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Article

ZmbHLH30 Enhances Cold Tolerance During Maize Germination

by
Xinguang Tang
,
Yitong Sun
,
Bangguo Zhang
,
Xinwen He
,
Lin Zhang
,
Ling Dong
,
Xing Zeng
,
Hong Di
,
Jiayue Zhang
,
Chunxiang Li
,
Jiapeng Xing
,
Qi Zhang
,
Zhenhua Wang
* and
Yu Zhou
*
Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Cold Region, Engineering Technology Research Center of Maize Germplasm Resources Innovation on Cold Land of Heilongjiang Province, Northeast Agricultural University, Harbin 150030, China
*
Authors to whom correspondence should be addressed.
Plants 2026, 15(4), 611; https://doi.org/10.3390/plants15040611
Submission received: 21 December 2025 / Revised: 21 January 2026 / Accepted: 5 February 2026 / Published: 14 February 2026
(This article belongs to the Section Plant Response to Abiotic Stress and Climate Change)
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Abstract

Low temperature is a major abiotic stress that affects maize across its entire growth cycle, with the germination stage being particularly sensitive. To investigate the genetic basis of early-stage cold tolerance, we used quantitative trait locus mapping and identified ZmbHLH30 as a candidate gene regulating maize responses to low temperature. The ZmbHLH30 protein is localized in the cytoplasm of maize protoplasts, and ZmbHLH30 promoter drives β-glucuronidase (GUS) expression in Arabidopsis thaliana leaves. The promoter region of ZmbHLH30 contains multiple environmental stress-responsive elements, including motifs associated with cold and auxin responses. Overexpression of ZmbHLH30 significantly enhanced cold tolerance at the germination, bud, and seedling stages, with the strongest effect observed during germination, where the cold-tolerance D-value increased by 0.366 relative to the control. In contrast, CRISPR/Cas9 knockout lines showed a 0.399 decrease in D-value. Under cold stress, ZmbHLH30 expression was markedly induced in overexpression lines but suppressed in knockout lines. Integrated transcriptomic and metabolomic analyses further identified ZmbHLH30 as a key regulator of cold tolerance in maize.

1. Introduction

Maize (Zea mays L.), a major global food crop and the most widely grown cereal in China, is highly sensitive to temperature fluctuations. As a species originating from tropical regions, maize is particularly vulnerable to low temperatures, which restrict the expansion of planting areas and pose a direct threat to global production and food security [1]. Low-temperature stress is a common agrometeorological disaster that can damage maize throughout its entire life cycle [2,3]. During germination, cold conditions inhibit key hydrolytic enzymes such as amylase, limit the mobilization of seed storage reserves, and increase the risk of imbibition injury, ultimately reducing germination efficiency [4]. At the seedling stage, low temperatures suppress photosynthesis, decrease antioxidant enzyme activity, impair membrane stability, reduce biomass accumulation, and delay growth and development [4]. In severe cases, cold stress can disrupt chloroplast integrity and compromise photosynthetic enzyme function. During grain formation, low temperatures reduce the synthesis of proteins and starches, thereby lowering overall yield quality [5,6]. Enhancing cold tolerance in maize is therefore essential for improving adaptation to high-latitude and high-altitude environments, overcoming latitude-associated cultivation limits, and addressing temperature variability driven by climate change. Identifying cold-tolerant genetic resources and elucidating the regulatory pathways that govern low-temperature responses are fundamental steps toward breeding maize varieties with improved cold tolerance [2].
Upon low-temperature stress, cold signals are first perceived by cell membrane sensory components and then transmitted via signaling pathways, including Ca2+ transduction and the mitogen-activated protein kinase (MAPK) cascade [7,8]. During this process, proteins including CAMTA and CRLK participate in cold signal relay through calmodulin binding or phosphorylation, while MAPK modules such as MEKK1–MKK2–MPK4 regulate downstream gene expression to modify membrane permeability and enhance cold adaptation [9,10]. Endogenous hormones act as central regulators of the maize cold response. Low-temperature stress markedly induces the synthesis of abscisic acid (ABA), which limits water loss by promoting stomatal closure and enhances the accumulation of soluble sugars and proline to protect membrane integrity [11,12]. Gibberellin (GA) levels exhibit cultivar-dependent responses under cold conditions: in cold-tolerant cultivars, GA3 content initially decreases and then increases, while in sensitive cultivars it shows the opposite trend. Overall, low temperature inhibits GA3 biosynthesis in maize seedlings [13]. Zeatin riboside (ZR), a cytokinin derivative, shows strongly reduced activity under cold stress, leading to suppressed cell division and subsequent effects on grain development and protein accumulation [14]. Maize also stabilizes membrane structure via synthesis of osmotic regulators (e.g., soluble sugars, proline, polyamines) and mitigates cold-induced damage by scavenging reactive oxygen species (ROS) through antioxidant enzymes, antifreeze proteins (AFPs), and late embryogenesis abundant (LEA) proteins [15]. Genome-wide association studies (GWAS) have identified cold-tolerance loci such as COOL1, which negatively regulates cold tolerance by suppressing DREB1 and TPS expression. The superior COOL1 haplotype is enriched in high-latitude cultivars, highlighting the role of natural selection in shaping maize cold adaptation [2]. Collectively, the synergistic action of these regulatory mechanisms provides essential protection against low-temperature stress.
Basic helix–loop–helix (bHLH) transcription factors, the second largest transcription factor family in plants after MYB, play central roles in cold responses in maize and other species. These transcription factors importance arises from conserved structural features and diverse regulatory functions that position them as key molecular switches controlling cold tolerance [16,17]. The bHLH domain contains approximately 60 amino acids: the N-terminal basic region binds specifically to E-box or MYC (CANNTG) cis-elements in the promoters of target genes, while the C-terminal helix–loop–helix region mediates homo- or heterodimer formation through hydrophobic interactions, enabling precise regulation of downstream gene expression [18,19]. In cold stress signaling, bHLH transcription factors act as upstream components of the ICE1–CBF–COR pathway. In Arabidopsis, ICE1 binding to the CBF3 promoter significantly enhances freezing tolerance. Homologous genes such as Vitis amurensis VaICE1 and VaICE2, Ipomoea batatas IbbHLH79, and Malus domestica MdCIbHLH1 improve cold tolerance after heterologous expression by inducing cold-responsive genes, increasing osmoprotectant accumulation, and reducing membrane damage [1,19,20,21]. Although this regulatory role is conserved, species-specific differences exist. For example, overexpression of AtICE1 in rice promotes CBF/DREB expression; transfer of Dimocarpus longan DlICE1 into Arabidopsis increases proline levels and decreases malondialdehyde accumulation; and Malus domestica MdbHLH3 responds to cold by elevating anthocyanin content [22,23,24]. In maize, ZmICE1 directly binds to the MYC element in the promoters of CBF/DREB1 genes to activate their transcription and contributes to cold tolerance by modulating amino acid metabolism and maintaining ROS homeostasis [25,26]. Members of the bHLH family also function outside the ICE1–CBF–COR pathway, for example by interacting with genes involved in ascorbic acid biosynthesis or by modulating the jasmonic acid signaling pathway during stress responses [26,27,28].
This study focuses on ZmbHLH30 [29], a low-temperature tolerance gene expressed during maize germination. We characterized the promoter activity of ZmbHLH30 and the subcellular localization of the ZmbHLH30 protein, assessed its contribution to cold tolerance at different developmental stages, and determined its expression pattern under low-temperature stress. By integrating transcriptomic and metabolomic analyses, we identified the metabolic pathways through which ZmbHLH30 participates in maize cold tolerance and provided initial insights into its regulatory mechanism during germination.

