Characterization of Hypoxia Induced Maize miRNAs Figure 6. Cluster miRNA targets expression profile. A: Model of miRNA target expression pattern. B: Heatmap of miRNA target expression profile. The cluster was done on the basis of log2 (expression level in treatment/expression level in control). Yellow shows down-regulation. Blue shows up-regulation. doi:10.1371/journal.pone.0039786.g006 genereg.net/) [63] (see methods). We found the occurrence of 18 stress and development associated transcription factor binding sites (TFBS) representing 8 TF families in the selected promoter region of miRNAs (Table S11). Out of 30 differentially expressed miRNAs in B73 that were studied for motif predictions, six miRNA genes (miR159h, miR159i, miR172c, miR398b, miR408b, miR528a) related to cluster 3 are induced whereas rest of the 24 miRNAs genes related to cluster 1 and 2 are repressed under hypoxia conditions. Computational predictions of the motifs in the promoters of the miRNAs of these three clusters showed that these miRNAs were found to have the binding sites of high mobility group proteins (HMG) [64], basic helix loop helix (bHLH) [65], homeodomain- leucine zipper proteins (HD-Zip) [66,67], AP2/ERF [68], bZIP [69,70,71], DOF [72], MYB [73,74,75] and MADS [76,77,78] TF families (Figure 9, Table S11 and S12). We have adopted the rigorous filtering methods to predict cis-regulatory elements, but cannot completely eliminate the false positive predictions; therefore these predictions should be experimentally verified for future studies. Cluster 1 and Cluster 2 motifs. The promoter regions of the majority of miRNAs present in cluster 1 and 2 were found to harbor the binding sites of TFs including HMG-I/Y, indeterminate Figure 7. Expression of zma-miR528a/b and one of its targets. A: SLRT-PCR results of miRNA528a/b at 3 biological replicates. B: qRT- PCR results of one of miRNA528a/b targets GRMZM2G039381 at 3 biological replicates. X-axis shows the inbred lines under different time point treatment compared to control. Y-axis shows the expression level. Error bars show the standard deviation. Student t test was performed on qRT-PCR results. ** denotes the p value ,0.01 and * denotes the p value ,0.05. doi:10.1371/journal.pone.0039786.g007 protein (ID1), HAT5 (also called as ATHB1), ABA insensitive protein (ABI4) and plastid envelope DNA binding protein (PEND) those belong to HMG, bHLH, HD-Zip, AP2/ERF, and bZIP TF families respectively (Figure 9, Table S12). Based on the computational analysis of the promoter regions, the cluster 2 miRNAs found to be regulated by more TFs than those from the cluster 1 miRNAs. The binding sites of TFs such as ATHB5 (HD- Zip), bZIP911 (bZIP), Em-binding protein -1 (EMBP-1, bZIP), HMG-1 (HMG), DOF and MYB TFs families were predominant in cluster 2 miRNA promoter regions whereas squamosa SQUA (MADS) binding sites were more present in cluster 1 miRNAs. The putative binding sites of EMBP-1 found to be enriched in the promoter regions of four miRNA families (miR156b, miR156k, miR164e and miR398b) and these miRNAs were demonstrated to be involved in different parts of plant development and stress [79]. Cluster 3 motifs. Expression levels of miR159h/i, miR172c, miR398b, miR408b, and miR528a were decreased in Mo17 but were increased in B73 and Hz32. The putative binding sites of ABI4 and HD-Zip domain binding motifs were present in miR408b and miR528a whereas putative motifs of bZIP911 and TGA1A of bZIP family were present in miR398b and miR408b. Additionally, putative binding sites of HMG, and SQUA TFs were present only in miR172c. The presence of binding sites of ABI4, HD-Zip and bZIP motifs on the miRNA promoter of this cluster suggests that these TFs are likely helping the induction of miRNA families of this cluster and may be involved in hormonal signal transduction which may repress the lateral root development and inhibit the root or shoot elongation in order to save limited oxygen supply and energy resources. miRNA-mediated Gene Regulatory Sub Network We constructed short-term hypoxia induced probable miRNA- mediated gene regulatory network using Cytoscape [80] (Figure 9). For the upstream miRNA promoter region (TF-miRNA), we predicted the putative binding sites of the TFs based on the experimentally derived PWMs that might be modulating the expression of differentially expressed miRNA genes implicated under waterlogging (Table S11 and S12). For the downstream analysis (miRNA-target), we selected the targets based on the expression profiles of miRNA gene target candidates by qRT- PCR, which were negatively correlated with their miRNAs (Table S8 and S13). The miRNA targets were functionally annotated based on the TF domains [http://www.ebi.ac.uk/interpro/], therefore individ- ual target genes were labeled based on their TF families. To gain the deeper biological insights from miRNA-mediated pathways, we constructed the regulatory network based on TF families for TF-miRNA and miRNA-TF interactions. miRNA-mediated regulatory interaction network consists of 47 nodes and 133 directed edges (Figure 9). The set of 47 nodes consists of 30 miRNAs (from 13 miRNA families), and 16 TF families. Out of these 16 TF families, TFs of 4 TF families (shown in teal color) putatively bind to the miRNA promoter region whereas target genes belonging to 8 TF families (light green color) are negatively regulated by miRNA genes, and the TFs of remaining 4 TF families (royal blue color) are involved in both upstream (TF- miRNA) and downstream (miRNA-target) interactions (Figure 9A). TF-miRNA interactions are shown as black arrows whereas downstream miRNA-target interactions are shown as red color PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39786