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Article

Genome-Wide Identification and Functional Characterization Under Abiotic Stress of Melatonin Biosynthesis Enzyme Family Genes in Poncirus trifoliata

by
Jian Zhu
2,
Ligang He
1,
Fang Song
1,3,
Zhijing Wang
1,
Xiaofang Ma
1,
Cui Xiao
1,
Xin Song
1,
Yanjie Fan
1,
Ce Wang
1,
Yun Xie
1,
Yingchun Jiang
1,3,
Liming Wu
1,3,* and
Yu Zhang
1,*
1
Hubei Key Laboratory of Germplasm Innovation and Utilization of Fruit Trees, Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
2
National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
3
Hubei Hongshan Laboratory, Wuhan 430070, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(10), 2246; https://doi.org/10.3390/agronomy15102246
Submission received: 18 August 2025 / Revised: 18 September 2025 / Accepted: 20 September 2025 / Published: 23 September 2025
(This article belongs to the Special Issue Fruit Tree Germplasm Innovation Driven by Molecular Breeding and Genomics)
Browse Figures
Versions Notes

Abstract

Plant melatonin is widely recognized as a pleiotropic regulator. As a growth-regulating hormone, it extensively participates in various growth and developmental processes and has significant functions in stress responses and disease resistance. Plant melatonin is synthesized primarily through the catalytic actions of five enzymes: TDC (tryptophan decarboxylase), T5H (tryptamine-5-hydroxylase), SNAT (serotonin N-acetyltransferase), ASMT (N-acetylserotonin methyltransferase), and COMT (caffeic acid-O-methyltransferase). There are multiple genes for each of these five enzymes in citrus genomes, however, with the exception of COMT5—whose function has recently been elucidated—and SNAT, which has only been preliminarily identified, the remaining genes have not been unequivocally characterized or functionally annotated. Hence, we carried out a genome-wide analysis of melatonin biosynthesis enzyme-related gene families in trifoliate orange (Poncirus trifoliata), one of the most common citrus rootstock varieties. Through bioinformatics approaches, we identified 96 gene family members encoding melatonin biosynthetic enzymes and characterized their protein sequence properties, phylogenetic relationships, gene structures, chromosomal distributions, and promoter cis-acting elements. Furthermore, by analyzing expression patterns in different tissues and under various stresses, we identified multiple stress-responsive melatonin synthase genes. These genes likely participate in melatonin synthesis under adverse conditions, thereby enhancing stress adaptation. Specifically, PtCOMT5, PtASMT11, and PtTDC9 were significantly induced by low temperature; PtSNAT1, PtSNAT14, PtSNAT18, and PtTDC10 were markedly responsive to drought; and PtASMT15, PtSNAT15, PtASMT16, and PtSNAT3 were strongly induced by ABA. Among them, PtASMT23 expression was induced up to 120-fold under low temperature, while PtSNAT18 showed over 100-fold upregulation under dehydration treatment. These findings strongly suggest that PtASMT23 and PtSNAT18 play critical roles in regulating melatonin biosynthesis in response to cold and drought stress, respectively. Collectively, these findings pinpoint novel genetic targets for enhancing stress resilience in citrus breeding programs and lay the foundation for the functional characterization of specific melatonin biosynthesis pathway gene family members in citrus and other horticultural crop species.

