Exogenous Melatonin Effects on Drought-Stressed Longan Plants: Physiology and Transcriptome Insights

Abstract

Drought stress severely constrains yield and quality stability in longan (Dimocarpus longan Lour.), an important medicine and food homology fruit in China. Melatonin (MT) shows potential for alleviating abiotic stress, but its mechanisms in drought-stressed longan remain unclear. Here, we investigated two cultivars (Shixia and Chuliang) under drought and exogenous MT treatments (CW, well-watered control; CM, exogenous MT application under well-watered control; DW, drought stress; DM, exogenous MT application under drought stress), revealing the following findings: (i) Drought treatment significantly reduced endogenous MT levels in both studied cultivars, and the reduction was reversed by exogenous foliar MT application. Specifically, under drought conditions, exogenous MT treatment increased endogenous MT content by 272.7% in Shixia and 53.6% in Chuliang, respectively. (ii) Drought and exogenous MT treatments modulated the activities of plant defense enzymes (superoxide dismutase, SOD; peroxidase, POD; phenylalanine ammonia lyase, PAL; and catalase, CAT) and the levels of related metabolites (malondialdehyde, MDA; proline, Pro). Across both cultivars, drought stress increased the activities of SOD, POD, and PAL, as well as the Pro content. Exogenous MT treatment, however, reduced the activities of SOD, POD, and PAL while increasing CAT activity and MDA content to some extent in both cultivars. Notably, the Pro content was significantly reduced in Shixia but significantly increased in Chuliang following exogenous MT application under drought stress. (iii) Drought and exogenous MT treatments regulated gene expression in longan cultivars. Relative to CW, 848, 3356, and 2447 differentially expressed genes (DEGs) were detected in CM, DW, and DM in Shixia, respectively. Relative to CW, 1349, 5260, and 5116 DEGs were identified in CM, DW, and DM in Chuliang. A gene ontology analysis indicated significant enrichment for abiotic stress defense and hormone-responsive processes. The KEGG pathway analysis showed significant enrichment in protein processing in the endoplasmic reticulum (ko04141), amino sugar and nucleotide sugar metabolism (ko00520), ascorbate and aldarate metabolism (ko00053), plant–pathogen interaction (ko04626), and starch and sucrose metabolism (ko00500). These findings provide physiological and transcriptomic insights into MT-regulated drought responses in longan, highlighting its potential for improving productivity in drought-prone regions.

1. Introduction

Longan (Dimocarpus longan Lour.) is an economically important subtropical fruit tree widely cultivated across southern China and Southeast Asian countries, including Thailand and Vietnam [1]. Renowned for its nutritional value and therapeutic properties [2,3], longan contributes substantially to regional agricultural economies. For example, Pingnan City in the Guangxi Zhuang Autonomous Region, known as the hometown of Longan in China, is estimated to yield 2.0 × 10⁸ kg of longan in 2025, with the elite cultivar Shixia accounting for the largest proportion. Despite its adaptation to warm, arid climates, longan growth and productivity are vulnerable to abiotic stresses, notably drought [4], during critical phenological stages, such as flowering and fruit enlargement [5].

Drought stress profoundly affects longan physiology. Although longan exhibits moderate drought tolerance, it generally requires >1200 mm annual precipitation [6], prolonged water deficit impairs photosynthetic efficiency, perturbs phytohormone homeostasis, and weakens antioxidant defenses [7]. Extensive studies show that drought lowers leaf water potential, stomatal conductance and chlorophyll content, leading to oxidative damage from excess reactive oxygen species and consequent lipid peroxidation and malondialdehyde (MDA) accumulation [8,9,10]. In Guangxi, a major longan-producing region, seasonal droughts have markedly reduced fruit yield and quality [11], underscoring the need for effective drought-mitigation strategies [12].

