Root-Specific Overexpression of the CmDUF239-1 Gene Enhances Heat Tolerance in Melon Seedlings by Upregulating Antioxidant Enzymes Activities, Proline Content, and Expression of Heat Shock Protein-Related Genes

Abstract

High temperature stress is a critical factor affecting the growth and yield of melons (Cucumis melo L.), and improving heat tolerance is therefore crucial for stable production. While the overexpression of the CmDUF239-1 gene is known to improve salt tolerance in melons, its impact on heat tolerance remains unexplored. The role of the CmDUF239-1 gene in enhancing heat tolerance and its underlying mechanisms was investigated in this study. Melon seedlings overexpressing CmDUF239-1 (OEDUF239-1), generated via root transformation, exhibited significantly lower reductions in fresh and dry mass under heat stress compared to controls, indicating enhanced heat tolerance. One day post-stress, antioxidant enzyme activities (SOD, POD, CAT, APX, and GR) increased significantly in OEDUF239-1, while malondialdehyde (MDA) levels decreased. Additionally, proline content and the activity of its synthesizing enzyme (P5CS) rose, whereas the activity of proline dehydrogenase (ProDH) dropped. Transcriptomic and qPCR analyses revealed that CmDUF239-1 overexpression upregulated antioxidant enzyme-related genes (e.g., CmCSD1, CmPOD1) and proline-related genes (e.g., CmP5CS), as well as Heat Shock Protein (HSP) genes (e.g., CmHSP17.6II, CmHSP18.2). In summary, the enhancement of heat tolerance in melon by the CmDUF239-1 gene was mediated through the upregulation of genes involved in antioxidant defense and proline metabolism, together with increased accumulation of HSPs, providing a mechanistic basis for heat-resilient breeding programs.

1. Introduction

As global warming intensifies, heat stress has emerged as a critical environmental factor that severely constrains plant growth and agricultural production [1]. Heat stress exerts detrimental effects on plants through multiple pathways. Firstly, elevated temperatures can induce protein denaturation and inactivation within plant cells, thereby disrupting normal enzymatic reactions [2]. Secondly, under high-temperature stress, the overaccumulation of reactive oxygen species (ROS) generates oxidative stress, which disrupts membrane integrity and permeability and negatively impacts cellular structure and function [3]. Moreover, heat stress significantly disrupts key physiological processes in plants, such as photosynthesis, respiration, and water balance. This disruption results in reduced photosynthetic efficiency, accelerated respiratory consumption, and ultimately impacts dry matter accumulation and yield formation [4,5,6].

Rising global temperatures impose substantial stress on plant growth and crop yields. To survive under such conditions, plants have gradually developed a range of long-term adaptive strategies that enhance their tolerance to high-temperature environments [7]. These mechanisms include the activation of antioxidant systems [such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)], the accumulation of osmotic regulatory substances (such as proline and betaine), and the synthesis and expression of Heat Shock Proteins (HSPs). The accumulation of HSPs enables plants to tolerate elevated temperatures by protecting proteins from thermal damage and ensuring the stability of the intracellular protein environment [8]. In specific crops, these adaptive mechanisms have been observed to confer significant heat tolerance. For instance, in potatoes, exogenous sucrose protects seedlings from heat stress by enhancing enzymatic antioxidant systems (SOD, POD, CAT, APX, and GR), reducing ROS accumulation, and maintaining membrane integrity [9]. Melatonin (MT) treatment in wheat strengthens the enzymatic antioxidant system [SOD, CAT, APX, GR, monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR)] and elevates non-enzymatic antioxidant levels. As a result, ROS accumulation and membrane damage are minimized, enhancing heat tolerance and stabilizing cellular osmotic balance in wheat seedlings [10]. In tomatoes, proline alleviates the impacts of heat stress by enhancing temperature resistance through improved morphological and physiological parameters [11]. In pumpkins, variations in the expressions of HSP genes (HSP70 and HSP70B) are closely associated with the heat stress resistance of different pumpkin varieties [12]. In rice, thermotolerance is established through the action of HSPs, which upregulate genes such as OsHsp16.9, OsHsp17.7, OsHsp18, OsHsp70, and OsHsp100 in conjunction with heat stress transcription factors including OsHsfA2a [13]. Similarly, in tomatoes, the regulation of HSP gene SlHSP21.5A expression enhances membrane stability and antioxidant capacity under high-temperature stress, thereby improving heat stress tolerance [14].

The DUF (Domain of Unknown Function) protein family is widely present in plants, although the functions of its structural domains have not been fully elucidated [15,16]. Recent evidence suggests that members of this family are involved in plant adaptation to abiotic stress, where they act in concert to control ROS levels and regulate osmotic balance [17]. Abiotic stresses such as salinity, drought, and oxidative conditions can compromise cell membrane stability and trigger ROS bursts. DUF proteins primarily alleviate damage through two main pathways: first, by enhancing the activity of antioxidant enzymes to eliminate excessive ROS; and second, by promoting the accumulation of osmotic regulatory substances, such as proline, to maintain water balance [18]. In rice, OsDUF2488 mediates drought tolerance through interaction with the peroxidase OsPrx1.1, which jointly regulates ROS metabolism [19]. Similarly, OsEP, which harbors a DUF-4057 domain, confers drought resistance by improving root architecture and antioxidant capacity via gene expression regulation [20]. In soybean, overexpression of GmDUF4228-70 significantly increases proline content under drought stress, enhancing its stress resistance [21]. In melon (Cucumis melo L.), CmDUF239-1 has been shown to participate in salt stress responses [22], suggesting it may engage in heat tolerance networks through similar mechanisms.

Rising global temperatures have made abiotic stresses, such as heat stress [23,24,25] and drought stress [26,27], significant factors restricting melon growth and yield, an economically important crop. Despite the known importance of heat tolerance in melons, the specific molecular mechanisms underlying this trait remain poorly understood. While recent studies have highlighted the role of antioxidant enzymes, proline metabolism, and HSPs in plant responses to heat stress, the specific contributions of these pathways in melon remain unexplored. Moreover, the CmDUF239-1 gene, previously shown to increase salt tolerance, has not been studied in relation to heat stress. Given its role in salt stress responses and the similar ROS and osmotic imbalance caused by heat stress, CmDUF239-1 is hypothesized to enhance heat tolerance in melon seedlings via the upregulation of genes associated with antioxidant defense, proline metabolism, and the accumulation of HSPs. To validate the hypothesis, the present study aims to investigate the role of the CmDUF239-1 gene in enhancing heat tolerance in melon seedlings, clarifies the molecular basis of its action, and examines the physiological impact of CmDUF239-1 overexpression under high-temperature conditions.

