Tissue-Specific Transcriptomic Profiling of <em>Sorghum propinquum</em> using a Rice Genome Array

<div><p>Sorghum (<i>Sorghum bicolor</i>) is one of the world's most important cereal crops. <i>S. propinquum</i> is a perennial wild relative of <i>S. bicolor</i> with well-developed rhizomes. Functional genomics analysis of <i>S. propinquum</i>, especially with respect to molecular mechanisms related to rhizome growth and development, can contribute to the development of more sustainable grain, forage, and bioenergy cropping systems. In this study, we used a whole rice genome oligonucleotide microarray to obtain tissue-specific gene expression profiles of <i>S. propinquum</i> with special emphasis on rhizome development. A total of 548 tissue-enriched genes were detected, including 31 and 114 unique genes that were expressed predominantly in the rhizome tips (RT) and internodes (RI), respectively. Further GO analysis indicated that the functions of these tissue-enriched genes corresponded to their characteristic biological processes. A few distinct <i>cis</i>-elements, including ABA-responsive RY repeat CATGCA, sugar-repressive TTATCC, and GA-responsive TAACAA, were found to be prevalent in RT-enriched genes, implying an important role in rhizome growth and development. Comprehensive comparative analysis of these rhizome-enriched genes and rhizome-specific genes previously identified in <i>Oryza longistaminata</i> and <i>S. propinquum</i> indicated that phytohormones, including ABA, GA, and SA, are key regulators of gene expression during rhizome development. Co-localization of rhizome-enriched genes with rhizome-related QTLs in rice and sorghum generated functional candidates for future cloning of genes associated with rhizome growth and development.</p> </div

The list of genes enriched specifically in rhizome tips relative to other tissues.

a<p>FC:Fold Change, represents the ratio of Avg_RT vs. MAX (Avg_ST, Avg_RI, Avg_SI, and Avg_YL), and q-value (%) ≤5%, while Avg_x represents the average ratio of the three biological replicates while RT for Rhizome tips/control, ST for Shoot tips/control, RI for Rhizome internodes/control, SI for Stem internodes/control and YL for Young leaves/control.</p

Identification of distinct <i>cis</i>-regulatory elements in the tissue-enriched genes.

a<p>W stands for [AT], ie A or T. ^bP value represents the significance between RT and ST, or RI and SI.</p

The list of up regulated genes in the comparison of the rhizome related DEGs.

a<p>A∼G represent the expression level of the 7 gene sets including DEGs of rhizome tip verse shoot tip in <i>O. longistaminata</i> from transcriptome sequencing data (A), microarray analysis data (C) and results of the present study in <i>Sorghum propinquum</i> (E), DEGs of underground tissues (rhizome tip and rhizome internode) verse above ground tissues (shoot tip, shoot internode and young leaf) in <i>O. longistaminata</i> from transcriptome analysis (B), microarray analysis (D) and results of the present study in <i>Sorghum propinquum</i> (F), and candidate rhizome-enriched genes in <i>S. Halepense</i> (pSH) and <i>S. propinquum</i> (G).</p

Validation of microarray data by <i>in situ</i> hybridization.

<p><i>In situ</i> localization of transcripts corresponding to the genes (a) Sb01g047010 and (c) Sb06g028820 in <i>S. propinquum</i> rhizome tips are illustrated; (b) and (d) represent the sense probe for control. Corresponding microarray-based expression profiles of these two genes are also shown as bar graphs for comparison.</p

Heat map showing the relationship between rhizome related differentially expressed genes (DEGs) and hormone target genes.

<p>The heat map was produced by analyzing genes comprising rhizome related DEGs for methyl jasmonate (MJ), ethylene (C<i>2</i>H<i>4</i>), abscisic acid (ABA), auxin (AUX), gibberellic acid (GA), zeatin, brassinosteroids (BR), and salicylic acid (SA). Subfigures a–g represent the seven gene sets analyzed: DEGs of RT vs. ST in <i>O. longistaminata</i> from (a) transcriptome sequencing and (c) microarray analysis, and (e) results of the present study in <i>S. propinquum</i>; DEGs of underground tissues (RT and RI) vs. above-ground tissues (ST, SI, and YL) in <i>O. longistaminata</i> from (b) transcriptome sequencing and (d) microarray analysis, and (f) results of the present study in <i>S. propinquum</i>; and (g) candidate rhizome-enriched genes in <i>S. halepense</i> (pSH) and <i>S. propinquum</i>. In the HORMONOMETER analysis, orange (1) = complete correlation, white (0) = no correlation, and blue (-1) = anti-correlation.</p

Identification of distinct <i>cis</i>-regulatory elements in the DEGs shown the same expression pattern in at least two different platforms.

a<p>P value represents the significance between up regulated and down regulated genes.</p

Real time PCR profiles of 12 selected tissue-enriched genes.

<p>RT, ST, RI, SI, and YL represent the rhizome tip, shoot tip, rhizome internodes, shoot internodes, and young leaves, respectively. Expression levels were calculated based on the expression level of YL genes set to 1. Expression profiles obtained by real time PCR for one gene, Sb01g036550, were not consistent with data obtained from microarray analysis. Correlation coefficients (r) for the remaining 11 genes were 0.74, 0.74, 0.74, 0.74, 0.77, 0.74, 0.82, 0.76, 0.76, 0.94, and 0.76, from left to right, respectively. Bars donate standard deviation.</p

GO slim categories in up- and down-regulated RT genes combined from this experiment and three other reported studies.

<p>Bars show number of genes with significantly higher relative transcript abundance. All GO slim categories significantly over- or underrepresented are calculated based on a hypergeometric distribution. Significant over- or under-represented categories are indicated by * for <i>p</i> ≤ 0.05, ** for <i>p</i> ≤ 0.01, and *** for <i>p</i> ≤ 0.001.</p

Comparative Transcriptome Profiling of Chilling Stress Responsiveness in Two Contrasting Rice Genotypes

<div><p>Rice is sensitive to chilling stress, especially at the seedling stage. To elucidate the molecular genetic mechanisms of chilling tolerance in rice, comprehensive gene expressions of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) with contrasting responses to chilling stress were comparatively analyzed. Results revealed a differential constitutive gene expression prior to stress and distinct global transcription reprogramming between the two rice genotypes under time-series chilling stress and subsequent recovery conditions. A set of genes with higher basal expression were identified in chilling-tolerant LTH compared with chilling-sensitive IR29, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulation in the chilling- tolerant genotype and strong repression in chilling-sensitive genotype. Early responses to chilling stress in both genotypes featured commonly up-regulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant genotype, while the chilling-sensitive genotype displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially-regulated TF genes and enriched <em>cis</em>-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3 regulons, were involved in chilling stress tolerance. A number of the chilling-regulated genes identified in this study were co-localized onto previously fine-mapped cold-tolerance-related QTLs, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for chilling tolerance in rice.</p> </div