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Home , Anthocyanidin, Anthocyanin, Biochemistry, Biosynthesis, Leucocyanidin

The , Vol. 7, 1071-1083, July 1995 O 1995 American Society of Plant Physiologists

Genetics and of Ant hocyanin

Timothy A. Holton’ and Edwina C. Cornish Florigene Proprietary Ltd., 16 Gipps Street, Collingwood, Victoria 3066, Australia

INTRODUCTION

Flavonoids represent a large class of secondary plant metab- centrate on the more recent developments in isolation olites, of which anthocyaninsare the most conspicuous class, and characterization.A review of the of bio- dueto the wide range of colors resulting from their synthesis. synthesis in other species was recently covered by Forkmann are important to many diverse functions within (1993). . Synthesis of anthocyanins in petals is undoubtedly in- The characterization of genetically defined mutations has tended to attract pollinators, whereas synthesis enabled the order of many reactions in anthocyanin synthesis in and may aid in dispersal. Anthocyanins and their modification to be elucidated. Some reactions have and other can also be important as feeding deter- been postulated only on the basis of genetic studies and have rents and as protection against damage from UV irradiation. not yet been demonstrated in vitro. Chemico-genetic studies The existence of such a diverse range of functions and types have been very important in determining the enzymatic steps of anthocyanins raises questions about how these compounds involved in anthocyanin biosynthesis and modification. The are synthesized and how their synthesis is regulated. generation of transposon-tagged mutations and the subse- The study of the genetics of anthocyanin synthesis began quent of the transposons provided a relatively last century with Mendel’s work on color in . Since straightforward means of isolating many from that time, there have been periods of intensive study into the (Wienand et al., 1990) and snapdragon (Martin et al., 1991). genetics and biochemistry of pigment production in a num- However, a number of genes in the pathway have not been ber of different species. In the early studies, genetic loci were amenable to transposon tagging. correlated with easily observable color changes. After the struc- Anthocyanin biosynthetic genes have been isolated using tures of anthocyanins and other flavonoids were determined, a range of methodologies, including purification, trans- it was possible to correlate single genes with particular struc- poson tagging, differential screening, and polymerase chain tural alterations of anthocyanins or with the presence or reaction (PCR) amplification. Functions of isolated anthocya- absence of particular flavonoids. Mutations in anthocyanin nin genes can be confirmed by restriction fragment length genes have been studied for many years because they are polymorphism (RFLP) mapping, complementation,or expres- easily identified and because they generally have no deleteri- sion in heterologoussystems. Reverse genetics has also been ous effect on plant growth and development. In most cases, used recently to identify gene ; this requires a well- mutations affecting different steps of the anthocyanin biosyn- defined pathway to correlate with gene function. thesis pathway were isolated and characterized well before Once a gene has been isolated from one species, it is usually their function was identified or the corresponding gene was a straightforward task to isolate the homologous gene from isolated. More recently, many genes involved in the biosyn- other species by using the original clone as a . thesis of anthocyanin pigments have been isolated and characterized using recombinant DNA technologies. Three species have been particularly important for elucidat- ing the anthocyanin biosynthetic pathway and for isolating ANTHOCYANIN BIOSYNTHETIC PATHWAY genes controlling the biosynthesis of flavonoids: maize (Zea mays), snapdragon (Anfirrhinum majus), and petunia (Wtunia The anthocyaninbiosynthetic pathway is well established (MOI hybrida).Petunia has more recently become the of et al., 1989; Forkmann, 1991). A generalized anthocyanin choice for isolating flavonoid biosynthetic genes and study- biosynthetic pathway is shown in Figure 1. Although the bio- ing their interactions and regulation. At least 35 genes are synthetic pathways in maize, snapdragon, and petunia share known to affect flower color in petuniawiering and de Vlaming, a majority of common reactions, there are some important 1984). Because this field of research has been reviewedfairly differences between the types of anthocyanins produced by extensively in the past (Dooner et al., 1991; van Tunen and each species. One major difference is that petunia does not MOI, 1991; Gerats and Martin, 1992), in this review we con- normally produce pigments, whereas snapdra- ‘To whom correspondence should be addressed. gon and- maize are incapable bf producing 1072 The Plant Cell

