100 μM S31-201 over 24 hours. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Genes identified in the inflammatory response and autoimmunity RT² Profiler Array.</p

Table2.XLSX

<p>Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.</p

Gene expression of NIH3T3 fibroblasts treated with different concentrations of purified p37 (0.5, 1, 5 and 25 μg ml^-1) for 24 hours.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 0.5, 1, 5 or 25 μg ml^-1 p37 for 24 hours. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Gene expression of NIH3T3 fibroblasts treated with different concentrations of purified p37 (0.5, 1, 5 and 25 μg ml^-1) for 24 hours.</p

Gene expression at different time points (2, 4, 8, 12 and 24 hours) following p37 (5 μg ml^-1) addition to NIH3T3 fibroblasts.

<p>Fold change (E^-ΔΔCt) of mRNA expression levels of NIH3T3 fibroblasts treated with 5 μg ml^-1 p37 at 2, 4, 8, 12 or 24 hours. The 25 μg ml^-1 p37 for 24 hours results are presented for comparison. Significant differences between treated and untreated cells were calculated by ANOVA analysis (p-values ≤0.05 are shown in bold).</p><p>Gene expression at different time points (2, 4, 8, 12 and 24 hours) following p37 (5 μg ml^-1) addition to NIH3T3 fibroblasts.</p

The effect of 0.5 μM VIPER and CP7 on p37-induced gene expression in NIH3T3 fibroblasts.

<p>Quantitative PCR (qPCR) analysis of NIH3T3 fibroblasts treated with 25 μg ml^-1 p37 for 24 hours (black) or pre-treated for 2 hours with VIPER (grey) or the control peptide CP7 (white) prior to 25 μg ml^-1 p37-treatment for 24 hours. Significant differences between CP7 or VIPER+p37 treatments and p37 treatment were calculated using ANOVA analysis (+p≤0.05, ++p≤0.01, +++p≤0.001).</p

Image1.JPEG

<p>Anther development progresses through 15 distinct developmental stages in wheat, and accurate determination of anther developmental stages is essential in anther and pollen studies. A detailed outline of the development of the wheat anther through its entire developmental program, including the 15 distinct morphological stages, is presented. In bread wheat (Triticum aestivum), anther developmental stages were correlated with five measurements, namely auricle distance, spike length, spikelet length, anther length and anther width. Spike length and auricle distance were shown to be suitable for rapid anther staging within cultivars. Anther length is an accurate measurement in determining anther stages and may be applicable for use between cultivars. Tapetal Programmed Cell Death (PCD) in wheat begins between late tetrad stage (stage 8) and the early young microspore stage (stage 9) of anther development. Tapetal PCD continues until the vacuolate pollen stage (stage 11), at which point the tapetum fully degrades. The timing of tapetal PCD initiation is slightly delayed compared to that in rice, but is two stages earlier than in the model dicot Arabidopsis. The MYB80 gene, which encodes a transcription factor regulating the timing of tapetal PCD, reaches its peak expression at the onset of tapetal PCD in wheat.</p

Purification of the p37 protein using Ni-affinity chromatography.

<p>Purified p37 was separated by 12% SDS-PAGE and stained with Coomassie blue <b>(A).</b> The purified protein was transferred to polyvinylidene fluoride membranes and probed with the T7-Tag monoclonal antibody and the goat anti-mouse IgG Horseradish Peroxide (HRP) conjugate (<b>B</b>). The molecular weight standards (MW) are in kilo Daltons (kDa) and indicated on the left of the figure. The purified p37 protein ran to the position of approximately 52 kDa. The p37 protein is predicted to be 43.5 kDa with an additional 8.5 kDa as a result of the 6x His Tag and Xpress Epitope of the pRSET A reading frame. The identity of the purified p37 protein was further confirmed using protein sequencing.</p