with miR393 (Figure 5). Up-regulation of miR393 only in Hz32 may inhibit lateral root production signal at the early stage of hypoxia. miR167 targets ARF6 and ARF8 TFs and these ARFs were also found to be conserved in rice [89]. The predicted targets of miR167 were ARF6/8 ortholog genes (Figure 6). ARF ortholog genes GRMZM2G028980, GRMZM2G035405 have been con- firmed to be cleaved by miR167 (Figure 8) and the expressions of all these ARF genes were negatively correlated with miR167 (Figure 5). Reduction of miR167 at the early stage of waterlogging (except after 4 h treatment in Hz32) suggested the over expression of its target ARF and that could induce the adventitious root development signal transduction. The third major pathway is the cupredoxin-mediated hypoxia- responding pathway. Cupredoxins are a family of copper proteins with redox activities [90]. Cupredoxins are also an important domain in numerous enzymes such as laccases, SOD, multicopper oxidases, metallothioproteins that catalyze several cellular and biological processes ranging from aerobic and anaerobic respira- tion to iron homeostasis [91]. Both miR408 and miR528 target cupredoxin. Both of these miRNAs are highly repressed in Mo17 and are induced in B73 and Hz32. It can be suggested that cupredoxin might be important in responding the early signal of waterlogging by mediating electron-transfer or oxidation homeo- stasis and thus preventing damage to cellular structures. In addition to these 3 pathways, other miRNAs might be involved in the waterlogging responses such as miR172 that serves as a negative regulator of AP2, which is involved in flowering development [92]. In our studies, an AP2/ERF domain protein (GRMZM2G076602) that is targeted by miR172b/c/d is reduced in Mo17 but induced in B73 and Hz32 at short-term waterlogging (Figure 6, cluster 3). However, the induced expression level of miR172b/c/d slowly decreases over time from 1 h to 4 h and shows down regulation at 4 h in B73 and Hz32. To better understand the molecular mechanisms regulating gene expression in response to waterlogging stress, our study has focused on the upstream (TF-miRNA) and downstream (miRNA- targets) interactions and their role in mediating stress responses. The identification of stress and development associated putative cis-regulatory elements in miRNA promoter region will advance our understanding of regulatory networks in which miRNAs play a crucial role. Majority of the miRNAs in three clusters have HMG, HAT5 (ATHB1), ATHB5, ABI4 and ID1 binding site motifs. HMG family proteins bind to an AT rich enhancer element [64,93]. These are involved in diverse nuclear processes and biological processes including growth, proliferation, differentiation and death and thus function as central hubs of nuclear function [94]. The functions of ATHB1, ATHB2 and ATHB5 belonging to HD-Zip family are mainly related to developmental events in response to abiotic stress, ABA and auxin signaling [95,96]. In Arabidopsis, putative motifs of ATHB1 and ATHB5 found to be present in the promoter region of hypoxia responsive miRNAs [31]. ABI4 (AP2/ ERF) is one of the key players that regulate ABA and sugar signaling pathways [68] and probably binds to GCC pathogenesis related promoter element [97]. ID1, a basic helix-loop-helix type (bHLH) protein, seems to play a key role in regulating the transition to flowering in maize by regulating floral inductive signals [98,99]. However, other TFs from bHLH family such as AtMYC2 and ATAIB function as transcriptional activators and found to be involved in the regulation of ABA signaling in Arabidopsis [100]. TFs like bZIP911, EMBP-1, TGA1A belonging to basic domain leucine zipper (bZIP) family were found enriched in cluster 2 and 3 miRNAs. bZIP 911 is known for activating ABA-responsive genes via binding to ABA-response element (ABRE) [101,102,103,104]. Characterization of Hypoxia Induced Maize miRNAs EMBP-1 is expressed in late embryogenesis and binds to G-box (CCACGTGG) motif on the promoter region [68,101] whereas TGA1A, induced by auxin and responsible for root specific expression in tobacco, binds to C-box (TGACGTCA) motifs [68,105]. The computational promoter analysis on up regulated genes in Arabidopsis showed the overrepresentation of G-box related sequence under waterlogging conditions [106]. The overrepresen- tation of the ABRE, G-box related and C-box related motif suggests a potential cross talk among different signaling transduction pathways under waterlogging stress. TFs from MYB, bZIP, AP2/ERF and bHLH families that bind to the miRNA promoter region are also negatively regulated by miRNAs (Figure 9). Based on their interactions and network analysis, the system might be employing these TFs as key developmental regulators. Arabidopsis response regulator 10 (ARR10), a type of MYB, was suggested to play an important role in the cytokinin signaling in roots [107] whereas GAMYB (MYB) plays role in GA signaling [108,109]. In Arabidopsis AtMYB2, found to be induced by hypoxia [22,110], dehydration, salt stress and exogenous ABA [111]. This TF binds specifically to the GT-motif of anaerobic response element ADH1 [22,110]. In wheat, a MYB TF called TaMYB1 was reported to be overexpressed in roots under waterlogging conditions [112] suggested the involvement of MYB in low oxygen signaling pathway and helps the survival of wheat plants under anaerobic conditions. miRNA-mediated regulatory sub-network based on miRNA-TF interactions suggests that one miRNA may be regulated by many TFs. The network also shows probable negative feedback loop between MYB and miR319b and MYB and miR159. miR319 and miR159 are highly conserved miRNAs and are involved in plant growth, morphogenesis and reproduction [113]. However, these putative interactions need to be confirmed by experimental data. We believe that our computationally predicted miRNA-mediated sub-network will be useful for future research studies and inspire research efforts aiming to better understand miRNA mediated regulatory mechanisms. Based on our studies, we propose a model of the regulation of differentially expressed miRNAs genes for sensitive, mid-tolerant and tolerant inbred lines under waterlogging stress. miR159, miR164, miR167,miR393, miR408 and miR528, which are involved in root cap formation, lateral root development, root/ shoot elongation and plant cell detoxification by scavenging the reactive oxygen species and thus protecting damage to cellular structure were induced under short waterlogging conditions in waterlogging tolerant line Hz32 and repressed in waterlogging sensitive line Mo17. In the tolerant line, the induction of miR159 might regulate ABA signal transduction pathway by repressing their MYB targets; induction of miR167 represses ARF that has been demonstrated to be involved in lateral root development, or adventitious rooting through the auxin signal [79]. miR164 regulates root or shoot development by repressing NAC/NAM proteins whereas miR393 is negative regulator of TIR1-like (F-box) and the cleavage of F-box gene down-regulate auxin signals. miR408 and miR528 target cupredoxin, which are involved in oxidative stress response signal by mediating electron-transfer or oxidation homeostasis and protecting cellular structures. Thus, in the tolerant line, the repression of hypoxia response signals at the early stage of waterlogging may suppress the signals for aerenchyma formation, coleoptiles growth, lateral root develop- ment and protection against oxidation to save limited oxygen and energy. Whereas, in waterlogging sensitive line, the repression of these miRNAs induces the overexpression of target genes, which will transduce hypoxia responding signals that will increase the signals for aerenchyma formation, coleoptiles growth, lateral root PLoS ONE | www.plosone.org 11 June 2012 | Volume 7 | Issue 6 | e39786