Betalains are pigments that replace anthocyanins in the majority of families of the plant order Caryophyllales. Betalamic acid is the common chromophore of betalains. The key enzyme of the betalain biosynthetic pathway is an extradiol dioxygenase that opens the cyclic ring of dihydroxy-phenylalanine (DOPA) between carbons 4 and 5, thus producing an unstable seco-DOPA that rearranges nonenzymatically to betalamic acid. A gene for a 4,5-DOPA-dioxygenase has already been isolated from the fungus Amanita muscaria, but no homolog was ever found in plants. To identify the plant gene, we constructed subtractive libraries between different colored phenotypes of isogenic lines of Portulaca grandiflora (Portulacaceae) and between different stages of flower bud formation. Using in silico analysis of differentially expressed cDNAs, we identified a candidate showing strong homology at the level of translated protein with the LigB domain present in several bacterial extradiol 4,5-dioxygenases. The gene was expressed only in colored flower petals. The function of this gene in the betalain biosynthetic pathway was confirmed by biolistic genetic complementation in white petals of P. grandiflora genotypes lacking the gene for color formation. This gene named DODA is the first characterized member of a novel family of plant dioxygenases phylogenetically distinct from Amanita sp. DOPA-dioxygenase. Homologs of DODA are present not only in betalain-producing plants but also, albeit with some changes near the catalytic site, in other angiosperms and in the bryophyte Physcomitrella patens. These homologs are part of a novel conserved plant gene family probably involved in aromatic compound metabolism.Betalains are vacuolar pigments that entirely replace anthocyanins in most plants of the order Caryophyllales, with the exception of Caryophyllaceae and Molluginaceae (for review, see Strack et al., 2003). Betalains are also present in some basidiomycete fungi belonging to the Amanita and Hygrocybe genera. Betalains provide a key taxonomic criterion in our understanding of Caryophyllales evolution (Cuenoud et al., 2002); the origin of the alternative pigment biosynthetic pathway is still unknown (Clement and Mabry, 1996), and the purpose of our work is to provide, through the characterization of a key biosynthetic enzyme, one element of the answer to this problem.Information on the biochemistry and genetics of the plant betalain pathway is scarce. Genetic analysis of the betalain biosynthetic pathway in the ornamental plant Portulaca grandiflora (Pg) indicates that four structural genes would be sufficient to account for the different color phenotypes observed in petals (Trezzini and Zrÿ d, 1990). A tyrosinase (Mueller et al., 1996;Steiner et al., 1999) is needed to convert the amino acid Tyr to DOPA, and a ring-opening extradiol DOPA-4,5-dioxygenase is crucial for the synthesis of the chromophore betalamic acid (see Fig. 1; Girod and Zrÿ d, 1991a;Hinz et al., 1997;Schliemann et al., 1998). The formation of cyclo-DOPA is needed for the bui...

The sections in this article are Betalain Pigments Chemistry Physiology of Betalains Genetics of Betalains Enzymology and Biotechnology Evolution of Betalains Social and Economic Value Prospectives Acknowledgements