Next Article in Journal
In Vitro Assessment of Cucumis sativus L. Growth Responses to Aqueous Extracts Derived from Vermicompost Produced from Vineyard, Winery and Sewage Sludge Residues
Previous Article in Journal
Valorization of Propolis Waste for Sustainable Agriculture: The Aqueous Extract Has a Unique Phytotoxic Profile
Previous Article in Special Issue
Magnesium Hydride Enhances Both Growth and Bioactive Compound Biosynthesis in Salvia miltiorrhiza: A Novel Eco-Friendly Approach for Medicinal Plant Cultivation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 12
Issue 6
10.3390/horticulturae12060694
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Melatonin-Induced Leaf Growth in Lithocarpus litseifolius: A Synergistic Interplay Among Hormone Homeostasis, Photosynthetic Enhancement, and Transcriptional Regulation

by
Huan Wang
1,
Wenlong Zhang
2,
Mingfeng Xu
2 and
Lingye Su
2,*
1
Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, China
2
Guangdong Provincial Key Laboratory of Silviculture Protection and Utilization, Guangdong Academy of Forestry, Guangzhou 510520, China
*
Author to whom correspondence should be addressed.
Horticulturae 2026, 12(6), 694; https://doi.org/10.3390/horticulturae12060694 (registering DOI)
Submission received: 5 May 2026 / Revised: 1 June 2026 / Accepted: 3 June 2026 / Published: 5 June 2026
(This article belongs to the Special Issue Chemical Regulators in Horticulture: Seed Germination, Propagation and Metabolic Enhancement of Medicinal Plants)
Browse Figures
Versions Notes

Abstract

Lithocarpus litseifolius is a medicinal tea plant recognized for its sweet flavor and anti-diabetic properties, but its limited leaf yield under cultivation restricts its economic sustainability. Melatonin (MLT) is a multifunctional plant growth regulator, but its roles in leaf growth under normal conditions remain not fully understood. Herein, we investigated the effects and mechanisms of foliar-applied MLT on L. litseifolius seedlings, including growth indices, phytohormone profiles, photosynthetic characteristics, and transcriptome alterations. The 100 μM MLT treatment significantly enhanced leaf dry weight by 33.8% and leaf dry matter content by 22.2% compared to the control group. MLT decreased both free and bound abscisic acid (ABA), while increasing gibberellins (GAs), 5-deoxystrigol, auxins (e.g., IAM), and cytokinins (e.g., cZ9G). Additionally, exogenous MLT improved photosynthetic rate, stomatal conductance, chlorophyll content, and soluble sugars in leaves. RNA-seq revealed that MLT up-regulated DEGs involved in hormone biosynthesis and signaling (CYP707A, BAK1, D14, CCD1, and IAA6), photosynthesis (PsbC/B, PetH, PsaB, and ATPase β), and sugar metabolism (WAXY, glgC, and otsB). Our results demonstrate that MLT promotes leaf dry matter accumulation through coordinated phytohormone homeostasis, photosynthetic enhancement, and transcriptional regulation, offering a cost-effective strategy to improve leaf yield in L. litseifolius.

1. Introduction

Lithocarpus litseifolius, an evergreen tree from the Fagaceae family, has been traditionally utilized as a valuable medicinal tea plant in southern China [1]. The leaves of L. litseifolius are glabrous, papery to subcoriaceous in texture, and have a compact layer of furfuraceous scales on the abaxial surface, without dense trichomes or a thick waxy cuticle [2]. Due to the high concentration of distinctive sweet-tasting chemicals (mainly belonging to dihydrochalcone groups), its leaves are commonly referred to as ‘sweet tea.’ Research has shown that sweet tea has multiple health advantages, such as anti-diabetic, anti-hypertensive, and anti-hyperlipidemic qualities [1,3,4]. As a result, L. litseifolius functions as a potential sugar-free tea drink for approximately 600 million diabetic patients worldwide, and its market demand has dramatically increased [5]. However, the wild resources are generally scattered in distribution in secondary forest areas, and the large-scale cultivation techniques of L. litseifolius are still immature, resulting in low leaf yields and an inability to fulfill market demand [6]. Consequently, it is vital to investigate effective methods to improve the production of leaves in L. litseifolius cultivation.
Plant growth regulators serve an important function in the development of leaves. Melatonin (N-acetyl-5-methoxytryptamine; MLT), an indoleamine compound ubiquitously synthesized in the cytoplasm, endoplasmic reticulum, and chloroplasts. It is generally distributed in leaves, flowers, seeds, and fruits, and performs multiple growth-regulatory functions in plants such as growth, development and stress tolerance [7,8]. Extensive research indicates that exogenous MLT may improve plant seed germination, root development, senescence delay, and stress tolerance [7,8]. Notably, MLT also plays a positive role in the production of leaves in horticultural crops, with major reports focusing on stress conditions [9]. For instance, MLT treatment for two months can maintain the leaf biomass of mature tea plants under cold stress; all levels of MLT applications improve the leaf area and weight in sunflowers under water deficit conditions [10,11]. Nonetheless, the effect and processes of MLT on leaf growth remain poorly understood under normal conditions, with no reports in L. litseifolius. Further investigation into the hormonal, physiological, and transcriptional pathways is still needed.
The molecular signaling network between MLT and other phytohormones collaborates in a variety of regulatory functions [12]. MLT regulates plant growth by modulating endogenous levels of key hormones such as abscisic acid (ABA), gibberellins (GA), strigolactone (SL), auxin, and cytokinin (CTK). Since these hormone families typically exist in various forms (such as free and bound forms), more research is needed into the interplay between multiple phytohormone types and MLT-induced leaf growth. Additionally, the photosynthesis system serves as the core biosynthetic pathway for carbon assimilation in plants [13]. Numerous studies have reported that MLT exerts beneficial effects on the photosynthesis system, mainly by improving photosystem II (PSII) efficiency [14,15], increasing stomatal conductance [16], and elevating chlorophyll and soluble sugar levels [17,18,19]. MLT inhibits ABA biosynthesis to protect PS II and delay leaf senescence in Arabidopsis [20]. Furthermore, MLT might encourage a dynamic molecular network in plants, resulting in MLT-mediated signal transduction cascades. Exogenous MLT application significantly up-regulates ABA-catabolism genes MdCYP707A1 and MdCYP707A2, leading to decreased ABA levels, enhanced photosynthetic rate (Pn) and stomatal conductance (Gs) in Malus plants [21]. MLT promotes the expression of WAXY genes in barley, which regulates leaf stomatal movement and carbon and nitrogen metabolism. Therefore, it is speculated that MLT might operate through the complex regulatory network of the hormonal, photosynthetic, and transcriptional processes.
Herein, this work investigated the regulatory effects and underlying mechanisms of foliar spraying MLT on leaf growth in L. litseifolius seedlings, owing to its leaf surface properties. Moreover, extensive investigation was performed on MLT-mediated alterations to growth-related hormone profiles (ABA, GA, SL, auxin, and CTK classes), photosynthetic parameters (Pn, Gs, intercellular CO2 concentration (Ci), transpiration rate (E), and SPAD), sugar metabolism (soluble sugar), and transcriptional profiling (RNA-seq). According to the current research, foliar-applied MLT represents an effective method for increasing leaf production in L. litseifolius cultivation, principally via modulating phytohormone homeostasis, photosynthetic capacity and gene expression.

2. Materials and Methods

2.1. Plant Materials

The L. litseifolius seeds used in the experiment were collected from 20-year-old mature trees in Luofu Mountain, Huizhou City, Guangdong Province. The seedling cultivation approach was based on previous work [22]. To promote germination, the collected seeds were maintained for 120 days in wet sand stratification conditions. Germinated seeds were then transplanted onto non-woven fabric culture pots within a greenhouse. The substrate in the pots was a 3:6:1 mixture of peat, rice husk, and loess (main composition of SiO2 and Al2O3). After approximately seven months of cultivation, the L. litseifolius seedlings with identical growth conditions were selected for further investigation.

