Identification of SNAT Gene Family and Their Response to Abiotic Stress in Citrus

Abstract

Serotonin N-acetyltransferase (SNAT) is a crucial enzyme in the melatonin synthesis pathway, playing an essential role in both melatonin biosynthesis and plant resistance to abiotic stress. A bioinformatics approach was employed to identify the members of the citrus SNAT gene family and to analyze their physicochemical properties, gene structure, conserved domains, phylogenetic relationships, and promoter cis-acting elements. Additionally, the tissue-specific expression of trifoliate orange SNAT family members and their expression patterns under stress conditions were examined. This study identified 21 members of the SNAT gene family across five citrus genomes, distributed over five chromosomes, with the majority predicted to localize within chloroplasts. These genes were characterized by having between 1 and 8 exons, 0 and 7 introns, 1 and 2 conserved domains, and 5 and 8 motifs. Phylogenetic analysis classified the genes into four subgroups, demonstrating significant collinearity with SNAT genes in rice. Analysis of the promoter regions revealed 32 cis-acting elements, with those responsive to light, abscisic acid, and drought being the most common. Expression analysis of SNAT genes in trifoliate orange indicated tissue specificity, with the highest expression levels detected in leaves. Quantitative real-time PCR analysis showed that the PtrSNAT1 gene was notably upregulated under various stress conditions, suggesting its role in stress response. Overall, these findings provide critical insights for further functional studies of citrus SNAT genes in relation to abiotic stress responses. Moreover, the PtrSNAT1 gene represents a potential target for developing rootstocks with enhanced resistance to abiotic stress.

1. Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is a highly conserved indole compound that has been the subject of scientific investigation since its initial isolation from the pineal gland of cattle in 1958 [1]. The groundbreaking identification of melatonin in plants in 1995 marked the inception of a novel area of melatonin research [2]. Subsequent studies have demonstrated that melatonin is ubiquitously distributed across various plant tissues [3]. Functioning as a plant growth regulator, melatonin plays a crucial role in modulating diverse aspects of plant growth and development, including seed germination, root formation, flowering, fruit maturation, leaf senescence, and circadian rhythms [4]. Extensive research has established that melatonin enhances plant tolerance to abiotic stresses such as low temperatures, drought, heavy metal exposure, and high salinity, as well as biotic stresses like pathogen attacks [5,6].

In plant organisms, akin to animals, melatonin synthesis utilizes tryptophan as a precursor, and the conversion of tryptophan to melatonin typically involves four enzymatic reaction steps [5]. Initially, tryptophan is converted to tryptamine by tryptophan decarboxylase (TDC). Subsequently, tryptamine is transformed into serotonin by tryptophan-5-hydroxylase (T5H). Following this, 5-hydroxytryptamine-N-acetyltransferase (SNAT) facilitates the conversion of serotonin to N-acetyl-serotonin. Finally, N-acetyl-serotonin is converted to melatonin by either N-acetyl-5-hydroxytryptamine methyltransferase (ASMT) or caffeic acid-O-methyltransferase (COMT). Concurrently, serotonin can also be converted by ASMT/COMT into 5-methoxytryptamine, which is subsequently transformed into melatonin by SNAT. Notably, SNAT functions as either the penultimate or the final enzyme in the melatonin synthesis pathway. Growing evidence indicates that SNAT plays a key role in regulating melatonin accumulation and in enhancing abiotic stress tolerance [7].

