Flavonoid Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell Biology, and Biotechnology
Schematic of the major branch pathways of flavonoid biosynthesis, starting with general phenylpropanoid metabolism and leading to the nine major subgroups: the colorless chalcones, aurones, isoflavonoids, flavones, flavonols, and flavandiols (gray boxes), and the anthocyanins, condensed tannins, and phlobaphene pigments (colored boxes). The first committed step is catalyzed by chalcone synthase (CHS), which uses malonyl CoA and 4-coumaroyl CoA as substrates. Only a few examples are shown of the enormous variety of end products that arise through terminal modification by the addition of sugars as well as methyl, ferulate, and other groups. P450 hydoxylases that may function as membrane anchors for multienzyme assemblies are indicated in red. The photographs illustrate the three major classes of pigments in the model plants, snapdragon, Arabidopsis, maize, and petunia. Root nodulation by rhizobia, which involves flavone as well as flavanone and isoflavone signal molecules, is also shown, in this case for sweet clover (Melilotus alba). Enzyme names are abbreviated as follows: cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone reductase (CHR), chalcone synthase (CHS), 4-coumaroyl:CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), 7,2′-dihydroxy, 4′-methoxyisoflavanol dehydratase (DMID), flavanone 3-hydroxylase (F3H), flavone synthase (FSI and FSII), flavonoid 3′ hydroxylase (F3′H) or flavonoid 3′5′ hydroxylase (F3′5′H), isoflavoneO-methyltransferase (IOMT), isoflavone reductase (IFR), isoflavone 2′-hydroxylase (I2′H), isoflavone synthase (IFS), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), Phe ammonia-lyase (PAL), rhamnosyl transferase (RT), stilbene synthase (STS), UDPG-flavonoid glucosyl transferase (UFGT), and vestitone reductase (VR). Photographs are courtesy of Cathie Martin (John Innes Centre, Norwich, UK; Antirrhinum), Francesca Quattrocchio (Free University, Amsterdam; petunia), Erich Grotewold (Ohio State University, Columbus; maize), and Yimei Lin and Ann Hirsch (University of California, Los Angeles; sweet clover). A well-known physiological function of the anthocyanin pigments and flavonol copigments is the recruitment of pollinators and seed dispersers. These compounds also have figured into some of the major scientific breakthroughs of the past 150 years, including Mendel's elucidation of genetics, seed coat color being one of the major characters followed in his experiments with peas (Pisum sativum), and McClintock's discovery of transposable elements, which moved in and out of flavonoid biosynthetic genes expressed in maize kernels. Anthocyanins more recently have aided in understanding the phenomenon of cosuppression, particularly in petunia (Petunia hybrida). But besides providing beautiful pigmentation in flowers, fruits, seeds, and leaves, flavonoids also have key roles in signaling between plants and microbes, in male fertility of some species, in defense as antimicrobial agents and feeding deterrents, and in UV protection. The “early” steps in the pathway are found even in the bryophytes (mosses) and it has been suggested that synthesis of flavones, flavanones, and flavonols may have evolved first to provide chemical messengers and then UV sunscreens (Stafford, 1991). Flavonoids also have significant activities when ingested by animals, and there is great interest in their potential health benefits, particularly for compounds such as isoflavonoids, which have been linked to the anticancer benefits of soy-based foods, and the stilbenes in red wine that are believed to contribute to reduced heart disease. In recent years, much effort has been directed at elucidating the flavonoid biosynthetic pathway from a molecular genetic point of view. Mutants affecting flavonoid synthesis have been isolated in a variety of plant species based on alterations in flower and seed pigmentation. Maize, snapdragon (Antirrhinum majus), and petunia were established as the first major experimental models in this system, and work in these species led to the isolation of many flavonoid structural and regulatory genes (for review, see Holton and Cornish, 1995; Mol et al., 1998). Arabidopsis more recently has helped facilitate analysis of the regulation and subcellular organization of the flavonoid pathway. One unique aspect of using Arabidopsis for studying flavonoid biosynthesis is that all but one of the enzymes of central flavonoid metabolism (leading to flavonols