Advances in Cold Stress Response Mechanisms of Cucurbits

Abstract

Cold stress can inhibit the growth of cucurbits, disrupt pollination and fertilization, induce fruit deformities, reduce plant resistance, and increase susceptibility to diseases, ultimately resulting in yield reduction, quality deterioration, or even complete crop failure. This review focuses on the main cucurbits, such as melon, cucumber, and watermelon, systematically expounding the roles of plant hormones, signaling molecules, soluble sugars, key regulatory factors, molecular mechanisms, and network interactions in their response to cold stress. Furthermore, it highlights future research directions and application potential. By analyzing existing challenges and prospective advancements in this field, the review aims to provide a comprehensive reference for facilitating genetic improvement in cold tolerance.

1. Introduction

Cucurbits, a diverse group of flowering plants, are predominantly cultivated in tropical and subtropical regions [1,2]. Cucumber (Cucumis sativus), melon (Cucumis melo), and watermelon (Citrullus lanatus) represent the three most economically significant cucurbit crops, valued for their nutritious fruits and cultivated worldwide across varied agroecological zones [3]. Cucumber originated in the South Asian subcontinent, while melon and watermelon trace their origins to Africa [4,5,6]. Like most tropical and subtropical plant species, all three species lack the ability to adapt to freezing temperatures [7]. They are thermophilic, requiring consistently warm temperatures throughout their life cycle, with seedlings being particularly sensitive to cold stress [8,9,10]. Cucurbit seedlings typically sustain injury when exposed to temperatures below 10 °C or 15 °C for durations exceeding a critical threshold under production conditions [11,12,13]. In northern China, these crops are predominantly cultivated in early spring, while cucumber is also utilized for overwintering production. However, cold stress during the seedling stage often impairs plant growth and development, delays fruit maturation, and reduces economic returns. To mitigate chilling injury, current agricultural practices, including grafting onto cold-tolerant rootstocks, cold acclimation, and application of hormone-based growth regulators and osmoprotective compounds, have demonstrated efficacy in enhancing early seedling resilience [14,15,16,17].

Cold acclimation has been demonstrated to significantly enhance freezing tolerance in Arabidopsis thaliana [18]. Subsequent research has revealed that plants employ complex regulatory networks integrating phytohormonal signaling and reactive oxygen species (ROS) homeostasis to adapt to temperature changes [19]. Under cold stress, small signaling molecules including calcium ions (Ca²⁺), ROS, nitric oxide (NO), hydrogen sulfide (H₂S), and cyclic guanosine monophosphate (cGMP) initiate downstream signaling cascades that modulate gene expression and hormonal pathways, thereby conferring cold tolerance [20]. Due to the chilling sensitivity of cucurbits, understanding the regulatory physiology and molecular mechanisms underlying their cold stress response is critical for developing strategies to mitigate seedlings chilling injury under field conditions.

Although the responses to cold stress have been extensively characterized in model plants [19,20,21,22,23,24], research on cucurbits remains limited. Recent advances in cucurbit genomics have accelerated the discovery of cold-tolerance genes, yet research on low-temperature adaptation focuses predominantly on three key species: cucumber, melon, and watermelon. In this review, we focus exclusively on cold adaptation mechanisms described in these three cucurbits, also incorporating comparisons with model species when evidence in cucurbits is limited. We discuss the current understanding of their physiological adaptations (including plant hormones, signaling molecules, and soluble sugars) and molecular regulatory networks, to establish a theoretical foundation for genetic improvement of cold tolerance in these economically important crops.

2. Phytohormone-Mediated Cold Stress Response in Cucurbits

Research in model plant species, including Arabidopsis thaliana, rice, and maize, has demonstrated that several phytohormones, i.e., abscisic acid (ABA), brassinosteroids (BRs), jasmonic acid (JA), salicylic acid (SA), auxin (AUX), gibberellins (GA), and ethylene (ETH), play critical roles in plant cold stress responses [25,26,27]. In cucurbits, the exogenous application of phytohormone-based growth regulators can alleviate cold-induced damage and improve chilling tolerance [15,16,28,29,30].