Since the initial cloning of SNAT gene in Oryza sativa, further SNAT genes have been identified in various plant species, such as Arabidopsis, tomato, and grape [8,9]. In plants, SNAT genes are encoded by a small gene family. For example, two SNAT gene members have been identified in five dicotyledonous plants, including Arabidopsis thaliana [10], tobacco [11], flowering Chinese cabbage [12], and Hypericum perforatum [13]. Additionally, three and seven SNAT gene members have been discovered in maize [14] and soybean [15], respectively. In numerous instances, plant SNAT genes are responsive to various abiotic stresses. For instance, BcSNAT1 from flowering Chinese cabbage (Brassica rapa ssp. chinensis var. parachinensis), VvSNAT1 from grape (Vitis vinifera L. cv. Merlot), and HpSNAT1/2 from Hypericum perforatum demonstrated upregulated expression under short-term salt treatment [10,12,16]. Similarly, drought conditions led to the upregulation of plant SNAT genes, including BcSNAT1/2, HpSNAT1, and ZmSNAT1/3 from Zea mays L. and MzSNAT5 from Malus zumi Mats [10,12,14,17]. Moreover, the transcription levels of ZmSNAT1/3 and NtSNAT1 from tobacco (Nicotiana tabacum) were significantly induced by heat stress [11,14]. Furthermore, the overexpression of SNAT genes generally enhanced stress tolerance, while their suppression diminished resistance [16,17,18]. Collectively, existing research indicated that the SNAT gene represents a promising target for improving plant resistance to abiotic stress.

As the leading global producer of citrus fruits, China’s citrus industry is the largest in the world, contributing approximately one-third of global production. However, as a subtropical fruit tree, citrus faces significant challenges in agricultural cultivation, particularly its susceptibility to low temperatures and poor cold tolerance [19]. Additionally, issues such as soil salinization and aridification further constrain the sustainable development of the citrus industry in China [20]. Consequently, the identification and utilization of resistant genes are crucial for advancing citrus-resistance breeding. Despite the critical role of SNAT genes in resistance responses, research on their application in citrus remains unexplored. Our study employed a comprehensive research approach that integrated in silico bioinformatic predictions with in vivo experimental validation. Firstly, we employed bioinformatics techniques to identify SNAT genes across five significant citrus species: sweet orange (Citrus sinensis), clementine (Citrus clementina), lemon (Citrus limon), pomelo (Citrus maxima), and trifoliate orange (Poncirus trifoliata). Subsequently, we conducted a comprehensive analysis of the physicochemical properties, gene structures, phylogenetic relationships, promoter cis-acting elements, and gene expression patterns of the SNAT gene family members in these citrus species. The objective was to investigate SNAT genes associated with stress response, thereby providing a theoretical foundation for further research on SNAT gene function in citrus. This study has identified a pivotal stress-responsive SNAT gene, which serves as a molecular target for the development of stress-resistant citrus varieties. Moreover, precise modification of this essential SNAT gene via transgenic methods or CRISPR/Cas9 gene-editing technology facilitates the generation of a novel stress-tolerant germplasm. These findings present a viable molecular breeding strategy to mitigate the environmental challenges confronting the citrus industry in the context of climate change.

2. Materials and Methods

2.1. Identification of SNAT Genes from Five Citrus Genome

The genome sequences, protein sequences, and associated annotation information for sweet orange, mandarin, lemon, pomelo, and trifoliate orange were obtained from the CPBD database (http://Citrus.hzau.edu.cn/ (accessed on 12 May 2024)) and citrus genome database (https://www.citrusgenomedb.org/ (accessed on 12 May 2024)). Additionally, genome-related data, including genome sequences and annotation information for Arabidopsis thaliana and rice, were sourced from the plant reference genome database Ensembl (http://plants.ensembl.org (accessed on 12 May 2024)). The three SNAT protein sequences from Arabidopsis thaliana were utilized as query sequences, with a search threshold (E-value) set at 10⁻⁵. A search of the citrus protein database was conducted using the BLAST function within the TBtools-II software, leading to the identification of candidate citrus SNAT protein sequences. The conserved domains were subsequently identified using the NCBI and SMART databases (http://smart.embl-heidelberg.de/ (accessed on 12 May 2024)). The physicochemical properties of citrus SNAT gene family members were predicted using the online tool Expasy-ProtParam (https://web.expasy.org/protparam/ (accessed on 12 May 2024)). Furthermore, the subcellular localization of Citrus SNAT family members was predicted using the online software Plant-mPLoc v2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 12 May 2024)) and CELLO v2.5 (http://cello.life.nctu.edu.tw/ (accessed on 12 May 2024)). The chloroplast signal peptides were predicted with TargetP (https://services.healthtech.dtu.dk/service.php?TargetP-2.0 (accessed on 12 May 2024)).

