Melatonin enhances salt stress tolerance in rubber tree (Hevea brasiliensis) seedlings

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Melatonin enhances salt stress tolerance in rubber tree (Hevea brasiliensis) seedlings Hong Yanga,b,1, Longjun Daia,b,1, Yongxuan Weia, Zhi Denga,b, Dejun Lia,b,* a Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China b State Key Laboratory Incubation Base for Cultivation & Physiology of Tropical Crops, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Melatonin Salinity stress Antioxidant Hydrogen peroxide Transcriptome Rubber tree

As a well-known agent, melatonin plays very important roles in animals. However, its possible function is less clear in plants, especially in rubber tree. In this study, we demonstrate that melatonin acts as a potential agent to enhance salt stress tolerance in rubber tree seedlings. Chlorophyll degradation and H2O2 accumulation in leaves of seedlings under salinity stress were significantly relieved by melatonin treatment. Transcriptome analyses suggested that melatonin was a potential antioxidant, and its exogenous application resulted in enhanced antioxidant protection mainly by regulating the genes involved in photosynthesis, ROS metabolism, flavonoids and melatonin biosynthesis. These results demonstrate that melatonin can enhance salt stress tolerance directly or indirectly by counteracting the cellular accumulation of H2O2 in rubber tree. Our findings are helpful for not only understanding melatonin’s roles in salinity stress, but also providing new insights into potential utilization of melatonin against abiotic stresses in rubber tree.

1. Introduction As a member of the spurge family (Euphorbiaceae), rubber tree (Hevea brasiliensis) is one of the most economically important plants throughout tropic zones. Natural rubber harvested from rubber tree is an excellent industrial raw material with unique physical and chemical properties, and it cannot be fully replaced by synthetic rubber (van Beilen and Poirier, 2007). At the same time, natural rubber is a very important strategic material. Salinity, one of the most common abiotic stresses, not only restricts plant growth and development, but also reduces plant productivity. Most plants are sensitive to soil salinity, and salt stress usually has negative effects on plants in many ways such as oxidative damage, Na+ toxicity, physiological water-deficit, nutritional imbalance, metabolic disorder, activity alteration of cytosolic enzymes, photoinhibition, etc (Chen et al., 2007; Cuin and Shabala, 2007; Munns and Tester, 2008; Pandolfi et al., 2012; Tang et al., 2015; Gharat et al., 2016). Mangroves are woody plants with unique ability to tolerate high salinity. Salt tolerance mechanisms of mangroves can be partially explained by its morphological, anatomical, physiological, biochemical, and molecular as well as genetic attributes. Morphological and

anatomical features contain salt-excreting leaves, viviparous water dispersed propagules, salt glands in their leaves, thick-walled epidermis with waxy cuticle, and sunken stomata. Physiological and biochemical mechanisms adopted by mangroves include salt excretion, salt accumulation, salt secretion, accumulation of compatible solutes, and induction of antioxidative enzymes. In addition, the salt-related genes involved in protein synthesis, defense, transport, ion homeostasis, protein destination, and signal transduction are closely associated to salt tolerance in mangroves (Parida and Jha, 2010). It was reported from FAO 2008 that more than 800 million hectares of land was subjected to salinity in the world. Additionally, about 1.5 million hectares of land was unsuitable for cultivation due to high levels of salinity in soil (Munns and Tester, 2008). Salinity has been a major abiotic stress limiting plant productivity. Under salinity stress, the root-to-shoot ratio and seed vigor of rubber tree significantly decreased; moreover, the growth of rubber tree was inhibited (Zeng et al., 2007). With the expansion of salinized land, rubber tree will be faced with the threat from salt stress. Therefore, improving salt tolerance of rubber tree will be benefit for reducing negative effect of salinity on rubber tree. Two strategies can be utilized to improve salt tolerance of rubber

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Corresponding author at: Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China. E-mail address: [email protected] (D. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2019.111990 Received 28 April 2019; Received in revised form 15 October 2019; Accepted 13 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Hong Yang, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111990

