Advances in the Molecular Regulatory Network of Low-Temperature Tolerance in Maize: Key Genes, Hormones, Signaling Pathways, and Genetic Improvement Strategies

Abstract

Maize (Zea mays L.) is a globally important crop, but its sensitivity to cold stress leads to substantial yield losses in temperate zones, where low temperatures, particularly below 10 °C, critically constrain cultivation in high-altitude and high-latitude regions. Recent progress has identified key cold-tolerance genes, dissected natural variations, elucidated signaling pathways, and enabled multi-omics network construction. Some studies have provided evidence for the proposed coupling of ZmICE1 with amino acid metabolism and COR gene expression, the association of COOL1 promoter variation with HY5 binding and high-latitude adaptation, and the involvement of HSF21 in lipid homeostasis without an apparent yield penalty. Several novel genes (ZmWAK3, ZmMAPK4, TIP4;3, ZmDUF966-10) have been identified as promising candidates, expanding the genetic reservoir for further validation. Mechanistically, an unconventional ZmBSK4-ZmMAPK4 cascade, calcium-MAPK crosstalk, histone modifications, and small RNA-mediated regulation have been implicated in cold responses, although the extent to which each mechanism has been experimentally validated varies. Integrative approaches (chromatin accessibility, WGCNA, multi-omics) have revealed hierarchical synergies among transcription factors, epigenetics, hormones, and metabolism. Favorable alleles (COOL1HapA, HSF21Hap1, and the TIP4;3 beneficial haplotype) represent potential targets for molecular design, while CRISPR/Cas9 offers opportunities to accelerate gene editing and pyramiding. This review synthesizes these advances, and offers a comprehensive framework for cold-tolerance breeding in maize.

1. Introduction

1.1. Severity and Urgency of Low-Temperature Stress in Maize

Maize (Zea mays L.) ranks among the most indispensable crops worldwide, serving as a primary source of food, animal feed, and industrial raw materials. Its sustained high productivity is therefore fundamental to global food security and the advancement of sustainable agriculture. In 2026, the Food and Agriculture Organization of the United Nations forecast a global maize harvest of 1.3 billion tons, with production spanning diverse agroecological zones from tropical to temperate latitudes [1,2]. As a thermophilic crop native to tropical and subtropical habitats, maize possesses inherently low tolerance to low-temperature stress. Low-temperature stress imposes detrimental effects on maize growth and development throughout its entire life cycle [3]. For instance, low-temperature stress (approximately 5 °C) significantly inhibited germination, growth, and survival, while increasing membrane permeability in maize seedlings of different breeding forms, with membrane permeability serving as a reliable physiological indicator for evaluating cold tolerance [4]. When temperatures drop below 10 °C, maize seedling growth slows markedly, and it stops entirely at 6–8 °C [5]. Moreover, cold tolerance is closely associated with the efficient modulation of sucrose/starch metabolism, as chilling-tolerant lines grown at 14 °C day/10 °C night maintain higher chlorophyll content, glucose-6-phosphate dehydrogenase activity, and sucrose-to-starch ratios under prolonged low-temperature stress [6]. Low-temperature conditions disrupt physiological and biochemical metabolism in maize, inhibiting seed germination, seedling establishment, photosynthetic efficiency, and reproductive development, with severe chilling causing plant mortality and substantial yield loss [7,8]. However, the molecular and metabolic mechanisms underlying maize acclimation to chilling remain poorly understood, particularly regarding the coordination among core regulatory components. Elucidating these mechanisms is therefore a prerequisite for developing cold-tolerant germplasm and enhancing grain yield.

Chilling injury in maize is usually caused by persistent low temperatures in the range of 0–15 °C [8]. From a physiological standpoint, low-temperature damage operates through four principal mechanisms: (i) reduction in membrane fluidity, accompanied by a phase transition of membrane lipids and consequent elevation of permeability; (ii) suppression of photosynthetic and respiratory activities, leading to perturbation of energy homoeostasis; (iii) overproduction of reactive oxygen species (ROS) and the ensuing oxidative injury; and (iv) inhibition of protein biosynthesis alongside induction of protein denaturation [9].

In the context of global climate change, the frequency of extreme temperature events—for instance, late-spring frosts—has increased markedly in northeast China and other high-latitude maize-growing regions [10,11]. Concurrently, the progressive northward (or southward) expansion of maize cultivation into high-latitude, colder regions has intensified the pressing need for chilling-tolerant cultivars [12]. Traditional breeding approaches, however, are hampered by the polygenic architecture of cold tolerance, as well as by genetic trade-offs between cold resilience and key agronomic traits such as early maturity and high yield [13]. Moreover, the accuracy of phenotypic selection remains vulnerable to environmental variability, and conventional evaluation of cold tolerance often necessitates destructive sampling, which precludes large-scale screening at early generational stages. Hence, dissecting the molecular regulatory networks that underpin cold tolerance pinpointing critical genes and favorable allelic variants, and implementing molecular design breeding strategies based on these genetic resources have become imperative to overcome the inherent limitations of conventional breeding process [14].

1.2. Research Background of Plant Low-Temperature Response

The response of plants to low-temperature stress is a multi-layered and complex process orchestrated through synergistic regulation, encompassing cold signal perception and transduction, transcriptional reprogramming, metabolic remodeling, and epigenetic modulation [15]. In Arabidopsis thaliana, the molecular underpinnings of low-temperature responses have been extensively elucidated: cold signals are initially perceived by plasma membrane-localized calcium channels and receptor kinases, which trigger a rapid rise in cytosolic Ca²⁺ concentration. This calcium transient subsequently activates calcium-dependent protein kinases (CPKs) and mitogen-activated protein kinase (MAPK) cascades. Following stepwise signal amplification, these pathways converge on the core ICE1–CBF–COR transcriptional regulatory module, which in turn induces broad expression of cold-responsive genes and enhances plant cold acclimation [16].

However, as a C4 crop, maize differs profoundly from the C3 model Arabidopsis in its low-temperature response mechanisms. First, the C4 photosynthetic pathway is intrinsically more vulnerable to low temperatures, and the activities of key C4 enzymes decline more sharply under chilling conditions [17,18]. Second, maize has undergone a relatively short domestication history, largely confined to warm regions, which has resulted in limited natural variation in cold-tolerance-related genes [19,20]. Third, the maize genome is large and characterized by extensive gene duplication and functional divergence, with many cold-responsive gene families having experienced notable expansion [21]. Consequently, the knowledge framework established in Arabidopsis cannot be directly transferred to explain the cold-response features unique to maize, underscoring the urgent need for systematic investigations specifically targeted at this crop [20].

Cold tolerance in maize is governed by a complex genetic architecture [20]. Although low-temperature exposure triggers differential expression of hundreds of genes, only a limited subset of major-effect loci and core regulatory hubs ultimately determines the natural variation in cold tolerance observed among diverse germplasm [22]. In recent years, the integration of genome-wide association study (GWAS), map-based gene cloning, transcriptome sequencing analysis, and gene editing-based functional verification has become a powerful technical system for mining maize cold-tolerance genes. Through these multi-omics and genetic validation approaches, a series of pivotal genes with practical breeding value for cold tolerance have been successfully identified and functionally characterized. These candidate genes cover diverse functional categories, including transcription factors, protein kinases, aquaporins, as well as functionally uncharacterized proteins with unique structural domains [20,23,24,25,26,27].

Although several previous reviews have addressed the physiological, genetic and breeding aspects of maize cold tolerance, most of them predate the recent wave of functionally validated genes and natural variants, and few have systematically integrated the newly identified modules, epigenetic regulation and multi-omics findings into a coherent regulatory framework. In particular, earlier reviews have not fully captured (i) recently identified and functionally validated cold-tolerance genes, such as ZmICE1, COOL1, HSF21, ZmWAK3, TIP4;3 and ZmDUF966-10, with emphasis on their molecular mechanisms rather than descriptive cataloguing [20,23,24,25,26,27]; (ii) the natural variation underlying cold adaptation, including elite haplotypes and their evolutionary and domestication history [20]; (iii) an integrated view of signaling networks, covering the atypical ZmBSK4–ZmMAPK4 cascade, calcium signaling, epigenetic and small-RNA regulation, and hormonal crosstalk [25]; and (iv) breeding applications, including molecular markers, gene pyramiding, genomic selection and the practical gap between molecular validation and cultivar development [28]. Centered on the main thread of “key gene discovery—signaling pathway dissection—genetic improvement application”, this review systematically synthesizes landmark progress in molecular research on maize cold tolerance over recent years. We highlight the identification of core cold-tolerance genes and the characterization of their natural allelic variations, summarize current advances in low-temperature signal transduction and regulatory networks, and evaluate genetic improvement strategies for cold resistance. In addition, we critically analyze existing bottlenecks and outline promising directions for future studies. Through this comprehensive synthesis, we aim to provide both theoretical insights and practical guidance for fundamental research and molecular breeding of chilling-tolerant maize.

2. Excavation of Key Genes and Dissection of Natural Variations

2.1. ZmICE1: A Dual-Function Regulator Coupling Transcriptional Control and Amino Acid Metabolism

ICE1 (INDUCER OF CBF EXPRESSION 1) functions as an upstream transcription factor within the evolutionarily conserved ICE–CBF–COR cold signaling cascade, and its orthologues are widely present across diverse plant species, including Arabidopsis, rice, and maize. Early work in Arabidopsis established that ICE1 specifically activates CBF gene transcription under low-temperature conditions [29]. Subsequent mechanistic studies refined the regulatory model of this pathway: at normal temperatures, ICE1 is subject to HOS1-mediated ubiquitination and subsequent proteasomal degradation; cold exposure, however, stabilizes the protein via SUMOylation, thereby sustaining downstream cold-responsive gene expression [30]. In contrast to this post-translational modification-centric regulation observed in Arabidopsis, maize ZmICE1 displays pronounced species-specific features, being governed by both transcriptional and post-translational controls. Whether this differentiated dual regulation is linked to the unique metabolic adaptability of the maize C4 photosynthetic system remains an open question that warrants further investigation.

