15 research outputs found
Transcriptomic Analysis of Responses to Imbalanced Carbon: Nitrogen Availabilities in Rice Seedlings
<div><p>The internal C:N balance must be tightly controlled for the normal growth and development of plants. However, the underlying mechanisms, by which plants sense and balance the intracellular C:N status correspondingly to exogenous C:N availabilities remain elusive. In this study, we use comparative gene expression analysis to identify genes that are responsive to imbalanced C:N treatments in the aerial parts of rice seedlings. Transcripts of rice seedlings treated with four C:N availabilities (1:1, 1:60, 60:1 and 60:60) were compared and two groups of genes were classified: high C:low N responsive genes and low C:high N responsive genes. Our analysis identified several functional correlated genes including <i>chalcone synthase</i> (<i>CHS</i>), <i>chlorophyll a-b binding protein</i> (<i>CAB</i>) and other genes that are implicated in C:N balancing mechanism, such as <i>alternative oxidase 1B</i> (<i>OsAOX1B</i>), <i>malate dehydrogenase</i> (<i>OsMDH</i>) and <i>lysine and histidine specific transporter 1</i> (<i>OsLHT1</i>). Additionally, six jasmonate synthetic genes and key regulatory genes involved in abiotic and biotic stresses, such as <i>OsMYB4</i>, <i>autoinhibited calcium ATPase 3</i> (<i>OsACA3</i>) and <i>pleiotropic drug resistance 9</i> (<i>OsPDR9</i>), were differentially expressed under high C:low N treatment. Gene ontology analysis showed that high C:low N up-regulated genes were primarily enriched in fatty acid biosynthesis and defense responses. Coexpression network analysis of these genes identified eight <i>jasmonate ZIM domain protein</i> (<i>OsJAZ</i>) genes and several defense response related regulators, suggesting that high C:low N status may act as a stress condition, which induces defense responses mediated by jasmonate signaling pathway. Our transcriptome analysis shed new light on the C:N balancing mechanisms and revealed several important regulators of C:N status in rice seedlings.</p></div
Coexpression network analysis of high C:low N up-regulated genes.
<p>(A) Module 1 extracted from coexpression analysis using 14 microarray identified genes. (B) Module 2 extracted from coexpression analysis using 9 microarray identified genes. Red and blue nodes indicate high C:low N up-regulated genes and the red ones are transcription factors. Other genes with known names or encode for transcription factors are marked on the nodes. Genes involved into KEGG pathways are marked with color dots beneath the nodes and the detailed information are listed on the tables.</p
Validation of microarray results by qRT-PCR.
<p>(A) <i>OsLOX</i>; (B) <i>OsAOS2</i>; (C) <i>OsOPR5</i>; (D) <i>OsCHS</i>; (E) <i>OsCAB2</i>; (F) <i>OsPERO</i>. <i>Actin6</i> was used as the internal reference. The gray bars indicated the fold change of the genes between treatments (1:60, 60:1 and 60:60) and the control (1:1). Values are shown as means ± SDs from three technical replicates. A representative experiment of two biological replicates is shown.</p
Experimental design to identify genes responsive to imbalanced C:N.
<p>(A) N starved rice seedlings were treated with four different C:N conditions: balanced C:N (1:1 and 60:60) or imbalanced C:N (1:60 and 60:1). (B) Hypothetical models of genes responsive to exogenous imbalanced C:N conditions. Genes responsive to imbalanced high C:low N (60:1) or low C:high N (1:60) are proposed to show higher or lower expression levels compared with 1:1 and 60:60 treatments.</p
Identification of genes responsive to imbalanced high C:low N and low C:high N.
