Single-Molecule Real-Time and Illumina Sequencing to Analyze Transcriptional Regulation of Flavonoid Synthesis in Blueberry

Blueberries (Vaccinium corymbosum) contain large amounts of flavonoids, which play important roles in the plant’s ability to resist stress and can also have beneficial effects on human health when the fruits are eaten. However, the molecular mechanisms that regulate flavonoid synthesis in blueberries are still unclear. In this study, we combined two different transcriptome sequencing platforms, single-molecule real-time (SMRT) and Illumina sequencing, to elucidate the flavonoid synthetic pathways in blueberries. We analyzed transcript quantity, length, and the number of annotated genes. We mined genes associated with flavonoid synthesis (such as anthocyanins, flavonols, and proanthocyanidins) and employed fluorescence quantitative PCR to analyze the expression of these genes and their correlation with flavonoid synthesis. We discovered one R2R3 MYB transcription factor from the sequencing library, VcMYB1, that can positively regulate anthocyanin synthesis in blueberries. VcMYB1 is mainly expressed in colored (mature) fruits. Experiments showed that overexpression and transient expression of VcMYB1 promoted anthocyanin synthesis in Arabidopsis, tobacco (Nicotiana benthamiana) plants and green blueberry fruits. Yeast one-hybrid (Y1H) assay, electrophoretic mobility shift assay, and transient expression experiments showed that VcMYB1 binds to the MYB binding site on the promoter of the structural gene for anthocyanin synthesis, VcMYB1 to positively regulate the transcription of VcDFR, thereby promoting anthocyanin synthesis. We also performed an in-depth investigation of transcriptional regulation of anthocyanin synthesis. This study provides background information and data for studying the synthetic pathways of flavonoids and other secondary metabolites in blueberries.

Foliar Spraying of 6-Benzylaminopurine Promotes Growth and Flavonoid Accumulation in Mulberry (Morus alba L.)

Mulberry (Morus alba L.) leaf, a “source of both medicine and food”, contains antioxidant ingredients such as flavonoids, alkaloids and polyphenols. The effects of 6-benzylaminopurine (6-BA) treatment on plant growth and flavonoid contents in mulberry leaves were investigated in this study. The expression of rutin (Rut), chlorogenic acid (ChA), isoquercitrin (IQ) and astragaloside IV (Ast) related genes in the flavonoid synthesis pathways was investigated in mulberry leaves. The results showed that 6-BA treatment significantly promoted mulberry differentiation and growth as well as, increased the numbers of new shoots and buds compared to the control. In addition, 30 mg/L 6-BA significantly increased the contents of Rut, IQ and Ast, and it strongly induced the expression of flavonoid biosynthesis-related genes, including flavonoid 3-O-glucosyltransferase (F3GT), 4-xoumarate-CoA ligase (4CL), phenylalanine (PAL) and chalcone synthase (CHS). The dietary risk assessment of mulberry leaves was based on hormone residues 5 days after treatment with 30 mg /L 6-BA, and the results showed that the dietary exposure risk of 6-BA was extremely low without causing any health concern. Thus, treatment with 30 mg/L 6-BA is a new method to improve the medicinal quality and development of high-value mulberry leaf foods without any potential risk.

Pre-sowing seeds stimulation by UV-C and X-ray irradiation on the content of antioxidants in inflorescence plants genotypes Matricia chammomila L.

The differences in the dynamics of the flavonoids and phenols content in plants of eight genotypes of matricaria in the control and at presowing UV-C and X-ray irradiation of seeds were studied. Groups of genotypes by the stimulating effect on the content of antioxidants were determined mainly by UV-C irradiation, as well as groups with a significant increase in the content of antioxidants during X-ray irradiation have been identified. A high significant correlation (R = 0.84) between stimulation of flavonoid synthesis by X-ray irradiation and the level of these antioxidants in the control group is shown. Above average (R = 0.64), but insignificant, the correlation is observed between the level of flavonoids in UV-C stimulation and the level of these antioxidants in the control group. No correlation was found between the content of phenols in the control group and the increase of this indicator after irradiation.

Genome-wide identification of WD40 superfamily genes and prediction of WD40 gene of flavonoid-related genes in Ginkgo biloba

The WD40 transcription factor family is a superfamily found in eukaryotes and implicated in regulating growth and development. In this study, 167 WD40 family genes are identified in the Ginkgo biloba genome. They are divided into 5 clusters and 16 subfamilies based on the difference analysis of a phylogenetic tree and domain structures. The distribution of WD40 genes in chromosomes, gene structures, and motifs is analyzed. Promoter analysis shows that five GbWD40 gene promoters contain the MYB binding site participating in the regulation of flavonoid metabolism, suggesting that these five genes may participate in the regulation of flavonoid synthesis in G. biloba. The correlation analysis is carried out based on FPKM value of WD40 gene and flavonoid content in 8 tissues of G. biloba. Six GbWD40 genes that may participate in flavonoid metabolism are screened. The biological functions of the WD40 family genes in G. biloba are systematically analyzed, providing a foundation for further elucidating their regulatory mechanisms. A number of WD40 candidate genes involved in the biosynthesis and metabolism of G. biloba also predicted. This study presents an important basis and direction for conducting further research on the regulatory network of flavonoid synthesis and metabolism.