2.2. Gene Structure, Conserved Domains, and Motif Analysis

The motif analysis was conducted utilizing the online tool MEME (https://meme-suite.org/meme/tools/meme (accessed on 12 May 2024)), with the number of motifs specified as 10, while all other parameters were maintained at their default settings. Conserved domains were retrieved using the NCBI-CDD online tool. Visualization and mapping of gene structure, conserved domains, and motifs were accomplished using the TBtools-II software [21].

2.3. Phylogenetic Tree Construction and Collinearity Analysis

Multiple sequence alignments of SNAT protein sequences from citrus, Arabidopsis thaliana, and rice were performed with MEGA X software. A phylogenetic tree was subsequently constructed employing the Neighbor-Joining (NJ) method, with the Bootstrap parameter set to 1000 and other parameters left at their default values. The phylogenetic tree was further refined and enhanced using the Evolview online platform. Comparative genomic analysis of citrus, Arabidopsis thaliana, and rice was carried out using the MCScanX program, and collinearity was assessed via the multiple synteny plot tool in TBtools software.

2.4. Identification of Cis-Acting Elements

The sequence of the citrus SNAT gene, extending 2000 base pairs upstream of the transcriptional start site, was selected as the primary promoter region for the analysis of cis-acting elements. All cis-acting elements within the promoter region of each citrus SNAT gene were identified using the online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 12 May 2024)). These elements were visualized and mapped using the TBtools software, followed by statistical analysis conducted in Excel.

2.5. Tissue-Specific Expression Analysis

The FPKM values for SNAT genes from trifoliate orange were retrieved from the CPBD database (http://Citrus.hzau.edu.cn/ (accessed on 12 May 2024)), and heatmaps were generated using TBtools.

2.6. Plant Material and Adverse Stress Treatments

One-year-old seedlings of trifoliate orange, which had attained a height of approximately 15 cm above the ground, were selected for this study and subsequently planted in pots containing nutrient-rich humus soil. Remarkably, it is likely that the seedlings used came from nucellar embryos. These seedlings were cultivated in a greenhouse environment maintained at a temperature of 25 °C. The light conditions were set to a full intensity of 20,000 lux during the day and complete darkness at night, with a photoperiod of 14 h of light followed by 10 h of darkness. After approximately one month of cultivation, the seedlings were deemed suitable for experimental treatments. Three distinct stress treatments were administered to the trifoliate orange seedlings: low-temperature stress at 4 °C, salt stress using a 200 mmol/L solution, and simulated drought stress achieved with a 20% polyethylene glycol (PEG) solution. The specific experimental conditions were as follows: low-temperature stress at 4 °C was applied in an adverse growth chamber; for salt stress, a 200 mmol/L salt solution was administered to each pot at a volume of 200 mL; and for drought stress, a 20% PEG solution was applied to each pot at a volume of 500 mL. Leaf samples were collected at intervals between 0 and 24 h post-treatment. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Three biological replicates were established for each experimental condition, with three seedlings sampled from each replicate.