3 x Malonyl-COA

p-Coumaroy I-COA CHS OH O hydroxychalcone

ÓH Ò

OHKaempferol O Homo&OH

/-- -0H Dihydrokaempferol 7-H 'OH / OH O \ OU A MyricetG

Quercetin - Ty.OH OH 0 Dihydroquercetin OH O Dihydromyricetin 1DFR 1DFR

II ÒH ÒH OH OH OH OH Leucocyanidin

ANS I 3GT i .OH

OH OH OH Pelargonidin-3-glucoside -3-glucoside Delphinidin-3-glucoside

Figure 1. Anthocyanin and Flavonol Biosynthetic Pathway. Anthocyanin Biosynthesis 1073 pigments. The extent of modificationof the anthocyanins also by flavonoid 3'-hydroxylase (F3'H) to produce dihydroquerce- varies among the three species. The reasons for these differ- tin (DHQ) or by flavonoid 3;5'-hydroxylase (F33'H) to produce ences are discussed in the following outline of the dihydromyricetin (DHM). F33'H can also convert DHQ to DHM. and genes involved in anthocyanin biosynthesis. At least three enzymes are required for converting the color- The precursors for the synthesis of all flavonoids, including less dihydroflavonols (DHK, DHQ, and DHM) to anthocyanins. anthocyanins, are malonyl-COA and p-coumaroyl-COA.Chal- The first of these enzymatic conversions is the reduction of cone (CHS) catalyzes the stepwise condensation of dihydroflavonols to flavan9,4-cis-diols () three units from malonyl-COA with p-coumaroyl-COA by dihydroflavonol4-reductase(DFR). Further oxidation, dehy- to yield tetrahydroxychalcone.Chalcone (CHI) then dration, and glycosylation of the different leucoanthocyanidins catalyzes the stereospecific isornerization of the yellow-colored produce the corresponding brick-redpelargonidin, cyani- tetrahydroxychalcone to the colorless naringenin. Naringenin din, and blue delphinidin pigments. Anthocyanidin3-glucosides is converted to dihydrokaempferol (DHK) by may be rnodified further in many species by glycosylation, 3-hydroxylase(F3H). DHK can subsequently be hydroxylated , and , as illustrated in Figure 2. There

.OH PH

"-0H "-0H Oclc "mHOHOClC

OH Cyanidin-Sglucoside Delphinidin-Sglucoside

PH

oH Cyanidin-3-rutinoside oH Delphinidin-3-rutinoide

1. OH PH "-0. "-0.

O-Glc-Rha OClc-ORh9H Glc-Ò Cyanidin-3-(pcoumaroyl)- "'-0 Delphinidin-b(p-coumaroyl)- ru tinoside-Sglucoside ru tinoside-5glucoside Mtf,MtZ IMt 1,MtZ Mf\ I,MfZ I PCH

O-Glc-Rha I Glc-Ò Glc-O HowoHPeonidin->(pcoumacoyI> Glc-o ->(pcoumaroyl)- -S(p-coumaroyl)- rutinoside-5-glucoside rutinoside-5-glucoside rutinoside-Sglucoside

Figure 2. Genetic Control of Anthocyanin Modifications in Petunia. Genetic loci controlling each enzymatic step of the pathway are shown in italics. No genetic locus encoding the 5GT gene has yet been identified. Glc, ; Rha, rhamnose. 1074 The Plant Cell

are both species and variety differences in the extent of modifi- Two genes encode CHS in maize: c2 is involved in anthocya- cation and the types of and acyl groups attached. nin biosynthesis in seed (Dooner, 1983; Wienand et al., 1986), and whp controls CHS activity in pollen (Coe et al., 1981). The maize c2 gene was isolated followingtransposon tagging using STRUCTURAL GENES the En (Spm) transposable element (Wienand et al., 1986). Precursorfeeding studies and activity measurements in different c2 lines indicatedthat c2 might encode CHS. Many genes encoding anthocyanin biosynthetic enzymes have A cDNA clone corresponding to the c2 locus was sequenced been characterized and cloned frorn maize, snapdragon, and and shown to have a high sequence similarity with the pars- petunia. Table 1 summarizes the genetic loci and structural ley CHS clone. A second CHS gene, whp, has subsequently genes isolated from each of these species. been isolated from maize (Franken et al., 1991). The nivea locus controls CHS enzyme activity in of snapdragon (Spribille and Forkmann, 1982). A CHS gene from Chalcone Synthase snapdragon was isolated by hybridization to the parsley CHS clone (Sommer and Saedler, 1986). The snapdragon The first flavonoid biosynthetic gene isolated was the CHS gene contains only one CHS gene. from parsley (Kreuzaler et al., 1983). A cornbinationof differen- screening and hybrid-arrested and hybrid-selected was used to identify a cDNA clone homologous to Chalcone lsomerase CHS mRNA. The parsley CHS clone was also used as a mo- lecular probe to isolate clones of two different CHS genes from The accumulationof chalcone in plant tissues is rare: chalcone petunia (Reif et al., 1985). It was later shown that there are is rapidly isomerized by CHI to form naringenin. Even in the 12 different CHS genes in the petunia genome, but only four absence of CHI, chalcone can spontaneously isomerizeto form of these (chsA, chsB, chsG, and chsJ) are known to be ex- naringenin, albeit at a slower rate. However, there are some pressed (Koes et al., 1989). All four genes are expressed in examples of chalcone accumulation in CHI . Corollas UV-irradiated seedlings, but only chsA and chsJ are expressed of Callisfephuschinensis (China aster) and Dianfhus caryoph- in floral tissues. yllus (carnation) are devoid of CHI activity, resulting in the

