Characterization of Hypoxia Induced Maize miRNAs Figure 4. Expression profiles of 24 candidate miRNAs under hypoxia stress. Blue bars show the results of small RNA library in Hz32 under 4 h treatment. Red bars show the results of SLRT-PCR in Hz32 under 4 h treatment at 3 biological replicates. Vertical red line divides the expression profiles of 24 candidate miRNAs with 4 miRNAs (control expression profiles). Error bars show the standard deviation. Chi-square was applied on the small RNA library data and student t-test was performed on SLRT-PCR results. ** denotes the p value ,0.01 and * denotes the p value ,0.05. doi:10.1371/journal.pone.0039786.g004 cupredoxin (GRMZM2G039381) that shows negative correlation with its miRNA, is shown in Figure 7. Cluster of novel miRNA targets: Out of eight targets of two novel miRNAs (miRn6 and miRn7), five targets showed negative correlation to their miRNAs in most of the three inbred lines. Based on the interpro [http://www.ebi.ac.uk/interpro/] annota- tions, two targets have a keratin-like (K-box) domain; one has catalase like domain; and remaining two are of uncharacterized function (Figure S5). K-box domain is one of domains of MADS genes that plays important roles in protein-protein interactions [57]. MADS genes are key developmental regulators of vegetative and reproductive development [58]. Catalase plays role in protecting maize roots from toxic effects of hydrogen peroxide (H2O2) under severe environmental conditions [61]. Based on the expression profiles of miRNA gene target candidates by qRT-PCR, we found fewer than 50% of the targets demonstrated expression levels that were negatively correlated with their miRNAs. There can be two possible reasons. First, recent studies have shown that, in addition to RNA cleavage, miRNAs in plants have widespread translational repressor capability [62], so many of these targets may in fact be regulated through this translational pathway. Second, the miRNA targets may be regulated by more than one miRNA or other TFs, which will lead to different expression pattern. However, the fact that miRNA targets express in different inbred lines in response to waterlogging treatment suggested that miRNAs play a role in regulating transcript expression. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39786