2.2. MLT Treatments

The seedlings of L. litseifolius were randomly divided into four groups, and each group included 20 plantlets. An MLT stock solution (0.2 M; Sangon Biotech, Shanghai, China) was prepared by dissolving 1.15 g of MLT in 25 mL of ethanol, supplemented with 0.05% (v/v) Tween-20 as a surfactant, and then diluted with distilled water to different final concentrations. These groups were foliar sprayed with 0, 50, 100, and 200 μM MLT solutions, referred to as CK, MLT 50, MLT 100, and MLT 200 groups, respectively. The experimental procedures followed the methodology of [22]. Different concentrations of MLT solution were evenly applied to both sides of leaves until completely covered with water droplets. The applications were carried out at 24 h intervals three times. At ten days after the final treatments, the seedlings were measured for growth parameters, photosynthetic parameters, and chlorophyll contents. Subsequently, the leaf samples were collected, flash-frozen in liquid nitrogen, and stored at −80 °C for analyses of plant hormones, soluble sugars, RNA-seq, and quantitative real-time PCR (qRT-PCR).

2.3. Determination of Growth Parameters

The plantlet height of each L. litseifolius seedling before and after four MLT treatments was first determined. Then the total leaves were picked for fresh weight (FW) measurements. After vacuum lyophilization, the dry weights (DW) and leaf dry matter contents (LDMC, LDMC = DW/FW × 100%) were finally measured.

2.4. Determination of Phytohormone Levels

A total of 17 metabolites from five phytohormone classes (ABA, GA, SL, auxin, and CTK) were qualified and quantified in the leaves of CK and MLT 100 (the optimal concentration for LDMC) groups. Each group contained three replicates. Frozen samples (0.5 g FW) were extracted with a mixture of methanol/water/formic acid (15:4:1, v/v/v) containing 10 μL of an isotopically labeled internal standard mix (100 ng/mL each). According to [23], the extracts were analyzed by UPLC-ESI-MS/MS using an AB Sciex QTRAP 6500+ platform (MetWare, Wuhan, China). Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 C18 column (100 mm × 2.1 mm, 1.8 μm) with mobile phases A (water + 0.04% acetic acid) and B (acetonitrile + 0.04% acetic acid) at 0.35 mL/min, 40 °C, and 2 μL injection. The gradient was: 0–1 min 5% B, 1–8 min 5–95% B, 8–9 min 95% B, 9.1–12 min 5% B. Mass spectrometry was operated in scheduled MRM mode with ESI± (ion spray voltage ±5500/−4500 V, source temperature 550 °C, CUR 35 psi). Hormones were quantified by the internal standard method using calibration curves (0.01–500 ng/mL, R2 > 0.995). The peak area ratio (analyte/IS) was plotted against the concentration ratio, and absolute content (ng/g FW) was calculated as (c × V)/(1000 × m), where c = interpolated concentration (ng/mL), V = 100 μL (final volume), m = 0.5 g (sample weight). Data were processed with Analyst 1.6.3 and MultiQuant 3.0.3 (AB SCIEX, Marlborough, MA, USA).

2.5. Measurements of Photosynthetic Parameters, Chlorophyll Levels, and Soluble Sugar Contents

The second to third mature leaves on L. litseifolius seedlings from all four MLT treatments were used for photosynthesis and chlorophyll measurements. The Li-6800 portable photosynthesis system (LI-COR, Lincoln, NE, USA) was applied to determine the photosynthesis parameters, including Pn, Gs, Ci and E. Chlorophyll levels were measured on the same leaves as those for photosynthesis measurements. The leaf-relative chlorophyll contents (SPAD values) were detected by the SPAD-502 chlorophyll meter (Minolta,Osaka, Japan). In addition, the soluble sugar contents were determined via anthrone colorimetric analysis in accordance with [24].

2.6. RNA-Seq Analysis

The RNA-seq analysis was carried out on the leaves from CK and MLT 100 (the optimal concentration for LDMC) groups, with three biological replicates for each group. Total RNA from the samples was extracted first with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The library building and sequencing were performed on the Illumina Hiseq platform. After eliminating low-quality reads, the expression levels of each gene were determined using FPKM values. The analysis of differentially expressed genes (DEGs) from different libraries (NR, NT, Pfam, KOG, Swiss-Prot, KEGG, and GO databases) was performed by the DESeq R package [25]. The heat map plotting of DEGs involved in hormone, photosynthesis, and sugar metabolism was carried out by the Metware Cloud (https://cloud.metware.cn).

2.7. qRT-PCR Validation of DEGs

Total RNA from the CK and MLT 100 groups was simultaneously extracted using the RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). The RNA samples were then utilized to synthesize cDNA with a Transcribed One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). qRT-PCR experiments were performed with Talent qPCR Premix (TIANGEN, Beijing, China) through application of the SYBR Green method. The 2−ΔΔCt method was employed to calculate the relative expression level of each gene. A detailed list of the qRT-PCR primers is provided in Table S1.

2.8. Statistical Analysis

The data in this study are represented as mean ± SDs. ANOVA was used to compare the results from various MLT treatments, and then Tukey’s post hoc test was conducted. The correlation of different physiological indicators was carried out by Sangerbox (http://sangerbox.com).

3. Results

3.1. Effects of MLT on Leaf Growth of L. litseifolius

To investigate the potential effects of MLT on plant growth in L. litseifolius seedlings, the physiological indicators of leaf growth (plant height, FW, DW, and LDMC) were determined. As shown in Figure S1, the plant height showed no significant difference among the four groups before MLT treatments. However, a significant increase in plant height only occurred at 50 μM MLT when compared with CK (Table 1, Figure 1). Although no significant differences in FW were detected between any MLT treatment and the control, all MLT concentrations (50, 100, and 200 μM) significantly enhanced both DW and LDMC levels compared with CK (Table 1). DW was elevated by 29.4%, 33.8%, and 12.3% under 50, 100, and 200 μM MLT, respectively, while LDMC was increased by 13.8%, 22.2%, and 11.9%, respectively (Table 1). These responses followed a clear trend of initial promotion at lower concentrations followed by inhibition at higher concentrations of MLT, with the peak values for both DW and LDMC observed at 100 μM MLT in L. litseifolius seedlings (Table 1). Taken together, these results demonstrate that 100 μM is the optimal concentration for promoting leaf dry matter accumulation in L. litseifolius.

3.2. Effects of MLT on Phytohormone Profile in L. litseifolius

To determine the phytohormone balance triggered by MLT, the endogenous levels of five growth-related phytohormone classes were measured in CK and MLT100, including ABA, GA, SL, auxin, and CTK. A total of two ABA, four GA, one SL, 14 auxin, and 24 CTK hormones were detected by a UPLC-ESI-MS/MS system (Table S2). As shown in Figure 2, exogenous MLT significantly reduced the contents of both ABA and ABA glucosyl ester (ABA-GE), with decreases of 29.92% and 16.00% compared to CK, respectively (Figure 2). Nevertheless, the levels of two active GA forms (GA1 and GA3) were both enhanced by MLT, exhibiting 18.64% and 25.24% enhancements compared with CK, respectively (Figure 2). Similarly, MLT encouraged the accumulation of 5-deoxystrigol (5DS) in L. litseifolius leaves (Figure 2). Five auxins and seven CTK classes had higher contents after MLT treatment (Figure 2). IAM showed the largest increase (2.59 fold) among these auxins, followed by IAA-Gly, MEIAA, IBA, and IAA-Glc (Figure 2). Additionally, mT9G, tZOG, mTR, cZROG, K9G, and BAP7G all increased in CTKs, with cZ9G exhibiting the largest 1.56-fold increase (Figure 2).

