Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism

Fischer, Thilo C.; Halbwirth, Heidi; Meisel, Barbara; Stich, Karl; Forkmann, G.

doi:10.1016/s0003-9861(03)00013-4

Cited by 104 publications

(85 citation statements)

References 20 publications

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“…All constructs were verified by sequencing with gene-specific primers. A dihydroflavonol reductase gene (MdDFR) from apple, which transforms dihydroflavonols to leucoanthocyanidins (Fischer et al, 2003), also was cloned using the above protocols into the Gateway-compatible expression vector pH9GW, a modified pET28a(+) vector (Novagen), which carries a sequence encoding a 9-His tag at the N terminus of the expressed protein (O'Maille et al, 2004). For PnLAR expression, chemically competent E. coli BL21 [DE3] (Invitrogen) was cotransformed with the PnLAR and MdDFR expression constructs.…”

Section: Heterologous Expression Of Pnlar and Pnanr Genes In Eschericmentioning

confidence: 99%

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

et al. 2017

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Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(2)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula 3 Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

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Section: Heterologous Expression Of Pnlar and Pnanr Genes In Eschericmentioning

confidence: 99%

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

et al. 2017

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“…As such it catalyses the last common step in the biosynthesis of the most abundant flavonoids, i.e. flavan-3-ols and condensed tannins [9][10][11][12][13][14][15][16][17][18]. Because of this strategic position, DFRs with distinct substrate specificities may exert an important control on the pattern of anthocyanins and proanthocyanidins which are produced downstream.…”

Section: Introductionmentioning

confidence: 99%

“…Because of this strategic position, DFRs with distinct substrate specificities may exert an important control on the pattern of anthocyanins and proanthocyanidins which are produced downstream. In recent years, several groups attempted to compare distinct DFRs in terms of substrate specificity and pH or temperature profiles, and to gather some kinetic information from in vitro assays [13,14,[19][20][21][22]. Although such studies did bring new and significant information, especially in terms of substrate specificity, they did not produce interpretable kinetic data.…”

Section: Introductionmentioning

confidence: 99%

Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from Vitis vinifera and its unusually strong substrate inhibition

Trabelsi¹,

d’Estaintot²,

Sigaud³

et al. 2011

JBPC

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Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30˚C, substrate inhibition was observed above 30 µM DHQ. At lower/non-inhibitory DHQ concentrations, NADP + behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP + released last. Binding-equilibrium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP + and DHQ. The mechanism which best accounts for substrate inhibition at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the accumulation of unstable leucoanthocyanidins within cells.

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“…Anthocyanin has diverse biological functions in plant and it attracts insects, birds and other pollinators for pollination and also provides protection against oxidative damage 2,3 .Anthocyanins are mainly synthesized via phenylpropanoid pathways 4 . Dihydroflavonol-4-reductase (DFR) is a pivotal enzyme for anthocyanin production 5 .Anthocyaninsare water soluble pigments whose colours depend 6 on pH. Due to its crucial role in anthocyanin production it has been intensively studied in various plants 5,7,8 .…”

Section: Introductionmentioning

confidence: 99%

“…Dihydroflavonol-4-reductase (DFR) is a pivotal enzyme for anthocyanin production 5 .Anthocyaninsare water soluble pigments whose colours depend 6 on pH. Due to its crucial role in anthocyanin production it has been intensively studied in various plants 5,7,8 . Apart from that its transcriptional regulation has also been documented in several studies 6,9 , recently even in Arabidopsis thaliana 10 .…”

Section: Introductionmentioning

confidence: 99%

Blood chemistry profile of Bali cattle fed silage rice straw and biosupplement of selected rumen and termites lignocellulolytic bacteria consortium

2017

JCBSC

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Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism

Cited by 104 publications

References 20 publications

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection

Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from Vitis vinifera and its unusually strong substrate inhibition

Blood chemistry profile of Bali cattle fed silage rice straw and biosupplement of selected rumen and termites lignocellulolytic bacteria consortium

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