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.

Brassica rapa L. is an important overwintering oilseed crop in Northwest China. Histone acetyltransferases (HATs) play an important role in epigenetic regulation, as well as the regulation of plant growth, development, and responses to abiotic stresses. To clarify the role of histone acetylation in the low-temperature response of B. rapa L., we identified 29 HAT genes in B. rapa L. using bioinformatics tools. We also conducted a comprehensive analysis of the physicochemical properties, gene structure, chromosomal localization, conserved structural domains and motifs, cis-acting regulatory elements, and evolutionary relationships of these genes. Using transcriptome data, we analyzed the expression patterns of BrHAT family members and predicted interactions between proteins; the results indicated that BrHATs play an important role in the low-temperature response of B. rapa L. HAT inhibitor (curcumin; CUR) and histone deacetylase inhibitor (Trichostatin A; TSA) were applied to four B. rapa L. varieties varying in cold resistance under the same low-temperature conditions, and changes in the physiological indexes of these four varieties were analyzed. The inhibitor treatment attenuated the effect of low temperature on seed germination, and curcumin treatment was most effective, indicating that the germination period was primarily regulated by histone acetylase. Both inhibitor treatments increased the activity of protective enzymes and the content of osmoregulatory substances in plants, suggesting that histone acetylation and deacetylation play a significant role in the response of B. rapa L. to low-temperature stress. The qRT-PCR analyses showed that the expression patterns of BrHATs were altered under different inhibitor treatments and low-temperature stress; meanwhile, we found three significantly differentially expressed genes. In sum, the process of histone acetylation is involved in the cold response and the BrHATs gene plays a role in the cold stress response.

N6-methyladenosine (m⁶A) is an mRNA modification considered essential in plants, and is a key player in gene regulation at the transcriptional and translational levels. In present study, we mapped Brassica rapa's whole transcriptome m⁶A profile under low-temperature stress in different cold tolerant varieties to elucidate the m⁶A methylation pattern. The distribution of m⁶A modifications changed significantly under low temperature stress. More 5'UTR m⁶A was deposited in strong cold-resistant varieties and responded positively to cold resistance by regulating mRNA expression abundance. The increase in m⁶A abundance was correlated with the increase in mRNA abundance after low temperature stress. ZAT12 might positively regulate its mRNA expression through m⁶A methylation. MYBC1 might be a negative regulator to cope with low-temperature stress. The hypothetical protein was involved in starch and sucrose metabolic pathways, and that the Low-quality protein was involved in the regulation of DNA binding, DNA-binding, transcription factor activity, and proline biosynthetic processes and leaf senescence pathways. In addition, a number of m⁶A methyltransferases and m⁶A demethylases play crucial roles in response to cold stress. These results revealed the critical role of m⁶A -modified genes under cold stress and provide new insights into the study of cold resistance in winter Brassica rapa.

Background: DNA methylation can change rapidly to regulate the expression of stress-responsive genes. Previous studies have shown that there are significant differences in the cold resistance of winter rapeseed (Brassica rapa L.) after being domesticated in different selection environments; however, little is known about the epigenetic regulatory mechanisms of its cold resistance formation. Methods: Four winter rapeseed materials (‘CT-2360’, ‘MXW-1’, ‘2018-FJT’, and ‘DT-7’) domesticated in different environments were selected to analyze the DNA methylation level and pattern changes under low temperature using methylation-sensitive amplified polymorphism technology with 60 primer pairs. Results: A total of 18 pairs of primers with good polymorphism were screened, and 1426 clear bands were amplified, with 594 methylation sites, accounting for 41.65% of the total amplified bands. The total methylation ratios of the four materials were reduced after low-temperature treatment, in which the DNA methylation level of ‘CT-2360’ was higher than that of the other three materials; the analysis of methylation patterns revealed that the degree of demethylation was higher than that of methylation in ‘MXW-1’, ‘2018-FJT’, and ‘DT-7’, which were 22.99%, 19.77%, and 24.35%, respectively, and that the methylation events in ‘CT-2360’ were predominantly dominant at 22.95%. Fifty-three polymorphic methylated DNA fragments were randomly selected and further analyzed, and twenty-nine of the cloned fragments were homologous to genes with known functions. The candidate genes VQ22 and LOC103871127 verified the existence of different expressive patterns before and after low-temperature treatment. Conclusions: Our work implies the critical role of DNA methylation in the formation of cold resistance in winter rapeseed. These results provide a comprehensive insight into the adaptation epigenetic regulatory mechanism of Brassica rapa L. to low temperature, and the identified differentially methylated genes can also be used as important genetic resources for the multilateral breeding of winter-resistant varieties.

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.

Hypocotyl length is a botanical trait that affects the cold tolerance of Brassica napus L. (B. napus). In this study, we constructed an F₂ segregating population using the cold-resistant short hypocotyl variety ‘16VHNTS158’ and the cold-sensitive long hypocotyl variety ‘Tianyou 2288’ as the parents, and BSA-seq was employed to identify candidate genes for hypocotyl length in B. napus. The results of parental differences showed that the average hypocotyl lengths of ‘16VHNTS158’ and ‘Tianyou 2288’ were 0.41 cm and 0.77 cm at the 5~6 leaf stage, respectively, after different low-temperature treatments, and ‘16VHNTS158’ exhibited lower relative ion leakage rates compared to ‘Tianyou 2288’. The contents of indole acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) in hypocotyls of ‘16VHNTS158’ and ‘Tianyou 2288’ increased with decreasing temperatures, but the IAA and GA contents were significantly higher than those of ‘Tianyou 2288’, and the BR content was lower than that of ‘Tianyou 2288’. The genetic analysis results indicate that the genetic model for hypocotyl length follows the 2MG-A model. By using SSR molecular markers, a QTL locus associated with hypocotyl length was identified on chromosome C04. The additive effect value of this locus was 0.025, and it accounted for 2.5% of the phenotypic variation. BSA-Seq further localized the major effect QTL locus on chromosome C04, associating it with 41 genomic regions. The total length of this region was 1.06 Mb. Within this region, a total of 20 non-synonymous mutation genes were identified between the parents, and 26 non-synonymous mutation genes were found within the pooled samples. In the reference genome of B. napus, this region was annotated with 24 candidate genes. These annotated genes are predominantly enriched in four pathways: DNA replication, nucleotide excision repair, plant hormone signal transduction, and mismatch repair. The findings of this study provide a theoretical basis for cloning genes related to hypocotyl length in winter rapeseed and their utilization in breeding.