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2.3. DAB staining and H2O2 content assay

tree. One is to breed rubber tree varieties or clones with high salt tolerance, which is very inefficient and time-consuming due to an average duration of 20–25 years of field experiments and unknown molecular mechanisms underlying salt tolerance in rubber tree (Priyadarshan, 2017). The other is to utilize growth-regulating substances to improve salt tolerance. It has been reported that some growth-regulating substances including glycine betaine, salicylic acid (SA), nitric oxide (NO), and N-acetyl-5-methoxytryptamine (melatonin) can improve salt tolerance of crop plants (Yang and Lu, 2005; Zhang et al., 2006; Bastam et al., 2013; Song et al., 2014; 2015; Wei et al., 2015; Kreslavski et al., 2017; Li et al., 2017; Chen et al., 2018). Melatonin, firstly discovered in the bovine pineal gland, is associated with numerous vital life processes in animals, including sleep, body temperature, mood, circadian rhythm, oncogenesis, and antioxidative activity (Calvo et al., 2013; Galano et al., 2013; Zhang et al., 2018). As an important and necessary functional molecule, endogenous melatonin widely exists in bacteria, fungi, animals, and plants (Arnao and Hernández-Ruiz, 2015). In accordance with animals, melatonin also plays vital roles in plant growth, development, plant-pathogen interactions, and various stress responses (Kolár and Machácková, 2005; Tan et al., 2012; Zhang et al., 2014; Shi et al., 2015; Wei et al., 2015; Li et al., 2016). Among these roles, melatonin was widely reported to be involved in plant response on abiotic stresses mainly including salinity (Jiang et al., 2016), drought (Wang et al., 2013), heavy metal (Posmyk et al., 2008), and low temperature (Shi et al., 2015). However, melatonin’s role on salinity stress remains largely unknown in rubber tree. In the present study, we investigated the potential roles of exogenous melatonin on salinity stress in rubber tree seedlings. We found that salinity stress caused leaf chlorophyll degradation and H2O2 accumulation in rubber tree seedlings, while melatonin partly alleviated the aforementioned adverse influences. Transcriptome analyses revealed that melatonin might play important roles against salinity stress mainly by regulating the genes involved in photosynthesis, ROS metabolism, flavonoids and melatonin biosynthesis. These results are helpful for not only understanding melatonin’s role in salinity stress, but also providing new insights into potential utilization of melatonin against stresses in rubber tree.

Seedling leaves from different treatments and the control were collected and soaked in 1 mg/mL DAB (3, 3′-diaminobenzidine) solution (50 mM Tris−HCl, pH 3.8). After the seedling leaves were incubated with DAB solution for one day, the incubated leaves were decolored to translucent with absolute ethanol. The pictures of the decolored leaves were taken, and the brown color shows the H2O2 presence in rubber tree leaves. H2O2 was extracted with 5 % (w/v) trichloroacetic acid and measured according to the methods of Patterson et al. (1984). 2.4. RNA preparation, transcriptome sequencing, and assembly Total RNAs of seedling leaves from each treatment and the control were extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and quantified using Qubit® RNA Assay Kit and the Qubit® 2.0 Flurometer (Life Technologies). RNA degradation and contamination were monitored on 1 % agarose gels; the purity and integrity were assessed using the NanoPhotometer® spectrophotometer (IMPLEN) and the RNA Nano 6000 Assay Kit of the Agilent Bio analyzer 2100 system (Agilent Technologies), respectively. Sequencing library construction and Illumina sequencing were performed by Novogene Corporation, China. After filtering out the adaptor sequences and deleting low-quality and contaminated reads, we assembled leaf transcriptomes using Trinity with min_kmer_cov set to 2 by default and all other parameters set default (Grabherr et al., 2011). 2.5. Gene functional annotation and analysis All assembled unigenes were searched against the following databases: Nr (NCBI non-redundant protein sequences), Nt (NCBI non-redundant nucleotide sequences), Pfam (Protein family), Swiss-Prot (A manually annotated and reviewed protein sequence database), KOG/ COG (Clusters of Orthologous Groups of proteins), KO (Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology) database, and GO (Gene Ontology) with blastx. The expression levels of the assembled genes were calculated via the RPKM (reads per kb per million reads) method by RSEM software (Li and Dewey, 2011). With the assembled unigenes as the reference transcriptome, the clean reads from the control and treated samples were separately realigned to the reference transcriptome. With qvalue < 0.005 and |log2FoldChange| > 1 as the thresholds, the DEGseq of the R package (Version 1.12.0) was applied to identify the differentially expressed genes (DEGs) between the control and treated samples (Wang et al., 2010). GO enrichment analyses of the DEGs were performed using the R package GOseq based Wallenius non-central hyper-geometric distribution (Young et al., 2010), which can adjust for gene length bias in the DEGs. KOBAS software was used to analyze the enriched KEGG pathway of the DEGs (Mao et al., 2005). In addition, hierarchical cluster analysis of the DEGs was performed using R (http://www.r-project.org).

