Functional Analysis of Two Flavanone-3-Hydroxylase Genes from Camellia sinensis: A Critical Role in Flavonoid Accumulation.
ABSTRACT: Flavonoids are major secondary metabolites in Camellia sinensis. Flavanone-3-hydroxylase (F3H) is a key enzyme in flavonoid biosynthesis in plants. However, its role in the flavonoid metabolism in C. sinensis has not been well studied. In this study, we cloned two F3Hs from C. sinensis, named CsF3Ha and CsF3Hb, where CsF3Ha containing 1107 bases encoded 368 amino acids, and CsF3Hb containing 1071 bases encoded 357 amino acids. Enzymatic activity analysis showed both recombinant CsF3H enzymes in Escherichia coli could convert naringenin and eriodictyol into dihydrokaempferol (DHK) and dihydroquercetin (DHQ), respectively. The expression profiles showed that CsF3Ha and CsF3Hb were highly expressed in the tender leaves of tea plants. Under different abiotic stresses, the two CsF3Hs were induced remarkably by ultraviolet (UV) radiation, sucrose, and abscisic acid (ABA). In the seeds of CsF3Hs transgenic Arabidopsis thaliana, the concentration of most flavonol glycosides and oligomeric proanthocyanidins increased significantly, while the content of monocatechin derivatives decreased. The present study revealed that CsF3Hs played critical roles in flavonoid biosynthesis in tea plants.
Project description:Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.
Project description:Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.
Project description:Dihydroquercetin (DHQ) is a flavonoid compound known for its anti-oxidant effects. Oxidative stress plays a dominant role in regulating the pathways associated with systemic inflammatory immune activation during endotoxemia. Whether and how DHQ regulates inflammatory responses in endotoxemia remains elusive. Here we show DHQ pretreatment effectively reduced the Ten-day mortality in bacterial endotoxin lipopolyssacharide (LPS)-challenged mice, suppressing LPS-induced inflammatory responses reflected by impaired production of tumor necrosis factor ? (TNF-?) and interleukin-6 (IL-6) in the serum of mice. In Raw 264.7 cells, DHQ pretreatment significantly inhibited the transcriptional upregulation of TNF-?, interferon-? (IFN-?), interleukin-10 (IL-10) and toll-like receptor 4 (TLR-4) after LPS stimulation. Additionally, knockdown of heme oxygenase-1 (HO-1), one of the most important DHQ induced antioxidant genes, cancelled the inhibition of DHQ treatment on LPS induced TNF-?, IFN-? production. Nuclear factor erythroid 2-related factor 2 (Nrf2) expression and AMP-activated protein kinase (AMPK) phosphorylation were both enhanced by DHQ in Raw 264.7 cells, indicating a DHQ induced AMPK/Nrf2/HO-1 signal axis. In conclusion, DHQ pretreatment could protect mice against the inflammation and mortality associated with endotoxemia.
Project description:A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harboring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3'-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g., by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower color. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism-absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes-allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.
Project description:Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 18.104.22.168) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 ?M, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.
Project description:Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase (IbDHKR) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway.
Project description:BACKGROUND:Commercially available poinsettia (Euphorbia pulcherrima) varieties prevalently accumulate cyanidin derivatives and show intense red coloration. Orange-red bract color is less common. We investigated four cultivars displaying four different red hues with respect to selected enzymes and genes of the anthocyanin pathway, putatively determining the color hue. RESULTS:Red hues correlated with anthocyanin composition and concentration and showed common dark red coloration in cultivars 'Christmas Beauty' and 'Christmas Feeling' where cyanidin derivatives were prevalent. In contrast, orange-red bract color is based on the prevalent presence of pelargonidin derivatives that comprised 85% of the total anthocyanin content in cv. 'Premium Red' and 96% in cv. 'Harvest Orange' (synonym: 'Orange Spice'). cDNA clones of flavonoid 3'-hydroxylase (F3'H) and dihydroflavonol 4-reductase (DFR) were isolated from the four varieties, and functional activity and substrate specificity of the corresponding recombinant enzymes were studied. Kinetic studies demonstrated that poinsettia DFRs prefer dihydromyricetin and dihydroquercetin over dihydrokaempferol, and thus, favor the formation of cyanidin over pelargonidin. Whereas the F3'H cDNA clones of cultivars 'Christmas Beauty', 'Christmas Feeling', and 'Premium Red' encoded functionally active enzymes, the F3'H cDNA clone of cv. 'Harvest Orange' contained an insertion of 28 bases, which is partly a duplication of 20 bases found close to the insertion site. This causes a frameshift mutation with a premature stop codon after nucleotide 132 and, therefore, a non-functional enzyme. Heterozygosity of the F3'H was demonstrated in this cultivar, but only the mutated allele was expressed in the bracts. No correlation between F3'H-expression and the color hue could be observed in the four species. CONCLUSIONS:Rare orange-red poinsettia hues caused by pelargonidin based anthocyanins can be achieved by different mechanisms. F3'H is a critical step in the establishment of orange red poinsettia color. Although poinsettia DFR shows a low substrate specificity for dihydrokaempferol, sufficient precursor for pelargonidin formation is available in planta, in the absence of F3'H activity.
