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Hosung Jang - One of the best experts on this subject based on the ideXlab platform.
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Correction: De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.).
PLOS ONE, 2015Co-Authors: Hosub Shin, Hosung JangAbstract:Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars ‘Anyang-jungji’ (AJ) and ‘Greenzoa’ (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of Dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.
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De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.).
PloS one, 2015Co-Authors: Hosub Shin, Hosung JangAbstract:Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars 'Anyang-jungji' (AJ) and 'Greenzoa' (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of Dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.
Karl Stich - One of the best experts on this subject based on the ideXlab platform.
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dahlia variabilis cultivar seattle as a model plant for anthochlor biosynthesis
Plant Physiology and Biochemistry, 2021Co-Authors: Benjamin Walliser, Karl Stich, Daria Nitarska, Calin Rares Lucaciu, Christian Molitor, Silvija Marinovic, Didem Aktas, Thomas Rattei, Ioannis Kampatsikas, Christian HaselmairgoschAbstract:We investigated the bi-colored dahlia cultivar 'Seattle', which exhibits bright yellow petals with white tips, for its potential use as a model system for studies of the anthochlor biosynthesis. The yellow base contained high amounts of the 6'-deoxychalcones and the structurally related 4-deoxyaurones, as well as flavones. In contrast, only traces of anthochlors and flavones were detected in the white tips. No anthocyanins, flavonols, flavanones or Dihydroflavonols were found in the petals. Gene expression studies indicated that the absence of anthocyanins in the petals is caused by a lack of flavanone 3-hydroxylase (FHT) expression, which is accompanied by a lack of expression of the bHLH transcription factor IVS. Expression of other genes involved in anthocyanidin biosynthesis such as dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) was not affected. The yellow and white petal parts showed significant differences in the expression of chalcone synthase 2 (CHS2), which is sufficient to explain the absence of yellow pigments in the white tips. Transcriptomes of both petal parts were de novo assembled and three candidate genes for chalcone reductase (CHR) were identified. None of them showed a significantly higher expression in the yellow base compared to the white tips. In summary, it was shown that the bicolouration is most likely caused by a bottleneck in chalcone formation in the white tip. The relative prevalence of flavones compared to the anthochlors in the white tips could be an indication for the presence of a so far unknown differentially expressed CHR.
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Re-investigating substrate specificity of dihydroflavonol 4-reductase with respect to the B-ring hydroxylation pattern of substrates
Acta Horticulturae, 2019Co-Authors: Heidrun Halbwirth, Karl Stich, Silvija Miosic, Malvina Milosevic, Daria Nitarska, Jana Thill, Christian GoschAbstract:Dihydroflavonol 4-reductase (EC 1.1.1.219, DFR) catalyses the reduction of (2R,3R)-(+)-Dihydroflavonols into (2R,3S,4S)-cis-3,4-leucoanthocyanidins. Depending on the plant species, DFR can be unspecific with regard to the B-ring hydroxylation pattern or selective, as in Petunia hybrida in which the DFR does not convert DHK, or in Fragaria species where a pair of DFRs are present that shows contrasting substrate specificity with regard to DHK. DFR substrate specificity has been largely investigated in many plant species. The amino acids determining DFR substrate specificity are not yet completely understood, but previous studies have identified a region of 26 amino acids putatively relevant and in particular, an aspartic acid in position 134, that seems to be responsible for the non-acceptance of DHK as substrates. The recently identified pair of Fragaria DFRs with contrasting substrate specificity was used to study putative regions responsible for the divergent substrate specificity. We demonstrate that neither the versatile C-terminus nor the DFR length nor two of three putative regions are of any relevance. In addition, we analyse previously published DFRs of Malus × domestica, Pyrus communis and Ginkgo biloba and the correlation between their substrate specificity and amino acid sequences. Technical constraints of DFR enzyme assays and potential putative substrate specificity bias is discussed.
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Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility
Plant Physiology and Biochemistry, 2011Co-Authors: Henryk Flachowsky, Christian Gosch, Heidrun Halbwirth, Karl Stich, Magda-viola Hanke, Iris Szankowski, Dieter Treutter, Klaus Richter, Thilo C. FischerAbstract:Abstract Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora . Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to Dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.
