• Molecules and Cells
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• v.25 no.2
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• pp.312-316
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• 2008

Flavanone $3{\beta}$-hydroxylases (F3H) are key enzymes in the synthesis of flavonol and anthocyanin. In this study, three F3H cDNAs from Oryza sativa (OsF3H-1 ~3) were cloned by RT-PCR and expressed in E. coli as gluthatione S-transferase (GST) fusion proteins. The purified recombinant OsF3Hs used flavanone, naringenin and eriodictyol as substrates. The reaction products with naringen and eriodictyol were determined by nuclear magnetic resonance spectroscopy to be dihydrokaempferol and taxifolin, respectively. OsF3H-1 had the highest enzymatic activity whereas the overall expression of OsF3H-2 was highest in all tissues except seeds. Flavanone $3{\beta}$-hydroxylase could be a useful target for flavonoid metabolic engineering in rice.

• Molecules and Cells
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• v.25 no.2
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• pp.247-252
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• 2008

There are several branch points in the flavonoid synthesis pathway starting from chalcone. Among them, the hydroxylation of flavanone is a key step leading to flavonol and anthocyanin. The flavanone 3-${\beta}$-hydroxylase (GmF3H) gene was cloned from soybean (Glycine max cultivar Sinpaldal) and shown to convert eriodictyol and naringenin into taxifolin and dihydrokaempferol, respectively. The major flavonoids in this soybean cultivar were found by LC-MS/MS to be kamepferol O-triglycosides and O-diglycosides. Expression of GmF3H and flavonol synthase (GmFLS) was induced by ultraviolet-B (UV-B) irradiation and their expression stimulated accumulation of kaempferol glycones. Thus, GmF3H and GmFLS appear to be key enzymes in the biosynthesis of the UV-protectant, kaempferol.

• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.85-91
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• 2006

Flavanone 3$\beta$-hydroxylase (FHT) is an enzyme acting in the central part of the flavonoid biosynthesis pathway. FHT catalyses the hydroxylation of flavanone to dihydroflavonols in the anthocyanin pathway. In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme FHT in Gypsophila paniculata L. A heterologous cDHA probe from Dianthus cavophyllus was used to isolate FHT-encoding cDHA clones from Gypsophila paniculata L.. Inspection of the 1471 bp long sequence revealed an open reading frame 1047 bp, including a 190 bp 5' leader region and 288 bp 3' untranslated region. Comparison of the coding region of this FHT cDHA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Petunia hybrida, Solanum tuberosum, Vitis vinifera reveals a identity higher than 69% at the nucleotide level. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRHA from wild type and mutant plants, by in vitro expression yielding and enzymatically active hydroxylase, as indicated by the small dihydrokaempferol peak. Genomic southern blot analysis showed the presence of only one gene for FHT in Gypsophila paniculata.

• Journal of Radiation Industry
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• v.2 no.3
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• pp.121-128
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• 2008

Flavanone-3-hydroxylase (F3H) is one of the key enzymes for the biosynthesis of flavonals, anthocyanins, catechins and proanthocyanins. F3H catalyzes the $3{\beta}$-hydroxylation of (2S)-flavonones to form (2R, 3R)-dihydroflavonols. In this report, we isolated a full-length cDNA of RocF3H from black raspberry (Rubus occidentalis L.) using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of RocF3H contains a 1,098 bp open reading frame (ORF) encoding a 365 amino acid protein with a calculated molecular weight of about 41.1 kDa and isoelectric point (pI) of 5.45. The genomic DNA analysis revealed that the RocF3H gene had three exons and two introns. Comparison of the deduced amino acid sequence of the RocF3H with other F3Hs revealed that the protein is highly homologous with various plant species. The conserved amino acids ligating the ferrous iron and the residues participating in the 2-oxoglutarate binding (R-X-S) were found in RocF3H at the similar positions to other F3Hs. Southern blot analysis indicated that RocF3H exist a multi-gene family. The isolation of RocF3H gene will be helpful to further study the role of F3H gene in the biosynthesis of flavonoids in R. occidnetalis.

• Journal of Microbiology and Biotechnology
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• v.20 no.9
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• pp.1295-1299
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• 2010

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone $3{\beta}$-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

• Journal of Bio-Environment Control
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• v.25 no.1
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• pp.9-15
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• 2016

High temperature is one of the important environmental factors limiting cultivation of apple (Malus domestica Borkh). The expression of genes related with anthocyanin synthesis and sugar accumulation in response to high temperature was studied in the 'Hongro' apple fruits at different developmental stages in different temperature conditions through real-time PCR. Expression of ${\hat{a}}$-amylase (BMY) and polygalacturonase (PG) genes related with sugar synthesis was higher in late ripening stages than in initial ripening stages. Expression of four genes such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), and malate dehydrogenase (MDH), which were related with fruit skin coloration, increased gradually in apple fruits of the middle and late ripening stages. Interestingly, the expressions of all genes were highly inhibited expressed at $30-35^{\circ}C$ compared to $25^{\circ}C$ in all ripening stages. In the further work, investigation of expression levels of various genes could be conducted in the level of transcriptomics in fruits at the middle ripening stages to get meaningful information of ripening metabolism in apple in high temperatures.

• Horticultural Science & Technology
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• v.34 no.5
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• pp.665-676
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• 2016

As high temperature during citrus growing season has caused a serious problems including inferior coloration in production of mandarins in Korea, we were to investigate the expression pattern of several genes related with coloration during the ripening in high temperature condition of citrus fruits. The expression of genes related with sugar metabolism, cell wall degradation, and flavonoid synthesis in high temperature conditions was investigated in fruits of 'Haryejosaeng' (Citrus unshiu) and 'Shiranuhi' mandarin (C. reticulata). While the expression of beta-amylase (BMY), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and flavanone 3-hydroxylase (F3H) was differently induced, expression of polygalacturonase (PG) decreased dependently on temperature conditions. In 'Haryejosaeng' mandarin, while the expression of genes related to the skin coloration, such as CHS and F3H genes increased at $25^{\circ}C$, the expression of PAL and stilbene synthase (STS) genes were induced at $30-35^{\circ}C$ in all ripening stages. In 'Shiranuhi' mandarin, the expression of the BMY gene decreased at early time point in all temperature condition and then increased at $30-35^{\circ}C$ than at $25^{\circ}C$ in the ripening stage 2 to 3 of fruits. F3H and STS genes also showed the tendency to decrease at $30-35^{\circ}C$. Although the expression levels of genes in ripening stage 1 and stage 2 of fruits showed similar patterns in both 'Haryejosaeng' and 'Shiranuhi', the expression levels of genes were down-regulated in late ripening stage of 'Shiranuhi' fruits compared to 'Haryejosaeng'. In general, the mRNA levels of seven tested genes were higher in 'Haryejosaeng' than in 'Shiranuhi' mandarin, and expression of genes by high temperature was regulated sensitively in 'Haryejosaeng' compared to 'Shiranuhi' mandarin. Further investigations of expression of various genes based on transcriptome analysis in early ripening stage can provide valuable information about the responses to climatic changes in ripening citrus fruits.