Integrated transcriptomic and metabolomic analyses elucidate the mechanism of flavonoid biosynthesis in the regulation of mulberry seed germination under salt stress.

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.

Metabolomic Study of Flavonoids in Camellia drupifera under Aluminum Stress by UPLC-MS/MS.

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.

Physiological and Proteomic Changes in Camellia semiserrata in Response to Aluminum Stress.

Camellia semiserrata is an important woody edible oil tree species in southern China that is characterized by large fruits and seed kernels with high oil contents. Increasing soil acidification due to increased use of fossil fuels, misuse of acidic fertilizers, and irrational farming practices has led to leaching of aluminum (Al) in the form of free Al3+, Al(OH)2+, and Al(OH)2+, which inhibits the growth and development of C. semiserrata in South China. To investigate the mechanism underlying C. semiserrata responses to Al stress, we determined the changes in photosynthetic parameters, antioxidant enzyme activities, and osmoregulatory substance contents of C. semiserrata leaves under different concentrations of Al stress treatments (0, 1, 2, 3, and 4 mmol/L Alcl3) using a combination of physiological and proteomics approaches. In addition, we identified the differentially expressed proteins (DEPs) under 0 (CK or GNR0), 2 mmol/L (GNR2), and 4 mmol/L (GNR4) Al stress using a 4D-label-free technique. With increasing stress concentration, the photosynthetic indexes of C. semiserrata leaves, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), soluble protein (SP), and soluble sugar (SS) showed an overall trend of increasing and then decreasing, and proline (Pro) and malondialdehyde (MDA) contents tended to continuously increase overall. Compared with the control group, we identified 124 and 192 DEPs in GNR2 and GNR4, respectively, which were mainly involved in metabolic processes such as photosynthesis, flavonoid metabolism, oxidative stress response, energy and carbohydrate metabolism, and signal transduction. At 2 mmol/L Al stress, carbon metabolism, amino sugar and nucleotide sugar metabolism, and flavonoid metabolism-related proteins were significantly changed, and when the stress was increased to 4 mmol/L Al, the cells accumulated reactive oxygen species (ROS) at a rate exceeding the antioxidant system scavenging capacity. To deal with this change, C. semiserrata leaves enhanced their glutathione metabolism, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, and other metabolic processes to counteract peroxidative damage to the cytoplasmic membrane caused by stress. In addition, we found that C. semiserrata resisted aluminum toxicity mainly by synthesizing anthocyanidins under 2 mmol/L stress, whereas proanthocyanidins were alleviated by the generation of proanthocyanidins under 4 mmol/L stress, which may be a special mechanism by which C. semiserrata responds to different concentrations of aluminum stress.