Winter rapeseed is not only an important oilseed crop, but also a winter cover crop in Northern China, where its production was severely limited by freezing stress. As an overwinter crop, the production is severely limited by freezing stress. Therefore, understanding the physiological and molecular mechanism of winter rapeseed (Brassica napus L.) in freezing stress responses becomes essential for the improvement and development of freezing-tolerant varieties of Brassica napus. In this study, morphological, physiological, ultrastructure and transcriptome changes in the Brassica napus line “2016TS(G)10” (freezing-tolerance line) that was exposed to –2 °C for 0 h, 1 h, 3 h and 24 h were characterized. The results showed that freezing stress caused seedling dehydration, and chloroplast dilation and degradation. The content of malondialdehyde (MDA), proline, soluble protein and soluble sugars were increased, as well as the relative electrolyte leakage (REL) which was significantly increased at frozen 24 h. Subsequently, RNA-seq analysis revealed a total of 98,672 UniGenes that were annotated in Brassica napus and 3905 UniGenes were identified as differentially expressed genes after being exposed to freezing stress. Among these genes, 2312 (59.21%) were up-regulated and 1593 (40.79%) were down-regulated. Most of these DEGs were significantly annotated in the carbohydrates and energy metabolism, signal transduction, amino acid metabolism and translation. Most of the up-regulated DEGs were especially enriched in plant hormone signal transduction, starch and sucrose metabolism pathways. Transcription factor enrichment analysis showed that the AP2/ERF, WRKY and MYB families were also significantly changed. Furthermore, 20 DEGs were selected to validate the transcriptome profiles via quantitative real-time PCR (qRT-PCR). In conclusion, the results provide an overall view of the dynamic changes in physiology and insights into the molecular regulation mechanisms of winter Brassica napus in response to freezing treatment, expanding our understanding on the complex molecular mechanism in plant response to freezing stress.

The largest gene families in plants were found to be Glutathione transferases (GSTs), which played significant roles in regulating plant growth, development, and stress response. Within the GSTs gene family, members were found to play a crucial role in the low-temperature response process of plants. A comprehensive study identified a total of 70 BraGSTs genes. Cluster analysis results demonstrated that the BraGSTs in Brassica rapa (B. rapa) could be categorized into eight sub-families and were unevenly distributed across ten chromosomes. The 39 BraGSTs genes were found to be organized into 15 tandem gene clusters, with the promoters containing multiple cis-elements associated with low-temperature response. Cold stress was observed to stimulate the expression of 15 genes, with the BraGSTF2 gene exhibiting the highest level of expression, suggesting its significant involvement in winter B. rapa’s response to low-temperature stress. Subcellular localization analysis of the BraGSTF2 protein indicated its potential expression in both the cell membrane and nucleus. The analysis of stress resistance in BraGSTF2 transgenic Arabidopsis thaliana lines demonstrated that the over-expression of this gene resulted in significantly elevated levels of SOD, POD activity, and SP content compared to the wild type following exposure to low temperatures. These levels reached their peak after 24 h of treatment. Conversely, the MDA content was lower in the transgenic plants compared to the wild-type (WT) Arabidopsis (Arabidopsis thaliana L.). Additionally, the survival rate of BraGSTF2 transgenic Arabidopsis was higher than that of the WT Arabidopsis thaliana, suggesting that the BraGSTF2 gene may play a crucial role in enhancing the cold stress tolerance of winter B. rapa. This study lays a foundation for further research on the role of the BraGSTs gene in the molecular regulation of cold resistance in winter B. rapa.

Sugars will eventually be exported transporter (SWEET), a class of glucose transport proteins, is crucial in plants for glucose transport by redistribution of sugars and regulates growth, development, and stress tolerance. Although the SWEET family has been studied in many plants, little is known about its function in winter B. rapa (Brassica rapa L.). Bioinformatics approaches were adopted to identify the SWEET gene (BraSWEETs) family in B. rapa to investigate its role during overwintering. From the whole-genome data, 31 BraSWEET genes were identified. Gene expansion was realized by tandem and fragment duplication, and the 31 genes were classified into four branches by phylogenetic analysis. As indicated by exon–intron structure, cis-acting elements, MEME (Multiple EM for Motif Elicitation) motifs, and protein structure, BraSWEETs were evolutionarily conserved. According to the heat map, 23 BraSWEET genes were differentially expressed during overwintering, revealing their potential functions in response to low-temperature stress and involvement in the overwintering memory-formation mechanism. BraSWEET10 is mainly associated with plant reproductive growth and may be crucial in the formation of overwintering memory in B. rapa. The BraSWEET10 gene was cloned into B. rapa (Longyou-7, L7). The BraSWEET10 protein contained seven transmembrane structural domains. Real-time fluorescence quantitative PCR (qRT-PCR) showed that the BraSWEET10 gene responded to low-temperature stress. BraSWEET10 was localized to the cell membrane. The root length of overexpressing transgenic A. thaliana was significantly higher than that of wild-type (WT) A. thaliana under low temperatures. Our findings suggest that this gene may be important for the adaptation of winter B. rapa to low-temperature stress. Overall, the findings are expected to contribute to understanding the evolutionary links of the BraSWEET family and lay the foundation for future studies on the functional characteristics of BraSWEET genes.