To shed light on this open question, the functional properties of ZmICE1 were systematically dissected, uncovering its distinctive dual transcriptional roles during chilling responses in maize. Under low temperature, ZmICE1 directly binds to and trans-activates ZmDREB1, the maize orthologue of CBF, thereby launching the canonical COR-dependent cold-responsive transcriptional program and positively modulating cold tolerance. Concurrently, ZmICE1 represses transcription of the asparagine synthetase gene family (ZmASs), thus sustaining amino acid metabolic homoeostasis under cold stress. These findings establish that maize ZmICE1 operates as a bifunctional transcription factor, possessing both activating and repressing capabilities—a mode of action that expands the previously recognized regulatory repertoire of plant ICE1 proteins [23].

Asparagine occupies a pivotal position in the glutamate/asparagine metabolic pathway. Metabolomic analyses revealed that cold-induced upregulation of ZmASs leads to substantial accumulation of glutamate and asparagine, which in turn provokes a mitochondrial reactive oxygen species (mtROS) burst. Excessive mtROS further represses the cold-induced expression of ZmDREB1. Biochemical evidence suggests that surplus amino acids entering mitochondria generate reducing equivalents via dehydrogenases, promoting electron transport chain leakage and subsequent ROS production. ZmICE1 directly targets the promoter regions of ZmASs and suppresses their transcription, thereby blocking mtROS overaccumulation triggered by excessive glutamate/asparagine and establishing a favorable intracellular redox milieu for efficient ZmDREB1 transcription [23]. This regulatory circuit—encompassing a transcription factor, a metabolic enzyme, and ROS—underscores the central importance of amino acid metabolic homoeostasis in maize cold acclimation.

Natural allelic variation in the ZmICE1 promoter constitutes a major genetic determinant of differential cold tolerance among maize inbred lines. A genome-wide association study (GWAS) performed on 245 inbred lines identified SNP −465 in the ZmICE1 promoter as a key functional variant, with two allelic forms (A and C). The cold-tolerant haplotype, carrying the A allele, creates a cis-element recognized by ZmMYB39, facilitating its binding; the cold-sensitive C allele weakens this interaction. Binding of ZmMYB39 to the A-type promoter elevates ZmICE1 transcription and sustains higher ZmICE1 protein abundance. The resulting increase in ZmICE1 concurrently activates ZmDREB1 to initiate the canonical cold signaling cascade and represses ZmASs to mitigate mtROS burst, thus synergistically enhancing cold tolerance through dual regulatory outputs. Transgenic complementation assays demonstrated that overexpression of ZmICE1 significantly improves seedling survival under cold stress and alleviates membrane damage. Collectively, this work not only refines the regulatory framework of maize ZmICE1 but also validates the utility of the ZmICE1 promoter functional variants as molecular markers, offering tangible targets for marker-assisted selection of chilling-tolerant maize germplasm [23].

2.2. COOL1: A Genetic Switch Governing High-Latitude Cold Adaptation

2.2.1. GWAS Identification and Haplotype Analysis

Complementary to ZmICE1, which governs the internal molecular regulatory pathways of maize cold tolerance, COOL1 is a newly identified gene that uncovers the evolutionary and domestication basis underlying maize adaptation to northern high-latitude cold habitats [31]. Using a diverse panel of 205 maize inbred lines encompassing tropical, subtropical, temperate, and cold-temperate germplasms, genome-wide association analysis successfully pinpointed COOL1 (COLD-RESPONSIVE OPERATION LOCUS 1). This gene is positioned on the short arm of chromosome 3 and encodes a basic helix-loop-helix (bHLH) transcription factor that participates in cold response modulation. Its cold-tolerant allele COOL1 HapA reaches 88% frequency in American land-races and rises sharply with increasing latitude, becoming nearly fixed in the United States. Previous studies have demonstrated that COOL1 represents a highly promising target for molecular design breeding of cold-resistant maize [20].

Low-temperature stress significantly represses the transcriptional level of COOL1, and the magnitude of such repression is tightly associated with the cold tolerance of different maize genotypes. Functional characterization has validated COOL1 as a negative regulator of maize cold tolerance. Higher transcript accumulation of COOL1 renders maize plants more vulnerable to cold damage. In contrast, COOL1 loss-of-function mutants generated via EMS mutagenesis or CRISPR/Cas9 editing exhibit markedly improved seedling survival at 4 °C relative to wild-type controls [20].

Nine single-nucleotide polymorphisms located in the promoter region of COOL1 stratify natural maize populations into two major haplotypes, namely HapA and HapB. Population haplotype analysis showed that HapA accessions are predominantly distributed in high-latitude frigid regions, including Northeast [20].

2.2.2. HY5-Mediated Integration of Light Signaling

Recent research, however, indicates that COOL1 integrates light and calcium signaling to fine-tune maize cold tolerance. Molecular dissection further revealed that natural sequence polymorphisms in the COOL1 promoter modulate the binding affinity of HY5 (ELONGATED HYPOCOTYL 5), a core transcription factor in light signalling, for A-box cis-elements. The physical interaction between HY5 and the A-box region of the COOL1 promoter was corroborated by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Notably, the nucleotide combinations characteristic of the HapA promoter remodel the sequence context surrounding the A-box, substantially enhancing HY5 binding [20].

In high-latitude regions, where spring daylight duration progressively increases under low-temperature conditions, light signaling activates HY5, leading to elevated protein abundance and activity. Enhanced HY5 binding to the HapA promoter represses COOL1 transcription under low-temperature stress, thereby alleviating its inhibitory effect on downstream targets and ensuring proper cold-induced expression of ZmDREB1 and ZmTPS (trehalose-6-phosphate synthase). This cascade ultimately culminates in improved cold tolerance. By contrast, the weaker HY5–promoter interaction in HapB lines fails to adequately repress COOL1 transcription during cold exposure. The consequent accumulation of COOL1 protein interferes with ZmDREB1 induction, rendering HapB inbred lines more susceptible to chilling injury [20].

Additional work has implicated the calcium-dependent protein kinase CPK17 in the regulatory hierarchy governing COOL1 activity. Upon cold stimulation, CPK17 translocates to the nucleus and phosphorylates COOL1, which impedes its degradation by the 26S proteasome and thus stabilizes the protein. This calcium-signaling module, acting in concert with the HY5-mediated light-signaling pathway, constitutes a multilayered regulatory network that integrates multiple inputs to determine the overall magnitude of the cold-response program in maize [20].

2.2.3. Adaptive Evolution from Teosinte to Modern Maize

Evolutionary analyses have traced the origin of the COOL1 HapA haplotype not to modern breeding efforts but to teosinte, the wild progenitor of maize. By genotyping teosinte, Mexican grass, and thousands of pre-Columbian maize landraces, researchers have reconstructed the evolutionary history of COOL1 allelic variants. Although the HapA allele occurs at low frequencies in wild teosinte populations, it has been subject to positive selection and has progressively accumulated in temperate and cold-temperate maize germplasm as cultivation expanded into high-latitude regions—an outcome driven by its adaptive advantage under chilling conditions [31].

The discovery of COOL1 represents a milestone in several respects: it not only provides a superior cold-tolerant allele (HapA) with direct applicability to maize breeding, but also offers the first molecular framework that integrates light and cold signaling to govern adaptation to high-latitude environments. Moreover, functional molecular markers developed from key polymorphic sites within the COOL1 promoter provide practical tools for the efficient screening of cold-resistant maize germplasm.

2.3. HSF21: A Novel Link Between Lipid Metabolic Homeostasis and Cold Tolerance

2.3.1. Functional Expansion from Heat to Cold Stress Response

Heat shock transcription factors (HSFs) have long been recognized as primary regulators of heat stress responses, maintaining cellular protein homeostasis via modulating heat shock proteins (HSPs) [32]. Cumulative research confirms that the HSF family broadly participates in responses to diverse abiotic stresses, emerging as central integrators of plant stress signaling networks [33]. Most Class B HSFs act as transcriptional repressors in heat-response pathways. However, recent research revealed that Class B transcription factor HSF21 (HEAT SHOCK TRANSCRIPTION FACTOR 21) functions as a critical positive regulator of maize cold tolerance [24]. This discovery expands the functional spectrum of the HSF family, suggesting evolutionary sharing of partial regulatory modules between heat and cold response pathways and offering novel clues for dissecting cross-stress regulatory networks of temperature.

2.3.2. The bZIP68–HSF21 Regulatory Module

A genome-wide association study (GWAS) conducted across diverse natural maize populations uncovered a superior cold-tolerant allelic variant, designated HSF21 Hap1, which harbors a −683 bp SNP and a −693 bp 5-bp InDel within its promoter. These polymorphisms alter the sequence of the A-box cis-element recognized by bZIP68, leading to a pronounced increase in promoter transcriptional activity. Molecular interaction assays further established that HSF21 expression is transcriptionally repressed by bZIP68 in cold-sensitive inbred lines, with electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) confirming direct binding of bZIP68 to the A-box region of the HSF21 promoter [24].

Notably, the key sequence variations defining the HSF21Hap1 promoter fall within the core bZIP68-binding domain, thereby weakening the physical interaction and relieving the transcriptional suppression of HSF21. Under low-temperature conditions, efficient cold induction of HSF21 occurs, and the resulting nuclear-localized HSF21 protein directly activates the transcription of core lipid-metabolism genes, including GPATs, SADs, and FADs. The composition of unsaturated fatty acids in cellular membranes critically determines membrane fluidity at low temperatures, representing a fundamental physiological determinant of plant cold resistance [34,35]. By orchestrating lipid metabolic reprogramming, HSF21 sustains membrane lipid homoeostasis under low-temperature stress and thereby enhances cold tolerance in maize.

2.3.3. Molecular Basis Coordinating Cold Tolerance and High Yield

A long-standing and universal challenge in crop stress breeding is the genetic trade-off between stress tolerance and agronomic performance: constitutive overexpression of most stress-responsive genes tends to penalize vegetative growth and reduce grain yield [36]. Field trials with transgenic lines, however, showed that overexpression of HSF21 not only significantly improves seedling cold tolerance but also increases grain yield under optimal temperature conditions, with no observable yield penalty. Lipidomic analyses confirmed that HSF21 upregulates unsaturated fatty acid biosynthetic pathways and helps stabilize membrane architecture under low-temperature stress [37].