and anthocyanins) are encoded by single-copy genes. The exception is flavonol synthase (FLS), which appears to be encoded by six genes, two of which may not be expressed (A. Bandara, D. Owens, and B. Winkel-Shirley, unpublished data). Genetic loci for both structural (TableI) and regulatory genes are scattered across the Arabidopsis genome and have been identified largely on the basis of mutations that abolish or reduce pigmentation in the seed coat. As a result, the loci were named transparent testa by Maarten Koornneef (Wageningen Agricultural University, The Netherlands), who isolated many of the first mutants in this class in the 1980s (for review, see Koornneef, 1990). The initial collection of 12 tt mutants has been expanded to include 21 members (tt1–19 plus ttg1 and ttg2), largely through directed searches in the Koornneef lab for new lines producing yellow or pale-brown seeds, and indirectly, by Loic Lepiniec and coworkers at the Institut National de la Recherche Agronomique (Versailles, France), in screens for plants exhibiting reduced seed dormancy. Transposon and activator tagging have been used to isolate additional mutations in genes either directly or indirectly involved in flavonoid biosynthesis (Wisman et al., 1998; Kubo et al., 1999; Borevitz et al., 2000). As a result most of the structural genes, as well as a number of regulatory genes, have now been correlated with specific mutant loci in Arabidopsis. Although Arabidopsis does not appear to use flavonoids in all of the same ways as some other species (for example, in defense or for male fertility), the Arabidopsis mutants are helping to define a role for these compounds in essential processes such as UV protection (Li et al., 1993; Landry et al., 1995) and the regulation of auxin transport (Murphy et al., 2000; Brown et al., 2001). Genetic loci for cloned flavonoid enzymes in Arabidopsis1-a Similar information for maize, petunia, and snapdragon is described by Holton and Cornish (1995). Based on the AGI map, 11/12/00; numbers in parentheses refer to P1 or bacterial artificial chromosome clones on which these sequences reside. Transposon-tagged mutant for FLS1 (Wisman et al., 1998). A. Tanaka (personal communication). Genetic loci for cloned flavonoid enzymes in Arabidopsis1-a Similar information for maize, petunia, and snapdragon is described by Holton and Cornish (1995). Based on the AGI map, 11/12/00; numbers in parentheses refer to P1 or bacterial artificial chromosome clones on which these sequences reside. Transposon-tagged mutant for FLS1 (Wisman et al., 1998). A. Tanaka (personal communication). Several important new genes required for flavonoid biosynthesis have been characterized in a variety of plant species over the past few years, including some with direct practical applications. One of these is the Arabidopsis BANYULS gene, which encodes a DFR-like protein that may be an LCR that catalyzes an early step in condensed tannin biosynthesis (Fig. 1; Devic et al., 1999). A locus identified independently as anthocyanin spotted testa (ast;Tanaka et al., 1997), with a very similar mutant phenotype, now appears to be identical to BAN (A. Tanaka, personal communication). Controlling condensed tannin levels in forage crops has long been of interest, both to improve nutritional value either by increasing or decreasing endogenous levels and to provide amounts sufficient to control pasture bloat. Some success has been achieved by modulating late steps in the central flavonoid pathway, such as the DFR reaction, for which cloned genes were previously available (Morris and Robbins, 1997). The LCR gene may provide an opportunity to direct metabolic engineering efforts more specifically to the proanthocyanidin branch pathway. Several breakthroughs have been made in the isoflavonoid pathway, including the isolation of the first IFS genes. Biochemical and genetic data have long suggested that this enzyme is a member of the cytochrome P450 oxygenase family of enzymes. This was confirmed by Shin-ichi Ayabe's laboratory (Nihon University, Fujisawa, Kanagawa, Japan) with the isolation of IFS from a licorice (Glycyrrhiza echinata) cell line that produces isoflavonoids upon elicitation (Akashi et al., 1999). At the same time, Richard Dixon's group (The Noble Foundation, Ardmore, OK) identified an IFS gene from soybean (Glycine max) by functional screening of candidate P450 cDNAs in insect cells (Steele et al., 1999), whereas a group at DuPont Wilmington, DE identified the same gene as well as a second IFS using a