Characterization of Hypoxia Induced Maize miRNAs Figure 3. Flowchart of the novel miRNA prediction pipeline. The flowchart shows the filtering criteria to reduce and refine the numbers of small RNAs to the most interesting subset based on the abundance, removal of repeats (uniqueness), removal of known miRNAs, other ncRNAs, and folding structure. 10 unique sequences were identified as potentially novel miRNA candidates. Light blue squares show the filtered out sequences. Blue squares show the sequences that passed the filter. doi:10.1371/journal.pone.0039786.g003 miRNAs do not correspond to the differences in the inbred sensitivity to waterlogging. In the sensitive line (Mo17), the 22 known mature miRNA candidates were generally repressed or showed late, weak, or transient induction. This contrasts with the tolerant line (Hz32) in which these miRNAs generally showed early, strong, and persistent induction. The mid-tolerant (B73) line exhibited a distinct pattern in which some miRNA genes were induced early and persistent while others were not induced or repressed. Cluster 3 miRNAs were induced in both mid-tolerant (B73) and tolerant (Hz32) lines and directly involved in stress response, which indicated that miRNAs from cluster 3 might contribute to B73’s tolerance under waterlogging and be involved in the initial signal transduction. miRNAs from cluster 1 and 2 showed significant differential changes only in the waterlogging-tolerant line (Hz32) and are basically involved in development and stress response. The differential expression of miRNAs among the three clusters showed that these miRNAs were very sensitive to hypoxia and altered at different time points even within a very short time treatment, indicating that miRNAs may be one of the initial signal transduction components in responding to hypoxia at early time. Differential Expression of miRNA Targets in Response to Short-term Waterlogging A large proportion of miRNA targets are proteins that encode TFs required for growth, development, and stress responses [21,49]. A total of 119 out of 159 (74.8%) computational predicted targets [11] of the known and novel hypoxia-induced miRNAs were assayed by qRT-PCR. Due to challenges associated with sequence paralogs in maize, only 77.3% (92 out of 119) of the predicted targets had single amplicon and these were used for further analysis. Many of the potential target genes assayed were TFs of the squamosal l-promoter binding protein (SBP), myeloblast (MYB), Cys3His zinc finger domain (C3H), Teosinte branched1/Cycloi- dea/Proliferating cell factors 1 and 2 (TCP), CCAAT-binding factor (CBF), auxin response factor (ARF), APETALA2-ethylene response factor (AP2/ERF), basic region/leucine zipper motif (bZIP), growth regulating factor (GRF) and no apical meristem (NAM) classes. The miRNA targets were sub-divided into four groups based on the previously defined miRNA expression clusters (Figures S2, S3, S4 and S5). However, only the targets that showed negative correlation of expression levels with their miRNAs in all three inbred lines considered to be significantly affected by the miRNA and these were selected from each clusters and grouped accordingly (Figure 6). Cluster 1. 11 out of 28 (42.9%) miRNA targets (from Figure S2) showed changes in expression that negatively correlated with respect to their corresponding miRNAs in all three inbred lines. These targets belonged to TF families MYB, ARF and GRF, which are involved in root development, plant secondary metabolism and cell fate [50,51,52] (Table S6). Cluster 2. 15 out of 41 (36.6%) miRNA targets (from Figure S3) showed changes negatively correlated with their miRNAs in all three inbred lines. These targets belonged to CBF, K-box, LRR (leucine-rich repeat), MYB, SBP, TCP, TIR1-like (F-box), Zinc-Finger families, methyltransferase and rhodopsin-like receptor, which are involved in signal transduction, protein interaction, meristem growth and plant development [53,54,55,56,57,58] (Table S6). Cluster 3. 16 out of 23 (69.6%) miRNA targets (from Figure S4) showed negative correlation to their miRNAs in all three inbred lines. These targets belonged to AP2/ERF, MYB, cupredoxin, and EF hand. AP2/ERF proteins have important functions in the transcriptional regulation of a variety of biological processes related to growth and development, as well as various responses to environmental stimuli [59]. Targets such as cupredoxin (miR408 and miR528 targets) are involved in oxidative stress response [60]. An example of miR528a/b and expression of one of its targets, PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39786