Exogenous plant growth regulators are recognized as promising tools to enhance plant drought tolerance. Among them, N-acetyl-5-methoxytryptamine (melatonin, MT) has attracted considerable interest for its diverse roles in modulating drought responses [13]. As a potent antioxidant, MT directly scavenges reactive oxygen species (ROS) and activates enzymatic defenses, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), to maintain cellular redox homeostasis [14]. In crops, MT also regulates key physiological processes, including germination, root development and leaf senescence, and promotes osmotic adjustment by enhancing the accumulation of proline and abscisic acid, thereby mitigating drought injury [15].

Recent work further reveals MT’s interactions with multiple phytohormones, flavonoids, and the plant microbiome, reinforcing its role in drought tolerance [16]. Nevertheless, the mechanisms by which exogenous MT confers drought tolerance in longan, whose fruit was officially included in China’s national list of medicine and food homology, remain poorly understood. Most previous studies have focused on herbaceous or model species, with limited research attention paid to woody fruit trees such as longan. In the current study, we investigate the effects of exogenous MT on longan under drought stress, integrating physiological measurements with transcriptomic profiling. The results provide a conceptual framework for enhancing longan drought resilience and optimizing cultivation practices in water-limited regions.

2. Materials and Methods

2.1. Experimental Site and Plant Materials

Pot experiments were conducted at the Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, Guangxi Zhuang Autonomous Region, China (25°4′ N, 110°18′ E) from July to September 2022. Two elite Chinese longan cultivars, Shixia and Chuliang, were selected as experimental materials. The Shixia cultivar, native to Pingnan County, Guangxi, was designated as a National Geographic Indication Product in China due to its industrial significance, high market recognition, and widespread cultivation. In contrast, the Chuliang cultivar boasts the largest cultivation area, attributed to its superior yield potential. Two-year-old grafted plants of both cultivars were sourced from the Institute of Horticulture, Guangxi Academy of Agricultural Sciences, Nanning, Guangxi Zhuang Autonomous Region, China (22°50′ N, 108°14′ E). For each cultivar, healthy and uniformly growing plants were selected and acclimatized in a greenhouse at the Guangxi Institute of Botany.

Soil substrate was prepared by thoroughly mixing red clay, peat, and sandy soil at a 1:1:1 ratio (v/v/v). The mixture was air dried under sunlight for three consecutive days to reach a constant weight. The dried substrate was then weighed and dispensed into plastic pots (20.0 cm height and 20.0 cm diameter), with each pot containing 6.00 kg of substrate, and thoroughly watered. Sixteen two-year-old longan plants of uniform height (approximately 60 cm) and fresh weight (1.52 ± 0.15 kg) were carefully stripped to minimize root damage. The longan plants were transplanted into the prepared pots on 16 July 2022 and acclimatized for one month. On 14 August 2022, the plants were subjected to water treatments and exogenous substance applications, with four replicates per group.

2.2. Water Treatments and Exogenous MT Application

Water treatments: Longan plants were subjected to two watering regimes over a 16-day period, namely a well-watered control (CW) maintained at approximately 20% (w/w) of dry soil weight and a drought treatment (DW) maintained at approximately 13% (w/w) of soil dry weight. Soil moisture was monitored using the gravimetric method. Specifically, soil samples were collected at a 0–20 cm depth from the pot rim every three days. For each sampling, approximately 30 g of fresh soil was collected using a soil auger, placed in pre-weighed aluminum foil containers, and immediately weighed the fresh weight (FW) of the sample. Subsequently, soil samples were oven-dried at 105 °C until constant weight to eliminate residual moisture, and the dry weight (DW) of each soil sample was measured. Soil moisture content was calculated as (FW − DW)/DW × 100%.