2. Results

2.1. Analysis of the Biological Characteristics of ZmbHLH30

Subcellular localization was examined using protoplasts isolated from stable maize lines expressing a GFP-tagged ZmbHLH30 fusion protein. Laser scanning confocal microscopy showed that fluorescence signals were confined to the cytoplasm, indicating that the ZmbHLH30 protein is cytoplasmic (Figure 1A). To assess promoter activity, two-week-old Arabidopsis thaliana seedlings were subjected to whole-plant GUS staining. Blue coloration was observed in the leaves (Figure 1B), demonstrating that the ZmbHLH30 promoter successfully drove leaf-specific GUS expression. Analysis of the ZmbHLH30 promoter revealed multiple cis-acting regulatory elements, including light-responsive motifs, maize zein metabolism regulatory elements, inducible response elements, MYB and MYC binding sites, hypoxia-inducible elements, circadian rhythm regulatory motifs, and hormone-responsive elements associated with abscisic acid, auxin, and general stress responses (Figure 1C).

2.2. ZmbHLH30 Enhances Cold Resistance in Maize

Low-temperature tolerance was assessed at the germination, bud, and seedling stages using optimal ZmbHLH30 overexpression (OE) and knockout (CR) lines. Genotypes were confirmed at the DNA, RNA, and protein levels (Table S1, Figure S1). Comprehensive cold tolerance was evaluated using D-values derived from the membership function method (Table S2 and Table 1). Based on the D-values, cold tolerance was categorized into five levels: high tolerance (I), tolerance (II), moderate tolerance (III), sensitivity (IV), and high sensitivity (V). At the germination stage, clustering analysis of D-values for the OE lines showed that all three lines displayed enhanced cold tolerance relative to the control, which had a D-value of 0.531 and belonged to grade III. The OE-7, OE-6, and OE-4 lines were classified as grades I, I, and II, with D-values of 0.897, 0.793, and 0.651, respectively. OE-7 exhibited the strongest cold tolerance, with a D-value 0.366 higher than the control. In contrast, the CR lines showed markedly reduced cold tolerance. CR-3, CR-6, and CR-7 were classified as grade V, with D-values of 0.139, 0.148, and 0.132, respectively. CR-7 had the lowest tolerance, with a D-value 0.399 lower than the control (Figure 2A).
At the bud stage, clustering analysis again revealed improved cold tolerance in OE lines. Relative to the control (D-value 0.463, grade III), the OE-2, OE-4, and OE-7 lines showed D-values of 0.782, 0.730, and 0.613, corresponding to grades I, I, and II, respectively. OE-2 exhibited the greatest improvement, with a D-value 0.319 higher than the control. The CR lines showed reduced tolerance: CR-5, CR-1, and CR-3 were assigned grades V, V, and IV, with D-values of 0.219, 0.272, and 0.332, respectively. CR-5 displayed the weakest performance, with a D-value 0.244 lower than the control (Figure 2B).
At the seedling stage, OE lines again showed improved tolerance. Compared with the control (D-value 0.448, grade III), the OE-6, OE-3, and OE-5 lines had D-values of 0.731, 0.557, and 0.514, corresponding to grades I, II, and II, respectively. OE-6 exhibited the strongest tolerance, with a D-value 0.283 higher than the control, and showed reduced leaf wilting under cold stress. CR lines displayed the opposite trend: CR-7, CR-4, and CR-3 were classified as grades V, V, and IV, with D-values of 0.293, 0.296, and 0.344, respectively. CR-7 had the weakest tolerance, with a D-value 0.155 lower than the control, and exhibited more severe leaf wilting under cold stress (Figure 2C).