2.1. Abscisic Acid (ABA)

Abscisic acid is a critical phytohormone regulating plant growth, development, and adaptive responses to abiotic stressors [31,32,33]. Exogenous ABA application has been reported to enhance chilling tolerance in various crops, such as rice [34], wheat [35], maize [27], and tomato [36]. Notably, ABA signaling exhibits conserved functionality in cucurbit species regarding cold stress adaptation. For instance, foliar application of 25 mg/L ABA significantly upregulated antioxidant enzyme activities, improved photosynthetic efficiency, and maintained metabolic homeostasis in cucumber seedlings, thereby mitigating cold-induced physiological impairments [8]. In melon, Li et al. [16] demonstrated that 75 μM ABA treatment significantly enhanced ROS scavenging capacity, as evidenced by increased activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), while simultaneously reducing membrane lipid peroxidation and improving cold stress tolerance. Genetic evidence further supports ABA’s role, as the silencing of ABA receptor genes CmPYL7 and CmPYL6 compromised cold tolerance, accompanied by accumulated H₂O₂ and malondialdehyde (MDA) content and decreased ROS scavenging capacity in melon seedlings [37,38]. Furthermore, ABA exhibits synergistic interactions with putrescine during cold stress response in melon [16,39]. ABA-mediated cold tolerance involves the regulation of endogenous GA and SA metabolism [29]. From an agronomic perspective, grafting can effectively improve plant stress resistance. Guo et al. [28] revealed that ABA, melatonin, and methyl jasmonate (MeJA) cooperatively regulate grafting-induced cold tolerance in watermelon. Moreover, the synergistic interaction of ABA with GA, SA, MeJA, melatonin, and putrescine suggests an integrated hormonal regulation that remains poorly characterized in cucurbits.

2.2. Brassinosteroids (BRs)

Brassinosteroids, a class of plant-specific steroidal phytohormones, are well documented enhancers of plant cold tolerance [40]. BR signaling is involved in both CBF-dependent and CBF-independent pathways in low-temperature responses [40,41]. Current research has identified 81 naturally occurring BR compounds, among which brassinolide (BL), 24-epibrassinolide (EBR), and 28-homobrassinolide (HBL) demonstrate the most potent biological activity [40]. Exogenous EBR application has been shown to significantly enhance chilling tolerance across multiple plant species, including maize [42], tobacco [43], peach [44], apple [45], grape [46], tomato [47], and cucumber [15,48]. In cucumber, foliar application of EBR enhanced cold tolerance by activating key enzymes in the Calvin cycle, enhancing cellular antioxidant capacity, and accelerating the recovery of photosystem II (PSII) from photoinhibition [48]. Anwar et al. [15] further demonstrated that under cold stress, exogenous EBR upregulated endogenous EBR levels by transcriptionally activating BR biosynthesis genes (CsDWF1, CsDWF2, and CsDWF4) while increasing the activities of antioxidant enzymes (SOD, POD, GR, CAT, and APX), reducing ROS accumulation, and ultimately enhancing cucumber seedling growth.

Beyond hormone content, the BR signaling transduction pathway is critical for cold stress adaptation in plants. The G protein is a core component of plant signal transduction [49]. The rice G-protein α subunit 1 (RGA1) can interact with chilling tolerance divergence 1 (COLD1) [50,51]. In cucumber, CsGPA1 silencing significantly reduced cold tolerance [51]. Under cold stress conditions, CsGPA1-RNA interference lines showed significant downregulation of key BR signaling components (including CsCDL1, CsBZR1, and CsBZR2), indicating that CsGPA1 functions as a positive regulator of BR-mediated cold stress responses in cucumber seedlings [51]. Furthermore, the basic pentacysteine (BPC) transcription factor family, particularly CsBPC2, is implicated in BR signaling [52,53]. In cucumber, CRISPR/Cas9-mediated knockout of CsBPC2 significantly impaired cold tolerance, coinciding with reduced transcript levels of both cold-responsive (CsICE1, CsCOR413IM2) and BR signaling-related genes (CsBZR1, CsBZR2) [54]. Collectively, these results delineate a crucial role for BR-mediated, CBF-dependent cold adaptation pathways in cucumber [51,54,55]. However, no functional BR pathway genes related to cold have been characterized in melon and watermelon, limiting the generalization of the model described in cucumber.

2.3. Jasmonic Acid (JA)

Jasmonic acid, a lipid-derived phytohormone, plays a pivotal role alongside methyl jasmonate (MeJA) in mediating plant responses to biotic and abiotic stresses [56,57]. JA regulated plant cold tolerance by mediating the interaction between JAZ and ICE proteins, thereby modulating the transcriptional activity of ICEs and the expression of CBF genes in the ICE–CBF–COR signaling pathway [56,58]. Additionally, JA enhanced plant resistance to low-temperature stress by inducing ROS-scavenging enzymes [58]. Accumulating evidence indicates that JA positively regulates cold tolerance across diverse plant species, including Arabidopsis thaliana [59], apple [60], tomato [61], cucumber [62], and watermelon [63,64]. For instance, Qi et al. [62] found that overexpression of CsHSFA1d and heat-shock pre-treatment increased endogenous JA levels in cucumber seedlings under cold stress, consequently enhancing their cold tolerance. Similarly, in watermelon, grafting onto pumpkin or fig leaf gourd rootstocks elevated endogenous levels of MeJA, melatonin, and hydrogen peroxide (H₂O₂) in the scion, enhancing seedling cold tolerance [63]. The exogenous application of MeJA also increased H₂O₂ and bolstered cold tolerance in watermelon seedlings [63]. Guo et al. [64] further revealed that MeJA-induced cold tolerance involves calcium (Ca²⁺) signaling: under cold stress, MeJA upregulated the expression of calcium-permeable channel genes (ClCNGC2 and ClCNGC20), triggering a transient Ca²⁺ influx and significantly elevating cytoplasmic free Ca²⁺ levels [64]. This, in turn, activated the CBF-dependent pathway, ultimately enhancing cold tolerance in watermelon seedlings. Although the exogenous application of ABA, EBR, or MeJA has shown benefits, it remains unclear whether high doses could have negative pleiotropic effects on growth or flowering under non-stress conditions.

