Comparative physiological, metabolomic, and transcriptomic analyses reveal mechanisms of improved abiotic stress resistance in bermudagrass [Cynodon dactylon (L). Pers.] by exogenous melatonin

Summary Exogenous melatonin application confers abiotic stress resistance in bermudagrass through modulation of antioxidants and metabolic homeostasis, and extensive transcriptional reprogramming such as the reorientation of photorespiratory, carbohydrate, and nitrogen metabolism.

To date, melatonin has also been found to be a ubiquitous modulator in multiple plant developmental processes and various stress responses (Kolář and Macháčkova, 2005;Arnao and Hernández-Ruiz, 2006;Tan et al., 2007aTan et al., , 2012. Changes in melatonin in plants may be involved in circadian rhythms, flowering, promotion of photosynthesis, preservation of chlorophyll (Arnao and Hernández-Ruiz, 2009a;Tan et al., 2012), stimulation and regeneration of root system architecture (Hernández-Ruiz et al., 2005;Pelagio-Flores et al., 2012;Zhang et al., 2014), delayed senescence of leaves (Byeon et al., 2012;Wang et al., 2012Wang et al., , 2013a, and alleviation of oxidative damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS) burst (Tan et al., 2012) Moreover, melatonin protects against multiple abiotic processes such as cold stress (Posmyk et al., 2009a;Kang et al., 2010;Bajwa et al., 2014), copper stress (Posmyk et al., 2008(Posmyk et al., , 2009b, high temperature (Byeon and Back 2014b), salt stress , osmotic stress (Zhang et al., 2013), drought stress (Wang et al., 2014), and pathogen infection (Yin et al., 2013). The mechanisms were partially characterized after the direct exogenous application of melatonin to plants (Posmyk et al., 2008(Posmyk et al., , 2009aZhao et al., 2011;Li et al., 2012;Pelagio-Flores et al., 2012;Wang et al., 2012Wang et al., , 2013aYin et al., 2013;Bajwa et al., 2014) or the creation of transgenic plants that produced either more or less melatonin through modulating its metabolic pathway (Kang et al., 2010;Byeon et al., 2013Byeon et al., , 2014Park et al., 2013;Byeon and Back, 2014a;Wang et al., 2014). Finally, the recent studies which showed the protective roles of melatonin in response to abiotic stress indicate that this indole might be a potentially ideal target for future genetic engineering technology to improve abiotic stress resistance in plants. Thus, transgenic plants with higher melatonin concentration might lead to breakthroughs to improve crop production in agriculture as well as the general health of humans (Tan et al., 2012).
Bermudagrass [Cynodon dactylon (L). Pers.] is a warmseason turfgrass for lawns, parks, and sport fields cultivated worldwide (Shi et al., 2012(Shi et al., , 2013a(Shi et al., , b, 2014b. In response to global changed environmental stresses, improvement of abiotic stress resistance is very important for grass engineering (Shi et al., 2012(Shi et al., , 2013a(Shi et al., , b, 2014b. As mentioned above, melatonin might be an ideal target for future genetic engineering of some plant species. However, the endogenous melatonin concentration and the possible role of melatonin in response to abiotic stress in bermudagrass is largely unknown. In this study, endogenous melatonin was examined after abiotic stress treatments in bermudagrass plants, and exogenous melatonin treatment was applied to investigate the in vivo role of melatonin in the response of bermudagrass to abiotic stress. In addition, the effects of exogenous melatonin treatment on ROS accumulation and cell damage, as well as underlying antioxidant responses, were determined. Moreover, comparative metabolomic and transcriptomic analyses were performed to identify differentially expressed metabolites and genes after exogenous melatonin treatment. This study provided some insights into the physiological and molecular mechanisms of melatonin in bermudagrass responses to multiple abiotic stresses.

Plant materials and growth conditions
Newly harvested common bermudagrass seeds were used in this study. After stratification in deionized water at 4 °C for 4 d in darkness, the bermudagrass seeds were sown in soil in the growth room, which was controlled at 28 °C, with an irradiance of ~150 μmol quanta m -2 s -1 , 16 h light and 8 h dark cycles, and ~65% relative humidity.

