•A functional characterization platform for putative Dihydroflavonol 4-reductase in Carthamus tinctorius L. was presented.•CtCYP45082C1 was able to catalyze Cis-3,4-leucopelargonidin and ...dihydromyricetin-dependent leucoanthocyanidin activities.•The expression of CtCYP45082C1 and other structural genes showed susceptibility against under variable stress conditions.•Dihydroflavonol 4-reductase (CtCYP45082C1) positively regulates leucoanthocyanidin biosynthesis in Carthamus tinctorius
Flavonoids are a distinctive class of phenolic compounds known to be involved in plant growth, development and floral pigmentation. During the natural phenylalanine pathway, dihydroflavonol-4-reductase is the first committed enzyme that catalyzes the stereo-specific reduction of dihydroflavonols into leucoanthocyanidins.Howsoever, less attention has been given to studies explaining the evolution and function of dihydroflavonol-4-reductases in Carthamus tinctorius L. This study explains the first comprehensive genome-wide identification and functional characterization of putative dihydroflavonol 4-reductase in Carthamus tinctorius L. Altogether, 20 CtCYP45082C enzyme encoding genes have been identified in the Carthamus tinctorius genome. Phylogeney analysis revealed the clustering of CtCYP45082C sequences into five major clades illustrating the significant effects of evolutionary divergence across the plant kingdom. Further in silico analyses indicated that all enzyme-encoding CtCYP45082Cs contain fundamental cis-regulatory units and protein domains/motifs that are specifically conserved throughout eukaryotic CYP450 s. In addition, the transient expression of CtCYP45082C1 fused with green fluorescent protein in onion epidermal cells and tobacco leaves confirmed a clearly distinct subcellular localization to plasma membrane. Biochemical characterization of CtCYP45082C1 using the heterologous protein expression assay indicated that CtCYP45082C1 effectively catalyzes the reduction of cis-3,4-leucopelargonidin and dihydromyricetin in leucoanthocyanidin biosynthesis. Moreover, the transcript expression of 20 Carthamus tinctorius derived CYP45082C genes has also been analyzed by real-time quantitative PCR. Each mRNA transcript was detected, in general, in all the investigated tissues, but with different patterns except for CtCYP45082C17-20, indicating the potential role of CtCYP45082C gene family in the secondary metabolite biosynthesis of Carthamus tinctorius. Additionally, the expression of CtCYP45082C1 and two downstream flavonoid pathway regulatory genes following induction of methyl jasmonate, cold, H202 and heat irradiation suggested that mRNA expressions of CtCYP45082C1, CtCHI and CtFLS are susceptible to various environmental changes. This study provides meaningful insights for further functional characterization studies of the CtCYP45082C encoding enzymes which may also be involved in biosynthesis of flavonoids.
MAIN CONCLUSION : In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3′ position by flavonoid ...3’-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3′ or 3′ and 5′, respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3′H and F3′5′H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3′H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3′-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3′,5′-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3′Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.
Remarkable progress toward the complete elucidation of the biosynthesis of flavanoids has been accomplished during the last decade, but the final steps presumably involving the transformation of ...leucoanthocyanidins, which are highly unstable when free in aqueous solution, into both anthocyanidins and proanthocyanidins still remain to be fully understood. Herein is described the synthesis of stabilized solid-supported leucoanthocyanidin variants that should serve as valuable tools for in vitro studies aimed at investigating the metabolism of these seemingly fleeting common precursors of two main classes of flavanoids.
Biosynthesis is well elucidated for 5-hydroxyflavonoids (phloroglucinol type), but for 5-deoxyflavonoids (resorcinol type) the knowledge is still limited. We provide detailed and optimized protocols ...for the synthesis of (
14C)-labeled 6′-deoxychalcones, 5-deoxyflavanones, 5-deoxydihydroflavonols and 5-deoxyleucoanthocyanidins. With the exception of the formation of 6′-deoxychalcones, all steps were performed enzymatically using enzymes normally involved in the formation of 5-hydroxyflavonoids. The availability of (
14C)-labeled substrates will facilitate future work on the hitherto largely unknown biosynthesis of 5-deoxyflavonoids. In particular, the 5-deoxyleucoanthocyanidins, which are more stable than the corresponding 5-hydroxy compounds, may provide excellent tools for investigating enzymes, which use the unstable 5-hydroxyleucoanthocyanidins as natural substrates. As a first example, the conversion of (
14C)-labeled 5-deoxyleucoanthocyanidins to dihydroflavonols in the presence of NADP
+ was shown. Studies with defined genotypes of
Matthiola incana possessing or lacking dihydroflavonol 4-reductase activity and genetically modified yeast expressing the
Matthiola enzyme confirmed that the reaction is catalyzed by the well-known dihydroflavonol 4-reductase, which catalyzes the conversion of dihydroflavonols to leucoanthocyanidins (forward reaction). Thus, the reverse reaction of dihydroflavonol 4-reductase could be demonstrated for the first time. The forward reaction shows an optimum at pH 6.25, the reverse reaction at pH 7.75. The impact of the results on the regulation of flavonoid accumulation is discussed.