2. Materials and Methods

2.1. Creation of CmDUF239-1 Overexpressing Melon Seedlings

Transient expression was successfully induced through the Agrobacterium-mediated hairy root transformation [2]. The melon variety ‘Xizhou Honey No. 17’ used in this experiment was purchased from Hebei Liertian Seed Industry Co., Ltd. Initially, the melon seeds were sterilized in pure water at 55 °C for 15 min, followed by a 6-h soaking at 25 °C. The seeds were then laid out on a Petri dish (10 cm in diameter) lined with three layers of moist filter paper and placed in an incubator set to 28 °C for germination. After seed germination, seedlings were transferred into a 50-cell tray and maintained in a growth chamber under controlled conditions: a day temperature of 30 °C, a night temperature of 20 °C, a light intensity of 250 μmol m⁻² s⁻¹, and a photoperiod of 12 h light/12 h dark. The melon seedlings were cultivated in an LED plant growth chamber (ZRX-460, Ningbo Kesheng Laboratory Instrument Co., Ltd., Ningbo, Zhejiang, China).

Next, the overexpression vector for CmDUF239-1 (MELO3C022991) was constructed according to the methods previously described by Peng et al. [28]. The pKSE403 plasmid, carrying the DsRed fluorescent marker, was digested with XabI and SacI to enable insertion of the CmDUF239-1 gene under the 35S promoter. The vector was subsequently transformed into Escherichia coli strain 5α. Following this, transformation of the empty pKSE403 plasmid and the pKSE403 plasmid containing the CmDUF239-1 coding sequence into Agrobacterium tumefaciens K599 was performed according to the instructions provided by K599 (Weidi, Shanghai, China). The Agrobacterium tumefaciens strain K599 used in this study was purchased from Weidi Biotechnology Co., Ltd., Shanghai, China. Competent Agrobacterium K599 cells were thawed on ice, after which 2 μL of plasmid DNA (50 ng/μL) was added and mixed gently to avoid bubble formation. The mixture was subjected to a heat shock at 42 °C for 90 s, followed by an ice bath for 2–3 min. Subsequently, 900 μL of antibiotic-free TY liquid medium (5 g/L tryptone, 3 g/L yeast extract, 0.5 g/L MgSO₄·7H₂O, pH 7.0) was added, and the cells were incubated at 30 °C with shaking at 200 rpm for 1 h to allow resistance marker expression. A volume of 50 μL of the culture was plated onto TY solid medium containing 50 μg/mL kanamycin and 50 μg/mL streptomycin, and incubated at 30 °C in the dark for 48–72 h to obtain single colonies. For transient transformation of melon cotyledons, positive clones were cultured in TY medium with streptomycin and kanamycin for 14 h, centrifuged at 4000× g rpm for 8 min, and resuspended in MS medium (0.1% sucrose) with 200 μM acetosyringone to an OD600 of 0.6. Melon seedlings with flattened cotyledons were obliquely cut 2–3 cm from the hypocotyl and immersed in bacterial suspension for 30 min for infection. Seedlings were placed in a culture box containing vermiculite and MS medium and incubated at 23 °C in darkness for 4–5 days to detect red fluorescence. Afterward, seedlings were moved to a tray with vermiculite supplemented with 1/2 Hoagland nutrient solution, and fluorescence was evaluated ten days following infection. The DsRed gene encoded a red fluorescent protein that could be visualized under blue light illumination (450–490 nm) with red glasses to filter out the excitation light. The blue light flashlight and red glasses were both produced by Orange Purple Lighting & Optoelectronics Co., Ltd. (Model: 12W Red Fluorescence RFP, Shenzhen, Guangdong, China). Every five days, roots lacking red fluorescence were discarded, and only fluorescent roots were maintained.

To confirm overexpression of CmDUF239-1, red fluorescent roots of melon seedlings with CmDUF239-1 overexpression in roots were analyzed by qRT-PCR. Verified seedlings with CmDUF239-1 overexpression in roots were then transplanted at the one-leaf, one-heart stage into hydroponic cups (90 mm top diameter, 57 mm bottom diameter, 135 mm height) containing 400 mL of half-strength modified Hoagland solution. The nutrient solution was formulated with 1 mM MgSO₄·7H₂O, 4 mM CaCl₂, 60 mM KNO₃, 0.5 mM Ca(H₂PO₄)₂·H₂O, and 74.93 mg/L of trace elements solid produced by Coolaber Company (Beijing, China) (DZPM0059), with pH adjusted to 6.5 using 1 M KOH [28].

2.2. Heat Stress Treatment

Upon reaching the one-leaf, one-heart stage, melon seedlings were subjected to either normal temperature conditions (control) or heat stress treatment. The seedlings were placed in an incubator, with the control group set at 30 °C during the day and 20 °C at night (12 h each). For the heat stress treatment, the temperatures were set at 45 °C during the day and 35 °C at night (12 h each) [29]. Each treatment was conducted with three repetitions, with six seedlings per repetition. After 24 h of heat stress, the first early sampling was performed. A portion of the root samples from the melon plants was stored at −80 °C for subsequent transcriptome analysis. On the fourth day of heat stress, random sampling was conducted for phenotypic photography.

2.3. Measurement of Phenotypic Indicators

On day 4 of the heat stress treatment, the shoots and roots of the melon plants were collected to measure fresh mass and dry mass using an electronic analytical balance (LICHEN, Model: LA-FC, Shanghai, China). To measure dry mass, samples were placed in an oven at 80 °C and dried until a constant weight was obtained [30].

2.4. Measurement of Physiological Indicators

On day 4 of the heat stress treatment, the REC of the leaves and roots of the melon seedlings was measured. For REC determination, 0.1 g of plant leaves or roots was weighed, rinsed three times with distilled water, and dried on filter paper to remove surface moisture. The samples were then placed in a 15 mL centrifuge tube, to which 5 mL of pure water was added. After vacuum treatment of the mixture at 0.8 MPa for 20 min, leachate electrical conductivity (R1) was determined using a DDSJ-308F conductivity meter (Leici, Shanghai, China). The samples were subsequently boiled for 30 min, cooled to room temperature, mixed thoroughly, and leachate conductivity (R2) was measured again. The relative conductivity was calculated as follows: Relative Conductivity = (R1/R2) × 100% [31].

