Search

Flavonols are one class of flavonoids that play a crucial role in regulating plant growth and promoting stress resistance. They are also important dietary components in horticultural crops due to their benefits for human health. However, flavonol levels in commonly consumed horticultural crops are insufficient to provide optimal benefits, and their role in regulating postharvest pathogen resistance in fruit remains poorly understood. In this study, overexpression of MrMYB12 or MrMYB12/MrF3'5'H from Morella rubra in tomato (Solanum lycopersicum) resulted in high accumulation of flavonols in fruit up to 3.2 g kg-1 by upregulating expression of genes involved in the phenylpropanoid pathway including SlPAL , SlC4H , Sl4CL , SlCHS1 , SlCHI-like , SlF3H , and SlFLS. Transgenic tomato fruit showed high free radical scavenging activities including 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and they also possessed excellent inhibitory effects against α -glucosidase. Postharvest storage experiments indicated that the flavonol-rich tomato fruit exhibited less surface shrinkage than wild type (WT), and they also showed decreased weight loss and increased antioxidant activity. Experiments of Botrytis cinerea infection showed that overexpression of MrMYB12 or MrMYB12 / MrF3'5'H in tomato improved their resistance against B. cinerea during postharvest storage. Improvement of B. cinerea resistance in the flavonol-rich tomato fruit was associated with increase of superoxide dismutase (SOD) and catalase (CAT) enzyme activities and upregulated expression of defense-related genes such as SlPAL , SlβCA3 , SlNPR1 , SlPR1a , SlPR1b1 , and SlPR3a. This study provides new insights for improving the nutritional value and postharvest pathogen resistance of horticultural crops by metabolic engineering. • Overexpression of MrMYB12 or MrMYB12/MrF3'5'H generates flavonol-rich tomato. • Flavonol-rich tomato has higher antioxidant and α -glucosidase inhibiting properties. • Flavonol accumulation improves resistance against Botrytis cinerea in tomato shelf life. • Increased B. cinerea resistance correlates with upregulated defense-related genes. • Increased B. cinerea resistance also correlates with high ROS scavenging activity. [ABSTRACT FROM AUTHOR]

Flavonoids, such as anthocyanins, proanthocyanidins, and flavonols, are widespread plant secondary metabolites and important for plant adaptation to diverse abiotic and biotic stresses. Flavonoids can be variously hydroxylated and decorated; their biological activity is partly dependent on the degree of hydroxylation of the B-ring. Flavonoid biosynthesis is regulated by MYB transcription factors, which have been identified and characterized in a diversity of plants. Here we characterize a new MYB activator, MYB117 , in hybrid poplar (Populus tremula×tremuloides). When overexpressed in transgenic poplar plants, MYB117 enhanced anthocyanin accumulation in all tissues. Transcriptome analysis of MYB117 -overexpressing poplars confirmed the up-regulation of flavonoid and anthocyanin biosynthesis genes, as well as two flavonoid 3′,5′-hydroxylase (F3′5′H) genes. We also identified up-regulated cytochrome b 5 genes, required for full activity of F3′5′H. Phytochemical analysis demonstrated a corresponding increase in B-ring hydroxylation of anthocyanins, proanthocyanidins, and flavonols in these transgenics. Similarly, overexpression of F3′5′H1 directly in hybrid poplar also resulted in increased B-ring hydroxylation, but without affecting overall flavonoid content. However, the overexpression of the cytochrome b 5 gene in F3′5′H1 -overexpressing plants did not further increase B-ring hydroxylation. Our data indicate that MYB117 regulates the biosynthesis of anthocyanins in poplar, but also enhances B-ring hydroxylation by up-regulating F3′5′H1. [ABSTRACT FROM AUTHOR]

