Seed propagation is the main method of mulberry expansion in China, an important economic forest species. However, seed germination is the most sensitive stage to various abiotic stresses, especially salinity stress. To reveal the molecular regulatory mechanism of mulberry seed germination under salt stress, flavonoid metabolomics and transcriptomics analyses were performed on mulberry seeds germinated under 50 and 100 mmol/L NaCl stress. Analysis of the flavonoid metabolome revealed that a total of 145 differential flavonoid metabolites (DFMs) were classified into 9 groups, 40 flavonols, 32 flavones, 16 chalcones and 14 flavanones. Among them, 61.4% (89) of the DFMs accumulated continuously with increasing salt concentration, reaching the highest level at a 100 mmol/L salt concentration; these DFMs included quercetin-3-O-glucoside (isoquercitrin), kaempferol (3,5,7,4'-tetrahydroxyflavone), quercetin-7-O-glucoside, taxifolin (dihydroquercetin) and apigenin (4',5,7-trihydroxyflavone), indicating that these flavonoids may be key metabolites involved in the response to salt stress. Transcriptional analysis identified a total of 3055 differentially expressed genes (DEGs), most of which were enriched in flavonoid biosynthesis (ko00941), phenylpropanoid biosynthesis (ko00940) and biosynthesis of secondary metabolites (ko01110). Combined analysis of flavonoid metabolomic and transcriptomic data indicated that phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavonol synthase (FLS), bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR) and anthocyanidin reductase (ANR) were the key genes involved in flavonoid accumulation during mulberry seed germination under 50 and 100 mmol/L NaCl stress. In addition, three transcription factors, MYB, bHLH and NAC, were involved in the regulation of flavonoid accumulation under salt stress. The results of quantitative real-time PCR (qRT‒PCR) validation showed that the expression levels of 11 DEGs, including 7 genes involved in flavonoid biosynthesis, under different salt concentrations were consistent with the transcriptomic data, and parallel reaction monitoring (PRM) results showed that the expression levels of 6 key enzymes (proteins) involved in flavonoid synthesis were consistent with the accumulation of flavonoids. This study provides a new perspective for investigating the regulatory role of flavonoid biosynthesis in the regulation of mulberry seed germination under salt stress at different concentrations.

To analyze the flavonoids in Coreopsis tinctoria and compare the differences in flavonoids among C. tinctoria of different origins, the chemical composition of C. tinctoria capitulum was analyzed by ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and the flavonoid metabolites were analyzed and identified based on their retention time, mass-to-charge ratio and fragment ions in the UPLC-QTOF-MS matrix. Capitulum samples of C. tinctoria were collected from three locations in the Xinjiang region at different altitudes. A total of 204 flavonoid compounds were identified, and 31 different flavonoid metabolites were then identified from flowers of C. tinctoria of different origins. Further analysis of these 31 significantly accumulated metabolites identified seven flavonoid metabolites, namely, homoplantaginin, kaempferol, quercetin, isorhamnetin, avicularin, quercetin 3-O-(6′-galloyl)-β-D-galactopyranoside and isorhamnetin 3-O-glucoside, with high accumulation only in sample collected from Tashkurgan Tajik (TX) and low expression in sample collected from Yutian County (YT) and Shaya County (SY). Moreover, 7,4′-dihydroxyflavone and 4,4′-dimethoxychalcone showed high accumulation only in SY, and afzelin was specifically highly accumulated in YT. In addition, the identified flavonoid metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and key pathways that might regulate the biosynthesis of these flavonoid compounds were analyzed. These findings provide key information for research on flavonoids and their biosynthesis in C. tinctoria and will provide a theoretical basis for studying the herbal quality and origin of C. tinctoria.

