Transcriptome Profile Analysis of Winter Rapeseed (Brassica napus L.) in Response to Freezing Stress, Reveal Potentially Connected Events to Freezing 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.

Genome-Wide Identification of GSTs Gene Family and Functional Analysis of BraGSTF2 of Winter Rapeseed (Brassica rapa L.) under Cold 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.

Corrigendum: Transcriptomic insights into root development and overwintering transcriptional memory of Brassica rapa L. grown in the field.

[This corrects the article DOI: 10.3389/fpls.2022.900708.].

Endothelial depletion of Atg7 triggers astrocyte-microvascular disassociation at blood-brain barrier.

Microvascular basement membrane (BM) plays a pivotal role in the interactions of astrocyte with endothelium to maintain the blood-brain barrier (BBB) homeostasis; however, the significance and precise regulation of the endothelial cell-derived BM component in the BBB remain incompletely understood. Here, we report that conditional knockout of Atg7 in endothelial cells (Atg7-ECKO) leads to astrocyte-microvascular disassociation in the brain. Our results reveal astrocytic endfeet detachment from microvessels and BBB leakage in Atg7-ECKO mice. Furthermore, we find that the absence of endothelial Atg7 downregulates the expression of fibronectin, a major BM component of the BBB, causing significantly reduced coverage of astrocytes along cerebral microvessels. We reveal Atg7 triggers the expression of endothelial fibronectin via regulating PKA activity to affect the phosphorylation of cAMP-responsive element-binding protein. These results suggest that Atg7-regulated endothelial fibronectin production is required for astrocytes adhesion to microvascular wall for maintaining the BBB homeostasis. Thus, endothelial Atg7 plays an essential role in astrocyte-endothelium interactions to maintain the BBB integrity.

Transcriptomic Insights Into Root Development and Overwintering Transcriptional Memory of Brassica rapa L. Grown in the Field.

As the only overwintering oil crop in the north area of China, living through winter is the primary feature of winter rapeseed. Roots are the only survival organ during prolonged cold exposure during winter to guarantee flowering in spring. However, little is known about its root development and overwintering memory mechanism. In this study, root collar tissues (including the shoot apical meristem) of three winter rapeseed varieties with different cold resistance, i.e., Longyou-7 (strong cold tolerance), Tianyou-4 (middle cold tolerance), and Lenox (cold-sensitive), were sampled in the pre-winter period (S1), overwintering periods (S2-S5), and re-greening stage (S6), and were used to identify the root development and overwintering memory mechanisms and seek candidate overwintering memory genes by measuring root collar diameter and RNA sequencing. The results showed that the S1-S2 stages were the significant developmental stages of the roots as root collar diameter increased slowly in the S3-S5 stages, and the roots developed fast in the strong cold resistance variety than in the weak cold resistance variety. Subsequently, the RNA-seq analysis revealed that a total of 37,905, 45,102, and 39,276 differentially expressed genes (DEGs), compared to the S1 stage, were identified in Longyou-7, Tianyou-4, and Lenox, respectively. The function enrichment analysis showed that most of the DEGs are significantly involved in phenylpropanoid biosynthesis, plant hormone signal transduction, MAPK signaling pathway, starch and sucrose metabolism, photosynthesis, amino sugar and nucleotide sugar metabolism, and spliceosome, ribosome, proteasome, and protein processing in endoplasmic reticulum pathways. Furthermore, the phenylpropanoid biosynthesis and plant hormone signal transduction pathways were related to the difference in root development of the three varieties, DEGs involved in photosynthesis and carbohydrate metabolism processes may participate in overwintering memory of Longyou-7 and Tianyou-4, and the spliceosome pathway may contribute to the super winter resistance of Longyou-7. The transcription factor enrichment analysis showed that the WRKY family made up the majority in different stages and may play an important regulatory role in root development and overwintering memory. These results provide a comprehensive insight into winter rapeseed's complex overwintering memory mechanisms. The identified candidate overwintering memory genes may also serve as important genetic resources for breeding to further improve the cold resistance of winter rapeseed.

Screening and verification of reference genes for analysis of gene expression in winter rapeseed (Brassica rapa L.) under abiotic stress.