The largest gene families in plants were found to be Glutathione transferase (GSTs), which played significant roles in regulating plant growth, development, and stress response. Within the GSTs gene family, members were found to play a crucial role in the low-temperature response process of plants. A comprehensive study identified a total of 70 BraGSTs genes. Cluster analysis results demonstrated that the BraGSTs in Brassica rapa(B.rapa) could be categorized into eight sub-families and were unevenly distributed across ten chromosomes. The 39 BraGSTs genes were found to be organized into 15 tandem gene clusters, with the promoters containing multiple cis-elements associated with low-temperature response. Cold stress was observed to stimulate the expression of 15 genes, with the BraGSTF2 gene exhibiting the highest level of expression, sug-gesting its significant involvement in winter B.rapa's response to low-temperature stress. Sub-cellular localization analysis of the BraGSTF2 protein indicated its potential expression in both the cell membrane and nucleus. The analysis of stress resistance in BraGSTF2 transgenic Arabidopsis thaliana lines emon-strated that the over-expression of this gene resulted in significantly elevated levels of SOD, POD activity, and SP content compared to the wild type following exposure to low temperatures. These levels reached their peak after 24 hours of treatment. Conversely, the MDA content was lower in the transgenic plants compared to the wild type. Additionally, the survival rate of BraGSTF2 transgenic Arabidopsis thaliana was higher than that of the wild type, suggesting that the BraG-STF2 gene may play a crucial role in enhancing the cold stress tolerance of winter B. rapa. This study lay a foundation for further study on the role of BraGSTs gene in the molecular regulation of cold resistance in winter B.rapa.

Glutathione S-transferases (GSTs) plays pivotal roles in plant growth, abiotic stress responses, detoxification of xenobiotics, and maintenance of redox homeostasis. In this study, transcriptomic analysis was employed to identify differentially expressed genes (DEGs) in pea of DX27 under low temperature stress(-4°C) at 3, 6, and 12 h. GO and KEGG enrichment analyses of global transcriptomic data further revealed that DEGs were significantly enriched in oxidation-reduction processes, stress responses, glutathione metabolism, and secondary metabolite biosynthesis—functional categories. A total of 52 PsatGST genes were identified and classified into nine subfamilies based on phylogenetic relationships. Chromosomal localization revealed a non-random distribution across seven chromosomes. Promoter cis-element analysis indicated that PsatGST genes harbor diverse regulatory elements associated with light responsiveness, hormone signaling (auxin, abscisic acid, gibberellin, salicylic acid, and MeJA), and abiotic/biotic stress responses (low-temperature, wounding, and anaerobic induction). PsatGSTU18 and PsatGSTF2 expression analysis showing pronounced up-regulation at 12 h under low temperature stress (-4°C) at 3, 6, and 12 h. These findings provide a foundational framework for understanding the evolutionary history and functional diversification of the PsatGSTs gene family and offer valuable candidate genes for breeding stress-tolerant pea varieties.

The pea weevil (Bruchus pisorum L.) causes severe yield losses in cultivated pea (Pisum sativum L.), yet the molecular basis of host resistance remains unclear. Here, we performed integrated transcriptomic and metabolomic analyses on pods and seeds of resistant (YWD) and susceptible (LW1) pea genotypes to characterize the constitutive defence network against weevils. Extensive transcriptional and metabolic reprogramming was observed in resistant materials, with upregulation of defence pathways and suppression of primary metabolism. Pods directed phenylpropanoid flux toward isoflavonoid biosynthesis and enhanced cutin/wax pathways, while seeds redirected flux to flavone/flavonol biosynthesis, accumulating high levels of flavone C-glycosides including isovitexin (5260.46-fold) and vitexin (3771.09-fold). We identified Dof21 as a key transcription factor putatively regulating flavonoid biosynthesis, with its Dof domain predicted to bind promoters of 4CL1, CHS3 and CYP75B. A seed-specific lectin gene LG16 showed 8.99-fold higher expression in resistant seeds with negligible expression in pods. The ABA catabolite dihydrophaseic acid and semi-volatile compounds vanilloloside and cimidahurinine were significantly enriched in resistant seeds, implicating ABA turnover and glycosylation in defence compound stabilization. This study provides a multi-dimensional molecular framework for weevil resistance in pea and offers key candidate genes including Dof21, LG16, 4CL1, CHS3 and CYP75B for resistant germplasm screening and marker-assisted breeding.