similar screen in yeast (Saccharamyces cerevisiae; Jung et al., 2000). Both isoforms of soybean IFS appear to be able to use both liquiritigenin and naringenin as substrates to produce genistein or daidzein, respectively (Fig. 1), although naringenin is used less efficiently. The DuPont group showed that soybean IFS1 can function to convert naringenin to genistein in transgenic Arabidopsis and, more recently, in tobacco (Nicotiana tabacum) and maize (Yu et al., 2000). They have also shown that introduction of IFS1 together with chalcone reductase, which provides the additional substrate, liquiritigenin, results in the synthesis of daidzein in maize. Nancy Paiva's laboratory (The Noble Foundation) is attempting to express VR in tobacco plants; this enzyme is one of several that will be required to engineer production of the isoflavonoid, medicarpin, the major phytoalexin produced by alfalfa (Medicago sativa) in response to fungal pathogens (Fig. 1;Watson and Paiva, 2000). A cDNA encoding the cytochrome P450 protein, I2′H, another enzyme required for medicarpin biosynthesis (Fig. 1), has also been isolated from licorice (Akashi et al., 1998). Together, these advances are laying the foundation for engineering isoflavonoid biosynthesis for agronomic and nutritional enhancement of a wide variety of crop plants that do not normally synthesize these compounds. There may also be more immediately feasible applications in the engineering of legumes, for example, to improve the palatability of soy milk by down-regulating isoflavonoid synthesis in soybean seeds. Efforts to engineer flower color have also led to some interesting developments in the last few years. The hydroxylation pattern of the B ring of anthocyanins is a major determinant of the color of these pigments. All flavonoids carry a hydroxyl group at the 4′ position, including the pink-to-red cyanidin-based pigments. Hydroxylation at two variable positions is controlled by the P450 enzymes, F3′H, which leads to brick-red to orange pelargonidin-based pigments, and F3′5′H, which is required for synthesis of purple and blue delphinidin-based pigments (Fig. 1). Several years ago, workers at Florigene isolated two F3′5′H genes from petunia based on sequence homology to other p450s, a pattern of high-level expression in flowers, and correlation with theHf1 and Hf2 loci (Holton et al., 1993). Cloning of the first F3′H gene took a bit longer, but a petunia gene was eventually isolated using a similar approach (Brugliera et al., 1999). Together with Chris Cobbett (The University of Melbourne, Parkville, Victoria, Australia), this group also identified the F3′H gene in Arabidopsis by chromosome walking to the tt7 locus (C. Cobbett, personal communication); the same gene has been identified independently based on information from the Arabidopsis Genome Project (Schoenbohm et al., 2000; Saslowsky and Winkel-Shirley, 2001). It is unfortunate that these genes are not, by themselves, sufficient for engineering altered flower color in horticulturally important species, for example, by overexpression in roses (Rosa spp.) and carnations (Dianthus caryophyllus) that normally lack F3′5′H activity and therefore do not produce blue or purple pigments (Brugliera et al., 2000). However, it has been shown that a specific cytochrome b5 is required for maximal activity of the petunia F3′5′H as an to the P450 reductase that is with cytochrome P450 et al., 1999). It is that the group at Florigene recently that when the petunia F3′5′H and b5 genes are together into flower color is from red to a purple (Brugliera et al., 2000). It appears that the long blue may be at are also being made in understanding the regulation of flavonoid biosynthesis, particularly as a result of molecular genetic such as tagging and This has led to the of a number of regulatory that are to in the between that the pathway and well-known flavonoid such as the and and of maize. In has been made in genes that expression of pathway enzymes, which are specific to proanthocyanidin and anthocyanin include a new class of flavonoid regulatory by in petunia et al., and in Arabidopsis et al., 1999), that and are to the of appears to be a protein that expression of the recently cloned protein, et al., 1999). from in several including role not in flavonoid synthesis but in cell and the production of seed A gene, has recently been isolated from maize personal which may some on the and function of this new class of regulatory In a of phenylpropanoid has been cloned in Arabidopsis, when results