3.3. Effects of MLT on the Photosynthetic Systems

To explore the involvement of photosynthetic systems in MLT-elicited networks, photosynthetic parameters were measured in L. litseifolius seedling leaves. All three MLT treatment groups showed notable improvements in all four primary parameters (Pn, Gs, Ci, and E) in leaves compared to CK (Figure 3a–d). Pn and Ci showed a concentration-dependent response after MLT applications, first rising and then falling with increasing MLT concentrations (Figure 3a,c). The MLT100 group had the highest Pn and Ci values, which were 1.31 (2.65 µmol m−2 s−1) and 1.45 (394.85 µmol mol−1) times greater than those of CK (Figure 3a,c). Following MLT treatment, the Gs exhibited an opposite response to the pattern shown for Pn and Ci. The MLT50 and MLT200 treatment groups showed the most noticeable changes in Gs, with values that were 1.56 and 1.61 times higher than those of the CK group (Figure 3b). Furthermore, all MLT groups exhibited comparable E enhancement of 1.47–1.59 fold compared to CK, respectively (Figure 3d).

3.4. Effects of MLT on the Chlorophyll Levels and Soluble Sugar Contents

To determine the involvement of sugar metabolism by MLT treatments, the chlorophyll levels and soluble sugar contents were also measured in L. litseifolius seedling leaves. In comparison to the control, MLT treatment considerably increased the chlorophyll SPAD values in all three treatment groups (50, 100, and 200 μM), with the MLT 100 group showing the largest augmentation by 22.53%, followed by MLT 200 (Figure 4a). Additionally, we found that only the MLT 100 group of the three MLT concentration treatments exhibited a significant rise in soluble sugars, up 28.3% in leaves compared to the control (Figure 4b).

3.5. DEGs Involved in MLT-Elicited Leaf Growth of L. litseifolius

To investigate the regulatory effect of MLT on phytohormone-related gene expression, the expression profiles of genes involved in ABA, GA, SL, auxin, and cytokinin pathways were analyzed. A total of 13 candidate DEGs associated with these hormone pathways were identified in MLT-treated groups compared with CK (Figure 5; Table S3). Regarding ABA-related genes, MLT suppressed the expression of genes AAO3 (1676/f5p0/4475) and ABA3 (3577/f2p0/3893), with decreases of 0.90- and 0.71-fold, respectively (Figure 5). On the contrary, two ABA catabolism-related CYP707As (28272/f4p0/1803 and 31093/f3p0/1750) were induced by 1.30- and 1.20-fold, respectively (Figure 5). Notably, three ABA signaling-related transcription factors ABIs (20553/f2p0/2286, 30023/f8p0/1731, and 33823/f2p0/1621) were consistently up-regulated after MLT treatments (Figure 5). For GA-related genes, MLT significantly induced the GA signaling-related genes BAK1 (20911/f3p0/2268) and PIF3 (14202/f2p0/2702), by 5.18- and 2.06-fold, respectively (Figure 5). Meanwhile, the SL receptor gene D14 (39887/f2p0/1238) was up-regulated by 2.06-fold, and CCD1 (22376/f2p0/2181), a gene putatively involved in SL biosynthesis, also exhibited a 3.41-fold increase in expression (Figure 5). Multiple auxin-responsive genes were induced by MLT, such as IAA6 (23868/f4p0/2094), SAUR36 (38817/f4p0/1255), and AUX22B (39402/f1p0/1210) (Figure 5). Auxin signaling-related genes AXR4 (27360/f7p0/1896), ARF2 (4628/f2p0/3697), and ARF6 (7906/f2p0/3273) also showed increased expression, with fold changes of 1.70, 2.98, and 1.94, respectively (Figure 5). Moreover, the CTK receptor gene AHK3 (1491/f2p0/4603) was up-regulated by 1.93-fold following MLT treatment (Figure 5).
According to the transcriptome results, exogenous MLT treatments had a significant influence on the molecular pathway in photosynthesis, with 93 DEGs participating in this KEGG pathway. We then examined 14 major up-regulated DEGs in the photosynthetic pathway compared to the CK (Figure 6; Table S4). Nine (64%) unigenes have been identified in photosystem II (PS II), including PsbB, PsbC, PsbK, PsbM, PsbS, and PsbW (Figure 6). The expression of PsbC (13861/f3p0/2721) and PsbB (31402/f7p0/1735) was more than nine times that of CK (Figure 6). Additionally, two, one, and two DEGs were found in the photosynthetic electron transport, photosystem I (PS I), and F-type ATPase sections, respectively (Figure 6). Among them, the greatest increase was found in PetH (4.14-fold), PsaB (7.79-fold), and ATPase β (3.37-fold), respectively (Figure 6). These unigenes might play crucial roles in MLT-elicited photosynthesis changes in L. litseifolius.
Additionally, in the MLT-induced leaf development process, the expression profiles of eighteen potential up-regulated unigenes implicated in starch and sucrose metabolism were examined (Figure 7; Table S5). Five WAXY unigenes were up-regulated by MLT among the starch biosynthesis-related genes, and WAXY (18818/f2p0/2391) expression levels were 9.18 times higher than CK (Figure 7). Notably, following MLT treatments, two glgC genes that encode ADP-glucose pyrophosphorylase were significantly up-regulated, with glgC (16522/f3p0/2539) showing a 99.5-fold increase over CK (Figure 7). Furthermore, in the MLT100 group, the gene otsB (33004/f3p0/1662), which codes for trehalose 6-phosphate phosphatase, was up-regulated by 9.81 times (Figure 7). We found that MLT stimulated the mobilization of carbohydrates in six members of the BGL family, with the highest expression of BGL (29948/f2p0/1732) being 181.6 times greater than CK (Figure 7). Compared to CK, two SPS and three INV unigenes were also consistently up-regulated, with the highest fold changes of 47.3 and 3.8, respectively (Figure 7). Collectively, these results suggest that MLT might trigger the specific gene expressions in starch and sucrose metabolism, potentially contributing to carbon reallocation and leaf growth in L. litseifolius.

3.6. qRT–PCR Validation of DEGs

Nine up-regulated DEGs mentioned above (CYP707A, BAK1, D14, ARF2, PsbC, PetH, ATPase, WAXY, and otsB) were chosen for qRT-PCR investigation to confirm the accuracy of the RNA-Seq data (Table S1). As shown in Figure 8, the majority of gene expression levels were strongly consistent with the RNA-seq results. Thus, the RNA-seq data could be used to further examine the molecular network of MLT-induced leaf development in L. litseifolius.

3.7. Correlation Analysis of the Above Indicators

To determine the degree of correlation between the aforementioned indicators, correlation coefficients were calculated. The growth indicator LDMC had a substantial positive association with several hormones (GA1, IAM, cZ9G, and tZOG), all photosynthetic parameters (E, Pn, Ci, and gs), SPAD, and soluble sugar, but a negative correlation with ABA and GA20. IAM showed a substantial positive link (at least p < 0.01) with all photosynthetic indicators, while ABA had a strong negative relationship (at least p < 0.001). These findings suggested that IAM and ABA could play important roles in MLT-induced leaf growth in L. litseifolius.