1. Introduction

Plants face persistent threats from both biotic and abiotic stresses throughout their life cycle [1]. Stressful environments severely constrain essential plant activities including growth and development. Therefore, plants have evolved complex and sophisticated regulatory networks to adapt to adversity [2]. Accumulating evidence demonstrated that plant hormones and secondary metabolites alleviate stress-induced damage through signal transduction and osmotic adjustment mechanisms, playing crucial roles in stress responses [2,3,4]. Among these compounds, melatonin, as a potent antioxidant, can participate in nearly all biotic and abiotic stresses [5,6,7]. However, current research in citrus has predominantly focused on the role of COMT5 in enhancing drought tolerance, while the functional characterization of other melatonin-related genes and their contributions to stress adaptation remains largely unexplored and warrants further investigation.
Melatonin, a pleiotropic plant hormone, widely participates in plant organ development and stress responses [7,8,9,10]. In terms of root development, studies have shown that exogenous melatonin promotes primary root growth in Arabidopsis by mimicking auxin (IAA) signaling pathways [8,11,12]. Notably, melatonin and IAA were moderately correlated at the transcriptome level, coregulating genes such as PIN7 and SAUR48, and their participation in IAA signal transduction was confirmed through activation of the DR5::GFP reporter gene. Research on herbaceous peony (Paeonia lactiflora) revealed that melatonin enhances stem bending resistance by increasing lignin deposition and secondary cell wall thickness. The overexpression of PITDC (tryptophan decarboxylase) and PICOMT1 in transgenic tobacco further confirmed that endogenous melatonin strengthens stem structure by regulating the S/G lignin monomer ratio [13].
The melatonin synthase-related genes primarily include tryptophan decarboxylase (TDC), tryptamine-5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), N-acetylserotonin methyltransferase (ASMT), and caffeic acid-O-methyltransferase (COMT) [14,15,16,17,18]. Apart from the T5H gene, which is highly conserved in multiple species (e.g., rice, Arabidopsis, and mulberry) and typically exists as a single homologous gene, other gene family members generally exhibit significant divergence in plants. For example, in longan (Dimocarpus longan), 2 SNAT, 18 ASMT, and 7 COMT family members were identified. The ASMT and COMT families are classified into the same evolutionary clade because of their high homology, and their promoter regions are enriched with hormone-responsive elements (e.g., auxin and gibberellin), suggesting their regulation of early somatic embryogenesis through melatonin synthesis [19]. Additionally, in mulberry (Morus spp.), 37 melatonin synthesis-related genes (including 1 TDC, 7 T5H, 6 SNAT, 20 ASMT, and 3 COMT) were identified. Among these, ASMT genes can be divided into three categories on the basis of functional differentiation: Class I (MnASMT12) and Class III (MnASMT20) simultaneously catalyze the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine, whereas Class II (MnASMT16) specifically catalyzes N-acetylserotonin methylation [20].
Regarding abiotic stress responses, melatonin enhances plant stress resistance through two primary mechanisms: ROS scavenging and chlorophyll preservation [7]. In apple (Malus spp.), melatonin pretreatment significantly reduces H2O2 accumulation and electrolyte leakage under drought stress while increasing SOD/POD/CAT enzyme activities [21]. Under high-temperature stress, MdASMT9 transgenic plants maintain the maximum quantum yield of photosystem II (Fv/Fm) and chloroplast ultrastructure integrity by scavenging ROS [22]. Under high-temperature conditions, this effect promotes stomatal opening through MdWRKY33-mediated transcriptional repression of MdNCED1/3 [23]. In citrus, previous studies have shown that the PtABF4-PtbHLH28-PtCOMT5 molecular module regulated melatonin accumulation and root development under drought stress [24].
As the world’s largest fruit category, citrus ranks first among all fruits in terms of cultivation area, yield, and import–export volume. Trifoliate orange (Poncirus trifoliata (L.) Raf) exhibits strong resistance to drought, cold, waterlogging, diseases and low tolerance. Due to its overall robust resilience, excellent graft compatibility, early fruiting, and high productivity, it is widely used as a rootstock for citrus grafting. So far, very few melatonin -related genes have been unraveled from trifoliate orange, such as the ABF4-bHLH28-COMT5 module for melatonin synthesis and drought tolerance [24].
In summary, melatonin plays a central role in plant organ development (roots, stems) and stress responses (drought, heat) by regulating IAA signaling, lignin metabolism, ROS homeostasis, and ABA-melatonin crosstalk [25,26]. Abiotic stresses are major factors affecting citrus yield and quality, with extensive impacts on citrus growth and development [27,28,29]. Analyzing citrus stress response mechanisms provides important guidance for citrus resistance breeding. Therefore, this study identified 96 melatonin synthase gene family members via conserved domain analysis, characterized their promoter cis-elements, and examined their expression patterns in tissues and abiotic stress. Several key stress-responsive melatonin synthase-related genes were identified, to uncover novel genetic elements that underlie its adaptability. The overarching goal of this research is to provide foundational insights and genetic resources for developing stress-tolerant citrus varieties, thereby contributing to sustainable agricultural and horticultural production systems in the face of increasing environmental challenges.

2. Materials and Methods

2.1. Identification of Melatonin Biosynthetic Pathway Genes and Proteins in Poncirus trifoliata (L.)

The genomic files of trifoliate orange (Poncirus trifoliata), Arabidopsis thaliana, and rice (Oryza sativa) were downloaded from the Sweet Orange Annotation Project (CAP) datasets (http://citrus.hzau.edu.cn, accessed on 1 August 2025) and the Ensembl Plants database (http://plants.ensembl.org/index.html, accessed on 1 August 2025). The longest transcript of each gene was extracted to represent the gene sequence. Protein sequences for enzymes mediating melatonin biosynthesis were obtained via domain-specific searches: Pyridoxal decarboxylase domain (PF00282) for tryptophan decarboxylase (TDC), P450 domain (PF00067) for tryptamine-5-hydroxylase (T5H), acetyltransferase domain (PF00583) for serotonin N-acetyltransferases (SNATs), and dimerization domain (PF08100) and methyltransferase domain (PF00891) for N-acetylserotonin methyltransferases (ASMTs).
Using HMMER (specifically hmmsearch, set E-value < 0.05), protein sequences of the three species were scanned to identify genes harboring the same domains. Candidate genes for the gene families were ultimately determined by comparing gene domains via the online tool HMMSCAN (https://www.ebi.ac.uk/Tools/HMMER/search/hmmscan, accessed on 1 August 2025).

2.2. Physical and Chemical Properties and Evolutionary Relationship Analysis of Melatonin Biosynthesis Family Genes

The theoretical isoelectric points (pIs) and molecular weights (MWs) of melatonin biosynthesis genes were calculated via the ProtParam tool (http://smart.embl-heidelberg.de/, accessed on 3 August 2025). The subcellular localization of proteins was predicted via WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 3 August 2025) and Localizer (http://localizer.csiro.au/, accessed on 1 August 2025) and named by the genes located on chromosomes. To study the affinities among gene family members and the genetic diversity of genes across species, the Muscle and Fasttree tools were used to align the multiple sequences in which genes are involved in melatonin biosynthesis, and phylogenetic trees were constructed on the basis of the genomes of P. trifoliata, Arabidopsis, and rice. Software with default settings was used, and bootstrapping with 1000 replicates for the Poisson correction model was performed to assess node support. The phylogenetic trees were visually edited via the website tool iTOL (https://itol.embl.de, accessed on 7 August 2025). The conserved motif of melatonin biosynthetic genes was identified via the Multiple EM for Motif Elicitation (MEME Suite 5.4.1) server under the default setting (https://meme-suite.org/meme/tools/meme, accessed on 7 August 2025). The gene structures and chromosomal locations of melatonin biosynthetic genes were designed via TBtools (v2.112).