Table 1. Structural Genes Encoding Anthocyanin Biosynthetic Enzymes

Maize Snapdragon Petunia Enzyme Locus Clonea Locus Clone Locus Clone Chalcone synthase (CHS) c2 nivea NAb + WhP Chalone isomerase (CHI) NA NA Po + Flavanone 3-hydroxylase (F3H) NA incolorata An3 + Flavonoid 3’-hydroxylase (F3’H) Pr eosina HtllHt2 +c Flavonoid 3‘5’-hydroxylase (F3’5‘H) NA NA Hfl + Hf2 + Dihydroflavonol reductase (DFR) A1 pallida An6 + synthase (ANS) A2 Candica NA + Flavonoid 3-glucosyltransferase(3GT) . Bzl NA NA + UDP rhamnose:anthocyanidin-3-gIucoside rhamnosyltransferase (3RT) NA NA Rt + Anthocyanin acyltransferase (AAT) NA NA Gf - Anthocyanin 5-0-glucosyltransferase (5GT) NA NA NA - Anthocyanin methyltransferase (AMT) NA NA MfllMf2 +d MtllMt2 Glutathione S- IGSTP 822 NA An13 + a + , a cDNA or gene has been cloned; - , a gene has not been cloned. NA, a genetic locus encoding the structural gene has not been identified. A clone encoding F3’H activity has been identified, but the gene does not correspond to Htl or Ht2. The genetic locus has yet to be identified. e Enzyme identity is inferred from . Anthocyanin Biosynthesis 1075 production of yellow flowers dueto the accumulation of chal- Flavonoid t’-Hydroxylase cone pigments (Kuhn et al., 1978; Forkmann and Dangelmayr, 1980). In flower extracts of defined genotypes of petunia, an enzyme A CHI cDNA was first isolated from French bean using anti- activity was demonstrated that catalyzes the of bodies made against the purified enzyme (Mehdy and Lamb, naringenin and dihydrokaempferol in the 3‘position (Stotz et 1987). Similarly, an antiserum raised against CHI enzyme pu- al., 1985). Two genetic loci, Ht7 and Ht2, control F3’H activity rified from petunia flower buds (van Tunen and MOI,1987) was in flowers of petunia. Hfl acts in the limb and tube of the corolla, used to screen a petuniapeta1 cDNA expression library to iso- whereas Hf2 acts only in the corolla tube (Wiering, 1974). The late CHI clones (van Tunen et al., 1988). P hybrida has two Hfl and Ht2 genes control 3’-hydroxylation of anthocyanins CHI genes (chiA and chiB), both of which have been isolated and . A cytochrome P-450 cDNA clone encoding F3‘H and characterized(van Tunen et al., 1988,1989). The two genes activity has been isolated from petunia, but the correspond- show different patterns of expression: chiA is expressed in all ing gene is not linked to either Htl or Hf2 (Y. Tanaka and TA. floral tissues and in UV-irradiatedseedlings, whereas chiB is Holton, unpublished results). expressed in immature anthers only (van Tunen et al., 1988). Flavonoid 3‘-hydroxylation in maize is controlled by the Pr In petunia, the PO gene controls the expression of CHI in locus (Coe et al., 1988). The of Pr plants is anthers. In polpo plants, tetrahydroxychalcone accumulates due to the accumulation of mostly cyanidin glucoside, whereas in pollen, which is therefore yellow or green. RFLP analysis the aleurone of pr plants is red due to accumulation of mostly showed a 100% linkage between chiA and RI (van Tunen et pelargonidin glucoside. Larson and Bussard (1986) detected al., 1991), suggesting that PO corresponds to chiA. Transfor- the presence of F3’H activity in microsomal preparations mation of apolpo line with a functional chiA gene resulted in obtained from maize seedlings. (a flavonol), narin- complementation of the mutation, as visualized by a shift in genin (a flavanone), and (a flavone) all serve as pollen color from yellow to white. It has been proposed that substrates for the hydroxylase. lnhibitor studies suggested that the po mutation is a mutation in the regulatory region of chiA the F3‘H enzyme belongs to the cytochrome P-450 superfamily. that abolishes in anthers but not in corollas (van In snapdragon, the gene eosina (eos) controls the 8-ring Tunen, 1990). hydroxylation of flavonoids in the 3‘ position (Forkmann and Snapdragon and maize CHI genes were isolated by homol- Stotz, 1981). Recessive genotypes (eosleos) are known to pro- ogy with previously cloned CHI genes (Martin et al., 1991; duce apigenin, kaempferol, and pelargonidin in the flowers, Grotewold and Peterson, 1994). RFLP mapping data indicated whereas the corresponding 3‘,4’-hydroxylated compounds are that some maize lines contain three loci with CHI-homologous found in €os plants. sequences. This may explain the observation that to date, no CHI mutants of maize have been identified. Flavonoid 3:5’-Mydroxylase

