Characterization of Hypoxia Induced Maize miRNAs Figure 9. miRNA-mediated gene regulatory sub-network of short-term waterlogging stress in B73. A. TF-miRNA and miRNA-target interactions are shown. B. Probable negative feedback loop between GAMYB and miR319b is shown. Upstream motifs and downstream targets are indicated as circular nodes whereas miRNA are denoted as diamond nodes. Teal color TF nodes represent the upstream motifs, light green nodes represent miRNA targets, whereas royal blue color nodes denote TFs which are involved in both upstream and downstream interactions. Yellow color miRNA shows down regulated miRNAs whereas violet color shows up regulated miRNAs. TF-miRNA interactions are shown as black arrows whereas downstream miRNA-target interactions are shown as red color lines. doi:10.1371/journal.pone.0039786.g009 polypeptides are synthesized leading to the accumulation of alcohol dehydrogenase [4]. Based on our observed differential expression of miRNAs, we hypothesize that these inbred lines might be employing two different miRNA-mediated energy-saving strategies to survive short-term waterlogging stress. The first strategy might be applicable for more sensitive inbred line (Mo17), where the down-regulation of most of the miRNAs would lead to over-expression of downstream targets. As the majority of the targets in our studies are transcriptional activator TFs, probably the rapid involvement of these miRNAs in different signaling pathways are using their entire limited store of O2 and sending signals for potentially enhancing the existing developmental regime. However, accelerated O2 consumption in this process is a short-term adaptation and might decrease their overall long- term efficiency and fitness and thus making them susceptible to waterlogging stress. An alternative strategy might be applicable for the more tolerant inbred line (Hz32), wherein specific miRNA genes are up regulated thereby reducing their respective target expression levels and causing repression of most of the growth and elongation signaling pathway genes during first few hours. This would shut down unnecessary gene expression and would reserve O2 and energy by only inducing genes necessary to withstand the hypoxia stress. The mid-tolerant line, B73 falls somewhere in the middle, with an intermediate response where majority of the miRNA genes are repressed like Mo17 while some miRNA genes are induced as in Hz32. Expression analysis of targets and promoter analysis in differentially expressed miRNAs subjected to short-term water- logging stress indicates that the responses might be involved in three major signal transduction pathways, which are ethylene and ABA-dependent signaling pathway, auxin-signaling pathway and cupredoxin-mediated oxidative stress responding pathway. To survive under waterlogging, low production of O2 might cause ethylene production which promotes death of parenchyma cells that results in the formation of aerenchyma in shoots and roots of a large number of plant species including maize, rice and Arabidopsis [82,83,84]. ABA is also known as growth inhibitor and the rapid decline in endogenous ABA under waterlogging appears to be due to an increase in ABA breakdown and suppression of its biosynthesis [85]. However, the removal of ABA itself is not the indicator of faster elongation, it merely unlocks ethylene-promoted extension [85]. In Arabidopsis, miR159 regulates ABA signaling by targeting MYB33 and MYB101 [17]. In our study, MYB33 homolog (GRMZM2G28054) and MYB101 homolog (GRMZM2G139688) are targeted by miR159 which have been validated by the 59-RLM-RACE (Figure 8). The induction of miR159 in waterlogging tolerant line Hz32 suggests miR159 repress the ABA signal by repressing MYB proteins. The second signal transduction pathway is an auxin-signaling pathway that is involved in plant root development. miR164, miR167 and miR393 have been shown to be involved in root cap formation, lateral root development, or adventitious rooting through the auxin signal which is further transduced by their downstream NAC/NAM, ARF and F-box targets respectively [79]. The NAC (NAM/ATAF/CUC) family is involved in embryo and shoot meristem development, lateral root formation, auxin signaling, defense and abiotic stress responses [86]. Auxin-induced expression of miR164 and subsequent cleavage of NAC1 provides a homeostatic mechanism to down-regulate auxin signals for lateral root development in Arabidopsis [87]. NAC-domain protein (GRMZM2G009892) and NAM protein (GRMZM2G146380) were predicted targets of miR164 in our study. qRT-PCR results showed the expression of GRMZM2G146380 was negatively correlated with miR164 in all three inbred lines (Figure 6) while GRMZM2G009892 only showed negative correlation in Hz32 (Figure S4). The induction of miR164 in Hz32 might suggest that miRNA mediated the breakdown of NAC/NAM mRNA to attenuate waterlogging signals, which led to reduce embryo/shoot meristem or lateral root production signals at the early stage of waterlogging. The regulatory pathway consisting of miR393 that targets TIR1, a negative regulator in auxin signaling [88], may participate in lateral root development in Arabidopsis [79]. In our study, miR393 targeted TIR1-like (F-box) gene (GRMZM2G135978) (Figure 6 and 8), which was co-expressed PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39786