2.3. Plant Materials and Stress Treatment

Fresh shoots and seedlings of trifoliate orange were cultivated in the Key Laboratory of Fruit Tree Germplasm Innovation and Utilization in Hubei Province. The seeds were harvested from the fresh trifoliate orange fruits then disinfected with 1 M NaOH (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). and NaClO (20% v/v) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). for 15 min respectively [24,28,29,30]. The disinfected seeds were germinated and sown in a growth chamber (light cycle: 16 h light, 8 h dark; temperature: 25 °C), and two-month-old seedlings were then used for further treatments. For the dehydration treatment, the seedlings were placed on filter paper in an illuminated incubator (light cycle: 16.0 h light, 8.0 h dark; temperature: 25 °C) for 0 h to 24 h before leaf sampling. For the ABA treatment, the seedlings were removed from the soil and placed in a 100 μM ABA (Shanghai yuanye Bio-Technology Co., Ltd, Shanghai, China) solution for 0 h to 24 h. For the cold stress treatment, the seedlings were placed at 4 °C for 0 h to 24 h. The leaves were collected and frozen immediately in liquid nitrogen and stored at −80 °C. The transcriptome data reference for low-temperature treatment of trifoliate orange seedlings [30], drought, and ABA treatment RNA-seq data have been deposited in the China National Center for Bioinformation (GSA: CRA028691).

2.4. RNA Extraction and Quantitative Real-Time PCR Validation

Total RNA was extracted via TRIzol reagent (RN33, Aidlab Biotechnologies Co., Ltd., Beijing, China) following the manufacturer’s protocol, and first-strand complementary DNA (cDNA) was synthesized via the RevertAid First Strand cDNA Synthesis Kit (PC7002; Keep Biotechnologies Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was conducted on a QuantStudio 7 Flex system (Applied Bios systems, (Applied Biosystems, Foster City, CA, USA) using AceQ SYBR Green Master Mix (Vazyme, Nanjing, China). The Actin gene was used as an internal reference control; the relative expression level was calculated via the 2(−ΔΔCT) method. Three technical replicates were performed for each biological replicate. Primers used in this study are listed in Table S1.

2.5. Statistical Analysis

The data were analyzed via GraphPad 8.0. Student’s t test and one-way ANOVA were conducted to determine significant differences between the two datasets at p < 0.05 and p < 0.01. The data are expressed as the means ± SDs of at least three biological replicates.

3. Results

3.1. Genome-Wide Mining Identified Genes Involved in Melatonin Biosynthesis in Trifoliate Orange

To identify melatonin biosynthetic genes in trifoliate orange, Arabidopsis, and rice, conserved domains were used to search for genes encoding enzymes, as described in the Materials and Methods section. We identified 9 and 18 TDC genes, 24 and 23 SNAT genes, 14 and 22 ASMT genes, and 2 and 8 COMT genes in Arabidopsis and rice, respectively. In addition, 13 TDC genes, 27 SNAT genes, and 48 ASMT genes were identified in trifoliate orange. T5H is present as a single gene in three species (Table 1). Among the melatonin biosynthetic genes in trifoliate orange, the protein length of the identified TDCs varies from 355 amino acids (aa) to 568 aa, and the molecular weights (MWs) range from 53.49 to 68.56 kDa, with the exception of PtTDC13, whose isoelectric points (pIs) range between 5.31 and 7.28. For the SNATs, the MWs were within the range of 10.22–49.07 kDa, and the isoelectric points ranged between 5.8 and 10.75, with protein lengths ranging from 92 to 431 aa being highly variable. The molecular weights of the identified ASMTs varied from 23.46 to 56.01, the pIs ranged from 4.95 to 9.28, and the protein lengths ranged from 212 to 511 aa. For the COMTs, the protein lengths ranged from 214 to 367 aa, and the MWs ranged from 23.26 to 40.02 kDa, with a pI ranging from 5.83 to 8.21. In addition, T5H has an MW of 58.34 kDa, a pI of 6.58, and a protein length of 514 aa (Table 2). In summary, protein physicochemical properties are highly diverse among members of different gene families. The genes involved in melatonin biosynthesis predicted in this study have diverse characteristics.
In addition to the above physical and chemical properties, we also predicted the subcellular localization of the genes. The majority of the gene family members involved in melatonin biosynthesis were predicted to be located in the chloroplast and cytoplasm, while most of the genes involved in melatonin biosynthesis were localized, indicating that they may also participate in multiple other growth and development processes.

