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

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

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

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 log₂ ratio values of probe sets were used for the analysis with R software. The colored bars represent the value (log₂(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

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

Vesicle trafficking in the <i>pgkc-1</i> mutant.

<p><b>(A)</b> EYFP-RABA4D signal in WT and <i>pgkc-1</i> pollen tubes subjected to mock or 0.4 μM BFA treatment. Scale bar = 5 μm. <b>(B)</b> EYFP-RABA4D signal intensity. Measurements were performed as described in Materials and Methods. Fifteen to twenty pollen tubes from each sample were measured. 0 μm indicates the position of apical tip. Error bars on curves indicate standard error. <b>(C and D)</b> WT and <i>pgkc-1</i> pollen tube morphology when subjected to <b>(C)</b> mock and <b>(D)</b> 0.4 μM BFA treatment. Scale bar = 50 μm. <b>(E to G)</b> WT and <i>pgkc-1</i> plant pollen germination <b>(E)</b>, pollen tube length <b>(F)</b>, and pollen tube width <b>(G)</b> when subjected to mock or 0.4 μM BFA treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus mock treatment as determined using Student’s <i>t</i>-test (** = p < 0.001).</p

Effects of disrupting GAPDH on pollen tube polarity.

<p><b>(A)</b> Glycolytic pathway of GAPDH and PGK. <b>(B to G)</b> Pollen tube morphology of WT <b>(B)</b>, <i>pgkc-1</i> <b>(C)</b>, and <i>ren1-3</i> <b>(D)</b> plants subjected to mock treatment; pollen tube morphology of WT <b>(E)</b>, <i>pgkc-1</i> <b>(F)</b>, and <i>ren1-3</i> <b>(G)</b> plants treated with 40 μM CGP 3466B. Both <i>pgkc-1</i> and <i>ren1-3</i> plants were dramatically depolarized by CGP medium. Scale bar = 50 μm. <b>(H and I)</b> Pollen tube length <b>(H)</b> and width <b>(I)</b> of WT, <i>pgkc-1</i>, and <i>ren1-3</i> pollen tubes subjected to mock and 40 μM CGP treatment. Bars represent mean ± SEM. Asterisks indicate significant differences versus either single mutant as determined using Student’s <i>t</i>-test with either single mutant (** = p < 0.001). <b>(J)-(M)</b> Average signal intensity along WT pollen tubes subjected to mock or CGP treatment of <b>(J)</b> GFP- REN1, <b>(K)</b> CRIB4-GFP, <b>(L)</b> Lifeact-mEGFP, <b>(M)</b> EYFP-RABA4D. Measurements were performed as described in Materials and Methods. Fifteen pollen tubes from each sample were measured. The 0 μm label indicates the position of the apical tip.</p

Glycolysis regulates pollen tube polarity via Rho GTPase signaling

<div><p>As a universal energy generation pathway utilizing carbon metabolism, glycolysis plays an important housekeeping role in all organisms. Pollen tubes expand rapidly via a mechanism of polarized growth, known as tip growth, to deliver sperm for fertilization. Here, we report a novel and surprising role of glycolysis in the regulation of growth polarity in <i>Arabidopsis</i> pollen tubes via impingement of Rho GTPase-dependent signaling. We identified a <i>cytosolic phosphoglycerate kinase</i> (<i>pgkc-1</i>) mutant with accelerated pollen germination and compromised pollen tube growth polarity. <i>pgkc-1</i> mutation greatly diminished apical exocytic vesicular distribution of REN1 RopGAP (Rop GTPase activating protein), leading to ROP1 hyper-activation at the apical plasma membrane. Consequently, <i>pgkc-1</i> pollen tubes contained higher amounts of exocytic vesicles and actin microfilaments in the apical region, and showed reduced sensitivity to Brefeldin A and Latrunculin B, respectively. While inhibition of mitochondrial respiration could not explain the <i>pgkc-1</i> phenotype, the glycolytic activity is indeed required for PGKc function in pollen tubes. Moreover, the <i>pgkc-1</i> pollen tube phenotype was mimicked by the inhibition of another glycolytic enzyme. These findings highlight an unconventional regulatory function for a housekeeping metabolic pathway in the spatial control of a fundamental cellular process.</p></div