Two genetic loci, Hfl and Hf2, control F3‘5‘H activity in flowers Flavanone 3-Hydroxylase of petunia(Wiering, 1974). Hf7 acts in the corolla, stigma, and pollen, whereas Hf2 acts only in the corolla limb. The F33’H A combination of differential screening and genetic mapping enzyme belongs to the cytochrome P-450 superfamily (Holton was used to isolate a cDNA clone corresponding to the in- et al., 1993a). More than 200 different P-450 sequences are colorata locus of snapdragon, which is known to encode F3H available, predominantly from (Nelson et al., 1993). (Martin et al., 1991). The petunia enzyme, which is encoded All P-450 sequences share a number of small regions of se- by the An3 locus (Froemel et al., 1985), has been purified to quence similarity. This sequence conservation was used to homogeneity and characterized as a typical 2-oxoglutarate- design degenerate oligonucleotide primers to isolate P-450 se- dependent dioxygenase (Britsch and Grisebach, 1986; Britsch, quences from petunia via PCR amplification (Holton et al., 1990). A cDNA encoding F3H was isolated from petals of petu- 1993a). Clones of two different P-450 genes were shown to nia (Britsch et al., 1992). The function of the clone was verified encode F33‘H activity by expression of full-length cDNA clones by comparison of the encoded sequence with se- in . RFLP mapping and complementation of mutant petu- quences obtained from the purified plant enzyme and by nia lines showed that these two genes correspond to the Hf7 prokaryotic expression, which yielded an enzymatically active and Hf2 genetic loci. hydroxylase. The snapdragon and petunia sequences share high sequence similarity (Britsch et al., 1993). Using the petunia F3H cDNA clone as a heterologousprobe, Dihydroflavonol 4-Reductase the corresponding cDNAs have been cloned from carnation, China aster, and stock (Britsch et al., 1993). The cDNAs of DFR genes have been isolated from maize and snapdragon China aster and carnation were verified as F3H clones by in by transposon tagging. Recessive mutations at the a7 gene vitro expression in a reticulocyte system. of maize lead to a colorless aleurone layer. The a7 gene was 1076 The Plant Cell