2.7. RNA Extraction and qRT-PCR Assays

The total RNA from each trifoliate orange treatment was extracted using the CTAB method [22]. RNA quality was assessed via gel electrophoresis (Beijing JunYi Electrophoresis Co., Ltd., Beijing, China) and a nanodrop microvolume spectrophotometer (Thermo Fisher Scientific Co., Ltd., Shanghai, China). Complementary DNA (cDNA) was synthesized through reverse transcription employing the Hiscript^® II Q RT SuperMix for qPCR (+gDNA wiper) Reverse transcriptase (Vazyme Biotech Co., Ltd., Nanjing, China), followed the protocol outlined in the manufacturer’s instruction manual. The gene-specific primer sequences for quantitative reverse transcription PCR (qRT-PCR) are detailed in Table S1. A 10 μL PCR reaction was conducted using 2 μL of cDNA as a template with ChamQ SYBR qPCR Master Mix, also from Vazyme, where each primer was used at a concentration of 10 μmol/L. Each well of a 96-well plate contained a reaction mixture comprising 5 μL of SYBR Mix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 2 μL of RNase-free double-distilled water (ddH₂O), and 2 μL of cDNA. Quantitative real-time PCR (qRT-PCR) was conducted utilizing a CFX96 real-time fluorescence quantitative PCR instrument (BIO-RAD), with the citrus β-actin gene serving as the internal control. The reaction protocol comprised an initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 10 s and annealing at a primer-specific temperature for 30 s. Subsequent to the amplification process, a melting curve analysis was carried out over a temperature range of 65 to 95 °C. Each qRT-PCR analysis was performed in triplicate. Gene expression levels were quantified using the 2^−ΔΔCT method. The cycle threshold (CT) values for both target and reference genes in control and experimental samples were determined from the amplification curve. Subsequently, the CT values of the reference genes were used to normalize the CT values of the target genes. This process ultimately yields the relative expression levels.

3. Results

3.1. Identification of Citrus SNAT Gene Family Members and Prediction of Physicochemical Properties of Their Corresponding Proteins

A comprehensive analysis utilizing BLAST and HMM methodologies identified 21 SNAT gene family members within the genomes of five distinct citrus species (

Table 1. Basic information on SNAT gene family members of citrus.

Gene Name	CPBD Registration Number	Chromosomes	Amino Acid Length	Molecular Weight (kDa)	Isoelectric Point (pI)	Instability Index (II)	Chloroplast Transit Peptide	Subcellular Localization Predicted
CsSNAT1	Cs_ont_5g027410.1	Chr5	271	31.15	9.25	44.99	absence	chloroplast
CsSNAT2	Cs_ont_6g012680.2	Chr6	288	32.67	8.70	45.44	absence	chloroplast
CsSNAT3	Cs_ont_7g003510.1	Chr7	614	67.53	8.72	50.06	absence	cytoplasm
CsSNAT4	Cs_ont_7g020400.1	Chr7	264	29.19	9.35	44.25	presence	cell membrane
CcSNAT1	Ciclev10021647m.1	Chr3	271	31.07	9.26	44.68	absence	chloroplast
CcSNAT2	Ciclev10033598m.1	Chr4	251	27.50	8.97	44.77	absence	chloroplast
CcSNAT3	Ciclev10032841m.1	Chr4	180	20.11	9.13	37.93	absence	chloroplast
CcSNAT4	Ciclev10012371m.1	Chr6	288	32.67	8.70	45.44	absence	chloroplast
ClSNAT1	CL5G054858012.t1_alt	Chr5	271	31.11	9.32	44.68	presence	chloroplast
ClSNAT2	CL6G059010012.t1_alt	Chr6	240	26.65	5.38	43.98	presence	chloroplast
ClSNAT3	CL7G061361012.t1_alt	Chr7	258	28.58	6.33	40.87	absence	chloroplast
ClSNAT4	CL7G063246012.t1_alt	Chr7	189	21.23	9.78	39.34	absence	chloroplast
CgSNAT1	Cg5g025450.1	Chr5	271	31.11	9.32	44.68	presence	chloroplast
CgSNAT2	Cg6g010930.1	Chr6	247	27.57	5.73	45.14	presence	chloroplast
CgSNAT3	Cg7g004160.1	Chr7	200	22.14	7.69	43.33	absence	nucleus
CgSNAT4	Cg7g015440.1	Chr7	189	21.22	9.78	39.89	absence	mitochondria
PtrSNAT1	Pt3g018760.1	Chr3	271	30.98	9.20	48.23	presence	chloroplast
PtrSNAT2	Pt3g018810.1	Chr3	271	30.96	9.13	47.35	absence	chloroplast
PtrSNAT3	Pt4g009400.1	Chr4	236	26.27	10.12	40.40	presence	chloroplast
PtrSNAT4	Pt4g020200.1	Chr4	603	65.92	7.96	47.99	absence	chloroplast
PtrSNAT5	Pt6g011220.2	Chr6	247	27.57	5.73	48.72	presence	chloroplast