On day 1 of the heat stress treatment, the content of malondialdehyde (MDA) and the activities of SOD, POD, CAT, APX, and GR were measured in the roots of melon plants. The assays were conducted using kits from Aidi Biological Co., Ltd. (Yangzhou, Jiangsu, China), with the following catalog numbers: MDA (ADS-W-YH002), SOD (ADS-W-KY011-48), POD (ADS-W-YH002), CAT (ADS-W-KY002-48), APX (ADS-F-VC005), and GR (ADS-W-FM029-48) [32,33]. For each measurement, three biological replicates were used, and samples were randomly selected from different plants to ensure representativeness.

Approximately 0.1 g of melon root tissue was weighed and homogenized in 1 mL of extraction buffer on ice. The homogenate was then centrifuged at 12,000× g rpm for 10 min at 4 °C to obtain the supernatant for subsequent analysis. For MDA content, 200 μL of the supernatant was mixed with 300 μL of the working solution in an EP tube and incubated in a water bath at 90–95 °C for 30 min. The cooled mixture was centrifuged at 12,000× g rpm for 10 min at 25 °C. Absorbance readings were taken at 532 nm and 600 nm from the supernatant, and ΔA (A532 − A600) was calculated. MDA content (nmol/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ ÷ V). The parameters are defined as follows: ΔA is the absorbance difference; ε is the molar extinction coefficient of MDA (155 × 10³ L/mol/cm); d is the light path length of the cuvette (0.5 cm); V₂ is the total reaction volume comprising the sample and working solution (5 × 10⁻⁴ L); V₁ is the volume of sample extract added to the reaction system (0.2 mL); V is the total volume of the sample extract (1 mL); and W is the sample weight (g). The supernatant was used directly to assess SOD activity. In a 96-well plate, the following reagents were added sequentially: Reagent 1 (70 μL), Reagent 2 (20 μL), sample (20 μL), Reagent 3 (10 μL), and Reagent 4 (80 μL). The reaction mixture was incubated in the dark at 25 °C for 30 min, and absorbance was measured at 450 nm. SOD activity (U/g fresh weight) was calculated using the formula: SOD activity (U/g fresh weight) = [Inhibition Percentage ÷ (1 − Inhibition Percentage) × V₂] ÷ (W × V₁ ÷ V) × D, which simplifies to 10 × Inhibition Percentage ÷ (1 − Inhibition Percentage) ÷ W × D. The parameters are defined as: V is the total volume of extract added (1 mL); V₁ is the volume of sample extract added to the reaction system (0.02 mL); V₂ is the total reaction volume (0.2 mL); D is the sample dilution factor (1 for undiluted samples); W is the sample weight (g); and Inhibition Percentage represents the percentage inhibition of the reaction. For POD activity, the supernatant was used directly. In a 96-well plate, the following reagents were added sequentially: sample (10 μL), Reagent 1 (40 μL), Reagent 2 (140 μL), and Reagent 3 (10 μL). The absorbance (A1) was immediately read at 470 nm, followed by a second reading (A2) after 1 min. Peroxidase (POD) activity was calculated according to the kit manufacturer’s protocol using the formula: POD activity (U/g fresh weight) = [ΔA ÷ (W × V₁ ÷ V) ÷ 1 ÷ T] × D, which simplifies to 100 × ΔA ÷ W × D. The parameters are defined as follows: ΔA is the change in absorbance per minute; V is the total volume of extraction buffer added (1 mL); V₁ is the volume of sample extract added to the reaction (0.01 mL); T is the reaction time (1 min); W is the sample weight (g); and D is the sample dilution factor (1 if undiluted). For the CAT activity, the supernatant was used directly. In an EP tube, the following reagents were added sequentially: sample (10 μL), Reagent 1 (70 μL), and Reagent 2 (20 μL). Samples were allowed to react at 25 °C for 5 min. Reagent 3 (100 μL) was added for color development, followed by Reagent 1 (900 μL) and Reagent 4 (290 μL). Absorbance at 510 nm was recorded after an additional 5 min reaction at room temperature (25 °C). The absorbance difference ΔA was calculated, and catalase (CAT) activity was determined according to the kit’s formula: CAT activity (U/g fresh weight) = {[(ΔA + 0.0135) ÷ 0.1413] ÷ (W × V₁ ÷ V) ÷ T} × D. The parameters are defined as follows: V is the total extraction volume (1 mL); V₁ is the sample volume added to the reaction (0.01 mL); T is the total reaction time (5 min); W is the sample weight (g); and D is the dilution factor (1 if undiluted). For APX activity, the supernatant was used directly. In a 96-well plate, the sample, extraction buffer, Reagent 1, Reagent 2, and Reagent 3 were added sequentially and mixed. The absorbance (A1) was read at 290 nm, followed by a second reading (A2) after a certain incubation period. ΔA (A2 − A1) was calculated, and APX activity was determined using the kit’s formula. APX activity (U/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁶] ÷ (W × V₁ ÷ V) ÷ T. Parameter definitions are as follows: ε is the molar extinction coefficient of AsA at 290 nm (2.8 × 10³ L/mol/cm); d is the cuvette light path length (1 cm); W is the sample mass (g); V is the total extraction volume (1 mL); V₁ is the volume of supernatant added to the reaction system (0.06 mL, equivalent to 60 μL); V₂ is the total reaction system volume (1 × 10⁻³ L, equivalent to 1000 μL); and T is the catalytic reaction time (5 min). For determination of GR activity, the supernatant was used directly. In a 96-well plate, Reagent 1 (100 μL), extraction buffer (60 μL), sample (20 μL), Reagent 2 (10 μL), and Reagent 3 (10 μL) were added sequentially and mixed immediately. The absorbance (A1) was read at 412 nm, followed by a second reading (A2) after incubation for 10 min at room temperature (25 °C). The absorbance difference ΔA (A₂ − A₁) was calculated, and glutathione reductase (GR) activity was determined using the kit’s formula: GR activity (nmol/min/g fresh weight) = [(ΔA ÷ ε ÷ d ÷ 2 × 10⁹ × V₂) ÷ (W × V₁ ÷ V) ÷ T] × D. The parameters are defined as: ε is the molar extinction coefficient of TNB (1.36 × 10⁴ L/mol/cm); d is the cuvette light path length (0.5 cm); V is the total extraction volume (1 mL); V₁ is the volume of sample added to the reaction system (0.02 mL, equivalent to 20 μL); W is the sample mass (g); V₂ is the total reaction volume (2 × 10⁻⁴ L, equivalent to 200 μL); D is the dilution factor (1 for undiluted samples); and T is the reaction time (10 min).

