MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

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.

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.

Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV-B irradiation.

Flavonol glycosides are bioactive compounds important for plant defence and human nutrition. Glycosylation and methylation play an important role in enriching the diversity of flavonols in response to the environment. Peach flowers and fruit are rich in flavonol diglycosides such as isorhamnetin 3-O-rutinoside (I3Rut), kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside, and flavonol monoglycosides such as I 3-O-glucoside and Q 3-O-galactoside. UV-B irradiation of fruit significantly induced accumulation of all these flavonol glycosides. Candidate biosynthetic genes induced by UV-B were identified by genome homology searches and the in vitro catalytic activities of purified recombinant proteins determined. PpUGT78T3 and PpUGT78A2 were identified as flavonol 3-O-glucosyltransferase and 3-O-galactosyltransferase, respectively. PpUGT91AK6 was identified as flavonol 1,6-rhamnosyl trasferase catalysing the formation of flavonol rutinosides and PpFOMT1 was identified as a flavonol O-methyltransferase that methylated Q at the 3'-OH-OH to form isorhamnetin derivatives. Transient expression in Nicotiana benthamiana confirmed the specificity of PpUGT78T3 as a flavonol 3-O-glucosyltransferase, PpUGT78A2 as a 3-O-galactosyltransferase, PpUGT91AK6 as a 1,6-rhamnosyltrasferase and PpFOMT1 as an O-methyltransferase. This study provides new insights into the mechanisms of glycosylation and methylation of flavonols, especially the formation of flavonol diglycosides such as I3Rut, and will also be useful for future potential metabolic engineering of complex flavonols.

Characterization and Purification of Bergamottin from Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou and Its Antiproliferative Activity and Effect on Glucose Consumption in HepG2 cells.

Bergamottin is a natural furanocoumarin compound with weak polarity. Characterization and quantification of bergamottin were carried out in different fruit tissues of various citrus cultivars. Among the four citrus tissues tested, i.e., flavedo, albedo, segment membrane (SM), and juice sacs (JS) in eight citrus cultivars, the highest bergamottin content was found in the flavedo of Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou (YJZXY, 666.54 µg·g-1 DW). A combination of silica gel column chromatography and high-speed counter-current chromatography (HSCCC) was established to efficiently purify bergamottin from the flavedo of YJZXY. Bergamottin showed significant antiproliferative activity on three cancer cell lines, i.e., human liver cancer HepG2, promyelocytic leukemia HL-60, and gastric cancer BGC-823 cells, which showed a marked inhibition effect on these cell lines in a dose-dependent manner. In addition, bergamottin significantly increased glucose consumption in HepG2 cells also in a dose-dependent manner, which is the first report of its potential in anti-diabetes applications.

Two Myricetin-Derived Flavonols from Morella rubra Leaves as Potent α-Glucosidase Inhibitors and Structure-Activity Relationship Study by Computational Chemistry.

Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia, and oxidative stress is an important cause and therapeutic target of DM. Phytochemicals such as flavonols are important natural antioxidants that can be used for prevention and treatment of DM. In the present study, six flavonols were precisely prepared and structurally elucidated from Morella rubra leaves, which were screened based on antioxidant assays and α-glucosidase inhibitory activities of different plant tissues. Myricetin-3-O-(2″-O-galloyl)-α-L-rhamnoside (2) and myricetin-3-O-(4″-O-galloyl)-α-L-rhamnoside (3) showed excellent α-glucosidase inhibitory effects with IC50 values of 1.32 and 1.77 µM, respectively, which were hundredfold higher than those of positive control acarbose. Molecular docking simulation illustrated that the presence of galloyl group altered the binding orientation of flavonols, where it occupied the opening of the cavity pocket of α-glucosidase along with Pi-anion interaction with Glu304 and Pi-Pi stacked with His279. Pi-conjugations generated between galloyl moiety and key residues at the active site of α-glucosidase reinforced the flavonol-enzyme binding, which might explain the greatly increased activity of compounds 2 and 3. In addition, 26 flavonols were evaluated for systematic analysis of structure-activity relationship (SAR) between flavonols and α-glucosidase inhibitory activity. By using their pIC50 (-log IC50) values, three-dimensional quantitative SAR (3D-QSAR) models were developed via comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA), both of which were validated to possess high accuracy and predictive power as indicated by the reasonable cross-validated coefficient (q 2) and non-cross-validated coefficient (r 2) values. Through analyzing 3D contour maps of both CoMFA and CoMSIA models, QSAR results were in agreement with in vitro experimental data. Therefore, such results showed that the galloyl group in compounds 2 and 3 is crucial for interacting with key residues of α-glucosidase and the established 3D-QSAR models could provide valuable information for the prediction of flavonols with great antidiabetic potential.

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.

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.