3.2. The Chromosomal Location of Melatonin Biosynthesis Gene Family Members in Trifoliate Orange

Chromosomal location analysis revealed that 96 melatonin biosynthesis gene family members were distributed on nine chromosomes of trifoliata orange (Figure 1). More than half, 49 gene family members, were found on Chr2 and Chr3; among these, PtASMT4/5/6/7/8 and PtSNAT7/8/9/10 may form gene clusters and work together according to their localizations. Notably, seven PtCOMT genes were localized on Chr 3 together, with the exception of PtCOMT7, and the other gene family members were evenly distributed on seven remaining chromosomes. These results indicate that the gene family members mentioned above participate in melatonin biosynthesis together and may have functional redundancy.

3.3. Phylogenetic Relationships Among Gene Family Members

To classify the phylogenetic relationships of gene family members and analyze the evolutionary relationships among them, we constructed phylogenetic trees among PtTDCs, PtSNATs, PtASMTs, and PtCOMTs from Oryza sativa, Arabidopsis thaliana, and trifoliate orange, respectively. This method is based on sequence similarity and the maximum likelihood (ML) method. Most of the TDC orthologs were more closely related to members identified within each species than to those from other plant species (Figure 2A). The SNAT genes from the three plant species were grouped into three clades according to genetic distance: ancient branches, middle branches, and modern branches (Figure 2B). According to previous studies, phylogenetic analyses revealed that COMT genes evolved from ASMT genes and have ASMT enzyme activity; therefore, we analyzed these two gene families together (Figure 2C). Moreover, the PtASMT3, PtTDC2, PtTDC3, and PtSNAT26 isoforms are direct tandem repeats, and their homologous genes located on ChrUn, PtASMT28, and PtASMT46 are located on Chr5 and Chr9, respectively.

3.4. Gene Structure Collinearity Analysis of Gene Family Members

For the gene structure analysis, five motifs were identified for each gene family member. In general, members with close phylogenetic relationships presented high sequence similarity and similar motifs (Figure 3, Figures S1 and S2). The presence of the same type of conserved motif might indicate functional similarity among each family member. Interestingly, some repeat sequences, such as PtASMT2, and repeat motifs, e.g., PtASMT5, were found. Collinearity analysis of gene family genes revealed that TDC family genes presented the most collinearity relationships, among which PtTDC2 and PtTDC11, and PtTDC3 and PtTDC12 presented collinearity relationships. In addition, PtASMT28 and PtASMT46, PtASMT3 and PtUn032310, and PtSNAT26 and Pt5g015690 also presented collinearity relationships (Figure 4), which may be due to multiple copies caused by chromosomal doubling events.

3.5. Expression of Melatonin Biosynthesis Genes in Tissues During Growth and Development in Trifoliate Orange

The expression levels of genes in different plant tissues are closely related to their roles in the regulation of growth and development. We analyzed the expression of genes involved in melatonin biosynthesis from a public database in seven samples. Interestingly, melatonin biosynthesis gene family members were expressed in all the tested tissues of trifoliate orange, including the roots, seeds, leaves, early-stage ovules, late-stage ovules, young fruits, and mature fruits (Figure 5). Differential expression was observed for most of the genes across various stages of plant development. Notably, PtTDC5, PtASMT11, PtCOMT5, and PtASMT26 presented moderate to high levels of expression in three tissues. We speculate that these genes may play important roles in plant development. On the other hand, some gene family members, such as PtASMT43/44/9/40, are specifically expressed in roots, and some genes, such as PtASMT12, PtASMT19, PtTDC5, and PtASMT15, have differential expression patterns between early-stage and late-stage ovules or young and mature fruits. These differential expression patterns and specific expression patterns in different stages or tissues may provide important clues for exploring their functions in different tissues in the future.

3.6. Gene Expression Analysis of Melatonin Biosynthesis-Related Genes Involved in the Abiotic Response

Citrus plants are highly susceptible to low-temperature and drought stress during their annual growth cycle [30]. To investigate the contribution of melatonin biosynthetic genes to abiotic stress responses in trifoliate orange, we analyzed transcriptome datasets from plants subjected to low-temperature and dehydration treatments. Specific members of the melatonin biosynthesis-related gene families were identified, and their expression patterns under these stresses were examined. Analysis revealed that PtCOMT5, PtASMT11/23, and PtTDC9 were significantly upregulated by low-temperature stress (Figure 6A). In addition, PtSNAT1/14/18 and PtTDC10 were strongly induced by dehydration treatment (Figure 6B). This differential expression highlights how gene family expansion and functional divergence enable tailored responses to distinct environmental challenges. As potential melatonin biosynthetic genes, these candidates likely drive melatonin accumulation under stress conditions, thereby enhancing adaptive responses in citrus.

3.7. Gene Expression Analysis of Melatonin Biosynthesis-Related Genes in Response to ABA Treatment

It has been reported that melatonin levels are strongly affected by unfavorable conditions such as cold, heat, and drought stress. The activities of melatonin biosynthesis enzymes appear to be tightly regulated by stress stimuli. Moreover, ABA extensively participates in various plant stress regulation processes. We analyzed the RNA-seq data of trifoliate orange seedlings before and after 3 h or 6 h of ABA treatment (Figure 6C), among the genes involved in melatonin biosynthesis. There were three gene expression patterns in the ABA-treated leaves. PtASMT15, PtASMT16, PtSNAT15, and PtSNAT3 induction persisted after 3 h and 6 h of ABA treatment. PtT5H1, PtSNAT17, and PtSNAT24 were downregulated after 3 h of ABA treatment but were slightly upregulated from 3 h to 6 h. In contrast, PtSNAT22, PtTDC5, and PtCOMT5/6 expression was significantly inhibited after ABA treatment for 6 h. These genes may potentially function in response to ABA treatment.