tagged with the transposable element SpmlEn (OReilly et al., with Ac (Fedoroff et al., 1984; Dooner et al., 1985). A putative 1985) and was shown to encode DFR following in vitro trans- 3GT clone, pJAM338, was isolated from snapdragon using the lation of an a7 cDNA clone (Reddy et al., 1987). maize gene as a probe (Martin et al., 1991). The snapdragon Mutations at thepallida @a/)locus of snapdragon block an- clone was confirmed to encode 3GT by expression in lisian- thocyanin biosynthesis, giving rise to uncolored or partially thus (Schwinn et al., 1993). A 3GT gene has recently been colored flowers. It was concluded that the pal gene encodes isolated from petunia (Y. Tanaka, personal ). DFR from feeding in which anthocya- In petunia and snapdragon as well as in many other spe- nin synthesis was observed when flowers ofpal mutants were cies, anthocyanidin 3-glucosides can be modified further by infused with leucocyanidin but not when they were supplied rhamnosylation to produce anthocyanidin 3-rutinosides. A com- with DHQ (Almeida et al., 1989). Thepallocus was cloned using bination of differential screening and genetic mapping was a transposable element (Tam3) probe to isolate an unstable used to isolate clones of UDP rhamnose:anthocyanidin-3-glu- insertion (Martin et al., 1985). Further evidence for the coside rhamnosyltransferase(3RT) from petunia(Brugliera et identity of the pal came from sequence analysis, which al., 1994; Kroon et al., 1994). The 3RT gene corresponds to showed amino acid homology between pal and the a7 gene the Rt locus in petunia. The 3RT gene product shares a low of maize. leve1 of sequence homology with 3GT and other glycosyl- A snapdragon DFR clone was used to isolate a homologous . gene from petunia (Beld et al., 1989). Petunia contains three Anthocyanins found in flowers of petunia are glucosylated different DFR genes (dfrA, dfrB, and dfrc), but only the dfrA at the 3 position (Figure 2). Dependingon genetic background, gene is transcribed in floral tissues. The dfrA gene was shown anthocyanins may also possess a glucose group at the 5 po- to correspond to the An6 locus using RFLP mapping and com- sition. Glucosylation of anthocyanins at the 5 position was plementation (Huits et al., 1994). The petunia DFR enzyme thought to be controlled by the gene Gf, based on the lack preferentiallyconverts DHM to leucodelphinidin; DHQ is a poor of 5-glucosylatedanthocyanins in Gf mutants (Wiering and de , and DHK is not converted to leucopelargonidin Vlaming, 1973). However, further analysis demonstrated that (Forkmann and Ruhnau, 1987). The distinct substrate speci- anthocyanin 5-0-g1ucosyltransferase(5GT) activity correlates ficity explains the preferential accumulation of delphinidin with theAn7 gene, not the Gf gene (Jonsson et al., 1984). An7 derivatives and the lack of pelargonidin pigments in petunia does not encode 5GT but is a regulatory gene controlling ex- (Gerats et al., 1982). pression of many anthocyanin structural genes, including 5GT Gf is therefore likely to encode an anthocyanin acyltransfer- ase (AAT) enzyme. Because the 5GT enzyme from petunia Anthocyanidin Synthase requires anthocyanidin-3-@-coumaroyl)rutinosides as sub- strates (Jonsson et al., 1984), mutation of an AAT gene results The enzymatic steps catalyzing the conversion of leucoantho- in the accumulation of anthocyanidin 3-rutinosides. to colored anthocyanidinsare not well characterized but involve an oxidation and a dehydration step (Heller and Forkmann, 1988). In maize, mutation of the A2 gene blocks Anthocyanin Methyltransferases the enzymatic conversion of leucoanthocyanidins to anthocy- anidins (Reddy and Coe, 1962). The snapdragon gene Candica In petunia, the genetic loci Mtl and Mt2 control methylation at (Candi) is also considered to be involved in the conversion the 3' position; Mf7 and Mf2 control methylation at the 3' and of leucoanthocyanidinsto (Bartlett, 1989). 60th 3',5'positions (Wiering, 1974; Jonsson et al., 1983). Quattrocchio the A2 and the Candi gene have been cloned (Menssen et et al. (1993) reported on the cloning of an anthocyanin-0- al., 1990; Martin et al., 1991). The deduced amino acid se- methyltransferase gene from petunia but did not identify its quence of the product of a petunia gene, ant77 (Weiss et al., corresponding genetic locus. 1993), is 48% identicalto the A2-encoded sequence of maize and 73% identical to the Candi-encoded sequence of snap- dragon. Because of the significant similarities between these Other Structural Genes three , it is likely that they have the same enzymatic activity (anthocyanidin synthase, or ANS). The ANS proteins Recessive mutations of the Bronze2 (822) gene of maize re- also share some sequence similarity with P-oxoglutarate- sult in bronze pigmentation of the aleurone layer and modify dependent dioxygenases, which include such enzymes as purple plant color to reddish brown (Nueffer et al., 1968). 822 F3H, flavonol synthase (FLS), and l-aminocyclopropane-lcar- sequences were cloned by transposon tagging (Theres et al., boxylate (ACC) oxidase. 1987) and by combining the techniques of transposon tagging and differentialhybridization (Mclaughlin and Walbot, 1987). lntertissue complementation studies (Reddy and Coe, 1962; Anthocyanin McCormick and Coe, 1977) provided evidence that the prod- uct of the 622 gene is involved in one of the last steps of The maize Bzl gene, encoding UDP g1ucose:flavonoid 3-Oglu- anthocyanin synthesis. The polypeptide encoded by 822 cosyltransferase (3GT), was isolated by transposon tagging shares homology with various -related proteins (Schmitz Anthocyanin Biosynthesis 1077

