Tao Yu

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.

Single-replicate field trials are common in early-stage maize hybrid screening, but raw plot observations contain field noise that can weaken genomic prediction. We evaluated whether pedigree and spatial adjustment improve the predictability of phenotypic targets and Top-N recovery, whether joint pedigree-spatial adjustment provides complementary gains, and whether target construction has a larger influence than baseline model choice. Across four sites, five agronomic traits, four phenotypic inputs, and six genomic prediction models, pedigree-corrected phenotypes (Ped) increased mean predictive ability from 0.465 ± 0.157 for raw observations (Raw) to 0.573 ± 0.131 and increased upper-tail Top-20 recall from 0.558 ± 0.146 to 0.656 ± 0.146 under a 20% predicted retention window. Spatial correction alone (Spa) was less predictable than Raw, whereas joint pedigree-spatial correction (Ped+Spa) improved over Raw but did not exceed Ped. Phenotypic input explained substantially more variation in predictive ability than model choice, whose main effect was not significant (F5428 = 1.59, p = 0.161). These results support pedigree correction as a useful target-construction strategy in the evaluated single-replicate trials while showing that spatial adjustment is conditional on model adequacy and trial structure. Top-N recall directly represents favorable direction recovery for grain yield; for grain moisture, plant height, ear height, and silking date, the archived upper-tail metric is interpreted as rank consistency because the favorable direction or ideotype is breeding-program-dependent.

Drought stress is a major abiotic factor that adversely affects plant growth, development, and crop productivity. Lipoxygenases (LOXs) are key enzymes in the jasmonic acid (JA) biosynthetic pathway and play crucial roles in plant responses to environmental stresses. In this study, the ZmLOX2 gene was cloned from maize (Zea mays L.), and was found to encode a chloroplast-localized 13-lipoxygenase (13-LOX) protein. Expression analysis revealed that ZmLOX2 was predominantly expressed in leaves and was strongly induced under osmotic stress conditions. Compared with wild-type plants, transgenic Arabidopsis thaliana overexpressing ZmLOX2 exhibited significantly higher survival rates, enhanced growth performance, and improved root development under water deficit conditions. Physiological and biochemical analyses showed that the overexpression lines displayed higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), accompanied by significantly reduced malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation. Further investigation demonstrated that the expression levels of JA biosynthesis- and signaling-related genes, including ALLENE OXIDE SYNTHASE (AtAOS), 12-OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3), MYC DOMAIN PROTEIN 2 (AtMYC2), and JASMONATE ZIM-DOMAIN 1 (AtJAZ1), were markedly upregulated in the transgenic lines. Protein–protein interaction analysis suggested that ZmLOX2 may interact with ALLENE OXIDE SYNTHASE 1 (AOS1), HYDROPEROXIDE LYASE 1 (HPL1), and ALPHA-DIOXYGENASE 1 (DOX1). Collectively, these results suggest that ZmLOX2 may contribute to plant adaptation to water limitation by regulating responses associated with JA, enhancing antioxidant defense, and maintaining ROS homeostasis.

Maize (Zea mays L.), a typical thermophilic crop originating from tropical regions, exhibits an inherent sensitivity to low-temperature stress. Cold stress severely restricts maize seed germination, seedling growth, the physiological metabolism, and the final grain yield, which greatly limits its geographical cultivation range and sustainable industrial development. Elucidating the molecular regulatory mechanisms underlying maize cold tolerance and excavating cold-resistant functional genes are essential for the molecular breeding of cold-tolerant maize varieties and expanding maize planting areas in high-latitude and low-temperature-prone regions. In this study, using the strongly cold-tolerant maize inbred line B144 as the experimental material, we cloned the ZmMAPKKKA gene (NCBI accession: LOC103651289) and systematically screened and verified its cold-stress-specific interacting proteins via multiple molecular biological assays. The full-length coding sequence (CDS) of ZmMAPKKKA is 1134 bp, encoding a 377-amino-acid protein with a predicted molecular weight of 40.37 kDa. The quantitative real-time PCR (qRT-PCR) results demonstrated that the ZmMAPKKKA expression was significantly upregulated by 16.56-fold in maize roots after 12 h of low-temperature treatment, indicating a tissue-specific and robust cold response in root tissues. A total of 25 interacting proteins were identified through yeast two-hybrid screening, among which three stress-responsive proteins, including a protein kinase (LOC100286253), a protein phosphatase 2C (PP2C) (LOC542176), and a NAC transcription factor (LOC118474710), were selected for subsequent verification. The Pull-Down, Co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays consistently confirmed that ZmMAPKKKA specifically interacts with these three proteins both in vitro and in vivo under cold stress conditions. This study is the first to construct a ZmMAPKKKA-centered protein interaction module in the maize mitogen-activated protein kinase (MAPK) cascade under cold stress, establishing a novel kinase–phosphatase–transcription factor regulatory cascade that improves the current understanding of cold signal transduction mechanisms in maize. Homologous genes of ZmMAPKKKA in gramineous crops including rice (Oryza sativa) and sorghum (Sorghum bicolor) have been proven to participate in diverse abiotic stress responses, suggesting the conserved functional roles of MAPKKK family genes across gramineous species. Collectively, our findings provide comprehensive insights into the molecular mechanism of the maize MAPK signaling pathway mediating cold stress adaptation and supply valuable functional gene resources for cold-tolerant maize germplasm innovation and molecular breeding.