Diversity of colors in flowers and fruits is largely due to anthocyanin pigments. The flavonoid/anthocyanin pathway has been most extensively studied. Dihydroflavonol 4-reductase (DFR) is a vital enzyme of the flavonoid pathway which displays major impact on the formation of anthocyanins, flavan 3-ols and flavonols. The substrate specificity of the DFR was found to play a crucial role in determination of type of anthocyanidins. Altering the flavonoid/ anthocyanin pathway through genetic engineering to develop color of our own choice is an exciting subject of future research. In the present study, comparison among four DFR genes (Gossypium hirsutum, Iris × hollandica, Ang. DFRI and DFRII), sequence alignment for homology as well as protein modeling and docking is demonstrated. Estimation of catalytic sites, prediction of substrate preference and protein docking were the key features of this article. For specific substrate uptake, a proline rich region and positions 12 plus 26 along with other positions emphasizing the 26-amino acid residue region (132-157) was tested. Results showed that proline rich region position 12, 26, and 132-157 plays an important role in selective attachment of DFRs with respective substrates. Further, "Expasy ProtParam tool" results showed that Iris × hollandica DFR amino acids (Asn 9: Asp 23) are favorable for reducing DHQ and DHM thus accumulating delphinidin, while Gossypium hirsutum DFR has (Asn 13: Asp 21) hypothesized to consume DHK. Protein docking data showed that amino acid residues in above mentioned positions were just involved in attachment of DFR with substrate and had no role in specific substrate uptake. Advanced bioinformatics analysis has revealed that all above mentioned positions have role in substrate attachment. For substrate specificity, other residues region is involved. It will help in color manipulations in different plant species. [ABSTRACT FROM AUTHOR]

The enzymes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3′5′H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3′H and two F3′5′H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3′Hs and F3′5′Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3′H and F3′5′H, respectively, and that the ancestor of A. majus lost F3′5′H activity after its speciation from the ancestor of A. kelloggii. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3′- or the 3′- and 5′-position, respectively. Sequence identity between F3′H and F3′5′H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3′5′H from an F3′H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3′H and F3′5′H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5′-hydroxylation activity to recombinant Gerbera hybrida F3′H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3′5′H into an F3′H with low remaining 5′-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3′- to 3′,5′-hydroxylation activity. [Copyright &y& Elsevier]

A novel cDNA for the flavonoid-3′,5′-hydroxylase ( F35H) gene was cloned from petals of Phalaenopsis, and designated as Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealed that its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3′5′H protein is classified into an α-domain which contains most of the α-helices with three small β-sheets, a β-domain that contains the larger β-sheets with three small α-helices by homology modeling. The substrate-binding site for dihydrokaempferol on PhF3′5′H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are involved in the biosynthetic pathway of cyanidin- and delphinidin-based anthocyanins, respectively, as well as quercetin and myricetin (flavonols) and procyanidin and prodelphinidin (flavan-3-ols). Grape (Vitis vinifera) accumulates these three classes of flavonoids and is presumed to have both F3′h and F3′5′h. Thus, we obtained the genomic sequences of F3′h and F3′5′h from grape and determined their mRNA levels as well as the flavonoid composition of several grape organs to investigate whether the transcription of these genes controls the flavonoid composition. The flower, stem, tendril, and seed, which accumulated a higher level of mRNA of F3′h than F3′5′h, showed reasonable compositions of flavonols and/or flavan-3-ols. The organs that accumulated a high level of F3′5′h mRNA contained a high level of delphinidin-based anthocyanins (berry skin at the harvest stage) or prodelphinidin (small leaf), consistent with the mRNA levels. However, not all classes of flavonoid showed a similar composition according to the mRNA levels of F3′h and F3′5′h. The compositions of anthocyanins and flavonols in the small leaf were inconsistent with the mRNA levels. Consequently, other mechanisms in addition to gene transcription are also expected to control the composition of each flavonoid class. [Copyright &y& Elsevier]

Abstract: The gene Hf1 plays a key role in the expression of floral color in petunias. Hf1 encodes a flavonoid-3′,5′-hydroxylase (F3′5′H). The recessive allele (hf1) in an inbred line of petunia is known to be generated by the insertion of a transposable element (Psl). We isolated a novel Mutator-like transposable element (named dTph9) from the hf1 allele of a commercial petunia with red flowers. Another novel transposable element (named rTph1) was found in hf1 of another red petunia. rTph1 shared features with the copia-like retrotransposable element family. These novel elements were inserted independently in the third exon of the Hf1 gene, at different positions. The hf1 allele harboring dTph9 or rTph1 cannot produce an active F3′5′H enzyme because there are stop codons in the dTph9 and rTph1 sequences. Southern analysis showed that these elements were present in relatively low copy numbers and that mutation of the Hf1 locus was associated with the transposition of both elements. We conclude that a loss-of-function mutation of the petunia Hf1 gene is caused by the insertion of at least two different transposable elements, other than Psl, within the Hf1 gene. [Copyright &y& Elsevier]

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3′,5′-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3′,5′-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3′,5′-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3′,5′-hydroxylase gene influences anthocyanin pigment synthesis. [ABSTRACT FROM AUTHOR]