2.4. Salicylic Acid (SA)

Salicylic acid is a naturally occurring phenolic compound that serves as a crucial regulator in plant growth, development, abiotic stresses, and defense responses [65,66,67,68,69]. Exogenous SA application mitigates chilling injury in postharvest horticultural products, including peach [70,71], pear [72], sweet pepper [73], broccoli [74], and cucumber [75], during cold storage, thereby extending shelf life. Furthermore, SA regulates grafting-induced cold resistance in cucumber. Studies by Fu et al. [76] revealed that under low-temperature stress, SA facilitates rootstock–scion communication by upregulating the expression of COR genes, thereby enhancing grafting-mediated cold tolerance. In higher plants, SA biosynthesis occurs primarily through two pathways: phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) [77]. Notably, the PAL pathway predominates in cucumber under cold stress [78]. The overexpression of the CsPAL1 gene enhances endogenous SA levels in cucumber, consequently improving the cold tolerance of grafted plants [79]. Additionally, SA-induced cold tolerance in cucumber is associated with ABA and hydrogen sulfide (H₂S) [80,81]. To date, SA has proven effective in enhancing postharvest preservation of various horticultural products during cold storage [70,71,72,73,74,75]. Nevertheless, research on its role in improving seedling cold tolerance and in grafting applications remains limited for other cucurbits, particularly melon and watermelon. This limitation has restricted its broader application in production.

2.5. Auxin (AUX)

Auxin is a critical regulator of plant growth and development, as well as a key integrator of abiotic stress signals [82,83,84]. In cucumber, Zhang et al. [85] found that cold stress could induce the accumulation of endogenous H₂S and indole-3-acetic acid (IAA). Exogenous application of 1.0 mM sodium hydrosulfide (NaHS) enhanced the activity of flavin monooxygenase (FMO) and upregulated the expression of FMO-like proteins (YUCCA2), promoting endogenous IAA biosynthesis and improving the cold tolerance in melon seedlings [85]. Further studies revealed that H₂S regulates low-temperature responses in cucumber via auxin signaling. Overexpression of the auxin response factor CsARF5 enhanced cold tolerance, and this regulatory mechanism was found to be H₂S-dependent [86]. Additionally, cold stress elevates endogenous melatonin (MT) levels in cucumber plants [87]. While exogenous MT application promoted IAA synthesis, exogenous IAA had no effect on endogenous MT content [87]. These results suggest that IAA acts as a downstream signaling molecule in H₂S- and MT-induced cold tolerance in cucumber seedlings [85,86,87]. Further evidence indicated that hydrogen peroxide (H₂O₂) functioned as a downstream signaling molecule of IAA, contributing to H₂S-mediated cold tolerance in cucumber seedlings [88]. However, whether H₂O₂ acts as a downstream component of IAA in MT-induced cold tolerance regulation remains unclear.

3. Signaling Molecules Involved in Cold Stress Response in Cucurbits

3.1. Calcium (Ca²⁺)

Calcium ions serve as pivotal secondary messengers in plant responses to various abiotic stresses [89,90]. Evidence from model species indicates that initial cold perception induces extracellular Ca²⁺ influx into the cytosol via Ca²⁺ exchangers (CAX), Na⁺/Ca²⁺ exchangers (NCX), and cyclic nucleotide gated channels (CNGC) [20,91]. Subsequently, Ca²⁺ signals are decoded by specific Ca²⁺ sensors, including calmodulins (CaMs), calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs), and Ca²⁺-dependent protein kinases (CPKs/CDPKs) [20,90,91]. This sensor network initiates a phosphorylation cascade that ultimately converts Ca²⁺ oscillations into specific cold-adaptive responses. In cucurbits, several CDPK and CRK family members (CmCDPK2/4/15, CmCRK2, and CsCDPK4/13/16) were activated under cold stress [92,93]. Moreover, the Ca²⁺/CPK signaling module played a critical role in melatonin-mediated cold tolerance in cucumber seedlings [94]. Notably, genetic and biochemical evidence has established ClCNGC2 and ClCNGC20 as nodal regulators that integrate MeJA- and MT-induced Ca²⁺ signaling and cold tolerance in watermelon [64,95].