). These genes were designated based on their chromosomal locations as follows: CsSNAT1-CsSNAT4 in sweet orange (Citrus sinensis), CcSNAT1-CcSNAT4 in clementine (Citrus clementina), ClSNAT1-ClSNAT4 in lemon (Citrus limon), CgSNAT1-CgSNAT4 in pomelo (Citrus maxima), and PtrSNAT1-PtrSNAT5 in trifoliate orange (Poncirus trifoliata). Specifically, CcSNAT1, PtrSNAT1, and PtrSNAT2 are situated on chromosome 3, while CcSNAT2, CcSNAT3, PtrSNAT3, and PtrSNAT4 are located on chromosome 4. Furthermore, CsSNAT1, ClSNAT1, and CgSNAT1 are positioned on chromosome 5; CsSNAT2, CcSNAT4, ClSNAT2, CgSNAT2, and PtrSNAT5 on chromosome 6; and CsSNAT3, CsSNAT4, ClSNAT3, ClSNAT4, CgSNAT3, and CgSNAT4 on chromosome 7 (Figure 1). The encoded proteins of these citrus SNAT genes exhibited amino acid lengths ranging from 180 to 614, molecular weights between 20.11 and 67.53 kDa, and isoelectric points spanning 5.38 to 10.12. Additionally, the instability indices of the citrus SNAT proteins ranged from 37.93 to 62.99, with CcSNAT3, ClSNAT5, and CgSNAT4 displaying indices below 40, thereby indicating their stability. The prediction of chloroplast-transfer peptides indicated that eight citrus SNAT proteins (CsSNAT4, ClSNAT1, ClSNAT2, CgSNAT1, CgSNAT2, PtrSNAT1, PtrSNAT3, and PtrSNAT5) possessed chloroplast-transfer peptides. Subcellular localization predictions, conducted using the online tools Plant-mPLoc and CELLO, revealed that the majority of citrus SNAT proteins were localized within chloroplasts. Exceptions included CsSNAT3, which was localized in the cytoplasm; CsSNAT4, which was associated with the cell membrane; CgSNAT3, which was located in the nucleus; and CgSNAT4, which was found in the mitochondria.

3.2. Gene Structure, Conserved Domains, and Motif Analysis of the SNAT Genes in Citrus

The examination of gene structure, conserved domains, and motif analysis among gene family members is widely acknowledged as an effective method for elucidating the functional characteristics of gene families. As illustrated in Figure 2, citrus SNAT genes exhibited a range of one to eight exons and zero to seven introns, with significant structural variation observed across different groups. Specifically, the first group of genes (CsSNAT4, CcSNAT3, ClSNAT4, CgSNAT4, and PtrSNAT3) was distinguished by the presence of only one to two exons. In contrast, the second group was characterized by the presence of eight exons. The third group exhibited a range of 3 to 8 exons, while the fourth group consistently contained four exons. An analysis of the conserved domains indicated that citrus SNAT proteins possessed one to two conserved domains, specifically featuring one SNAT-conserved domain (either Acetyltransf_1 or Acetyltransf_7). Notably, only PtrSNAT4 and CsSNAT3, which belong to the third group, possess two conserved domains. Motif analysis further revealed that all citrus SNAT proteins contained five to eight motifs. Within the same group, citrus SNAT proteins exhibited similar motifs, suggesting the potential for identical functional roles.

3.3. Phylogenetic Tree and Collinearity Analysis

To elucidate the evolutionary relationships of citrus SNAT with those of other model species, protein sequences from Arabidopsis SNAT (3 sequences), rice SNAT (2 sequences), and citrus SNAT (21 sequences) were subjected to analysis, culminating in the construction of a phylogenetic tree (Figure 3). The phylogenetic analysis delineated four distinct subgroups. Group 1 comprised CgSNAT1, CsSNAT1, ClSNAT1, CcSNAT1, PtrSNAT1, and PtrSNAT2; Group 2 included PtrSNAT4, CgSNAT3, CsSNAT3, CcSNAT2, and ClSNAT3; Group 3 consisted of CgSNAT2, ClSNAT2, CsSNAT2, CcSNAT4, and PtrSNAT5; and Group 4 encompassed CcSNAT3, ClSNAT4, CgSNAT4, PtrSNAT3, and CsSNAT4. These findings suggest the potential for functional diversity within the citrus SNAT gene family. Furthermore, our analysis revealed that the majority of Arabidopsis and rice SNAT were predominantly clustered within Groups 3 and 4, with the exception of AtSNAT3, which was exclusively located in Group 1.

