Arabidopsis Transcriptome Analysis Reveals Key Roles of Melatonin in Plant Defense Systems Sarah Weeda1, Na Zhang2, Xiaolei Zhao3, Grace Ndip4, Yangdong Guo2*, Gregory A. Buck5, Conggui Fu3, Shuxin Ren1* 1 Agriculture Research Station, Virginia State University, Petersburg, Virginia, United States of America, 2 College of Agriculture and Biotechnology, China Agricultural University, Beijing, P. R. China, 3 Tianjin Crop Science Institute, Tianjin Academy of Agricultural Science, Tianjin, P. R. China, 4 Department of Chemistry, Virginia State University, Petersburg, Virginia, United States of America, 5 Center of the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia, United States of America Abstract Melatonin is a ubiquitous molecule and exists across kingdoms including plant species. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. Much less attention has been drawn to its affect on genome-wide gene expression. To comprehensively investigate the role(s) of melatonin at the genomics level, we utilized mRNA-seq technology to analyze Arabidopsis plants subjected to a 16-hour 100 pM (low) and 1 mM (high) melatonin treatment. The expression profiles were analyzed to identify differentially expressed genes. 100 pM melatonin treatment significantly affected the expression of only 81 genes with 51 down-regulated and 30 up-regulated. However, 1 mM melatonin significantly altered 1308 genes with 566 up-regulated and 742 down-regulated. Not all genes altered by low melatonin were affected by high melatonin, indicating different roles of melatonin in regulation of plant growth and development under low and high concentrations. Furthermore, a large number of genes altered by melatonin were involved in plant stress defense. Transcript levels for many stress receptors, kinases, and stress-associated calcium signals were up-regulated. The majority of transcription factors identified were also involved in plant stress defense. Additionally, most identified genes in ABA, ET, SA and JA pathways were up-regulated, while genes pertaining to auxin responses and signaling, peroxidases, and those associated with cell wall synthesis and modifications were mostly down- regulated. Our results indicate critical roles of melatonin in plant defense against various environmental stresses, and provide a framework for functional analysis of genes in melatonin-mediated signaling pathways. Citation: Weeda S, Zhang N, Zhao X, Ndip G, Guo Y, et al. (2014) Arabidopsis Transcriptome Analysis Reveals Key Roles of Melatonin in Plant Defense Systems. PLoS ONE 9(3): e93462. doi:10.1371/journal.pone.0093462 Editor: Cynthia Gibas, University of North Carolina at Charlotte, United States of America Received January 8, 2014; Accepted March 6, 2014; Published March 28, 2014 Copyright: ß 2014 Weeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is partially supported by USDA Evans Allen Fund. No additional external funding received for this study. 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: [email protected] (YG); [email protected] (SR) Introduction demonstrated a peak in melatonin levels late in the day [19], indicating its biosynthesis in light. Furthermore, melatonin Melatonin (N-acetyl-5-methoxytryptamine) is a versatile mole- biosynthesis occurred under constant light in senescent rice leaves cule that, since its 1958 discovery in bovine pineal glands [1], has and was nearly undetectable under constant darkness [22]. Other been found across kingdom lines including bacteria, fungi, and reports found no significant correlation with melatonin levels and plants [2–5]. Its numerous and diverse functions include regulating day/night cycles [18]. Interestingly, developing sweet cherries circadian rhythms and seasonal reproduction cycles, promoting exhibited a dual peak of melatonin levels, one nocturnal and one sleep, preventing diabetes, attenuating cancer cell proliferation, in late day [20]. Contradicting reports of melatonin levels in and enhancing immunity [6–10]. Melatonin has also been ripening fruits add to the variation observed among plant species; characterized as a potent antioxidant capable of scavenging melatonin levels decreased in ripening cherries [20], but increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) in ripening tomatoes [18]. The possible role of melatonin in [11–14]. regulating flowering has also been investigated [23–25]; however Since the relatively recent discovery of melatonin in plants, an unequivocal role of melatonin in photoperiod-dependent investigations