3.2. Hydrogen Peroxide (H₂O₂)

Hydrogen peroxide is a non-free radical reactive oxygen species characterized by relatively higher stability compared to other ROS [96]. While high concentrations of H₂O₂ induce oxidative damage and phytotoxicity, low concentrations function as a key signaling molecule, activating stress-responsive genes and enabling rapid plants to adapt to adverse environmental conditions [96,97]. In cucumber, the exogenous application of 1.0 mM H₂O₂ significantly enhances cold tolerance in seedlings [88]. Conversely, the H₂O₂ scavenger N,N′-dimethylthiourea (DMTU) and the H₂O₂ synthesis inhibitor diphenyleneiodonium chloride (DPI) attenuate cold tolerance induced by IAA and H₂S, underscoring a critical signaling role for H₂O₂ in these pathways [88]. Furthermore, H₂O₂ acts as a downstream signaling molecule in the regulation of MT-mediated cold tolerance in both cucumber and watermelon [10,63,98]. According to Meng et al. [98], exogenous application of 100 µM MT upregulated CmRBOH1 expression in cucumber seedlings, leading to elevated H₂O₂ levels. Accumulated H₂O₂ activated antioxidant defense mechanisms, reducing malondialdehyde (MDA) content and electrolyte leakage (EL), thereby alleviating cold-induced damage [98]. Similarly, Li et al. [63] demonstrated that MT enhances graft-induced cold tolerance by promoting endogenous MeJA and H₂O₂ accumulation in watermelon. During early cold stress responses, MT induced ClRBOHD-dependent H₂O₂ accumulation, which elevated cytosolic free Ca²⁺ levels [10]. The increased Ca²⁺ further stimulated H₂O₂ generation via ClRBOHD, establishing a reciprocal positive-regulatory loop that synergistically enhanced cold tolerance [10]. H₂O₂ also plays a signaling role in trehalose-induced cold tolerance in melon seedling [99]. Additionally, H₂O₂ and ABA coordinately regulate proline homeostasis through a fine bidirectional closed-loop system under cold stress in melon seedlings [30]. The integration of redox signals with hormonal pathways (e.g., ABA-H₂O₂, MT-H₂O₂, IAA-H₂O₂, JA-Ca²⁺, and MT-Ca²⁺) suggests a multifactorial response architecture that has yet to be fully mapped in cucurbits.

3.3. Nitric Oxide (NO) and Hydrogen Sulfide (H₂S)

Nitric oxide and hydrogen sulfide function as pivotal signaling molecules in low-temperature responses, playing crucial roles in enhancing plant cold tolerance and alleviating postharvest cold damage in fruits [100,101,102,103]. Cold acclimation stimulates endogenous NO production, which modulates proline biosynthesis to improve freezing tolerance in Arabidopsis thaliana [104]. In cucumber, endogenous NO is primarily generated through two independent pathways: nitric oxide associated 1 (CsNOA1) and nitrate reductase (NR) [105]. The overexpression of CsNOA1 increased accumulation of soluble sugars and starch in leaves, upregulated CBF3 expression, and reduced the chilling injury index (CII) [105]. Short-term cold exposure significantly enhanced plasma membrane-bound nitrate reductase (PM-NR) activity and induced the expression of CsNR1, CsNR2 and CsNR3 [106]. Prolonged cold stress simultaneously enhanced cytoplasmic cytosolic NR activity and the expression of the amidoxime-reducing component gene (CsARC) [106]. Additionally, exogenous application of sodium nitroprusside (SNP, an NO donor) enhanced nitric oxide synthase-like (NOS-like) activity, as well as the content of glutathione (GSH) and polyamines (PAs) [107]. Conversely, treatment with buthionine sulfoximine (BSO, a glutathione synthetase inhibitor) significantly attenuated NO-mediated effects. These findings indicate that NO and GSH can synergistically improve the cold tolerance of cucumber seedlings [107].

H₂S improves plant stress resistance by enhancing antioxidant capacity and mitigating oxidative damage [108,109,110]. As a key component of sulfur and cysteine metabolism, endogenous H₂S is synthesized via enzymatic and non-enzymatic pathways [108]. Cold stress upregulated L-/D-cysteine desulfhydrase (L/D-CD) gene expression and activity, inducing the accumulation of endogenous H₂S [85]. Moreover, enhancement of plant cold tolerance induced by H₂S is closely related to the auxin pathway [85,86]. NaHS (an H₂S donor) treatment significantly increased both endogenous GSH levels and the GSH/GSSG ratio [111]. These changes accompanied by improved photosynthetic efficiency and enhanced chilling tolerance, suggesting GSH as a downstream signal in H₂S-induced plant cold tolerance [111]. Furthermore, exogenous H₂S treatment upregulated the expression of cucurbitacin C (CuC) biosynthesis genes, increased CuC content, and enhanced plant stress resistance [112]. MT is a key bioactive molecule that enhances plant cold tolerance. In watermelon and cucumber, NO and H₂S acted as downstream signals in MT-induced cold tolerance by activating the CBF signaling pathway [113,114]. Therefore, comprehensive investigation of hormone-signaling molecule crosstalk in cold tolerance regulation is essential to improve cold resistance in cucurbit seedlings. This represents a critical research priority for future studies.

