development and cell detoxification at the very beginning of waterlogging, which will lead to rapid consuming limited oxygen and energy. In B73, the mid-tolerant line, miR159, miR408 and miR528 (similar to Hz32) are induced while miR164, miR167 and miR393 (similar to Mo17) are reduced, suggested the repression of ABA and cupredoxin signals and the induction of auxin signals at the initial stages of waterlogging. In conclusion, our study suggests that the early response to waterlogging is not linear and crosstalk takes place between different biochemical pathways. The signal transduction pathways regulate different plant hormone cascades, provides significant clues for understanding the regulation of gene expression and metabolic adaptation under hypoxia stress. At early hours of waterlogging, plants sense the lack of oxygen around the root system and trigger initial changes to gene expression, and over time at later stage, signal transduction pathways are activated to provide the morphological and metabolic adaptation. Materials and Methods We selected the waterlogging-tolerant maize line, Hz32, the mid-tolerant line, B73, and the waterlogging-sensitive line, Mo17, and used them for this study. Seeds of three inbred lines were sown in sand filled 3-inch pots (8 seeds per pot). The day/night temperatures during seedling development ranged from 25uC to 33uC that was similar to field temperatures during maize seedling stage development [24]. Experiment design is shown in Figure 1. Waterlogging Treatment Waterlogging treatment was given to eight two-week-old plants of each of three maize inbred lines by transferring to a container that filled with water 2–3 cm above the sand surface [23,24,25,26] subjecting these plants to short-term waterlogging (1 h, 2 h and 4 h). The controlled plants were grown in the same condition without waterlogging treatment. Just after treatment, plants were removed from the container. The roots were cleaned and immediately frozen in liquid nitrogen for further biochemical and molecular studies. Waterlogging Phenotype Screen Waterlogging tolerance coefficient (WTC) was used to measure the waterlogging tolerance of three inbred lines that were subjected to stress [26]. The WTC of samples was calculated using the following formula: WTC = the mean value of treated seedling/the mean value of control seedlings. Where the mean value of treated seedling is the measurement values of leaf length (LL), root length (RL), leaf dry weight (LW) and root dry weight (RW) of waterlogged plants. The total dry weight (TW = LW+RW) was used to calculate the tolerance of the inbred lines. RNA Extraction Frozen root tissue was ground into a fine white powder. Total RNA extraction was performed using Trizol (Invitrogen, Carls- bad, CA) following manufacturer’s instructions. High Throughput Sequencing High-throughput sequencing of small RNAs (18–28 nt) was done from total RNA by size fractionation and sequenced by Cold Spring Harbor Laboratory Woodbury Genome Research Center (Woodbury, NY, USA) using high-throughput pyrosequencing technology developed by Illumina, using an Illumina 1G Genome Analyzer (Illumina, San Diego, CA) [28], following the manufac- Characterization of Hypoxia Induced Maize miRNAs turers instructions. All small RNA sequences have been deposited in the Gene Expression Omnibus in GenBank under the identifier (GSE32983). Data Analysis of Small RNAs Ilumina sequencing data were received in FASTQ format with all the reads shown as 36-mers prior to the removal of the 39 adapter sequences. A custom script was used to trim the adaptor sequence from each sequence. We used the Vmatch large-scale sequence analysis software (www.vmatch.de) to map unique consensus sequences tags to the maize reference genome (B73 RefGen v1 [27]). A summary of the origin of the small RNA fragments can be found in Table S2. Reads that mapped with known maize miRNA [11] from miRBase release 18 [32] were selected. Prediction of Waterlogging Induced Novel miRNAs and their Targets The unique sequence reads obtained after combining water- logging treatment and control libraries with appropriate abun- dance ($100 reads, around over 10RPM) were selected and mapped with the maize reference genome (B73 RefGen v1 [27]). Those sequences that overlapped with repetitive elements and transposable elements were removed. Further, the sequences that matched with known maize miRNAs or other ncRNAs with two mismatches were also removed. miRcheck (http://web.wi.mit. edu/bartel/pub/software.html) and RNAfold (http://www.tbi. univie.ac.at/˜ ivo/RNA/RNAfold.html) were used to predict the miRNA secondary structures [37]. For the target prediction of novel miRNAs, the miRNA target transcript prediction pipeline was developed using Vmatch [www.vmatch.de] and indexed using mkvtree. These mature miRNA sequences were reverse comple- mented and matched against the indexed maize transcript database with the parameters relaxed to allow up to six mismatches [11]. The matched miRNA target transcripts were filtered by applying empirical rules defined by Schwab et al. [114]. Perfect matches were given a score of 0, and all other mismatches were scored 1. Only one mismatch score was allowed between positions 2 to 12 inclusive. However, no mismatches were allowed at position 10 and 11 and no more than 2 consecutive mismatches were allowed after position 12. A maximum of four mismatches was allowed across the length of the mature miRNA. Statistical Analysis of miRNA Frequency To determine whether differences in miRNA frequency between control and waterlogging-treated samples were signifi- cant, a x-square test was performed using total sequence numbers as previously described by Qiu et al. [115]. The candidate mature miRNA signatures were selected after applying the filter (P value ,0.01; cut off over 1.5 fold; reads .50 reads per million (RPM)) and were selected for validation using SLRT-PCR. Validation of Mature miRNA Expression by Stem-loop Real Time PCR (SLRT-PCR) The stem-loop reverse transcriptase primer for each miRNA consisted of a selfed stem-loop sequence (GTCGTATCCAGTG- CAGGGTCCGAGGTATTCGCACTGGATACGAC) with the specificity conferred by a six nucleotide extension at the 39 end, which is complementary to the last six nucleotides at the 39 end of mature miRNA [34]. The RT reactions were performed on 100 ng RNA using Superscript III (Invitrogen, Carlsbad, CA) and carried out according to the manufacturer’s instructions. For each RNA sample, two cDNA reactions were performed. The first had PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39786