2.3. Cold Stress Response Analysis of ZmbHLH30

Expression profiling of ZmbHLH30 in embryos at the germination stage under cold stress (Figure 3A) showed that, except at 2 h and 12 h, transcript levels were significantly higher than those of the control at all sampling points. The strongest induction occurred at 6 h, where expression reached an average value of 6.96 and differed significantly from the control (p < 0.01). In the three overexpression lines, ZmbHLH30 expression showed a pattern of increasing and then decreasing with prolonged stress exposure. In contrast, the knockout lines exhibited significantly reduced expression at 4 h, 6 h, and 8 h, with the largest reduction observed at 6 h. At this time point, average expression in knockout lines was 0.60-fold that of B104. At the bud stage, cold stress altered ZmbHLH30 expression in both plumules and radicles (Figure 3B). In plumules, expression was significantly higher than that of the control at all time points except 2 h (p < 0.05). In radicles, expression at 4 h and 8 h was significantly higher than that of the control (p < 0.01). Transcript levels increased and then decreased over time, with the strongest induction observed at 8 h, reaching average values of 5.50 in plumules and 6.17 in radicles (p < 0.01). In knockout lines at the bud stage, expression in plumules was significantly lower than that of B104 at 8 h. In radicles, expression was significantly lower at 4 h and 8 h, reaching a minimum at 8 h, where average levels were 0.69-fold and 0.56-fold those of B104. Analysis of gene expression at the seedling stage under cold stress (Figure 3C) revealed strong induction of ZmbHLH30 across tissues. In leaves, expression at 4 h and 8 h was significantly higher than that of the control. In stems, expression was significantly higher than that of the control at all time points except 12 h. In roots, expression was significantly higher than that of the control except at 2 h. The strongest induction occurred at 8 h, where average expression reached 6.97 in leaves, 5.79 in stems, and 6.86 in roots (p < 0.01). Expression increased and then decreased over time, with the most pronounced differences observed in leaf tissue. In knockout lines at the seedling stage, expression in leaves, stems, and roots at 8 h was significantly lower than that in B104, with average values of 0.55-fold, 0.67-fold, and 0.74-fold those of the control.

2.4. ZmbHLH30 Enhances Transcriptional Responses That Improve Maize Cold Stress Resistance

After confirming that the transcriptome sequencing data met quality requirements (Figure S2), a total of 2585 cold-responsive differentially expressed genes (DEGs) were identified by comparing ZmbHLH30 overexpression lines with B104. Among these, 468 genes were significantly upregulated and 2117 were significantly downregulated (Figure 4A). GO enrichment analysis of the 2585 DEGs revealed enrichment in terms related to protein phosphorylation, flavonoid biosynthesis, cell wall organization, and chitinase activity (Figure 4B,C). KEGG pathway enrichment showed that DEGs were distributed across five major functional categories, mainly within metabolism and cellular processes. The metabolism category contained the largest number of enriched genes, with 868 genes involved in pathways such as peroxisome function, plant hormone signal transduction, phenylpropanoid biosynthesis, RNA degradation, and glycolysis or gluconeogenesis (Figure 4D). Focusing on cold-related biological functions, GO annotations highlighted pathways associated with response to stress, response to cold, response to abscisic acid, response to gibberellin, calcium ion binding, plasma membrane function, chloroplast-associated processes, transporter activity, and antioxidant enzyme activity such as superoxide dismutase and peroxidase activity. Representative DEGs included Zm00001d053894, Zm00001d039094, Zm00001d019078, and Zm00001d036135 (Figure 4E). Core cold-related KEGG pathways included phenylpropanoid biosynthesis, galactose metabolism, linoleic acid metabolism, MAPK signaling pathway, brassinosteroid biosynthesis, and glutathione metabolism. Phenylpropanoid biosynthesis had 56 enriched DEGs, including 10 associated with flavonoid biosynthesis. Linoleic acid metabolism contained 5 DEGs, with Zm00001d033624 as a representative gene. The MAPK signaling pathway–plant contained 57 DEGs, brassinosteroid biosynthesis contained 6, and glutathione metabolism contained 27. Transcription factors such as AP2/ERF and MYB, including Zm00001d040651 and Zm00001d046517, may be implicated in the modulation of cold-responsive signaling via the ICE-CBF-COR pathway (Figure 4F).

2.5. ZmbHLH30-Mediated Regulatory Mechanisms of Cold Tolerance Through Galactose and Phenylpropanoid Metabolic Pathways in Maize

Metabolomic data quality was confirmed by consistent TIC profiles, stable m/z and retention time characteristics, and a permutation test that verified that the PLS–DA model was not overfitted (Figure S3). Identified secondary metabolites were classified into 14 MS2 superclasses, including 81 amino acids, peptides, and analogues such as proline betaine. In total, 21926 primary metabolites were detected, among which 1977 were upregulated and 2039 were downregulated, along with 1101 secondary metabolites. Among the secondary metabolites, 32 were upregulated, including 3,4-dihydroxybenzoic acid, raffinose, and trans-4-hydroxy-L-proline, whereas 121 were downregulated, including arginine, caproic acid, and adenine (Figure 5A). KEGG enrichment analysis showed that among the 1101 secondary metabolites, 610 positive metabolic ions were annotated to 237 pathways. Ninety-four significantly different metabolites were enriched in 39 pathways, mainly including linoleic acid metabolism, phenylpropanoid biosynthesis, glycerophospholipid metabolism, galactose metabolism, and oxidative phosphorylation. Additionally, 491 negative metabolic ions were annotated to 190 pathways, with 59 significantly differentially abundant metabolites enriched in pathways such as phenylpropanoid biosynthesis, flavonoid biosynthesis, tryptophan metabolism, beta-alanine metabolism, glycine, serine, and threonine metabolism, and carbon metabolism (Figure 5B). Notably, galactose metabolism and phenylpropanoid biosynthesis, both strongly enriched, are closely associated with plant stress resistance.