Figure 4 illustrates collinearity analysis results, revealing collinearity between SNAT genes of various citrus species and Arabidopsis or rice. Specifically, sweet orange has two collinear pairs with Arabidopsis and three with rice; clementine has one with Arabidopsis and two with rice; lemon has two with both Arabidopsis and rice; pomelo has one with Arabidopsis and two with rice; trifoliate orange has two with Arabidopsis and three with rice. These findings suggest a common ancestral origin for these SNAT genes, with citrus SNAT genes showing closer relation to rice SNAT genes.

3.4. Analysis of Cis-Acting Elements in the Promoter Regions of Citrus SNAT Genes

To enhance our understanding of the function and transcriptional regulation of the citrus SNAT gene, we conducted an analysis of the cis-acting elements within the 2000 base pair region upstream of the transcriptional start site of the citrus SNAT gene, utilizing the PlantCARE software. The analysis of the cis-acting elements revealed the presence of 32 distinct elements within the promoter region of the citrus SNAT gene. These elements can be categorized into three primary types: growth and development elements, hormone-responsive elements, and stress-responsive elements (Figure 5). Notably, stress-responsive elements were the most prevalent, with a total of 20 identified. The elements associated with growth and development predominantly comprised the meristem-specific element (CAT-box, present in 85.7% of SNAT members), the circadian-regulated element (circadian, found in 52.4% of SNAT members), and the zein-metabolism-regulated element (O2-site, identified in 47.6% of SNAT members). The hormone-response elements were primarily represented by the abscisic acid-response element (ABRE, observed in 85.7% of SNAT members), the methyl jasmonate-response element (CGTCA-motif or TGACG-motif, detected in 66.7% of SNAT members), and the salicylic-acid response element (TCA-element, found in 28.6% of SNAT members). This indicates that the expression of citrus SNAT genes may be regulated by multiple hormones. The principal stress-response elements included the light-response element (such as G-box and box 4, present in 90.5% of SNAT members), the drought-response element (MBS, found in 85.7% of SNAT members), the anaerobic-inducible element (ARE, detected in 66.7% of SNAT members), and the low-temperature-responsive elements (LTR, present in 42.9% of SNAT members). This suggests that citrus SNAT genes may be responsive to various stress conditions. Notably, cell cycle regulatory elements (MSA-like) were only identified on the PtrSNAT1 promoter.

3.5. Expression Analysis of PtrSNAT Genes in Different Tissues

The trifoliate orange is a critical rootstock for citrus cultivation due to its ability to enhance the resistance of grafted scions. Consequently, this study utilized trifoliate orange as the experimental material to explore the tissue-specific expression of SNAT genes and their expression dynamics under stress conditions. Leveraging the citrus pan-genome database, we analyzed the tissue-specific expression profiles of the five SNAT genes in trifoliate orange. The findings revealed distinct tissue-specific expression patterns (Figure 6). Notably, PtrSNAT1, PtrSNAT2, PtrSNAT3, and PtrSNAT5 were predominantly expressed in leaves, with PtrSNAT1 being exclusively expressed in this tissue. PtrSNAT4 showed high expression levels in the flesh of young fruit, while PtrSNAT3 was specifically expressed in early-stage ovules. Furthermore, PtrSNAT4 and PtrSNAT5 were the only genes expressed, albeit at low levels, in the flesh of mature fruit. Overall, all five SNAT genes exhibited high expression levels in leaves, aligning with their predicted subcellular localization to chloroplasts. Interestingly, consistent with the findings in trifoliate orange, all four SNAT genes from Citrus sinensis demonstrated high expression levels in leaves (Supplementary Table S2).