Plant abiotic stress treatment
To test the effect of exogenous melatonin on plant physiological responses and abiotic stress resistance, 21-day-old bermudagrass plants were irrigated with water or with different concentrations of melatonin solutions for 7 d, respectively. After melatonin pretreatment, all control and melatonin-pre-treated 28-day-old bermudagrass plants were subjected to subsequent salt, drought, or cold stress treatments. For salt stress treatment, 28-day-old bermudagrass plants were irrigated with NaCl solutions for 25 d; the NaCl concentration was increased stepwise by 50 mM every 5 d to a final concentration of 300 mM. For drought stress treatment, 28-day-old plants were subjected to a drought condition by withholding water for 21 d and then re-watered for 4 d. For cold stress treatment, 28-day-old bermudagrass plants were subjected to 4 °C treatment for 21 d, and then transferred to -10 °C for 8 h. The freezing stress-treated plants were then recovered overnight at 4 °C and transferred to a standard growth room (28 °C) for 4 d. In each independent experiment, three pots with ~40 plants in each pot were used for each treatment in one concentration of melatonin, and at least three independent experiments were performed to obtain the results.
The survival rate of the salt-, drought-, or freezing-stressed plants was recorded at 25 d after stress treatments. The plant leaf samples from melatonin-pre-treated 28-day-old plants were collected at the indicated time points after salt, drought, or cold treatment for the assays of multiple of physiological parameters.

Quantification of melatonin by enzyme-linked immunosorbent assay (ELISA)
Melatonin from plant leaves was extracted using the acetonemethanol method as described by Pape and Lüning (2006). Briefly, 1 g of plant leaf samples was ground in liquid nitrogen, and then transferred to 5 ml of extraction mixture (acetone:methanol:wa ter=89:10:1) and homogenized extensively on ice, and the homogenate was centrifuged at 4500 g for 5 min at 4 °C. The supernatant was moved to a new centrifuge tube containing 0.5 ml of 1% trichloric acid for protein precipitation. After centrifugation at 12 000 g for 10 min at 4 °C, the extract was used for quantification of melatonin using the Melatonin ELISA Kit (EK-DSM; Buhlmann Laboratories AG, Schonenbuch, Switzerland) according to the manufacturer's instruction as described in Shi and Chan (2014a).

Quantifications of chlorophyll content
Plant leaf chlorophyll was extracted using 80% (v/v) acetone for 6 h with shaking in the dark. The concentration of chlorophyll was then calculated by examining the absorbance at 645 nm and 663 nm of the centrifuged supernatant.

Quantification of electrolyte leakage (EL)
The EL of plant leaves under control and abiotic stress conditions was assayed using a conductivity meter (Leici-DDS-307A, Shanghai, China) as previously described (Shi et al., 2012(Shi et al., , 2013a(Shi et al., , b, 2014b. The relative EL was expressed as the ratio of initial conductivity to the conductivity after boiling.

Determination of malondialdehyde (MDA) content
The MDA content was extracted using chilled thiobarbituric acid (TBA) reagent, and was quantified via determining the absorbance of the supernatant at 450, 532, and 600 nm as previously described (Shi et al., 2012(Shi et al., , 2013a(Shi et al., , b, 2014b.
Extraction, identification, and quantification of metabolites Extraction, identification, and quantification of metabolites from plant leaves were performed as in Shi et al. (2014d). Briefly, the metabolite extraction and sample derivatization were performed as in Lisec et al. (2006), then the derivatizated extract was injected into a DB-5MS capillary cloumn (30 m×0.25 mm×0.25 μm; Agilent J&W GC column, California, USA) using gas chromatography timeof-flight mass spectrometry (GC-TOF-MS) (Agilent 7890A/5975C) according to the protocol described by Shi et al. (2014d). After the GC-TOF-MS assay, the various metabolites were identified via comparing every retention time index-specific mass with reference spectra in mass spectral libraries (NIST 2005, Wiley 7.0). The numerous metabolites were then quantified based on the pre-added internal standard (ribitol) in the process of metabolite extraction.
For cluster analysis, all metabolites were quantified as fold change relative to the wild-type bermudagrass plants under control conditions, which was set as 1.0.