Exogenous application: MT was purchased from Macklin (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China). A 100 μmol L⁻¹ MT solution was prepared for foliar spraying by accurately weighing 23.28 mg MT, dissolved in 20 mL ethanol, and diluted to 1.0 L with double-distilled H₂O. Two drops of 0.01% (v/v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) were added as a surfactant, and the solution was mixed thoroughly. For foliar application, the MT solution was sprayed onto the leaves of plants in both CW and DW groups using a pressure sprayer with a nozzle diameter 0.3 mm until complete runoff. These treatments were designated as CM (CW + MT) and DM (DW + MT), respectively. Foliar spraying was initiated at 19:00, three days prior to the onset of water treatments, and reapplied at two-day intervals throughout the experimental period. During each application, treated plants were temporarily transferred to a separate area to prevent cross-contamination from spray drift and returned to their original positions afterward. Plants in the non-sprayed groups (CW, DW) received equal volumes of the vehicle control, which consisted of ddH₂O containing 0.01% Tween 20 without MT.

Experimental treatments were terminated on 30 August 2022. The changes in soil moisture content under different water and exogenous MT treatments for the two studied longan cultivars are presented in Figure 1. Specifically, the mean soil moisture contents under CW and CM treatments were 20.6 ± 2.23% and 20.7 ± 2.11% (mean ± SD), respectively, while the corresponding values under the DW and DM treatments were 13.3 ± 7.21% and 13.2 ± 7.27% (mean ± SD), respectively.

2.3. Sampling and Determination

At the end of the watering period (30 August 2022), fresh leaf samples were collected from the second to fourth mature functional leaves (counting from the shoot apex). Samples were frozen in liquid nitrogen for 30 s and stored at −80 °C for enzyme activity assays, phytohormone quantification and transcriptome profiling. Superoxide dismutase (SOD) activity was assayed by the nitroblue tetrazolium (NBT) photochemical reduction method [17]. Peroxidase (POD) activity was measured by the guaiacol oxidation method [18]. Catalase (CAT) activity was measured by the ultraviolet absorption method [19]. Phenylalanine ammonia-lyase (PAL) activity was determined by a spectrophotometric assay as described previously [20]. Proline (Pro) was quantified by an improved acid-ninhydrin method [21], and malondialdehyde (MDA) by the thiobarbituric acid method [22].

Endogenous MT in longan leaves was quantified by high-performance liquid chromatography (HPLC) following a procedure similar to our previous study [23]. Briefly, 0.50 g of leaf tissue was ground in liquid nitrogen and transferred to a 15 mL amber centrifuge tube. A total of 5 mL of cooled methanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added; the mixture was vortexed for 5 min and ultrasonically extracted at 4 °C for 30 min. After extraction, samples were centrifuged at 12,000× g for 5 min, and the supernatant transferred to a new amber centrifuge tube. The extraction was repeated twice, and supernatants from all three extractions were combined.

The pooled supernatant was concentrated to 3 mL via nitrogen evaporation at ambient temperature, followed by adjustment to a final volume of 6 mL using methanol. After thorough vortexing, the solution was filtered through a membrane filter before HPLC analysis. Chromatographic separation was performed on a 250 × 4.6 mm C18 column (5 μm, WondaCract ODS-2 C18 column, Shimadzu-GL, Kyoto, Japan). Detection wavelength was 222 nm and column temperature were maintained at 25 °C. Injection volume was 10 μL; the mobile phase was methanol: water (90:10, v/v) at a constant flow of 1.0 mL min⁻¹ under isocratic elution. The MT content was calculated from a standard curve generated using serial dilutions of MT standards.

2.4. RNA Extraction

Total RNA was extracted from longan leaf tissue using TRIzol^® Reagent (Plant RNA Purification Reagent) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), and genomic DNA was removed with DNase I (TaKaRa). RNA integrity and purity were assessed with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Only high-quality RNA samples (OD260/280 = 1.8–2.2; OD260/230 ≥ 2.0; RIN ≥ 8.0; 28S:18S ≥ 1.0; >1 μg) were used to construct sequencing libraries.