These findings demonstrate that the antagonism between stress resistance and yield is not insurmountable; rather, precise modulation of lipid metabolic homeostasis can reconcile cold tolerance with yield formation. As a rare natural allelic variant that confers both enhanced cold tolerance and yield stability under favorable conditions, HSF21Hap1offers valuable genetic resources for breaking the trade-off between stress resilience and productivity. It also represents a promising candidate gene and a directly usable germplasm resource for molecular design breeding aimed at developing cold-resistant maize varieties [37].

2.4. ZmWAK3: A Potential Regulatory Locus for Stress Responses Mediated by Wall-Associated Kinase

2.4.1. Gene Characteristics and Stress-Responsive Clues of ZmWAK3

Wall-associated kinases (WAKs) are plant-specific receptor-like kinases localized at the cell wall-plasma membrane interface [38]. They sense alterations in cell-wall structural integrity and trigger intracellular signaling cascades, serving as critical molecular hubs linking extracellular environmental stimuli to intracellular stress-adaptive responses, and play vital roles in multiple plant abiotic-stress adaptations [39]. In maize, WAK family members are extensively involved in transcriptional reprogramming under diverse abiotic stresses including drought, salinity and temperature fluctuations, representing promising candidate gene families for dissecting stress-sensing mechanisms in maize [40].

ZmWAK3 is a typical member of the maize WAK family, encoding an intact membrane-bound receptor protein containing an extracellular WAK domain, a transmembrane domain, and an intracellular serine/threonine-protein kinase domain, possessing the conserved structural basis for extracellular signal perception, transmembrane signal transduction and intracellular phosphorylation activation of WAK family proteins. Previous studies have validated the definite biological function of ZmWAK3 under drought stress: it acts as a negative regulator of seedling-stage drought tolerance in maize and mediates drought responses by modulating cell-wall pectin metabolism and stomatal dynamics [41].

Genome-wide transcriptomic analyses of the maize WAK family indicate that ZmWAK3 expression is modulated by temperature stress and exhibits distinct tissue-specific expression preferences. These observations imply that, beyond drought adaptation, ZmWAK3 may also participate in cold-stress signal perception. Further studies have demonstrated that genetic evidence from overexpression lines and loss-of-function mutants confirms that ZmWAK3 directly governs maize cold tolerance, and its exact biological roles in maize cold-response pathways have been further genetically validated [42].

2.4.2. The ZmNAC100-ZmWAK3 Transcriptional Regulatory Module

Transcriptional dissection identified the NAC-family transcription factor ZmNAC100 as the core upstream activator driving cold-induced expression of ZmWAK3. Multiple molecular interaction assays including yeast one-hybrid, dual-luciferase reporter and ChIP-qPCR validated specific binding of ZmNAC100 to cis-elements in the ZmWAK3 promoter to directly activate its cold-triggered transcription. ZmNAC100 itself is strongly induced by low temperature; overexpression lines display enhanced cold tolerance while gene-silenced lines show cold-sensitive phenotypes highly consistent with ZmWAK3 mutants, confirming a tight upstream-downstream transcriptional regulatory module [42].

Integrative physiological and transcriptomic analyses further revealed that this module orchestrates maize cold adaptation through multiple convergent pathways. On the one hand, it strongly upregulates the expression of key antioxidant enzyme genes, including SOD, POD, CAT, and APX, thereby reinforcing ROS scavenging capacity and alleviating oxidative damage and membrane injury triggered by cold exposure. On the other hand, it sustains the expression of genes central to photosynthetic function, preserving photosynthetic electron transport efficiency and the structural integrity of the photosynthetic apparatus, thus mitigating the suppressive effects of low temperature on photosynthetic metabolism. Collectively, these findings delineate a mechanistic framework for WAK-mediated cold responses in maize and establish a previously unrecognized cold-regulatory cascade—spanning cell-wall signal perception, transcriptional activation, and homoeostatic control of antioxidant and photosynthetic systems—thereby substantially enriching our current understanding of the cold-signal sensing network in this crop [42].

2.4.3. Stress-Specific Functional Divergence of ZmWAK3

An intriguing observation is that ZmWAK3 exerts opposite effects on stress responses depending on the nature of the challenge: it acts as a positive regulator under cold stress, yet functions as a negative regulator under drought conditions. Under water deficit, ZmWAK3 enhances polygalacturonase activity, which reduces pectin content, relaxes the cell wall, and modulates stomatal aperture, ultimately leading to impaired seedling drought tolerance. Its drought-induced expression is directly activated by the transcription factor ZmWRKY44 [41]. This functional duality of ZmWAK3 across different stresses underscores the high environmental specificity of WAK-mediated signaling pathways. Consequently, in breeding applications, genetic manipulation strategies must be tailored to the target stress type, rather than relying on a one-size-fits-all approach [42].

2.5. TIP4;3 and ZmDUF966-10: Functional Characterization of Novel Regulatory Factors

2.5.1. TIP4;3: Epigenetic Regulation of Aquaporins

Aquaporins are core membrane proteins that regulate intracellular water transport, osmotic homoeostasis, and stress adaptability, and have been broadly implicated in maize responses to cold, drought, and other abiotic challenges. Among them, the tonoplast-localized aquaporin encoded by TIP4;3 has been identified as a key negative regulator of cold tolerance during seed germination and seedling establishment. Under low-temperature conditions, TIP4;3 transcript abundance is markedly higher in cold-sensitive inbred lines than in tolerant ones, and its expression level correlates negatively with cold resistance. Disruption of TIP4;3 via CRISPR/Cas9-mediated knockout significantly improves both germination efficiency under cold and seedling survival, confirming its repressive function in maize cold tolerance [26].

Analysis of natural genetic variation revealed that the insertion of a CACTA-family MITE transposon within the TIP4;3 promoter constitutes a key functional polymorphism underlying differential cold tolerance among maize populations. This transposon insertion substantially elevates DNA methylation levels in the promoter region, leading to epigenetic suppression of TIP4;3 transcription and providing a genetic basis for cold-tolerant germplasm in natural populations. Mechanistic dissection has further uncovered two distinct pathways through which TIP4;3 negatively modulates cold adaptability. First, TIP4;3 localizes to the tonoplast of guard cells, where it modulates vacuolar water homoeostasis and influences stomatal opening, thereby accelerating water loss under low temperature and exacerbating chilling injury. Second, TIP4;3 promotes excessive ROS accumulation by modulating NADPH oxidase activity, which in turn represses the cold induction of core DREB-family cold-responsive genes and hinders activation of the canonical ICE–CBF cold cascade, ultimately compromising plant cold acclimation [26]. Collectively, these findings broaden our understanding of aquaporin functions in maize cold responses and integrate epigenetic regulation, ROS homoeostasis, and cold-signal crosstalk into a more comprehensive framework of the multilayered molecular network governing cold tolerance in maize.

2.5.2. ZmDUF966-10: A Novel Member of the Unknown Functional Domain Family

The DUF966 family comprises plant-specific proteins whose biological functions remain largely uncharacterized, and their involvement in stress responses—particularly in maize cold adaptation—has been poorly documented. A genome-wide identification and sequence analysis revealed that the promoters of maize ZmDUF966 family members are enriched with cold- and abscisic acid (ABA)-responsive cis-elements, hinting at a possible role in cold adaptation through ABA signaling cascades. Transcriptional profiling identified ZmDUF966-10 as the predominant family member responsive to low temperature, with stable cold-induced upregulation observed in both leaves and roots. Post-transcriptional regulation analysis further showed that ZmDUF966-10 is targeted by the conserved microRNA zma-miR159, which exerts negative translational control and contributes to multilayered regulation of its expression. Protein–protein interaction assays confirmed physical association between ZmDUF966-10 and regulatory proteins implicated in ABA- and osmotic stress-related signaling, providing initial evidence that it functions as an auxiliary component of ABA cascades in mediating cold responses in maize [27]. Collectively, this work adds a new case study to the scarce literature on DUF966 family members in plant cold responses and expands our understanding of cold regulatory mechanisms governed by proteins with previously unknown functional domains.

2.6. Other Newly Identified Cold-Tolerance Genes and Natural Variations

2.6.1. ZmNTL1 and ZmNTL5: Dual-Functional NAC Transcription Factors

Membrane-anchored NAC transcription factors represent a major gene family involved in plant abiotic stress responses, with documented functions in signal transduction and adaptation to cold, drought, and salinity. Through cold transcriptome screening coupled with genetic functional validation, two NAC-family regulators—ZmNTL1 and ZmNTL5—were identified as positive modulators of cold tolerance in maize. These two highly homologous proteins localize to the nucleus and are markedly upregulated by low-temperature exposure [43]. Loss-of-function double mutants of ZmNTL1 and ZmNTL5 exhibited drastically impaired survival under chilling conditions, confirming their synergistic and positive contribution to cold adaptation.

Transcriptome enrichment analysis revealed that ZmNTL1 and ZmNTL5 primarily govern two core cold-adaptive pathways—osmoprotectant biosynthesis and ROS scavenging—while also contributing to cell wall remodeling. Accordingly, these transcription factors modulate the expression of key cold-responsive target genes, including P5CS, TPS, SOD, and POD, as well as cell wall-related genes such as XTH and EXP. By promoting the accumulation of osmoprotective compounds, eliminating excessive ROS, and fine-tuning cell wall structural homeostasis, they orchestrate a multidimensional and synergistic cold-regulatory network. This work not only expands the functional repertoire of membrane-bound NAC factors in low-temperature responses but also provides valuable genetic resources and regulatory targets for molecular design breeding of cold-resistant maize [43].