and Theres, 1992), including glutathione S-transferase (GST). on their relative substrate specificities. Stafford (1991) proposed A gene corresponding to the An73 locus of petunia has recently that during biosynthesis, the flavonoid biosynthetic enzymes been isolated (Quattrocchio et al., 1993). TheAn73 gene prod- are organized into an ordered sequence as an aggregate or uct also shares homology with GSTs, but it has not been shown linear multienzyme complex, but no data support this idea. why this enzyme is required for anthocyanin synthesis. It may Most of the genes necessary for the synthesis of anthocya- be that conjugation to glutathione is required for transport of nin pigments have now been isolatedffom a number of species. anthocyanins into the vacuole. These genes can be used to help study the interactions among individual enzymes in the pathway as well as to characterize further each of the enzymes in isolation. When individual Competition between Flavonoid-Metabolizing Enzymes cloned genes are expressed in a heterologous host, they are generally functional. Petunia provides an ideal model system Dihydroflavonols are the precursors of both anthocyanins and for testing competition and interactions among flavonoid- flavonols. In most plants, both anthocyanins and flavonols are modifying enzymes because of the availability of many defined synthesized within the same cell and usually accumulate in mutants and a simple transformation system. the Same subcellular location. The dihydroflavonols are sub- strates for DFR, FLS, F3’H, and F3’5’H. Therefore, the potential exists for competition between each of these enzymes for com- REGULATORY GENES mon substrates. Lack of specificity for dihydroflavonols and of the flavonoid modification enzymes permits a grid-type pathway, or multiple paths to the same intermediate Genetics (Stafford, 1990). If both F3’H and F33’H enzymes are present in petunia, pre- Regulatory genes that control expression of the structural dominantly 3’,5’-hydroxylated anthocyanins are produced, genes of the anthocyanin biosynthetic pathway have been iden- presumably due to the substrate specificity of enzymes involved tified in many plants. These genes influencethe intensity and in the conversion of dihydroflavonols to anthocyanins (Beld pattern of anthocyanin biosynthesis and generally control ex- et al., 1989). Mutant petunia lines that have no F35’H activity pression of many different structuralgenes. Evidence for this but possess F3’H and FLS activity accumulate flavonols at the regulation can be obtained by either enzyme assays or mRNA expense of anthocyanins derived from cyanidin (Gerats, 1985). assays of structural gene activity. Table 2 summarizes the High levels of cyanidin derivatives accumulate only in petunia regulatory genes described in maize, snapdragon, and petunia. lines that possess F3’H activity but lack F33’H and FLS activ- The R gene family determines the timing, distribution, and ity. However, in the presence of F3’H, F3‘5’H, and FLS activities, amount of anthocyanin pigmentation in maize. This family com- 4‘- and 3’,4’-hydroxylated flavonols (kaempferol and querce- prises a set of regulatory genes, consisting of the R locus (which tin, respectively) accumulate. The accumulation of specific includes S, Lc, and Sn) and the B locus (Dooner et al., 1991). flavonoids is dependent both on the enzymes produced and Each gene determines pigmentation of different parts of the

~ Table 2. Regulatory Genes of Anthocyanin Biosynthesis Species Locus Cloned” Genes Regulatedb Gene Cloning Reference Maize R + CHS, DFR, 3GT Dellaporta et al. (1988) RfS) + CHS, DFR, 3GT Perrot and Cone (1989) WSn) + CHS, DFR Tonelli et al. (1991) RfW + CHS, DFR Ludwig et al. (1989) B + DFR, 3GT Chandler et al. (1989) c1 + CHS, DFR, 3GT Cone et al. (1986); Paz-Ares et al. (1986, 1987) PI + CHS, DFR, 3GT Cone and Burr (1989) VP 1 + c7 McCarty et al. (1989) Snapdragon Delila + F3H, DFR, ANS, 3GT Goodrich et al. (1992) €luta - F3H, DFR, ANS, 3GT Rosea - F3H, DFR, ANS, 3GT Petunia An 1 - chsJ, DFR, ANS, 3GT, 3RT, AMT, F3‘5’H, GST An2 + chsJ, DFR, ANS, 3GT, 3RT, AMT, GST Quattrocchio (1994) An4 +c chsJ, DFR, ANS, 3GT, 3RT, AMT, GST Quattrocchio (1994) Anl7 - chsJ, DFR, ANS, 3RT, AMT, GST