2.5. Library Preparation, and Illumina Hiseq X Ten/NovaSeq 6000 Sequencing

RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) following the manufacturer’s protocols (Illumina, San Diego, CA, USA). RNA-seq libraries from longan leaves were prepared using the Illumina TruSeq™ RNA Sample Preparation Kit (San Diego, CA, USA). Poly(A) mRNA was purified from total RNA using oligo(dT)-attached magnetic beads and fragmented with fragmentation buffer. Using these short fragments as templates, double-stranded cDNA was synthesized with a SuperScript cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) and random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation and A-tailing according to Illumina library construction protocols. Libraries were size-selected for 200–300 bp cDNA fragments on 2% Low Range Ultra Agarose and PCR-amplified using Phusion DNA polymerase (New England Biolabs, Boston, MA, USA) for 15 cycles. After quantification with a TBS380 fluorometer, two RNA-seq libraries were sequenced in a single lane on an Illumina HiSeq X Ten/NovaSeq 6000 platform (Illumina, San Diego, CA, USA) to generate 2 × 150 bp paired-end reads.

2.6. Statistics Analysis

The experiment used a completely randomized 2 × 2 × 2 factorial design (two cultivars, two water treatments and two exogenous treatments), resulting in eight treatment combinations, each with four biological replicates. The analysis of variance (ANOVA) was performed using Statistix 9.0 software (Analytical Software, Tallahassee, FL, USA) to evaluate the effects of water treatment, exogenous treatment, and cultivar on physiological variables. Since this study focused on the effects of exogenous treatment under drought stress, Duncan’s multiple range test was used for post-hoc comparisons, with significance levels set at p-value < 0.05, p-value < 0.01, and p-value < 0.001 to examine differences among combinations of water and exogenous treatments. For each cultivar, pairwise comparisons were conducted across water and exogenous treatments. Figures were generated using Metware Cloud [24], a free online data-analysis platform (https://cloud.metware.cn, accessed on 30 September 2022).

For transcriptome data, raw paired-end reads were trimmed and quality-controlled with Sickle (https://github.com/najoshi/sickle, accessed on 30 September 2022) and SeqPrep (https://github.com/jstjohn/SeqPrep, accessed on 30 September 2022) using default parameters. Clean reads from longan leaf samples were de novo assembled with Trinity (http://trinityrnaseq.sourceforge.net/, accessed on 30 September 2022) [25]. All assembled transcripts were searched against the NCBI non-redundant protein (NR), COG and KEGG databases using BLAST (v2.16.0, https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/, accessed on 30 September 2022) to retrieve functional annotations, with an E-value cut-off of <1.0 × 10⁻⁵. Gene Ontology (GO) annotations were obtained using Blast2GO (http://www.blast2go.com/b2ghome, accessed on 30 September 2022) [26] to describe biological process, molecular function and cellular component categories. Metabolic pathway analysis was performed using the Kyoto Encyclopediaof Genes and Genomes (KEGG; http://www.genome.jp/kegg/, accessed on 30 September 2022) [27].

To identify differentially expressed genes (DEGs), transcript abundance was quantified as transcripts per million (TPM). Gene abundances were estimated with RSEM (v1.3.3, https://github.com/deweylab/RSEM, accessed on 30 September 2022) [28]. Differential expression analysis was performed with DESeq2 [29], DEGseq [30] or edgeR [31]. Genes with |log2FC| > 1 and Q ≤ 0.05 (DESeq2/edgeR) or Q ≤ 0.001 (DEGseq) were considered significantly differentially expressed. In addition, functional enrichment analyses (GO and KEGG) were performed to identify DEGs significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected p-value ≤ 0.05 relative to the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out with GOATOOLS (https://github.com/tanghaibao/Goatools, accessed on 30 September 2022) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do, accessed on 30 September 2022) [32].