2.6.2. ZmbHLH30: A Critical Regulator of Cold Tolerance During Seed Germination

Low temperature during germination imposes a major constraint on seedling emergence and establishment in spring maize production regions. Through QTL mapping coupled with fine screening in recombinant inbred line (RIL) populations derived from a cross between cold-tolerant and cold-sensitive inbred lines, a major-effect QTL for germination-stage cold tolerance was delimited to chromosome 10, and the underlying causal gene was identified as ZmbHLH30. Functional validation confirmed that ZmbHLH30 acts as a positive regulator of cold tolerance across germination, sprouting, and seedling stages; transgenic lines overexpressing ZmbHLH30 displayed markedly improved germination efficiency and seedling growth under chilling conditions compared with wild-type controls [44].

Integrated multi-omics analyses revealed that ZmbHLH30 enhances cold resistance through synergistic activation of antioxidant defense and osmotic adjustment systems. Specifically, it strongly upregulates the expression of antioxidant enzyme genes, thereby reinforcing ROS scavenging capacity, while simultaneously promoting the accumulation of proline, soluble sugars, and other osmoprotective compounds to stabilize cellular osmotic potential and membrane integrity under low temperature, collectively alleviating chilling injury. Subcellular localization and heterologous expression assays further pointed to species-specific regulatory features of ZmbHLH30, providing fresh insights into the mechanistic basis of cold regulation in monocots [44].

2.6.3. Other Candidate Genes

In addition to the aforementioned core regulators, recent studies have uncovered a growing number of novel genes and pathways involved in cold responses, further enriching the genetic repertoire for cold tolerance in maize. One such example is ZmBARK1, a kinase associated with brassinosteroid (BR) signaling, which negatively modulates cold tolerance during germination and early seedling development. Loss-of-function mutants of ZmBARK1 exhibit significantly improved germination capacity and seedling survival under low-temperature conditions, positioning this gene as an important negative regulator governing chilling adaptation at early developmental stages [45].

2.7. Summary

Taken together, the key chilling-tolerance genes characterized in this section can be grouped into five functional classes: transcriptional regulators (ZmICE1, COOL1, HSF21, ZmNAC100, ZmNTL1/5, and ZmbHLH30); membrane sensors and signal transducers (ZmWAK3); metabolic homoeostasis modulators (ZmICE1 and HSF21); osmotic and water balance controllers (TIP4;3); and proteins with as-yet-uncharacterized domains (ZmDUF966-10) (Table 1) [20,23,24,25,26,27,43]. Collectively, these components form a multilayered regulatory network that governs the cold-response program in maize, with extensive cross-talk among distinct pathways. ROS homoeostasis serves as a hub linking metabolic and transcriptional outputs; light and cold signals converge through COOL1; and multiple cascades operate in concert to shape the overall chilling-tolerance phenotype [20]. This growing repertoire of functionally validated genes provides a rich repository of precisely defined targets for molecular design breeding aimed at enhancing cold resistance in maize.

Table 1. Summary of Functions, Natural Variations and Breeding Applications of Cloned Core Maize Cold-Tolerance Genes.

Gene Name	Encoded Protein Type	Regulatory Function	Key Natural Variations	Core Regulatory Pathways	Breeding Advantages/Limitations	References
COOL1	bHLH transcription factor	Negative	9 promoter SNPs forming HapA/HapB	HY5 light signaling + CPK17 calcium signaling	HapA adapts to high-latitude cold regions; wild-origin allele may link with unfavorable agronomic traits	[20]
ZmICE1	bHLH transcription factor	Positive	Promoter SNP -465 A/C	ICE1-ZmDREB1/ZmASs amino acid metabolic circuit	No yield penalty, suitable for seedling improvement; effects dependent on genetic background	[23]
HSF21	Class B heat shock transcription factor	Positive	Promoter SNP + 5-bp InDel forming Hap1	Lipid metabolic pathways (GPAT/FAD)	Rare allele simultaneously enhancing cold tolerance and yield, eliminating stress-yield trade-offs	[24]
TIP4;3	Tonoplast aquaporin	Negative	CACTA transposon insertion triggering methylation-mediated silencing	Water homeostasis + ROS suppression of DREB cascades	Transposon markers enable easy screening; potential reduction in water use efficiency	[26]
ZmDUF966-10	Protein with unknown functional domain	Positive	None identified	miR159-ABA osmotic signaling pathway	Limited research foundation, unsuitable for large-scale breeding at present	[27]
ZmWAK3	Wall-associated receptor kinase	Positive	No major-effect natural variations	ZmNAC100-mediated antioxidant and photosynthetic homeostasis	Function diverges across stresses; cannot simultaneously improve cold and drought resistance	[41,42]
ZmNTL1/5	Membrane-bound NAC transcription factor	Positive	None identified	Osmoprotectant accumulation + ROS scavenging + cell wall remodeling	Synergistic effects; dual-gene pyramiding generates stronger cold tolerance	[43]

These candidate genes have all been implicated in cold tolerance, yet the evidence supporting them is uneven in both quality and type. ZmICE1, COOL1, HSF21 and TIP4;3 are supported by GWAS or population-level natural variation evidence, whereas ZmWAK3, ZmNTL1/5, ZmbHLH30 and ZmBARK1 rest mainly on QTL mapping or reverse-genetic approaches and have not been validated at the population scale [20,23,24,25,26,27,43,44,45]. Most transcription-factor-centered modules have been biochemically validated for protein–DNA or protein–protein interactions, but comparable evidence is still lacking for several membrane-localized or uncharacterized-domain proteins. Among all candidates, only HSF21 has been subjected to limited field evaluation for yield performance, while most phenotypic validations remain confined to controlled-environment seed-ling or germination assays [32]. Caution is therefore warranted when extrapolating laboratory-derived mechanistic findings directly to field breeding applications. Nevertheless, this expanding set of functionally validated genes constitutes a valuable resource of well-defined targets for molecular design breeding aimed at enhancing cold resistance in maize.

3. Multi-Layered Molecular Regulatory Networks Governing Maize Cold Responses

3.1. The Atypical MAPK Cascade: ZmBSK4–ZmMAPK4–ZmbHLH111/181

3.1.1. Intrinsic Limitations of Canonical Three-Tier MAPK Cascades

Canonical MAPK signaling cascades operate through a three-tiered phosphorylation relay—MAPKKK→MAPKK→MAPK—that amplifies stress signals via sequential phosphorylation events. Although this stepwise architecture is well suited for signal amplification and specificity, its multi-step nature may limit the speed of signal transmission, potentially constraining the plant’s capacity for rapid responses to abrupt cold episodes such as late-spring frosts. In maize, nine MAPKK-encoding genes were identified, and subsequent genome annotations have not substantially expanded this gene family [44]. This relatively limited repertoire of upstream kinases may not fully accommodate the diverse and specific inputs triggered by low-temperature stress. These considerations have led to the hypothesis that maize may possess additional, atypical MAPK activation routes that bypass the conventional MAPKK-dependent intermediate steps, thereby providing an extra rapid signaling mechanism that operates along-side—rather than necessarily replacing—canonical MAPK cascades [46].

3.1.2. Molecular Mechanism of Direct ZmBSK4-Mediated ZmMAPK4 Activation

AMAPKK-independent activation cascade for MAPKs, thereby expanding the known regulatory repertoire of these kinases in plant cold responses. Under chilling conditions, the brassinosteroid-signaling kinase ZmBSK4 directly associates with ZmMAPK4 and phosphorylates it specifically at Ser-171. Importantly, phosphorylation at this single residue is sufficient to fully activate ZmMAPK4 kinase activity, bypassing the requirement for MAPKK-mediated phosphorylation. In vitro kinase assays demonstrated that purified ZmBSK4 exhibits catalytic efficiency towards ZmMAPK4 comparable to that of canonical MAPKK kinases. Time-course experiments under cold stress revealed that ZmMAPK4 phosphorylation occurs rapidly at early time points, with response kinetics substantially faster than those of conventional three-tier cascades. This rapid activation provides a molecular basis for timely defense against abrupt chilling injury in maize [25].

3.1.3. ZmbHLH111/181-Mediated Transcriptional Activation of Downstream Cold-Responsive Genes

Upon activation, ZmMAPK4 directly phosphorylates two downstream bHLH transcription factors, ZmbHLH111 and ZmbHLH181. Mass spectrometry combined with in vitro phosphorylation assays identified the modified residues: Thr-42 and Ser-48 in ZmbHLH111, and Thr-38 and Ser-44 in ZmbHLH181. Phosphorylation at these sites markedly promotes nuclear translocation of both factors. Once inside the nucleus, they bind specifically to E-box (CANNTG) cis-elements in the promoters of cold-responsive genes, thereby inducing the expression of core chilling-tolerance genes, including ZmDREB1.2, ZmDREB1.4, ZmDREB1.9, ZmDREB1.10, SOD, and POD. This transcriptional output synergistically enhances seedling cold adaptation. The positive regulatory role of this pathway was corroborated by a series of indoor cold-phenotype assays using transgenic materials: overexpression lines of ZmBSK4 and ZmMAPK4 exhibited reduced cold injury and improved seedling survival, whereas ZmbHLH111 knockout mutants displayed aggravated chilling damage [25]. In addition, canonical MAPKKs are also known to participate in maize cold responses, and their functional framework provides a useful comparative context for this atypical cascade.

3.2. Calcium Signaling in Cold Responses: Conserved Modules, Maize-Specific Branches, and MAPK Crosstalk

Calcium-dependent protein kinases (CDPKs) are unique in that they integrate Ca²⁺-binding and kinase catalytic domains within a single polypeptide, positioning them as central hubs that translate calcium signals into transcriptional outputs. In Arabidopsis, cold-activated CPK28 phosphorylates NLP7, promoting its nuclear translocation and subsequent activation of CBF/COR transcription, thereby affecting cold tolerance. The maize genome contains homologous CPK28 and NLP7 sequences, suggesting potential conservation, but direct in planta evidence in maize is lacking [47]. This module should therefore be regarded as an Arabidopsis-derived candidate pathway rather than a validated maize cold-regulatory mechanism.