4. Discussion

4.1. MLT Promotes Leaf Growth in L. litseifolius

Recently, as the global incidence of diabetes has increased year after year, the development and supply of L. litseifolius (sweet tea) have garnered worldwide attention due to its natural sweetener flavor and anti-diabetic properties [26]. Despite rising market demand, large-scale production of L. litseifolius remains restricted by insufficient leaf yield. At present, the majority of studies have focused on component extraction and pharmacological activity [26,27], with little attention given to leaf development management in this species. Sun et al. [6] found that selenium-enriched yeast increased tender leaf generation by 52%, while Ye et al. [28] found that combining biochar with nitrogen, phosphate, and potassium fertilizers improved the plantlet growth and quality of L. litseifolius. However, additional research into the approach and regulatory mechanisms of leaf growth in L. litseifolius is still needed, particularly on plant growth regulators.
Over the past decade, MLT has been thoroughly investigated as a multifunctional regulator of plant growth and development [9]. Exogenous MLT application has been shown in various studies to boost biomass accumulation in various plant species, including wheat [29], tea [30], cotton [31], and potato [32]. Notably, the effects of MLT on plant growth are typically dose-dependent. For example, 0.2 mM MLT generated the optimal improvements in tea plants, whereas higher concentrations were less efficient or even inhibiting [30]. Our research revealed that foliar application of 100 μM MLT significantly enhanced leaf DW and LDMC at maximum levels, but did not affect plantlet height or FW in L. litseifolius. The distinction indicates that rather than simply improving water intake or cell elongation, MLT might primarily encourage dry matter accumulation. One possible explanation is that MLT may promote photosynthetic carbon absorption and assimilate partitioning toward leaf structural components, which is consistent with a study conducted in maize plants [33]. Additionally, based on its industrial pricing of $70–90/kg in China, the cost of MLT used in the aforementioned improvement is only $0.14–0.18 per 100 seedlings.

4.2. MLT Modulates Phytohormone Homeostasis in L. litseifolius

The growth-promoting effects of MLT are closely associated with its ability to modulate phytohormone networks [8]. As a major environmental hormone, ABA is essential in plant stress responses [34,35]. MLT and ABA generally exhibit the opposite interactions, suggesting an appropriate balance in plant stress responses and growth regulation [8,12]. Bychkov et al. [36] found that ABA and MLT inhibit each other’s signaling and metabolic gene expression in Arabidopsis. Li et al. [37] discovered that MLT pre-treatment specifically down-regulated the biosynthetic gene MdNCED3 while up-regulated the catabolic genes MdCYP707A1 and MdCYP707A2 in apple, resulting in a significant decline in ABA levels during drought. Herein, this study detected that both free and bound forms of ABA decreased in non-stressed MLT-treated L. litseifolius leaves (Figure 2). MLT also inhibited the transcriptions of AAO3 and ABA3, as well as significantly up-regulated CYP707As, which encode ABA 8′-hydroxylase (Figure 5). Under normal conditions, high ABA is not necessary for defense, but rather inhibits cell division and assimilate partitioning [38]. These results suggested that MLT may reduce ABA synthesis by promoting its oxidative breakdown through CYP707As, and make it feasible for more resources to be allocated to leaf dry matter accumulation. This interpretation is consistent with [39].
MLT also interacts positively with GA and SL metabolism [12]. Zhang et al. [40] found that MLT treatment raised GA3 levels in kiwifruit, leading to improved fruit growth. Broader metabolomic research by [41] revealed that MLT impacts hormonal crosstalk involving auxins, GAs, and jasmonates during growth and stress adaptation. Our findings are consistent with those reported. Treatment with 100 μM MLT increased active GA1 and GA3 levels by 18.6% and 25.2% in L. litseifolius, respectively (Figure 2). The increase in GAs is consistent with their reported involvement in cell division and elongation [12], which probably contributes to further leaf growth. Furthermore, this study found that the SL content significantly increased following MLT treatments (Figure 2), along with the up-regulation of CCD1 and D14, known to be involved in SL production and perception (Figure 5). SLs are generated from carotenoids and are known for their function in shoot branching and leaf growth [42]. Up-regulation of CCD1 and D14 suggests MLT may activate the carotenoid cleavage pathway, leading to SL synthesis [43]. Under the non-stress conditions utilized here, this increase may not result in the normal reduction in branching seen with nutrient limitation; rather, it may help modify source-sink connections in a way that promotes leaf development. The MLT-induced GA and SL accumulation offers a unique perspective on how MLT influences plant architecture.
MLT and IAA both share tryptophan (Trp) as the common precursor [44]. Earlier studies have typically discovered that exogenous MLT leads to a moderate increase in endogenous IAA [12], although concentration-dependent effects have also been reported [45]. According to [46], MLT up-regulates a number of genes related to signaling pathways and auxin production. In the current study, the most significant change induced by MLT was a 2.59-fold increase in IAM, an intermediate in the ‘Trp to IAA’ biosynthetic pathway [47]. Several conjugated auxin forms, including IAA-Gly, MEIAA, IBA, and IAA-Glc, showed increased levels following MLT treatment. This pattern indicates that MLT may drive flux via the IAM branch of auxin biosynthesis in L. litseifolius, rather than simply competing with IAA for Trp. Yang et al. [48] found that MLT enhances primary root growth in an IAA-dependent way, and that MLT co-regulates the majority of IAA-regulated genes. The rise in conjugated auxins probably acts as a buffer, storing sufficient active auxin to avoid disrupting development while maintaining a reserve pool for use when necessary. Notably, the endogenous levels of MLT in L. litseifolius leaves were undetectable under our experimental conditions,owing to levels below the UPLC-ESI-MS/MS quantification limit, which was consistent with the generally low basal levels of MLT observed in non-stressed plants [49,50]. This potentially explains why exogenous application of MLT is necessary to elicit the observed physiological and molecular effects. Taken together, the hormonal changes, including reduced ABA, increased GAs and SL, and altered auxin metabolism, create conditions favorable for the growth-promoting effects of MLT in L. litseifolius.

4.3. MLT Enhances Photosynthetic Capacity and Sugar Metabolism

Photosynthesis acts as the unique source of carbon needed for plant growth [51]. Numerous research studies have revealed the beneficial impact of MLT on photosynthetic systems and biomass production, such as in rice and Carya cathayensis [52,53]. In the current study, MLT enhanced photosynthetic efficiency in L. litseifolius leaves at all concentrations, potentially leading to a more significant accumulation of leaf dry matter (Figure 1 and Figure 6), consistent with previous findings [54]. Notably, this study discovered that ABA exhibited a strong negative relationship with photosynthetic indices and SPAD value, suggesting the higher stomatal conductances and CO2 influx might have occurred due to the lower ABA levels in MLT-treated L. litseifolius (Figure 4 and Figure 9). Furthermore, previous studies demonstrated that the PS II reaction center is extremely susceptible to elicitors, and MLT can regulate multiple PS II subunit genes (Psbs) in wheat and Avena sativa [14,15]. Herein, we discovered that PS II contained more than 60% of MLT-induced genes in the photosynthetic pathway, with four Psbs (three PsbCs and one PsbD) exhibiting at least five-fold increases over CK (Figure 7). These findings demonstrated that MLT improved photosynthetic efficiency primarily by enhancing PS II activity, which was similar to the earlier report [55].
Soluble sugars act both as energy sources and signaling molecules that regulate plant growth [56]. Zhang et al. [40] identified that MLT treatment raised soluble sugar levels in kiwifruit, which was accompanied by transcriptional alterations in starch and sucrose metabolism. We discovered that MLT dramatically increased soluble sugars at 100 μM, which is also the optimal concentration for leaf growth in L. litseifolius. This highlights the importance of sugar metabolism in MLT-induced leaf growth. Additionally, WAXY encodes granule-bound starch synthase, while glgC encodes ADP-glucose pyrophosphorylase, which represents the rate-limiting step in starch synthesis. We found that MLT significantly increased the expression of genes related to starch synthesis, with WAXY expression rising up to 9.18-fold and glgC expression rising up to 99.5-fold (Figure 7). Furthermore, trehalose-6-phosphate acts as a critical signal that regulates carbon allocation between the source and sink tissues [57], and the gene encoding it, otsB, is also up-regulated by 9.81-fold, which was coincident with this finding [58]. This finding shows that MLT drives a dynamic carbon flow in which the plant increases its capacity for starch storage while also mobilizing soluble sugars. This reprogramming of carbohydrate metabolism provides a molecular illustration for the increased leaf growth in L. litseifolius.