3. Results

3.1. Efffects of Exogenous MT on Endogenous MT Content of Longan Cultivars Under Drought Stress

Endogenous MT concentrations in leaves differed significantly among water and exogenous MT treatments and longan cultivars (Figure 2). Across water and exogenous MT treatments, Shixia exhibited significantly higher MT levels than Chuliang. In Shixia, endogenous MT contents in the control (CW, CM) and drought (DW, DM) groups were 225.16, 75.42, 18.17 and 67.72 μg g⁻¹, respectively, with CW significantly higher than the others. Pairwise comparisons showed highly significant differences for CW vs. DW, CW vs. DM and DW vs. DM (p-value < 0.001). In Chuliang, MT contents in control (CW and CM) and drought (DW and DM) groups were 7.52, 1.72, 1.44 and 2.22 μg g⁻¹, respectively. Significant differences were observed for CW vs. DW and DW vs. DM (p-value < 0.01), and for CW vs. DM (p-value < 0.001). These results indicate that exogenous MT increased endogenous MT under drought in both cultivars, as evidenced by higher values in sprayed (DM) than unsprayed (DW) groups.

3.2. Effects of Exogenous MT on Plant Defense Enzymes and Related Compound Under Drought Stress

Exogenous MT differentially altered antioxidant enzyme activities in the two cultivars across watering regimes (Figure 3). SOD activity did not differ significantly among the four treatments in either Shixia or Chuliang (Figure 3a). POD activity showed cultivar-specific responses. In Shixia, drought groups (DW, DM) exceeded CW by 26.20% and 37.88% (p-value < 0.01), with no difference between DW and DM. Conversely, in Chuliang, POD did not differ between CW and DW, but DM was 24.86% lower than CW (p-value < 0.01) and 27.04% lower than DW (Figure 3b). PAL activity displayed marked cultivar differences under drought. In Shixia, DW increased PAL relative to CW, whereas DM reduced PAL relative to DW; in Chuliang, DM exceeded CW with no other differences among groups (Figure 3c). The CAT activity also differed by cultivar: in Shixia there was no difference between drought and control groups, whereas in Chuliang DM increased CAT relative to CW and DW by 39.86% and 34.09%, respectively (Figure 3d).

MDA and Pro accumulation varied among treatments (Figure 3e,f). In Shixia, MDA under CW was 79.29 nmol g⁻¹, increasing to 92.96 nmol g⁻¹ under DW. In Chuliang, the drought groups (DW and DM) showed MDA trends opposite to the controls. In both cultivars, drought groups (DW and DM) generally had significantly higher Pro than controls (CW and CM). In Shixia, Pro was 44.41 μg g⁻¹ in CW, rose to 180.29 μg g⁻¹ under DW, then decreased to 73.08 μg g⁻¹ after MT (DM). In Chuliang, Pro was 21.93 μg g⁻¹ in CW, increased to 276.98 μg g⁻¹ under DW, and further to 387.78 μg g⁻¹ after MT. These results indicate that drought induces significant MDA and Pro accumulation in longan. Exogenous MT reduced MDA while modulating Pro, specifically, decreasing it in Shixia and increasing it in Chuliang under drought condition.

3.3. Transcriptional Responses to Exogenous MT Application Under Drought Stress

3.3.1. Principal Component Analysis (PCA) and Differential Gene Expression Analysis

Results of PCA showed distinct clustering among treatment groups, with PC1 and PC2 explaining 70% and 18% of the variance, respectively. Replicates exhibited good within-group reproducibility and clear between-group separation (Figure 4a). Numerous DEGs were detected between control (CW and CM) and drought (DW) groups (Figure 4b), with marked inter-cultivar differences in the DEG counts. In Shixia, 848 DEGs (567 up, 281 down) were identified in CM vs. CW. DW exhibited 3356 DEGs (2395 up, 961 down), whereas DM displayed 2447 DEGs (1751 up, 696 down). Under drought, 1632 DEGs were detected between DW and DM (823 up, 809 down). Furthermore, 171 DEGs were shared among CW, DW, and DM in Shixia. Specifically, 503 DEGs were unique to DW vs. DM, 1119 to CW vs. DW, and 644 to CW vs. DM (Figure 4c,d).