2. Materials and methods 2.1. Plant materials and treatments One-year-old seedlings of rubber tree clone (Reyan 7-33-97) were used as experimental materials. According to the paper published by Wei et al (2015), we determined the dosage and duration of treatments in this study. The seedlings were treated with water (H, the control), 100 μM melatonin solution (M), 1 % NaCl (N) or 1 % NaCl plus 100 μM melatonin solutions (NM), respectively. After a three-day treatment, the seedling leaves from each treatment were separately collected and equivalently pooled for total RNA extraction. Three biological replicates were set for each treatment in this study, and the leaves collected from five seedlings were referred as one biological replicate. One and three replicates were separately for transcriptome sequencing and quantitative real-time PCR (qRT-PCR) experiments.

2.6. qRT-PCR analyses

2.2. Chlorophyll content assay

To validate the DEGs deduced from RNA-Seq analyses, we selected twenty-five DEGs to analyze their expression patterns by qRT-PCR with HbYLS8 as the internal control. All the primers used for qRT-PCR were designed using Primer premier 5 software (PREMIER Biosoft, Palo Alto, CA, USA) and synthesized by Sangon Biotechnology (Guangzhou, China). The primer pairs are listed in Supplementary Table S1. Total RNAs were reversely transcribed using PrimeScrip RT reagent Kit with gDNA eraser (Takara). The amplified PCR products were monitored by a CFX96 Real-Time PCR detection System (Bio-Rad). All qRT-PCR experiments were repeated 3 times in independent runs for all the references and selected genes. The Ct values were means ± SD of three

After a three-day treatment, the leaves from rubber tree seedlings treated with H, M, N, and NM were collected for chlorophyll measurement, respectively. Chlorophyll was extracted with 95 % ethanol, and the content was determined spectrophotometrically according to the method of Lichtenthaler (1987). Three biological replicates were set for measuring chlorophyll content, and the leaves from five seedlings of a treatment were equivalently pooled as one biological replicate. Statistical analysis was performed by Student’s t-test in SPSS 22.0 (IBM, Armonk, New York). 2

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Fig. 1. Melatonin effects on salinity stress in leaves of rubber tree seedling. (A) Fresh and DAB staining leaves of rubber tree seedlings. Brown colour indicates accumulation of H2O2. The “F” and the “D” columns show the fresh leaves and leaves stained by DAB, respectively. (B) H2O2 contents in rubber tree leaves before and after treatments. Different letters labeled above the bars indicate significant difference at P < 0.05 (Tukey's test). (C) Relative Chlorophyll contents in rubber tree leaves after treatments. Asterisks indicate a significant diff ;erence (*P < 0.05, t-test) between each treatment and the control. H: treated with water (the control), M: treated with 100 μM melatonin, N: treated with 1 % NaCl and NM: treated with 100 μM melatonin plus 1 % NaCl.

biological repeats and gene expression was evaluated by the 2-ΔΔCt method (Livak and Schmittgen, 2001). 3. Results

which was significantly lower than the NM-treated ones (Fig. 1C). These findings implied that salinity stress might lead to chlorophyll degradation and H2O2 accumulation whereas melatonin could partly alleviate negative effects of salinity stress in rubber tree seedlings.

3.1. Melatonin increases salt tolerance of rubber tree seedlings

3.2. Melatonin-regulated gene identified by transcriptome analysis

To investigate melatonin’s roles on NaCl stress, we treated one-yearold rubber tree seedlings with H, M, N and NM, respectively. Compared with the control, the leaves from rubber tree seedlings treated with N turned yellow three days later, whereas the leaves treated with M were almost as green as the control samples; the color of the leaves treated with NM was somewhere in between the leaves treated with M and N treatments (Fig. 1A). DAB staining indicated that the leaves with N treatment had the highest H2O2 levels, followed by the leaves treated with NM; the leaves with M treatment and the control had relatively lower H2O2 levels (Fig. 1A). Theese results from DAB staining were further confirmed by the H2O2 contents measured in the four samples (Fig. 1B). In accordance with the results mentioned above, the leaves of rubber tree seedlings treated with M had similar chlorophyll content as the control, whereas the chlorophyll contents in the seedlings treated with NM were lower than the control and the M-treated ones. The leaves from the N-treated seedlings had the lowest chlorophyll content