Project description:Among secondary metabolites, flavonoids are particularly crucial for plant growth, development, and reproduction, as well as beneficial for maintenance of human health. As a flowering plant, safflower has synthesized a striking variety of flavonoids with various pharmacologic properties. However, far less research has been carried out on the genes involved in the biosynthetic pathways that generate these amazing flavonoids, especially characterized quinochalcones. In this study, we first cloned and investigated the participation of a presumed flavanone 3-hydroxylase gene (F3H) from safflower (CtF3H) in a flavonoid biosynthetic pathway.Bioinformation analysis showed that CtF3H shared high conserved residues and confidence with F3H from other plants. Subcellular localization uncovered the nuclear and cytosol localization of CtF3H in onion epidermal cells. The functional expressions of CtF3H in Escherichia coli BL21(DE3)pLysS cells in the pMAL-C5x vector led to the production of dihydrokaempferol when naringenin was the substrate. Furthermore, the transcriptome expression of CtF3H showed a diametrically opposed expression pattern in a quinochalcone-type safflower line (with orange-yellow flowers) and a flavonol-type safflower line (with white flowers) under external stimulation by methyl jasmonate (MeJA), which has been identified as an elicitor of flavonoid metabolites. Further metabolite analysis showed the increasing tendency of quinochalcones and flavonols, such as hydroxysafflor yellow A, kaempferol-3-O-β-D-glucoside, kaempferol-3-O-β-rutinoside, rutin, carthamin, and luteolin, in the quinochalcone-type safflower line. Also, the accumulation of kaempferol-3-O-β-rutinoside and kaempferol-3-O-β-D-glucoside in flavonols-typed safflower line showed enhanced accumulation pattern after MeJA treatment. However, other flavonols, such as kaempferol, dihydrokaempferol and quercetin-3-O-β-D-glucoside, in flavonols-typed safflower line presented down accumulation respond to MeJA stimulus.Our results showed that the high expression of CtF3H in quinochalcone-type safflower line was associated with the accumulation of both quinochalcones and flavonols, whereas its low expression did not affect the increased accumulation of glycosylated derivatives (kaempferol-3-O-β-rutinoside and rutin) in flavonols-typed safflower line but affect the upstream precursors (D-phenylalanine, dihydrokaempferol, kaempferol), which partly revealed the function of CtF3H in different phenotypes and chemotypes of safflower lines.
Project description:The phytohormone salicylic acid (SA) is a secondary metabolite that regulates plant growth, development and responses to stress. However, the role of SA in the biosynthesis of flavonoids (a large class of secondary metabolites) in tea (<i>Camellia sinensis</i> L.) remains largely unknown. Here, we show that exogenous methyl salicylate (MeSA, the methyl ester of SA) increased flavonoid concentration in tea leaves in a dose-dependent manner. While a moderate concentration of MeSA (1 mM) resulted in the highest increase in flavonoid concentration, a high concentration of MeSA (5 mM) decreased flavonoid concentration in tea leaves. A time-course of flavonoid concentration following 1 mM MeSA application showed that flavonoid concentration peaked at 2 days after treatment and then gradually declined, reaching a concentration lower than that of control after 6 days. Consistent with the time course of flavonoid concentration, MeSA enhanced the activity of phenylalanine ammonia-lyase (PAL, a key enzyme for the biosynthesis of flavonoids) as early as 12 h after the treatment, which peaked after 1 day and then gradually declined upto 6 days. qRT-PCR analysis of the genes involved in flavonoid biosynthesis revealed that exogenous MeSA upregulated the expression of genes such as <i>CsPAL</i>, <i>CsC4H</i>, <i>Cs4CL</i>, <i>CsCHS</i>, <i>CsCHI</i>, <i>CsF3H</i>, <i>CsDFR</i>, <i>CsANS</i> and <i>CsUFGT</i> in tea leaves. These results suggest a role for MeSA in modulating the flavonoid biosynthesis in green tea leaves, which might have potential implications in manipulating the tea quality and stress tolerance in tea plants.
Project description:Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways.RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis.Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.