5. Conclusions

In this work, our findings suggest that foliar-applied MLT may be a cost-effective and practical strategy for increasing leaf dry matter production in L. litseifolius under normal conditions. MLT impacted growth-related phytohormone homeostasis, increasing GAs, 5DS, IAM, and cZ9G while decreasing free and bound ABA forms. In addition, MLT treatment stimulated the photosynthetic system as well as sugar biosynthesis. RNA-seq research suggests that essential DEGs involved in hormone biosynthesis and signaling (CYP707A, BAK1, D14, CCD1, and IAA6), photosynthesis (PsbC/B, PetH, PsaB, and ATPase β), and sugar metabolism (WAXY, glgC, and otsB) may be crucial in MLT-induced leaf development. Collectively, we have preliminarily illustrated the physiological effects and molecular processes of MLT in plants, laying the basis for promoting leaf production in L. litseifolius.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae12060694/s1, Table S1: Sequence of primer pairs used in qRT-PCR analysis; Table S2: Effect of melatonin on phytohormone level in L. litseifolius leaves; Table S3: DEGs associated with the biosynthesis of multiple phytohormone classes; Table S4: DEGs associated with the photosynthesis process; Table S5: DEGs associated with the sugar metabolism; Figure S1: The plantlet height in four groups before MLT treatments.