Additionally, the contents of proline (Pro), the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS), and proline dehydrogenase (ProDH) were measured using specific assay kits from Aidi Biological Co., Ltd. [34,35], employing the Pro content assay kit (catalog number ADS-W-AJS004-48), the P5CS enzyme activity assay kit (catalog number ADS-F-AJS011), and the ProDH enzyme activity assay kit (catalog number ADS-F-AJS014).

Approximately 0.1 g of melon root tissue was weighed and homogenized in 1 mL of extraction buffer on ice. The homogenate was then processed according to the specific requirements for each assay. For Pro content determination, the homogenate was transferred to a 1.5 mL EP tube and extracted by shaking in a water bath at 90 °C for 10 min. The mixture was subsequently centrifuged at 12,000× g rpm for 10 min at 25 °C, and the supernatant was cooled for further analysis. In a 96-well plate, 150 μL of the supernatant, 150 μL of glacial acetic acid, and 300 μL of Reagent 1 were mixed and heated in a water bath at 95 °C for 30 min (sealed to prevent evaporation). After cooling to room temperature, 200 μL of the clear liquid was transferred to a 96-well plate, and the absorbance (A) at 520 nm was recorded. Proline (Pro) content was determined according to the kit protocol, where ΔA represents the absorbance difference between test and blank measurements. The content was calculated using the formula: Pro content (μg/g fresh weight) = [((ΔA + 0.0052) ÷ 0.162) ÷ (W × V₁ ÷ V)] × D. Parameter definitions are: V is the total extract volume (1 mL); V₁ is the volume of extract added to the reaction system (0.15 mL); W is the sample mass (g); and D is the dilution factor (1 for undiluted samples). To assess P5CS activity, the homogenate was centrifuged at 12,000× g rpm for 10 min at 4 °C, and the supernatant was used as the test solution. A portion of the supernatant was boiled at 95 °C for 10 min, cooled, and centrifuged again to prepare the control supernatant. In a 1 mL quartz cuvette (path length 1 cm), 120 μL of sample, 20 μL of Reagent 1, 20 μL of Reagent 2, 520 μL of Reagent 3, and 20 μL of Reagent 4 were added and mixed. The absorbance (A) was measured at 340 nm immediately (A1) and after 10 min (A2). ΔA was calculated as (A1 − A2)test − (A1 − A2)control, and P5CS activity was determined according to the kit protocol using the formula: P5CS activity (U/g fresh weight) = [ΔA × V₂ ÷ (ε × d) × 10⁹] ÷ (W × V₁ ÷ V) ÷ T, which simplifies to 93.8 × ΔA ÷ W. The parameters are defined as follows: V is the total extraction volume (1 mL); V₁ is the volume of sample extract added to the reaction system (0.12 mL); V₂ is the total reaction volume (7 × 10⁻⁴ L); d is the cuvette path length (1 cm); T is the reaction time (10 min); W is the sample mass (g); and ε is the molar extinction coefficient of NADPH (6.22 × 10³ L/mol/cm). For ProDH activity assay, the homogenate was centrifuged at 12,000× g rpm for 10 min at 4 °C, and the supernatant was used as the test solution. In a 1 mL quartz cuvette (path length 1 cm), 120 μL of sample, 20 μL of Reagent 1, 560 μL of Reagent 2, and 20 μL of Reagent 3 were added and gently mixed. The mixture was incubated at room temperature (25 °C) for 6 min, and the absorbance (A) at 340 nm was measured immediately (A1) and after 30 min (A2). ΔA was calculated as A2 − A1, and ProDH activity was quantified according to the manufacturer’s protocol using the formula: ProDH activity (U/g fresh weight) = [ΔA × V₂ ÷ (ε × d) × 10⁹] ÷ (W × V₁ ÷ V) ÷ T, which simplifies to 32.2 × ΔA ÷ W. Parameter definitions include: V is the total extraction volume added (1 mL); V₁ is the volume of sample extract added to the reaction system (0.12 mL); V₂ is the total reaction volume (7.2 × 10⁻⁴ L); d is the cuvette light path length (1 cm); ε is the molar extinction coefficient of NADH (6.22 × 10³ L/mol/cm); W is the sample mass (g); and T is the reaction time (30 min).

2.5. Transcriptome Analysis

On day 1 of the heat stress treatment, transcriptome sequencing was performed on the roots of the melon seedlings. The RNA sequencing was conducted by Beijing Qingke Biotechnology Co., Ltd., China. Genome data were sourced from the Cucurbit Genomics Database (http://cucurbitgenomics.org/) and aligned using the Melon DHL92 genome v4. Quality control analysis of the sequencing data revealed that all samples had Q20 values exceeding 85% and Q30 values exceeding 80%. Subsequently, gene expressions were calculated based on transcripts per million (TPM), and differential expression analysis was performed using the DESeq2 software (version 1.26) [36]. The criteria for selecting differentially expressed genes were set as follows: Log2(Fold change value) > 1.0 and p < 0.05 [37]. The transcriptome data have been uploaded to NCBI (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1302446/, accessed on 20 July 2025).

2.6. Quantitative Fluorescent PCR Analysis

The roots of melon seedlings were sampled after 1 day of heat stress treatment. The roots were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent RNA extraction. Each treatment consisted of three biological replicates, each containing three seedlings. Following the method of Huang et al. [38], total RNA was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China). The integrity of the RNA was verified through 1% agarose gel electrophoresis and with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), ensuring that A260/A280 ratios were approximately 2.0–2.2 and A260/A230 ratios were ≥ 1.8 [38]. Subsequently, 2 μg of RNA was used as a template to synthesize cDNA via reverse transcription utilizing the Hifair^® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The synthesized cDNA was diluted to 200 ng µL⁻¹ with nuclease-free water and stored at −20 °C for further use. qRT-PCR was performed on an ABI 6500 HT system (Applied Biosystems, Foster City, CA, USA) with the following reaction setup: a total volume of 10 μL consisting of 1 μL of template cDNA, 0.5 μL each of forward and reverse primers (final concentration of 0.25 µM), 5 μL of TransStart^® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China), and 3 μL of nuclease-free water. The thermal cycling program was as follows: initial denaturation at 94 °C for 30 s; followed by 40 cycles of denaturation at 94 °C for 5 s, annealing at 56 °C for 30 s, and extension at 72 °C for 10 s. A melt curve analysis was conducted to confirm the specificity of the products. The relative gene expressions were calculated using the 2^−ΔΔCt method, with CmActin (MELO3C025848) serving as the internal reference gene [39]. The primer sequences used are listed in Supplementary Table S1. The genes selected for evaluation, including SOD, POD, CAT, APX, GR, Pro, and HSP-related genes, were chosen based on their known roles in antioxidant defense and stress response in plants. These genes were identified through the Cucurbit Genomics Database (http://cucurbitgenomics.org/v2/search/genome/23) using annotations for melon (Cucumis melo L.).

