Characterization of miRNAs in Response to Short-Term Waterlogging in Three Inbred Lines of Zea mays Zhijie Liu1,2, Sunita Kumari2, Lifang Zhang 2 , Yonglian Zheng1*, Doreen Ware2,3* 1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of China, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America, 3 United States Department of Agriculture – Agriculture Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca, New York, United States of America Abstract Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways. Citation: Liu Z, Kumari S, Zhang L, Zheng Y, Ware D (2012) Characterization of miRNAs in Response to Short-Term Waterlogging in Three Inbred Lines of Zea mays. PLoS ONE 7(6): e39786. doi:10.1371/journal.pone.0039786 Editor: Frank G. Harmon, USDA-ARS, United States of America Received November 17, 2011; Accepted May 31, 2012; Published June 29, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by a USDA grant (1907-21000-030, www.usda.gov), and project 111 (B07041, Ministry of Education of China and State Administration of Foreign Experts Affairs of China) (http://www.moe.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yonglianzheng@gmail.com (YZ); ware@cshl.edu (DW) Introduction Land plants, being strictly aerobic, receive freely diffused molecular oxygen from aerial or underground tissues. Oxygen deficiency due to short-term waterlogging (hypoxia) or its complete absence (anoxia) often damages roots under transient or sustained flooding conditions [1]. Long-term waterlogging can cause crop yield losses up to 30% when it occurs early in the season [2,3]. Maize survival is mainly dependent on its metabolic, physiological and morphological adaptation strategies. In hypoxia, anaerobical- ly induced polypeptides (ANPs) are selectively synthesized, such as aldolase, enolase, alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) [4]. Physiologically, anaerobic stress induc- es alteration of metabolic pathways, leading to the accumulation of metabolites such as ethanol, lactates, and CO2, as well as changes in cytosolic pH, reactive oxygen species (ROS) and hormone homeostasis [5,6,7,8]. Small RNAs have been identified as important post-transcrip- tional regulators of gene expression [9,10,11]. Many studies suggest important roles for miRNAs in plant responses to abiotic and biotic stresses such as cold [12,13,14], salt [12,13,15,16], heat [13], dehydration [12,17], oxidative stress [18], mechanical stress [13], pathogen infection [19], and submergence [20]. In addition to studying the effects of stress on miRNA levels, the identification of targets of miRNAs can provide clues about the roles of miRNAs in stress responses [21]. The known targets of post-transcriptional regulation include transcription factors (TFs), suggesting roles of miRNAs as early signaling components that could ultimately lead to broader changes in gene expression following stress. The response of a plant to hypoxia can be conceptually divided into three stages [22]. The first stage (0–4 h) consists of the rapid induction of signal transduction components, which then activates the second stage (4–24 h), a metabolic adaptation. The third stage (24–48 h) involves the formation of gas-filled air spaces (aeren- chyma) in the root [22]. In this paper, we have focused on the first stage because it determines the switch from normal to low-oxygen metabolism and plays an essential role in the survival of seedlings. To characterize early responses, we used three maize inbred lines having different sensitivities to waterlogging when assayed as seedlings under controlled experimental conditions. We verified previous research work showing that inbred Mo17 is highly sensitive to waterlogging, while Hz32 is relatively tolerant [23,24,25,26]. We also included B73, whose genome serves as the complete reference sequence in maize [27] and found it mid- PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39786