Author Contributions

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

Funding

This work was funded by Guangdong Basic and Applied Basic Research Foundation (2024A1515010516), National Natural Science Foundation of China (32301590), Forestry Science and Technology Innovational Specific Project of Guangdong Province (2023KJCX027), and Guangdong Science and Technology Program Project (2025WDZC-LKY02 and 2026WDZC-LKY01).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Shang, A.; Liu, H.-Y.; Luo, M.; Xia, Y.; Yang, X.; Li, H.-Y.; Wu, D.-T.; Sun, Q.; Geng, F.; Gan, R.-Y. Sweet Tea (Lithocarpus polystachyus Rehd.) as a New Natural Source of Bioactive Dihydrochalcones with Multiple Health Benefits. Crit. Rev. Food Sci. Nutr. 2022, 62, 917–934. [Google Scholar] [CrossRef]
  2. Flora of China; Wu, Z.Y., Raven, P.H., Hong, D.Y., Eds.; Science Press: Beijing, China; Missouri Botanical Garden Press: St. Louis, MO, USA, 1994. [Google Scholar]
  3. Liu, Y.; Liu, H.-Y.; Xia, Y.; Guo, H.; He, X.-Q.; Li, H.; Wu, D.-T.; Geng, F.; Lin, F.-J.; Li, H.-B.; et al. Screening and Process Optimization of Ultrasound-Assisted Extraction of Main Antioxidants from Sweet Tea (Lithocarpus litseifolius [Hance] Chun). Food Biosci. 2021, 43, 101277. [Google Scholar] [CrossRef]
  4. Huang, W.; Zhang, C.; Gu, Z.; Li, C.; Fang, Z.; Zeng, Z.; Zhang, Z.; Hu, B.; Chen, H.; Wu, W.; et al. Effect of Microbial Fermentation on the Sensory Characteristics and Chemical Compositions of Chinese Sweet Tea (Lithocarpus litseifolius (Hance) Chun). Food Biosci. 2022, 46, 101567. [Google Scholar] [CrossRef]
  5. Mata-Torres, G.; Andrade-Cetto, A.; Espinoza-Hernández, F. Approaches to Decrease Hyperglycemia by Targeting Impaired Hepatic Glucose Homeostasis Using Medicinal Plants. Front. Pharmacol. 2021, 12, 809994. [Google Scholar] [CrossRef]
  6. Sun, Y.; Xu, M.; Zhang, W.; He, C.; Wang, C.; Wang, H.; He, L.; Zhang, Z.; Su, L. Selenium-Enriched Yeast Enhances Dihydrochalcone Biosynthesis, Leaf Growth, and Heavy Metal Antagonism in Lithocarpus litseifolius by Physiological, Hormonal, and Transcriptional Analyses. Ind. Crops Prod. 2025, 227, 120826. [Google Scholar] [CrossRef]
  7. Nawaz, M.A.; Huang, Y.; Bie, Z.; Ahmed, W.; Reiter, R.J.; Niu, M.; Hameed, S. Melatonin: Current Status and Future Perspectives in Plant Science. Front. Plant Sci. 2016, 6, 01230. [Google Scholar] [CrossRef]
  8. Arnao, M.B.; Hernández-Ruiz, J. Melatonin as a Regulatory Hub of Plant Hormone Levels and Action in Stress Situations. Plant Biol. 2021, 23, 7–19. [Google Scholar] [CrossRef] [PubMed]
  9. Sun, C.; Liu, L.; Wang, L.; Li, B.; Jin, C.; Lin, X. Melatonin: A Master Regulator of Plant Development and Stress Responses. J. Integr. Plant Biol. 2021, 63, 126–145. [Google Scholar] [CrossRef] [PubMed]
  10. Mao, K.; Li, J.; Wu, S.; Qian, J.; Liao, Y.; Zeng, L.; Gu, D. Melatonin Treatment Promotes Cold Adaptation and Spring Growth of Tea Plants. Ind. Crops Prod. 2023, 200, 116834. [Google Scholar] [CrossRef]
  11. Mahmood, S.; Afzal, B.; Bashir, R.; Shakoor, M.B.; Nisa, Z.U.; Rizwan, M.; Awais, M.; Azeem, M.; Wahid, A.; Yong, J.W.H. Melatonin Priming Could Modulate Primary and Secondary Metabolism of Sunflower with Better Nutraceutical Value and Tolerance against Water Deficit Environment. Plant Stress 2024, 13, 100533. [Google Scholar] [CrossRef]
  12. Arnao, M.B.; Hernández-Ruiz, J. Melatonin and Its Relationship to Plant Hormones. Ann. Bot. 2018, 121, 195–207. [Google Scholar] [CrossRef]
  13. Yang, S.; Zhao, Y.; Qin, X.; Ding, C.; Chen, Y.; Tang, Z.; Huang, Y.; Reiter, R.J.; Yuan, S.; Yuan, M. New Insights into the Role of Melatonin in Photosynthesis. J. Exp. Bot. 2022, 73, 5918–5927. [Google Scholar] [CrossRef] [PubMed]
  14. Lin, S.; Song, X.-F.; Mao, H.-T.; Li, S.-Q.; Gan, J.-Y.; Yuan, M.; Zhang, Z.-W.; Yuan, S.; Zhang, H.-Y.; Su, Y.-Q.; et al. Exogenous Melatonin Improved Photosynthetic Efficiency of Photosystem II by Reversible Phosphorylation of Thylakoid Proteins in Wheat under Osmotic Stress. Front. Plant Sci. 2022, 13, 966181. [Google Scholar] [CrossRef]
  15. Varghese, N.; Alyammahi, O.; Nasreddine, S.; Alhassani, A.; Gururani, M.A. Melatonin Positively Influences the Photosynthetic Machinery and Antioxidant System of Avena Sativa during Salinity Stress. Plants 2019, 8, 610. [Google Scholar] [CrossRef] [PubMed]
  16. Dai, L.; Li, J.; Harmens, H.; Zheng, X.; Zhang, C. Melatonin Enhances Drought Resistance by Regulating Leaf Stomatal Behaviour, Root Growth and Catalase Activity in Two Contrasting Rapeseed (Brassica napus L.) Genotypes. Plant Physiol. Biochem. PPB 2020, 149, 86–95. [Google Scholar] [CrossRef]
  17. Ahmad, I.; Munsif, F.; Mihoub, A.; Jamal, A.; Saeed, M.F.; Babar, S.; Fawad, M.; Zia, A. Beneficial Effect of Melatonin on Growth and Chlorophyll Content in Wheat (Triticum aestivum L.) Grown Under Salt Stress Conditions. Positive Wirkung von Melatonin Auf Wachstum Und Chlorophyllgehalt von Weizen (Triticum aestivum L.) Unter Salzstress. Gesunde Pflanz. 2022, 74, 997–1009. [Google Scholar] [CrossRef]
  18. Zoufan, P.; Zare Bavani, M.R.; Tousi, S.; Rahnama, A. Effect of Exogenous Melatonin on Improvement of Chlorophyll Content and Photochemical Efficiency of PSII in Mallow Plants (Malva parviflora L.) Treated with Cadmium. Physiol. Mol. Biol. Plants 2023, 29, 145–157. [Google Scholar] [CrossRef]
  19. Khattak, W.A.; He, J.; Abdalmegeed, D.; Hu, W.; Wang, Y.; Zhou, Z. Foliar Melatonin Stimulates Cotton Boll Distribution Characteristics by Modifying Leaf Sugar Metabolism and Antioxidant Activities during Drought Conditions. Physiol. Plant. 2022, 174, e13526. [Google Scholar] [CrossRef] [PubMed]
  20. Guo, Y.; Zhu, J.; Liu, J.; Xue, Y.; Chang, J.; Zhang, Y.; Ahammed, G.J.; Wei, C.; Ma, J.; Li, P.; et al. Melatonin Delays ABA-Induced Leaf Senescence via H2 O2 -Dependent Calcium Signalling. Plant Cell Environ. 2023, 46, 171–184. [Google Scholar] [CrossRef]
  21. Muhammad, I.; Ahmad, S.; Shen, W. Melatonin-Mediated Molecular Responses in Plants: Enhancing Stress Tolerance and Mitigating Environmental Challenges in Cereal Crop Production. Int. J. Mol. Sci. 2024, 25, 4551. [Google Scholar] [CrossRef]
  22. Zhang, W.; Sun, Y.; Wang, H.; Xu, M.; He, C.; Wang, C.; Yu, Y.; Zhang, Z.; Su, L. Exogenous Melatonin Enhances Dihydrochalcone Accumulation in Lithocarpus litseifolius Leaves via Regulating Hormonal Crosstalk and Transcriptional Profiling. Int. J. Mol. Sci. 2024, 25, 4592. [Google Scholar] [CrossRef]
  23. Niu, Q.; Zong, Y.; Qian, M.; Yang, F.; Teng, Y. Simultaneous Quantitative Determination of Major Plant Hormones in Pear Flowers and Fruit by UPLC/ESI-MS/MS. Anal. Methods 2014, 6, 1766. [Google Scholar] [CrossRef]
  24. Buysse, J.; Merkx, R. An Improved Colorimetric Method to Quantify Sugar Content of Plant Tissue. J. Exp. Bot. 1993, 44, 1627–1629. [Google Scholar] [CrossRef]
  25. Love, M.I.; Huber, W.; Anders, S. Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [PubMed]
  26. Chen, X.-M.; Yang, W.-Q.; Wang, X.; Chen, C.; Qian, Z.-M.; Wang, S.-M.; Tang, D. Effects of Natural Dihydrochalcones in Sweet Tea (Lithocarpus polystachyus) on Diabetes: A Systematical Review and Meta-Analysis of Animal Studies. Food Funct. 2022, 13, 5899–5913. [Google Scholar] [CrossRef]
  27. Zhang, Q.; Wang, L.; Zhao, Y. An Overview of Lithocarpus polystachyus, with Dihydrochalcones as Natural-Derived Bioactive Compounds. Food Rev. Int. 2022, 39, 5934–5947. [Google Scholar] [CrossRef]
  28. Ye, Z.; Zhang, H.; Lin, X.; Huang, S.; Zou, S.; Zou, X. Effect of Biochar Using N, P, and K Fertilisers on Growth and Quality of Lithocarpus litseifolius. Agronomy 2024, 14, 728. [Google Scholar] [CrossRef]