2.7. Data Statistical Analysis and Graphical Presentation

All data were subjected to one-way ANOVA to assess treatment effects, with a significance level set at p < 0.05. The main factor and its respective levels were as follows: The treatment condition, with four levels—T1: control (normal growth conditions) with empty vector (EV); T2: control with overexpression of CmDUF239-1 (OEDUF239-1); T3: heat stress treatment with empty vector (EV); T4: heat stress treatment with overexpression of CmDUF239-1 (OEDUF239-1). The statistical analysis was conducted in the R programming language (version 4.0.3) using the RStudio integrated development environment (RStudio Team, (2020). RStudio: Integrated Development Environment for R. Boston, MA, USA: RStudio, PBC). Data cleaning and preprocessing were performed using the dplyr and tidyr packages to handle missing values and transform the data as needed. Additionally, the effect of CmDUF239-1 overexpression shown in Figure 1b was analyzed using a t-test. For Principal Component Analysis (PCA), the ‘FactoMineR’ package was employed to analyze the standardized complete transcriptome data matrix. The results were visualized using the ‘factoextra’ package. Graphical outputs were generated and optimized with the ‘ggplot2’ and ‘RColorBrewer’ packages to enhance color schemes and layout.

3. Results

3.1. Effects of Overexpressing the CmDUF239-1 on Phenotype and Relative Conductivity in Melon Seedlings Under Heat Stress

The Effect of CmDUF239-1 gene overexpression melon heat tolerance was assessed by transforming roots with an empty vector or a CmDUF239-1 overexpression construct, producing two groups: one with the empty vector (EV) and another with the CmDUF239-1 overexpressing seedlings (OEDUF239-1) (Figure 1a). Subsequent quantitative PCR analysis revealed that the expression of the CmDUF239-1 gene in the OEDUF239-1 roots was upregulated by 11.3 times compared to the EV group (Figure 1b). The seedlings were then subjected to heat stress treatment. Following 4 days of heat exposure, OE-DUF239-1 seedlings exhibited enhanced phenotypic performance relative to EV seedlings under control and heat stress conditions (Figure 1c). Specifically, under control conditions, the fresh mass of the shoot and roots of OEDUF239-1 increased significantly by 98.7% and 87.1%, respectively, compared to EV. Under heat stress, OEDUF239-1 showed even greater increases of 137.7% and 149.5% for the shoot and roots, respectively (Figure 1d,e). In contrast, the fresh mass of the shoot and roots of EV decreased by 67.9% and 67.2% under heat stress, while OEDUF239-1 only experienced reductions of 61.6% and 56.2% (Figure 1d,e). Additionally, under control conditions, the dry mass of the shoot and roots in OEDUF239-1 significantly increased by 112.2% and 94.4%, respectively, while under heat stress, these values rose by 168.4% and 137.5% (Figure 1f,g). Compared to controls, the dry mass of the shoot and roots in EV decreased by 43.2% and 77.8% under heat stress; in contrast, OEDUF239-1 showed declines of only 28.1% and 72.9% (Figure 1f,g). Furthermore, under heat stress, the relative electrolyte leakage in the leaves and roots of OEDUF239-1 was significantly reduced by 40.1% and 32.8%, respectively, compared to EV (Figure 1h,i). These results indicated that overexpressing the CmDUF239-1 gene could significantly alleviate the damage caused by heat stress in melon seedlings.

Figure 1. Effects of CmDUF239-1 overexpression on growth and relative conductivity in melon seedlings. (a) Observation of the DsRed-tagged protein in the root systems. (b) Relative expressions of the CmDUF239-1 gene in the OEDUF239-1 root systems. (c) Phenotype of melons after 4 days of heat stress treatment, including fresh mass of the aerial parts (d), fresh mass of the roots (e), dry mass of the aerial parts (f), dry mass of the roots (g), relative electrical conductivity (REC) of leaves (h), and REC of roots (i). Mean ± SE (n = 3). In Figure 1b, * indicates a significant difference between the EV and OEDUF239-1 (p < 0.05). Different lowercase letters indicate significant differences between treatments (p < 0.05). BF: bright field; DF: dark field; EV: empty vector-transformed melon seedlings; OEDUF239-1: melon seedlings overexpressing CmDUF239-1 in the roots; T1: control (normal growth conditions) with empty vector (EV), T2: control with overexpression of CmDUF239-1 (OEDUF239-1), T3: heat stress treatment with empty vector (EV), and T4: heat stress treatment with overexpression of CmDUF239-1 (OEDUF239-1).

Figure 1. Effects of CmDUF239-1 overexpression on growth and relative conductivity in melon seedlings. (a) Observation of the DsRed-tagged protein in the root systems. (b) Relative expressions of the CmDUF239-1 gene in the OEDUF239-1 root systems. (c) Phenotype of melons after 4 days of heat stress treatment, including fresh mass of the aerial parts (d), fresh mass of the roots (e), dry mass of the aerial parts (f), dry mass of the roots (g), relative electrical conductivity (REC) of leaves (h), and REC of roots (i). Mean ± SE (n = 3). In Figure 1b, * indicates a significant difference between the EV and OEDUF239-1 (p < 0.05). Different lowercase letters indicate significant differences between treatments (p < 0.05). BF: bright field; DF: dark field; EV: empty vector-transformed melon seedlings; OEDUF239-1: melon seedlings overexpressing CmDUF239-1 in the roots; T1: control (normal growth conditions) with empty vector (EV), T2: control with overexpression of CmDUF239-1 (OEDUF239-1), T3: heat stress treatment with empty vector (EV), and T4: heat stress treatment with overexpression of CmDUF239-1 (OEDUF239-1).

3.2. Differential Gene Analysis

Transcriptome sequencing of melon roots was conducted after one day of heat stress to examine the impact of CmDUF239-1 overexpression on gene expression. PCA revealed that the three replicates of each treatment group were contained within the 95% confidence ellipse, confirming good sample reproducibility (Figure 2a). Differential expression analysis revealed that, relative to the control, 1890 genes were upregulated and 2525 were downregulated in EV roots after one day of heat stress, while 2885 were upregulated and 2729 were downregulated in OEDUF239-1 (Figure 2b). Finally, Gene Ontology (GO) enrichment analysis of the 5614 differentially expressed genes from OEDUF239-1 revealed significant enrichment in molecular functions related to oxidoreductase activity and peroxidase activity (Figure 2c). These findings suggested that CmDUF239-1 may confer resistance to heat stress by regulating the antioxidant enzyme activity in the roots.

