Cold stress is regarded as a key factor limiting widespread use for bermudagrass (Cynodon dactylon). Therefore, to improve cold tolerance for bermudagrass, it is urgent to understand molecular mechanisms of bermudagrass response to cold stress. However, our knowledge about the molecular responses of this species to cold stress is largely unknown. The objective of this study was to characterize the transcriptomic response to low temperature in bermudagrass by using RNA-Seq platform. Ten cDNA libraries were generated from RNA samples of leaves from five different treatments in the cold-resistant (R) and the cold-sensitive (S) genotypes, including 4 °C cold acclimation (CA) for 24 h and 48 h, freezing (−5 °C) treatments for 4 h with or without prior CA, and controls. When subjected to cold acclimation, global gene expressions were initiated more quickly in the R genotype than those in the S genotype. The R genotype activated gene expression more effectively in response to freezing temperature after 48 h CA than the S genotype. The differentially expressed genes were identified as low temperature sensing and signaling-related genes, functional proteins and transcription factors, many of which were specifically or predominantly expressed in the R genotype under cold treatments, implying that these genes play important roles in the enhanced cold hardiness of bermudagrass. KEGG pathway enrichment analysis for DEGs revealed that photosynthesis, nitrogen metabolism and carbon fixation pathways play key roles in bermudagrass response to cold stress. The results of this study may contribute to our understanding the molecular mechanism underlying the responses of bermudagrass to cold stress, and also provide important clues for further study and in-depth characterization of cold-resistance breeding candidate genes in bermudagrass.

Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2‐type zinc‐finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α‐branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β‐branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one‐hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4‐mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4‐mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops.

The global citrus industry is under severe threat from devastating diseases, including Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), and Citrus tristeza disease, caused by Citrus tristeza virus (CTV). Establishing efficient pathogen elimination techniques is crucial for developing virus-free citrus nursery systems and helps facilitate the prevention and control of devastating diseases. This study systematically evaluated the multiple effects of hot water treatment and plant growth regulator application on bud germination, pathogen elimination, antioxidant enzyme activities, and CsWUS expression in citrus scion. The results showed that moderate hot-water treatment at 40 °C for 5 min promoted bud germination, while higher temperatures or extended durations were inhibitory. Both 6-benzylaminopurine (6-BA) and melatonin (MT) significantly enhanced germination, with 50 mg/L 6-BA and 100 μM MT being most effective. All treatments significantly enhanced pathogen elimination and increased the activities of antioxidant enzymes SOD, POD, and CAT, with melatonin concentrations of 100 μM and above reducing CLas and CTV titers by over 93.64% and 96.07%. Furthermore, hot-water treatment and MT, but not 6-BA, upregulated the expression of the virus-resistance-related gene CsWUS. A combined treatment of hot water and MT did not yield synergistic benefits. In conclusion, melatonin treatment, particularly at 100 μM, optimally balances high bud germination with effective pathogen elimination, likely through activating antioxidant defenses and CsWUS-mediated immunity. This study provides a practical and efficient strategy for citrus pathogen-free seedling production.

Global climate change is leading to more frequent extreme cold events, underscoring the need to study citrus cold tolerance to support breeding and enable potential northward expansion of citrus cultivation. In this study, the ‘Miyagawa’ wild type and its cold-tolerant mutant were selected for systematic comparison across cold-resistant phenotypes, leaf tissue structure, physiological and biochemical characteristics, and Cor8 gene expression. The mutant exhibited 50% lower relative conductivity and malondialdehyde (MDA) content under −6 °C stress compared to the wild type, indicating reduced membrane damage. Antioxidant enzyme activities were significantly higher in the mutant: superoxide dismutase (SOD) activity increased by 10–30%, peroxidase (POD) by 28%, and catalase (CAT) by up to 2-fold. Proline content was 57% higher in the mutant at peak levels, supporting stronger osmotic regulation. Moreover, Cor8 gene expression in the mutant was up to 2.98 times higher than in the wild type during natural overwintering. These findings confirm that the ‘Miyagawa’ mutant possesses distinct physiological, anatomical, and molecular advantages for low-temperature adaptation and provides valuable germplasm for breeding cold-tolerant citrus varieties.

Cold stress is a critical abiotic factor that severely limits plant growth and agricultural productivity in subtropical regions. Poncirus trifoliata exhibits exceptional cold hardiness and is widely used as a rootstock in Citrus. However, the key genes and mechanisms conferring this resilience remain largely unexplored. Here, we characterized PtCOR8, a cold-induced gene isolated from P. trifoliata. Phylogenetic and subcellular localization analyses confirmed that PtCOR8 encodes a plasma membrane-localized protein belonging to the WCOR413 family. Functional validation revealed that heterologous overexpression of PtCOR8 in tomato significantly enhanced cold tolerance, concomitant with reduced malondialdehyde (MDA) content, elevated peroxidase (POD) activity, and upregulation of cold-responsive genes (e.g., CIN8). Notably, expression profiling of COR8 in 16 citrus accessions under natural overwintering conditions indicated a strong positive correlation between its expression level and cold tolerance of different genotypes. Transgenic tomato plants with PtCOR8 driven by its native promoter also presented enhanced cold tolerance, confirming that the native promoter is sufficient to drive functional expression under cold stress in the tomato system. Through promoter deletion and β-glucuronidase (GUS) staining experiments, the ATCT motif was further identified as a cis-acting element capable of mediating cold-induced promoter activity. Our findings uncover a dual-layered mechanism in which the PtCOR8 protein alleviates membrane lipid peroxidation and oxidative damage, while its transcription level is precisely modulated by a novel promoter regulatory mechanism, thereby improving freezing tolerance. This study provides important genetic insights and a valuable gene resource for cold-resistant citrus breeding.