It Takes a Garden. How Work on Diverse Plant Species Has Contributed to an Understanding of Flavonoid Metabolism

Abstract

Flavonoid biosynthesis is one of the most extensively studied areas of plant secondary metabolism. Modern-day reports date back to 1664 and Robert Boyle's description of the effects of acids and bases on plant pigments. Today, through the study of flavonoid metabolism in diverse plant systems, a great deal is known about the genes involved in this pathway and the variety of biological functions in which flavonoids participate. What is abundantly clear from the sum of this work is that the central flavonoid pathway has been highly conserved during the course of plant evolution, but that there has been considerable divergence in the functional roles of its end products and, perhaps related to this, the mechanisms by which expression of the pathway is controlled. For this reason, the use of a variety of species in these studies has contributed much to developing a general view of flavonoid metabolism and function across the plant kingdom, in addition to providing a variety of experimental tools to facilitate the characterization of this complex system. The biochemistry of flavonoid metabolism has been elucidated over the course of many years through the careful identification and characterization of numerous enzymes. This work has taken advantage of tissues from which flavonoid enzymes could be easily isolated in large quantities. Examples include irradiated parsley (Petroselinum hortense) cells for chalcone synthase (Kreuzaler et al., 1979), soybean (Glycine max) seeds, and bean (Phaesoleus vulgarus) cell suspension cultures for chalcone isomerase (Moustafa and Wong, 1967; Dixon et al., 1982), and matthiola (Matthiola incana), petunia (Petunia hybrida), and carnation (Dianthus caryophyllus) flowers for flavanone 3-hydroxylase, flavonol synthase, flavonoid 3′-hydroxylase, and dihydroflavonol reductase (Forkmann et al., 1980; Spribille and Forkmann, 1984; Britsch and Grisebach, 1986; Stich et al., 1992). These experiments uncovered the complex network of biochemical reactions that mediate the synthesis of flavonoids in plants and laid the foundation for efforts to isolate the corresponding genes. An overview of the history of gene cloning in the flavonoid pathway (Table I) shows that most of the genes encoding enzymes of the central pathway were first isolated based on biochemical approaches (e.g. information from enzyme characterization or the use of antibodies raised against the purified protein). Mutations resulting from the insertion of transposable elements into flavonoid genes were also useful in this regard. In fact, isolation of the bronze 1 gene, which encodes UDP-Glc:flavonoid 3-O-glucosyl transferase, was the first example of gene cloning using transposon tagging (Federoff et al., 1984). Cloning of the original flavonoid structural genes was carried out, for the most part, in maize and petunia, although the first flavonoid gene, for chalcone synthase, was isolated from parsley (Kreuzaler et al., 1983). Identification of genes encoding enzymes of the isoflavonoid branch pathway, which is found primarily in legumes, has come from recent work in soybean and alfalfa (Medicago sativa), again largely based on biochemical approaches. Arabidopsis is a bit of a late bloomer in the effort to identify genes involved in flavonoid biosynthesis. Although Arabidopsis is not particularly amenable to biochemical approaches, mutations in all of the major structural genes have been identified in this species and are proving useful for filling in a few remaining gaps. This includes the recent identification of a gene that may be involved in the synthesis of condensed tannins (Devic et al., 1999). These are major pigments in many seeds that are also of agronomic importance in the vegetative tissues of forage crops, having the beneficial property of reducing bloat in grazing ruminants and at the same time the potential to act as antifeedants and antinutrients (Morris and Robbins, 1997). Moreover, the use of maize genes to complement Arabidopsis flavonoid mutants has recently provided evidence that flavonoid enzymes have been functionally conserved over large evolutionary distances (Dong et al., 2001). Transposon and T-DNA tagging approaches in maize, petunia, and Arabidopsis are also providing long-awaited information on the genes involved in transporting flavonoids from the site of synthesis in the cytoplasm to the vacuole (Marrs et al., 1995; Alfenito et al., 1998;Debeaujon et al., 2001). These studies underscore the limitations of homology-based approaches because petunia and maize glutathioneS-transferases with only 12% amino acid identity functionally complement each other in vacuolar transport of flavonoids, whereas an Arabidopsis cDNA with 50% identity to the petunia protein cannot. History of flavonoid gene isolation Only the original genes, identified by methods other than homology-based cloning, are included. Entries in each section are listed in chronological order. History of flavonoid gene isolation Only the