in purple et al., 2000). In addition to providing into regulation of phenylpropanoid the lines may provide a new into Arabidopsis genes that function the branch to condensed tannins, a of genes based on the The Arabidopsis which has a similar to has identified another class of flavonoid regulatory a member of the family that two and 1998). the regulatory pathways flavonoid synthesis and involves at two This of has been in one other species, a of Arabidopsis et al., It will be interesting to of are involved in flavonoid gene expression in species petunia and maize. additional identified by the petunia regulatory et al., and the Arabidopsis et al., new members of the family of and have to in maize and in directly expression of a DFR gene as well as an protein, whereas is required for expression of DFR and that these may be although there is from both sequence and experimental data to that are not of and et al., 2000). of genes identified by regulatory loci such as petunia and maize and Arabidopsis as well as analysis of the in flavonoid regulatory plant species, in some of the many that in the flavonoid gene regulation The of the activity of regulatory species is another important of example, Grotewold et recently shown that the of maize and with is by a number of specific in the and an essential aspect of regulation in this are for helping the of both structural and involved in flavonoid biosynthesis and for the of plant examples have been In one at and showed that genes exhibiting altered expression in maize cell lines flavonoid pathway both maize flavonoid genes as well as sequences et al., 2000). In the second example, and his have expression of both and candidate phenylpropanoid pathway genes and as the of this et al., 2000). Together with genetic and these efforts are to understanding of flavonoid biosynthesis is controlled and this information may be used to engineer flavonoid metabolism in plant The of the and organization of flavonoid enzymes was first by more years ago, together with the that the enzymes of general and flavonoid biosynthesis were to function as multienzyme (Stafford, The that these pathways may be as enzyme that facilitate the direct or of between is for a number of example, there is for substrates at the branch these the are and and the of these compounds appears to be there is the for these pathways to to and to the amounts of end products that are The first to this largely from group University, who used cell and experiments to that and were in the with the of the for review, see This group also data from experiments an of with the of the but not with or in The results of this work to a model for phenylpropanoid and flavonoid synthesis a of of enzymes that is through with membrane that include and has now been in of a flavonoid enzyme and experiments that there are direct between and DFR in Arabidopsis and Winkel-Shirley, 1999). However, the data do not point to a of enzymes, but to a in which not the enzyme in the pathway, and also and In the of with the first described by has now also been in Arabidopsis, together with of this enzyme with the that both are enzymes and Winkel-Shirley, 2001). a in the Arabidopsis F3′H gene that most of the of this P450 enzyme results in altered of and that F3′H may function as of a membrane for other enzymes of the flavonoid pathway, as first suggested by There have also been that the isoflavonoid branch pathway as an enzyme This pathway two cytochrome P450 that function as membrane IFS and (Fig. 1). first for the of an enzyme from this pathway, isoflavone reductase, with the in cell experiments with in alfalfa together with for in and in in the for have that isoflavone O-methyltransferase is of a metabolic et al., 1998; and 2000). It is interesting that this enzyme between the two P450 IFS and I2′H, in isoflavonoid These recent together with for between and in the general phenylpropanoid pathway and and and 1999), that the organization of these may an important in understanding plant metabolism is Although the flavonoid pathway is to be an of a a enzyme it is also that this organization metabolic engineering by the of to enzymes and 1999), at the same providing for the of into or branch A great of work is in this including efforts to define protein to plant cells or a variety of to the production of specific and to in the organization of enzyme in response to and information is also the transport of flavonoids from the of synthesis in the to in the or cell of pigments to the in the maize and petunia and also