RNA extraction, library construction, and sequencing
For RNA extraction, 28-day-old bermuagrass plants in pots that were irrigated with water or 20 μM melatonin for 7 d were used. Each treatment was represented by two replicate leaf samples, and each sample contained leaves from at least 30 seedlings. Total RNA was extracted with TRIzol (Invitrogen) and was quantified as previously described (Shi et al., 2013c). RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol. The cDNA libraries were constructed with the mRNA-Seq Sample Preparation Kit™ (Illumina, San Diego, CA, USA) and the DNA yield and fragment insert size distribution of each library were determined on the Agilent Bioanalyzer. The cDNA libraries were then sequenced on an Illumina HiSeq2500 sequencing instrument using the 100 bp single end protocol.

Quantitative real-time PCR
The above RNA samples were also used for synthesis of first-strand cDNA with reverse transcriptase (BIO-RAD, Hercules, CA, USA), and the cDNAs were used for quantitative real-time PCR using a CFX96™ Real Time System (BIO-RAD) as previously described (Shi et al., 2013c). The specific primers of the analysed genes for real-time PCR are listed in Supplementary Table S1 available at JXB online, and the housekeeping genes have been described in Hu et al. (2012).

Bioinformatics analyses of RNA-Seq data
Raw RNA-Seq reads were first trimmed for low quality regions using clean reads with length longer than 25 bp, obtained after trimming low quality bases (Q<17) using the SolexQA tool (v2.2) and removing adaptor sequences using the cutadapt tool (v1.3) (Martin, 2011). A total of 679 million clean RNA-Seq reads from 20 libraries, and four libraries were used for transcriptome profiling in this study. Transcriptome analyses of RNA-Seq data were used for transcriptome assembly using Trinity software (v r20131110) (Haas et al., 2013). The resulting pre-assembled transcriptome were refined according to the methods described by Ranjan et al. (2014). After transcripts expressed at a low level and redundant sequences were removed, 28 456 high quality transcripts were retained as the final reference transcriptome for bermudagrass.
To obtain putative annotations, the final transcriptome sequences were compared with the NCBI nr protein database by BlastX using an E-value of 1e-5 as the cut-off. Blast2GO (v 2.5.0) (Gotz et al., 2008) was used to assign GO terms to each transcript. The final transcriptome sequences were also compared with Arabidopsis (TAIR10) and rice (MSU release7) protein database using BlastX with an E-value cut-off of 1e-5. The best hit from these two well-annotated species was used to annotate each bermudagrass transcript.
To evaluate the abundance of each transcript, reads from individual libraries were mapped to the final reference transcripts using Bowtie, and the read counts on each transcript were estimated by the software RSEM with default parameters (Li and Dewey, 2011). Differentially expressed transcripts were identified by the R package edge R (Robinson et al., 2010.).
GO term enrichment analysis of differentially expressed genes was carried out using the topGO Bioconductor package (v 2.16.0) (Alexa et al., 2006) for up-and down-regulated genes, respectively. The Classification SuperViewer Tool (http://bar.utoronto.ca/ntools/cgi-bin/ ntools_classification_superviewer.cgi) was used to generate an overview of the enriched pathways (Provart and Zhu, 2003), and MapMan was used as the classification source to assign functional pathways for each gene (Thimm et al., 2004;Yang et al., 2013). The normalized frequency (NF) of each functional category was calculated as described in Chan et al. (2011): NF=sample frequency of each category in this experiment/ background frequency of each category in the Arabidopsis genome.

Statistical analysis
The experiments in this study were repeated three times and the data shown are the means ±SEs. The means are the average of three independent experiments. Each independent experiment was a pooled sample from at least 30 bermudagrass plants. Bars with different letters above the columns in the figures indicate significant differences at P<0.05 (Duncan's range test).

Abiotic stress induced the endogenous melatonin level in bermudagrass
To investigate how abiotic stress affected the melatonin content, endogenous melatonin levels in bermudagrass leaves were quantified after treatments with 300 mM NaCl, drought, or cold (4 °C) stresses for 0, 7, 14, and 21 d. Melatonin content remained steady at ~50 pg g -1 fresh weight (FW) in nontreated control plants (Fig. 1). After abiotic stress treatments, the melatonin content in bermudagrass leaves significantly increased (Fig. 1). The induction of melatonin content by multiple abiotic stress treatments indicated the in vivo role of melatonin in bermudagrass response to abiotic stress.