To elucidate the possible mechanism underlying melatonin’s roles on salinity stress of rubber tree seedlings, we further carried out comparative transcriptome analyses of rubber tree leaves with the four treatments including H, M, N, and NM. Total RNAs were isolated from the aforementioned materials, and transcriptome sequencing was performed using the Illumina paired-end sequencing approach. Before assembling transcriptome data, we removed the adaptors, low-quality, and contaminated reads. In total, 149,301,830 high-quality reads from four samples were obtained and assembled into leaf transcriptome with Trinity (Grabherr et al., 2011). In the end, 89,038 unigenes comprising ∼61.60 Mb were generated in this study. The raw data of RNA-Seq was uploaded to NCBI (SRA accession number: PRJNA566067), and the statistics for the assembled transcriptome of rubber tree leaves were shown in Table 1. With the 89,038 assembled unigenes as reference, the high-quality reads from the four samples were separately realigned to the assembled 3

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Table 1 Statistics of transcriptomic sequencing and assembly of rubber tree leaves. Item

Reads

Total number of raw reads Total number of clean reads Average Q20 (%) N50 length (bp) N90 length (bp) Maximum sequence length (bp) Average length (bp) Total number of transcripts/unigenes

152,178,110 149,301,830 95.88

Transcripts

Table 2 Numbers of the DEGs in different comparisons.

Unigenes Upregulated Downregulated Total

1728 344 11993 927 123,368

1162 275 11,993 692 89,038

MH

NH

NMH

105 67 172

112 103 215

114 171 285

rubber tree seedlings (Fig. 1), therefore the genes regulated by melatonin and salt were further analyzed. As shown in Fig. 2B, 27 and 50 DEGs were commonly up- or down-regulated by M and NM, respectively; whereas 69 and 61 DEGs were commonly down- or up-regulated by N and NM, respectively. To verify the accuracy of the DEGs identified from the RNA-seq analyses, twenty-five DEGs selected from NH, MH, and NMH comparisons were further analyzed with qRT-PCR method. As shown in Supplementary Fig. S1, there was a strong positive correlation (R2 = 0.9851) between RNA-seq and qRT-PCR results. Moreover, the regression slope of RNA-seq versus qRT-PCR was close to 1, indicating that the DEGs deduced from RNA-seq data were accurate, credible, and reproducible in this study. Next, GO and KEGG of the DEGs from the three comparisons were systematically analyzed in this study. Compared with the control, the DEGs from N-, M- and NM-treated samples were separately involved in 34, 32, and 37 GO terms. For the DEGs in all the three comparisons, obvious over-representation GO term was metabolic process in biological process category. Moreover, the DEGs in NH comparison were significantly enriched in catalytic activity, enzyme regulator activity, and antioxidant activity; whereas catalytic activity and transporter

unigenes, and gene expression was calculated based on mapped reads in each sample. As shown in Fig. 2A, 54,347, 56,387, 59,721, and 55,925 genes were expressed in H-, N-, M-, and NM-treated samples, respectively. Moreover, 2973, 4808, 4220, and 3436 transcripts were uniquely expressed in H, M, N, and NM, respectively. In total, 39,498 genes were co-expressed in all the four samples. Next, three comparisons were further conducted, including N VS H (NH), M VS H (MH), and NM VS H (NMH). 112 and 103 DEGs were separately up- and downregulated in NH comparison, whereas 105 and 67 in MH comparison; the corresponding numbers were separately 114 and 171 in NMH comparison (Table 2). Venn diagrams were constructed to analyze the relationship among different treatments. As shown in Fig. 2B and Supplementary Table S2, there were separately 12 and 13 DEGs up- or down-regulated by all three treatments in contrast to the control, indicating that the 25 genes may be associated with the response on environmental changes. According to the findings mentioned above, we presumed that melatonin might mitigate the negative effects of salt on

Fig. 2. Differentially expressed gene number affected by various treatments. (A) Comparison of expression gene numbers among the various treatments using Venn diagram. (B) Comparison of gene numbers affected by different treatments using Venn diagram. Up- and down-regulated genes (|log2FoldChange|≥1) were examined for common genes using Venn diagram. Overlapping areas represent common gene numbers. 4

