Physiological responses and transcriptome analysis of floral organs to low-temperature stress in Yuluxiang pear

【Objective】Global climate change has led to increased frequency of spring frost events during the flowering period of pear trees, causing severe damage to flower organs and significant economic losses. Yuluxiang pear (Pyrus bretschneideri) is an important cultivar in northern China. Current research on the cold resistance mechanisms of its floral organs has primarily focused on freezing injury investigations, disaster loss assessments, field protection techniques, and physiological responses under temperature gradients. However, the physiological and molecular response mechanisms of floral organs under prolonged duration of low-temperature stress remain unclear. Low-temperature stress during the flowering period in major growing regions of Yuluxiang pear, such as Xixian County and Taigu County occurs frequently. This study aimed to comprehensively analyze the physiological and transcriptomic re- sponses of Yuluxiang pear flower organs under sustained low-temperature stress, identify key genes involved in cold stress response and regulatory pathways, and provide candidate genes and a theoretical foundation for molecular breeding of cold- tolerant pear varieties.【Methods】Flower organs at full blooming stage were collected and subjected to 0 ℃ for different durations (1 h, 4 h, 8 h, 12 h, and 24 h). The control group (CK) was maintained at room temperature. The physiological indicators, including malondialdehyde (MDA) content, relative electrical conductivity, activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as soluble sugar (SS) and soluble protein (SP) content, were measured. The transcriptome sequencing was performed on CK and low- temperaturetreated samples (CT1, CT4, CT8) using Illumina high- throughput sequencing. The differentially expressed genes (DEGs) were screened with thresholds of FDR ＜ 0.05 and |log2FC| ≥ 2. The functional enrichment analysis (GO and KEGG), weighted gene co-expression network analysis (WGCNA), and qRT- PCR validation were conducted to identify key genes and pathways involved in cold stress response.【Results】Under low-temperature stress, the floral organs began to exhibit visible damage over time: at 4 h, the ovary started to blacken; at 8 h, the style showed browning; at 12 h, pronounced browning was observed in both the style and stigma, while the petals gradually turned translucent; by 24 h, the ovary was severely frozen and browned, the style and stigma were fully browned, and some stamens began to wilt.With prolonged low- temperature stress, MDA content and relative electrical conductivity showed a fluctuating upward trend, with peaks at 8 h and 4 h, respectively. The antioxidant enzyme activities (SOD, CAT, POD) increased significantly in the early stage (1 h) and then decreased, indicating progressively severe oxidative damage that exceeded the floral organs’self-tolerance capacity, led to a notable decrease in antioxidant defense ability. The later rise in enzyme activities might reflect adaptive adjustments by the floral organs in the late stage of stress. Moreover, SOD activity showed a significant positive correlation with the activities of CAT and POD, as well as with stress duration, suggesting that Yuluxiang pear mitigated oxidative damage through the synergistic action of multiple antioxidant enzymes. The soluble sugar content peaked at 12 h, while the soluble protein content peaked at 1 h and then declined. The transcriptome analysis identified 1836, 1125, and 1512 DEGs in CK_vs_CT1, CK_vs_CT4, and CK_vs_CT8, respectively. The DEGs were significantly enriched in plant hormone signal transduction, MAPK signaling pathway, starch and sucrose metabolism, and lipid metabolism. A significant upregulation was observed in the expression of the plant hormone signal transduction-related genes PP2C06 and PP2C51. The key transcription factors, especially from the WRKY family (WRKY53), were significantly upregulated at CT1. The analysis of the hub genes within the module highly correlated with SOD and POD activities revealed that the genes in the lightcyan module exhibited the highest expression levels and were significantly upregulated at CT1. The core genes, including the pspp.Chr03.01170 (CAF1), the pspp.Chr07.00236 (SQE1), were identified as potential key regulators. These genes likely enhance the cold tolerance of yuluxiang pear floral organs through the regulation of antioxidant enzyme activiy. The qRT-PCR validation of ten selected DEGs confirmed the reliability of the RNA-seq data.【Conclusion】The Yuluxiang pear flower organs responded to low-temperature stress through a complex regulatory network involving antioxidant defense, osmotic adjustment, and transcriptional reprogramming. The key genes and pathways, including members of the PP2C gene family、WRKY transcription factors and the lightcyan module genes, would play crucial roles in the response to low- temperature stress. These findings would provide valuable insights into the molecular mechanisms of cold resistance in pear flowers and offer candidate genes for future molecular breeding efforts aiming at improving cold tolerance in pear and other fruit trees. The subsequent research might be carried out on cold resistance evaluations of floral organs across different pear varieties and integrated transcriptomic and metabolomic analyses to elucidate the molecular basis of varietal differences in cold tolerance. Concurrently, it is essential to validate the biological functions of genes such as WRKY53, CAF1, and SQE1 using gene editing or transgenic technologies, in order to clarify their specific roles in the low-temperature signaling pathway and further investigate their downstream target genes.