3.8. qRT-PCR Analysis of Candidate Genes Under Stress Treatments

qRT-PCR analysis was performed on 12 candidate genes identified through transcriptome data screening, including PtCOMT5, PtASMT11/23, and PtTDC9 (responsive to cold stress), PtSNAT1/14/18 and PtTDC10 (responsive to drought stress), and PtASMT15, PtASMT16, PtSNAT15, and PtSNAT3 (induced by ABA). Consistent with the transcriptome data, PtCOMT5, PtASMT11/23, and PtTDC9 were significantly upregulated under cold stress, PtSNAT1/14/18 and PtTDC10 were markedly induced by drought stress, and PtASMT15, PtASMT16, PtSNAT15, and PtSNAT3 were strongly activated by ABA treatment. Notably, the expression of PtASMT23 increased by up to 120-fold under cold stress, while PtSNAT18 exhibited over 100-fold induction under drought conditions (Figure 7A–L), suggesting that these two genes are key candidates involved in melatonin metabolism pathways in response to cold and drought stress, respectively.

4. Discussion

Our genome-wide analysis identified 96 putative melatonin biosynthesis genes in trifoliate orange, revealing remarkable expansion of the ASMT family (48 members) compared to Arabidopsis (14) and rice (22). The dramatic expansion of the ASMT family (48 members), compared to Arabidopsis and rice, may provide a genetic basis for the enhanced stress resilience in trifoliate orange. This is strongly supported by our finding that PtASMT23 was induced up to 120-fold under cold stress, suggesting its paramount role in mediating cold tolerance. The divergence of gene family members is closely related to their functional specificity. In rice (Oryza sativa), 33 COMT genes are classified into Group I and Group II. Group I members, including OsCOMT8, OsCOMT9, and OsCOMT15, utilize conserved substrate-binding sites for lignin synthesis and exhibit high expression levels in stem tissues. Additionally, 14 OsCOMTs respond to salt stress, whereas 13 respond to drought stress, indicating their role in enhancing stress resistance through phenotypic plasticity [31]. In tobacco (Nicotiana tabacum), the SNAT family diverges into typical SNAT genes (NtSNAT1/2) and SNAT-like genes (10 members). Among these genes, NtSNAT1 is significantly upregulated under high-temperature and cadmium stress, whereas SNAT-like genes exhibit organ-specific expression, suggesting that subfunctionalization plays a key role in regulating development and stress responses [32].
On the basis of conserved domains and other characteristics, we identified 96 melatonin synthase family genes in the trifoliate orange genome. Compared with those of Arabidopsis and rice, the trifoliate orange genome exhibited significant expansion of 48 ASMT genes. Among these genes, PtASMT11/26 are highly expressed in roots, leaves, and seeds; PtASMT23 was significantly induced by low temperature; PtSNAT18 was significantly induced by drought, and PtASMT15/16 were induced by ABA, indicating functional diversity. Similar functional diversification has been observed in other horticultural crops. In tomato (Solanum lycopersicum), melatonin biosynthesis is regulated by synthetic genetic circuits involving COMT and SNAT, which enhance salinity tolerance by improving antioxidant defense and ion homeostasis [10]. Similarly, in soybean (Glycine max), seed-specific overexpression of COMT and SNAT via synthetic promoters increased melatonin accumulation, concomitantly improving salt tolerance during germination and elevating protein content while reducing oil content [33]. These findings align with our observations in trifoliate orange, where ASMT and COMT family expansions may underpin enhanced stress adaptability. In addition, we performed protein sequence alignment and modeling of candidate genes PtASMT23 and PtSNAT18 are provided in Supplemental Figure S3 [34].
Notably, gene divergence and epigenetic regulatory mechanisms jointly influence the melatonin synthesis pathway [15,16]. Genome-wide analysis revealed that ASMT is the only gene family that displays monocot/dicot-specific clustering trends, with its expression regulated by miRNA-mediated posttranscriptional mechanisms. For example, in rice, miR6249a and miR-1846e fine-tune melatonin synthesis under light and stress conditions by targeting OsTDC5 and OsASMT18, respectively [17]. Furthermore, silencing GhSNAT3D in cotton (Gossypium hirsutum) reduces the melatonin content and salt tolerance, whereas exogenous melatonin restores the phenotype, indicating that SNAT-mediated melatonin synthesis is a critical mechanism for plant stress adaptation [35]. Within the trifoliate orange genome, we identified multiple pairs of syntenic relationships, likely resulting from chromosome duplication events.
Studies on molecular regulatory networks reveal multiple signaling cascades. In citrus, the ABA signaling core factor PtABF4 activates PtbHLH28 expression by binding to the ABRE (ACGTG) in its promoter [24]. PtbHLH28 further binds to the TAAGAGA motif in the PtCOMT5 promoter, forming an ABF4-bHLH28-COMT5 module that coordinately regulates melatonin synthesis, root development, and drought resistance [24]. Genetic manipulation of this module confirmed that overexpressing PtCOMT5 significantly increased the number, length, and area of roots, whereas CRISPR mutants presented the opposite phenotype [24]. This provides a molecular breeding strategy to increase plant environmental adaptability by modulating endogenous melatonin levels. Similar regulatory mechanisms have been identified in other species. In alfalfa (Medicago sativa), MsSNAT1 overexpression increased endogenous melatonin levels and salt tolerance by promoting antioxidant systems and maintaining ion homeostasis [33]. Conversely, MsbZIP55 acts as a negative regulator by repressing MsSNAT1 expression, reducing melatonin biosynthesis and salt tolerance [36].
In pigeon pea (Cajanus cajan), melatonin pre-treatment induced flavonoid accumulation, particularly luteolin, via upregulation of F3′H and the transcription factor PCL1, enhancing tolerance to salt, drought, and heat stresses [37]. This suggests cross-talk between melatonin and secondary metabolic pathways, which may also be relevant in trifoliate orange given the expansion of ASMT genes potentially involved in flavonoid modulation.
Through genome-wide identification, structural analysis, and expression profiling, we identified multiple candidate genes potentially involved in melatonin accumulation under stress. These findings identify target genes for future functional characterization and molecular network dissection. In summary, the functional diversity of melatonin synthase genes is reflected in phylogenetic divergence, expression regulation specificity, and enzymatic versatility. The divergence of these gene families—particularly SNAT, ASMT, and COMT—provides a molecular foundation for understanding plant development and stress adaptation. While our study provides a comprehensive genomic identification and expression profiling of melatonin biosynthetic genes in trifoliate orange, the precise enzymatic activities and in vivo functions of these candidates, especially PtASMT23 and PtSNAT18, require further validation through genetic transformation and metabolomic assays in the future.