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Supplementary Table S7). Two DEGs, TSB2 and COMT1 (c53265_g1), were separately down- and up-regulated in the three comparisons including MH, NH, and NMH. T5H (c48038_g1) was downregulated in MH comparison, whereas upregulated in NH one. In addition, another T5H (c5850_g1) and COMT3 were increased in NH and NMH comparisons; COMT1s, c26217_g1 and c53265_g2, were increased in NH and NMH comparisons, respectively. The expression patterns of the DEGs in melatonin biosynthesis necessarily affect melatonin content in rubber tree. Compared with the control, most of the genes in melatonin biosynthesis were upregulated by N and NM treatments, but not by M treatment; therefore, the rubber tree seedlings with N and NM treatments might possess higher melatonin content.

activity in NMH and growth in MH comparison (Supplementary Table S3). Interestingly, a relatively high percentage of the DEGs involved in response to stimulus were also noticed in all the three comparisons (Supplementary Table S3). Being consistent with GO results, KEGG analysis results indicated that the DEGs identified in all the three comparisons were exclusively enriched in metabolism, which mainly contains photosynthesis, flavonoids biosynthesis, cyanoamino acid metabolism, phenylalanine metabolism, photosynthesis-antenna proteins, and phenylpropanoid biosynthesis (Supplementary Table S4). These DEGs associated with different functional categories clearly indicated that molecular and cellular events including metabolic process, antioxidant activity, response to stimulus, etc occurred in rubber tree seedlings under N, M, or NM treatments.

3.6. Genes involved in ROS metabolism 3.3. Genes involved in photosynthesis

Flavonoids play a variety of roles in plants. Besides their influence on auxin transport (Peer et al., 2011), flavonoids are related to defense (Treutter, 2005), ROS balance (Taylor and Grotewold, 2005; Bais et al., 2006), and allelopathy (Bais et al., 2006). All the 13 DEGs in flavonoids biosynthesis pathway were significantly upregulated by M, N, or NM treatments, and their expression profiles were different from those of photosynthesis-related genes. As shown in Fig. 4 and Supplementary Table S6, one FDR gene, one 4 H L gene, one CHI gene, two CHS genes, one LAR gene, one F3'H gene, and one F3H gene were upregulated in NH and NMH comparisons; the expression of two PAL genes were only increased in NH comparison. Besides the DEGs including 4 H L, LAR, and CHI, two DEGs, PAL6 and CCoAOMT, were also upregulated by M treatment in contrast to the control. As important antioxidants, flavonoids content might increase in rubber tree seedlings treated with M, N or NM due to upregulating the genes in flavonoids biosynthesis pathway. Therefore we suggest that rubber tree seedlings may alleviate salinity-induced oxidant stress by increasing flavonoids content.

It was reported that salt stress can influence plant physiological, biochemical, cellular, and molecular processes (Jouyban, 2012). To avoid harmful effect of salt stress, plants have developed effective defense mechanisms (Gupta and Huang, 2014). Compared with the control, a total of 41 genes were identified to be the DEGs in M, N, and NM treatments. The DEGs encoding scopoletin glucosyltransferase and TT12-2 MATE transporter were increased in three comparisons, whereas the DEG encoding reticuline oxidase-like protein was decreased. The DEG encoding Cytochrome P450 90B1 was separately down- and up-regulated in MH, and NH as well as NMH, while the DEGs encoding MLP-like protein 43 and hypothetical protein POPTR_0014s17480 g were up- and down-regulated in MH, and NH as well as NMH, respectively. In addition, 6 and 14 DEGs were significantly up- and down-regulated in N-treated samples, respectively (Supplementary Table S8). Among six upregulated DEGs, the expressions of the DEGs encoding superoxide dismutase, lignin-forming anionic peroxidase, transparent testa 12 (c86627_g1), and major allergen Pru ar were also increased in NMH comparison; while the ones encoding pectin acetylesterase and transparent testa 12 (c49574_g1) were upregulated in MH comparison. Fourteen DEGs encoding ATP synthase subunit b, mitochondrial chaperone BCS1-B, disease resistance protein RGA3, LRR receptor-like serine/threonine-protein kinase GSO2, catalase 1, peroxidase 12, CBL-interacting serine/threonine-protein kinase 25, NAD(P)H-quinone oxidoreductase subunit 2 A, ASR-like protein 1, protease inhibitor protein, coatomer subunit epsilon-1, dynamin-related protein 4C, universal stress protein A-like protein, and aldehyde dehydrogenase family 3 member I1, were downregulated in NH comparison. Besides being downregulated in NH comparison, the first two DEGs were separately up- and down-regulated in MH comparison; the last six DEGs were also downregulated in NMH comparison. Moreover, three DEGs encoding tetrapyrrole-binding protein, Fibronectin, and calcium-binding protein CML44 were upregulated in MH comparison; seven DEGs encoding phytosulfokines 3 precursor family protein, mitoferrin, ABC transporter C family member 3-like, inactive poly [ADPribose] polymerase SRO2, MLP-like protein 423, Rho GTPase-activating protein 3, and MLO-like protein 12 were evidently downregulated in MH comparison. The last five were simultaneously downregulated in NMH comparison (Supplementary Table S8). The expression profiles of the aforementioned genes might alleviate harmful effects of salt stress by reducing or eliminating ROS accumulation.

