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.].

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.

iTRAQ-based quantitative proteome analysis insights into cold stress of Winter Rapeseed (Brassica rapa L.) grown in the field.

Winter rapeseed (Brassica rapa L.) is a major oilseed crop in Northern China, where its production was severely affected by chilling and freezing stress. However, not much is known about the role of differentially accumulated proteins (DAPs) during the chilling and freezing stress. In this study, isobaric tag for relative and absolute quantification (iTRAQ) technology was performed to identify DAPs under freezing stress. To explore the molecular mechanisms of cold stress tolerance at the cellular and protein levels, the morphological and physiological differences in the shoot apical meristem (SAM) of two winter rapeseed varieties, Longyou 7 (cold-tolerant) and Lenox (cold-sensitive), were explored in field-grown plants. Compared to Lenox, Longyou 7 had a lower SAM height and higher collar diameter. The level of malondialdehyde (MDA) and indole-3-acetic acid (IAA) content was also decreased. Simultaneously, the soluble sugars (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, soluble protein (SP) content, and collar diameter were increased in Longyou 7 as compared to Lenox. A total of 6330 proteins were identified. Among this, 98, 107, 183 and 111 DAPs were expressed in L7 CK/Le CK, L7 d/Le d, Le d/Le CK and L7 d/L7 CK, respectively. Quantitative real-time PCR (RT-qPCR) analysis of the coding genes for seventeen randomly selected DAPs was performed for validation. These DAPs were identified based on gene ontology enrichment analysis, which revealed that glutathione transferase activity, carbohydrate-binding, glutathione binding, metabolic process, and IAA response were closely associated with the cold stress response. In addition, some cold-induced proteins, such as glutathione S-transferase phi 2(GSTF2), might play an essential role during cold acclimation in the SAM of Brassica rapa. The present study provides valuable information on the involvement of DAPs during cold stress responses in Brassica rapa L, and hence could be used for breeding experiments.

Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress.

Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two ω-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), δ-ornithine aminotransferase (δ-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.

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.

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.

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.