4. Soluble Sugars Involved in Cold Stress Response in Cucurbits

In higher plants, various soluble sugars, such as glucose, sucrose, fructose, raffinose, stachyose, trehalose, and chitosan, are closely associated with cold tolerance [115]. These sugars act as osmoprotectants, regulating intracellular osmotic potential to lower the freezing point of cells [116]. They also interact with lipid bilayers to maintain membrane and cellular structure stability, thereby enhancing cold resistance [116].

Raffinose family oligosaccharides (RFOs), including raffinose, stachyose, and verbascose, serve as both osmoprotectants and desiccation protectants during abiotic stress [117,118]. Cold stress induced the accumulation of raffinose, stachyose, galactinol, and sucrose in the vacuoles of cucumber seedlings, with partial accumulation in the cytoplasm and chloroplasts, effectively lowering the freezing point of cell sap [119]. Galactinol synthase (GolS) catalyzes a key step in RFO biosynthesis [120]. In cucumber, CsGolS1 played a dual role in assimilate loading and cold stress response by increasing RFO concentrations in phloem sap, improving cucumber performance under cold stress [121]. Stachyose synthase (CsSTS) is crucial for phloem loading and carbohydrate transport in cucumber leaves [122]. Overexpression of CsSTS enhanced stachyose synthesis and transport, reduced starch accumulation, and significantly improved cold tolerance in cucumber seedlings [122].

In addition to RFOs, hexoses, such as glucose, fructose, and sucrose, also play a vital role in cold tolerance. In cucumber seedlings under cold stress, root hexose accumulation is linked to elevated invertase activity. Overexpression of CsVI1 could improve root growth, enhance vacuolar invertase activity, and promote glucose and fructose accumulation, significantly boosting cold tolerance [123]. CsSWEET2 encodes an energy-independent hexose/H⁺ uniporter; its overexpression significantly increased the glucose and fructose content in Arabidopsis thaliana, enhancing their cold tolerance [124]. Similarly, exogenous glucose irrigation elevated sucrose, fructose, and glucose levels, enhancing the cold tolerance of melon seedlings [125]. Moreover, glucose-mediated cold stress was achieved by regulating soluble sugars, ABA, and photosynthesis rather than utilizing the antioxidant enzyme system [125].

Furthermore, trehalose, fucoidan, and chitosan play important regulatory roles in enhancing the cold tolerance of cucurbits during the seedling stage and mitigating chilling injury during cold storage. Known as the “sugar of life”, trehalose enhanced cold tolerance by triggering antioxidant responses. In melon, trehalose treatment significantly induced the expression of the respiratory burst oxidase homolog (CmRBOHD) gene, generating extracellular H₂O₂ [126]. Moreover, the transcription factor CmTGA8 mediates this process, strengthening antioxidant defenses and cold tolerance [126]. Tan et al. [127] revealed that 50 mg·L⁻¹ chitosan effectively promoted cucumber seedling growth under cold stress by increasing chlorophyll content, photosynthetic capacity, osmoprotectants, and antioxidant enzyme activity while reducing electrolyte leakage (EL) and MDA accumulation. During postharvest storage, chitosan treatment regulates starch and sucrose metabolism in melons, reducing softening and chilling injury [128]. Fucoidan and chitosan also alleviate chilling injury in cucumbers and extend their shelf life by modulating ROS homeostasis and energy metabolism, extending the postharvest shelf life [129,130]. Consequently, sugar modulation not only improves osmotic status but also appears to regulate pathways such as ABA and ROS, indicating possible hormonal-metabolic feedback under cold conditions.

5. Molecular Regulatory Networks of Cucurbits in Response to Cold Stress

Chilling conditions during early spring severely limit the cultivation and yield of cucurbit crops, including melon, watermelon, and cucumber [8,9,30,131]. Elucidating the molecular regulatory networks underlying cold stress responses is essential for developing cold-resistant varieties. Although the specific receptors mediating cold perception in cucurbit crops remain unclear, conserved signaling pathways have been implicated in these processes.

5.1. CBF-Dependent Pathways

The CBF transcription factor acts as “molecular switch” in the plant cold response regulatory network [132,133], and their signaling pathway plays a critical role in the cold stress response of plants [134,135]. There are two primary cold-signaling mechanisms in plants: the CBF-dependent and CBF-independent pathways. The CBF-dependent pathway predominantly regulates cold tolerance through the ICE-CBF-COR regulatory cascade [21,22]. Upon cold stress, ICE transcription factors bind to the promoter of CBFs, activating their expression and subsequently inducing downstream COR, thereby enhancing plant cold tolerance [21,23]. In Arabidopsis thaliana, ICE1 undergoes post-translational modifications, including ubiquitination, SUMOylation, and phosphorylation, which collectively regulate its protein stability and DNA-binding capacity, thereby modulating CBF expression under cold stress [24,136,137,138,139].