3.5. Genes involved in melatonin biosynthesis

4. Discussion

Besides acting as important antioxidant itself, melatonin also works together with other antioxidants to improve their overall antioxidative effectiveness (Arnao and Hernández-Ruiz, 2006). Five groups of key genes in melatonin biosynthesis including isochorismate synthase (ICS), tryptamine-5-hydroxylase (T5H), anthranilate synthase (AS), tryptophan synthase (TS), and caffeic acid-O-methyltransferase (COMT) were regulated by M, N, or NM treatments, but only seven genes were identified as the DEGs in the three comparisons (Fig. 5 and

Melatonin, with plenty of functions in animals, also shows great potential roles in plants. Besides regulating plant growth and development, melatonin, as an anti-stress agent, enhanced salt tolerance of the crop plants (Yang and Lu, 2005; Zhang et al., 2006; Bastam et al., 2013; Song et al., 2014; Kostopoulou et al., 2015; Wei et al., 2015; Arora and Bhatla, 2017; Kreslavski et al., 2017; Li et al., 2017; Chen et al., 2018). In addition, exogenous application of melatonin enhanced drought tolerance and delayed drought-induced leaf senescence in

In contrast with the control, 18 photosynthesis-related genes were regulated in the leaves of rubber tree seedlings treated with M, N, or NM. Among the 18 photosynthesis-related genes, 15 were identified as the DEGs in three comparisons including MH, NH, and NMH. Interestingly, the DEGs in photosynthesis were upregulated by M treatment, whereas downregulated by N or NM treatments (Fig. 3 and Supplementary Table S5). Three DEGs, PsbK, PsbS, and PsbY in photosystem, were upregulated in the MH comparison, whereas PsaA and PsbA in photosystem were downregulated in the NH comparison (Fig. 3 and Supplementary Table S5). As electron transporters, F-type ATPase genes, ATPF0A, ATPF0B, and ATPF1B were upregulated in MH comparison. In contrast, ATPF0B and ATPF1B were downregulated in the NH comparison; ATPF0A and ATPF1A were downregulated in the NH and NMH comparisons (Fig. 3 and Supplementary Table S5). Moreover, light-harvesting complex II chlorophyll a/b binding (LHCB) genes including LHCB1, LHCB3, LHCB4, LHCB5, and LHCA4 were upregulated in MH comparison (Fig. 3 and Supplementary Table S5). These results suggest that salinity deteriorates photosynthesis process whereas melatonin partly improves this process. 3.4. Genes involved in flavonoids biosynthesis

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Fig. 3. The expression profiles of the genes in photosynthesis pathway. The pathway of photosynthesis was mapped based on the KEGG pathway ko00195 and ko00196. Fold changes of genes related to F-type ATPase gene, photosystem I and II and light-harvesting complex are indicated by log2 (Fold Change) value. The fold change and annotation of the genes can be found in Supplementary Table S5.

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Fig. 4. The expression profiles of the genes in flavonoids biosynthesis pathway. Arrow shows the metabolic stream of flavonoids biosynthesis. Enzyme names are abbreviated as follows; PAL: phenylalanine ammonia lyase; C4H: cinnamic acid 4-hydroxylase; 4CL: 4 coumarate CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; F3H: flavanone 3-hydroxylase; F3′H: flavanone 3 -hydroxylase; DFR: dihydroflavonol reductase; FLS: flavonol synthase; ANS/LDOX: anthocyanidin synthase/ leucoanthocyanidin dioxygenase; UFGT: UDPflavonoid glucosyltransferase; ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase. Enzyme names, unigene IDs and expression patterns are indicated on the side of each step. The fold change and annotation of the genes can be found in Supplementary Table S6.