5. Conclusions

This study provides a comprehensive genome-wide analysis of melatonin biosynthesis enzyme genes in trifoliate orange, identifying 96 members involved in this pathway. Through phylogenetic, structural, and promoter analyses, coupled with expression profiling, multiple stress-responsive genes were uncovered, notably PtASMT23 and PtSNAT18, which were drastically upregulated under cold and drought stress, respectively. These results highlight key genetic components regulating melatonin-mediated stress adaptation in citrus. Our findings offer valuable targets for molecular breeding aimed at enhancing abiotic stress resilience in citrus and related crops.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15102246/s1. Figure S1. Analysis of gene structure of melatonin biosynthesis gene TDC members in trifoliate orange, including motif (A), domain (B), and CDS (C). Figure S2. Analysis of gene structure of melatonin biosynthesis gene SNAT members in trifoliate orange, including motif (A), domain (B), and CDS (C). Figure S3. Protein structure prediction of (A), PtSNAT18 and (B), PtASMT23. Table S1. Primers of gene expression for qRT-PCR analysis.

Author Contributions

Conceptualization, J.Z. and Y.Z.; Data curation, J.Z., C.X., X.S., Y.F., and Y.Z.; Formal analysis, Y.Z.; Funding acquisition, Y.X., Y.J., L.W., and Y.Z.; Investigation, J.Z. and Y.Z.; Methodology, J.Z., L.H., and L.W.; Project administration, Y.J., L.W., and Y.Z.; Resources, L.H., F.S., Z.W., Y.F., C.W., Y.X., and L.W.; Software, J.Z.; Supervision, Y.X., Y.J., and L.W.; Validation, Y.Z.; Visualization, J.Z. and Y.Z.; Writing—original draft, J.Z. and Y.Z.; Writing—review and editing, J.Z., Z.W., X.M., C.X., and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Hubei Provincial Natural Science Foundation of China (2023AFB553), National Natural Science Foundation of China (32302475), Post-doctoral Innovation Practice Project of Hubei province (ERSH-2023-48), the Youth Foundation of Hubei Academy of Agricultural Sciences (2024NKYJJ22), and Innovation Team Project of Hubei Provincial Agricultural Science and Technology Innovation Center (2025-620-000-001-019).

Data Availability Statement

Sequence data from this article can be found in the CPBD (Citrus Pan-genome to Breeding Database, http://citrus.hzau.edu.cn/).