3.3. The Effect of CmDUF239-1 Overexpression on Antioxidant Enzyme Activity in Roots

Antioxidant enzyme activities and proline content were measured in roots following one day of heat stress to assess the contribution of CmDUF239-1 overexpression to heat tolerance. MDA content in OEDUF239-1 was reduced by 3.9% relative to EV under control conditions and exhibited a pronounced decrease of 27.8% under heat stress (Figure 3a). The SOD activity in OEDUF239-1 exhibited a 19.0% reduction relative to EV under control conditions, while exposure to heat stress resulted in a pronounced enhancement of 138.3%. (Figure 3b). Peroxidase (POD) activity was increased by 5.3% in OEDUF239-1 compared to EV under control conditions and rose by 52.8% under heat stress (Figure 3c). Catalase (CAT) activity was elevated by 20.5% in OEDUF239-1 relative to EV under control conditions and increased by 23.0% under heat stress (Figure 3d). Ascorbate peroxidase (APX) activity exhibited an increase of 9.4% in OEDUF239-1 when compared to EV under control conditions, with a notable rise of 48.9% during heat stress (Figure 3e). Glutathione reductase (GR) activity increased by 11.8% in OEDUF239-1 compared to EV under control conditions and further increased by 30.6% under heat stress (Figure 3f). These results demonstrated that the overexpression of the CmDUF239-1 gene could enhance the antioxidant enzyme activity in the root systems of melon seedlings, thereby contributing to their resistance against heat stress.

3.4. The Effect of CmDUF239-1 Overexpression on the Expression of Antioxidant Enzyme-Related Genes

Gene expression analysis was performed after one day of heat stress to evaluate the regulatory effect of CmDUF239-1 on genes related to antioxidant enzyme activities in melon roots. Among the SOD genes, CmCSD1 and CmMSD1 showed the highest transcript abundance. These genes were repressed by heat stress in EV but significantly upregulated in OEDUF239-1 (Figure 4a). In the case of POD genes, CmPOD1 and CmPRX25 exhibited the highest expressions, and both were induced by heat stress in EV and OEDUF239-1 (Figure 4b). For CAT genes, CmCAT2 showed the highest expression level, which was also upregulated by heat stress in both EV and OEDUF239-1 (Figure 4c). Among the APX genes, CmAPX1 and CmAPX3 had the highest expressions; both were similarly upregulated by heat stress in EV and OEDUF239-1. For glutathione reductase (GR) genes, CmGR1 had the highest expression (Figure 4d), which was also induced by heat stress in both groups. Further quantitative PCR (qPCR) analysis indicated that after one day of heat stress, the expressions of antioxidant enzyme-related genes significantly increased compared to the control. Specifically, the expression of SOD genes CmCSD1 and CmMSD1 increased by 101.3% and 63.6%, respectively (Figure 4f,g). Expression analysis of POD genes revealed significant upregulation, with CmPRX25 and CmPOD1 increasing by 49.8% and 45.8%, respectively (Figure 4h,i). For the CAT gene, the expression of CmCAT2 rose significantly by 34.5% (Figure 4j). Regarding APX genes, CmAPX3 and CmAPX1 increased by 119.8% and 82.6%, respectively (Figure 4k,l). Finally, the expression of the GR gene, CmGR1, significantly increased by 35.7% (Figure 4m). In summary, overexpressing CmDUF239-1 significantly enhanced the expressions of these antioxidant enzyme-related genes, and the changes in gene expression were consistent with the alterations observed in the activities of SOD, POD, CAT, APX, and GR. These results suggested that the overexpression of the CmDUF239-1 gene could enhance the heat tolerance of melon seedlings by increasing the expression of antioxidant enzyme-related genes, corroborating the earlier findings related to antioxidant enzyme activities.

3.5. The Effect of CmDUF239-1 Overexpression on Proline Accumulation and Related Gene Expression

The influence of CmDUF239-1 overexpression on proline metabolism in melon roots was examined by determining proline content, P5CS activity, ProDH activity, and transcript levels of related genes. A significant increase of 27.4% in proline content was observed in OEDUF239-1 compared with EV under heat stress (Figure 5a). Additionally, P5CS activity was enhanced by 13.2% (Figure 5b), while ProDH activity decreased significantly by 32.7% (Figure 5c). Further analysis revealed that among the proline synthesis and degradation-related genes, CmP5CS1 and CmProDH exhibited the highest expressions. Under heat stress, the expression of the CmP5CS1 gene was upregulated in both EV and OEDUF239-1, whereas the expression of the CmProDH gene was downregulated (Figure 5d). Quantitative PCR analysis further demonstrated that after one day of heat stress, the expression of the CmP5CS1 gene increased significantly by 31.3% and 95.8% in EV and OEDUF239-1, respectively, compared to controls (Figure 5e). Under heat stress conditions, CmProDH expression exhibited a significant 12.0% increase in EV, while a marked reduction of 44.6% was observed in OEDUF239-1 (Figure 5f). These results suggested that overexpression of the CmDUF239-1 promoted proline accumulation through stimulation of proline synthesis genes and repression of proline degradation genes, contributing to enhanced heat tolerance in melon seedlings.

3.6. The Effect of CmDUF239-1 Overexpression on HSP Gene Expression

Considering the critical role of HSPs in plant responses to high-temperature stress, this study further analyzed the expressions of HSP-related genes. The results indicated that under heat stress conditions, three HSP genes—CmHSP17.6II, CmHSP17.6C, and CmHSP18.2—were significantly upregulated in melon plants overexpressing the CmDUF239-1 gene (OEDUF239-1) compared to the control group (EV) (Figure 6a). Further quantitative PCR analysis revealed that after one day of heat stress, the expression of CmHSP17.6II increased significantly by 5.5-fold in EV and 20.7-fold in OEDUF239-1 compared to the control (Figure 6b). The expression of CmHSP17.6C increased by 7.7-fold in EV and 51.1-fold in OEDUF239-1 (Figure 6c), while the expression of CmHSP18.2 rose by 8.2-fold in EV and 49.8-fold in OEDUF239-1 (Figure 6d). Notably, after one day of heat stress, the expressions of CmHSP17.6II, CmHSP17.6C, and CmHSP18.2 in OEDUF239-1 were significantly enhanced by 129.9%, 162.9%, and 169.8%, respectively, compared to EV (Figure 6b–d). These findings suggested that the overexpression of the CmDUF239-1 gene could enhance the heat tolerance of melon seedlings by significantly promoting the expression of HSP genes, particularly CmHSP17.6II, CmHSP17.6C, and CmHSP18.2.