consistent with the protective role against chlorophyll degradation, melatonin may also increase photosynthetic efficiency in apple tree (Wang et al., 2013), cucumber (Zhang et al., 2013), cherry (Sarropoulou et al., 2012), and soybean (Wei et al., 2015) under stress conditions. Transcriptome analyses revealed that melatonin and salinity regulated the expression of the genes related to photosynthesis in soybean (Wei et al., 2015). In our study, the expression profiles of the genes associated with photosynthesis were altered and overrepresented in KEGG pathway. The genes in photosystem were regulated by three treatments including salinity, melatonin, and salinity plus melatonin in our present study. Generally speaking, salinity, or salinity plus melatonin downregulated some photosynthesis-related genes; on the contrary, most of their expressions were upregulated by melatonin (Fig. 3). The changed expression patterns of the aforementioned genes likely have impact on photosynthetic efficiency. F-type ATPases are responsible for synthesizing ATP that drives many processes in living cells (Muench et al., 2011). In present study, some F-type ATPase genes were upregulated by melatonin treatment, while downregulated by salinity, or melatonin plus salinity, which necessarily affected ATP synthesis and life processes including photosynthesis, growth, and salinity stress response. Moreover, the members of the LHCB family played an important role in plant adaptation to environmental stresses (Andersson et al., 2001; Ganeteg et al., 2004; Kovács et al., 2006). In Arabidopsis, downregulation or disruption of any member of the LHCB family resulted in tolerance decrease in drought stress. The expressions of

apple tree (Wang et al., 2013). Our present study indicated that exogenous melatonin treatment increased chlorophyll contents and reduced H2O2 levels in contrast to the leaves from rubber tree seedlings under salt treatment (Fig. 1), which is consistent with the results from Wang et al (2013) and Wei et al (2015). These results further suggested that melatonin has its potential application to enhance stress tolerance in plants. To elucidate the possible mechanisms that melatonin promotes salinity tolerance, we systematically carried out leaf transcriptome analyses of rubber tree seedlings with H, M, N, and NM treatments. Based on our results, we suggest that melatonin might mitigate the negative effects of salt on rubber tree seedlings by regulating the expression of many genes. Being consistent with our speculation, some genes, especially in photosynthesis pathway, were upregulated by melatonin while they were downregulated by salinity. GO analysis also showed that melatonin mainly regulated the expressions of the DEGs significantly enriched in metabolic process (Supplementary Table S3). Interestingly, the DEGs regulated by melatonin were significantly overrepresented in four KEGG pathways including phenylpropanoid biosynthesis, cyanoamino acid metabolism, photosynthesis-antenna proteins, and photosynthesis (Supplementary Table S4), which are likely associated with relatively higher chlorophyll contents and lower H2O2 levels in M-treated samples. It was reported that both salt and drought stresses led to downregulation of some photosynthetic genes (Chaves et al., 2009). In 7

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Fig. 5. The expression profiles of the genes in proposed melatonin pathway. Green arrows indicate an established biosynthetic pathway in plants, and broken green arrows indicate two or more steps between substrate and product. Black and brown arrows separately indicate a step shared by both the animals and plants established pathways and a newly proposed alternate step in the biosynthetic pathway. The fold change and annotation of the genes are shown in Supplementary Table S7.

antioxidants, can be induced by stresses to inhibit ROS generation in plants (Mouradov and Spangenberg, 2014). In consistent with higher H2O2 contents in N-treated samples and lower H2O2 contents in Mtreated ones, the expressions of most important genes involved in flavonoids biosynthesis such as DFR, CHS, LAR, F3'H, F3H, CHI, etc., were changed under different treatments and significantly enriched in KEGG pathway. Interestingly, the genes in flavonoids biosynthesis were upregulated by N treatment, while most of them were downregulated by M treatment (Fig. 4). In rice, OsDfr and OsAns are induced by dehydration, high salt, and ABA (Ithal and Reddy, 2004). It was suggested that stress-tolerance in Reaumuria soongorica might be explained by the combination of increases in RsF3H expression, RsF3H enzyme activity, and corresponding flavonoids production (Liu et al., 2013). As one of the well-known secondary metabolic pathway in plants, many key genes involved in flavonoids biosynthesis pathway in response to environment stress have been investigated (Vasquez-Robinet et al., 2008; Lenka et al., 2011). Our results indicated that melatonin might alleviate salinity stress by regulating the genes involved in flavonoids biosynthesis and ROS metabolism to decrease H2O2 level in rubber tree seedlings. Besides acting as a direct antioxidant, melatonin can work together with other antioxidants to improve their overall antioxidant effectiveness in plants (Jouyban, 2012). In consistent to the aforementioned