Melon genomes encode four CmCBFs and five CmABFs. CmABF1 and CmCBF4 can bind to the CmADC promoter to activate its transcription, promoting putrescine accumulation and improving cold tolerance in seedlings [39]. Silencing CmRR6 or CmPRR3 significantly enhanced cold tolerance in melon seedlings, concomitant with upregulated expression of CmCBF1, CmCBF2 and CmCBF3, suggesting that these components may regulate cold response through the CBF-dependent pathway [140].

G protein-mediating signaling also contributes to plant cold resistance. In cucumber, CsGPA1 mediates BR signaling in response to cold stress, with the CsGPA1-CsCOR413PM2-Ca²⁺ axis regulating CsICE-CsCBF transcriptional activation under cold stress [51]. Conversely, CsSGR negatively regulates cold tolerance, as CsCBF1 can bind to dehydration-responsive elements (DREs) in the CsSGR promoter to activate its expression [141]. Furthermore, the heat-shock transcription factor HSFA1d promotes JA synthesis in cucumber under low-temperature conditions. The increase of JA levels in the plants triggers the degradation of the CsJAZ5 protein, releasing its interaction protein CsICE1, which activates the ICE-CBF-COR signaling pathway and improves the tolerance of cucumber plants to cold stress [62].

Ca²⁺, as an important second messenger, regulates diverse plant growth and development processes while participating in response to adverse conditions [142,143,144]. Various environmental stresses induce distinct spatiotemporal patterns of cytosolic Ca²⁺ concentration fluctuations. Cold stress rapidly elevates intracellular Ca²⁺ levels through the activation of Ca²⁺ influx channels, subsequently triggering a downstream phosphorylation cascade and transcriptional regulatory pathways (e.g., the CBF-dependent pathway) to enhance cold tolerance [10,145,146]. In watermelon, cyclic nucleotide-gated ion channel (CNGC) plays a critical role in mediating MT-, MeJA-, and NO-induced Ca²⁺ influx, enhancing cold tolerance [64,95,147]. Overexpression of the MT synthesis gene COMT1 increases the cytoplasmic Ca²⁺ concentration to enhance the cold tolerance, while silencing CNGC20 reduces this adaptive response. CaM7 negatively regulates cold resistance by directly interacting with CNGC20 [95]. MeJA activates the CBF regulatory pathway by regulating the CNGC channel to mediate Ca²⁺ signals [64].

5.2. Non-CBF Regulatory Factors

In addition to the CBF-dependent pathway, multiple regulatory factors, including receptor-like kinases, phosphatases, and transcription factors, have been demonstrated to participate in plant cold responses [148,149,150]. Recent evidence highlights CmPYL7, a cold-induced gene in melon, as a key regulator of cold tolerance via its interaction with the protein phosphatase CmPP2C24-like, implying that the ABA signaling pathway is involved in cold tolerance regulation in melon [38]. Targeting the ABA signaling pathway represents a promising strategy for enhancing cold tolerance in melon breeding programs [37]. Additionally, CmEAF7 has been shown to enhance both cold tolerance of seedlings and fruit quality in melon [151].

In watermelon, 57 ClWRKYs have been identified, among which ClWRKY20 exhibits the highest sensitivity to cold stress. Overexpression of ClWRKY20 enhanced the low-temperature tolerance of transgenic Arabidopsis thaliana [152,153]. Transcriptome analysis over time revealed that ClMYB14, which interacts with ClHSF1 and ClFAD1, functions as a cold-induced repressor in watermelon [154]. Although several genes like CmPYL7 or ClWRKY20 have shown positive functions under controlled conditions, their agronomic impact under field conditions and across different genetic backgrounds remains to be validated.

CmABFs participate in trehalose-mediated cold tolerance in melon. Except for CmABF4, all CmABFs respond to trehalose. Notably, CmABF2 and CmABF3 can bind to the ABRE motif in the promoter of CmPIP2;3, activating its expression to regulate aquaporin-mediated H₂O₂ translocation from apoplast to cytoplasm. This process facilitates downstream antioxidant responses and enhances cold tolerance in melon seedlings [99]. A novel CmTGA8-CmAPX1/CmGSTU25 cascade has been identified in melon’s trehalose-mediated cold stress response [126].

Cold stress promotes proline accumulation by upregulating the expression of proline synthesis genes CmP5CS1, CmP5CR, and CmOAT while simultaneously inhibiting the expression of its catabolic genes (CmProDH and CmP5CDH), thereby regulating cold tolerance in melon [30]. In Rosa multiflora, the C₂H₂-type zinc finger transcription factor RmZAT10 binds to and activates the promoter of RmP5CS to regulate cold resistance [155]. However, the specific regulatory mechanisms underlying cold tolerance in cucurbit crops remain to be fully elucidated.