2.6. Screening of Cold Tolerance-Associated Differentially Expressed Genes in Galactose and Phenylpropanoid Metabolic Pathways

Significantly different metabolites and DEGs associated with ZmbHLH30 were screened using the predefined thresholds. Integrated transcriptomic and metabolomic analysis identified 332 co-associated DEGs and 31 co-associated differential metabolites. GO enrichment analysis of these DEGs produced 552 GO terms. Among them, genes such as Zm00001d047582 were annotated to terms including response to cold and response to oxidative stress, while genes such as Zm00001d015459 were associated with cold tolerance-related terms including phenylpropanoid metabolic process (Figure 6A). MapMan enrichment analysis showed that these DEGs were broadly enriched in pathways including phenylpropanoid biosynthesis, galactose metabolism, and linoleic acid metabolism. Notably, the DEG Zm00001d025958 was upregulated and enriched in seven metabolic pathways, including pyruvate metabolism and tryptophan metabolism, and Zm00001d012144 participated in jasmonic acid and auxin biosynthetic processes. The 31 co-associated differential metabolites were enriched in 70 KEGG pathways, including core pathways such as metabolic pathways and biosynthesis of amino acids (Figure 6B). Further integrated KEGG enrichment analysis combining DEGs and differential metabolites identified 33 related metabolic pathways. Using p < 0.05 as the significance threshold, galactose metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis were identified as the key pathways responding to cold stress (Figure 6C). Within these pathways, Zm00001d047582 was significantly upregulated in the galactose metabolism pathway in overexpression lines.

3. Discussion

3.1. Cold Tolerance During Germination Is a Critical Determinant of Maize Growth and Development

Maize, native to tropical and subtropical regions, is highly cold-sensitive, especially during germination—a process involving rapid water uptake, physiological lag, and embryonic regrowth [30,31]. This sensitivity substantially limits its geographical distribution and cultivation potential. In high-latitude regions with a short 4- to 5-month growing season, early-spring sowing is required to ensure sufficient development time; however, recurrent late-spring cold events and extended periods of low temperature expose germinating seeds to strong cold stress [32,33]. Such stress reduces the emergence rate, compromises seedling uniformity, and can cause irreversible injury to the embryo, in severe cases leading to germination failure [34,35]. Cold stress below 15 °C triggers a broad physiological response in maize, and development nearly ceases below 10 °C [36,37,38]. Low temperature disrupts membrane fluidity through lipid phase separation, decreases the activity of key metabolic enzymes such as amylase and protease, and restricts water and nutrient uptake by inhibiting root hair formation [39,40]. In parallel, cold stress accelerates reactive oxygen species accumulation, damaging DNA, proteins, and membrane lipids [41]. Lipid peroxidation generates reactive aldehydes such as malondialdehyde and methylglyoxal, and the latter can also accumulate due to glycolytic imbalance under stress [42,43]. These aldehydes further exacerbate cellular injury by forming DNA adducts and promoting protein cross-linking, leading to germination arrest, stunted seedling growth, chlorosis, and even seed abortion [44,45]. Methylglyoxal signaling can also enhance ROS and activate kinases, transcription factors, and downstream effector genes involved in the maize cold response network [46,47]. While some seedling-stage cold-responsive genes (e.g., COOL1, a negative regulator; CPK17, which stabilizes COOL1; ZmICE1, which enhances ZmDREB1 expression) have been characterized [2], as well as ZmICE1, which enhances expression of ZmDREB1. However, research focused specifically on germination-stage cold tolerance remains limited [47].
In this study, overexpression of ZmbHLH30 markedly enhanced cold tolerance at the germination, bud, and seedling stages. ZmbHLH30 showed strong induction in embryonic tissue at 6 h under cold stress, highlighting its involvement in early cold signaling. Moreover, transcriptomic and metabolomic integration demonstrated that ZmbHLH30 regulates multiple cold-responsive pathways, including galactose metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. Differentially expressed genes and metabolites in these pathways support a regulatory role for ZmbHLH30 in coordinating metabolic and transcriptional adjustments required for cold tolerance.

