Low ambient air temperature limits the growth and selection of crops in cold regions, and cold tolerance is a survival strategy for overwintering plants in cold winters. Studies of differences in transcriptional levels of winter rapeseed (Brassica rapa L.) under cold stress can improve our understanding of transcript-mediated cold stress responses. In this study, two winter rapeseed varieties, Longyou-7 (cold-tolerant) and Lenox (cold-sensitive), were used to reveal morphological, physiological, and transcriptome levels after 24 h of cold stress, and 24 h at room temperature, to identify the mechanism of tolerance to cold stress. Compared to Lenox, Longyou-7 has a shorter growth period and greater belowground mass, and exhibits stronger physiological activity after cold stress. Subsequently, more complete genomic annotation was obtained by sequencing. A total of 10,251 and 10,972 differentially expressed genes (DEG) were identified in Longyou-7 and Lenox, respectively. Six terms closely related to cold stress were found by the Gene Ontology (GO) function annotation. Some of these terms had greater upregulated expression in Longyou-7, and the expression of these genes was verified by qRT-PCR. Most of these DEGs are involved in phenylpropanoid biosynthesis, plant hormone signal transduction, ribosome biogenesis, MAPK signaling pathway, basal transcription factors, and photosynthesis. Analysis of the genes involved in the peroxisome pathway revealed that Longyou-7 and Lenox may have different metabolic patterns. Some transcription factors may play an important role in winter rapeseed tolerance to cold stress, and Longyou-7 is slightly slower than Lenox. Our results provide a transcriptome database and candidate genes for further study of winter rapeseed cold stress.

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.

Improving the soil structure and fertility of saline–alkali land is a major issue in establishing a sustainable agro-ecosystem. To explore the potential of different straw returning in improving saline–alkaline land, we utilized native saline–alkaline soil (SCK), wheat straw-returned saline–alkaline soil (SXM) and rapeseed straw-returned saline–alkaline soil (SYC) as our research objects. Soil physicochemical properties, fungal community structure and diversity of saline–alkaline soils were investigated in different treatments at 0–10 cm, 10–20 cm and 20–30 cm soil depths. The results showed that SXM and SYC reduced soil pH and total salinity but increased soil organic matter, alkali-hydrolyzable nitrogen, available phosphorus, total potassium, etc., and the enhancement effect of SYC was more significant. The total salinity of the 0–10 cm SCK soil layer was much higher than that of the 10–30 cm soil layers. Fungal diversity and abundance were similar in different soil layers in the same treatment. SXM and SYC soil had higher fungal diversity and abundance than SCK. At the genus level, Plectosphaerella, Mortierella and Ascomycota were the dominant groups of fungal communities in SXM and SYC. The fungal diversity and abundance in SXM and SYC soils were higher than in SCK soils. Correlation network analysis of fungal communities with environmental factors showed that organic matter, alkali-hydrolyzable nitrogen and available phosphorus were the main environmental factors for the structural composition of fungal communities of Mortierella, Typhula, Wickerhamomyces, Trichosporon and Candida. In summary, straw returning to the field played an effective role in improving saline–alkaline land, improving soil fertility, affecting the structure and diversity of the fungal community and changing the interactions between microorganisms.

Zinc-finger protein (ZFP) transcription factors are among the largest families of transcription factors in plants. They participate in various biological processes such as apoptosis, autophagy, and stemness maintenance and play important roles in regulating plant growth and development and the response to stress. To elucidate the functions of ZFP genes in the low-temperature response of winter (Brassica rapa L.) B. rapa, this study identified 141 members of the C2H2 ZFP gene family from B. rapa, which are heterogeneously distributed on 10 chromosomes and have multiple cis-acting elements related to hormone regulation and abiotic stress of adversity. Most of the genes in this family contain only one CDS, and genes distributed in the same evolutionary branch share mostly the same motifs and are highly conserved in the evolution of cruciferous species. The genes were significantly upregulated in the roots and growth cones of ‘Longyou-7’, indicating that they play a role in the stress-response process of winter B. rapa. The expression level of the Bra002528 gene was higher in the strongly cold-resistant varieties than in the weakly cold-resistant varieties after low-temperature stress. The survival rate and BrZAT12 gene expression of trans-BrZAT12 Arabidopsis thaliana (Arabidopsis) were significantly higher than those of the wild-type plants at low temperature, and the enzyme activities in vivo were higher than those of the wild-type plants, indicating that the BrZAT12 gene could improve the cold resistance of winter B. rapa. BrZAT12 expression and superoxide dismutase and ascorbate peroxidase enzyme activities were upregulated in winter B. rapa after exogenous ABA treatment. BrZAT12 expression and enzyme activities decreased after the PD98059 treatment, and BrZAT12 expression and enzyme activities were higher than in the PD98059 treatment but lower than in the control after both treatments together. It is speculated that BrZAT12 plays a role in the ABA signaling process in which MAPKK is involved. This study provides a theoretical basis for the resolution of cold-resistance mechanisms in strong winter B. rapa.

TCP transcription factors are important during plant growth and stress responses. However, their role in the cold stress response of Brassica rapa L. remains poorly understood. In this research, we identified the TCPs gene family in B. rapa to learn the features of the BrTCP gene family, functionally annotating the interacting proteins of TCP4 and analyzing their expression levels. Our results illustrated the presence of 19 members of the BrTCPs family in B. rapa, exhibiting molecular weights ranging from 27,367.45 to 59,433.64 Da. All identified proteins were classified as unstable, with isoelectric points ranging from 5.5 to 9.48. Subcellular localization forecasted that TCP proteins were all positioned in the nucleus. The BrTCP gene structure is relatively simple, with only seven members possessing introns, and none of the members contain UTR regions. BrTCPs comprise hormone-, light-, and stress-responsive elements. We found that the frequency of photoresponsive elements was greatest in the promoter region, suggesting that BrTCP genes are regulated by light signals and function synergistically with plant growth and development. In addition, five candidate interaction proteins of BrTCP4 were identified using yeast two-hybrid screening. RNA-Seq and q-PCR analyses of the interacting genes revealed differential expression of BrTCP family genes across various tissues following cold stress. Significant responses were observed under low-temperature stress, drought stress, and rehydration treatment, suggesting that these genes play crucial roles as regulators of the molecular network mechanisms responding to stress. This study enhances our understanding of the BrTCP family and provides significant insights into the stress tolerance mechanisms of B. 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.