3.6. Expression Profiling of PtrSNAT Genes Under Different Abiotic Stress Conditions

Research has demonstrated that SNAT genes in plants are typically upregulated in response to abiotic stress. Furthermore, the previous analysis on cis-acting elements suggested that the citrus SNAT genes may respond to a variety of stress conditions. Consequently, this study investigated the expression profiles of five SNAT genes from trifoliate orange under various abiotic stress conditions, including low-temperature, salt, and drought stress, utilizing qRT-PCR analysis. As illustrated in Figure 7, the application of low-temperature stress treatment resulted in upregulation of PtrSNAT1 and PtrSNAT2 expression within 12 h post-treatment. Notably, the expression level of PtrSNAT1 at 12 h post-treatment was elevated to 4.25 times that observed at 0 h post-treatment, whereas the expression of PtrSNAT2 increased to 7.07 times its initial value. Conversely, the expression levels of PtrSNAT3, PtrSNAT4, and PtrSNAT5 either remained constant or were downregulated by low temperature treatment. Figure 8 demonstrates that salt stress treatment significantly upregulated the expression of all genes, with the exception of PtrSNAT4. After 3 h post-treatment, the expression levels of PtrSNAT1, PtrSNAT2, PtrSNAT3, and PtrSNAT5 were elevated to 16.82-fold, 14.29-fold, 11.78-fold, and 10.19-fold of their respective initial values. As shown in Figure 9, following drought stress treatment, only PtrSNAT1 exhibited significant upregulation at both 3 h and 24 h post-treatment, with expression levels reaching 2.92-fold and 4.55-fold of the initial values, respectively. The expression of the remaining genes did not exhibit significant changes. In conclusion, these findings suggest that PtrSNAT1 may play a pivotal role as a key SNAT gene in the response to abiotic stress in trifoliate orange.

4. Discussion

SNAT, a crucial enzyme in the biosynthesis of melatonin, is integral to plant growth, development, and stress response mechanisms [7]. To date, investigations into SNAT genes within citrus species remain unexplored. In this study, we employed bioinformatics methodologies to identify 21 SNAT genes across five principal citrus species. Of these, four species each harbored four SNAT gene members, whereas trifoliate orange contained five. Previous studies have indicated that the number of SNAT gene members in plants typically ranges from 2 to 7 [10,11,12,13,14,15]. The chromosomal distribution of citrus SNAT genes is relatively uniform, although a majority are located on chromosomes 6 and 7. Importantly, only eight citrus SNAT genes are equipped with chloroplast-transfer peptides. In comparison, within soybean, only three out of seven SNAT gene members possess chloroplast-transfer peptides [15]. The structural analysis of the genes indicated that the citrus SNAT genes comprised between one and eight exons and zero to seven introns. Interestingly, significant variations in gene structure were observed among the gene family members within each citrus species, a phenomenon also noted in the SNAT gene family of soybean [15]. This observation suggests potential functional divergence among SNAT gene members. Subcellular localization predictions indicated that the majority of citrus SNAT were localized to chloroplasts, with a minority found in the nucleus, cytoplasm, cell membrane, and mitochondria. These findings are generally consistent with the results of previous studies [12,13].

Phylogenetic tree analysis categorized the citrus SNAT into four distinct subgroups, while collinearity analysis indicated a closer evolutionary relationship between the citrus SNAT and those of rice. Significantly, certain SNATs from Arabidopsis, rice, and citrus, including CgSNAT2, ClSNAT2, CsSNAT2, CcSNAT4, PtrSNAT5, AtSNAT1, and OsSNAT1, are grouped within the same clade. This clustering suggests that SNAT genes originated before the evolutionary divergence of monocots and dicots.