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synthesis of 14c labeled 5 deoxyflavonoids and their application in the study of dihydroflavonol leucoanthocyanidin interconversion by dihydroflavonol 4 reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.
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Synthesis of (14C)-labeled 5-deoxyflavonoids and their application in the study of dihydroflavonol/leucoanthocyanidin interconversion by dihydroflavonol 4-reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.
Hosub Shin - One of the best experts on this subject based on the ideXlab platform.
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Correction: De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.).
PLOS ONE, 2015Co-Authors: Hosub Shin, Hosung JangAbstract:Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars ‘Anyang-jungji’ (AJ) and ‘Greenzoa’ (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of Dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.
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De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.).
PloS one, 2015Co-Authors: Hosub Shin, Hosung JangAbstract:Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars 'Anyang-jungji' (AJ) and 'Greenzoa' (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of Dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.
Gert Forkmann - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of (14C)-labeled 5-deoxyflavonoids and their application in the study of dihydroflavonol/leucoanthocyanidin interconversion by dihydroflavonol 4-reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.
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synthesis of 14c labeled 5 deoxyflavonoids and their application in the study of dihydroflavonol leucoanthocyanidin interconversion by dihydroflavonol 4 reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.
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Heterologous expression of dihydroflavonol 4‐reductases from various plants
FEBS Letters, 2002Co-Authors: Stefan Martens, Teemu H. Teeri, Gert ForkmannAbstract:Abstract Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of Dihydroflavonols to the respective flavan 3,4-diols (leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway. Several cDNA clones encoding DFR have been isolated from different plant species. Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far. Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids. Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker’s yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR. These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences. Furthermore, this tool allows the stereospecific synthesis of 14C-labeled leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.
Heidrun Halbwirth - One of the best experts on this subject based on the ideXlab platform.
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Re-investigating substrate specificity of dihydroflavonol 4-reductase with respect to the B-ring hydroxylation pattern of substrates
Acta Horticulturae, 2019Co-Authors: Heidrun Halbwirth, Karl Stich, Silvija Miosic, Malvina Milosevic, Daria Nitarska, Jana Thill, Christian GoschAbstract:Dihydroflavonol 4-reductase (EC 1.1.1.219, DFR) catalyses the reduction of (2R,3R)-(+)-Dihydroflavonols into (2R,3S,4S)-cis-3,4-leucoanthocyanidins. Depending on the plant species, DFR can be unspecific with regard to the B-ring hydroxylation pattern or selective, as in Petunia hybrida in which the DFR does not convert DHK, or in Fragaria species where a pair of DFRs are present that shows contrasting substrate specificity with regard to DHK. DFR substrate specificity has been largely investigated in many plant species. The amino acids determining DFR substrate specificity are not yet completely understood, but previous studies have identified a region of 26 amino acids putatively relevant and in particular, an aspartic acid in position 134, that seems to be responsible for the non-acceptance of DHK as substrates. The recently identified pair of Fragaria DFRs with contrasting substrate specificity was used to study putative regions responsible for the divergent substrate specificity. We demonstrate that neither the versatile C-terminus nor the DFR length nor two of three putative regions are of any relevance. In addition, we analyse previously published DFRs of Malus × domestica, Pyrus communis and Ginkgo biloba and the correlation between their substrate specificity and amino acid sequences. Technical constraints of DFR enzyme assays and potential putative substrate specificity bias is discussed.
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Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility
Plant Physiology and Biochemistry, 2011Co-Authors: Henryk Flachowsky, Christian Gosch, Heidrun Halbwirth, Karl Stich, Magda-viola Hanke, Iris Szankowski, Dieter Treutter, Klaus Richter, Thilo C. FischerAbstract:Abstract Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora . Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to Dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.
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synthesis of 14c labeled 5 deoxyflavonoids and their application in the study of dihydroflavonol leucoanthocyanidin interconversion by dihydroflavonol 4 reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.
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Synthesis of (14C)-labeled 5-deoxyflavonoids and their application in the study of dihydroflavonol/leucoanthocyanidin interconversion by dihydroflavonol 4-reductase
Plant Science, 2006Co-Authors: Heidrun Halbwirth, Gert Forkmann, Stefan Kahl, Walter Jager, Gottfried Reznicek, Karl StichAbstract: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.