Flavonoid-3P P,5P P-hydroxylase (F3P P5P PH), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3P P,5P P-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3P P5P PH. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3P P,5P P-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3P P,5P P-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3P P5P PH activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3P P5P PH activity between tobacco and petunia is discussed.z 1999 Federation of European Biochemical Societies.

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The octopine tumor-inducing (Ti) plasmid pTiA66 has an insertion mutation in its T region (the DNA region incorporated into the plant genome) that results in the slow growth of crown gall tumors. These tumors exhibit hormonal autonomy different from that of the crown gall tumors caused by wild-type Ti plasmids. In the present study, the nucleotide sequences of both the DNA segment inserted into pTiA66 and its target site have been determined. The inserted segment is 2548 base pairs long and has 20-base-pair terminal inverted repeats. An 8-base-pair sequence at the target site is duplicated at both integration junctions. These structural features of the insert suggest that it is a bacterial insertion sequence (IS) element, which we have named IS66. Blot-hybridization analyses using IS66 probes revealed that genomes of octopine Ti plasmids contain at least three sequences homologous to IS66: two homologues are located in the virulence region and one is located between the left-hand (TL-DNA) and right-hand (TR-DNA) portions of T-DNA. The chromosome of Agrobacterium tumefaciens A66 also contains two sequences highly homologous to IS66. These results suggest that the mutant pTiA66 plasmid was generated by translocation of one of the sequences showing homology with IS66 into the T region. The fact that a sequence homologous to IS66 is present between TL-DNA and TR-DNA also suggests that the octopine T region was split into two portions, TL-DNA and TR-DNA, by translocation of IS66 or its relatives. Thus, IS66 may cause genetic and structural variations of the T region and the vir region of the octopine Ti plasmids.Tumor-inducing (Ti) plasmids harbored by oncogenic strains of Agrobacterium tumefaciens cause formation of tumors on dicotyledonous plants, called crown galls (1). Cells isolated from the crown gall tumors are characterized by their unlimited proliferation in medium lacking phytohormones, such as cytokinins and auxins, that are required for the culture of normal plant cells (2). It has been shown that a specific DNA region of the Ti plasmid, called the T-DNA region (Fig. 1), causes tumor formation when transferred into plant chromosomes and that the T-DNA persists in the chromosomes of the tumor cells (3-6). The virulence (vir) region of the Ti plasmid (Fig. 1) is located outside the T region and is also required for tumorigenesis by the Ti plasmid. However, this region is probably not stably maintained in the tumor cells (7-10).The Ti plasmids are classified on the basis of the novel amino acid metabolites, such as octopine or nopaline, whose syntheses are directed by T-DNA genes in the tumor cells (11-15). The octopine T region is composed of twQ DNA segments near each other in the Ti plasmid (16) (Fig. 1), whereas the nopaline T region consists of one contiguous DNA segment (17, 18). The chromosomes of the tumor cells caused by the octopine strain contain either one or two portions of the T region (16). All tumor cells that have been analyzed contained the left portion of T-DNA (TL-DNA) but not all...

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