3.2. ZmbHLH30 Is a Key Regulator That Enhances Maize Cold Tolerance

Plant cold tolerance is controlled by complex genetic networks in which transcription factors integrate stress signals and activate downstream protective pathways. In maize, ZmICE1 is a well-characterized cold-responsive transcription factor that binds directly to promoters of genes such as ZmASs and ZmASN2 to regulate cold tolerance and amino acid metabolism [19]. These findings illustrate the central regulatory role of bHLH family members in plant cold stress responses. Low temperature has severe impacts on maize germination, early seedling growth, and final yield, and susceptible varieties may experience yield losses of 20% to 50% when exposed to early-season cold stress [48]. Transgenic approaches have proven effective for improving cold tolerance in crops. For example, overexpression of PsLEA4 in tomato enhanced cold resilience by modulating proline metabolism and antioxidant enzyme activities, thereby improving both stress adaptation and productivity [49]. In this study, ZmbHLH30 overexpression significantly improved cold tolerance at germination, bud, and seedling stages: At germination, OE-7 and OE-6 lines improved by 2 grades to Grade I, OE-4 by 1 grade to Grade II; at bud stage, OE-2 and OE-4 improved by 2 grades to Grade I, OE-7 by 1 grade to Grade II; at seedling stage, OE-6 improved by 2 grades to Grade I, OE-3 and OE-5 by 1 grade to Grade II. Expression analysis showed strong induction of ZmbHLH30 in embryos at 6 h of cold treatment during germination, in radicles and plumules at 8 h during the bud stage, and in leaves at 8 h during the seedling stage, with the largest differences in leaf tissue. Knockout experiments confirmed ZmbHLH30′s functional importance: At germination, CR-3, CR-6, and CR-7 lines decreased by 2 grades to Grade V; at bud stage, CR-5 and CR-1 decreased by 2 grades to Grade V, CR-3 by 1 grade to Grade IV; at seedling stage, CR-7 and CR-4 decreased by 2 grades to Grade V, CR-3 by 1 grade to Grade IV. Knockout lines also exhibited significantly reduced expression at all sampling points, with the lowest levels detected at 8 h after cold treatment in embryos, radicles, plumules, leaves, stems, and roots. Collectively, these results demonstrate that ZmbHLH30 acts as a positive regulator of maize cold tolerance across key developmental stages. Moreover, for the individual traits of root length and shoot length, the D-values of all three overexpression lines were higher than those of the control, whereas those of all three gene-edited lines were lower than the control. These findings demonstrate that elevated expression of this gene can alleviate the detrimental effects of low-temperature stress on maize. Its strong cold-induced transcriptional activation and the distinct phenotypic differences between overexpression and knockout lines indicate that ZmbHLH30 is a valuable genetic resource for breeding cold-tolerant maize varieties, with important potential for molecular design breeding and sustainable agricultural production in cold-prone areas.

3.3. Effects of ZmbHLH30 on Transcriptional and Metabolic Pathways

ZmbHLH30 influences the synthesis of osmotic regulators such as proline and betaine, as well as secondary metabolites including lignin and flavonoids, by regulating hormone signaling and key metabolic pathways such as galactose metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. These coordinated changes contribute to enhanced cold tolerance through metabolic reprogramming. In this study, transcriptomic analysis identified 2585 differentially expressed genes between ZmbHLH30 overexpression lines and the control, including 468 significantly upregulated and 2117 significantly downregulated genes. These genes were enriched in GO terms related to abscisic acid response, calcium ion binding, and cold response, and were involved in major signaling and metabolic pathways such as plant hormone signal transduction, MAPK signaling pathway–plant, and linoleic acid metabolism. Within hormone signaling, ZmbHLH30 may enhance cold tolerance by modulating hormone homeostasis and downstream transcriptional activation. Metabolomic profiling detected 1101 secondary metabolites, including osmotic regulators such as proline and betaine. Among these metabolites, 32 were upregulated and 121 were downregulated in the overexpression lines. These compounds were significantly enriched in stress-associated pathways, including linoleic acid metabolism, phenylpropanoid biosynthesis, and glycerophospholipid metabolism. Phenylpropanoid biosynthesis is a major metabolic route in plants and produces secondary metabolites such as lignin, flavonoids, and phenolic acids [50], which enhance cold tolerance by scavenging reactive oxygen species, stabilizing membrane structures, and strengthening the cell wall. Previous studies have shown that galactose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis contribute to cold tolerance regulation in peach [51], overexpression of cold-induced galactoside synthase and raffinose synthase in rice enhances galactose and raffinose accumulation, alleviating cold damage [52]. Integrated transcriptomic and metabolomic analyses identified 332 co-associated differentially expressed genes enriched in 33 metabolic pathways. Core pathways included galactose metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis, indicating that ZmbHLH30 exerts a synergistic regulatory role across these stress-related processes. Within galactose metabolism, Zm00001d047582 encodes galactinol synthase 2, a type II Poaceae enzyme that enhances cold and drought tolerance when overexpressed by promoting raffinose family oligosaccharide biosynthesis, which reduces ROS accumulation and mitigates oxidative injury [53]. In the phenylpropanoid biosynthesis pathway, Zm00001d027948 encodes hydroxycinnamoyltransferase 2, a key enzyme in chlorogenic acid biosynthesis. This enzyme is highly expressed under low-temperature conditions in the cold-tolerant citrus variety Citrus ichangensis, where it promotes chlorogenic acid accumulation. Silencing CiHCT2 reduces chlorogenic acid content, weakens reactive oxygen species scavenging and membrane stability, and decreases cold tolerance [54]. Together, these findings demonstrate that ZmbHLH30 regulates maize cold tolerance through the coordinated control of multiple metabolic pathways, enabling an integrated transcriptional and metabolic response to cold stress.
Although ZmbHLH30 has been identified as a bHLH transcription factor, its localization differs from the nuclear localization characteristic of classic transcription factors, which is a key unresolved issue in this study. Transcriptomic and metabolomic data indicate that it is involved in the maize cold tolerance transcriptional regulatory network, and it is speculated that cytoplasm-localized ZmbHLH30 may regulate transcription indirectly. To resolve the discrepancy between localization and function, subsequent experiments will verify cold-induced nuclear translocation by optimizing the localization detection system, and screen its cytoplasmic interacting proteins using Co-IP combined with mass spectrometry.

4. Materials and Methods

4.1. Plant Materials and Promoter Element Analysis

The maize (Zea mays L.) inbred line B104 was used as the recipient genotype for genetic transformation. T2 generation ZmbHLH30 overexpression lines carrying a GFP tag and CRISPR/Cas9-mediated ZmbHLH30 knockout lines were generated, with B104 serving as the wild-type control. Arabidopsis thaliana ecotype Columbia-0 was used for promoter-driven GUS expression assays. All plant materials were provided by the Maize Genetics and Breeding Team of Northeast Agricultural University.
A 2000 bp region upstream of the ZmbHLH30 translation start site was selected for promoter analysis. Cis-acting regulatory elements were predicted using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 4 February 2026). TBtools-II software was used to visualize and compile the promoter element map.