In Chuliang, 1349 DEGs (557 up, 792 down) were identified in CM vs. CW. DW contained 5260 DEGs (3304 up, 1956 down), and DM had 5116 DEGs (3278 up, 1838 down). Between DW and DM, 794 DEGs were identified (556 up, 238 down). Cross-cultivar comparisons revealed 9709 DEGs between Shixia and Chuliang under DW (8218 up, 1491 down), which decreased to 8502 DEGs under DM (7301 up, 1201 down). In Chuliang, 246 DEGs were shared among CW, DW, and DM; 231 were unique to DW vs. DM, 863 to CW vs. DW, and 718 to CW vs. DM (Figure 4). These results suggest that exogenous MT differentially modulates drought-responsive gene expression in longan, with Chuliang showing greater MT responsiveness.

3.3.2. GO and KEGG Functional Classification

To assess DEG functions, we performed Gene Ontology (GO) annotation and enrichment analyses for DEGs from CW, DW, and DM in Shixia and Chuliang. GO annotations classified DEGs into three categories: biological process, cellular component and molecular function. In Shixia, GO enrichment for CW vs. DW highlighted biological processes including response to stimulus (GO:0050896), response to stress (GO:0006950), response to chemical (GO:0042221), response to abiotic stimulus (GO:0009628) and defense response (GO:0006952). Cellular component terms were enriched for membrane (GO:0016020), extracellular region (GO:0005576) and cell wall (GO:0005618). Molecular function terms were enriched for transferase activity (phosphorus-containing groups; GO:0016772), phosphotransferase activity (alcohol group as acceptor; GO:0016773) and carbohydrate binding (GO:0030246). For DW vs. DM in Shixia, enrichment patterns for biological process and molecular function were similar to CW vs. DW, with molecular functions enriched for ADP/ATP binding (GO:0043531, GO:0005524), adenyl ribonucleotide binding (GO:0032559) and adenyl nucleotide binding (GO:0030554). GO enrichment profiles for CW, DW, and DM in Chuliang mirrored those in Shixia, with DEGs predominantly enriched in abiotic-stress defense responses and hormone-signaling pathways (Figure 5).

To characterize pathways associated with MT-mediated drought responses, DEGs from CW, DW, and DM were mapped to KEGG for functional annotation and enrichment. Numerous DEGs were annotated to carbohydrate metabolism, amino-acid metabolism and other categories. In Shixia, DEGs for CW vs. DW were significantly enriched in glycolysis/gluconeogenesis (ko00010), tryptophan metabolism (ko00380), amino sugar and nucleotide sugar metabolism (ko00520), and starch and sucrose metabolism (ko00500). In Shixia, DEGs for DW vs. DM were significantly enriched in protein processing in the endoplasmic reticulum (ko04141), amino sugar and nucleotide sugar metabolism (ko00520), ascorbate and aldarate metabolism (ko00053), plant–pathogen interaction (ko04626) and starch and sucrose metabolism (ko00500). Both GO and KEGG analyses indicated enrichment of nitrogen-metabolism and amino-acid-metabolism pathways under drought and MT treatments in both cultivars (Figure 5).

4. Discussion

Drought stress causes excessive accumulation of reactive oxygen species (ROS), which attack biological macromolecules, including membranes, proteins and nucleic acids [33]. Such ROS-mediated damage leads to membrane lipid peroxidation, protein denaturation and nucleic-acid damage, ultimately disrupting cellular structure and function and causing oxidative injury [34,35,36]. To counteract this, plants deploy enzymatic and non-enzymatic antioxidant defenses that scavenge excess ROS and maintain intracellular redox homeostasis [37,38]. In this study, exogenous MT increased Pro and MDA contents and CAT activity while reducing POD activity in Chuliang under drought (Figure 3). These findings suggest that MT regulates antioxidant-enzyme activities, enhances ROS-scavenging capacity and alleviates membrane lipid peroxidation, thereby mitigating oxidative damage. Consistent with our results, previous studies report that exogenous MT enhances drought tolerance in wheat through coordinated antioxidant defense and osmotic regulation [39]. The protective actions of MT include direct scavenging of radicals and their products, induction of antioxidant-enzyme expression, attenuation of pro-oxidant-enzyme activation and preservation of mitochondrial homeostasis [40].