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Chromosomal localization of the melatonin synthase gene in trifoliate orange. Genes from the same family are represented in the same color, where blue represents the SNAT family (e.g., Pt1g006020), red represents the ASMT family (e.g., Pt1g016200), green represents the TDC family (e.g., Pt2g002670), pink represents the T5H family (e.g., Pt3g004620), and brown represents the COMT family (e.g., Pt2g002670).
Figure 1. Chromosomal localization of the melatonin synthase gene in trifoliate orange. Genes from the same family are represented in the same color, where blue represents the SNAT family (e.g., Pt1g006020), red represents the ASMT family (e.g., Pt1g016200), green represents the TDC family (e.g., Pt2g002670), pink represents the T5H family (e.g., Pt3g004620), and brown represents the COMT family (e.g., Pt2g002670).
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Figure 2. Phylogenetic relationships of enzymes related to the melatonin synthesis pathway related to the TDC (A), SNAT (B), ASMT and COMT (C) gene families in Arabidopsis, rice and trifoliate orange.
Figure 2. Phylogenetic relationships of enzymes related to the melatonin synthesis pathway related to the TDC (A), SNAT (B), ASMT and COMT (C) gene families in Arabidopsis, rice and trifoliate orange.
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Figure 3. Analysis of the gene structure of the melatonin biosynthetic genes ASMT and COMT in trifoliate orange, including motifs (A), domains (B), and CDSs (C).
Figure 3. Analysis of the gene structure of the melatonin biosynthetic genes ASMT and COMT in trifoliate orange, including motifs (A), domains (B), and CDSs (C).
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Figure 4. Collinearity analysis of melatonin synthesis gene family members in the genome of trifoliate orange. Genes with collinearity are connected by red lines.
Figure 4. Collinearity analysis of melatonin synthesis gene family members in the genome of trifoliate orange. Genes with collinearity are connected by red lines.
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Figure 5. Expression patterns of melatonin biosynthesis genes in different tissues. (A) Expression level (log2 (FPKM + 1) of the DEGs in the roots, stems and (B) leaves during two different periods (early ovule and mature ovule, young fruit and mature fruit).
Figure 5. Expression patterns of melatonin biosynthesis genes in different tissues. (A) Expression level (log2 (FPKM + 1) of the DEGs in the roots, stems and (B) leaves during two different periods (early ovule and mature ovule, young fruit and mature fruit).
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Figure 6. Different expression gene response patterns of melatonin biosynthesis under stress treatment. (A) The log2(FPKM + 1) of differential gene expression before and after low-temperature treatment; (B) the log2(TPM + 1) of differential gene expression after 3 h and 6 h of dehydration treatment; (C) the log2(TPM + 1) of differential gene expression analysis after 3 h and 6 h of ABA treatment.
Figure 6. Different expression gene response patterns of melatonin biosynthesis under stress treatment. (A) The log2(FPKM + 1) of differential gene expression before and after low-temperature treatment; (B) the log2(TPM + 1) of differential gene expression after 3 h and 6 h of dehydration treatment; (C) the log2(TPM + 1) of differential gene expression analysis after 3 h and 6 h of ABA treatment.
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Figure 7. The expression patterns of 12 candidate genes under stress treatment. The stress treatment included low-temperature (4 °C, (A–D)), dehydration (E–H), and ABA (I–L) treatment. Error bars = ±SD (n = 3).
Figure 7. The expression patterns of 12 candidate genes under stress treatment. The stress treatment included low-temperature (4 °C, (A–D)), dehydration (E–H), and ABA (I–L) treatment. Error bars = ±SD (n = 3).
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Table 1. Details of the proteins involved in melatonin biosynthesis in Arabidopsis, rice, and trifoliate orange.
Table 1. Details of the proteins involved in melatonin biosynthesis in Arabidopsis, rice, and trifoliate orange.
NameArabidopsisRiceTrifoliate Orange
TDC91813
T5H111
SNAT242327
ASMT142248
COMT287
Total507296
Table 2. Information on genes involved in melatonin biosynthesis in trifoliate orange.
Table 2. Information on genes involved in melatonin biosynthesis in trifoliate orange.
Gene NameGene IDMolecular Weight (kDa)PIProtein Length (aa)Predicted Subcellular LocalizationGene NameGene IDMolecular Weight (kDa)PIProtein Length (aa)Predicted Subcellular Localization
PtASMT1Pt1g01618041.135.86367cyskPtTDC1Pt2g00267058.426.44520chlo
PtASMT2Pt1g01619047.286.3427cytoPtTDC2Pt2g01291055.46.51500cyto
PtASMT3Pt1g01620033.838.17300cytoPtTDC3Pt2g01748056.295.31495cyto
PtASMT4Pt2g00092031.496.95287cytoPtTDC4Pt2g02311055.886.44507nucl
PtASMT5Pt2g00095031.466.95287cytoPtTDC5Pt3g01764056.286.27499cyto
PtASMT6Pt2g00100031.477.66287cytoPtTDC6Pt3g03000068.566.47611plas
PtASMT7Pt2g00105031.477.66287cytoPtTDC7Pt4g01194057.836.86518chlo
PtASMT8Pt2g00107031.466.72287cytoPtTDC8Pt4g01195054.827.28492chlo
PtASMT9Pt2g00602040.714.95363cyskPtTDC9Pt5g00501063.446.77568chlo
PtASMT10Pt2g00603041.995.81377nuclPtTDC10Pt5g00666053.496.3477cyto
PtASMT11Pt2g01205039.46.13357cytoPtTDC11PtUn02280055.46.51500cyto
PtASMT12Pt2g01623039.665.7357cytoPtTDC12PtUn02316056.35.31495cyto
PtASMT13Pt2g02382040.225.25360cytoPtTDC13PtUn02506038.897.23355nucl
PtASMT14Pt2g02383038.836.83347cytoPtT5H1Pt3g00462058.346.58514chlo
PtASMT15Pt2g02524040.475.43360cytoPtSNAT1Pt1g00602018.4710.15165cyto
PtASMT16Pt2g02525049.146.4437cyskPtSNAT2Pt1g01634032.8210.29282chlo
PtASMT17Pt3g00006039.236.51354cyskPtSNAT3Pt1g02063031.257.04280chlo
PtASMT18Pt3g00008039.236.51354cyskPtSNAT4Pt2g00904023.716.51206cyto
PtASMT19Pt3g01845038.865.89353cytoPtSNAT5Pt3g01876030.989.45272chlo
PtASMT20Pt3g01902025.916.72237cytoPtSNAT6Pt3g01881030.969.34272chlo
PtASMT21Pt3g01904039.446.24358cytoPtSNAT7Pt3g02313040.938.3359cyto
PtASMT22Pt3g01906039.055.55353cytoPtSNAT8Pt3g02327010.226.5192chlo
PtASMT23Pt3g02556026.989.28250cytoPtSNAT9Pt3g02330016.4610.15146cyto
PtASMT24Pt3g02623029.75.59271chloPtSNAT10Pt3g02333049.149.56431chlo
PtASMT25Pt3g02624056.015.19511plasPtSNAT11Pt3g02589028.769.28254chlo
PtASMT26Pt3g02626039.324.99357cytoPtSNAT12Pt3g02685028.769.28254chlo
PtASMT27Pt3g02630045.667.71405chloPtSNAT13Pt3g03211025.199.64217chlo
PtASMT28Pt3g03446039.86.51359cytoPtSNAT14Pt4g00148023.57.95209chlo
PtASMT29Pt3g03447023.465.55212cytoPtSNAT15Pt4g00406034.718.92309nucl
PtASMT30Pt4g01722030.716.25279cytoPtSNAT16Pt4g00940026.2810.75237chlo
PtASMT31Pt5g01421040.36.37357cytoPtSNAT17Pt4g01740031.4410.39286chlo
PtASMT32Pt5g02595045.586.24411chloPtSNAT18Pt4g01761022.8510.38207nucl
PtASMT33Pt6g00684039.335.83354cytoPtSNAT19Pt6g01122027.575.8248chlo
PtASMT34Pt6g01641027.255.87249cytoPtSNAT20Pt7g00586046.249.45408cyto
PtASMT35Pt6g01866037.365.7334chloPtSNAT21Pt7g01531027.649.7244nucl
PtASMT36Pt7g00246036.376.4325cytoPtSNAT22Pt7g01610045.728.31417cyto
PtASMT37Pt7g00248036.376.4325cytoPtSNAT23Pt7g01636047.158.76420cyto
PtASMT38Pt8g00107038.565.8347cyskPtSNAT24Pt8g01215044.199.23387cyto
PtASMT39Pt9g00922033.666.96308nuclPtSNAT25Pt9g00131045.548.88400chlo
PtASMT40Pt9g00924028.246.83255cyskPtSNAT26Pt9g00548019.665.11175nucl
PtASMT41Pt9g00925032.485.95296cytoPtSNAT27PtUn02811049.079.46431cyto
PtASMT42Pt9g00926032.466.12296cytoPtCOMT1Pt3g01453025.557.13233cyto
PtASMT43Pt9g01076039.495.63357cytoPtCOMT2Pt3g01454031.716.95290cyto
PtASMT44Pt9g01191039.685.63357cyskPtCOMT3Pt3g01455023.266.67214extr
PtASMT45PtUn02675041.246.06373cytoPtCOMT4Pt3g01456030.466.51280cyto
PtASMT46PtUn02925039.86.51359cytoPtCOMT5Pt3g01705040.025.83367cyto
PtASMT47PtUn03018030.076.3275cytoPtCOMT6Pt3g01738034.068.21310cyto
PtASMT48PtUn03404039.035.55353cytoPtCOMT7Pt5g00719033.836.79312nucl
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Zhu, J.; He, L.; Song, F.; Wang, Z.; Ma, X.; Xiao, C.; Song, X.; Fan, Y.; Wang, C.; Xie, Y.; et al. Genome-Wide Identification and Functional Characterization Under Abiotic Stress of Melatonin Biosynthesis Enzyme Family Genes in Poncirus trifoliata. Agronomy 2025, 15, 2246. https://doi.org/10.3390/agronomy15102246