5.3. Post-Transcriptional Regulation

Mitogen-activated protein kinase (MAPK) cascades represent a class of evolutionarily conserved protein kinases that amplify signal cascades through phosphorylation, thereby participating in plant biotic and abiotic stresses signal transduction pathways [156,157,158].

In Arabidopsis thaliana and rice, MAPK3, MAPK4, MAPK6, and their upstream regulators MKK2/4 have been implicated in cold stress response. In Arabidopsis, MAPK3/MAPK6 negatively regulates cold tolerance by interacting with and phosphorylating ICE1, thereby suppressing the expression of CBF genes [138,159]. Conversely, in rice, OsMAPK3 phosphorylates OsbHLH002/OsICE1, enhancing its stability to positively regulate cold tolerance [160]. In maize, ZmMAPK8 phosphorylates ZmRR1 to negatively regulate cold tolerance [161]. Previous research on watermelon demonstrates that ClTRX h2 interacts with ClMPKK5 to inhibit ClMPK3 phosphorylation, modulating the CBF-COR signaling pathway under cold stress [162]. A total of 14 CmMAPKs, 6 CmMAPKKs, and 64 CmMAPKKKs were identified in melon [163]. Differential expression analyses revealed that CmMAPK3 and CmMAPK7 exhibit elevated expression under drought, salt, SA, MeJA, redlight, and Podosphaera xanthii (P. xanthii) treatments compared to controls [163], although their specific responses to cold stress require further experimental validation.

The mitogen-activated protein kinase (MAPK/MPK) cascades serve as a central transducer of cold stress signals, while epigenetic reprogramming (e.g., histone modification) establishes a molecular memory of cold exposure [164,165,166]. Research on epigenetic regulation of cold stress responses in melon, watermelon, and cucumber remains limited. H3K4me3 has been identified as an activation marker of some stress-memory genes, promoting their transcription under abiotic stress [167]. The expression of some key H3K4me3 methyltransferase genes and the accumulation of H3K4me3 at memory genes depend on CsRBOH5.1. A transient increase in CsRBOH5.1 expression is a necessary condition for cucumbers to maintain NADPH activity and exosomal H₂O₂ content, thereby conferring cold tolerance and sustaining cold stress adaption [168]. The persistence of epigenetic memory (via H3K4me3) could explain the differential plasticity between cultivars in response to repeated chilling events, opening new avenues for its use in cucurbits molecular phenotyping.

6. Conclusions and Perspectives

Cold stress severely impairs normal plant growth and development, limits their geographical distribution, and negatively affects agricultural production. In this review, we have systematically examined the role of plant hormones, signaling molecules, soluble sugars, and key regulatory factors, as well as the molecular mechanisms underlying cold response, in melon, watermelon, and cucumber.

Table 1. The phytohormones and genes that regulate cucurbits’ (cucumber, melon, and watermelon) response to cold stress.