The transcriptomic analysis provides a powerful approach to elucidate how exogenous MT alleviates drought stress. Sequencing across treatment groups generated extensive gene-expression data, offering insight into MT’s modes of action under drought [41,42]. Here, we analyzed DEGs for DW vs. CW, DW vs. DM and CM vs. CW. DEGs for DW vs. CW were enriched in pathways related to photosynthesis, carbon metabolism and plant-hormone signal transduction, indicating broad effects of drought on these processes in longan (Figure 4 and Figure 5). Within photosynthesis-related pathways, genes linked to electron transport and carbon assimilation were associated with net photosynthetic rate [43,44]. We infer that drought suppresses photosynthesis-related gene expression, thereby impairing the photosynthetic apparatus and reducing photosynthetic efficiency [45]. In phytohormone signaling pathways, drought regulated genes associated with abscisic acid (ABA) signaling [46]. As a key drought-response hormone, ABA regulates stomatal movement, promotes root growth and induces drought-responsive gene expression [47,48]. These changes indicate activation of ABA signaling under drought, triggering physiological responses that help longan adapt to arid conditions.

DEGs between DM and DW were significantly enriched in plant-hormone signal transduction, phenylpropanoid biosynthesis and glutathione metabolism. In hormone signaling, MT markedly affected the expression of multiple pathway genes, for example, MT can activate auxin biosynthesis and signaling [49]. By upregulating auxin-signaling genes, MT likely enhances root growth and development, improving water and nutrient uptake to increase drought resistance. In the phenylpropanoid pathway, enzymes and genes involved in lignin and flavonoid synthesis are modulated by drought [50,51]. MT-mediated regulation of phenylpropanoid genes promotes lignin and flavonoid biosynthesis, which may enhance cell-wall rigidity and antioxidant capacity to alleviate drought injury [52,53,54]. In the glutathione-metabolism pathway, genes involved in glutathione synthesis are modulated under stress. As a major antioxidant, glutathione participates in redox reactions to scavenge ROS and protect cells [55,56]. MT increases glutathione content and activity by upregulating related genes, thereby reinforcing antioxidant capacity and mitigating oxidative damage under drought [57,58].

Plant responses to exogenous MT vary across species and depend on plant type, growth environment and developmental stage. As a subtropical evergreen fruit tree, longan exhibits growth characteristics and physiological traits that differ from those of many other plants. Our transcriptomic analysis shows that MT and drought affect gene expression across multiple biological processes and metabolic pathways in longan, potentially reflecting species-specific drought-resistance mechanisms and MT-response pathways. Indeed, MT and MT-mediated phytohormonal crosstalk play multifaceted roles in improving drought tolerance through molecular mechanisms and biochemical interactions in horticultural plants [59]. We speculate that longan may possess distinct signal-transduction pathways and gene-regulatory networks under drought compared with other plants. Our study provides a preliminary basis for further investigation of longan’s drought-resistance mechanisms and the actions of exogenous MT.

5. Conclusions

Application of exogenous MT under drought stress significantly promoted the secretion of endogenous MT, and alleviated drought injury to some extent, in longan plants by regulating the antioxidant system. The gene expression of longan under drought stress was differentially regulated by exogenous MT, and there were inter-varietal differences, and the differentially expressed genes were significantly enriched in abiotic stress defense responses and hormone-responsive processes. Our study establishes a preliminary framework for subsequent investigations into the drought-resistance mechanisms of longan and the regulatory role of exogenous MT.