to elucidate its function in plants has been driven by processes in plants has not yet been established what is known in animals. One such area of focus is the Melatonin has been studied extensively as an antioxidant in involvement of melatonin in modulating circadian rhythms and mammals. Many studies demonstrate the ability of melatonin to photoperiod-dependent processes. While melatonin levels do protect against many human diseases, including those linked to appear to be affected by light/dark cycles in some plants, the oxidative stress [26–27]. Melatonin was able to attenuate pattern varies among species, tissues, and organs [15–21]. The nocturnal peak of melatonin characteristic of humans and animals paraquat-induced lung and liver damage in rats [28–29] and has been observed in Chenopodium rubrum [16], grape skins [17] and Parkinson’s disease in mice [30]. Furthermore, exogenously Ulva sp [21]. Conversely, studies conducted in water hyacinth applied melatonin can enhance the production of antioxidative PLOS ONE | www.plosone.org 1 March 2014 | Volume 9 | Issue 3 | e93462 MT-Mediated Stress Defense in Arabidopsis enzymes such as glutathione peroxidase and superoxide dismutase Arabidopsis will provide us basic information to genetically dissect [31]. Melatonin may similarly play a protective role against melatonin-mediated signaling pathway(s) in regulating plant oxidative stress in plants. Oxidative stress is capable of inducing growth and development. elevated melatonin levels in various plant species [17,32–34]. Indeed, the daytime peak of melatonin levels found in sweet cherry Materials and Methods was associated with high temperature and light intensity, suggesting melatonin was synthesized in response to oxidative Plant materials and growth condition stress [17]. Transgenic rice seedlings with elevated levels of Arabidopsis thaliana ecotype Columbia (Col-0) was used for RNA- melatonin were more resistant to herbicide induced oxidative seq experiments and oxidative stress experiments. All plants were stress than their wild type counterparts [35]. Furthermore, grown in a growth-chamber under 23uC and 14/10 hours light/ oxidative stress induced the expression of genes involved in dark cycle. Three-week-old seedlings were removed from soil, melatonin biosynthesis, leading to increased melatonin production rinsed, and submerged in100 pM, or 1 mM melatonin for in both wild type and transgenic rice [35]. Melatonin also appears 16 hours with gentle shaking. Mock solution was used as control. to protect plants against UV and ozone damage [36–40], All experiments were duplicated for statistical analysis. attenuate photo-oxidation of the photosynthetic system, and, at moderate levels, protect chlorophyll during senescence [39–42]. In RNA extraction, library construction and sequencing addition, melatonin can promote low temperature and osmotic After melatonin treatment, total RNA was extracted using the stress tolerance [43–48], alleviate copper damage [49–50], and QIAGEN RNeasy Mini Kit according to the manufacturer’s improve salt tolerance [51] and fungal disease resistance [52] in a instructions (QIAGEN). Purification of mRNA from total RNA diversity of plant species. was conducted using an Oligotex mRNA Mini Kit (QIAGEN). The structure of melatonin is another feature that has driven The mRNA was then used to construct cDNA libraries using the investigations into its function in plants. Melatonin is structurally mRNA-Seq Sample Preparation KitTM (Illumina) following similar to the plant hormone indole-3-acetic acid (IAA) and has standard protocols. Briefly, the mRNA was fragmented by many features that make it a candidate for a functional auxin [53– exposure to divalent cations at 94uC and the fragmented mRNA 54]. In addition, melatonin and auxin biosynthetic pathways share was converted into double stranded cDNA. The cDNA ends were the same precursor, tryptophan [55]. Since auxins play critical polished, the 39-hydroxls extended with A bases, and ligated to roles as growth regulators during plant development such as shoot Illumina-specific adapter-primers. The adaptor ligated DNA was elongation, lateral root formation, and cell expansion, much work amplified by 15 cycles of PCR followed by purification using has focused on the effect of melatonin on these processes QiagenTM PCR purification kit to obtain the final library for [42,48,56–63]. Investigations have shown that melatonin and its sequencing. The DNA yield and fragment insert size distribution precursor serotonin