In contrast to this conserved candidate module, a maize-specific calcium regulatory branch has been functionally dissected. Cold-evoked calcium signals activate CPK17 kinase and trigger its translocation to the nucleus. Once inside, the activated CPK17 physically associates with COOL1 and phosphorylates it at Thr-124, a residue identified by LC-MS/MS; substitution of this site markedly attenuates CPK17-mediated phosphorylation. This phosphorylation event reduces the recognition and subsequent degradation of COOL1 by the 26S proteasome, thereby elevating COOL1 protein ac-cumulation. Given that COOL1 functions as a negative transcriptional regulator of cold tolerance, its increased abundance represses the transcription of core cold-resistance genes, including ZmDREB1 and ZmTPS, ultimately compromising cold adaptation [20]. This regulatory circuit exemplifies how calcium signaling fine-tunes the intensity of cold responses by stabilizing a negative regulator, thus establishing a maize-specific calcium-signaling balancing cascade.

Beyond these calcium-sensing modules, calcium signaling does not operate in isolation; instead, it engages in extensive cross-talk with both the atypical ZmBSK4–MAPK cascade and the canonical three-tier MAPK cascades at multiple levels. In model systems such as Arabidopsis, calcium signals often serve as upstream switches that modulate the activity of MAPKKK–MAPKK–MAPK kinase hierarchies [25]. Whether this calcium–MAPK crosstalk operates in the same manner in maize is less clear, and direct evidence for the interconnection of these layers in maize is still limited. The interconnected layers of calcium perception, MAPK-mediated signal amplification, and bHLH-dependent transcriptional regulation have been proposed to constitute the complete cold-signal transduction network in maize, but further in planta validation is required to confirm this framework.

3.3. Systematic Identification of Core Cold-Responsive Gene Sets and Co-Expression Networks

3.3.1. Overview of Shared Core Genes Across Multiple Stresses

Maize deploys a set of shared core stress-responsive genes when challenged by cold, drought, salinity, and other abiotic stresses. Integrative analyses of multi-stress transcriptomic datasets have pinpointed the AP2/ERF, NAC, bZIP, HSF, and C2C2-CO-like families as the predominant transcription factor groups orchestrating stress regulation. Operating as upstream signal switches, these transcription factors drive the widespread expression of downstream functional genes, and typically exhibit larger expression fluctuations than their target genes—a pattern consistent with signal amplification at the transcriptional level [48].

From the perspective of cold-regulatory cascades, cold-responsive genes in maize can be divided into two major categories: those operating through CBF-dependent pathways and those functioning independently of CBF. The CBF/DREB1 cascade constitutes the core cold-acclimation branch, while supplementary routes mediated by NAC and bHLH transcription factors also contribute to cold transcriptional reprogramming [48]. The coordinated action of both classes of pathways ensures successful cold adaptation in maize—a layered regulatory architecture that has been validated across species, including Arabidopsis and maize.

3.3.2. The “Core–Satellite” Hierarchical Co-Expression Network Model

Weighted gene co-expression network analysis (WGCNA) of transcriptomic datasets derived from cold-stressed maize consistently reveals a “core–satellite” topological architecture. In this framework, core transcription factors establish strong co-expression links with a broad set of stress-responsive genes, while maintaining only weak connections with stress-specific satellite genes, collectively giving rise to multilayered regulatory networks. The core hubs—including ZmERF, ZmNAC, and ZmbZIP—integrate diverse environmental signals, whereas satellite genes execute physiological functions tailored to particular stresses. This network topology confers a degree of buffering capacity against environmental challenges; the functional redundancy and multiplicity of core nodes ensure a basal level of stress tolerance, thereby providing a critical genetic foundation for polygenic resistance traits [48].

3.3.3. Developmental Stage Specificity of Maize Cold Responses

Maize deploys distinct regulatory strategies in response to low temperatures at different developmental stages. During seed germination, stress-responsive genes are significantly enriched in pathways related to ABA and gibberellin (GA) hormone metabolism, as well as cell wall remodeling [44]. In contrast, seedlings predominantly activate cascades that govern antioxidant enzymes (including SOD, POD, and CAT), alongside the accumulation of proline, trehalose, and other osmoprotective metabolites [48].

This stage-specific transcriptional divergence dictates distinct priorities for breeding improvement. Enhancing cold tolerance at the germination stage centers primarily on the amino acid metabolic cascade mediated by ZmICE1, whereas improving seedling cold resistance focuses on the COOL1 light–temperature module and the atypical ZmBSK4–ZmMAPK kinase cascade [20,23]. In a related study, seedling-stage chilling phenotypes were evaluated, and GWAS was performed to identify multiple candidate genes and molecular markers associated with seedling cold tolerance, thereby furnishing valuable resources for molecular breeding. These candidate genes include those encoding MYB and GRAS transcription factors, leucine-rich repeat (LRR) proteins, and protein kinases [49].

3.3.4. Mining Regulatory Hub Genes via WGCNA

Transcriptomic profiles generated from cold-tolerant and cold-sensitive inbred lines at both germination and seedling stages were employed to construct weighted gene co-expression networks under low-temperature stress. Module-trait association analyses revealed that modules enriched in hormone signaling, redox homeostasis, and MAPK pathways exhibited strong positive correlations with cold-tolerance phenotypes. In contrast, modules associated with cell wall synthesis and ubiquitin-mediated protein degradation showed negative correlations with cold resistance. Notably, the co-expression module centered on ZmICE1 contains multiple kinase and transcription factor hubs, including ZmMAPK, ZmWRKY, and ZmERF, suggesting their synergistic involvement in the cold-response network of maize [23].

3.4. Epigenetic and Chromatin-Level Regulation of Cold Responses

3.4.1. Cold-Induced Remodeling of Chromatin Accessibility

Dynamic remodeling of chromatin accessibility represents a critical epigenetic mechanism underpinning transcriptional reprogramming under cold stress. Extensive studies in model species such as Arabidopsis and rice have established that low temperature induces genome-wide rearrangements of DNase I-hypersensitive sites (DHSs) and accessible chromatin regions (ACRs) [50,51]. In maize, however, systematic profiling of time-series and tissue-specific changes in chromatin accessibility at the genome scale remains limited, despite the availability of a few ATAC-seq and DNase-seq datasets. Notably, a large proportion of DHSs in plants are located in intergenic non-coding regions, implicating distal cis-regulatory elements in cold-signal perception and pointing to unexplored avenues for investigating epigenetic regulation of cold responses in maize [52]. Histone acetylation also participates extensively in plant cold responses; shifts in acetylation status influence chromatin configuration and modulate the transcription of cold-responsive genes [51]. Nevertheless, the regulatory modes governing these acetylation dynamics in maize await thorough experimental validation.

3.4.2. Motif Characteristics of Transcription Factor Binding in Cold-Remodeled Accessible Chromatin

Chromatin regions that transition to an accessible state under low temperature are typically enriched with cis-binding motifs recognized by diverse transcription factors, which mediate the transcriptional output of stress signals [52]. Motifs for the ERF, NAC, and bZIP families are frequently overrepresented in cold-responsive ACRs [51,52]. Multiple transcription factors often bind concurrently to individual open chromatin loci, enabling the formation of synergistic regulatory modules that integrate signals from upstream cascades to orchestrate downstream cold-responsive gene expression [52]. Among these, the CBF/DREB, NAC, and bZIP families are considered core regulators of plant cold responses—a functional paradigm that has been validated through integrative multi-species transcriptomic analyses [53,54,55].

3.4.3. Dynamic Reprogramming of Histone Modifications

The activating histone mark H3K4me3 and the repressive mark H3K27me3 undergo genome-scale dynamic reprogramming during plant cold adaptation, and their bivalent configurations jointly shape transcriptional regulatory networks linked to stress resilience [56,57]. In Arabidopsis, ChIP-seq data revealed that cold exposure rapidly and specifically alters the abundance of H3K4me3 and H3K27me3 at cold-regulatory loci. Under normal conditions, cold-responsive genes are generally marked by H3K27me3; however, removal of this repressive modification is not an obligatory prerequisite for cold-induced transcriptional activation. Instead, low temperature simultaneously triggers nucleosome depletion and the redeposition of bivalent histone marks, initiating cold transcriptional program through the reshaping of chromatin accessibility [58].

In maize, cold responses similarly rely on the coordinated action of chromatin openness and histone modifications [52,59]. In addition, hormone balance and sugar metabolic homoeostasis are known to participate in physiological adaptation to cold. Yet the upstream–downstream regulatory links connecting these physiological cascades to chromatin remodeling and histone modification events remain to be integrated into a unified framework. Furthermore, sustained cold-induced reprogramming of histone modifications may contribute to plant adaptive regulation in response to recurring low-temperature environments.

3.4.4. Epigenetic Regulatory Hub Genes in Cold Co-Expression Networks

Weighted gene co-expression networks constructed from transcriptomic data of maize under low-temperature stress at germination and seedling stages can be partitioned into modules that correlate either positively or negatively with cold-tolerance phenotypes. Positively associated modules are enriched in genes involved in ABA and jasmonic acid (JA) signaling, redox homoeostasis, and MAPK cascades. In contrast, negatively correlated modules are predominantly composed of pathways related to cell wall synthesis and ubiquitin-mediated protein degradation. Hub genes identified through module connectivity screening—including ZmMAPK3, ZmPP2C, ZmHSP90, ZmWRKY24, ZmbZIP23, ZmNAC72, and ZmERF7—have had their roles in cold tolerance validated by virus-induced gene silencing (VIGS) assays [60].

3.5. Post-Transcriptional Regulation Mediated by Small RNAs

3.5.1. Roles of miRNAs in Cold Responses

In addition to protein-coding genes and epigenetic regulation, small RNAs execute critical post-transcriptional fine-tuning within the cold-regulatory networks of maize. Cross-species investigations have established that miR169 targets NF-YA and miR393 targets TIR1, mediating targeted mRNA degradation to precisely modulate the intensity of cold responses [61].

High-throughput small RNA sequencing of cold-stressed maize seedlings revealed differential cold-induced expression of several miRNA families, including miR156, miR159, miR164, miR167, miR169, miR172, miR319, and miR396. Their predicted target genes are predominantly enriched in auxin, GA, and ABA signaling cascades, NAC/MYB/SBP transcriptional regulatory pathways, and ROS-scavenging cascades mediated by SOD and POD [62].

