Co-expression analysis of tissue-specific DEGs and MAPKKK genes.

<p>The weight value obtained from the WGCNA package was used as a parameter for the parametric analysis of gene co-expression levels. A cutoff of 0.2 was used to select highly co-expressed genes in all three tissues. The left side of the heat map represents the Z-scores obtained from a parametric analysis of gene co-expression. The lower left bar represents the degrees of the Z-score scale. The right side of the heat map represents the expression patterns of the DEGs co-expressed with MAPKKKs that were enriched in the corresponding pathways on the left. The lower right bar represents the log2 of the drought/control ratio.</p

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

<div><p>The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (<i>Zea mays</i> L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.</p></div

Relative gene expression of the 8 MAPKKK genes in various inbred lines based on qRT-PCR analysis.

<p>To determine whether the relative expression levels of the drought stress-responsive MAPKKK genes differed among varieties induced by drought, ZD619 and the 6 inbred maize lines J24, J853, X178, E28, C8605-2, 200B, Q319 and B73 were used. Lines X178, J24 are drought-resistant lines; 200B and E28 have poor drought tolerance. The eight MAPKKK genes are <i>GRMZM2G305066</i> (<i>ZmMAPKKK18</i>), <i>GRMZM2G165099</i> (<i>ZmMAPKKK19</i>), <i>GRMZM2G476477</i> (<i>ZmMAPKKK20</i>), <i>GRMZM2G173965</i> (<i>ZmMAPKKK21</i>), <i>GRMZM2G041774</i> (<i>ZmMAPKKK22</i>), <i>GRMZM2G021416</i> (<i>ZmMAPKKK26</i>), <i>GRMZM2G063069</i> (<i>ZmMAPKKK56</i>), <i>GRMZM2G474546</i> (<i>ZmMAPKKK73</i>). * indicates significant differences in comparison with the control at P < 0.05 respectively. Error bars indicate standard deviation for three replicates.</p

Differentially expressed genes (drought vs. control).

<p>Differentially expressed genes (drought vs. control).</p

Differential expression of MAPKKKs identified by RNA-seq and qRT-PCR.

<p>(a, b and c). Expression patterns of 71 MAPKKK genes in the three tissues. (a). ZIK family genes. (b). MEKK family genes. (c). Raf family genes. (d). Relative expression levels of the MAPKKK genes in various tissues based on qRT-PCR analysis. The expression in leaf is shown on the left. The expression in stem is shown on the right.</p

A model of drought stress effects on maize.

<p>The colored shapes represent groups of glyphs with similar processes or events. The arrows ending in a solid triangle indicate positive effects. The arrows ending in a transverse line indicate a clear negative influence. The arrows ending in a hollow triangle indicate co-expression. The curved arrows indicate positive feedback.</p

The correlation between relative expression levels of the 8 MAPKKK genes based on qRT-PCR analysis and relative fresh and dry biomass in 8 maize inbred lines under well-watered and drought-stress conditions.

<p>* indicate significant correlation at P<0.05 respectively.</p><p>The correlation between relative expression levels of the 8 MAPKKK genes based on qRT-PCR analysis and relative fresh and dry biomass in 8 maize inbred lines under well-watered and drought-stress conditions.</p

Pathway enrichment of differentially expressed genes involved in different regulatory processes under drought stress.

<p>A q-value cutoff of 0.05 was used to select enriched gene sets in all three tissues. The heat map represents the Z-scores obtained from a parametric analysis of gene set enrichment q-values for term enrichment. Red represents enriched genes in the treatment group that were over-represented compared with the control set. Blue represents the enriched genes in the treatment group that were under-represented compared with the control set. The absolute values represent the enrichment level. The bar represents the Z-score region from -3 to 3.</p

Phylogenetic tree and genomic locations of maize, rice and Arabidopsis MAPKKK family genes.

<p>The different colors represent three species. Red represents maize, blue represents rice, and pink represents Arabidopsis. (a). Raf subfamily. (b). MEKK subfamily. (c). ZIK subfamily. (d). Physical locations of MAPKKK genes on maize chromosomes.</p

Functional categorization of differentially expressed genes (DEGs) in three tissues.

<p>The top four GO terms are functional categorizations (biological process) of the DEGs that were enriched in all three tissues, and the lower seven GO terms are functional categorizations (biological process) of the DEGs enriched in two tissues. The enrichment figure was constructed from significantly enriched GO terms only. The X-axis is the -log₁₀ (p-value), which represents the level of enrichment, and the cutoff of the p-value is 0.05. Blue represents leaf tissue, red represents stem tissue, and green represents root tissue.</p