Castanopsis fissa is a native, broadleaf tree species in Guangdong with characteristics of barrenness and fast growth and is often used as a pioneer species for vegetation restoration with excellent ecological benefits. To explore the response of C.fissa to drought, this study investigated the drought tolerance mechanism of C.fissa using physiological and proteomic assessments. Using a potted continuous drought experimental method with normal water supply as a control, we measured photosynthetic parameters, antioxidant enzyme activities, and osmoregulatory substances of C. fissa in response to drought stress for 1 to 4 weeks, respectively. In addition, we used TMT quantitative proteomics to identify differentially expressed proteins (DEPs) between the drought-stress-treated C. fissa leaves and the control leaves. With the extension of drought stress time, the photosynthetic indexes and peroxidase (POD) activity of C. fissa leaves showed a decreasing trend. The malondialdehyde (MDA) content; superoxide Dismutase (SOD) and catalase (CAT) activities; and proline (Pro), soluble sugar (SS) and soluble protein (SP) contents showed an overall increasing trend, all of which reached significant differences at 4 w of stress. We identified 177 and 529 DEPs in the 2 and 4 weeks drought-stress leaves, respectively, in reference to the control leaves. These DEPs were closely related to physiological metabolic processes such as photosynthesis, energy and carbohydrate metabolism, stress response and defense, transcriptional regulation, and signal ion transduction. Drought stress mainly affects photosynthesis, carbohydrate metabolism, and protein synthesis and degradation in C. fissa leaves. At 2 weeks of stress, the expression of carbon metabolism, pyruvate metabolism and ribosome-related proteins was significantly changed, however, and at 4 weeks of stress, protein processing in the endoplasmic reticulum and spliceosome-related proteins were significantly increased in plant leaves. To alleviate the effect of water unavailability, the drought-stressed C.fissa leaves increased its oxidative protective enzyme system to eliminate excess reactive oxygen species (ROS) and also increased its Pro and SP contents to maintain the intracellular osmotic potential balance.

Korla fragrant pear (Pyrus sinkiangensis Yü) fruits have a unique flavor and are rich in phenolic acids, flavonoids, amino acids, and other nutrients. At present, the molecular basis of the quality differences among Korla fragrant pear fruits with a convex calyx and rough skin (RS), calyx shedding (SD), and a convex calyx (CV) remains unknown. To analyze the main metabolic components of Korla fragrant pear fruits and compare the antioxidant activities of these three fruits with different qualities, we used nutrient composition analysis and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS)-based widely targeted metabolomics approaches to analyze the changes in the quality characteristics of the pulp of these three Korla fragrant pear fruits with different appearances. The nutrient composition analysis showed that the fructose and glucose contents were not significantly different, and sucrose and vitamin C contents were significantly higher in SD fruits compared with CV and RS fruits. However, the levels of flavor substances such as titratable acids, total phenols, and total flavonoids were high in the pulp of RS fruits. The metabolomics results identified 1976 metabolites that were clustered into 12 categories, and phenolic acid and flavonoid metabolites were the most abundant. The differentially accumulated metabolites (DAMs) in the fruits with different appearances were screened by multivariate statistical methods, and a total of 595 DAMs were detected. The analysis identified 300 DAMs between the CV and SD fruits, 246 DAMs between the RS and CV fruits, and 405 DAMs between the RS and SD groups. SD fruits contained the most metabolites with a high relative content, especially phenolic acids, lipids, amino acids and derivatives, alkaloids, and organic acids. Compared with CV fruits, flavonoid metabolism was more active in RS fruits, which also had a higher content of flavonoids, whereas the fewest metabolites were found in CV fruits, which also displayed less flavonoid accumulation. KEGG pathway enrichment analysis revealed that the DAMs were mainly enriched in the metabolic pathways of flavone and flavonol biosynthesis, confirming that CV fruits have decreased flavone and flavonol biosynthesis and accumulate fewer flavonoids than RS fruits, which may explain the less bitter and astringent flavor of CV fruits. However, the flavonoid content in RS fruits was very high, which may be one of the reasons why RS fruits have a harder pulp and are less juicy, more slaggy, and less flavorful. Moreover, the analysis of the antioxidant activity showed that during fruit development and maturation, RS fruits had stronger antioxidant activity than SD and CV fruits. These results provide a theoretical basis for improving the fruit quality of Korla fragrant pears and the processing of pear pulp.