To elucidate the mechanisms by which the rhizosphere microbial community influences cold tolerance in maize, this study employed the metagenomic technology to systematically analyze the community composition, functional characteristics, and their association with host cold tolerance in the rhizosphere of maize genotypes with different cold tolerance (cold-tolerant material B144 and cold-sensitive material Q319, among others) (n = 3 biological replicates per genotype). The results revealed that the rhizosphere microbial community of the cold-tolerant genotype B144 exhibited higher species diversity and more complex genomic features. LEfSe analysis indicated that the rhizosphere soil microbiota of B144 was significantly enriched in two major phyla, Firmicutes and Actinobacteria, as well as microbial taxa with stress tolerance potential, such as the Bacillus and Streptomyces. Further functional analysis revealed that the microbial community was specifically enriched in metabolic pathways related to glycan biosynthesis and metabolism, as well as coenzyme and vitamin metabolism. We hypothesize that the physiological stability of maize under low temperatures can be enhanced through mechanisms such as the synthesis of extracellular polysaccharides to reduce the freezing point and the provision of vitamins and antioxidant substances. In contrast, the rhizosphere microorganisms of the cold-sensitive material Q319 were more enriched in basic metabolic functions. The present study elucidates the pivotal mechanisms by which rhizosphere microorganisms facilitate maize resistance to low-temperature stress from a functional perspective. This provides theoretical support and new strategies for enhancing crop stress resistance by regulating the rhizosphere microbiome.

Maize (Zea mays L.) originated in tropical and subtropical regions. During its growth and development, cold stress severely threatens seedling survival rates and final yield by inducing oxidative stress, compromising cell membrane integrity, and causing “physiological drought.” The Domain of Unknown Function 966 (DUF966) gene family comprises a class of regulatory factors containing conserved domains of undetermined function. Although they are considered to be extensively involved in plant growth, development, and stress response, their specific roles within the maize cold-tolerance regulatory network remain to be explored. In this study, 10 ZmDUF966 family members were identified via genome-wide analysis, and their phylogenetic relationships, gene structures, conserved motifs, chromosomal localizations, and cis-acting elements were systematically analyzed. The results indicate that the ZmDUF966 family is highly conserved among Poaceae species, and its promoters are enriched with stress-responsive elements such as LTR and ABRE. The core gene, ZmDUF966-10, was significantly up-regulated (approximately 35-fold at 48 h, p < 0.05) as validated by RT-qPCR under cold stress and is post-transcriptionally regulated by conserved miRNAs such as zma-miR159. Further yeast two-hybrid experiments revealed a preliminary physical interaction between the ZmDUF966-10 protein and an ABA/WDS-induced protein, suggesting its potential involvement in ABA-mediated stress signaling, though functional validation remains to be conducted. In conclusion, this study identifies ZmDUF966-10 as a promising candidate gene that responds to cold signals through multi-level regulatory networks, providing a valuable gene resource for further functional characterization and potential application in cold-tolerant maize improvement.

Sucrose phosphate synthase (SPS) is a key rate-limiting enzyme that regulates carbon partitioning and stress tolerance in plants. In this study, we systematically characterized the SPS gene family in maize (Zea mays L.) and identified key members and their interaction networks involved in drought responses. A total of seven ZmSPS genes were identified through genome-wide bioinformatics analyses. Motif composition, gene structure, phylogenetic relationships, and synteny analyses indicated that the ZmSPS gene family is highly conserved among monocot species. Promoter analysis revealed that the upstream regions of ZmSPS genes are enriched with multiple stress responsive cis-acting elements. Drought stress treatments combined with quantitative real-time PCR (RT-qPCR) analyses showed that the expression of ZmSPS3 was significantly upregulated with increasing stress duration. Furthermore, yeast two-hybrid assays demonstrated that ZmSPS3 physically interacts with protein kinases and F-box proteins. These interactions suggest a potential involvement of ZmSPS3 in post-translational modification and protein stability regulation during osmotic stress. As a potential candidate gene responsive to drought, ZmSPS3 provides a preliminary basis for understanding the complex drought-response networks in maize.