4.2. Subcellular Localization

Protoplasts were isolated from T2 ZmbHLH30–GFP overexpression lines using the Maize Protoplast Preparation and Chemical Transformation Kit (Coolaber, Beijing, China). Fluorescence microscopy was used to observe the subcellular localization of the protein in maize cells. Primer sequences are provided in Table S1.

4.3. Tissue Expression Localization Analysis

The pCAMBIA1301–ZmbHLH30 Pro–GUS vector was digested with EcoR I and Nco I, and the ZmbHLH30 promoter fragment was ligated into the vector. The recombinant construct was transformed into Agrobacterium tumefaciens strain EHA105 and used to infect A. thaliana via the floral dip method. Seeds were surface-sterilized and germinated on Murashige and Skoog medium supplemented with 30 g/L sucrose and 8 g/L agar. Seedlings with 4–6 true leaves were transplanted into soil and grown under standard conditions (22 °C, 16 h light/8 h dark, 50% humidity, 10,000 lux light intensity). Healthy seedlings with normal growth were selected for GUS staining, with wild-type plants of similar growth status cultured on standard MS medium used as controls. The selected plants were immersed in GUS staining solution and incubated at 37 °C in the dark overnight, followed by decolorization in 75% ethanol. Staining patterns were then observed.

4.4. Construction of Recombinant Vectors

The overexpression plasmid CUB–ZmbHLH30–GFP–3×Flag and the CRISPR/Cas9 plasmid pXUE411C–ZmbHLH30–gRNA were constructed for functional analysis. Both vectors were introduced into Agrobacterium tumefaciens strain EHA105 and subsequently transformed into the maize inbred line B104, which served as the recipient genotype.

4.5. Functional Verification of Cold Tolerance

This experiment adopted a three-replicate design. Seeds from each treatment group were surface-sterilized with 75% ethanol for 10 min and rinsed three times with sterile water to remove residual ethanol. The seeds were then soaked in a constant-temperature water bath at 25 °C for 6 h to ensure full imbibition. For cold tolerance evaluation during germination, seeds in the control group were incubated at 25 °C for 7 days, whereas seeds in the treatment group were cultured at 10 °C for 7 days and then allowed to recover at 25 °C for 3 days. At each developmental stage and for each treatment, 10 plants with uniform growth vigor were selected for measurement, and the relative values of each trait were calculated. The measured indicators included germination rate (GR), simple vigor index (SVI), germ length (GL), average root diameter (ARD), root length (RL), root surface area (RSA), and root volume (RV). For cold tolerance assessment at the bud stage, seedlings with buds approximately 1 cm in length and exhibiting uniform GL were selected. They were transferred to a 6 °C incubator for 7 days, while the control group germinated and grew at 25 °C for the same period. The recorded indicators included GL, RL, total length, root fresh weight, bud fresh weight, total fresh weight, and the corresponding dry weights. For cold tolerance assessment at the seedling stage, seedlings were grown at 25 °C under a 16 h light/8 h dark cycle until the two-leaf-one-heart stage. The treatment group was then transferred to a 4 °C incubator for 2 to 4 days, followed by 2 days of recovery at 25 °C, while the control group remained at 25 °C. Measurements included seedling length, RL, total length, seedling fresh weight, root fresh weight, total fresh weight, and the corresponding dry weights. Additional parameters, including RV, RSA, and ARD, were obtained using a root scanner. Data analysis was performed using SPSS 22.0, and cold tolerance at the germination stage was comprehensively evaluated using the membership function method [55]. Five low-temperature tolerance grades were classified based on the D-value.
μ X i = X i − X m i n X m a x − X m i n ,   i = 1 ,   2 ,   3 …   … ,   n
W e i g h t : W i = C V i ∑ i = 1 n C V i
Comprehensive   evaluation : D = ∑ i = 1 n [ μ ( X i ) × W i ]

4.6. qRT-PCR

Samples were collected from embryos at the germination stage, from radicles and plumules at the bud stage, and from roots, stems, and leaves at the seedling stage of ZmbHLH30 overexpression lines, knockout lines, and wild-type controls used for phenotypic identification. Sampling criteria were as follows. For the germination stage, seeds were soaked at 25 °C for 6 h and then exposed to 10 °C or 25 °C for 2, 4, 6, 8, 10, and 12 h. Each treatment included 3 replicates, and each replicate consisted of 5 individual samples. For the bud stage, seeds were germinated at 25 °C until buds were approximately 1 cm long, then treated at 6 °C or 25 °C for 2, 4, 8, and 12 h. Each treatment again included 3 replicates of 5 samples. For the seedling stage, plants at the two-leaf-one-heart stage were treated at 25 °C or 4 °C for 2, 4, 8, and 12 h, using the same replicate and sample numbers [55]. Total RNA was extracted from pooled tissue using the Coolaber Plant RNA Rapid Extract Kit. All primer sequences are listed in Table S1.