<p>(A-D) The Volcano Plots for differentially expressed genes between treatments. The two vertical lines are the 1.5-fold change boundaries and the horizontal lines are the statistical significance boundaries (<i>p</i><0.05). Genes with fold change>1.5 and statistical significance are marked with red dots. A, 60:1 compared with 1:1; B, 60:60 compared with 60:1; C, 1:60 compared with 1:1; D, 60:60 compared with 1:60. (E) Venn diagram of rice genes (probe sets) responded to C:N treatments. (F-H) Hierarchical cluster analysis of high C:low N and low C:high N responsive genes. The log<sub>2</sub> ratio values of probe sets were used for the analysis with R software. The colored bars represent the value (log<sub>2</sub>(fold change)) of the transcripts in each bin after C:N treatments. Green represents down-regulated probe sets, red represents up-regulated probe sets, and dark indicates no significant difference in gene expression. F, High C:low N up-regulated genes; G, High C:low N down-regulated genes; H, Low C:high N up-regulated genes. “vs” represents “compared with”.</p
qRT-PCR analysis of CN metabolic genes at different time points after C:N treatments.
<p>Expression patterns of <i>NR</i>, <i>GOGAT</i>, <i>GS</i>, <i>PEPCase</i> and <i>PK</i> were analyzed in rice seedlings treated with four different C:N conditions (A 1:1; B 1:60; C 60:1; D 60:60) for 1, 2, 3 and 4 h. The beginning of the treatment (0 h) was used as the control and <i>Actin6</i> served as the internal reference. Values are shown as means ± SDs from three technical replicates. A representative experiment of two biological replicates is shown.</p
Gene ontology (GO) enrichment analysis of genes up-regulated by high C:low N.
<p>The differentially expressed probe sets were analyzed by SEA (singular enrichment analysis) using AgriGO, and the comparison is displayed in graphical mode. Each box contains GO term number, the false discovery rate (FDR) value, GO term and item number associated with the GO term in the query list and background as well as total number of query list and background. The degree of color saturation of a box is positively correlated to the enrichment level of the term (the yellow-to-red represents the term is up-regulated while non-significant terms are shown as white boxes). Solid, dashed and dotted lines represent two, one and zero enriched terms at both ends connected by the line, respectively. (A) Biological process category analysis of high C:low N up-regulated genes; (B) Molecular function category analysis of high C:low N up-regulated genes; (C) List of screened genes in “fatty acid biosynthesis”, “defense response” and “oxidoreductase activity” categories with <i>p</i>-values.</p
Identification of <i>OsMPH1</i>-regulated genes through RNA-seq analysis.
<p><b>A</b> Type one Venn diagram showing the overlapping profile of up-regulated genes in the <i>OsMPH1V</i> transgenic line and down-regulated genes in the <i>OsMPH1E</i> line in comparison with the WT, as determined via RNA-seq; Type two Venn diagram showing the overlapping profile of down-regulated genes in the <i>OsMPH1V</i> transgenic line and up-regulated genes in the <i>OsMPH1E</i> line in comparison with the WT, as determined via RNA-seq. <b>B</b>, Type one and Type two gene function categories.</p
Transcription activation analysis of OsMPH1 in the yeast GAL4 system.
<p>Each transformed yeast strain was dropped onto SD/-W and SD/-W-H-Ade plates and allowed to grow for 48 hours before taking photographs. β-galactosidase activity was quantified through a liquid culture assay using CPRG as the substrate. Data are shown as the means ± s.d. (Student’s t tests, **P < 0.01, n = 3).</p
<i>OsMPH1</i> regulates plant height and improves grain yield in rice
<div><p>Plant height is a major trait affecting yield potential in rice. Using a large-scale hybrid transcription factor approach, we identified the novel MYB-like transcription factor OsMPH1 (MYB-like gene of Plant Height 1), which is involved in the regulation of plant height in rice. Overexpression of <i>OsMPH1</i> leads to increases of plant height and grain yield in rice, while knockdown of <i>OsMPH1</i> leads to the opposite phenotypes. Microscopy of longitudinal stem sections indicated that a change in internode cell length resulted in the change in plant height. RNA sequencing (RNA-seq) analysis of transgenic rice lines showed that multiple genes related to cell elongation and cell wall synthesis, which are associated with plant height and yield phenotypes, exhibited an altered expression profile. These results imply that <i>OsMPH1</i> might be involved in specific recognition and signal transduction processes related to plant height and yield formation, providing further insights into the mechanisms underlying the regulation of plant height and providing a candidate gene for the efficient improvement of rice yield.</p></div