Exogenous melatonin improved abiotic stress resistance in bermudagrass
After 7 d pre-treatment with different concentrations of melatonin (0, 4, 20, and 100 μM melatonin, respectively), no significant differences were observed between non-treated and melatonin pre-treated plants (28 d old) ( Fig. 2A). When salt, drought, or cold (4 °C) stresses were applied, the endogenous melatonin levels were activated, and 20 μM and 100 μM melatonin-pre-treated bermudagrass had significantly higher levels than non-melatonin-treated plants (Fig. 2B). Growth and physiological parameters including chlorophyll, EL, survival rate, plant height, and plant biomass (weight) of melatoninpre-treated 28-day-old bermudagrass plants were generally equivalent to those of non-treated plants under well-watered conditions for the following 25 d (Fig. 2C-G). After salt, drought, or freezing stress treatments, growth of both melatonin-pretreated and non-treated plants was inhibited, but 20 μM and 100 μM melatonin-pre-treated plants had greener leaf tissues than those of non-treated bermudagrass plants ( Fig. 2A). Consistently, 20 μM or 100 μM melatonin-pre-treated plants exhibited a significantly higher chlorophyll content, lower EL, and higher survival rate than did non-treated bermudagrass plants ( Fig. 2C-E). Moreover, 20 μM and 100 μM melatoninpre-treated plants exhibited healthy growth in comparison with non-treated and 4 μM melatonin-pre-treated plants, with significantly higher plant height and weight (Fig. 2F, G). These results indicate that exogenous melatonin application improved salt, drought, and freezing stress resistance in bermudagrass.

Exogenous melatonin alleviated abiotic stress-induced ROS accumulation in bermudagrass
As the major indicators of the stress-triggered ROS level and oxidative damage, H 2 O 2 , O 2 · -, and MDA contents were assayed among control and 20 μM melatonin-pre-treated plants during abiotic stress treatments. Under control conditions, melatonin had no significant effect on H 2 O 2 , O 2 · -, and MDA contents ( Fig. 3A-C). When salt, drought, and cold (4 °C) stresses were applied, melatonin-pre-treated plants showed significantly lower levels of H 2 O 2 , O 2 · -, and MDA in comparison with non-treated bermudagrass plants, conferring less oxidative damage ( Fig. 3A-C). These results indicated that exogenous application of melatonin could modulate abiotic stress-triggered ROS accumulation and related oxidative damage in bermudagrass.

Effects of exogenous melatonin on ROS-related antioxidants in bermudagrass response to abiotic stress
To alleviate abiotic stress-triggered ROS burst, plants have developed complex antioxidant defence system, including several antioxidant enzymes and non-enzymatic glutathione antioxidant pool. Under control conditions, no significant differences in antioxidant enzymes and the non-enzymatic glutathione antioxidant pool were found between non-treated and melatonin-pre-treated bermudagrass (Fig. 4A-F). Under abiotic stress conditions, the activities of antioxidant enzymes (SOD, CAT, and POD) and the GSSG content were greatly induced, while GSH content was significantly decreased (Fig. 4A-F). Additionally, melatonin-pre-treated plants Fig. 1. The endogenous melatonin level was induced by salt, drought, and cold stresses in bermudagrass. Twenty-eight-day-old bermudagrass plants were treated with control, 300 mM NaCl, drought, and cold (4 °C) stresses for 0, 7, 14, and 21 d, respectively. Bars with different letters above the columns of figures indicate significant differences at P<0.05 (Duncan's range test).
showed significantly higher activities of antioxidant enzymes (SOD, CAT, and POD) and a higher GSH redox state in comparison with non-treated plants, conferring more effective antioxidants (Fig. 4A-F). These results indicated that melatonin had significant effects on both antioxidant enzymes and the non-enzymatic glutathione antioxidant pool, which might be consistent with alleviated abiotic stress-induced ROS accumulation and related oxidative damage in bermudagrass.