a + , gene or cDNA has been cloned; - , gene has not been cloned. See Table 1 for full names of enzymes. Expression patterns and RFLP analysis suggest that the clone jaf73 corresponds to An4, but this has not been confirmed. 1078 The Plant Cell plant. Accumulation of anthocyanins in competent tissues also binding and transcriptionalactivation domains were found to requires the presence of either C7 (in the seed) or PI (in the interact when synthesized and assayed in yeast (Goff et al., plant ) (Coe et al., 1988). Viviparous-7 (Vp7)controls the 1992). These studies suggest that the regulationof the maize anthocyanin pathway in the developing maize seed primarily anthocyanin pathway involves a direct interaction between through regulation of the C7 gene (Hattori et al., 1992). How- members of two distinct classes of transcriptionalactivators. ever, the anthocyanin-lessphenotype of the vp7 mutant is also The De/ gene from snapdragon was isolated by transposon associated with a general failure of seed maturation, result- tagging using the Tam2 transposable element. De/ shares ex- ing in viviparous development of the embryo. tensive sequence similarity with the R gene family of maize In snapdragon flowers, three anthocyanin regulatory genes- (Goodrich et al., 1992), indicating that a homologous regula- Delila (De/), €/ufa, and Rosea- have been identified.The first tor controls anthocyanin synthesis in both plants. two steps, CHS and CHI, show minimal regulation, but sub- A gene fragment of An2 from petunia was cloned by tag- sequent steps have an absolute requirement for the Delgene ging with a Tphl transposon, and a full-length cDNA clone was product and show quantitative regulationby €luta and Rosea. isolated (Quattrocchio, 1994). The encoded amino acid se- Mutation of the Delgene reduces the leve1 of anthocyanin bio- quence of An2 shows the highest similarity with the proteins synthetic gene transcripts of DFR (Almeida et al., 1989), F3H, encoded by the maize C7 and P/ genes. The An2 gene is ex- Candi, and 3GT (Martin et al., 1991) in the flower tube. This pressed in the corolla and tube but not in the anthers. A PCR suggests that the De/ gene product acts by transcriptionalac- approach was used to isolate a petunia homolog (jaf73) of the tivation of these structural genes. The De/ gene product also maize R genes (Quattrocchio, 1994). The encoded amino acid acts as a repressor of CHS in the flower lobe sequence of jaf73 shares extensive homology with the prod- mesophyll (Jackson et al., 1992). Both €/ufa and Rosea mu- ucts of R and De/. Expression patterns and RFLP analysis tants have decreased expression of F3H, DFR, Candi, and 3GT suggest that jaf73 corresponds to the An4 gene. (Bartlett, 1989). Transformation of tobacco and Arabidopsis with a maize Lc In petunia, mutations at four loci, An7, An2, An4, and An77, gene leads to increased pigmentation in severa1tissues (Lloyd have similar regulatory effects on transcription of at least six et al., 1992), indicating that Lc can activate anthocyanin bio- structural anthocyanin biosynthetic genes, including DFR, synthesis genes from species other than maize. Transformation ANS, An73, 3RT, anthocyanin methyltransferase (AMT), and of with De/ leads to increased anthocyanin synthesis chsJ (Quattrocchio et al., 1993; Huits et al., 1994). An7 and in flowers and vegetative tissues, whereas transformation of An77 are required for transcription of this set of structural an- tobacco increased anthocyanin biosynthesis in flowers only thocyanin biosynthetic genes in all pigmented tissues of the (Mooney et al., 1995). However, in Arabidopsis, De/ has no plant. In addition, An2 controls the transcription of these an- strong phenotypic effect. Although there are some minor differ- thocyanin genes in the flower limb and An4 controls expression ences, the control of anthocyanin biosynthesis appears to be of the same set of genes in the anthers. Enzymatic activity mediated by similar factors in different species. of 3GT is controlled byAn7, An2, and An4 (Tabak et al., 1981); An7 also controls F33’H activity associated with Hf7 (Gerats, 1985). Therefore, it appears that the petunia regulatory genes APPLICATIONS: MOLECULAR FLOWER BREEDING control expression of essentially all of the anthocyanin genes after F3H, excluding F3’H. Recent advances in , especially in gene iso- lation and gene transfer between species, have made it Cloning and Characterization of Regulatory Genes possible to alter flower color in a highly directed fashion. Genetic techniques have been used in two ways Transposon tagging has proven very useful in isolating an- to change flower color: (1) introductionof genes encoding novel thocyanin regulatory genes for which no prior information enzyme activities, and (2) inactivationof endogenous genes. existed concerning the of the gene products. Other Figure 3 shows some of the flower color changes that have regulatory genes have been isolated based on their homol- been genetically engineered in petunia. ogy to previously characterized regulatory genes. The generation of transgenic petunia plants with pelargoni- The proposed duplicated function of the R, Sn, Lc, and B din-producingflowers was the first example of the creation of loci is reflected in their sequence similarity. The R gene fam- a novel flower color by . In general, DFR ily encodes proteins with homology with the basic helix-loop- catalyzes the conversion of DHK, DHQ, and DHM to the respec- helix motif in the myc transcription activator (Consonni et al., tive leucoanthocyanidins. However, petunia DFR cannot convert 1993). C7 and P/ were also shown to be homologous (Cone DHK to leucopelargonidin, which explains the natural lack of and Burr, 1989). The C7 and PIgene products share sequence pelargonidin pigments in petunia (Forkmann and Ruhnau, similarity with the DNA binding domains from the myb on- 1987). Transformation of a DHK-accumulating petunia line with cogenes. All of the regulatory genes have been shown to affect a DFR gene (A7) from maize, which is able to convert DHK the expression of multiple anthocyanin structural genes, as to leucopelargonidin, enables the production of pelargonidin detailed in Table 2.6 and C7 fusions with the yeast GAL4 DNA by the flowers and leads to a change in flower color from pale Anthocyanin Biosynthesis 1079

