Melatonin Confers Heavy Metal Tolerance of a Dark Septate Endophyte (DSE) Exophiala Pisciphila Via Lowering Oxidative Stress and Heavy Metal Accumulation

Background: The high antioxidant capacity of melatonin contributing to heavy metal tolerance for plants and animals is widely studied, while researches on microorganisms especially in lamentous fungi are rare. One typical dark septate endophyte (DSE), Exophiala pisciphila, showed signicant resistance to heavy metals. Results: In this study, exogenous melatonin was veried to reduce heavy metal damage via relieving oxidative stress, activating antioxidant systems, and decreasing heavy metal accumulation in E. pisciphila. Melatonin biosynthesis enzyme genes were upregulated under heavy metal stress. Furthermore, the overexpression of E. pisciphila TDC1 (EpTDC1) and E. pisciphila ASMT1 (EpASMT1) responsible for melatonin biosynthesis in Escherichia coli and Arabidopsis thaliana, enhanced heavy metal stress tolerance for the two organisms by lowering the oxidative stress and reducing the Cd accumulation in the whole plants, especially in the roots. Conclusions: Our results indicate that melatonin confers heavy metal resistance in E. pisciphila by lowering oxidative stress and heavy metal accumulation.


Background
Melatonin is an ancient indole molecule that dates back to 2.5 billion years ago, and exists in almost all organisms include animals, plants, and microorganisms [1][2][3]. The original function of melatonin is scavenging the excess reactive oxygen (ROS) and reactive nitrogen species (RNS) produced in the original respiration and photosynthesis [4,5].
Under heavy metal stress conditions, the levels of ROS and RNS signi cantly increase, then cause serious oxidative stress damage to the organism [6,7]. Melatonin levels were raised by various heavy metals such as cadmium (Cd), vanadium (V), and Zinc (Zn) treatment [7][8][9]. It is imperative for organisms to utilize elevated melatonin levels to improve their heavy metal stress tolerance by clearing excessive ROS and RNS. Consistent with this hypothesis, melatonin production was promoted through upregulating the expression of biosynthesis enzyme genes containing tryptophan decarboxylase (TDC), tryptophan hydroxylase (TPH), serotonin N-acetyltransferase (SNAT), and N-acetylserotonin O-methyltransferase (ASMT), and then enhanced heavy metal stress resistance for plants [3,9,12,[10][11][12].
Although various functions of melatonin in varied animals and plants, and partial microorganisms have been studied via regulating endogenous melatonin levels, little investigation in lamentous fungi is available [13]. Melatonin originated from the primitive bacteria (cyanobacteria and α-proteobacteria) and has been retained throughout the evolution of all organisms [14,15]. Therefore, it can't be ignored that lamentous fungi have a signi cant position in the melatonin evolution process [5].
Dark septate endophytes (DSEs), a group of dark pigmented and septate hyphae fungi, widely colonize in the plant's roots [16]. Notably, these fungi wildly inhabit in most roots of plants that grow in the high concentrations of Pb, Zn, and Cd amine smelting region [17,18]. We isolated one highly heavy metal tolerant DSE strain, Exophiala pisciphila, from the roots of Arundinella bengalensis living an old mine smelting site in Yunnan province, southwest China. The plumbum (Pb), Cd, and Zn concentrations in the dry weight of E. pisciphila hyphae reach over 25%, 4.9%, and 16.0% respectively [16]. Signi cant adaption to the high heavy metal environment for the isolate suggested its particular metal tolerance mechanisms. Ground on the primary anti-oxidation function of melatonin, the compound possibly participates in heavy metal tolerance formation of E. pisciphila. Moreover, our transcriptome data of E. pisciphila [19] showed that melatonin biosynthetic enzyme genes (TDC, SANT, and ASMT) were upregulated under Cd stress, which indicated the involvement of melatonin in heavy metal tolerance.
The objective of this study was to investigate the effects of melatonin in improving various heavy metals tolerance, genes controlling melatonin biosynthesis, and the behind mechanisms in terms of relieving oxidative stress and reducing heavy metal accumulation.