4.7. Transcriptome–Metabolome Integration Analysis

Seeds of the ZmbHLH30 overexpression lines and wild-type B104 were imbibed at 25 °C for 6 h and then subjected to cold stress at 10 °C. Embryonic tissue was collected at the time point corresponding to the peak relative expression of ZmbHLH30, which occurred 6 h after the onset of cold treatment. Three biological replicates were collected per line. The embryo samples were used for both transcriptomic and metabolomic sequencing. Transcriptome sequencing was performed on the Illumina NovaSeq™ 6000 platform (LC Bio Technology Co., Ltd., Hangzhou, China) following the standard operating protocol, using paired-end sequencing with a read length of 150 bp (PE150). After library construction and sequencing, data quality assessment, sequence assembly, expression quantification, and differential gene expression analysis were performed. Differentially expressed genes were identified using a p value < 0.05 and a fold change ≥ 2. GO and KEGG enrichment analyses were conducted using the LC Bio Cloud Platform. For metabolomic analysis, maize embryos were sampled as described above, with 6 biological replicates per line, and immediately frozen in liquid nitrogen. Differential metabolites were identified using the criteria ratio > 2 or ratio ≤ 1/2, p < 0.05, and variable importance in projection (VIP) ≥ 1. DEGs were screened using |log2(fold change)| ≥ 1 and p < 0.05. GO enrichment analysis was performed on the integrated DEG set. Metabolic pathway overviews were generated using the MapMan pathway annotator (https://mapman.gabipd.org/mapman/, accessed on 4 February 2026). Regulatory pathways and metabolic pathways associated with DEGs were analyzed using MapMan KEGG pathway enrichment tools, with maize X4.2 selected as the reference dataset for grouping and visualization.

5. Conclusions

The expression pattern of ZmbHLH30 showed significant induction at specific time points under cold stress, and overexpression of this gene markedly enhanced maize cold tolerance at the germination, bud, and seedling stages, with the strongest effect observed during germination. Integrated transcriptomic and metabolomic analyses indicate that ZmbHLH30 functions as a key regulator of maize cold tolerance.

Supplementary Materials

The transcriptomic data in this study is available at https://ngdc.cncb.ac.cn/gsub/submit/gsa/subPRO079554, accessed on 4 February 2026; the metabolomic data is available at https://ngdc.cncb.ac.cn/gsub/submit/gsa/subPRO079556, accessed on 4 February 2026. The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040611/s1.

Author Contributions

L.Z., L.D., X.Z. and H.D. provided experimental ideas. J.Z., C.L. and J.X. designed the experiments. X.T. performed the experiments, analyzed the data and drafted the manuscript. Y.S. and B.Z. participated in the experiments. X.H. and Q.Z. participated in phenotypic assays. Z.W. and Y.Z. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

Natural Science Excellent Young Scholars Fund of Heilongjiang Province (YQ2022C009).

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors have no conflicts of interest to declare.