The miR164–NAC regulatory module appears to be functionally conserved across wheat and Arabidopsis. In the winter wheat, low temperature downregulates tae-miR164 expression while concomitantly upregulating its target gene TaNAC6A. Heterologous transformation assays in Arabidopsis demonstrated that overexpression of tae-miR164 attenuates cold tolerance, whereas silencing miR164 or overexpressing TaNAC6A enhances antioxidant enzyme activity, reduces ROS and MDA accumulation, and activates cold-responsive cascade genes including AtDREB1, AtDREB2, and AtABI5 [61]. This module thus modulates cold responses through both CBF-dependent and CBF-independent pathways.

In maize, however, current evidence is limited to sequencing datasets documenting cold-responsive expression changes in zma-miR164—which vary depending on germplasm and treatment conditions—and genetic validation of its regulatory relationship with NAC targets, as well as its biological function in cold tolerance, remains absent [61]. A comprehensive review highlighted extensive cross-talk between miRNAs and hormone signaling cascades, which collectively orchestrate multilayered molecular net-works governing cold responses in crops via signaling and hormonal crosstalk, with implications for translational [62].

3.5.2. The miR159-ZmDUF966-10 Regulatory Module

Genome-wide analysis of the maize DUF966 gene family identified conserved complementary binding sites for zma-miR159 within the 3′ untranslated region (3′UTR) of ZmDUF966-10, the core cold-responsive member of this family. Bioinformatic predictions support a targeted interaction between the two molecules, suggesting that miR159 represses ZmDUF966-10 expression at the post-transcriptional level—likely through mRNA cleavage or translational inhibition [27].

In rice and wheat, long non-coding RNAs (lncRNAs) have been shown to function as miRNA sponges that competitively sequester small RNAs, thereby participating in post-transcriptional regulation under cold stress [63,64]. However, investigation into non-coding RNA functions in maize cold responses remains limited. The multilayered synergistic interplay among coding genes, miRNAs, and lncRNAs collectively constitutes the sophisticated molecular regulatory system that underpins cold tolerance in maize.

3.6. Crosstalk and Integration of Hormonal Signaling Pathways

3.6.1. Overall Characteristics of Hormonal Networks

Plant hormones function as both “switches” and “amplifiers” during cold responses. Plants coordinate their low-temperature adaptation through cross-talk among multiple hormonal signals, including ABA, JA, BR, salicylic acid (SA), GA, and ethylene (ET). Among these, ABA, JA, BR, and SA are generally regarded as classical stress-responsive hormones whose cascades are activated under adverse conditions. GA primarily regulates growth processes and is typically suppressed under low temperature, whereas ethylene exerts bidirectional effects on cold regulation that depend on environmental conditions and crop genotypes [65].

3.6.2. ABA Signaling Pathway

Cold-induced ABA accumulation enhances cold tolerance in maize by activating downstream transcription factors (AREB/ABF, NAC, bZIP) and antioxidant defense systems. Exogenous ABA application significantly elevates antioxidant enzyme activity and osmoprotectant accumulation in cold-stressed maize seedlings, thereby mitigating membrane lipid peroxidation injury [66]. Moreover, ABA signaling engages in extensive cross-talk with MAPK cascades: MAPK pathways suppress the activity of PP2C phosphatases—core negative regulators of ABA signaling—thereby amplifying the output intensity of ABA signals.

3.6.3. BR Signaling Pathway

In addition to the atypical ZmBSK4–ZmMAPK4 signaling module described earlier, exogenous application of 24-epibrassinolide (EBR) enhances cold tolerance in maize seedlings by modulating sugar signaling and carbon metabolism. EBR upregulates the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS), promoting sucrose synthesis and translocation while inhibiting sucrose catabolism, ultimately increasing soluble sugar accumulation in plant tissues [67].

Comparative experiments have examined the phenotypic effects of exogenous ABA, exogenous BR, and combined ABA + BR treatments on maize seedling cold tolerance. While individual applications of either ABA or BR effectively improve cold resistance, the combined treatment fails to produce synergistic benefits; in fact, composite application performs worse than single-hormone treatments with respect to indicators such as proline accumulation and SOD activity. Transcriptomic data revealed that ABA and BR co-regulate genes enriched in cell wall and secondary metabolic pathways, whereas their uniquely regulated genes participate in distinct signal transduction processes. These observations suggest competitive cross-talk between ABA and BR cascades during cold-signal integration, or mutual interference in the differential regulation of downstream target proteins such as PP2C and SnRK2, thereby eliminating synergistic phenotypic outcomes. From a broader perspective on cold-adaptation research in cereal crops, hormonal cross-talk constitutes a core frontier, with ABA–BR signal interaction occupying a central position in the cold-regulatory system of maize. Endogenous soluble sugar profiles in maize seedlings are strongly correlated with the level of cold tolerance, and optimal concentrations of exogenous EBR remodel sugar signaling and carbon metabolic homoeostasis to alleviate cold-induced injury [66].

3.6.4. Recent Advances in JA Signaling Pathways

Recent work has identified the E3 ubiquitin ligase NLA as a key activator of JA signaling under low temperature, acting through the proteasomal degradation of JAZ11—a core repressor of the JA cascade. Activation of JA signaling remodels downstream transcriptional and metabolic networks to mediate cold adaptation in maize [68]. There exists complex reciprocal cross-talk between JA cascades and both CBF-dependent and CBF-independent pathways: JA signals positively promote CBF gene transcription, while the CBF cascade reciprocally modulates JA biosynthesis, jointly constructing the hormonal regulatory network that governs cold responses. CDPKs, central components of calcium signaling cascades, function as convergence hubs interconnecting multiple pathways that regulate maize adaptation to diverse abiotic stresses. A systematic review has dissected the molecular mechanisms through which CDPKs integrate distinct signaling cascades to govern stress adaptability in maize [69]. Beyond CDPKs, other signaling modules also play critical roles. Collectively, these pathways—ranging from the canonical three-tier MAPK cascade and the atypical ZmBSK4-ZmMAPK4 module to calcium signaling, the core ZmICE1-ZmDREB1 cascade, and hormonal networks—form a multilayered regulatory system that governs maize cold responses. A comparative overview of their signal transduction modes, response speeds, maize-specific features, and crosstalk nodes is presented in Table 2.

Table 2. Comparative Characteristics of Core Cold Signaling Pathways in Maize.

Pathway Name	Signal Transduction Mode	Cold Response Speed	Maize-Specific Features	Core Biological Functions	Crosstalk Nodes with Other Pathways	References
CPK17-COOL1 calcium branch	Ca2+ influx → nuclear translocation of CPK17 to phosphorylate COOL1	Rapid	Maize-specific balancing cascade modulating cold response intensity	Preventing excessive activation of cold defense pathways	Interconnected with HY5 light signaling via COOL1	[20]
Core ZmICE1-ZmDREB1 cascade	Dual transcriptional and post-translational regulatory layers	Moderate speed	Distinct from Arabidopsis which relies solely on post-translational modification	Global cold response coordination and maintenance of metabolic homeostasis	Convergence hub for ROS signaling, amino acid metabolism and MAPK cascades	[23]
Atypical ZmBSK4-ZmMAPK4	Direct phosphorylation of MAPK4 by BSK4	Extremely rapid (adapted to abrupt late spring cold snaps)	Unique to maize, brassinosteroid-dependent	Rapid short-term defense against sudden chilling injury	Downstream bHLH111/181 activate DREB cascades	[25]
Canonical three-tier MAPK	MAPKKK-MAPKK-MAPK	Slow (multi-step phosphorylation)	Conserved with Arabidopsis	Long-term adaptive responses during sustained cold acclimation	Calcium signals upstreamly regulated by CPKs	[48]
ABA/BR hormonal pathways	Hormone accumulation → activation of SnRK2/BSK kinases	Moderate speed	ABA and BR exhibit no synergistic cold-protective effects	Osmotic adjustment and soluble sugar accumulation	Upstream master switches governing all transcription factor cascades	[67]

3.7. Progress in Integrative Multi-Omics Analysis

3.7.1. Transcriptome-Metabolome Integration Strategies

In recent years, the integration of transcriptomics, metabolomics, and proteomics has provided powerful tools for dissecting the topological architecture of cold-regulatory networks in maize. This combinatorial strategy offers complementary insights into the molecular and metabolic changes underlying cold responses [70]. Integrating these datasets facilitate the identification of core regulatory network nodes and helps generate hypotheses about which molecular shifts may contribute to cold responses.

3.7.2. Sugar Metabolic Reprogramming in Cold-Stressed Maize Roots

In maize, combined transcriptomic and metabolomic profiling of B73on roots 10 °C cold treatment at 0, 6, 12, 24, and 48 h, revealed numerous differentially expressed genes and differential metabolites [71]. The latter were predominantly involved in starch–sucrose metabolism (fructose, glucose, sucrose, maltose), the tricarboxylic acid cycle (citrate, malate, succinate), and amino acid metabolism (proline, GABA, asparagine). Genes enriched in starch–sucrose and phenylpropanoid pathways exhibited prominent cold-induced differential expression in treated roots.

Fructose, glucose, and other soluble sugars accumulated substantially in maize roots under low temperature, and multiple genes governing starch and sucrose metabolism were concurrently upregulated. These observations suggest that sugar metabolic reprogramming may constitutes a core adaptive strategy of maize roots under low temperature stress [71]. Soluble sugars may serve dual roles—as osmoprotectants that lower the intracellular freezing point and as signaling molecules that activate downstream defensive responses [72].

3.7.3. Flavonoid Metabolism During Cold Germination of Waxy Maize

Multi-omics investigation of cold germination in waxy maize revealed that cold-tolerant inbred lines exhibit stronger transcriptional induction of genes involved in plant hormones and MAPK signaling pathway than their cold-sensitive counterparts. At the metabolic level, tolerant germplasm accumulated substantially higher levels of flavonoids—including luteolin, apigenin, kaempferol, and quercetin—under low temperature, with markedly greater fold changes relative to sensitive lines [73].

