R EPORTS dase-catalyzed oxidation of chalcones (Fig. Aureusidin Synthase: A 1A, 2) yields a 2-(␣-hydroxybenzyl)couma- ranone derivative, a hydrated form of aurone, Polyphenol Oxidase Homolog which is then dehydrated (Fig. 1C, arrows a and b) (5). In this study, we establish that aurone biosynthesis is catalyzed by a ho- Responsible for Flower molog of plant polyphenol oxidase (PPO). PPOs ubiquitously occur in higher plants and Coloration are responsible for the browning of plant tissues exposed to air. However, the physio- T. Nakayama,1* K. Yonekura-Sakakibara,2* T. Sato,1 S. Kikuchi,1 2 2 3 2 2 logical function of PPO in plants remains to Y. Fukui, M. Fukuchi-Mizutani, T. Ueda, M. Nakao, Y. Tanaka, be established (6). This report demonstrates 2 1 T. Kusumi, T. Nishino the participation of a PPO homolog in flower coloration. Aurones are plant flavonoids that provide yellow color to the flowers of some The yellow coloration of snapdragon flow- popular ornamental plants, such as snapdragon and cosmos. In this study, we ers is mainly provided by the glucosides of have identified an enzyme responsible for the synthesis of aurone from chal- aurones (aureusidin and bracteatin). Aureusidin cones in the yellow snapdragon flower. The enzyme (aureusidin synthase) is a can be produced from either 2Ј,4Ј,6Ј,4-tetrahy- 39-kilodalton, copper-containing glycoprotein catalyzing the hydroxylation droxychalcone (THC) or 2Ј,4Ј,6Ј,3,4-pentahy- and/or oxidative cyclization of the precursor chalcones, 2Ј,4Ј,6Ј,4-tetrahy- droxychalcone (PHC), whereas bracteatin aris- droxychalcone and 2Ј,4Ј,6Ј,3,4-pentahydroxychalcone. The complementary es from PHC (7) (Fig. 1C, arrows e, f, and g). DNA encoding aureusidin synthase is expressed in the petals of aurone-con- A single enzyme, which should not be a perox- taining varieties. DNA sequence analysis revealed that aureusidin synthase idase, catalyzes dual chemical transformations, belongs to the plant polyphenol oxidase family, providing an unequivocal i.e., hydroxylation and oxidative cyclization (2Ј, example of the function of the polyphenol oxidase homolog in plants, i.e., flower ␣-dehydrogenation), of THC and PHC into au- coloration. reusidin and bracteatin, respectively (7). There- Most floral colors present in nature arise from snapdragon (Antirrhinum majus) (Fig. 1B). 1Department of Biomolecular Engineering, Graduate flavonoids (1). Genetic and biochemical Although the aurone biosynthetic gene(s) is School of Engineering, Tohoku University, Aoba-yama knowledge of flavonoid biosynthesis in an attractive tool to engineer yellow flowers, 07, Sendai 980-8579, Japan. 2Institute for Fundamen- plants has provided a basis for controlling the biochemical and genetic details of aurone tal Research, Suntory Ltd., Wakayamadai 1-1-1, Shi- mamoto-cho, Mishima-gun, Osaka 618-8503, Japan. floral color through genetic engineering ap- biosynthesis have remained unclear (4). One 3Kobe Gakuin University, Kobe 651-2180, Japan. proaches (2). Aurones (Fig. 1A, 1) (3), a class mechanism proposed for aurone biosynthesis *To whom correspondence should be addressed. E- of plant flavonoids, confer bright yellow col- in plants (soy seedling) is a two-step path- mail: [email protected]; yskeiko@ or to flowers such as cosmos, coreopsis, and way, in which an H2O2-dependent peroxi- postman.riken.go.jp Fig. 1. (A) General structures of aurones, 1, and chalcones, 2. The positional numbering in the aurones and chalcones is different. A and B shown in the aromatic rings indicate A- and B-rings in the flavonoid structures, respectively. (B) Photograph of yellow snapdragon flowers. (C) Possible pathways for aurone biosynthesis from chalcones in the snapdragon flowers. Dotted arrows indicate the putative pathway for aurone biosynthesis according to Rathmell et al. (5). Thick arrows indicate the pathway for aurone biosynthesis identified in this study. www.sciencemag.org SCIENCE VOL 290 10 NOVEMBER 2000 1163 R EPORTS fore, we attempted to purify the enzyme respon- num majus (cv. Yellow Butterfly) that had the purified aureusidin synthase (39 kD). sible for aurone biosynthesis, which we call been prepared as described previously (11). It However, such a difference is consistently aureusidin synthase, from crude extracts of yel- contained an open reading frame of 1686 explained in terms of the proposed biogenesis low snapdragon flowers. base pairs encoding 562 amino acids. The of known plant PPOs; plant PPOs are gener- To purify a sufficient amount of the en- deduced amino acid sequence showed high ally synthesized as an ϳ65-kD precursor pro- zyme, we used 32 kg of snapdragon buds (8) similarity to plant PPOs such as those from tein (15), which is subsequently processed as starting material. Moreover, the addition of apple fruit (identity, 51%), grape berry into a mature PPO