Exogenous melatonin alleviated oxidative stress under heavy metal stresses
Under heavy metals stress, oxidative stress is one of the most serious damages for organisms [1]. In this study, we investigated the in uence of different doses melatonin on oxidative stress caused by assessing the increases in malondialdehyde (MDA) and oxygen free radical (OFR) content in E. pisciphila. The MDA content of the isolate gradually decreased as the exogenous melatonin concentration application increased under Cd, Zn, and Pb stresses (Fig. 1A Exogenous melatonin increased superoxide dismutase (SOD) activity and decreased heavy metals accumulation Given that melatonin relieved the oxidative stress, the activity of superoxide dismutase (SOD), one of the antioxidant enzymes, was examined. The application of melatonin increased the activity of SOD under Zn and Pb stresses, and signi cantly increased by 30.01% and 33.45% respectively pretreated with 200.0 µM melatonin ( Fig. 2C and D). However, melatonin did not signi cantly in uence the SOD activity under Cd and Cu stresses ( Fig. 2A and B).
Preventing excessive heavy metal accumulation is an important way to limit the deleterious impact on organisms [1]. Thus, we explored whether melatonin changed the Cd, Cu, Zn, and Pb contents in E. pisciphila. Intriguingly, the heavy metal accumulation in E. pisciphila gradually decreased with elevated melatonin levels (Fig. 2E, F, G, and H). Pretreated with 200.0 µM melatonin, the content of Cd, Zn, and Pb signi cantly reduced by 32.24%, 46.7%, and 31.55% respectively (Fig. 2E, G, and H). In summary, exogenous melatonin enhanced heavy metal tolerance by increasing SOD activity and reducing heavy metals accumulation in E. pisciphila.
EpTDC1, EpSNAT1, and EpASMT1 differentiated from that of plants and animals Based upon the transcriptome data of E. pisciphila (Zhao et al., 2015), three melatonin biosynthetic enzymes cDNA sequences (EpTDC1, EpSNAT1, and EpASMT1) were obtained. According to amino acid BLAST search, the EpTDC1, EpSNAT1, and EpASMT1 homologs were found in various animals and plants. The phylogenetic tree indicated that EpTDC1, EpSNAT1, and EpASMT1 all gathered in the fungi cluster which formed a clade separately from the animals and plants ( EpTDC1 and EpASMT1 conferred heavy metals tolerance for E. coli and A. thaliana The expression of EpTDC1, EpSNAT1, and EpASMT1 in response to heavy metal stress suggested that it is involved in the physiological process of conferring heavy metal resistance. We transferred EpTDC1 and EpASMT1 into E. coli and A. thaliana to further investigate the role of melatonin in the heavy metal stress resistance. The abundance of E. coli in liquid culture was measured by the optical density at 600 nm (OD 600 ), which is a widely used method in bacteria [20]. The OD 600 of the E. coli overexpressing EpTDC1 and EpASMT1 were signi cantly enhanced under Cd Cu, Zn, and Pb stresses (Fig. 5). Hence, both EpTDC1 and EpASMT1 conferred E. coli heavy metal stress resistance. . Asterisks indicate signi cant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.
For transgenic Arabidopsis, EpTDC1-1/EpTDC1-2 and EpASMT1-1/EpASMT1-2 were used for investigating the heavy metal tolerance. Under 10.0 µM Cd 2+ , the root length and fresh weight signi cantly increased by 19.2% 29.6% in EpTDC1-1 and 14.0% 15.5% in EpTDC1-2 in comparison with the wild-type plants ( Fig. 6B and C). The overexpression of EpASMT1 also enhanced the growth of Arabidopsis under Cu stress (Fig. 6D). The root length and fresh weight signi cantly increased by 31.46% and 78.82% in EpASMT1-1, and 36.52% and 63.53% in EpASMT1-2 under 10.0 µM Cu 2+ , respectively ( Fig. 6E and F). In this section, it has been indicated that the overexpression of EpTDC1 and EpASMT1 relieved heavy metal stresses for transgenic Arabidopsis.   (Fig. 7A and B). These data suggested that EpTDC1 and EpASMT1 enhanced Cd resistance was associated with decreasing Cd accumulation in root tissues of Arabidopsis.