4. Discussion

4.1. Positive Regulation of Heat Tolerance in Grafted Melon by CmDUF239-1

Plants frequently encounter various abiotic stresses during their growth and development, with high-temperature stress significantly affecting crop yield and quality. Plants respond to these adverse conditions through intricate regulatory networks encompassing antioxidant defenses, osmotic regulation, and the modulation of heat stress-related protein expression. DUF proteins are critical regulators of heat tolerance and other abiotic stresses across different species. For example, in rice, the overexpression of OsDUF6 significantly mitigated salt stress-induced reductions in plant height, root length, and biomass, suggesting that sustained growth and biomass accumulation constitute the phenotypic basis for OsDUF6-mediated salt tolerance [18]. Overexpression of CsDUF966 in cucumber alleviated stress-induced reductions in plant height and fresh and dry mass, maintaining growth vigor and biomass accumulation and highlighting its role as a positive regulator of crop tolerance to salt and drought stresses [40]. In cotton, the overexpression of GhDUF668 significantly reduced the inhibitory effects of heat stress on plant height and the loss of fresh and dry mass, supporting the growth vigor and biomass accumulation of cotton seedlings and providing direct phenotypic evidence for the heat tolerance imparted by GhDUF668 [41].

In this study, the overexpression of the CmDUF239-1 gene significantly alleviated the inhibitory effects of heat stress on the fresh and dry mass of both the aboveground parts and root systems of melon seedlings (Figure 1d,g). Under heat stress conditions, the reductions in fresh and dry mass of OEDUF239-1 plants were notably less than those of the empty vector (EV) control, aligning with findings from previous studies on rice OsDUF6 [18], cucumber CsDUF966 [40], and cotton GhDUF668 [41]. However, a distinct observation in this research was that the overexpression of the CmDUF239-1 gene also significantly reduced the REC of the leaves and roots of melon seedlings under heat stress (Figure 1h,i), a change not mentioned in studies of other crops. This discrepancy may be attributed to the greater susceptibility of melon cell membrane stability to damage under heat stress, and the overexpression of the CmDUF239-1 gene appears to enhance cell membrane stability, thereby reducing REC. This suggested that DUF genes may operate through different mechanisms in various plants when responding to stress, and that melon’s response to heat stress was particularly sensitive in terms of cell membrane stability.

4.2. CmDUF239-1 Enhances Heat Tolerance in Melon Seedlings by Increasing Antioxidant Enzyme Activity and Proline Accumulation

Plants respond to high-temperature stress not only through changes in growth and phenotype but also by activating their antioxidant systems and accumulating osmotic regulators to maintain cellular homeostasis [42]. DUF proteins may synergistically enhance plant heat tolerance by regulating the activity of antioxidant enzymes and the expression of proline metabolism-related genes [43]. Specifically, DUF proteins are likely involved in modulating the activity of antioxidant enzymes such as SOD, POD, and CAT, thereby reducing ROS accumulation and alleviating membrane lipid peroxidation damage. Furthermore, DUF proteins may facilitate proline accumulation by stimulating the expression of key proline synthesis genes like P5CS, thereby strengthening osmotic regulation and protein stabilization, which collectively enhance plant heat tolerance. Rice under heat stress enhances antioxidant defense by synchronizing the expression of OsDUF2488 with OsPrx1.1, a key peroxidase gene. This is evidenced by a significant increase in POD activity, effectively clearing ROS and reducing membrane lipid peroxidation damage, resulting in a marked decrease in MDA content. The coordinated expression of OsDUF2488 and OsPrx1.1 significantly upregulated the expression of antioxidant-related genes, including OsPrx1.1, OsSOD, and OsCAT. The increased expression of these genes further bolstered the ability of rice seedlings to eliminate ROS, thereby enhancing their heat tolerance and drought resistance [19]. Moreover, DUF proteins can also regulate drought resistance in rice; for instance, OsDUF4057 was found to continuously downregulate the expression of OsSOD, OsPOD, and OsCAT under 20% PEG-6000 drought stress over 7 days, leading to corresponding decreases in enzyme activities of 34.6%, 28.1%, and 31.4%, respectively. This resulted in reduced proline accumulation and exacerbated membrane lipid peroxidation, thus adversely affecting rice drought resistance [20]. In cotton (Gossypium hirsutum L.), exposure to 42 °C for 6 h induced oxidative stress responses through the regulation of GhDUF668 gene and its downstream antioxidant enzyme-related genes. Notably, the expression of the GhDUF668 gene was significantly upregulated, accompanied by increased expressions of antioxidant enzyme genes such as SOD, POD, and CAT. These enzymes effectively mitigated membrane lipid peroxidation damage by clearing ROS, resulting in a significant reduction in MDA content [41].

The results of this study exhibited notable similarities and differences compared to findings from other crops regarding the regulation of antioxidant enzyme activity and gene expression. The similarities are evident in that DUF proteins enhance heat stress tolerance across crops—whether in rice [19,20], cotton [41], or melon—through the regulation of antioxidant enzyme activity and the expression of related genes. For instance, after the overexpression of CmDUF239-1 in melons, the activities of antioxidant enzymes such as SOD, POD, CAT, APX, and GR were significantly increased (Figure 3a–f), alongside substantial upregulation of antioxidant enzyme-related gene expression (Figure 4a–m), akin to the mechanism by which OsDUF2488 regulates OsPrx1.1 and other antioxidant-related genes to enhance antioxidant defense in rice [19]. Additionally, the upregulation of GhDUF668 in cotton coincided with increased transcript levels of antioxidant enzymes (SOD, POD, CAT), enhancing ROS clearance and alleviating lipid peroxidation damage, a pattern that aligns with the enhanced antioxidant enzyme expression observed in CmDUF239-1-overexpressing melon seedlings [41]. However, there were differences in the specific regulatory patterns of DUF proteins on antioxidant enzyme activity and gene expression among different crops. In rice, OsDUF4057 negatively regulated antioxidant enzyme gene expression and enzyme activity under drought stress, whereas CmDUF239-1 positively regulated antioxidant enzyme gene expression and activity under heat stress (Figure 3a–f and Figure 4a–m), indicating that DUF proteins may function differently depending on crops and stress conditions [20]. This discrepancy may arise from the unique gene regulatory networks formed during the evolution of different crops, as well as variations in intracellular signaling pathways and metabolic processes under different stress conditions.