Figur . Modificatioe3 f Petunino a Flower Colo Genetia vi r c Engineering. Flowers (A) to (D) are from nontransgenic plants; flowers (E) to (H) are from transgenic ones. The genotypes (where known) are shown in parentheses. (A) cv Old Glory Blue. ) Skr(B x 4SW6 3 (hflhfl, hf2hf2, htlhtt, ht2ht2). (C) VR (Hf1hf1, Hf2hf2, Htlhtl, Flfl). ) Skr(D 4x SW6 3 (hflhfl, hf2hf2, htlhtl, M2M2). Glord Ol yv c Blu ) e(E transformed wit antisensn ha geneS eCH . (F) Skr4 x SW63 transformed with a rose DFR gene. transformeR V ) (G d wit genha e that suppresses bot activityS h F3'5'FL d .Han (H) Skr4 x SW63 transformed with a sense petunia F3'5'H gene.

to brick red (Meyer et al., 1987). A similar change in flower reduced pigmentation. Increased anthocyanin accumulation colo bees rha n achieve transformatioy db petunif no a witR hDF in flowers has been achieved by cosuppression of an FLS gene genes from gerbera (Helariutta et al., 1993) and rose (Tanaka tobaccn i o (Holto t al.ne , 1993b). et al., 1995; Figure 3). CHS activity can be reduced or eliminated in plants by trans- formation with an antisense version of the CHS gene (van der FUTURE RESEARCH Kro t al.e l , 1988). Antisense suppressio f genno e expression has been shown to occur in a range of . Experiments performed at DMA Plant Technology Corporation (Oakland, Isolatio f Flavonoino d Modification Genes CA) (Napol al.t discovere ie ,th 1990o t phenomenod a )f le y o n known as sense suppression or cosuppression. Cosuppression In petunia lines with a mutation at the regulatory An1 locus, occun ca r when extra "sense" copie gena introducef e so e ar d transcriptioe th f severano l anthocyanin biosynthesis genes into an organism. Therefore, both antisense and cosuppression is strongly reduced. Kroon et al. (1994) used a differential technologies can be used to change flower color by reducing screening strateg isolato yt e cDNA clone genef so s regulated or eliminating expression of genes affecting the synthesis of by the An1 gene. The differential clones represent seven dis- flower pigments. Antisens cosuppressior eo geneS CH sf no tinct classes largese th ; t cDNA clone from each class swa has also been achieved in petunia, gerbera (Elomaa et al., designated dlfA, difC, difE, difF, difG, difH, and difl. Expres- 1993), chrysanthemum (Courtney-Gutterson et al., 1994), and genee sioth f no s correspondin eaco gt f thesho e classes rose (Elomaa and Holton, 1994) to produce flowers with displays a similar spatial, temporal, and genetic control. Further 1080 The Plant Cell characterization of these clones indicates that difA encodes regulatory genes may lead to useful increases in anthocyanin ANS, difD encodes CHS, difE encodes an AMT, difF encodes synthesis in important cut-flower species. It should also be pos- an anthocyanidin glucosyltransferase, difG encodes 3Wr, and sible to modify color using similar strategies. Many difl encodes a GST (R.E. Koes, unpublished results). The re- anthocyanin biosyntheticgenes have been isolated from maining classes may represent as-yet-unidentified clones of (Sparvoli et al., 1994) and apple (Davies, 1993; Podivinsky et ant hocyanin biosynthetic genes. al., 1993). The availability of genetically defined mutants and genetic Blue-flowering varieties are missing from a number of import- maps enables the identification of gene function in two ways- ant ornamental plants, including carnations, chrysanthemums, by genetic complementation and gene suppression. Such and roses. None of these plants is capable of producing blue methods can be used even when the gene product cannot be delphinidin pigments, presumablydue to the absence of a gene assayed directly. The first of these methods relies on trans- encoding F33‘H. Transformation of these species with an forming a genetically defined mutant with the gene of interest F3’5’H gene should overcome this limitation and allow the and relies on expression of the introduced gene to overcome production of delphinidin derivatives, thereby increasing the the effects of a genetic lesion. The second approach utilizes possibility of producing blue flowers. Other genes that may the ability of an introduced gene to suppress expression of be required for creation of blue flowers have also been recently an endogenous gene homolog. The effect on phenotype may isolated (Holton and Tanaka, 1994). then be used to predict the function of the gene product (re- verse genetics). Gene mapping, complementation, and reverse genetics have played an important part in identifying the DFR REFERENCES (Beld et al., 1989; Huits et al., 1994), F33’H (Holton et al., 1993a), FLS (Holton et al., 1993b), and 3RT (Brugliera et al., 1994) genes from petunia. Application of similar methods may Almeida, J., Carpenter, R., Robbins, T.P., Martin, C., and Coen, enable identification of function of the petunia dif genes. The E.S. (1989). Genetic interactions underlying flower color patterns development of a straightforward method for cloning genes in . Genes Dev. 3, 1758-1767. tagged by the multicopy Tphl transposon (Souer et al., 1995) Bartlett, N.J.R. (1989). 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