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Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

Miosic S, Thill J, Milosevic M, Gosch C, Pober S, Molitor C, Ejaz S, Rompel A, Stich K, Halbwirth H - PLoS ONE (2014)

Bottom Line: One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate.Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar.The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

View Article: PubMed Central - PubMed

Affiliation: Vienna University of Technology, Institute of Chemical Engineering, Vienna, Austria.

ABSTRACT
During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

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Neighbour-joining tree of DFR amino acid sequences of Fragaria and various species published in the NCBI GenBank.DFR1 F. vesca cv Alexandria, early stage (KC894042), late stage 1 (KC894043), late stage 2 (KC894044), cv Red Wonder early stage (KC894045), late stage (KC894046), F.×ananassa cv. Elsanta, early stage (KC894047), late stage (KC894048), DFR2 F. vesca cv Alexandria, early stage (KC894049), late stage 1 (KC894050), late stage 2 (KC894051), cv Red Wonder early stage (KC894052), late stage (KC894053), F.×ananassa cv. Elsanta, early stage (KC894054), late stage (KC894055), Angelonia×angustifolia (KF285561), Camellia sinensis (AAT84073), Citrus sinensis (AAS00611), Crataegus monogynae (AAX16491), Delphinium belladonna (BAF49325), Garcinia mangostane (ACM62744), Gerbera hybrida (CAA78930), Malus×domestica (AAO39816, AAO39817), Medicago truncatula (AAR27014, AAR27015), Onobrychis viciifolia (AEF14420), Petunia×hybrida (AAF60298), Pyrus communis (AAO39818), Rosa hybrida (AAX1242), Triticum aestivum (AAQ77347), Vitis vinifera (AAX12423, XP002275531), Vaccinium macrocarpon (AAL89715, AAL8971, AAX12420). The bootstrap values are indicated next to the relevant nodes (1000 replicates).
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pone-0112707-g003: Neighbour-joining tree of DFR amino acid sequences of Fragaria and various species published in the NCBI GenBank.DFR1 F. vesca cv Alexandria, early stage (KC894042), late stage 1 (KC894043), late stage 2 (KC894044), cv Red Wonder early stage (KC894045), late stage (KC894046), F.×ananassa cv. Elsanta, early stage (KC894047), late stage (KC894048), DFR2 F. vesca cv Alexandria, early stage (KC894049), late stage 1 (KC894050), late stage 2 (KC894051), cv Red Wonder early stage (KC894052), late stage (KC894053), F.×ananassa cv. Elsanta, early stage (KC894054), late stage (KC894055), Angelonia×angustifolia (KF285561), Camellia sinensis (AAT84073), Citrus sinensis (AAS00611), Crataegus monogynae (AAX16491), Delphinium belladonna (BAF49325), Garcinia mangostane (ACM62744), Gerbera hybrida (CAA78930), Malus×domestica (AAO39816, AAO39817), Medicago truncatula (AAR27014, AAR27015), Onobrychis viciifolia (AEF14420), Petunia×hybrida (AAF60298), Pyrus communis (AAO39818), Rosa hybrida (AAX1242), Triticum aestivum (AAQ77347), Vitis vinifera (AAX12423, XP002275531), Vaccinium macrocarpon (AAL89715, AAL8971, AAX12420). The bootstrap values are indicated next to the relevant nodes (1000 replicates).

Mentions: The phylogenetic relationship between the DFRs of the various Fragaria species was analyzed via a neighbor-joining tree that includes further amino acid sequences of DFRs accessible in the GenBank (Figure 3). In this tree, the DFRs of the Rosaceae family formed a separate cluster. Within this cluster the DFR2s from Fragaria form a group together with the DFRs of rose (Rosa×hybrida, AAX12422), apple (Malus×domestica, AAO39816, AAO39817), pear (Pyrus communis AAO39818) and hawthorn (Crataegus monogynae, AAX1649). Fragaria DFRs1 and DFRs2 are clearly revealed in different clusters with DFR2s more closely related to the other Rosaceous DFRs than to DFR1.


Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

Miosic S, Thill J, Milosevic M, Gosch C, Pober S, Molitor C, Ejaz S, Rompel A, Stich K, Halbwirth H - PLoS ONE (2014)

Neighbour-joining tree of DFR amino acid sequences of Fragaria and various species published in the NCBI GenBank.DFR1 F. vesca cv Alexandria, early stage (KC894042), late stage 1 (KC894043), late stage 2 (KC894044), cv Red Wonder early stage (KC894045), late stage (KC894046), F.×ananassa cv. Elsanta, early stage (KC894047), late stage (KC894048), DFR2 F. vesca cv Alexandria, early stage (KC894049), late stage 1 (KC894050), late stage 2 (KC894051), cv Red Wonder early stage (KC894052), late stage (KC894053), F.×ananassa cv. Elsanta, early stage (KC894054), late stage (KC894055), Angelonia×angustifolia (KF285561), Camellia sinensis (AAT84073), Citrus sinensis (AAS00611), Crataegus monogynae (AAX16491), Delphinium belladonna (BAF49325), Garcinia mangostane (ACM62744), Gerbera hybrida (CAA78930), Malus×domestica (AAO39816, AAO39817), Medicago truncatula (AAR27014, AAR27015), Onobrychis viciifolia (AEF14420), Petunia×hybrida (AAF60298), Pyrus communis (AAO39818), Rosa hybrida (AAX1242), Triticum aestivum (AAQ77347), Vitis vinifera (AAX12423, XP002275531), Vaccinium macrocarpon (AAL89715, AAL8971, AAX12420). The bootstrap values are indicated next to the relevant nodes (1000 replicates).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231056&req=5

pone-0112707-g003: Neighbour-joining tree of DFR amino acid sequences of Fragaria and various species published in the NCBI GenBank.DFR1 F. vesca cv Alexandria, early stage (KC894042), late stage 1 (KC894043), late stage 2 (KC894044), cv Red Wonder early stage (KC894045), late stage (KC894046), F.×ananassa cv. Elsanta, early stage (KC894047), late stage (KC894048), DFR2 F. vesca cv Alexandria, early stage (KC894049), late stage 1 (KC894050), late stage 2 (KC894051), cv Red Wonder early stage (KC894052), late stage (KC894053), F.×ananassa cv. Elsanta, early stage (KC894054), late stage (KC894055), Angelonia×angustifolia (KF285561), Camellia sinensis (AAT84073), Citrus sinensis (AAS00611), Crataegus monogynae (AAX16491), Delphinium belladonna (BAF49325), Garcinia mangostane (ACM62744), Gerbera hybrida (CAA78930), Malus×domestica (AAO39816, AAO39817), Medicago truncatula (AAR27014, AAR27015), Onobrychis viciifolia (AEF14420), Petunia×hybrida (AAF60298), Pyrus communis (AAO39818), Rosa hybrida (AAX1242), Triticum aestivum (AAQ77347), Vitis vinifera (AAX12423, XP002275531), Vaccinium macrocarpon (AAL89715, AAL8971, AAX12420). The bootstrap values are indicated next to the relevant nodes (1000 replicates).
Mentions: The phylogenetic relationship between the DFRs of the various Fragaria species was analyzed via a neighbor-joining tree that includes further amino acid sequences of DFRs accessible in the GenBank (Figure 3). In this tree, the DFRs of the Rosaceae family formed a separate cluster. Within this cluster the DFR2s from Fragaria form a group together with the DFRs of rose (Rosa×hybrida, AAX12422), apple (Malus×domestica, AAO39816, AAO39817), pear (Pyrus communis AAO39818) and hawthorn (Crataegus monogynae, AAX1649). Fragaria DFRs1 and DFRs2 are clearly revealed in different clusters with DFR2s more closely related to the other Rosaceous DFRs than to DFR1.

Bottom Line: One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate.Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar.The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

View Article: PubMed Central - PubMed

Affiliation: Vienna University of Technology, Institute of Chemical Engineering, Vienna, Austria.

ABSTRACT
During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

Show MeSH
Related in: MedlinePlus