Phytohormone	Plant Species	Phytohormone Functions	Regulation of Cold-Tolerance Genes	References
Abscisic acid	Cucumber	Upregulates antioxidant enzyme activities; Reduces the chilling injury index, relative electrical conductivity, and MDA content; Positively regulates seedling cold tolerance		[8]
	Melon	Increases activities of SOD, CAT, and APX; Reduces membrane lipid peroxidation; Positively regulates chilling tolerance		[16]
	Melon	Increases endogenous GA4 and SA content; Positively regulates seedling cold tolerance		[29]
	Melon	Enhances activities of antioxidant enzymes (SOD, CAT, and APX) and limited H2O2; Reduces electrolyte leakage and MDA content; Increases proline and soluble sugar content; Positively regulates seedling cold tolerance	CmPYL7 and CmPYL6 positively regulate seedling cold tolerance; CmPP2C24-like negatively regulates seedling cold tolerance	[37,38]
	Melon	Positively regulates early-stage cold stress resistance	CmABF1/3/4/5, CmCBF1/2/4, and CmADC positively regulate seedling cold tolerance	[39]
	Watermelon	Induces antioxidant potential; Mediates grafting-induced cold tolerance		[28]
Brassinosteroids	Cucumber	Exogenous EBR upregulates endogenous EBR levels; Increases the activities of SOD, POD, GR, CAT, and APX; reduces ROS and MDA content; Positively regulates chilling tolerance		[15]
	Cucumber	Activates of enzymes in Calvin cycle; Increases the antioxidant capacity; Accelerates the recovery of PSII; Positively regulates seedling cold tolerance		[48]
	Cucumber	CsGPA1 positively regulates the brassinolide signal to affect cold stress	CsGPA1 and CsCOR413PM2 positively regulate seedling cold tolerance	[51]
	Cucumber	CsBPC2 is associated with BR signaling transduction	CsBPC2 positively regulates seedling cold tolerance	[54]
	Cucumber	Exogenous EBR promotes BR synthesis and expression of CsICE-CsCBF-CsCOR genes under cold stress; Positively regulates early-stage cold stress resistance		[55]
Jasmonic acid	Cucumber	CsHSFA1d positively regulates endogenous JA content after cold treatment; JA positively regulates seedling cold tolerance	CsHSFA1d positively regulates seedling cold tolerance	[62]
	Watermelon	Induces H2O2 accumulation and activates the antioxidant system; Positively regulates chilling tolerance		[63]
	Watermelon	Upregulates the expression of ClCNGC2 and ClCNGC20; Triggers Ca2+ influx; Positively regulates seedling cold tolerance	ClJMT, ClCNGC2, and ClCNGC20 positively regulate seedling cold tolerance	[64]
Salicylic acid	Cucumber	Increases antioxidant enzymes concentrations; Alleviates fruit chilling injury during cold storage		[75]
	Cucumber	Enhances actual photochemical efficiency, maximum photochemical efficiency, and photosynthetic rate; Decreases EL, MDA, and CI; Upregulates the expression level of COR genes; Improving the chilling tolerance of grafted cucumber		[76]
	Cucumber	Precise induction of cellular H2O2 levels; Enhances the expression of cold-responsive genes; Positively regulates seedling cold tolerance		[78]
	Cucumber	Decreases EL, H2O2, and O2− contents; Upregulates the expression of cold-responsive genes; Positively regulates the cold tolerance of grafted plants	CsPAL and CsNPR1 positively regulate the cold tolerance of grafted plants	[79]
	Cucumber	Stimulates the biosynthesis of ABA and H2O2; Upregulates the expression of CBF1, COR47, NCED, and RBOH1; Induces chilling tolerance in grafted cucumber plants		[80]
	Cucumber	Induces endogenous H2S content; Improves the activities and mRNA level of L-/D-cysteine desulfhydrase and antioxidant enzymes (SOD, POD, CAT, APX, and GR); Upregulates the expression of ICE, CBF1, and COR; Induces chilling tolerance of cucumber seedlings		[81]
Auxin	Cucumber	Endogenous IAA system is triggered by cold stress; Acts as a downstream signaling molecule in H2S-induced cold tolerance; Positively regulates early-stage cold stress resistance		[85]
	Cucumber	H2S regulates cold stress response by mediating auxin signaling; Positively regulates seedling cold tolerance	CsARF5 and CsDREB3 positively regulate seedling cold tolerance	[86]
	Cucumber	Decreases MDA and ROS contents; Upregulates the expression of cold response genes; Improves Pn, Jmax, Vcmax, P700(I/I0), and photosynthetic electron transport; Acts as a downstream signaling molecule in MT-induced cold tolerance; Positively regulates seedling cold tolerance	CsASMT and CsYUCCA10 positively regulate seedling cold tolerance	[87]
	Cucumber	Decreases CI, EL, and MDA content; Improves photosynthesis and the expression of COR genes under cold stress; Positively regulates chilling tolerance		[88]

summarizes the plant hormones, genes, and their functions in terms of cold stress tolerance in cucurbit crops.

While considerable progress has been made in elucidating the physiological mechanisms of cold stress responses in cucurbits, the molecular basis of these responses remains relatively underexplored compared to model plants such as Arabidopsis thaliana, rice, and tomato. Current knowledge is largely confined to a limited set of transcription factors, and the regulatory networks governing downstream target genes have not been comprehensively characterized (Figure 1). Although several cold-tolerance-related genes have been identified, their practical application in breeding remains limited, primarily due to the unclear breeding value of these genes, such as their contribution to cold-tolerance phenotype under different genetic backgrounds, potential pleiotropic effects, or linkage drag. Moreover, traditional hybrid breeding is inadequate for genetic manipulation of these environment-adaption-related genes [169].

With the completion of genome sequencing for cucurbit crops like cucumber, melon, and watermelon, there has been substantial progress in uncovering the molecular regulatory mechanisms that govern complex traits such as fruit shape, plant architecture, and fruit quality. These advancements have also shed light on the molecular mechanisms involved in cold stress responses. By fully utilizing the rich genomic data and genetic resources available for cucurbit crops, along with modern biotechnological and bioinformatic tools, future research can be enhanced in several key areas to achieve significant breakthroughs in understanding how these plants respond to cold stress: (1) Functional validation of candidate genes under real cultivation conditions. (2) Development of stable transformation systems in cucurbits. This allows us not only to validate the functions of identified cold-related genes within their native species, thereby revealing their biological roles, but also to directly perform genetic modifications (e.g., CRISPR-based gene editing) on target genes to assess their potential in breeding programs. (3) Integration of omics data into multiscale response networks. The integration of transcriptomics, epigenomics, and metabolomics will enable the design of cold response networks specific to species, organs, and phenological stages. This will advance our understanding of the environmental adaptation mechanisms in cucurbit crops and their genetic improvement.