Genetics and Biochemistry of Anthocyanin Biosynthesis

Flavonoids represent a large class of secondary plant metabolites, of which anthocyanins are the most conspicuous class, dueto the wide range of colors resulting from their synthesis. Anthocyanins are important to many diverse functions within plants. Synthesis of anthocyanins in petals is undoubtedly intended to attract pollinators, whereas anthocyanin synthesis in seeds and fruits may aid in seed dispersal. Anthocyanins and other flavonoids can also be important as feeding deterrents and as protection against damage from UV irradiation. The existence of such a diverse range of functions and types of anthocyanins raises questions about how these compounds are synthesized and how their synthesis is regulated. The study of the genetics of anthocyanin synthesis began last century with Mendel’s work on flower color in peas. Since that time, there have been periods of intensive study into the genetics and biochemistry of pigment production in a number of different species. In the early studies, genetic loci were correlated with easily observable color changes. After the structures of anthocyanins and other flavonoids were determined, it was possible to correlate single genes with particular structural alterations of anthocyanins or with the presence or absence of particular flavonoids. Mutations in anthocyanin genes have been studied for many years because they are easily identified and because they generally have no deleterious effect on plant growth and development. In most cases, mutations affecting different steps of the anthocyanin biosynthesis pathway were isolated and characterized well before their function was identified or the corresponding gene was isolated. More recently, many genes involved in the biosynthesis of anthocyanin pigments have been isolated and characterized using recombinant DNA technologies. Three species have been particularly important for elucidating the anthocyanin biosynthetic pathway and for isolating genes controlling the biosynthesis of flavonoids: maize (Zea mays), snapdragon (Anfirrhinum majus), and petunia (Wtunia hybrida). Petunia has more recently become the organism of choice for isolating flavonoid biosynthetic genes and studying their interactions and regulation. At least 35 genes are known to affect flower color in petuniawiering and de Vlaming, 1984). Because this field of research has been reviewed fairly extensively in the past (Dooner et al., 1991; van Tunen and MOI, 1991; Gerats and Martin, 1992), in this review we concentrate on the more recent developments in gene isolation and characterization. A review of the genetics of flavonoid biosynthesis in other species was recently covered by Forkmann (1993). The characterization of genetically defined mutations has enabled the order of many reactions in anthocyanin synthesis and their modification to be elucidated. Some reactions have been postulated only on the basis of genetic studies and have not yet been demonstrated in vitro. Chemico-genetic studies have been very important in determining the enzymatic steps involved in anthocyanin biosynthesis and modification. The generation of transposon-tagged mutations and the subsequent cloning of the transposons provided a relatively straightforward means of isolating many genes from maize (Wienand et al., 1990) and snapdragon (Martin et al., 1991). However, a number of genes in the pathway have not been amenable to transposon tagging. Anthocyanin biosynthetic genes have been isolated using a range of methodologies, including protein purification, transposon tagging, differential screening, and polymerase chain reaction (PCR) amplification. Functions of isolated anthocyanin genes can be confirmed by restriction fragment length polymorphism (RFLP) mapping, complementation, or expression in heterologous systems. Reverse genetics has also been used recently to identify gene function; this requires a welldefined pathway to correlate phenotype with gene function. Once a gene has been isolated from one species, it is usually a straightforward task to isolate the homologous gene from other species by using the original clone as a molecular probe.