Modulation of metabolic homeostasis by exogenous melatonin treatment under control and abiotic stress conditions
To gain more insights into the modulation of metabolic homeostasis by exogenous melatonin treatment under control and abiotic stress conditions, GC-TOF-MS was performed to identify differentially expressed metabolites. In total, 54 metabolites, comprising 16 amino acids, 13 organic acids, 18 sugars, five sugar alcohols, and two aromatic amines, were reproducibly examined in non-treated and melatonin-pretreated plants under control and abiotic stress conditions  Table S2 at JXB online). Under control conditions, no significant regular pattern of these metabolites was shown in non-treated and melatonin-pre-treated plants ( Fig. 5; Supplementary Table S2). When salt, drought, and cold (4 °C) stresses were applied, melatonin-pre-treated plants exhibited higher concentrations of almost all the 54 metabolites than non-treated plants ( Fig. 5; Supplementary  Table S2). Additionally, many of these metabolites were commonly regulated by salt, drought, and cold (4°C) stresses  Table S2). Interestingly, 18 metabolites, comprising 10 amino acids, six sugars, and two sugar alcohols, were assigned to the carbon metabolic pathway comprising glycolysis, oxidative pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle, indicating the direct link between melatonin and the carbon metabolic pathway in bermudagrass response to abiotic stress. Melatonin-pretreated plants exhibited significantly higher levels of these metabolites than non-treated plants under abiotic stress conditions (Fig. 6A).

Transcriptome analysis: GO annotation and enrichment analysis
Since melatonin pre-treatment increased salt, drought, and freezing stress resistance in bermudagrass, the 28-day-old plants (without and with 7 d of pre-treatment) were used for transcriptomic analysis to reveal the effect of melatonin pre-treatment on global transcriptional reprogramming. Approximately 679 million RNA-Seq reads were used for de novo assembly of the bermudagrass transcriptome. After removing poorly expressed and redundant transcripts, 28 456 high quality transcripts were retained as the final reference transcriptome. These annotated transcripts were used to search various protein databases. In total, 22 137 (78%) had BLASTX hits in the well-annotated Arabidopsis and/or rice protein database (E-value >1e-5). GO annotation was performed using the Blast2GO pipeline, and 18 701 (66%) transcripts were assigned with at least one GO term. Among the three GO categories, 13 402 transcripts were annotated in Biological Process, 14 052 transcripts in Molecular Function, and 14 685 in Cellular Component.
Using fold change >2 and false discovery rate (FDR) <0.05 as thresholds, 3933 transcripts (2361 up-regulated and 1572 down-regulated by exogenous melatonin treatment) were identified as differentially expressed genes (Supplementary  Tables S4, S5 at JXB online). Many stress-responsive genes were highly induced by exogenous melatonin treatment in bermudagrass (Table 1). Interestingly, several C-REPEAT-BINDING FACTORS/DEHYDRATION-responsive ELEMENT-BINDING PROTEIN (CBF/DREB) genes and target genes, heat shock transcription factors (TFs), zinc finger TFs, WRKY, MYB, bHLH genes, and hormone-related genes were highly induced >16-fold after melatonin treatment (Table 2). GO enrichment analysis in the biological process domain suggested that genes related to the cysteine biosynthetic process, response to light signal, and the photosynthetic process were down-regulated. In particular, the studies of Wang et al. (2012) showed that melatonin can lower ROS damage of many photosynthetic components. Therefore, the expression of genes involved in the photosystem might been suppressed through a negative feedback mechanism. The up-regulated genes were greatly enriched with the GO terms involved in gene expression regulatory process, such as protein phosphorylation, DNA-dependent transcription, regulation of circadian rhythm, etc. (Fig. 7).
To confirm the reliability of the RNA-Seq data, the expression of 18 genes (nine up-regulated and nine down-regulated by exogenous melatonin treatment) that were differentially expressed between control and melatonin-treated plants was assessed via quantitative real-time PCR. Consistently, the results of the real-time PCR assay exhibited the same trend and were correlated with the RNA-Seq data ( Supplementary  Fig. S1 at JXB online), confirming the reproducibility of RNA-Seq data.

Pathway and GO term enrichment analyses
The transcriptome data were submitted to the Mercator web tool to align with the public protein database, and 14 288 transcripts were located in at least one point in plant biological pathways. As shown in Table 1, pathway analysis revealed that melatonin affected the expression of many genes involved in N metabolism, minor carbohydrate metabolism, TCA/org transformation, transport, hormone metabolism, metal handling, redox, and secondary metabolism (Table 1,   showed that melatonin altered many genes involved in plant defence ( Supplementary Fig. S2), and these changes might contribute to the enrichment of stress-related GO terms (Fig. 7).