several members of the LHCB family were also increased by melatonin treatment, suggesting that these genes might be involved in salinity stress response. Photosynthesis can absorb light and transfer energy and electrons, therefore we speculate that melatonin may alleviate salinity stress by regulating the photosynthesis-related genes. Stress including salinity can cause deregulation, overflow, and disruption of electron transport chains (ETC) in plants. As an electron acceptor, oxygen can lead to ROS accumulation under stress conditions. The strong oxidizing compounds including H2O2 are potentially harmful for cell integrity (Groß et al., 2013), whereas antioxidant metabolism, including antioxidant enzymes and nonenzymatic compounds, plays critical roles in detoxifying ROS induced by stress conditions. It was reported that salinity tolerance is positively correlated with antioxidant enzyme activities and the accumulation of nonenzymatic antioxidant compounds (Asada, 1999; Gupta et al., 2005). In our study, the genes involved in ROS metabolism, such as superoxide dismutase, peroxidase, lignin-forming anionic peroxidase, catalase isozyme 2, etc., were regulated by N or NM treatments (Supplementary Table S8). In addition, flavonoids have been involved in diverse processes, such as pigmentation, redox and UV protection, plant-microbe interactions, development, regulation of auxin transport, etc. (Kuhn et al., 2011; Peer et al., 2011; Buer et al., 2013; Emiliani et al., 2013; Ishihara et al., 2016). Most importantly, flavonoids, as vital 8

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results, the expressions of the genes related to melatonin biosynthesis were significantly altered under salinity/melatonin treatments (Supplementary Tables S7), which necessarily change melatonin level in rubber tree seedlings. Moreover, the genes in ROS metabolism were also changed in our present study (Supplementary Table S8), which was consistent with the results from Ma et al (2016). In summary, salinity stress leads to an oxidative burst, and rubber tree percept ROS signals. Salinity stressor probably acts as negative effectors to regulate many cellular and physiological processes including ROS metabolism, chlorophyll degradation, and photosynthesis, etc. ROS burst is likely responsible for regulating the genes involved in photosynthesis, ROS metabolism, flavonoids and melatonin biosyntheses, whereas endogenous melatonin can partly attenuate or reverse the negative effects of salinity by changing the expression of the aforementioned genes.

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5. Conclusion As a well-known agent, melatonin has several important roles in plants. However, its possible functions have not been reported in rubber tree. In this study, we showed that melatonin enhanced salt stress tolerance in rubber tree seedlings. In contrast to rubber tree seedlings treated with salinity, the seedlings treated with melatonin indicated lower chlorophyll degradation and H2O2 accumulation in leaves. Transcriptome analyses suggested that antioxidant protection was caused by exogenous melatonin application via regulating the genes involved in photosynthesis, ROS metabolism, flavonoids and melatonin biosynthesis. These findings demonstrate that melatonin can enhance salt stress tolerance directly or indirectly by counteracting H2O2 accumulation in rubber tree. Our findings are helpful for not only understanding melatonin’s role in salinity stress, but also providing new insights into potential utilization of melatonin against abiotic stresses in rubber tree. Declaration of Competing Interest We confidently confirm that this manuscript has never been submitted for publication elsewhere. No conflict of interest exits in the submission of this manuscript, and the manuscript is approved by all authors for publication. Acknowledgments This research was supported by the earmarked funds from National Natural Science Foundation of China (31570684and31270651). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111990. References Andersson, J., Walters, R.G., Horton, P., Jansson, S., 2001. Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation. Plant Cell 13 (5), 1193–1204. Arnao, M.B., Hernández-Ruiz, J., 2006. The physiological function of melatonin in plants. Plant Signal. Behav. 1 (3), 89–95. Arnao, M.B., Hernández-Ruiz, J., 2015. Functions of melatonin in plants: a review. J. Pineal Res. 59 (2), 133–150. Arora, D., Bhatla, S.C., 2017. Melatonin and nitric oxide regulate sunflower seedling growth under salt stress accompanying differential expression of Cu/Zn SOD and Mn SOD. Free Radic. Biol. Med. 106, 315–328. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Bio. 50, 601–639. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in rhizosphere interations with plants and other organisms. Annu. Rev. Plant Biol. 57, 233–266. Bastam, N., Baninasab, B., Ghobadi, C., 2013. Improving salt tolerance by exogenous application of salicylic acid in seedlings of pistachio. Plant Growth Regul. 69 (3), 275–284.

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