Background: Salinity is the main abiotic stress that affects seed germination, plant growth and crop production. Plant growth begins with seed germination, which is closely linked to crop development and final yields. Morus alba L. is a well-known saline-alkaline tree with economic value in China, and the most prominent method of expanding mulberry tree populations is seed propagation. Understanding the molecular mechanism of Morus alba L. salt tolerance is crucial for identifying salt-tolerant proteins in seed germination. Here, we explored the response mechanism of mulberry seed germination to salt stress at physiological and protein omics levels. Methods: Tandem mass tag (TMT)-based proteomic profiling of Morus alba L. seeds germinated under 50 mM and 100 mM NaCl treatment for 14 days was performed, and the proteomic findings were validated through parallel reaction monitoring (PRM). Results: Physiological data showed that salt stress inhibited the germination rate and radicle length of mulberry seeds, decreased the malondialdehyde (MDA) content and significantly increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Then, a TMT marker technique was used to analyze the protein groups in mulberry seeds with two salt treatment stages, and 76,544 unique peptides were detected. After removing duplicate proteins, 7717 proteins were identified according to TMT data, and 143 (50 mM NaCl) and 540 (100 mM NaCl) differentially abundant proteins (DAPs) were screened out. Compared with the control, in the 50 mM NaCl solution, 61 and 82 DAPs were upregulated and downregulated, respectively, and in the 100 mM NaCl solution, 222 and 318 DAPs were upregulated and downregulated, respectively. Furthermore, 113 DAPs were copresent in the 50 mM and 100 mM NaCl treatments, of which 43 were upregulated and 70 were downregulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DAPs induced by salt stress during mulberry seed germination were mainly involved in photosynthesis, carotenoid biosynthesis and phytohormone signaling. Finally, PRM verified five differentially expressed proteins, which demonstrated the reliability of TMT in analyzing protein groups. Conclusions: Our research provides valuable insights to further study the overall mechanism of salt stress responses and salt tolerance of mulberry and other plants.

Aluminum (Al) affects the yield of forest trees in acidic soils. The oil tea plant (Camellia drupifera Lour.) has high Al tolerance, with abundant phenolic compounds in its leaves, especially flavonoid compounds. The role of these flavonoids in the Al resistance of oil tea plants is unclear. In this metabolomic study of C. drupifera under Al stress, ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify metabolites, while principal component analysis, cluster analysis, and orthogonal partial least squares discriminant analysis were applied to analyze the data on the flavonoid metabolites. The leaf morphology of C. drupifera revealed significant damage by excess aluminum ions under each treatment compared with the control group. Under Al stress at 2 mmol/L (GZ2) and 4 mmol/L (GZ4), the total flavonoid content in C. drupifera leaves reached 24.37 and 35.64 mg/g, respectively, which are significantly higher than the levels measured in the control group (CK) (p < 0.01). In addition, we identified 25 upregulated and 5 downregulated metabolites in the GZ2 vs. CK comparison and 31 upregulated and 7 downregulated flavonoid metabolites in GZ4 vs. CK. The results demonstrate that different levels of Al stress had a significant influence on the metabolite profile of C. drupifera. It was found that the abundance of the 24 differential flavonoid metabolites was gradually elevated with increasing concentrations of Al stress, including catechin, epicatechin, naringenin-7-glucoside, astilbin, taxifolin, miquelianin, quercitrin, and quercimeritrin. Moreover, the most significant increase in antioxidant activity (about 30%) was observed in C. drupifera precultured in leaf extracts containing 7.5 and 15 μg/mL of active flavonoids. The qRT-PCR results showed that the expression levels of key genes involved in the synthesis of flavonoids were consistent with the accumulation trends of flavonoids under different concentrations of Al. Therefore, our results demonstrate the key role of flavonoid compounds in the oil tea plant C. drupifera in response to Al stress, which suggests that flavonoid metabolites in C. drupifera, as well as other aluminum-tolerant plants, may help with detoxifying aluminum.