The structures of pro-/leuco-anthocyanidins with bonding positions other than those linking flavan-3-ol moieties with resorcinol- and/or phloroglucinol-type A-rings are discussed.
The ...proanthocyanidin pool in the floral kingdom usually involves the presence of carbon–carbon bonds linking predominantly flavan-3-ol constituent moieties. Such an ensemble of flavan-3-ol units originates via electrophilic aromatic substitution of flavan-4-yl carbocations (or their equivalents) derived from flavan-4-ols and/or flavan-3,4-diols and the nucleophilic centers of the m-oxygenated A-rings of flavan-3-ol nucleophiles. In the absence of these potent flavan-3-ol nucleophiles with their aptitude for the formation of carbon–carbon bonds, alternative centers emerge as participants in interflavanyl bond formation. Such a phenomenon is demonstrated for the distribution of various profisetinidin-, prorobinetinidin-, proguibourtinidin-, promelacacinidin- and proteracacinidin-type pro- and leuco-anthocyanidins in several southern hemisphere heartwood species.
Biosynthesis of food constituents: Natural pigments. Part 2 - a review Velisek, J.,Vysoka Skola Chemicko-technologicka, Prague (Czech Republic). Ustav Chemie a Analyzy Potravin; Davidek, J.,Vysoka Skola Chemicko-technologicka, Prague (Czech Republic). Ustav Chemie a Analyzy Potravin; Cejpek, K..,Vysoka Skola Chemicko-technologicka, Prague (Czech Republic). Ustav Chemie a Analyzy Potravin
Czech Journal of Food Sciences,
01/2008, Letnik:
26, Številka:
2
Journal Article
Recenzirano
Odprti dostop
This review article is a part of the survey of the generally accepted biosynthetic pathways that lead to the most important natural pigments in organisms closely related to foods and feeds. The ...biosynthetic pathways leading to xanthones, flavonoids, carotenoids, and some minor pigments are described, including the enzymes involved and reaction schemes with detailed mechanisms.
The first triflavanoids with both C–C and C–O–C interflavanyl bonds, epioritin-(4β→6)-epioritin-(4α→4)-epioritin-4β-ol, epioritin-(4β→3)-epioritin-(4β→6)-epioritin-4β-ol and ...epioritin-(4β→3)-epioritin-(4β→6)-epimesquitol-4α-ol, were identified in the heartwood of
Acacia caffra. The ethereal interflavanyl bond is readily susceptible to reductive cleavage with sodium cyanoborohydride in trifluoroacetic acid/dichloromethane which hence permits the unequivocal assignment of the absolute configuration of constituent flavanyl units.
Australian blackwood is known to be an important cause of allergic contact dermatitis in Australia. Previous investigations have shown that 2,6-dimethoxy-1,4-benzoquinone and acamelin are 2 of the ...responsible, but weak, sensitizers. When these 2 quinones are lacking, which is occasionally found, the wood still possesses allergenic properties. The present re-examination led to the isolation and identification of 4 hydroxyflavans, of which 3 proved to be allergens. Melacacidin, known to be the main constituent of these flavan derivatives in the heartwood, was isolated and its sensitizing capacity in guinea pigs determined. It showed a moderate sensitizing power. Melacacidin occurs abundantly in 125 Australian and 3 African Acacia species.
Cytochemical observations indicate the occurrence of leucoanthocyanidins in the protein bodies of Cercis siliquastrum endosperm; the endosperm cell walls give a positive Mayer's test, indicating ...mucopolysac-charides. These results are discussed in relation to the endosperm-imposed dormancy of the seed.
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