Winter rapeseed (Brassica rapa L.) is the main oilseed crop in northern China and can safely overwinter at 35 (i.e., Tianshui, China) to 48 degrees north latitude (i.e., Altai, Heilongjiang, Raohe, and Xinjiang, China). In order to identify stable reference genes to understand the molecular mechanisms of stress tolerance in winter rapeseed, internal reference genes of winter rapeseed under four abiotic stresses were analyzed using GeNorm, NormFinder, BestKeeper, and RefFinder software. The most stable combinations of internal reference genes were ß-actin and SAND in cold-stressed leaves, ß-actin and EF1a in cold-stressed roots, F-box and SAND in high temperature-stressed leaves, and PP2A and RPL in high temperature-stressed roots, SAND and PP2A in NaCl-stressed leaves, RPL and UBC in NaCl-stressed roots, RPL and PP2A in PEG-stressed leaves, and PP2A and RPL in PEG-stressed roots. Expression profiles of PXG3 were used to verify these results. The stable reference genes identified in this study are useful tools for identifying stress-responsive genes to understand the molecular mechanisms of stress tolerance in winter rapeseed.

A Cryophyte Transcription Factor, CbABF1, Confers Freezing, and Drought Tolerance in Tobacco.

Abscisic acid responsive element binding factors (ABFs) play crucial roles in plant responses to abiotic stress. However, little is known about the roles of ABFs in alpine subnival plants, which can survive under extreme environmental conditions. Here, we cloned and characterized an ABF1 homolog, CbABF1, from the alpine subnival plant Chorispora bungeana. Expression of CbABF1 was induced by cold, drought, and abscisic acid. Subcellular localization analysis revealed that CbABF1 was located in the nucleus. Further, CbABF1 had transactivation activity, which was dependent on the N-terminal region containing 89 residues. A Snf1-related protein kinase, CbSnRK2.6, interacted with CbABF1 in yeast two-hybrid analysis and bimolecular fluorescence complementation assays. Transient expression assay revealed that CbSnRK2.6 enhanced the transactivation of CbABF1 on ABRE cis-element. We further found that heterologous expression of CbABF1 in tobacco improved plant tolerance to freezing and drought stress, in which the survival rates of the transgenic plants increased around 40 and 60%, respectively, compared with wild-type plants. Moreover, the transgenic plants accumulated less reactive oxygen species, accompanied by high activities of antioxidant enzymes and elevated expression of stress-responsive genes. Our results thus suggest that CbABF1 is a transcription factor that plays an important role in cold and drought tolerance and is a candidate gene in molecular breeding of stress-tolerant crops.

Ecological and economic benefits of planting winter rapeseed (Brassica rapa L.) in the wind erosion area of northern China.

Winter and early spring wind soil erosion have considerable impacts on ecosystems, human well-being and agricultural production in the low precipitation zones of northern China. Little is known about the impact of growing winter rapeseed on ecological cropping systems and the associated economic benefits in the wind erosion area. To explore the winter rapeseed cover effect, we conducted a field experiment in which we covered the soil with winter rapeseed, winter wheat and wheat stubble at different plant density levels and used the spring bare ground as the control (CK). The effects of wind erosion, the "winter rapeseed + " multiple cropping system, and the economic benefits were compared. There was a large difference in the dry matter, the maximum water absorption, the maximum water storage, the soil evaporation and total wind erosion, the amount of sediment transported in the stratum and the wind erosion modulus. Among them, the mean wind erosion modulus of spring sowing bare land was as high as 490.9 kg·hm-2·h-1, which was 7 and 13 times that of winter wheat and winter rapeseed, respectively. As the wind speed increased from 14 to 22 m·s-1, from a small density to a large density, the mean wind erosion modulus decreased from 68 to 17 kg·hm-2·h-1 for winter rapeseed, and 150 to 31 kg·hm-2·h-1 for winter wheat. Total wind-erosion of sediment transport of CK was 18.6 g·m-2 min-1, which was 16 and 31 times the mean value of winter wheat and winter rapeseed, respectively. "Winter rapeseed + " replanting peanuts, potatoes, rice, seed melons and other crops generally increased the production value by 5-74% compared with wheat and corn intercropping, which was 98-255% higher than the traditional wheat single crop. Our results suggested that the suitable area for planting winter rapeseed in northern China was approximately 3.3 × 106 hm2, and in terms of the best economic and ecological effects, the appropriate density was 5 × 105 plants·hm-2 in northern China. Our results indicated that Chinese winter rapeseed was the best choice for preventing wind erosion and improving ecological and economic benefits in winter and spring in northern China; additionally, winter rapeseed has important impacts on agricultural sustainability in semi-arid and arid climates.