RESUMEN
We have isolated a cDNA clone that corresponds to the Ht1 locus of petunia which controls the hydroxylation of dihydrokaempferol to dihydroquercetin and of naringenin to eriodictyol by the action of flavonoid 3'-hydroxylase (F3'H). The cDNA encodes a 512 amino acid polypeptide with regions of similarity to petunia flavonoid 3',5'-hydroxylases (F3'5'H). Both F3'H and F3'5'H are cytochromes P450 and are key enzymes in the flavonoid pathway leading to the production of the coloured anthocyanins. The F3 'H transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the ovaries, sepals, peduncles, stems and anthers of the petunia plant. No or very reduced levels of transcripts are detected in ht1/ht1 lines. This is the first report of isolation of a F3 'H cDNA clone from any species.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Flavanonas , Genes de Plantas/genética , Oxigenasas de Función Mixta/genética , Solanaceae/genética , Secuencia de Aminoácidos , Antocianinas/metabolismo , Secuencia de Bases , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Flavonoides/metabolismo , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Ligamiento Genético/genética , Hibridación Genética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Estructuras de las Plantas/enzimología , Estructuras de las Plantas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Solanaceae/enzimología , Levaduras/genéticaRESUMEN
We have isolated, via differential screening of a Petunia hybrida petal cDNA library, a cDNA clone that corresponds to the Rt locus which controls the conversion of anthocyanidin-3-glucosides to anthocyanidin-3-rutinosides by the UDP rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The cDNA encodes a 469 amino acid long polypeptide with regions of similarity to the UDP glucose: flavonoid glucosyltransferases (3GT) from barley and maize. Some sequence similarity was also observed with non-plant glycosyltransferases. Two aberrant transcripts are present in most of the rt/rt petunia lines examined. Excision of a transposon from an unstable Rt locus of one petunia line (Tr38) is associated with a change in transcript size back to wild-type. The Rt transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the style and anthers but not in leaf, stem, root, petiole, ovary or sepals. Incubation of leaves in glucose under high light conditions induces the expression of the Rt gene as well as other flavonoid pathway genes. In situ hybridization revealed that the Rt transcript predominantly accumulates in the epidermal cells of the petal, the site of anthocyanin accumulation.
Asunto(s)
Antocianinas/genética , Hexosiltransferasas/genética , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN sin Sentido , ADN Complementario/aislamiento & purificación , Regulación de la Expresión Génica , Hexosiltransferasas/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Plantas/enzimología , Homología de Secuencia de Aminoácido , Supresión GenéticaRESUMEN
Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl, suggesting that Fl is the structural gene for FLS.
Asunto(s)
Oxidorreductasas/genética , Proteínas de Plantas , Plantas/genética , Agrobacterium tumefaciens , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN , Cartilla de ADN , ADN sin Sentido , Datos de Secuencia Molecular , Oxidorreductasas/biosíntesis , Plantas/enzimología , Polimorfismo de Longitud del Fragmento de Restricción , Saccharomyces cerevisiaeRESUMEN
Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally 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'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.