Discussion
As sessile organisms, plants have developed sophisticated strategies to respond to diverse environmental stresses. The stress signals are perceived by several receptors at the cell membrane level, followed by their transduction to multiple second messengers such as abscisic acid (ABA), H 2 O 2 , nitric oxide (NO), etc. These activate downstream stress-responsive genes and physiological responses, eventually leading to protective responses at the whole-plant level (Shi et al., 2012(Shi et al., , 2013a(Shi et al., , b, 2014bShi and Chan, 2014a, b). Although no direct evidence had indicated that melatonin could serve as a second messenger, the induction of melatonin by multiple stress treatments ( Fig. 1) indicates an in vivo role for melatonin in bermudagrass response to abiotic stress Tan et al., 2007aTan et al., , 2012.
In this study, the protective role of melatonin on the response of bermudagrass to abiotic stress was revealed. Under control conditions, melatonin had no significant effect on bermudagrass growth or its physiological responses (Fig. 2). Under abiotic stress conditions, however, melatoninpre-treated plants exhibited significantly higher chlorophyll content, lower EL, and higher survival rate than did nontreated bermudagrass plants (Fig. 2C-E). After recovery from abiotic stress treatments, melatonin-pre-treated plants exhibited better growth status than non-treated plants, with higher biomass (plant height and weight) (Fig. 2F, G). These results indicate that exogenous melatonin application improved salt, drought, or freezing stress resistance in bermudagrass, in accordance with the enhanced resistance to cold stress (Posmyk et al., 2009a;Kang et al., 2010;Bajwa et al., 2014), copper stress (Posmyk et al., 2008(Posmyk et al., , 2009b, high temperature (Byeon and Back, 2014b), salt stress , osmotic stress (Zhang et al., 2013), drought stress (Wang et al., 2014), and pathogen infection (Yin et al., 2013) due to melatonin in various plant species.
As an antioxidant in animals, melatonin scavenges free radicals directly, stimulates the activities of antioxidant enzymes including CAT, SOD (both MnSOD and CuSOD), glutathione reductase (GR), and glutathione peroxidase (GPX), and increases the efficiency of mitochondrial oxidative phosphorylation (Tan et al., 1993(Tan et al., , 1999(Tan et al., , 2003(Tan et al., , 2007aReiter et al., 2000;Kolář and Macháčkova, 2005). In plants, melatonin is also an important antioxidant and a radical scavenger (Arnao et al., 1996(Arnao et al., , 2001Cano et al., 2003Cano et al., , 2006. Li et al. (2012) also found that exogenous melatonin modulates salinity-induced oxidative damage by directly scavenging H 2 O 2 and enhancing the activities of antioxidative enzymes in Malus hupehensis. Consistently, exogenous application of melatonin significantly activated ROS detoxification of antioxidants, including enzymatic antioxidant enzymes (SOD, CAT, and POD) and non-enzymatic glutathione (GSH redox state), to maintain cellular ROS (mainly including H 2 O 2 and O 2 · -) at a relatively low level. This results in the alleviation of abiotic stress-induced oxidative damage and further conferred improved abiotic stress resistance (Figs 3, 4). Consistently, RNA-Seq found that many genes involved in redox, many POD genes and glutathione S-transferases (GST) genes were significantly modulated by exogenous melatonin treatment (Table 1; Supplementary Fig. S2 at JXB online). In summary, the positive modulation by exogenous melatonin of the ROS detoxification system might contribute greatly to enhanced abiotic stress resistance in bermudagrass.
Moreover, comparative metabolomic analysis showed the actions of melatonin treatment on carbon metabolites and amino acid metabolism under abiotic stress conditions. Notably, melatonin-pre-treated plants exhibited higher concentrations of 54 metabolites compared with non-melatonintreated plants (Fig. 5; Supplementary Table S2 at JXB online). Among these metabolites, proline and some carbohydrates (fructose, sucrose, glucose, maltose, cellobiose, trehalose, galactose, and galactinol) are important compatible solutes to respond to abiotic stress for osmotic adaptation (Krasensky and Jonak, 2012). Thus, higher levels of proline and carbohydrates (glucose, maltose, fructose, sucrose, and trehalose) in melatonin-pre-treated plants provided beneficial effects under abiotic stress conditions. In addition, higher levels of other metabolites including multiple amino acids, organic acids, and sugars in melatonin-pre-treated plants indicate the beneficial physiological processes in melatonin-pre-treated plants during abiotic stress treatment; the data further confirm the protective role of melatonin in response to abiotic stress. Notably, 18 metabolites comprising 10 amino acids, six sugars, and two sugar alcohols of the carbon metabolic pathway exhibited significantly higher levels in melatoninpre-treated plants under abiotic stress conditions (Fig. 6A). These results indicate the widespread effects of melatonin treatment in carbon metabolism and amino acid metabolism; these metabolites might play some role in melatonin-mediated abiotic stress resistance in bermudagrass.
