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[学术文献 ] A high-efficiency PEG-Ca2+-mediated transient transformation system for broccoli protoplasts 进入全文
Front Plant Sci
Transient transformation of plant protoplasts is an important method for studying gene function, subcellular localization and plant morphological development. In this study, an efficient transient transformation system was established by optimizing the plasmid concentration, PEG4000 mass concentration and genotype selection, key factors that affect transformation efficiency. Meanwhile, an efficient and universal broccoli protoplast isolation system was established. Using 0.5% (w/v) cellulase R-10 and 0.1% (w/v) pectolyase Y-23 to hydrolyze broccoli cotyledons of three different genotypes for 3 h, the yield was more than 5×106/mL/g, and the viability was more than 95%, sufficient to meet the high standards for protoplasts to be used in various experiments. The average transformation efficiency of the two plasmid vectors PHG-eGFP and CP507-YFP in broccoli B1 protoplasts were 61.4% and 41.7%, respectively. Using this system, we successfully performed subcellular localization of the products of three target genes (the clubroot resistance gene CRa and two key genes regulated by glucosinolates, Bol029100 and Bol031350).The results showed that the products of all three genes were localized in the nucleus. The high-efficiency transient transformation system for broccoli protoplasts constructed in this study makes it possible to reliably acquire high-viability protoplasts in high yield. This research provides important technical support for international frontier research fields such as single-cell sequencing, spatial transcriptomics, plant somatic hybridization, gene function analysis and subcellular localization.
[学术文献 ] The analysis of lysine succinylation modification reveals the mechanism of oxybenzone damaging of pakchoi (Brassica rapa L. ssp. chinensis) 进入全文
Front Plant Sci
Oxybenzone (OBZ), one of a broad spectrum of ultraviolet (UV) absorbents, has been proven to be harmful to both plants and animals, while omics analysis of big data at the molecular level is still lacking. Lysine succinylation (Ksuc) is an important posttranslational modification of proteins that plays a crucial role in regulating the metabolic network in organisms under stress. Here, we report the changes in intracellular Ksuc modification in plants under OBZ stress. A total of 1276 succinylated sites on 507 proteins were identified. Among these sites, 181 modified proteins were hypersulfinylated/succinylated in OBZ-stressed pakchoi leaves. Differentially succinylated proteins (DSPs) are distributed mainly in the chloroplast, cytoplasm, and mitochondria and are distributed mainly in primary metabolic pathways, such as reactive oxygen species (ROS) scavenging, stress resistance, energy generation and transfer, photosynthetic carbon fixation, glycolysis, and the tricarboxylic acid (TCA) cycle. Comprehensive analysis shows that Ksuc mainly changes the carbon flow distribution, enhances the activity of the antioxidant system, affects the biosynthesis of amino acids, and increases the modification of histones. The results of this study first showed the profiling of the Kusc map under OBZ treatment and proposed the adaptive mechanism of pakchoi in response to pollutants and other abiotic stresses at the posttranslational level, which revealed the importance of Ksuc in the regulation of various life activities and provides a reference dataset for future research on molecular function.
[学术文献 ] A Comparative Study on the Nutrients, Mineral Elements, and Antioxidant Compounds in Different Types of Cruciferous Vegetables 进入全文
Agronomy
Studies on the diversity within and among cabbage (Brassica oleracea L. var. capitata L.), cauliflower (Brassica oleracea var. botrytis), and Chinese cabbage (Brassica rapa L. ssp. pekinensis) variants are essential for the development of healthy diets. However, most studies on them have been limited to a single species, with little integrated analysis between them. In this study, the diversity within and among these species and varieties is assessed by determining the contents of 15 major characteristic nutrients, antioxidants, and minerals in 12 varieties of cabbage, 9 varieties of cauliflower, and 12 varieties of Chinese cabbage cultivated under the same conditions. The results show that there are significant differences in the compositional distributions of cabbage, cauliflower, and Chinese cabbage. Cabbage has the highest contents of soluble sugars (27.73 mg·kg−1 FW), flavonoids (5.90 mg·g−1 FW), and Fe (46.90 mg·kg−1 DW). Cauliflower is an ideal source of soluble protein (603.04 mg·kg−1 FW), polyphenols (1.53 mg·g−1 FW), glucosinolates (25.27 μmol·g−1 FW), and Cu (4.25 mg·kg−1 DW). Chinese cabbage is rich in vitamin C (0.45 mg·g−1 FW) and minerals (K, Ca, Mg, P, Mn, and Zn, at 9206.67 mg·kg−1 DW, 3297.00 mg·kg−1 DW, 3322.79 mg·kg−1 DW, 5614.78 mg·kg−1 DW, 15.36 mg·kg−1 DW, and 21.87 mg·kg−1 DW, respectively). There is a correlation between the quality, antioxidant properties, and minerals of the three species. In principal component analysis, a wide distribution of cabbage varieties and a high degree of overlap with the confidence ellipse of cauliflower are observed, indicating that certain cabbage varieties share compositional characteristics with cauliflower. These findings provide a reference for selecting varieties with higher nutritional value and antioxidant properties, as well as breeding new varieties.