Transcriptome and metabolome analyses revealing the potential mechanism of seed germination in Polygonatum cyrtonema

Polygonatum cyrtonema Hua (Huangjing, HJ) has medicinal and edible value in China. However, the seeds of this plant are naturally difficult to germinate. Therefore, to elucidate the mechanism underlying the germination of this plant in order to meet the market demand, the metabolomic and transcriptomic analyses were performed in this study. We observed that plant hormones and α-amylase activity were differentially regulated when comparing germinated and un-germinated seeds. In addition, the metabolites related to phenylpropanoid and flavonoid biosynthesis were significantly up-accumulated in germinated seeds. Hydroxycinnamoyl derivatives and organic acids were observed to be significantly decreased during germination. The results of this study suggested that compared to un-germinated seeds, germinated seeds promote flavonoid synthesis and inhibit lignin synthesis which could be beneficial to the germination of HJ seeds. Furthermore, these results suggested that starch if hydrolyzed into glucose, which could provide the necessary energy for germination. Our results may help to establish a foundation for further research investigating the regulatory networks of seed germination and may facilitate the propagation of HJ seeds.

Flavonoids Modulate Bacterial Endophyte Communities of Ginkgo Biloba Leaves

Plant-specialized secondary metabolites have ecological functions in mediating interactions between plants and their entophytes. Here, we aimed to reveal the interaction between flavonoid synthesis and endophytic bacterial communities in wild Ginkgo trees spanning 100-800 years. We found that flavonoids including quercetin, kaempferol and isorhamnetin decreased while the microbial diversity in leaves increased with the age of sampled trees. Older trees had more unique genera and shifted their endophytic bacterial community structure. Also, Mantel tests and correlation analysis indicated a generally significant (p < 0.05) negative correlation between endophytic bacterial communities and flavonoids. Additionally, both deterministic and stochastic processes could play roles in the assembly of endophytic bacterial communities in Ginkgo trees with a progressive increase in stochastic processes as flavonoid concentrations decreased. This study provides a mechanistic understanding of how flavonoids modulate the endophytic microbial community assembly.

Molecular Mechanisms of Antiproliferative Effects of Natural Chalcones

Although great progress has been made in the treatment of cancer, the search for new promising molecules with antitumor activity is still one of the greatest challenges in the fight against cancer due to the increasing number of new cases each year. Chalcones (1,3-diphenyl-2-propen-1-one), the precursors of flavonoid synthesis in higher plants, possess a wide spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant, and anticancer. A plethora of molecular mechanisms of action have been documented, including induction of apoptosis, autophagy, or other types of cell death, cell cycle changes, and modulation of several signaling pathways associated with cell survival or death. In addition, blockade of several steps of angiogenesis and proteasome inhibition has also been documented. This review summarizes the basic molecular mechanisms related to the antiproliferative effects of chalcones, focusing on research articles from the years January 2015–February 2021.

Physiological Responses of the Tetrastigma hemsleyanum Plant under Different Color Films

Tetrastigma hemsleyanum is a traditional Chinese medicine herb, commonly used for its anti-inflammatory and antitumor properties. Flavonoids are the main functional constituents of T. hemsleyanum, and their production in the herb is affected by light quality. T. hemsleyanum is a shade-loving plant and is usually covered by black shade nets during cultivation. However, there are only a few studies on the effects of using color films on growth and flavonoid synthesis in T. hemsleyanum. In this study, we measured the influence of five different color films on growth indexes—sugar, soluble amino acid, soluble protein, and flavonoid content—and flavonoid-synthesizing enzyme activities in T. hemsleyanum. The films used were colorless plastic film as the control group (CK-W), red film (RF), yellow film (YF), green film (GF), and blue film (BF). BF promoted plant growth and increased yield, as evidenced by the highest growth indexes, soluble amino acid content, and chalcone isomerase (CHI) enzyme activity. RF increased the content of secondary metabolites, thereby enhancing herb quality, as evidenced by the highest phenylalanine ammonia lyase (PAL) activity and increased flavonoid content.

Synthetic Biology towards Improved Flavonoid Pharmacokinetics

Flavonoids are a structurally diverse class of natural products that have been found to have a range of beneficial activities in humans. However, the clinical utilisation of these molecules has been limited due to their low solubility, chemical stability, bioavailability and extensive intestinal metabolism in vivo. Recently, the view has been formed that site-specific modification of flavonoids by methylation and/or glycosylation, processes that occur in plants endogenously, can be used to improve and adapt their biophysical and pharmacokinetic properties. The traditional source of flavonoids and their modified forms is from plants and is limited due to the low amounts present in biomass, intrinsic to the nature of secondary metabolite biosynthesis. Access to greater amounts of flavonoids, and understanding of the impact of modifications, requires a rethink in terms of production, more specifically towards the adoption of plant biosynthetic pathways into ex planta synthesis approaches. Advances in synthetic biology and metabolic engineering, aided by protein engineering and machine learning methods, offer attractive and exciting avenues for ex planta flavonoid synthesis. This review seeks to explore the applications of synthetic biology towards the ex planta biosynthesis of flavonoids, and how the natural plant methylation and glycosylation pathways can be harnessed to produce modified flavonoids with more favourable biophysical and pharmacokinetic properties for clinical use. It is envisaged that the development of viable alternative production systems for the synthesis of flavonoids and their methylated and glycosylated forms will help facilitate their greater clinical application.