Characterization of Hypoxia Induced Maize miRNAs Figure 8. 59-RLM-RACE verification of predicted miRNA targets. Each top strand depicts a miRNA complementary site (targets), and each bottom strand depicts the miRNA. miR159 and miR319 shares the target sequence. Watson-Crick pairing (vertical dashes) and G:U wobble pairing (circles) are indicated. Arrows indicate the 59termini of mRNA fragments isolated from roots of the maize seedlings, as identified by cloned 59-RACE products, with the frequency of clones shown. Only cloned sequences that matched the correct target gene and had 59 ends within a 100 nt window centered on the miRNA complementary site are counted. doi:10.1371/journal.pone.0039786.g008 lines. The miRNA-mediated gene regulated sub-network also shows probable negative feedback circuits between MYB and miR319b (Figure 9B). This suggests that TFs of MYB TF family might be regulating the expression of these miRNA genes and might also be repressed by these miRNA genes post-transcrip- tionally. Based on our computational analysis of homology, we found the GAMYB TF (belonging to MYB, derived from Hordeum vulgare HvGAMYB) binding sites in the promoter region of miR319b. To find the Zea mays homologs, the sequence of HvGAMYB was BLASTed and two GAMYB genes (GRMZM2G139688, GRMZM2G028054,) were top hits (77% and 68% homology). We found these GAMYB target genes among miR319b target genes. Therefore, it can be suggested that GAMYB might be regulating miR319b and is also regulated by miR319b post- transcriptionally forming a probable negative feedback loop. We also found the probable feedback loop between three miRNA genes of miR159 (miR159j, miR159h, miR159k), and MYB family. However, based on upstream and downstream homology analysis of miR159 (miR159j, miR159h, miR159k), we found MYB.ph3 binding sites in the promoter region of these three genes of miR159, whereas targets of these genes belong to GAMYB family. However, based on this analysis, we can’t assign a negative feedback loop between miR159 and MYB, though a probable feedback loop between miR159 and MYB33 was found in Arabidopsis where feedback involved the down-regulation of AtMYB33 activity by miR159 and compensatory up-regulation of miR159 by AtMYB33 [81]. Both miR159 and miR319 are down regulated in B73 under hypoxia conditions. These miRNAs are therefore likely key developmental regulators within the system, and thus, under- standing the regulatory control of their upstream regulators will allow us to identify putative cases of negative feedback regulation within the system. Discussion Waterlogging is a complex trait that alters pattern of protein synthesis and metabolic pathways such as ethanol, lactates, and CO2, as well as changes in cytosolic pH, ROS, hormone homeostasis, antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase [5,6,7,8]. During the initial hours of hypoxia treatment to maize roots, aerobic protein synthesis is stopped, and gradually anaerobic PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39786