[学术文献 ] Genome-Wide Identification and Expression Analysis of BrATGs and Their Different Roles in Response to Abiotic Stresses in Chinese Cabbage 进入全文
Agronomy
Autophagy is an important degradation pathway that maintains cellular homeostasis in living organisms and plays a key role in plant fitness and immunity. To date, more than 30 autophagy-related genes (ATGs) have been identified in model plants such as Arabidopsis. However, autophagy in Chinese cabbage, the largest cultivated vegetable crop in China, has scarcely been studied. We identified 64 Chinese cabbage autophagy-associated genes, named BrATGs, at the genome-wide level. The majority of the BrATGs were highly conserved over a long evolutionary period, and the expression patterns indicated that BrATGs were most highly expressed in the healing tissues and flowers. Furthermore, BrATGs responded to the stresses of the heavy metal Cd, drought, salt, and low and high temperatures to varying degrees. Among them, BrATG8c/8j was specifically induced in response to drastic temperature changes; BrATG4c was upregulated only in response to drought and salt stress; and BrATG8f/10/VTI12C was highly expressed only in response to Cd stress. This work will advance the understanding of the molecular mechanisms underlying the abiotic stress response in Chinese cabbage.
[学术文献 ] Metabolomics Analysis Reveals Dynamic Accumulation of Sugar and Acid during Stem Development of Brassica juncea 进入全文
Agronomy
The composition and content of sugar and acid are important indicators of organ develop-ment and the quality of horticultural products. B. juncea is an important vegetable in the cruciferous family, with a swollen fleshy stem as the edible organ. Elucidating the characteristics of sugar and acid accumulation during stem expansion of stem mustard and its regulatory mechanism could enhance stem quality. In this study, physiological indicators such as dry matter content, sugar-acid content and related enzyme activities were measured in eight stages of the tumorous stem. The results showed that the sugar and sucrose contents initially increased and then decreased during stem development: Sucrose exhibited a positive correlation with sucrose synthase and sucrose phosphate synthase, while acid content was highly positively correlated with malate dehydrogenase. Further analysis of the dynamic patterns of sugar and acid metabolite contents using metabolomics showed that 1097 metabolites were detected, including 229 organic acids and derivatives, 109 lipids, and other metabolites. Metabolic pathway enrichment analysis showed that metabolites were significantly enriched in organic acids, amino acids, glycolysis/gluconeogenesis, starch, and sucrose metabolism. Analysis of the sugar pathway and the tricarboxylic acid cycle revealed obvious differences in the content and type of metabolites, with most upregulated metabolites in S3 and S4. The expression patterns of enzyme genes associated with the biosynthesis and accumulation of sugar and acid metabolites were found based on differentially expressed genes at different developmental stages, and gene expression levels were verified by qPCR, which showed that the expression patterns of enzyme genes associated with this pathway were highly consistent with the metabolite accumulation. These results suggest that amino acids, sugars, and acids play a critical role in regulating the development of tumorous stems. Results of the present study offer a theoretical basis to improving the quality of mustard by using a metabolomics approach to determine the dynamics of metabolites and related regulatory enzymes during development and the correlation between these levels, which provides novel insights into the potential mechanisms underlying sugar and acid metabolism.
[学术文献 ] Single-cell transcriptome reveals dominant subgenome expression and transcriptional response to heat stress in Chinese cabbage 进入全文
Genome Biology
Chinese cabbage (Brassica rapa ssp. pekinensis) experienced a whole-genome triplication event and thus has three subgenomes: least fractioned, medium fractioned, and most fractioned subgenome. Environmental changes affect leaf development, which in turn influence the yield. To improve the yield and resistance to different climate scenarios, a comprehensive understanding of leaf development is required including insights into the full diversity of cell types and transcriptional net-works underlying their specificity. Here, we generate the transcriptional landscape of Chinese cabbage leaf at single-cell resolution by performing single-cell RNA sequencing of 30,000 individual cells. We characterize seven major cell types with 19 transcriptionally distinct cell clus-ters based on the expression of the reported marker genes. We find that genes in the least fractioned subgenome are predominantly expressed compared with those in the medium and most fractioned subgenomes in different cell types. Moreover, we gener-ate a single-cell transcriptional map of leaves in response to high temperature. We find that heat stress not only affects gene expression in a cell type-specific manner but also impacts subgenome dominance. Our study highlights the transcriptional networks in different cell types and provides a better understanding of transcriptional regulation during leaf develop-ment and transcriptional response to heat stress in Chinese cabbage.
