Comparison of transcript accumulation of the four wheat miRNAs in the KU-2075-type homozygous bulks and KU-2075/KU-2025 heterozygous bulks under normal temperature.

<p>Total RNA extracted from crown tissues of each genotype was bulked for qRT-PCR. Each bulked RNA sample was extracted from the crown tissues of five F₂ individuals. The miRNA levels are shown as values relative to those in a KU-2075-type homozygous bulk. Means ± SD were calculated from data obtained in three experiments. 18S rRNA was used as an internal control.</p

Top 15 transcription factor genes identified by microarray analysis as down-regulated in crown tissues of the type II necrosis line (Ldn/KU-2025) under LT relative to under normal temperature.

<p>Top 15 transcription factor genes identified by microarray analysis as down-regulated in crown tissues of the type II necrosis line (Ldn/KU-2025) under LT relative to under normal temperature.</p

Global gene expression profiling related to temperature-sensitive growth abnormalities in interspecific crosses between tetraploid wheat and <i>Aegilops tauschii</i>

<div><p>Triploid wheat hybrids between tetraploid wheat and <i>Aegilops tauschii</i> sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat <i>APETALA1</i>-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes <i>SPL</i>s, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/<i>SPL</i>s module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.</p></div

Expression analysis of the miR156-targeted wheat <i>SPL</i>s.

<p>(A) Complementary relationship between wheat miR156 molecules and their putative target sequences in <i>TaSPL</i>s. The reverse complement of five tae-miR156 targeting sites to the four <i>TaSPL</i> sequences is represented in bold letters. (B) Comparison of transcript accumulation levels of the four <i>TaSPL</i>s in crown tissues of the WT and type II necrosis/grass-clump dwarf lines by qRT-PCR. The transcript levels are shown as values relative to those in crown tissues of the WT line for each comparison. Means ± SD were calculated from data obtained in three experiments. The <i>Actin</i> gene was used as an internal control. Student’s <i>t</i>-test was used to test for statistical significance (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001) between WT and type II necrosis/grass-clump dwarf synthetic plants.</p

Comparison of transcript accumulation of the two defense-related genes in crown tissues of the WT and type II necrosis/grass-clump dwarf lines.

<p>The transcript accumulation levels were analyzed by qRT-PCR. The transcript levels are shown as values relative to those in crown tissues of Ldn/KU-2059 under normal temperature. Means ± SD were calculated from data obtained in three experiments. The <i>Actin</i> gene was used as an internal control. Student’s <i>t-</i>test was used to test for statistical significance (***<i>P</i><0.001) between WT and type II necrosis/grass-clump dwarf synthetic plants.</p

Top 13 transcription factor genes identified by microarray analysis as down-regulated in crown tissues of the type II necrosis line (crt/KU-2025) under normal temperature compared with WT.

<p>Top 13 transcription factor genes identified by microarray analysis as down-regulated in crown tissues of the type II necrosis line (crt/KU-2025) under normal temperature compared with WT.</p

Top 13 transcription factor genes identified by microarray analysis as up-regulated in crown tissues of the type II necrosis line (Ldn/KU-2025) under LT relative to normal temperature.

<p>Top 13 transcription factor genes identified by microarray analysis as up-regulated in crown tissues of the type II necrosis line (Ldn/KU-2025) under LT relative to normal temperature.</p

Heat map for the altered gene expression levels of the selected 9,974 probes in the three examined crown tissues; WT (Ldn/KU-2059) and grass-clump dwarf (Ldn/KU-2025), WT (Ldn/KU-2059) and grass-clump dwarf (Ldn/KU-2025), and grass-clump dwarf (Ldn/KU-2025) under the normal temperature and LT.

<p>The branches of the trees are arranged according to the Pearson correlation coefficients. The distance threshold adjustment resulted in six major clusters. The colour-scale shows log₂ fold change values.</p

Comparison of transcript accumulation of the three MADS-box genes in crown tissues of the WT and type II necrosis/grass-clump dwarf lines.

<p>(A) Phylogenetic tree of the deduced MADS-domain amino acid sequences in the <i>AP1</i>/<i>FUL</i>-type MADS-box genes from <i>Arabidopsis</i>, rice and wheat. Bootstrap values after 1,000 replicates are shown at nodes. (B) Transcript accumulation levels of the three MADS-box gene transcripts from qRT-PCR. The transcript levels are shown as values relative to those in crown tissues of Ldn/KU-2059 under normal temperature. Means ± SD were calculated from data obtained in three experiments. The <i>Actin</i> gene was used as an internal control. Student’s <i>t-</i>test was used to test for statistical significance (***<i>P</i><0.001) between WT and grass-clump dwarf synthetic plants.</p

Comparison of transcript accumulation of the three miR156 molecules in the WT and type II necrosis/grass-clump dwarf lines.

<p>Means ± SD were calculated from data obtained in three qRT-PCR experiments. 18S rRNA was used as an internal control. (A) The miR156 levels in Ldn/KU-2159 and Ldn/KU2012 under normal temperature. Student’s <i>t</i>-test was used to test for statistical significance (***<i>P</i><0.001) between crown tissues of the two lines under normal temperature. (B) The miR156 levels in crown tissues of Ldn/KU-2059 and Ldn/KU-2025. Student’s <i>t</i>-test was used to test for statistical significance (***<i>P</i><0.001) in the two lines between growth at the normal temperature and LT.</p