Correlation network analysis integrating genes and metabolites revealed strong positive associations between the expression of key phenylpropanoid and flavonoid biosynthetic enzymes (PAL, C4H, 4CL, CHS, CHI, F3H, FLS) and flavonoid accumulation. MAPK signaling cascades and flavonoid biosynthetic pathways appear to interact synergistically, potentially forming adaptive mechanisms that contribute to seed germination under low temperature [74]. Multi-omics analytical platforms have been widely adopted to mine candidate cold-tolerance genes in maize under abiotic stress, providing reliable technical tools for functional characterization of core cold-regulatory factors [70]. Flavonoids may alleviate membrane lipid peroxidation injury through ROS scavenging, offering potentially viable targets for engineering cold-resistant maize via metabolic manipulation [74].

3.7.4. Integrated Model of Multi-Layered Maize Cold Regulatory Networks

Following cold signal perception at the plasma membrane, calcium signaling, MAPK cascades (including the atypical ZmBSK4–ZmMAPK4 pathway), and protein phosphorylation/stability control relay signals to transcription factor networks involving ZmICE1, COOL1, HSF21, and NAC-family members [20,23,24,25,47]. Concurrently, transcriptional output is modulated by chromatin remodeling, histone modifications, and miRNA-mediated post-transcriptional regulation [52,56,62]. Collectively, these interconnected layers are proposed coordinate metabolic reprogramming, antioxidant defense, and membrane lipid homoeostasis to enhance cold tolerance in maize [20,23,24,25,42].

4. Genetic Improvement and Breeding Strategies for Cold Tolerance

4.1. Excavation of Elite Allelic Variants and Development of Molecular Markers

4.1.1. Breeding Applications of COOL1 HapA

The discovery of elite allelic variants constitutes a critical first step in molecular breeding. Among the currently available alleles, COOL1 HapA represents one of the most promising resource for improving cold tolerance. Natural polymorphisms in the COOL1 promoter divide maize germplasm into two haplotypes, HapA and HapB, with HapA conferring significantly enhanced cold tolerance and therefore being particularly suitable for the genetic improvement of germplasm intended for high-latitude, cold-season cultivation. High-throughput haplotyping can be achieved using KASP markers developed from functional variants in the COOL1 promoter. Marker-assisted backcrossing is currently underway to introgress COOL1 HapA into early-maturing elite inbred lines and commercial cultivars from northern regions, with the goal of generating novel cold-resistant germplasm. Preliminary field observations have confirmed improved seedling emergence stability under low-temperature conditions in early spring for the introgressed materials. However, these observations remain limited, and the stability of this effect across genetic backgrounds, locations, and years has yet to be systematically evaluated. Moreover, potential pleiotropic effects and yield penalties associated with COOL1 HapA re-quire further assessment before large-scale cultivar development [20].

4.1.2. Enhancer Promoter Markers for ZmICE1

A functional SNP (−465 A/C) within the MYB39-binding site of the ZmICE1 promoter alters the binding affinity of ZmMYB39, thereby affecting ZmICE1 expression and maize cold tolerance, making this variant highly relevant for breeding. In natural populations, inbred lines carrying the high-expression promoter allele is generally associated with superior cold tolerance Among 203 of 219 inbred lines classified as HapA and HapB, the accumulation of ROS was generally higher in the HapA than in the HapB subgroup under cold treatment, suggesting that HapB may confers the cold-tolerance advantage [23]. Locus-specific molecular markers developed for this SNP facilitate high-throughput screening of elite cold-tolerant haplotypes in breeding populations through marker-assisted selection. Nevertheless, the predictive value of this marker across different genetic backgrounds and environments requires further validation, and its potential effects on yield and other agronomic traits should be carefully evaluated through multi-environment field trials before large-scale deployment in breeding programs.

4.1.3. Elite Haplotypes of HSF21 and TIP4;3

HSF21 Hap1represents a promising allelic variant for cold-tolerance improvement, as it has been reported to enhances cold resistance during both germination and seedling stages without incurring yield penalties. Molecular markers developed from promoter SNPs and InDels of HSF21 have been deployed across multiple breeding populations to track and select this beneficial haplotype [24]. The favorable haplotype of TIP4;3 arises from a CACTA transposon insertion in its promoter region, which epigenetically suppresses TIP4;3 transcription and elevates cold tolerance [26]. However, the stability of these effects across diverse environments and genetic back-grounds, as well as possible trade-offs with water-use efficiency and other traits, re-quires further investigation through multi-environment field trials. Their performance in elite breeding backgrounds and potential pleiotropic effects or yield penalties also remain to be systematically assessed.

4.1.4. Optimization Strategies for Marker-Assisted Selection

Marker-assisted selection (MAS) has proven highly effective for cold-tolerance breeding in several crop species. Nevertheless, single-locus MAS delivers limited gains for polygenic traits such as cold tolerance. Recently developed genomic selection (GS) strategies, which predict breeding values using genome-wide markers evenly distributed across the genome, hold considerable promise for maize cold-resistance breeding. Comprehensive reviews have systematically summarized the application of DNA markers, GS, and gene-editing technologies in crop genetic improvement, providing theoretical guidance for refining breeding strategies in maize. However, their practical effectiveness depends heavily on phenotyping accuracy, genotype × environment interactions, and the representativeness of training populations, and GS prediction accuracy is often population-specific and may decline in genetically divergent materials [75]. Integrating these approaches with multi-environment phenotyping and long-term yield evaluation will therefore be essential for translating molecular predictions into practical cultivar improvement.

4.2. Excavation and Utilization of Genetic Resources from Wild Relatives

The Circular Economy Corn Agriculture (CERCA) framework has recently been proposed. A core objective of this initiative is to develop cold-tolerant maize varieties capable of being sown when spring soil temperatures fall below 10 °C, thereby extending the growing season and more fully exploiting the yield potential of C4 photosynthesis. Model projections suggest that overcoming seedling-stage cold-tolerance constraints could advance sowing dates by 15–20 days, boost grain yields by more than 10%, and reduce spring nitrogen leaching losses from soil by 20%. The initiative advocates mining cold-adaptive genetic resources from wild maize relatives, deploying gene editing to introduce beneficial variants into cultivated germplasm, and pyramiding genes for cold tolerance and nitrogen-use efficiency—such as ZmDof1, ZmNAC4, and ZmGS1—to achieve the dual goals of early sowing stability and reduced nitrogen fertilizer input [76]. These projections are based on model simulations and have not yet been validated through multi-environment field trials; the actual performance of such varieties under diverse environmental conditions remains to be determined.

Perennial maize is recognized as a promising source of cold-tolerant wild germplasm. Comparative transcriptomic approaches have been applied to screen for cold-specific responsive genes in this material, and the identification of associated elite genes is expected to provide novel resources for improving cold resistance in cultivated maize. However, the introgression of wild alleles into elite germplasm often faces challenges such as linkage drag, genetic incompatibility, and unpredictable epistatic interactions, which may limit the practical utility of these resources.

4.3. Gene Pyramiding for Cold Tolerance and Prospects of Super Cold-Resistant Varieties

4.3.1. Theoretical Basis of Gene Pyramiding

Cold tolerance is a polygenic trait, and modification of single genes generally yields only moderate phenotypic improvements. Pyramiding multiple elite cold-tolerant alleles can produce additive or synergistic effects. Theoretically, in the absence of unfavorable genetic linkage and with compatible genetic backgrounds, combining COOL1 HapA (which attenuates negative regulation), the high-expression ZmICE1 promoter variant (which strengthens positive cold-signaling cascades), and the beneficial TIP4;3 haplotype could generate germplasm with superior cold tolerance compared with single-gene lines [20,23,26]. However, these predicted effects are largely theoretical; the actual performance of pyramided lines depends on complex epistatic interactions, genetic background effects, and environmental conditions, all of which require extensive field validation.

4.3.2. Optimization of Pyramiding Strategies

In practice, however, gene pyramiding faces several challenges. First, genetic trade-offs exist between cold tolerance and agronomic traits such as early maturity and yield. Notably, the elite HSF21 Hap1 allele simultaneously enhances cold resistance without yield loss, confirming that precise modulation of lipid metabolic homoeostasis can reconcile the antagonism between stress resistance and yield formation [24]. Constitutive overexpression of certain cold-resistance genes often triggers undesirable pleiotropic effects, including reduced biomass and extended growth cycles; for example, ZmCBF-overexpressing lines exhibit dwarfism and delayed flowering [30]. This bottleneck can be mitigated by using tissue-specific promoters—for instance, a germination-specific LEA promoter driving ZmCBF expression—which enhances cold tolerance while avoiding developmental defects through spatially and temporally restricted transgene activity. Second, complex interconnections exist between distinct stress-response pathways: cold tolerance and drought/disease resistance sometimes act synergistically (e.g., ABA signaling governs both cold and drought adaptation) and sometimes antagonistically (e.g., aquaporin regulation: suppression of TIP4;3 improves cold tolerance but may compromise water-use efficiency) [26,77]. Future molecular design breeding must adopt a full-cycle, multi-stress perspective to construct gene networks conferring resilience to multiple stresses [70]. In addition, the performance of pyramided lines under field conditions is influenced by genotype × environment interactions and may vary across locations and years, highlighting the need for multi-environment testing before cultivar release [78].

4.4. Integrative Breeding Coordinating Cold Tolerance and Other Stress Responses

4.4.1. Dual Improvement of Cold Tolerance and Phosphorus Use Efficiency

The regulatory mechanisms underlying coordinated improvement of cold tolerance and other stress responses have become a prominent frontier in breeding research. A genetic trade-off exists between cold tolerance and phosphorus-acquisition efficiency: low temperature reduces soil phosphorus diffusion rates and microbial activity, thereby lowering phosphorus bioavailability, while sustained activation of certain cold-resistance pathways further inhibits phosphorus uptake. The molecular basis of this antagonism has been elucidated: under low temperature, the E3 ligase NLA simultaneously activates JA-mediated cold tolerance through JAZ11 degradation and suppresses phosphorus uptake by degrading the phosphate transporter PT4. Gene-editing-mediated functional remodeling of NLA to generate the NLAΔ12 variant eliminates this negative genetic correlation, simultaneously enhancing cold tolerance and phosphorus-use efficiency, thereby establishing a theoretical paradigm for coordinated improvement of multiple traits in maize. However, this strategy has so far been tested mainly under controlled conditions; its effectiveness in the field, potential pleiotropic effects, and compatibility with diverse genetic backgrounds remain to be determined [68].