of ϳ40 kD after removal THC to the buffers used during purification (47%), and potato tuber (39%) (Fig. 3) (13, of a 10-kD NH2-terminal peptide containing somewhat stabilized enzyme activity. Finally, 14). The predicted Mr (64 kD) of the AmAS1 transit sequences (15) and a 15-kD COOH- the enzyme (90 ␮g) was purified 107-fold to product was substantially larger than that of terminal peptide of unknown function (16). homogeneity by a combination of nine puri- fication steps in an activity yield of 1.4% (Fig. 2) (8). The mean value of the final specific activities under the standard assay conditions with THC as a substrate was 578 units mgϪ1 (9). However, the specific activ- ity of the enzyme and the actual degree of purification might have been considerably higher, given the instability of this enzyme during the purification. The relative molecu- lar mass (Mr) of the purified aureusidin syn- thase was estimated to be 40 kD by gel permeation fast protein liquid chromatogra- phy on a Superdex 200HR column. The sub- unit Mr of the enzyme was estimated to be 39 kD by SDS–polyacrylamide gel electrophore- sis (SDS-PAGE) (Fig. 2). These results show that the enzyme was monomeric. The aureu- sidin synthase band in the SDS-PAGE gels was positive to sugar staining (10). In addi- tion, the aureusidin synthase was adsorbed on a ConA Sepharose column and was specifi- cally eluted with methyl ␣-glucoside, indicat- ing that the enzyme is a glycoprotein. We then determined the amino acid se- quences of peptide fragments obtained by lysyl-endopeptidase digestion of the purified synthase essentially as described (11). The sequences coincided with the deduced amino acid sequences of a clone that was specifical- ly expressed in the aurone-containing petals, as obtained through a subtractive hybridiza- tion approach (12). Using this clone as a probe, we isolated a cDNA (termed AmAS1) that encodes the precursor of aureusidin syn- thase from a petal cDNA library of Antirrhi- Fig. 2. SDS-PAGE of purified aureusidin synthase. The purified enzyme (right lane) (8) was simultaneously subjected to electro- phoresis with standard proteins (left lane). Molecular sizes (in kD) Fig. 3. Alignment of the deduced amino acid sequence of AmAS1 protein with those of plant PPOs. are indicated by ar- Amino acid residues identical to that of AmAS1 are shown in red. The dashed underline below the rows. AmAS1 sequence indicates the identification of purified peptides derived from the purified aureusidin synthase. The histidine ligands for the active-site binuclear Cu center, identified in potato PPO by x-ray crystallography (17), are shown with black circles. Putative Cu-binding domains (A and B) of AmAS1 are underlined (shown as a and b, respectively). The NH2-terminal processing sites identified in the PPO sequences of grape and potato and the COOH-terminal processing site identified in that of grape are indicated by vertical lines in the sequence. Sequences of known PPOs representing the “n region” and the “thylakoid transfer domain” are boxed and indicated by I and II, respectively. The nucleotide sequence of AmAS1 has been submitted to the DNA Data Bank of Japan under the accession number AB044884. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 1164 10 NOVEMBER 2000 VOL 290 SCIENCE www.sciencemag.org R EPORTS The processing sites in the sequence of the product, as follows, with an optimum pH of To confirm the role of the AmAS1 gene AmAS1 protein remain to be determined be- 5.4: in flower coloration, we analyzed the spa- cause the NH -terminus of the purified au- tial and temporal expressions of the AmAS1 2 THC ϩ O 3 aureusidin ϩ H O reusidin synthase was blocked. The deduced 2 2 gene in the snapdragon plants (Fig. 4). amino acid sequence of the AmAS1 protein The rate of aureusidin formation from THC Northern blot analysis revealed that AmAS1 contained two putative Cu-binding domains was greatly enhanced by H2O2, which should was expressed in the aurone-accumulating that are conserved in those sequences of the act as an enzyme activator. This observation petals. More AmAS1 transcripts were found plant PPOs that are binuclear Cu enzymes is consistent with the nature of PPO catalysis, in the petals of yellow varieties than in the (17). Atomic absorption spectrophotometric where the monophenol monooxygenase ac- petals of pink varieties, which contain analysis of the purified aureusidin synthase tivity of PPO is activated by H2O2 (19). PHC small amounts of aurones; and no tran- allowed us to confirm that it was indeed a served as a much better substrate than THC scripts were detectable in the aurone-lack- binuclear Cu enzyme (18). (relative activity, 2210%) for the purified ing petals of white, pink, and red varieties In accordance with previous observa- aureusidin synthase to produce aureusidin (Fig.