in both a and a to the family of et al., 1995; et al., 1998). The enzymes, in petunia and in maize, are and are able to in of flavonoids in a variety of plant et have recently suggested that and may function as and that transport is by a with reduced to the transport of in the A is in Arabidopsis, and of the gene has that transport of proanthocyanidin into the in the seed coat involves a protein to the and family of in this species et al., 2001). Erich group has new from analysis of maize cells with a of flavonoid synthesis (Fig. 1), for the transport of yellow or compounds to the and cell respectively (for review, see 2001). These are of the subcellular that in cells and 1990). for of flavonoid enzymes in in Arabidopsis cells and Winkel-Shirley, may be to this transport between the of genes that are essential for this and the analysis of the cell of the system, elucidation of the molecular the of flavonoid compounds in of the cell may be on the This is an of central in understanding flavonoid biosynthesis is controlled and may provide additional into engineering this metabolic pathway. The elucidation of the of and from alfalfa by (The and Richard Dixon's is one of the most recent developments in understanding the flavonoid pathway in three et al., 1999; et al., In addition to providing new information the of flavonoid as of the of and the synthase et al., are providing a great of information the of plant This is for engineering these enzymes to produce new similar to has been with the bacterial It is that homology of the can be used to activities of enzymes in at some (for review, see and 1999). of the Arabidopsis enzyme has also into specific mutations reduce not by the but also by the protein or with et al., 2000). on the other has a and enzyme activity that are unique to the has the first information on the alfalfa enzyme naringenin chalcone and and catalyzes the of The also that may be important in the of enzymes in and other plant Efforts are to the of Arabidopsis flavonoid enzymes to facilitate the of experiments to define the of these in the enzyme It is that the that have been are providing information for studying a variety of of flavonoid biosynthesis, from to and subcellular The of flavonoid biosynthesis plants has long the of the pathway and structural and regulatory Biochemical data first the that this pathway been from In the of the enzymes of flavonoid biosynthesis are members of three classes of enzymes found in all the flavonol and and and cytochrome P450 F3′5′H, and 1; and 1999). and on the other appear to have a more in appears to be unique to plants in both sequence and et al., is a member of the plant synthase which also and This family of enzymes uses similar and similar or substrates to produce a wide variety of Although these enzymes are not to the bacterial or fungal other are in In recent work has a sequence with plant including the enzyme identified by in Arabidopsis, which the first step in biosynthesis et al., 1999; et al., 2000). on the gene family in point to gene and of this enzyme over the of et al., 2000). It has also been suggested that has evolved from in plant species, based on the results of and structural and the that is found in a number of plant species (for review, see 1997). for of specific enzyme activities from flavone which is as a dioxygenase in and a P450 in snapdragon 1990). In some of the of the flavonoid pathway may have evolved from the enzymes, as suggested for which may in flavonoid transport as directly these compounds to et al., 2000). It is interesting to that the genes in the flavonoid pathway appear to have evolved more the genes et al., 1999). The of genome sequence and protein information additional on the of the flavonoid pathway and also provide into such as the of in enzymes that to control at major branch The flavonoid biosynthetic pathway has been one of the most metabolic in As with new of information appears to a number of and At the same time, new are providing the opportunity to flavonoid biosynthesis, not as an of but as of a and metabolic The to now flavonoid enzymes, for the very first time, in three and to the of the pathways of metabolism using and metabolic are to much more this The of for that include Arabidopsis, the and the model are also to this metabolic model from new It is even for pathways flavonoid biosynthesis, these are The the members of and two for on the are also to Chris Cobbett, Erich Tanaka, and for information to and to Erich Ann Cathie and Francesca Quattrocchio for providing