<i>CBF</i> (<i>C-REPEAT/DRE BINDING FACTOR</i>) co-expression cluster is induced by submergence in maize.

<p>(a.) <i>CBF</i> co-expression network generated form genes differentially expressed at 24 h in comparisons between submerged and control plants. Each gene in the network is represented as a circle. (b.) Genes highlighted in the heatmap are represented by grey circles, while that with a red outline indicates the seed gene for the network. The heatmap highlights the expression of a subset of genes in B73, B97, Mo18W and M162W after 24 h of submergence. (c.) Expression of highlighted genes at 72 h after submergence. All genes showed significant differences in expression between submerged and control plants (FDR <0.001). The scale indicates log₂ fold-change. Up-regulation indicates higher expression in submerged samples. <i>CBF2</i>: <i>C-REPEAT/DRE BINDING FACTOR2</i>; <i>CBF3</i>: <i>C-REPEAT/DRE BINDING FACTOR3</i>; <i>ERF7</i>: <i>ETHYLENE RESPONSE FACTOR7</i>; <i>RRTF1</i>: <i>REDOX REPONSIVE TRANSCRIPTION FACTOR1</i></p

Summary of differential expression analysis.

<p>Number of differentially expressed transcripts from comparisons between submerged and control plants. All transcripts showed significant differences in expression (FDR <0.001).</p><p>Summary of differential expression analysis.</p

Real-time quantitative PCR of <i>PYROPHOSPHATE-DEPENDANT FRUCTOSE-6-PHOSPHATE 1-PHOSPHOTRANSERASE</i> (<i>PFP</i>) and anaerobic glycolysis genes during submergence.

<p>(a.) The expression of an unannotated <i>PYROPHOSPHATE-DEPENDANT FRUCTOSE-6-PHOSPHATE 1-PHOSPHOTRANSERASE</i> (<i>PFP</i>) gene in submerged shoot tissue during submergence. (b.) <i>PYRUVATE DECARBOXYLASE3</i> (<i>PDC3</i>) expression in shoot tissues during submergence. (c.) <i>ALCOHOL DEHYDROGENASE1</i> (<i>ADH1</i>) transcript levels in submerged shoot tissue. All sample were collected at 24 h and 72 h after submergence. Expression levels are relative to shoot tissue of control plants at 24 h. Letters above bars indicate nonsignificant differences in expression determined using Tukey’s HSD (<i>p</i> < 0.05). Statistical tests were performed separately for each timepoint. Plots without letters (ie 3a 72 h and 3c 72 h) indicate that all comparisons are significantly different. Error bars display standard error where <i>n</i> = 3.</p

Detection of ROS and expression of ROS marker genes in inbreds with contrasting levels of submergence tolerance.

<p>(a.) Detection of H₂O₂ and superoxide in selected submergence tolerant and sensitive maize genotypes. A subset of four nested association mapping panel parents were selected based on visual leaf scoring for ROS staining assays after submergence. Hydrogen peroxide was detected using stain 3,3′-diaminobenzidine tetrahydrochloride (DAB), while superoxide was detected using nitroblue tetrazolium (NBT). Both assays were performed after 96 h of submergence. In B97, the third leaf was barely emerged and not used for the assay. (b-d) Real-time quantitative PCR of ROS genes. ROS genes were selected based on the study by Gadjev, Vanderauwera and Gechev [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120385#pone.0120385.ref030" target="_blank">30</a>] and showed a significant induction in response to at least two stresses that induce ROS formation in Arabidopsis. (b.) <i>ALTERNATIVE OXIDASE 1a</i> (<i>AOX1a</i>); (c.) <i>WRKY6</i> (d.) <i>CYTOCHROME P81 D8</i> (<i>CYP81D8</i>). All sample were collected at 24 h and 72 h after submergence. Expression levels are relative to shoot tissue of control plants at 24 h. Letters above bars indicate nonsignificant differences in expression determined using Tukey’s HSD (<i>p</i> < 0.05). Statistical tests were performed separately for each time point. Plots without letters (ie 2c 24 h) indicate that all comparisons are significantly different. Error bars display standard error where <i>n</i> = 3.</p

<i>Subtol6</i> is associated with submergence tolerance in maize.

<p>(a.) Distribution of mean leaf scores for Mo18W x B73 RIL mapping population and parental inbreds. Leaf score was averaged across leaves 1 to 3. Scores for parental inbreds are indicated with red arrows. (b.) Interval mapping results for QTL identified in the Mo18W x B73 RIL family from the NAM population. LOD threshold was determined using 10,000 permutations. (c.) Observed genotypes of chromosome 6 for a subset of tolerant and sensitive RILs. Markers with the B73 allele are represented by open ovals, while Mo18W alleles are represented by closed ovals. (d.) Expression of a subset of genes that fall within the Bayes credible interval for the most significant marker. Expression data was derived from RNA sequencing of B73 and Mo18W after 24 h and 72 h after submergence. All genes between ∼141.4 to 162.8 Mb on chromosome 6 and showing significant differences in expression in comparisons between B73 and Mo18W were considered (FDR <0.001). The scale indicates log₂ fold-change. Up-regulation indicates higher expression Mo18W relative to B73. <i>TH9</i>: <i>THIOREDOXIN9</i> (GRMZM2G701204); <i>WRKY33</i> (GRMZM2G169966); <i>HB2</i>: <i>HEMOGLOBIN2</i> (GRMZM2G168898); <i>GSTU8</i>: <i>GLUTATHIONE S-TRANSFERASE8</i> (GRMZM2G097989); <i>RAV1</i>: <i>RELATED TO ABI3/VP1 1</i> (GRMZM2G059939); <i>ERF4</i>: <i>ETHYLENE RESPONSE FACTOR4</i> (GRMZM2G020150)</p