  29. Khan, M.; Hussain, A.; Yun, B.-W.; Mun, B.-G. Melatonin: The Multifaceted Molecule in Plant Growth and Defense. Int. J. Mol. Sci. 2024, 25, 6799. [Google Scholar] [CrossRef] [PubMed]
  30. Yang, N.; Han, M.-H.; Teng, R.-M.; Yang, Y.-Z.; Wang, Y.-H.; Xiong, A.-S.; Zhuang, J. Exogenous Melatonin Enhances Photosynthetic Capacity and Related Gene Expression in A Dose-Dependent Manner in the Tea Plant (Camellia sinensis (L.) Kuntze). Int. J. Mol. Sci. 2022, 23, 6694. [Google Scholar] [CrossRef]
  31. Ahmad, I.; Guanglong, Z.; Zhou, G.; Younas, M.U.; Yan, X. Melatonin and Mepiquat Chloride Impact on Cotton Growth, Physiological and Yield at Different Growth Stages. Ind. Crops Prod. 2025, 226, 120702. [Google Scholar] [CrossRef]
  32. Liu, S.; Wang, Y.; Wang, X.; Zhang, X.; Li, Y.; Chen, Y.; Yang, J.; Liu, L.; Zhai, X. Integrative WGCNA Analysis Uncovers the Molecular Framework of Melatonin-Mediated Drought Stress Mitigation in Potato. BMC Plant Biol. 2026, 26, 477. [Google Scholar] [CrossRef] [PubMed]
  33. Zhao, C.; Guo, H.; Wang, J.; Wang, Y.; Zhang, R. Melatonin Enhances Drought Tolerance by Regulating Leaf Stomatal Behavior, Carbon and Nitrogen Metabolism, and Related Gene Expression in Maize Plants. Front. Plant Sci. 2021, 12, 779382. [Google Scholar] [CrossRef]
  34. Fu, J.; Wu, Y.; Miao, Y.; Xu, Y.; Zhao, E.; Wang, J.; Sun, H.; Liu, Q.; Xue, Y.; Xu, Y.; et al. Improved Cold Tolerance in Elymus Nutans by Exogenous Application of Melatonin May Involve ABA-Dependent and ABA-Independent Pathways. Sci. Rep. 2017, 7, 39865. [Google Scholar] [CrossRef]
  35. Singh, A.; Banerjee, A.; Roychoudhury, A. Fluoride Tolerance in Rice Is Negatively Regulated by the ‘Stress-Phytohormone’ Abscisic Acid (ABA), but Promoted by ABA-Antagonist Growth Regulators, Melatonin, and Gibberellic Acid. Protoplasma 2022, 259, 1331–1350. [Google Scholar] [CrossRef] [PubMed]
  36. Bychkov, I.; Kudryakova, N.; Pojidaeva, E.S.; Doroshenko, A.; Shitikova, V.; Kusnetsov, V. ABA and Melatonin: Players on the Same Field? Int. J. Mol. Sci. 2024, 25, 12266. [Google Scholar] [CrossRef]
  37. Li, C.; Tan, D.-X.; Liang, D.; Chang, C.; Jia, D.; Ma, F. Melatonin Mediates the Regulation of ABA Metabolism, Free-Radical Scavenging, and Stomatal Behaviour in Two Malus Species under Drought Stress. J. Exp. Bot. 2015, 66, 669–680. [Google Scholar] [CrossRef]
  38. Humplík, J.F.; Bergougnoux, V.; Van Volkenburgh, E. To Stimulate or Inhibit? That Is the Question for the Function of Abscisic Acid. Trends Plant Sci. 2017, 22, 830–841. [Google Scholar] [CrossRef] [PubMed]
  39. Chen, M.; Yang, Y.; Cao, X.; Wang, Y.; Liang, X.; Wei, J.; Xin, Z.; Li, H. Auxin Response Factor 4 Regulates the ABA Catabolism Gene CYP707A2 to Reduce the Accumulation of ABA and Inhibit Anthocyanin Synthesis in Malus “Pinkspire” Fruits. J. Agric. Food Chem. 2025, 73, 24701–24712. [Google Scholar] [CrossRef] [PubMed]
  40. Zhang, X.; Wang, Y.; Wang, T.; Wen, X.; Lv, Z.; Li, Y.; Liang, D.; Xia, H. Exogenous Melatonin Affects Kiwifruit Growth and Quality through Hormone Signaling Pathway. Plant Physiol. Biochem. PPB 2025, 228, 110277. [Google Scholar] [CrossRef]
  41. Bargunam, S.; Roy, R.; Shetty, D.; Amisha, S.H.; Shukla, V.S.; Babu, V.S. Melatonin-Governed Growth and Metabolome Divergence: Circadian and Stress Responses in Key Plant Species. Plant Physiol. Biochem. PPB 2025, 221, 109635. [Google Scholar] [CrossRef] [PubMed]
  42. Gomez-Roldan, V.; Fermas, S.; Brewer, P.B.; Puech-Pagès, V.; Dun, E.A.; Pillot, J.-P.; Letisse, F.; Matusova, R.; Danoun, S.; Portais, J.-C.; et al. Strigolactone Inhibition of Shoot Branching. Nature 2008, 455, 189–194. [Google Scholar] [CrossRef]
  43. Hao, J.; Yang, Y.; Futrell, S.; Kelly, E.A.; Lorts, C.M.; Nebie, B.; Runo, S.; Yang, J.; Alvarez, S.; Lasky, J.R.; et al. CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase (CCD) Genes in Sorghum Alters Strigolactone Biosynthesis and Plant Biotic Interactions. Phytobiomes J. 2023, 7, 339–351. [Google Scholar] [CrossRef]
  44. Arnao, M.B.; Hernández-Ruiz, J. Melatonin Promotes Adventitious- and Lateral Root Regeneration in Etiolated Hypocotyls of Lupinus albus L. J. Pineal Res. 2007, 42, 147–152. [Google Scholar] [CrossRef]
  45. Wen, D.; Gong, B.; Sun, S.; Liu, S.; Wang, X.; Wei, M.; Yang, F.; Li, Y.; Shi, Q. Promoting Roles of Melatonin in Adventitious Root Development of Solanum lycopersicum L. by Regulating Auxin and Nitric Oxide Signaling. Front. Plant Sci. 2016, 7, 718. [Google Scholar] [CrossRef]
  46. Wei, W.; Tao, J.-J.; Yin, C.-C.; Chen, S.-Y.; Zhang, J.-S.; Zhang, W.-K. Melatonin Regulates Gene Expressions through Activating Auxin Synthesis and Signaling Pathways. Front. Plant Sci. 2022, 13, 1057993. [Google Scholar] [CrossRef]
  47. Pollmann, S.; Neu, D.; Weiler, E.W. Molecular Cloning and Characterization of an Amidase from Arabidopsis Thaliana Capable of Converting Indole-3-Acetamide into the Plant Growth Hormone, Indole-3-Acetic Acid. Phytochemistry 2003, 62, 293–300. [Google Scholar] [CrossRef]
  48. Yang, L.; You, J.; Li, J.; Wang, Y.; Chan, Z. Melatonin Promotes Arabidopsis Primary Root Growth in an IAA-Dependent Manner. J. Exp. Bot. 2021, 72, 5599–5611. [Google Scholar] [CrossRef] [PubMed]
  49. Hardeland, R. Melatonin in Plants—Diversity of Levels and Multiplicity of Functions. Front. Plant Sci. 2016, 7, 00198. [Google Scholar] [CrossRef] [PubMed]
  50. Shi, H.; Tan, D.-X.; Reiter, R.J.; Ye, T.; Yang, F.; Chan, Z. Melatonin Induces Class A1 Heat-Shock Factors (HSFA1s) and Their Possible Involvement of Thermotolerance in Arabidopsis. J. Pineal Res. 2015, 58, 335–342. [Google Scholar] [CrossRef]
  51. Evans, J.R. Improving Photosynthesis. Plant Physiol. 2013, 162, 1780–1793. [Google Scholar] [CrossRef]
  52. Yan, F.; Zhang, J.; Li, W.; Ding, Y.; Zhong, Q.; Xu, X.; Wei, H.; Li, G. Exogenous Melatonin Alleviates Salt Stress by Improving Leaf Photosynthesis in Rice Seedlings. Plant Physiol. Biochem. 2021, 163, 367–375. [Google Scholar] [CrossRef]
  53. Sharma, A.; Wang, J.; Xu, D.; Tao, S.; Chong, S.; Yan, D.; Li, Z.; Yuan, H.; Zheng, B. Melatonin Regulates the Functional Components of Photosynthesis, Antioxidant System, Gene Expression, and Metabolic Pathways to Induce Drought Resistance in Grafted Carya Cathayensis Plants. Sci. Total Environ. 2020, 713, 136675. [Google Scholar] [CrossRef]
  54. Ralmi, N.H.A.A.; Khandaker, M.M.; Mohd, K.S.; Majrashi, A.; Fallatah, A.M.; Badaluddin, N.A.; Yusoff, N.; Mahmud, K.; Saifuddin, M.; Osman, N.; et al. Influence of Rhizopheric H2O2 on Growth, Mineral Absorption, Root Anatomy and Nematode Infection of Ficus deltoidea. Agronomy 2021, 11, 704. [Google Scholar] [CrossRef]
  55. Wan, J.; Zhang, P.; Wang, R.; Sun, L.; Ju, Q.; Xu, J. Comparative Physiological Responses and Transcriptome Analysis Reveal the Roles of Melatonin and Serotonin in Regulating Growth and Metabolism in Arabidopsis. BMC Plant Biol. 2018, 18, 362. [Google Scholar] [CrossRef] [PubMed]
  56. Rolland, F.; Baena-Gonzalez, E.; Sheen, J. Sugar Sensing and Signaling in Plants: Conserved and Novel Mechanisms. Annu. Rev. Plant Biol. 2006, 57, 675–709. [Google Scholar] [CrossRef]
  57. Figueroa, C.M.; Lunn, J.E. A Tale of Two Sugars: Trehalose 6-Phosphate and Sucrose. Plant Physiol. 2016, 172, 7–27. [Google Scholar] [CrossRef] [PubMed]
  58. Supriya, L.; Dake, D.; Muthamilarasan, M.; Padmaja, G. Tuning the Circadian Clock and Sugar Metabolism: Divergent Roles of Adequate and Excessive Melatonin under Salt Stress in Upland Cotton. Plant Physiol. Biochem. PPB 2025, 227, 110132. [Google Scholar] [CrossRef] [PubMed]
Horticulturae 12 00694 g001
Figure 1. The seedling phenotypes before and after MLT treatments in L. litseifolius.
Figure 1. The seedling phenotypes before and after MLT treatments in L. litseifolius.
Horticulturae 12 00694 g001