4.4.2. Synergistic Networks Governing Cold, Drought and Disease Resistance

Cold and drought tolerance share synergistic regulatory foundations at the level of ABA signaling [55,79]. The relationship between cold tolerance and disease resistance is more complex: defense-related transcription factors such as NPR1 and WRKY also participate in cold responses [80,81]. and in maize, the cold-responsive factor ZmDREB1A negatively regulates disease resistance, revealing a trade-off between cold tolerance and immunity [82].

Dissecting the genetic correlations and molecular convergence points that govern multiple abiotic and biotic stresses will facilitate the breeding of “climate-resilient” maize varieties capable of withstanding cold, drought, low phosphorus, and other field adversities in combination. Yu and colleagues combined SLAF-seq with bulked segregant analysis (BSA) to dissect QTLs governing cold tolerance during maize germination, providing molecular marker resources for coordinated multi-trait breeding [83]. Nevertheless, translating these findings into practical cultivars requires integrating multi-environment phenotyping, yield evaluation, and agronomic performance, which remain the key bottlenecks in molecular breeding for complex stress tolerance.

5. Conclusions and Future Perspectives

5.1. Major Research Advances and Contributions

Recent years have witnessed substantial progress in the molecular dissection of cold tolerance in maize. At the level of core gene discovery, the field has accumulated a growing repertoire of functionally validated genes with clear breeding potential. ZmICE1 modulates cold adaptability by reshaping intracellular amino acid metabolic balance [23]; natural variants of COOL1 have illuminated the evolutionary trajectory underlying maize adaptation to high-latitude cold environments [20]; and HSF21 enhances cold tolerance through the stabilization of membrane lipid homoeostasis without incurring yield penalties [24]. Additional regulators—including ZmWAK3, the tonoplast aquaporin TIP4;3, and the functionally uncharacterized protein ZmDUF966-10—have been sequentially identified, substantially expanding the genetic resource pool for cold tolerance [26,27,41]. These genes span diverse functional categories, from transcription factors and receptor kinases to heat-shock regulators and aquaporins, collectively illustrating the multifaceted complexity of the cold-response regulatory network in maize.

In terms of signaling transduction and network dissection, the discovery of the atypical ZmBSK4–ZmMAPK4 kinase cascade has challenged the conventional view of canonical three-tier MAPK phosphorylation cascades [25]. Multiple layers of regulation have now been systematically characterized, including cross-talk between calcium and MAPK signaling, epigenetic modulation via histone modifications, post-transcriptional fine-tuning by small RNAs, and extensive hormonal pathway interactions [47,56,62,65]. These interconnected regulatory strata together constitute a comprehensive signaling system that underpins cold adaptation in maize, deepening our overall understanding of the plant cold-response program.

On the translational front, elite natural haplotypes of COOL1, HSF21, TIP4;3, and other genes have emerged as priority targets for molecular breeding [20,26,32]. Continuous advances in CRISPR/Cas9 technology now enable not only simple gene knockout but also precise allelic replacement and targeted modification of protein function [84]. Novel breeding frameworks—including multi-gene pyramiding and genomic selection—offer feasible strategies for the simultaneous improvement of complex agronomic traits such as cold tolerance and grain yield [75,76]. Collectively, these theoretical and technological advances have advanced fundamental plant cold-biology knowledge while providing clearly defined gene targets and mature molecular tools for breeding cold-resistant maize varieties [25].

5.2. Persistent Bottlenecks and Unresolved Challenges

Despite considerable progress, several bottlenecks and critical scientific questions remain unresolved.

First, the phenotypic effects of elite cold-tolerant alleles are strongly dependent on genetic background. Introgression of major-effect genes such as ZmICE1 and COOL1 into different inbred lines produces markedly variable cold-tolerance gains [20,23]; for example, introduction of the COOL1 elite haplotype confers substantially greater improvement in tropical germplasm than in temperate materials [20]. The underlying causes include differences in intrinsic cascade activation intensity, metabolic network architecture, and epigenetic modification patterns across germplasm backgrounds. Consequently, backcross-based introgression alone cannot reliably achieve predicted cold-tolerance improvements across all breeding materials, necessitating background-tailored genomic selection strategies.

Second, natural cold-adaptation mechanisms that evolved during maize domestication are often at odds with modern high-yield breeding objectives. The cold-tolerant COOL1 HapA haplotype originated in wild teosinte and accumulated in temperate and cold-temperate germplasm through natural and artificial selection [20]. Yet most widely cultivated high-yield hybrids carry cold-sensitive haplotypes, indicating that cold-tolerance traits have been unintentionally counter-selected during modern breeding. Cold-resistance loci frequently display unfavorable linkage with key yield-related traits such as early maturity, plant architecture, and growth duration. Simple introgression of wild-derived alleles often introduces deleterious agronomic traits, and conventional backcrossing cannot efficiently break linkage drag. Molecular-level redesign of regulatory pathways is therefore needed to reconcile the antagonism between cold resistance and high yield.

Third, most existing studies have focused on isolated cold stress, whereas field environments commonly involve combinations of multiple concurrent stresses. Low temperature often coincides with drought, nutrient limitation, and pest or disease pressure, generating complex synergistic or antagonistic interactions whose regulatory logic remains poorly understood. For instance, ABA signaling enhances both cold and drought tolerance, yet sustained overactivation suppresses normal growth and reduces final yield. Clarifying the genetic trade-offs among diverse agronomic traits is a prerequisite for coordinated improvement of multi-stress resilience and high yield [85,86].

Fourth, a substantial gap persists between laboratory findings and field application. Most cold-tolerance genes have been validated under controlled greenhouse conditions, whereas field settings introduce confounding variables such as temperature fluctuations, uneven light, variable soil nutrients, and biotic interference [87]. Cold-resistance advantages observed indoors often attenuate considerably under field complexity [88,89]. Establishing standardized phenotyping platforms that mimic authentic field environments and conducting adaptive screening of pyramided lines across multiple ecological zones remain critical bottlenecks in bridging basic research and breeding practice.

5.3. Future Research Directions and Prospects

To address these gaps, five frontier research directions are proposed for future investigation. First, synthetic biology offers the potential to artificially reconstruct cold-regulatory cascades. Logic-gated synthetic gene circuits can be constructed by assembling COOL1 light-signaling promoter elements, ZmICE1 cold-responsive enhancer sequences, antifreeze protein genes, and antioxidant biosynthetic genes, enabling coordinated activation of cold-defense responses triggered by dual cold and light signals [20,23]. Liquid–liquid phase separation has recently emerged as a novel cold-sensing mechanism in plants: transcription factors form biomolecular condensates through phase separation to initiate cold transcriptional program in rice, but this mode of regulation remains unexplored in maize [20]. Future work could investigate whether phase separation contributes to maize cold responses and exploit synthetic biology to engineer artificial phase-separation systems for targeted enhancement of cold tolerance.

Second, the integration of multi-stress and full-growth-cycle research with high-throughput phenomics should be advanced [90]. Phenotyping platforms covering the entire life cycle—from germination to grain filling—could be equipped with visible, infrared, hyperspectral, and chlorophyll fluorescence imaging to enable automated, high-throughput quantification of cold-tolerance traits and their genetic relationships with drought and disease resistance [91]. Large-scale multi-environment, multi-genotype datasets can be used to train genomic selection models, accelerating the pyramiding of multiple elite cold-tolerance alleles and facilitating translational breeding [75].

Third, single-cell and spatial transcriptomics can resolve cell-type-specific cold responses that are masked by bulk tissue analyses. Single-cell RNA-seq and spatial transcriptomics technologies can delineate the unique cold-response program operating in guard cells, root apical meristem cells, bundle sheath cells, and other specialized populations, and can dissect intercellular signal transmission networks. Such knowledge would enable targeted enhancement of cold tolerance in critical tissues while avoiding global growth suppression [92].

Fourth, fuller exploitation of wild maize germplasm and the development of de novo domestication strategies represent a promising avenue. Conventional breeding typically introduces cold-tolerance genes from wild teosinte into cultivated maize, often accompanied by unfavorable linked genomic segments. De novo domestication—using gene editing to directly modify wild relatives—could rapidly generate novel germplasm that combines intact native cold-resistance networks with high-yield characteristics, circumventing linkage drag and opening new routes for cold-resistance breeding [93].

Fifth, artificial intelligence and multi-omics big-data integration should be harnessed to accelerate gene discovery. The growing wealth of maize genomic, transcriptomic, metabolomic, and phenomic datasets provides powerful predictive capacity when analyzed with deep learning and graph neural networks [94]. These tools can forecast gene function and regulatory relationships, predict the cold-tolerance functions of uncharacterized genes, and organize vast literature into knowledge graphs linking genes to agronomic traits—thereby supporting intelligent decision-making in breeding program design [95].

5.4. Concluding Remarks

In summary, research on the molecular regulatory networks governing cold tolerance in maize is transitioning from single-gene characterization towards an era of artificial network reconstruction and molecular design breeding. The coordinated advancement of fundamental mechanistic studies and translational breeding applications, empowered by cutting-edge technologies—including gene editing, synthetic biology, multi-omics integration, artificial intelligence, and high-throughput phenotyping—is expected to accelerate the development and deployment of cold-resistant maize varieties over the next 5–10 years, providing robust support for global food security and sustainable agricultural development. At the same time, sustained investigation into fundamental questions—such as early cold-signal perception, epigenetic stress memory, and cross-stress adaptive mechanisms—will continue to drive progress across the broader field of plant stress biology.