Additionally, comparative transcriptomic analysis identified 2361 up-regulated and 1572 down-regulated transcripts as a consequence of exogenous melatonin treatment. Quantitative real-time PCR of 18 genes supported the reliability of the RNA-Seq data ( Supplementary Fig. S1 at JXB online). Pathway enrichment analysis indicated that eight pathways were over-represented among differentially expressed genes between control and melatonin-treated bermudagrass plants, including N metabolism, major carbohydrate metabolism, TCA/org transformation, transport, hormone metabolism, metal handling, redox, and secondary metabolism (Table 1, group I). The enrichment of redox-related genes affected by melatonin (Table 1, group I) was consistent with the effects of exogenous melatonin on ROS detoxification in bermudagrass (Figs 4, 5). In animals, melatonin is known to be involved in circadian rhythms (Tal et al., 2011). Interestingly, the GO enrichment analysis also showed that transcripts that function in regulation of rhythms and flowering were over-represented (Fig. 7). Notably, the pathway analysis results were consistent with those of the study carried out in Arabidopsis by Weeda et al. (2014). Thus, melatonin altered many genes involved in plant defence in bermudagrass ( Supplementary Fig. S2); these changes probably contributed to the enrichment of stressrelated GO terms (Fig. 7). Additionally, exogenous melatonin treatment had significant effects on various signalling pathways including primary metabolism, secondary metabolism, photosynthesis, large enzyme families, receptor-like kinases, proteolysis, and autophagy pathways in bermudagrass as determined using MapMan software ( Fig. 6B; Supplementary  Fig. S2). Those genes modulated by exogenous melatonin treatment might also contribute to melatonin-enhanced abiotic stress resistance in bermudagrass.
Asparagine accumulation shows that nitrogen re-distribution and mobilization were important features of the melatonin response (Lea et al., 2007;Maaroufi-Dguimi et al., 2011). Jia et al. (2001) also suggested that asparagine may be a signalling molecule involved in sensing the nitrogen status. In addition, asparagine is an amino group donor for the synthesis of the photorespiratory intermediate glycine. Nagy et al. (2013) and Shi et al. (2014) found that this is also a good Fig. 5. Hierarchical cluster analysis of metabolites modulated by exogenous melatonin in bermudagrass response to abiotic stress. Hierarchical cluster analysis of 54 metabolites of 28-day-old plants by melatonin effect (melatonin versus control, NaCl+melatonin versus NaCl, drought+melatonin versus drought, 4 °C+melatonin versus 4c°C) and by stress effect (NaCl versus control, drought versus control, 4 °C versus control). The log 2 ratios and scale bars are shown in the resulting tree figure, which was obtained using the CLUSTER software package and the Java Treeview. The concentrations of these metabolites are listed in Supplementary Table S2  indicator of drought stress in drought-tolerant and sensitive wheat and bermudagrass cultivars. At the same time, some carbohydrate metabolism compounds increased: these included fructose, glucose, and trehalose, but not sucrose. Such differential dynamics of carbohydrates could reflect modifications of carbon balance and carbon utilization. Moreover, asparagine synthetase genes that are involved in asparagine synthesis are regulated by the level of carbohydrates (Lam et al., 1998;Foito et al., 2009). TCA/org transformation is important for the Calvin cycle for CO 2 assimilation and separation of initial carbon fixation by contact with air and secondary carbon fixation into sugars (Selwood and Jaffe, 2011). Glycolysis is an important metabolic pathway in carbohydrate metabolism, and the central role of glycolysis in the plant metabolic pathway is to provide energy such as ATP and generates precursors for anabolism such as fatty acids and amino acids (Plaxton, 1996). In accordance with the metabolic profiles, transcriptomic analysis found that many genes involved in TCA/org transformation and N metabolism were modulated by melatonin treatment (Table 1; Fig. 6B). Genes which functioned in sucrose and amino acid metabolism were also greatly changed after melatonin treatment (Fig. 6B), leading to altered sucrose and amino acid contents revealed by metabolite analysis (Figs 5,6). These results showed that the underlying mechanisms related to melatonin may involve major reorientation of photorespiratory and carbohydrate and nitrogen metabolism.