Horticulturae 12 00694 g002
Figure 2. The effects of MLT on the levels of growth-related phytohormones in L. litseifolius. The heat graph shows the amounts of five phytohormone classes in the leaves after the treatment with water (CK) and MLT at 100 μM (MLT 100). The values (ng g−1) were log2 transformed.
Figure 2. The effects of MLT on the levels of growth-related phytohormones in L. litseifolius. The heat graph shows the amounts of five phytohormone classes in the leaves after the treatment with water (CK) and MLT at 100 μM (MLT 100). The values (ng g−1) were log2 transformed.
Horticulturae 12 00694 g002
Horticulturae 12 00694 g003
Figure 3. Effect of MLT on the photosynthetic parameters in L. litseifolius. The photosynthetic rate (Pn, (a)), stomatal conductance (Gs, (b)), intercellular CO2 concentration (Ci, (c)) and transpiration rate (E, (d)) in seedling leaves following 10 days of final treatments at 0 (CK), 50 (MLT 50), 100 (MLT 100) and 200 μM (MLT 200). Values are means ± SDs. *, ***, **** indicated significant (p < 0.05, p < 0.001 and p < 0.0001) differences compared to CK, respectively.
Figure 3. Effect of MLT on the photosynthetic parameters in L. litseifolius. The photosynthetic rate (Pn, (a)), stomatal conductance (Gs, (b)), intercellular CO2 concentration (Ci, (c)) and transpiration rate (E, (d)) in seedling leaves following 10 days of final treatments at 0 (CK), 50 (MLT 50), 100 (MLT 100) and 200 μM (MLT 200). Values are means ± SDs. *, ***, **** indicated significant (p < 0.05, p < 0.001 and p < 0.0001) differences compared to CK, respectively.
Horticulturae 12 00694 g003
Horticulturae 12 00694 g004
Figure 4. Effect of MLT on the chlorophyll levels and soluble sugar contents in L. litseifolius. The SPAD value (a) and soluble sugar contents (b) in seedling leaves following 10 days of final treatments at 0 (CK), 50 (MLT 50), 100 (MLT 100) and 200 μM (MLT 200). Values are means ± SDs *, ****, and ns indicated significant (p < 0.05 and p < 0.0001) and no significant differences compared to CK, respectively.
Figure 4. Effect of MLT on the chlorophyll levels and soluble sugar contents in L. litseifolius. The SPAD value (a) and soluble sugar contents (b) in seedling leaves following 10 days of final treatments at 0 (CK), 50 (MLT 50), 100 (MLT 100) and 200 μM (MLT 200). Values are means ± SDs *, ****, and ns indicated significant (p < 0.05 and p < 0.0001) and no significant differences compared to CK, respectively.
Horticulturae 12 00694 g004
Horticulturae 12 00694 g005
Figure 5. Expression profiles of MLT-induced phytohormone-related genes in L. litseifolius. The heat graph shows the expression profiles of up-regulated DEGs related to the phytohormone pathway in the MLT 100 group over CK. The FPKM values were log2 transformed.
Figure 5. Expression profiles of MLT-induced phytohormone-related genes in L. litseifolius. The heat graph shows the expression profiles of up-regulated DEGs related to the phytohormone pathway in the MLT 100 group over CK. The FPKM values were log2 transformed.
Horticulturae 12 00694 g005
Horticulturae 12 00694 g006
Figure 6. Expression profiles of photosynthesis-related genes in L. litseifolius. (a): The heat graph shows the expression profiles of up-regulated DEGs related to the photosynthesis pathway in the MLT 100 group over CK. The FPKM values were log2 transformed. (b): Metabolic pathway map of the photosynthesis system. The white words in red solid boxes represent the up-regulated unigenes from MLT 100 treatments. The red dotted boxes represent the four major parts of the photosynthesis system.
Figure 6. Expression profiles of photosynthesis-related genes in L. litseifolius. (a): The heat graph shows the expression profiles of up-regulated DEGs related to the photosynthesis pathway in the MLT 100 group over CK. The FPKM values were log2 transformed. (b): Metabolic pathway map of the photosynthesis system. The white words in red solid boxes represent the up-regulated unigenes from MLT 100 treatments. The red dotted boxes represent the four major parts of the photosynthesis system.
Horticulturae 12 00694 g006
Horticulturae 12 00694 g007
Figure 7. Expression levels of genes related to sugar metabolism pathway under MLT treatment. The heat graph shows the expression profiles of up-regulated DEGs related to the starch and sucrose metabolism in the MLT 100 group over CK. The FPKM values were log2 transformed.
Figure 7. Expression levels of genes related to sugar metabolism pathway under MLT treatment. The heat graph shows the expression profiles of up-regulated DEGs related to the starch and sucrose metabolism in the MLT 100 group over CK. The FPKM values were log2 transformed.
Horticulturae 12 00694 g007
Horticulturae 12 00694 g008
Figure 8. qRT–PCR validation of candidate key DEGs from RNA-seq. The blue and red columns show the expression levels of genes (MLT vs. CK) based on RNA-seq data and qRT-PCR measurements. Values are means ± SDs.
Figure 8. qRT–PCR validation of candidate key DEGs from RNA-seq. The blue and red columns show the expression levels of genes (MLT vs. CK) based on RNA-seq data and qRT-PCR measurements. Values are means ± SDs.
Horticulturae 12 00694 g008
Horticulturae 12 00694 g009
Figure 9. Heatmap of correlation analysis between indicators under MLT treatment. The color of the square represents the correlation coefficient value. *, **, ***, and **** denote the significant at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 level, respectively.
Figure 9. Heatmap of correlation analysis between indicators under MLT treatment. The color of the square represents the correlation coefficient value. *, **, ***, and **** denote the significant at p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 level, respectively.
Horticulturae 12 00694 g009
Table 1. Effect of MLT on leaf growth of L.litseifolius seedlings. Different letters besides data denote significant differences (p < 0.05) among four MLT treatment groups.
Table 1. Effect of MLT on leaf growth of L.litseifolius seedlings. Different letters besides data denote significant differences (p < 0.05) among four MLT treatment groups.
MLT (μM)Plant Height (cm)FW (g)DW (g)LDMC (%)
022.80 ± 0.52 b4.92 ± 0.42 a2.28 ± 0.06 c46.38 ± 0.51 c
5024.10 ± 0.43 a5.59 ± 0.24 a2.95 ± 0.24 a52.78 ± 1.21 b
10023.05 ± 0.35 b5.38 ± 0.30 a3.05 ± 0.18 a56.66 ± 1.25 a
20023.35 ± 0.28 b4.94 ± 0.48 a2.56 ± 0.12 b51.89 ± 0.89 b
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, H.; Zhang, W.; Xu, M.; Su, L. Melatonin-Induced Leaf Growth in Lithocarpus litseifolius: A Synergistic Interplay Among Hormone Homeostasis, Photosynthetic Enhancement, and Transcriptional Regulation. Horticulturae 2026, 12, 694. https://doi.org/10.3390/horticulturae12060694

AMA Style

Wang H, Zhang W, Xu M, Su L. Melatonin-Induced Leaf Growth in Lithocarpus litseifolius: A Synergistic Interplay Among Hormone Homeostasis, Photosynthetic Enhancement, and Transcriptional Regulation. Horticulturae. 2026; 12(6):694. https://doi.org/10.3390/horticulturae12060694

Chicago/Turabian Style

Wang, Huan, Wenlong Zhang, Mingfeng Xu, and Lingye Su. 2026. "Melatonin-Induced Leaf Growth in Lithocarpus litseifolius: A Synergistic Interplay Among Hormone Homeostasis, Photosynthetic Enhancement, and Transcriptional Regulation" Horticulturae 12, no. 6: 694. https://doi.org/10.3390/horticulturae12060694

APA Style

Wang, H., Zhang, W., Xu, M., & Su, L. (2026). Melatonin-Induced Leaf Growth in Lithocarpus litseifolius: A Synergistic Interplay Among Hormone Homeostasis, Photosynthetic Enhancement, and Transcriptional Regulation. Horticulturae, 12(6), 694. https://doi.org/10.3390/horticulturae12060694

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop