Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra.

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 µg g-1 FW) than MrMYB5-MrbHLH2 (7.43 µg g-1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols.

Transcriptome Analysis Reveals the Molecular Mechanism and Responsive Genes of Waterlogging Stress in Actinidia deliciosa Planch Kiwifruit Plants.

Waterlogging stress is one of the major natural issues resulting in stunted growth and loss of agricultural productivity. Cultivated kiwifruits are popular for their rich vitamin C content and unique flavor among consumers, while commonly sensitive to waterlogging stress. The wild kiwifruit plants are usually obliged to survive in harsh environments. Here, we carried out a transcriptome analysis by high-throughput RNA sequencing using the root tissues of Actinidia deliciosa (a wild resource with stress-tolerant phenotype) after waterlogging for 0 d, 3 d, and 7 d. Based on the RNA sequencing data, a high number of differentially expressed genes (DEGs) were identified in roots under waterlogging treatment, which were significantly enriched into four biological processes, including stress response, metabolic processes, molecular transport, and mitotic organization, by gene ontology (GO) simplify enrichment analysis. Among these DEGs, the hypoxia-related genes AdADH1 and AdADH2 were correlated well with the contents of acetaldehyde and ethanol, and three transcription factors Acc26216, Acc08443, and Acc16908 were highly correlated with both AdADH1/2 genes and contents of acetaldehyde and ethanol. In addition, we found that there might be an evident difference among the promoter sequences of ADH genes from A. deliciosa and A. chinensis. Taken together, our results provide additional information on the waterlogging response in wild kiwifruit plants.

Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae.

Flavonols are health-promoting bioactive compounds important for plant defense and human nutrition. Quercetin (Q) and kaempferol (K) biosynthesis have been studied extensively while little is known about myricetin (M) biosynthesis. The roles of flavonol synthases (FLSs) and flavonoid 3',5'-hydroxylase (F3'5'H) in M biosynthesis in Morella rubra, a member of the Myricaceae rich in M-based flavonols, were investigated. The level of MrFLS transcripts alone did not correlate well with the accumulation of M-based flavonols. However, combined transcript data for MrFLS1 and MrF3'5'H showed a good correlation with the accumulation of M-based flavonols in different tissues of M. rubra. Recombinant MrFLS1 and MrFLS2 proteins showed strong activity with dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) as substrates, while recombinant MrF3'5'H protein preferred converting K to M, amongst a range of substrates. Tobacco (Nicotiana tabacum) overexpressing 35S::MrFLSs produced elevated levels of K-based and Q-based flavonols without affecting M-based flavonol levels, while tobacco overexpressing 35S::MrF3'5'H accumulated significantly higher levels of M-based flavonols. We conclude that M accumulation in M. rubra is affected by gene expression and enzyme specificity of FLS and F3'5'H as well as substrate availability. In the metabolic grid of flavonol biosynthesis, the strong activity of MrF3'5'H with K as substrate additionally promotes metabolic flux towards M in M. rubra.

Dynamics of a modified Leslie-Gower predator-prey model with double Allee effects.

In this paper, we investigate the dynamic behavior of a modified Leslie-Gower predator-prey model with the Allee effect on both prey and predator. It is shown that the model has at most two positive equilibria, where one is always a hyperbolic saddle and the other is a weak focus with multiplicity of at least three by concrete example. In addition, we analyze the bifurcations of the system, including saddle-node bifurcation, Hopf bifurcation and Bogdanov-Takens bifurcation. The results show that the model has a cusp of codimension three and undergoes a Bogdanov-Takens bifurcation of codimension two. The system undergoes a degenerate Hopf bifurcation and has two limit cycles (the inner one is stable and the outer one is unstable). These enrich the dynamics of the modified Leslie-Gower predator-prey model with the double Allee effects.

Hydroxylation decoration patterns of flavonoids in horticultural crops: chemistry, bioactivity and biosynthesis.

Flavonoids are the most widespread polyphenolic compounds and are important dietary constituents present in horticultural crops such as fruits, vegetables, and tea. Natural flavonoids are responsible for important quality traits, such as food colors and beneficial dietary antioxidants and numerous investigations have shown that intake of flavonoids can reduce the incidence of various non-communicable diseases (NCDs). Analysis of the thousands of flavonoids reported so far has shown that different hydroxylation modifications affect their chemical properties and nutritional values. These diverse flavonoids can be classified based on different hydroxylation patterns in the B, C, A rings and multiple structure-activity analyses have shown that hydroxylation decoration at specific positions markedly enhances their bioactivities. This review focuses on current knowledge concerning hydroxylation of flavonoids catalyzed by several different types of hydroxylase enzymes. Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3'5'-hydroxylase (F3'5'H) are important enzymes for the hydroxylation of the B ring of flavonoids. Flavanone 3-hydroxylase (F3H) is key for the hydroxylation of the C ring, while flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H) are key enzymes for hydroxylation of the A ring. These key hydroxylases in the flavonoid biosynthesis pathway are promising targets for the future bioengineering of plants and mass production of flavonoids with designated hydroxylation patterns of high nutritional importance. In addition, hydroxylation in key places on the ring may help render flavonoids ready for degradation, the catabolic turnover of which may open the door for new lines of inquiry.

Genome-wide analysis of UDP-glycosyltransferase gene family and identification of members involved in flavonoid glucosylation in Chinese bayberry (Morella rubra).

Glycosylation was catalyzed by UDP-glycosyltransferase (UGT) and was important for enriching diversity of flavonoids. Chinese bayberry (Morella rubra) has significant nutritional and medical values because of diverse natural flavonoid glycosides. However, information of UGT gene family was quite limited in M. rubra. In the present study, a total of 152 MrUGT genes clustered into 13 groups were identified in M. rubra genome. Among them, 139 MrUGT genes were marked on eight chromosomes and 13 members located on unmapped scaffolds. Gene duplication analysis indicated that expansion of MrUGT gene family was mainly forced by tandem and proximal duplication events. Gene expression patterns in different tissues and under UV-B treatment were analyzed by transcriptome. Cyanidin 3-O-glucoside (C3Glc) and quercetin 3-O-glucoside (Q3Glc) were two main flavonoid glucosides accumulated in M. rubra. UV-B treatment significantly induced C3Glc and Q3Glc accumulation in fruit. Based on comprehensively analysis of transcriptomic data and phylogenetic homology together with flavonoid accumulation patterns, MrUFGT (MrUGT78A26) and MrUGT72B67 were identified as UDP-glucosyltransferases. MrUFGT was mainly involved in C3Glc and Q3Glc accumulation in fruit, while MrUGT72B67 was mainly involved in Q3Glc accumulation in leaves and flowers. Gln375 and Gln391 were identified as important amino acids for glucosyl transfer activity of MrUFGT and MrUGT72B67 by site-directed mutagenesis, respectively. Transient expression in Nicotiana benthamiana tested the function of MrUFGT and MrUGT72B67 as glucosyltransferases. The present study provided valuable source for identification of functional UGTs involved in secondary metabolites biosynthesis in M. rubra.

Identification of UDP-rhamnosyltransferases and UDP-galactosyltransferase involved in flavonol glycosylation in Morella rubra.

Flavonol glycosides are health-promoting phytochemicals important for human nutrition and plant defense against environmental stresses. Glycosylation modification greatly enriches the diversity of flavonols. Morella rubra, a member of the Myricaceae, contains high amounts of myricetin 3-O-rhamnoside (M3Rha), quercetin 3-O-rhamnoside (Q3Rha), and quercetin 3-O-galactoside (Q3Gal). In the present study, MrUGT78R1 and MrUGT78R2 were identified as two functional UDP-rhamnosyltransferases, while MrUGT78W1 was identified as a UDP-galactosyltransferase. Site-directed mutagenesis identified Pro143 and Asn386 as important residues for rhamnosyl transfer activity of MrUGT78R1, while the two corresponding positions in MrUGT78W1 (i.e. Ser147 and Asn370) also play important roles in galactosyl transfer activity. Transient expression data for these three MrUGTs in Nicotiana benthamiana tested the function of MrUGT78R1 and MrUGT78R2 as rhamnosyltransferases and MrUGT78W1 as a galactosyltransferase in glycosylation of flavonols. This work enriches knowledge of the diversity of UDP-rhamnosyltransferase in planta and identifies two amino acid positions important for both rhamnosyltransferase and galactosyltransferase.

Genome-Wide Analysis of MYB Gene Family in Chinese Bayberry (Morella rubra) and Identification of Members Regulating Flavonoid Biosynthesis.

Chinese bayberry (Morella rubra), the most economically important fruit tree in the Myricaceae family, is a rich source of natural flavonoids. Recently the Chinese bayberry genome has been sequenced, and this provides an opportunity to investigate the organization and evolutionary characteristics of MrMYB genes from a whole genome view. In the present study, we performed the genome-wide analysis of MYB genes in Chinese bayberry and identified 174 MrMYB transcription factors (TFs), including 122 R2R3-MYBs, 43 1R-MYBs, two 3R-MYBs, one 4R-MYB, and six atypical MYBs. Collinearity analysis indicated that both syntenic and tandem duplications contributed to expansion of the MrMYB gene family. Analysis of transcript levels revealed the distinct expression patterns of different MrMYB genes, and those which may play important roles in leaf and flower development. Through phylogenetic analysis and correlation analyses, nine MrMYB TFs were selected as candidates regulating flavonoid biosynthesis. By using dual-luciferase assays, MrMYB12 was shown to trans-activate the MrFLS1 promoter, and MrMYB39 and MrMYB58a trans-activated the MrLAR1 promoter. In addition, overexpression of 35S:MrMYB12 caused a significant increase in flavonol contents and induced the expression of NtCHS, NtF3H, and NtFLS in transgenic tobacco leaves and flowers and significantly reduced anthocyanin accumulation, resulting in pale-pink or pure white flowers. This indicates that MrMYB12 redirected the flux away from anthocyanin biosynthesis resulting in higher flavonol content. The present study provides valuable information for understanding the classification, gene and motif structure, evolution and predicted functions of the MrMYB gene family and identifies MYBs regulating different aspects of flavonoid biosynthesis in Chinese bayberry.

Functional analysis of PpRHM1 and PpRHM2 involved in UDP-l-rhamnose biosynthesis in Prunus persica.

UDP-l-rhamnose (UDP-Rha) is an important sugar donor for glycosylation of various cell molecules in plant. Rhamnosides are widely present in different plant tissues and play important biological roles under different developmental or environmental conditions. However, enzymes involved in UDP-Rha biosynthesis and their encoding genes have been identified in few plants, which limits the functional analysis of plant rhamnosides. Here, two UDP-Rha biosynthesis genes, named PpRHM1 (2028 bp) and PpRHM2 (2016 bp), were isolated and characterized from Prunus persica, which is rich sources of flavonol rhamnosides. Both recombinant RHM proteins can catalyze the transformation from UDP-d-glucose (UDP-Glc) to UDP-Rha, which was confirmed by LC-MS and formation of flavonol rhamnosides. Biochemical analysis showed that both recombinant RHM proteins preferred alkaline conditions in pH range of 8.0-9.0 and had optimal reaction temperature between 25 and 30 °C. PpRHM1 showed the better UDP-Glc substrate affinity with Km of 360.01 µM. Gene expression analysis showed different transcript levels of both RHMs in all plant tissues tested, indicating the involvement of rhamnosides in various tissues in plant. Such results provide better understanding of UDP-Rha biosynthesis in fruit tree and may be helpful for further investigation of various rhamnose derivatives and their biological functions.

Involvement of MdUGT75B1 and MdUGT71B1 in flavonol galactoside/glucoside biosynthesis in apple fruit.

Apple is rich in flavonol glycosides, which are believed to contribute to putative health benefits associated with apple consumption. Glycosylation, catalyzed by uridine diphospho-glycosyltransferases (UGTs), is the last step in flavonol biosynthesis, which confers molecular stability and solubility to the flavonol. In the present study, the involvement of two UGTs, MdUGT75B1 and MdUGT71B1, in flavonol biosynthesis in apple was investigated. The major flavonols are quercetin 3-O-glycosides, and UV-B and blue light treatment significantly enhanced the accumulation of quercetin 3-O-galactoside, quercetin 3-O-glucoside, and kaempferol 3-O-galactoside. Transcript levels of MdUGT75B1 and MdUGT71B1 in fruit subjected to different treatments were correlated well with flavonol accumulation. MdUGT75B1 showed flavonol-specific activity with a preference for UDP-galactose as the sugar donor, while MdUGT71B1 using UDP-glucose exhibited a wider substrate acceptance. Thus, MdUGT75B1 and MdUGT71B1 are key UGTs involved in flavonol biosynthesis and may have important roles in regulating accumulation of these health-promoting bioactive compounds in apple.

PpMYB15 and PpMYBF1 Transcription Factors Are Involved in Regulating Flavonol Biosynthesis in Peach Fruit.

Flavonoids are major polyphenol compounds in plants and contribute substantially to the health-promoting benefits of fruit and vegetables. Peach is rich in polyphenols with flavonols as the main flavonoids. To investigate the regulation of flavonol biosynthesis in peach fruit, two R2R3-MYB transcription factor (TF) genes, PpMYB15 and PpMYBF1, were isolated and characterized. Sequence analysis revealed that the PpMYB15 and PpMYBF1 proteins are members of the flavonol clade of the R2R3-MYB family. Real-time quantitative PCR analysis showed that PpMYB15 and PpMYBF1 transcript levels correlated well with the flavonol content and the expression of flavonol synthase ( PpFLS1) in different fruit samples. Dual-luciferase assays indicated that both PpMYB15 and PpMYBF1 could trans-activate promoters of flavonoid biosynthesis genes, including chalcone synthase ( PpCHS1), chalcone isomerase ( PpCHI1), flavanone 3-hydroxylase ( PpF3H), and PpFLS1. Transient overexpression of 35S::PpMYB15 or 35S::PpMYBF1 both triggered flavonol biosynthesis but not anthocyanin and proanthocyanidin biosynthesis in tobacco leaves. In transgenic tobacco flowers, overexpression of 35S::PpMYB15 or 35S::PpMYBF1 caused a significant increase in flavonol levels and significantly reduced anthocyanin accumulation, resulting in pale-pink or pure white flowers. These results suggest that PpMYB15 and PpMYBF1 are functional flavonol-specific positive regulators in peach fruit and are important candidates for biotechnological engineering flavonol biosynthesis in plants.