In the current study, many TFs were significantly regulated by exogenous melatonin treatment (Table 2; Supplementary  Tables S4, S5 at JXB online), and these TFs might contribute to the enhanced stress tolerance of melatonin-treated plants, thus indicating that melatonin might pre-condition to be resistant to abiotic stresses. Some protein kinases [such as mitogen-activated protein kinase (MAPK)] and calcium signalling kinases [including calcium-dependent protein kinases (CDPKs), calcineurin B-like (CBL)-interacting protein kinases (CIPKs), and calcium-related protein kinases (CRKs)] were also transcriptionally regulated by exogenous melatonin treatment (Table 1; Supplementary Tables S4,  S5). This suggests that kinase signalling might play critical roles in melatonin-mediated stress responses. As sessile organisms, plants cannot avoid unfavourable stress conditions by adjusting their location; thus, they have evolved complex strategies to perceive stress signals and further translate the perception into effective responses, which might largely depend on various protein kinases and TFs (Shi et al., 2014a, e).
Recently, it was found that one cysteine2/histidine2-type zinc finger TF, zinc finger of Arabidopsis thaliana 6 (ZAT6), is involved in melatonin-mediated freezing stress response, and the AtZAT6-activated CBF pathway was essential for melatonin-mediated freezing stress response in Arabidopsis (Shi and Chan, 2014a). This study together with others in sunflower (Helianthus annuus) (Mukherjee et al., 2014) and in Arabidopsis (Shi and Chan, 2014a;Weeda et al., 2014) indicate that melatonin is involved in long-distance signal transduction in plants. Moreover, some important genes in plant hormone signalling [RCAR/PYR/PYL, SNF1-related protein kinases 2 (SnRK2), and nine-cis-epoxycarotenoid dioxygenase (NCED) genes in ABA signalling, and jasmonate (JA)-JIM-domain proteins (JAZs) in JA signalling] that were significantly regulated by exogenous melatonin treatment (Table 1; Supplementary Tables S4, S5 at JXB online) might also have some function in melatonin-mediated crosstalk among plant hormones, as well as in stress responses. Kolář and Macháčkova (2005) also found that melatonin might function as an auxin to promote vegetative growth. These results suggested that melatonin might serve as a plant hormone that cross-talks with other plant hormones. Thus, melatonin triggered extensive transcriptional reprogramming and pre-conditioned resistance to multiple abiotic stresses. Further investigation of the in vivo roles of these genes will shed additional light on melatonin-mediated stress responses in bermudagrass.  Taken together, this study provides the first evidence of the protective roles of exogenous melatonin in bermudagrass response to multiple abiotic stresses. This involved the activation of antioxidants, modulation of metabolic homeostasis, and extensive transcriptional reprogramming.

Supplementary data
Supplementary data are available at JXB online. Figure S1. Validation of differentially expressed genes by quantitative real-time PCR. Figure S2. Effect of exogenous melatonin treatment on stress-related pathways in bermudagrass.
Table S1. The specific primers used for real-time PCR. Table S2. Concentrations of 54 metabolites in 28-day-old bermudagrass plants under control conditions and different treatments [20 μM melatonin, 300 mM NaCl, drought, and cold (4 °C)] stress conditions for 14 d. Table S3. Summary of RNA-Seq data. Table S4. List of up-regulated genes in melatonin-treated bermudagrass plants. Table S5. List of down-regulated genes in melatonintreated bermudagrass plants. Byeon Y, Back K. 2014a. An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield. Journal of Pineal Research 56, 408-414.  Twenty-one-day-old bermuagrass plants in pots were irrigated with water or 20 μM melatonin for 7 d, then the 28-day-old plants (with and without 7 d of pre-treatment) were used for transcriptomic analysis. logFC, log2 fold change; FDR, false discovery rate. e E-value, expected value for putative annotation in Arabidopsis or rice.