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Figure 1. Analysis of biological characteristics of ZmbHLH30. (A) Subcellular localization. DIC shows the differential interference contrast image of maize protoplasts. RFP indicates the red fluorescent protein signal. GFP shows the green fluorescent protein signal of the ZmbHLH30–GFP fusion. Merge presents the combined DIC, RFP, and GFP channels. (B) Tissue expression analysis. The left and middle panels display GUS-stained transgenic Arabidopsis thaliana seedlings observed under 4× and 10× magnification. The right panel shows unstained wild-type A. thaliana seedlings under 4× magnification. (C) Promoter element profile of ZmbHLH30, showing predicted cis-acting regulatory elements within the 2000 bp promoter region.
Figure 1. Analysis of biological characteristics of ZmbHLH30. (A) Subcellular localization. DIC shows the differential interference contrast image of maize protoplasts. RFP indicates the red fluorescent protein signal. GFP shows the green fluorescent protein signal of the ZmbHLH30–GFP fusion. Merge presents the combined DIC, RFP, and GFP channels. (B) Tissue expression analysis. The left and middle panels display GUS-stained transgenic Arabidopsis thaliana seedlings observed under 4× and 10× magnification. The right panel shows unstained wild-type A. thaliana seedlings under 4× magnification. (C) Promoter element profile of ZmbHLH30, showing predicted cis-acting regulatory elements within the 2000 bp promoter region.
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Figure 2. Functional verification of ZmbHLH30-mediated cold tolerance in maize. (A) Phenotypic evaluation of the control, ZmbHLH30 overexpression lines, and knockout lines at the germination stage at 4 °C and 25 °C. (B) Phenotypic evaluation of the same genotypes at the bud stage under cold and control conditions. (C) Phenotypic evaluation of the same genotypes at the seedling stage under cold and control conditions.
Figure 2. Functional verification of ZmbHLH30-mediated cold tolerance in maize. (A) Phenotypic evaluation of the control, ZmbHLH30 overexpression lines, and knockout lines at the germination stage at 4 °C and 25 °C. (B) Phenotypic evaluation of the same genotypes at the bud stage under cold and control conditions. (C) Phenotypic evaluation of the same genotypes at the seedling stage under cold and control conditions.
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Figure 3. Spatio-temporal expression analysis of ZmbHLH30 during maize cold stress. (A) Relative expression of ZmbHLH30 in embryos at the germination stage under cold treatment. (B,C) Relative expression of ZmbHLH30 in plumules and radicles at the bud stage under cold treatment. (D–F) Relative expression of ZmbHLH30 in leaves, stems, and roots at the seedling stage under cold treatment. Note: * indicates a significant difference at p < 0.05, and ** indicates an extremely significant difference at p < 0.01.
Figure 3. Spatio-temporal expression analysis of ZmbHLH30 during maize cold stress. (A) Relative expression of ZmbHLH30 in embryos at the germination stage under cold treatment. (B,C) Relative expression of ZmbHLH30 in plumules and radicles at the bud stage under cold treatment. (D–F) Relative expression of ZmbHLH30 in leaves, stems, and roots at the seedling stage under cold treatment. Note: * indicates a significant difference at p < 0.05, and ** indicates an extremely significant difference at p < 0.01.
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Figure 4. Comprehensive analysis of differentially expressed genes regulated by maize ZmbHLH30 under cold stress. (A) Volcano plot showing differentially expressed genes between ZmbHLH30 overexpression lines and B104 under cold stress. (B) GO functional enrichment analysis of differentially expressed genes. (C) Histogram of GO term enrichment for differentially expressed genes. (D) Bar plot of KEGG pathway enrichment for differentially expressed genes. (E) Scatter plot of GO functional enrichment for cold stress-related differentially expressed genes. (F) Scatter plot of KEGG pathway enrichment for cold stress-related differentially expressed genes.
Figure 4. Comprehensive analysis of differentially expressed genes regulated by maize ZmbHLH30 under cold stress. (A) Volcano plot showing differentially expressed genes between ZmbHLH30 overexpression lines and B104 under cold stress. (B) GO functional enrichment analysis of differentially expressed genes. (C) Histogram of GO term enrichment for differentially expressed genes. (D) Bar plot of KEGG pathway enrichment for differentially expressed genes. (E) Scatter plot of GO functional enrichment for cold stress-related differentially expressed genes. (F) Scatter plot of KEGG pathway enrichment for cold stress-related differentially expressed genes.
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Figure 5. Metabolomic analysis of cold tolerance in maize mediated by the ZmbHLH30 gene. (A,B) Volcano plots of differential metabolites identified at the MS1 level and MS2 level, showing the distribution of metabolites involved in the maize cold stress response regulated by ZmbHLH30. (C) Chord diagram showing KEGG pathway enrichment of differential metabolites in ZmbHLH30 overexpression lines.
Figure 5. Metabolomic analysis of cold tolerance in maize mediated by the ZmbHLH30 gene. (A,B) Volcano plots of differential metabolites identified at the MS1 level and MS2 level, showing the distribution of metabolites involved in the maize cold stress response regulated by ZmbHLH30. (C) Chord diagram showing KEGG pathway enrichment of differential metabolites in ZmbHLH30 overexpression lines.
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Figure 6. Integrated transcriptional–metabolic pathways regulated by ZmbHLH30 during maize cold tolerance. (A) Chord diagram showing GO enrichment of differentially expressed genes in ZmbHLH30 overexpression lines. (B) MapMan visualization of enriched metabolic and regulatory pathways associated with differentially expressed genes. (C) Correlation diagram linking key ZmbHLH30-associated differentially expressed genes with enriched metabolic pathways.
Figure 6. Integrated transcriptional–metabolic pathways regulated by ZmbHLH30 during maize cold tolerance. (A) Chord diagram showing GO enrichment of differentially expressed genes in ZmbHLH30 overexpression lines. (B) MapMan visualization of enriched metabolic and regulatory pathways associated with differentially expressed genes. (C) Correlation diagram linking key ZmbHLH30-associated differentially expressed genes with enriched metabolic pathways.
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Table 1. Comprehensive evaluation of cold tolerance in ZmbHLH30 transgenic maize lines.
Table 1. Comprehensive evaluation of cold tolerance in ZmbHLH30 transgenic maize lines.
LineGermination
D-Value		Germination
Grade		Bud
D-Value		Bud GradeSeedling
D-Value		Seedling
Grade
OE-70.897I0.613II0.357IV
OE-60.793I0.524II0.731I
OE-50.563II0.567II0.514II
OE-40.651II0.730I0.413III
OE-30.479III0.552II0.557II
OE-20.623II0.782I0.483III
OE-10.610II0.482III0.379IV
CK0.531III0.463III0.448III
CR-10.247IV0.272V0.429III
CR-20.258IV0.428IV0.353IV
CR-30.139V0.332IV0.344IV
CR-40.162V0.364IV0.296V
CR-50.191V0.219V0.499II
CR-60.148V0.402IV0.547II
CR-70.132V0.398IV0.293V
D-values and corresponding cold tolerance grades at the germination, bud, and seedling stages for overexpression (OE) lines, knockout (CR) lines, and the wild-type control (CK). D-values were calculated using the membership function method, and cold tolerance grades were assigned according to established classification criteria.
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Tang, X.; Sun, Y.; Zhang, B.; He, X.; Zhang, L.; Dong, L.; Zeng, X.; Di, H.; Zhang, J.; Li, C.; et al. ZmbHLH30 Enhances Cold Tolerance During Maize Germination. Plants 2026, 15, 611. https://doi.org/10.3390/plants15040611

AMA Style

Tang X, Sun Y, Zhang B, He X, Zhang L, Dong L, Zeng X, Di H, Zhang J, Li C, et al. ZmbHLH30 Enhances Cold Tolerance During Maize Germination. Plants. 2026; 15(4):611. https://doi.org/10.3390/plants15040611

Chicago/Turabian Style

Tang, Xinguang, Yitong Sun, Bangguo Zhang, Xinwen He, Lin Zhang, Ling Dong, Xing Zeng, Hong Di, Jiayue Zhang, Chunxiang Li, and et al. 2026. "ZmbHLH30 Enhances Cold Tolerance During Maize Germination" Plants 15, no. 4: 611. https://doi.org/10.3390/plants15040611

APA Style

Tang, X., Sun, Y., Zhang, B., He, X., Zhang, L., Dong, L., Zeng, X., Di, H., Zhang, J., Li, C., Xing, J., Zhang, Q., Wang, Z., & Zhou, Y. (2026). ZmbHLH30 Enhances Cold Tolerance During Maize Germination. Plants, 15(4), 611. https://doi.org/10.3390/plants15040611

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