Discussion
Excessive heavy metals lead organisms to produce excessive reactive oxygen species (ROS) via inhibiting the antioxidant system, disrupting the electron transport chain, and disturbing the metabolism of essential elements [21][22][23]. Organisms have evolved kinds of mechanisms to relieve oxidative stress caused by heavy metals [24,25].
As one ancient antioxidant, melatonin plays key roles in decreasing oxidative stress [13,26]. Therefore, melatonin could potentially confer heavy metal stress resistance. Consistent with this hypothesis, melatonin production was promoted to ease heavy metal stress in plants and animals [1,2]. Signi cantly enhanced melatonin accumulation was also observed in E. pisciphila challenged with Cd, Cu, and Zn at 2 days (Fig. 3D). While Pb had no mitigating effect on melatonin production similar to that observed in rice [27], which indicated that E. pisciphila have distinct stress responses to the different type heavy metals [28]. The application of exogenous melatonin reduced OFR and MDA contents of E. pisciphila under Cd, Zn and Pb stresses (Fig. 1) suggested that melatonin may act as an antioxidant to relieve the deleterious impact [29].
Except to directly scavenged ROS, melatonin indirectly activated antioxidant enzymes and decreased heavy metal accumulation to relieve heavy metal stress [27,30]. For instance, melatonin decreased excessive ROS caused by heavy metals via the activation of antioxidant enzymes such as SOD in rice, watermelon, and wheat [6,8,9]. Our ndings identi ed that 200.0 µM melatonin signi cantly increased SOD activity under Zn and Pb stresses ( Fig. 2C and D). Hence, melatonin lowered oxidative stress through enhanced the activity of SOD under heavy metal stresses in E. pisciphila.
Another important way to relieve heavy metal toxicity is by decreasing heavy metal accumulation [1]. Melatonin application signi cantly lowered vanadium (V) in Citrullus lanatus, and Cd in rice and Arabidopsis [9,6,12]. Given that 200.0 µM melatonin signi cantly reduced the Cd, Zn, and Pb content of E. pisciphila (Fig. 2), it is likely that melatonin decreased the heavy metal accumulation to enhance heavy metal tolerance for this DSE (Fig. 1). In general, melatonin enlaced heavy metal tolerance was closely related to increase SOD activity and decrease heavy metal accumulation in E. pisciphila ( Figs. 1 and 2).
Establishing the precise roles of melatonin on heavy metal resistance in E. pisciphila requires an investigation of biosynthetic pathways [11]. Although the biosynthetic mechanisms of melatonin have been well characterized in animals and plants, little related research was carried out in microorganisms especially in lamentous fungi. In plants, the rst committed step for melatonin biosynthesis is TDC, and SNAT and ASMT contribute to the two nal catalyses [3]. In our previous transcriptome analysis of E. pisciphila [19], three genes annotated as TDC, SNAT, and ASMT expression were upregulated by Cd. The phylogenetic tree showed that EpTDC1, EpSNAT1, and EpASMT1 differentiated from that of plants and animals ( Fig. 3S1 and S2). Under Cd, Cu, and Zn stresses (2d), EpTDC1 and EpSNAT1 were transcriptionally upregulated with elevated melatonin levels (Fig. 4). Similar to our ndings, the upregulation of TDC and SNAT expression induced melatonin production with Cd treatment in rice [7]. Our results identi ed the essential role of EpTDC1 and EpSNAT1 at melatonin levels for heavy metal resistance in E. pisciphila. The reason for the downregulation of EpASMT1 (Fig. 4C) is not clear, but maybe important for the induction of melatonin production. Under various scenarios, transcript levels are not su cient to predict protein levels and to thus explain genotype-phenotype relationships [31]. Byeon et al. (2015) also founded that the melatonin production did not consist with the expression of its biosynthesis enzyme genes under Cd stress [27].
Furthermore, we overexpressed EpTDC1 and EpASMT1 in E. coli and A. thaliana, which rescued the growth inhibition caused by heavy metal (Figs. 5 and 6). Consistent with the nding of alfalfa SNAT overexpressed in Arabidopsis [12], EpTDC1 and EpASMT1 decreased Cd content both the total and roots of Arabidopsis under 40 mg kg -1 Cd 2+ (Fig. 7). These data indicated that EpTDC1 and EpASMT1 played key roles in decreasing excessive heavy metals accumulation.

Conclusions
In short, we rst demonstrated melatonin alleviated heavy metal stresses in one lamentous fungi. Melatonin biosynthesis enzyme genes responsible for melatonin biosynthesis relieved oxidative stress via directly clearing and indirectly enhancing SOD activity, and decreased heavy metal accumulation in E. pisciphila.