In addition to enhancing antioxidant defense via modulation of antioxidant enzyme-related genes, DUF proteins facilitate proline accumulation by upregulating P5CS, collectively contributing to improved heat tolerance [44]. In common beans (Phaseolus vulgaris L.), exposure to 42 °C for 6 h enhances osmotic regulation capabilities by modulating the expression of members of the DUF221 gene family and their downstream target genes. Notably, the expression of the GmDUF4228-70 gene within the DUF221 family was significantly upregulated, accompanied by a marked increase in the expression of P5CS, the key proline synthesis gene, which facilitates proline accumulation [21]. In this study, melons overexpressing the CmDUF239-1 gene demonstrated a significant increase in proline content under heat stress conditions (Figure 5a), which is consistent with findings on the regulation of proline metabolism by the GmDUF4228-70 gene in common beans [21]. Specifically, both cases promoted proline accumulation through the upregulation of the proline synthesis gene P5CS, thereby enhancing plant heat tolerance (Figure 5a–d). However, species-specific differences were observed. In common beans, the regulation of the DUF221 gene primarily manifests through the significant upregulation of its member GmDUF4228-70, which subsequently affects the expression of the downstream target gene P5CS [21]. In contrast, in this study, the overexpression of the CmDUF239-1 gene not only significantly upregulated the expression of the proline synthesis gene CmP5CS1 but also markedly downregulated the expression of the proline degradation gene CmProDH (Figure 5a,d). This dual regulatory mechanism resulted in a more pronounced accumulation of proline in melons, thereby enhancing the heat tolerance under stress. This divergence may be attributed to the specificity of gene regulatory networks and metabolic pathways among species, as well as the broader regulatory capacity of CmDUF239-1, which appeared to optimize the entire proline metabolic pathway beyond individual gene regulation.

4.3. CmDUF239-1 Enhances HSP-Related Gene Expression in Melon Seedlings to Mitigate Heat Stress

HSPs play crucial roles in plants’ responses to elevated temperatures, acting as molecular chaperones that protect proteins from heat-induced damage and maintain intracellular protein stability [45]. High-temperature treatment at 42 °C for 6 h in rice seedlings primarily affected the leaves, triggering the regulation of HSPs and their associated genes in response to heat stress. Notably, the expressions of heat shock transcription factors HSFA2a and HSFA3 were significantly upregulated, alongside substantial increases in the expression of HSP genes HSP70, HSP90, and HSP17.7. The heightened expression of these genes effectively protected proteins from heat damage, thereby maintaining cellular homeostasis. Additionally, the activities of antioxidant enzymes SOD and CAT were significantly elevated, which reduced the accumulation of ROS and alleviated lipid peroxidation, as evidenced by a notable decrease in MDA content [46]. In tomato, high-temperature treatment was performed at 38 °C/28 °C (day/night) for 7 days, revealing that under heat stress, tomato seedlings also regulated the expression of HSPs and their related genes. Similar to rice, the expressions of heat shock transcription factors HSFA2 and HSFA3 were significantly increased, along with notable upregulation of HSP70, HSP90, and HSP17.6. This increase in gene expression effectively safeguarded proteins from heat-related damage and preserved cellular stability. Furthermore, the activities of antioxidant enzymes SOD and CAT in tomato were markedly enhanced, leading to reduced ROS accumulation and diminished lipid peroxidation, characterized by significantly lower MDA levels. Thus, tomatoes improved the thermotolerance through a synergistic interaction between the HSP pathway and the antioxidant system [47].

In this study, the expression pattern of HSP genes in the root systems of melon seedlings showed both similarities and differences compared to findings in other crops such as rice [46] and tomato [47]. A common finding across all studies was that HSPs and their associated genes were markedly upregulated in response to heat stress. This suggested that HSPs performed a conserved molecular chaperone role in plants, safeguarding proteins from thermal damage and maintaining cellular homeostasis under high-temperature conditions. For instance, the HSP70, HSP90, and HSP17.7 in rice [46], HSP70, HSP90, and HSP17.6 in tomato [47], and CmHSP17.6II, CmHSP17.6C, and CmHSP18.2 in melon (Figure 6a,d) were all significantly upregulated under heat stress. Conversely, differences were observed in the specific HSP gene that exhibited the highest expressions among the crops. In rice, the most highly expressed were HSP70 and HSP90 [46], while in tomato, HSP70 and HSP17.6 were the most abundantly expressed [47]; in contrast, the predominant gene in melon was CmHSP18.2 (Figure 6a). Such discrepancies may stem from unique gene regulatory networks formed during the evolutionary process of different plant species, as well as the specific response mechanisms of different tissues (e.g., leaves versus roots) to heat stress. Moreover, this research focused solely on root systems, whereas studies on rice and tomato primarily concentrated on leaf tissue, which may partly explain the observed differences in gene expression patterns. Therefore, although HSPs exhibited a universal protective function in plants facing heat stress, their specific expression patterns and regulatory mechanisms differed significantly among various plant species and tissues, necessitating further cross-species and cross-tissue investigations for deeper insights.

In addition, roots are essential for plant responses to heat stress, functioning as the main organs for nutrient and water uptake and directly impacting the plant’s overall growth and performance. During heat stress, roots are exposed to elevated temperatures and must maintain their functionality to support the plant’s survival [48,49]. It is important to note that soil has the capacity to absorb heat, which may affect the roots, especially during the seedling stage [50]. The overexpression of CmDUF239-1 in the roots enhances the ability to cope with heat stress through the upregulation of antioxidant defense mechanisms, proline metabolism, and HSP-related gene expression. These molecular changes in the roots contribute to improved cellular stability and functionality, ultimately enhancing the plant’s overall heat tolerance.

5. Conclusions

In summary, our study provided valuable insights into the role of the CmDUF239-1 gene in enhancing heat tolerance in melon seedlings. Our findings supported our hypothesis that CmDUF239-1 enhanced heat tolerance by upregulating key genes involved in antioxidant defense, proline metabolism, and the accumulation of HSPs. Specifically, melon seedlings overexpressing CmDUF239-1 exhibited marked upregulation of genes related to antioxidant enzyme activities (e.g., CmCSD1, CmPOD1), proline content (e.g., CmP5CS), and expression of Heat Shock Proteins (e.g., CmHSP17.6II, CmHSP18.2) under heat stress conditions. These molecular changes were accompanied by improved physiological performance, as evidenced by higher fresh and dry mass, lower MDA contents, and increased antioxidant enzyme activities. This study addressed gaps in understanding the specific molecular mechanisms underlying heat tolerance in melon and provided a foundation for future research focused on improving heat resistance in this important crop. The findings suggested that CmDUF239-1 could serve as a valuable genetic resource for breeding programs focused on improving heat tolerance in melon.