AMA Style

Zhu J, He L, Song F, Wang Z, Ma X, Xiao C, Song X, Fan Y, Wang C, Xie Y, et al. Genome-Wide Identification and Functional Characterization Under Abiotic Stress of Melatonin Biosynthesis Enzyme Family Genes in Poncirus trifoliata. Agronomy. 2025; 15(10):2246. https://doi.org/10.3390/agronomy15102246

Chicago/Turabian Style

Zhu, Jian, Ligang He, Fang Song, Zhijing Wang, Xiaofang Ma, Cui Xiao, Xin Song, Yanjie Fan, Ce Wang, Yun Xie, and et al. 2025. "Genome-Wide Identification and Functional Characterization Under Abiotic Stress of Melatonin Biosynthesis Enzyme Family Genes in Poncirus trifoliata" Agronomy 15, no. 10: 2246. https://doi.org/10.3390/agronomy15102246

APA Style

Zhu, J., He, L., Song, F., Wang, Z., Ma, X., Xiao, C., Song, X., Fan, Y., Wang, C., Xie, Y., Jiang, Y., Wu, L., & Zhang, Y. (2025). Genome-Wide Identification and Functional Characterization Under Abiotic Stress of Melatonin Biosynthesis Enzyme Family Genes in Poncirus trifoliata. Agronomy, 15(10), 2246. https://doi.org/10.3390/agronomy15102246

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