Low temperature severely restricts maize seedling establishment and yield in northern China, but the proteomic basis of low-temperature tolerance in maize remains unclear. This study used TMT-labeled quantitative proteomics combined with data-independent acquisition (DIA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) to analyze dynamic proteome changes in two maize inbred lines (low-temperature-tolerant B144 and low-temperature-sensitive Q319) at the three-leaf stage under 5 °C treatment. A total of 4367 non-redundant proteins were identified. For differentially expressed proteins (DEPs, fold change >2.0 or p < 0.05, false discovery rate [FDR] < 0.05), B144 showed exclusive upregulation under stress (6 DEPs at 24 h; 16 DEPs at 48 h), while Q319 exhibited mixed regulation (9 DEPs at 24 h: 6 upregulated, 3 downregulated; 21 DEPs at 48 h: 19 upregulated, 2 downregulated). Functional annotation indicated that ribosomal proteins, oxidoreductases, glycerol-3-phosphate permease, and actin were significantly upregulated in both lines. Pathway enrichment analysis revealed associations with carbohydrate metabolism, amino acid biosynthesis, and secondary metabolite synthesis. Weighted gene co-expression network analysis (WGCNA) identified genotype-specific expression patterns: B144 showed differential expression of proteins related to acetyl-CoA synthetase and fatty acid β-oxidation at 24 h and of proteins related to D-3-phosphoglycerate dehydrogenase at 48 h; Q319 showed differential expression of proteasome-related proteins at 24 h and of proteins related to elongation factor 1α (EF-1α) at 48 h. Venn analysis found no shared DEPs between the two lines at 24 h but four overlapping DEPs at 48 h. These results clarify proteomic differences underlying low-temperature tolerance divergence between maize genotypes and provide candidate targets for molecular breeding of low-temperature-tolerant maize.

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial and rate-limiting step of glycerolipid biosynthesis, yet their contribution to salt tolerance in the soybean (Glycine max (L.) Merr.) plants remains largely uncharacterized. In this study, a total of 27 GmGPAT genes were identified, and their evolutionary relationships, chromosomal distribution, conserved motifs, and cis-regulatory elements were comprehensively analyzed. Through transcriptomic and qPCR analyses, many GmGPATs were found to be predominantly expressed in roots, with GmGPAT1, a plastid-targeted isoform, displaying the most rapid and pronounced transcriptional activation under salt stress. GFP-fusion experiments in transient expression assays confirmed plastid localization of GmGPAT1. Heterologous expression in Escherichia coli together with enzyme kinetics analyses validated its enzymatic function as a GPAT family member. The soybean hairy-root lines overexpressing GmGPAT1 exhibited enhanced root elongation, increased biomass, and improved photosynthetic efficiency under 120 mM NaCl stress, while CRISPR/Cas9 knockout mutants showed pronounced growth inhibition. Physiological assays demonstrated that GmGPAT1 overexpression mitigated oxidative damage by limiting reactive oxygen species (ROS) accumulation and lipid peroxidation, increasing antioxidant enzyme activities (CAT, SOD, POD), and elevating the ratios of AsA/DHA and GSH/GSSG. These changes contributed to redox homeostasis and improved Na⁺ extrusion capacity. A genome-wide association study (GWAS) involving 288 soybean accessions identified a single nucleotide polymorphism in the GmGPAT1 promoter that was significantly correlated with salt tolerance, and the beneficial Hap1 allele emerged as a promising molecular marker for breeding. Together, these analyses emphasize the status of GmGPAT1 as a major regulator of salt stress adaptation through the coordinated modulation of lipid metabolism and redox balance, extend the functional annotation of the soybean GPAT family, and highlight new genetic resources that can be leveraged to enhance tolerance to salt stress in soybean cultivars.

Maize (Zea mays L.) seedlings are highly susceptible to low-temperature stress, which significantly impacts maize yield and quality. A zinc finger protein transcription factor (ZmZFP69) mutant and a control (B73) maize inbred line were subjected to low-temperature treatment, and changes in the phenotypic characteristics, hormone levels, and other indicators before and after the treatment were systematically identified. Subsequently, a combined RNA-seq and DAP-seq analysis was conducted to explore the influence of ZmZFP69 on the promoters of downstream genes. Finally, the proteins interacting with ZmZFP69 were examined using InterProDesign combined with BiFC and subcellular localization. The zmzfp69 homozygous mutant maize inbred line exhibited enhanced low-temperature tolerance compared to the control. RNA-seq and DAP-seq analyses revealed that ZmZFP69 binds to the ZmAOX2 gene promoter, significantly suppressing its expression. The interaction between ZmZFP69 and the downstream protein ZmBG6 was confirmed by InterProDesign, subcellular localization, and BiFC assays. ZmZFP69 negatively regulates maize seedling low-temperature tolerance by inhibiting ZmAOX2 expression and interacting with ZmBG6.