Fungal materials, growth conditions and treatments
Exophiala pisciphila was isolated from the roots of Arundinella bengalensis (authenticated by Pro. Shugang Lu from Yunnan University, and the voucher specimen was preserved in Yunnan University), Quanti cation Of Melatonin By High-performance Liquid Chromatography (hplc) Hyphae (0.1 g) were ground in liquid nitrogen and extracted with 1.0 mL chloroform for 1 hour at room temperature before melatonin quanti cation. Chloroform extracts (200.0 µL) were completely evaporated and dissolved in 0.1 mL 40% methanol, and 10.0 µL aliquots were subjected to HPLC using a uorescence detector system (Waters, Milford, MA, USA). The samples were separated on a Sun re C18 column (Waters; 4.6 × 150 mm) using the following gradient elution pro le: from 42-50% methanol in 0.1% formic acid for 27 minutes, followed by isocratic elution with 50% methanol in 0.1% formic acid for 18 minutes at a ow rate of 0.15 mL min -1 . Melatonin was detected at 280 nm (excitation) and 348 nm (emission). All measurements were taken in triplicate.
Identi cation and phylogenic tree construction of EpTDC1, EpSNAT1, and EpASMT1 Based on the transcriptome database of E. pisciphila [19], the nucleic acid sequences of putative EpTDC1, EpSNAT1, and EpASMT1 were obtained. The open reading frame nucleotide and amino acid sequences of these unigenes were predicted using Open Reading Frame Finder (ORF) in National Center for Biotechnology Information (NCBI). Then BLAST search was performed using the EpTDC1, EpSNAT1, and EpASMT1 amino acid sequences. Phylogenetic trees were generated from various amino acid sequences after alignment with Clustal X (version 1.83), and phylograms were constructed using the Neighborjoining method algorithm with branch length (MEGA 5.0).

Expression analysis of EpTDC1, EpSNAT1, and EpASMT1
Total RNA was extracted from hyphae using the RNAiso Plus (TaKaRa, Japan, 9108) according to the manufacturer's protocol. The isolated RNA (1.0 µg) was synthesized cDNA via the PrimeScriptII 1st Strand cDNA Synthesis Kit (TaKaRa, Japan, 6210A). The quantitative real-time PCR (qRT-PCR) analysis was performed with a LightCycler® 480 II Real-Time PCR Detection System (Roche, Basel, Swiss) using SYBR Premix Ex Taq (TaKaRa, Japan, RR820A), and β-tubulin gene as the internal control. Primers used in this study were listed in Table S1. All of the reactions were performed with three biological and technical replicates independently.

Determination of Cd accumulation in Arabidopsis thaliana
The transgenic Arabidopsis and wild-type plant seeds were germinated on 1/2MS plates. When the second true leaves were fully expanded, seedlings were transferred into the soil containing 0, 20.0, 40.0 mg kg -1 Cd 2+ . Plants grown in the soil for 30 days were collected and the Cd contents of the total, shoot, and root tissues were detected by the method in the determination of Cd accumulation in E. pisciphila. Each treatment was conducted independently four times.

Statistical Analysis
Independent-samples t-test and one-way ANOVA with Duncan's multiple range test (SPSS 16.0) were used to detect the signi cant differences between means, P-values < 0.05 were considered statistically signi cant.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.
funding bodies had no contribution in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request 8.     Data are means ± SD (n=4). Asterisks indicate signi cant differences (p < 0.05*, p < 0.01**, p < 0.001***) compared to the control according to Independent-Samples T test.

Figure 6
The overexpression of EpTDC1 and EpASMT1 enhanced the heavy metal tolerance of Arabidopsis. Seeds of the transgenic EpTDC1 (EpTDC1-1 and EpTDC1-2) and EpASMT1 (EpASMT1-1 and EpASMT1-2) Arabidopsis grown on 1/2 MS medium plates contained 0, 5.0, and 10.0 μM Cd2+, and 0, 10.0, and 30.0 μM Cu2+ respectively for 10 days with the wild-type (WT) Arabidopsis as the control. The corresponding pictures were respectively taken (A, D), Scale bars = 1 cm. Meanwhile, the root length (B, E) and fresh weight (C, F) were measured. Data are means ± SD (n=6). Columns with different letters denote signi cant differences at P < 0.05 according to Duncan's multiple range test.

Figure 7
The overexpression of EpTDC1 and EpASMT1 decreased Cd accumulation in Arabidopsis. The Arabidopsis seedlings at the four-leaf stage of wild-type (WT), EpTDC1-1, EpTDC1-2, EpASMT1-1, and EpAMST1-2 were transferred to soil containing 0, 20.0, 40.0 mg kg-1 Cd2+ for 30 days. The Cd contents in the shoot, root, and the whole plant of A. thaliana were measured. Data are means ± SD (n=4).
Columns with different letters denote signi cant differences at p < 0.05 according to Duncan's multiple range test.