The analysis of cis-acting elements within the promoter region of the citrus SNAT gene revealed the presence of 32 distinct types of elements associated with growth and development, hormone responsiveness, and stress response. In flowering Chinese cabbage, the BcSNAT1 promoter also exhibited a high density of cis-acting elements responsive to phytohormones (including auxin, GA, MeJA, salicylic, and abscisic acid), stress (such as anaerobic induction, defense and stress responses, light, low temperature, and drought inducibility), and development (meristem) [12]. Interestingly, BcSNAT1 expression was upregulated in response to various hormone treatments (GA, 6BA, IAA, and ABA) as well as under abiotic stress conditions (Al, Cd, PEG, and NaCl) [12]. These findings suggest that citrus SNAT genes may play a role in regulating plant growth and development, as well as in mediating responses to various hormones and environmental stresses. Notably, among the growth and development-related elements, a circadian-regulated element was identified, present in 52.4% of SNAT gene members. Interestingly, similar circadian-regulated elements were also found in HpSNAT1 from Hypericum perforatum and GmSNAT1 from soybean [13,15]. Moreover, the expression of HpSNAT1 genes follows a diurnal rhythm, with elevated levels during the day and reduced levels at night, paralleling fluctuations in endogenous melatonin concentrations [23]. The findings indicate that the SNAT gene plays a crucial role in the synthesis of melatonin in plants. Additionally, our analysis of the SNAT1 genes across five citrus species revealed the presence of circadian-regulated elements. This observation leads us to hypothesize that SNAT1 is integral to melatonin synthesis in citrus. Nonetheless, further experimental validation is necessary to substantiate this hypothesis.

Previous studies have demonstrated that SNAT genes are predominantly highly expressed in leaves. Notable examples include BcSNAT1 in flowering Chinese cabbage, NtSNAT1/2 in tobacco, ZmSNAT1/3 in Zea mays, and HpSNAT1/2 in Hypericum perforatum [11,12,13,14]. Additionally, a few SNAT genes showed high expression in roots, flowers, or seeds [11,12,15]. In the present study, we investigated the tissue-specific expression patterns of SNAT genes using trifoliate orange, a citrus rootstock, as the experimental model. The results revealed that that all SNAT genes in trifoliate orange exhibit high expression levels in the leaves, suggesting a significant role in leaf growth and development. This expression pattern is consistent with observations in the majority of plant species [11,12,13,14]. The tissue specific expression of profile of SNAT genes in plants aligns with the tissue distribution of melatonin, suggesting a possible role of SNAT genes in melatonin synthesis [3,4,24,25].

Numerous studies have demonstrated that SNAT genes play a positive role in regulating plant resistance to abiotic stress [16,17,18]. It is well-established that SNAT genes, across various plant species, are activated in response to abiotic stresses, such as drought, salinity, and elevated temperatures [10,11,12,14,16,17]. In this study, trifoliate orange was employed as the experimental model to examine the expression patterns of SNAT genes under various abiotic stress conditions, including low-temperature, salt, and drought stress, with the objective of identifying key candidate genes. The results revealed that, among the five SNAT genes in trifoliate orange, only PtrSNAT1 exhibited significant upregulation across all three abiotic stress conditions. Additionally, PtrSNAT2 was notably induced under low-temperature and salt stress, whereas PtrSNAT3 and PtrSNAT5 were significantly induced under salt stress. These observations suggest that these genes, particularly PtrSNAT1, may be involved in the response to abiotic stress. Our research group has successfully cloned the PtrSNAT1 gene, and its functional characterization is currently in progress.

5. Conclusions

The study identified 21 SNAT genes across five citrus genomes, mostly located in chloroplasts and spread over five chromosomes. These genes have 1–8 exons, 0–7 introns, 1–2 conserved domains, and 5–8 motifs. Phylogenetic analysis grouped them into four subgroups, showing strong similarity with rice SNAT genes. Promoter analysis found 32 cis-acting elements, with light-, abscisic acid-, and drought-response elements being most common. In trifoliate orange, SNAT genes showed tissue-specific expression, with the highest in leaves. PtrSNAT1 was significantly upregulated under stress, indicating its role in stress response. These findings offer insights for further research on citrus SNAT genes and suggest PtrSNAT1 as a target for developing stress-resistant rootstocks.