Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9033-9038, August 1996 Evolution Duplication and functional divergence in the chalcone synthase gene family of Asteraceae: Evolution with substrate change and catalytic simplification (anthocyanin/flavonoid genetics/gene phylogeny/secondary metabolism/stilbene synthase) YRJO HELARIUTA*t*, MiKA KOTILAINEN*, PAULA ELOMAA*, NISSE KALKKINEN*, KARE BREMER§, TEEMU H. TEERI*, AND VICTOR A. ALBERTt *Institute of Biotechnology, University of Helsinki, P.O. Box 45, FIN-00014 Helsinki, Finland; *The New York Botanical Garden, Bronx, NY 10458-5126; and §Department of Systematic Botany, Uppsala University, Villavagen 6, S-752 36 Uppsala, Sweden Communicated by Michael T Clegg, University of California, Riverside, CA, May 20, 1996 (received for review November 29, 1995) ABSTRACT Plant-specific polyketide synthase genes con- SS genes at the level of deduced amino acid sequence. Its stitute a gene superfamily, including universal chalcone syn- expression pattern at both organ and cellular levels is not thase [CHS; malonyl-CoA:4-coumaroyl-CoA malonyltrans- correlated with anthocyanin pigmentation, for which CHS ferase (cyclizing) (EC 2.3.1.74)] genes, sporadically distrib- provides the first committed biosynthetic step. Furthermore, uted stilbene synthase (SS) genes, and atypical, as-yet- the catalytic properties of the corresponding enzyme differ uncharacterized CHS-like genes. We have recently isolated from CHS and SS, although the GCHS2 catalytic reaction and from Gerbera hybrida (Asteraceae) an unusual CHS-like gene, its role in vivo are not yet completely understood. GCHS2, which codes for an enzyme with structural and In this study we show that the GCHS2-like genes in Aster- enzymatic properties as well as ontogenetic distribution dis- aceae constitute a gene family, whose corresponding amino tinct from both CHS and SS. Here, we show that the GCHS2- acid sequences share some consensus residues. Phylogenetic like function is encoded in the Gerbera genome by a family of parsimony analysis of (i) the GCHS2 nucleotide sequence, (ii) at least three transcriptionally active genes. Conservation further GCHS2-like genes screened from a Gerbera cDNA within the GCHS2 family was exploited with selective PCR to library, (iii) gene fragments amplified from various Asteraceae study the occurrence ofGCHS2-like genes in other Asteraceae. using GCHS2 family-specific primers, and (iv) CHS superfam- Parsimony analysis of the amplified sequences together with ily genes isolated from other angiosperms of subclass Asteri- CHS-like genes isolated from other taxa of angiosperm sub- dae indicates that GCHS2 probably evolved from CHS via a class Asteridae suggests that GCHS2 has evolved from CHS single gene duplication event that occurred before the diver- via a gene duplication event that occurred before the diversifi- sification of Asteraceae. Structural and functional variation cation of the Asteraceae. Enzyme activity analysis of proteins observed among the members of the GCHS2 gene family produced in vito indicates that the GCHS2 reaction is a non-SS suggests that subsequent diversification has also taken place. A variant of the CHS reaction, with both different substrate comparison of the catalytic properties of GCHS2 to parsley specificity (to benzoyl-CoA) and a truncated catalytic profile. CHS shows that both substrate specificity and progressivity of Together with the recent results of Durbin et al. [Durbin, M. L., catalytic reaction steps have changed during GCHS2 evolution. Learn, G. H., Jr., Huttley, G. A. & Clegg, M. T. (1995) Proc. Natl. Acad. Sci. USA 92, 3338-3342], our study confirms a gene MATERIALS AND METHODS duplication-based model that explains how various related func- tions have arisen from CHS during plant evolution. Plant Material. G. hybrida is a hybrid of two species (G. jamessonii and G. viridifolia) belonging to the tribe Mutisieae Plant-specific polyketide synthase genes constitute a gene (Asteraceae subfamily Cichorioideae; ref. 16). We chose for superfamily. Genes encoding chalcone synthase [CHS; malo- analysis Leibnitzia (a closely related genus) and Onoseris (a nyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing) more distantly related genus) from Mutisieae, Taraxacum (EC 2.3.1.74)] and flavonoids, their corresponding reaction (tribe Lactuceae) from Cichorioideae, and Dahlia (tribe He- products, seem to be universally distributed in plants (1-3). liantheae) from subfamily Asteroideae (16). CHS genes have been isolated from a wide taxonomic spec- Mature plants of G. hybrida var. Regina (obtained from trum from nonflowering seed plants to dicots and monocots (4, Terra Nigra, De Kwakel, The Netherlands) and seedlings of 5). Stilbene synthase (SS) genes coding for enzymes with a Leibnitzia anandria and Onoseris sagittatis were grown under related activity to CHS have been isolated from species that standard greenhouse conditions. Leaf material of Dahlia sp. accumulate stilbene phytoalexin (5-8). In addition to SS genes, and Taraxacum sp. (collected from gardens in Helsinki) were other structurally unusual CHS-like genes or gene products also used as sources of DNA. have been reported (9-12). Recently, Tropf et al. (13) have Isolation of GCHS17 and GCHS26 from a Genomic A provided evidence that SS genes have evolved from CHS genes Library. Nuclear DNA from Gerbera leaves was prepared by via independent gene duplication events several times during the method ofJofuku and Goldberg (17). A genomic library seed plant evolution. Durbin et al. (14) have demonstrated an was constructed with LambdaGEM-11 vector (Promega) and analogous mechanism leading to the evolution of structurally was screened using GCHS1-3 cDNA clones as probes (15, 18). unusual genes in the genus Ipomoea. Short fragments (181 bp) from the clones were amplified using We have recently isolated a structurally unusual CHS-like primers designed from the conserved region of the CHS genes gene, GCHS2, from Gerbera hybrida (Asteraceae; ref. 15). GCHS2 is '70% identical to typical CHS genes and the related Abbreviations: CHS, chalcone synthase; SS, stilbene synthase; 2-ME, 2-mercaptoethanol. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge deposited in the GenBank data base (accession nos. X91339-X91345). payment. This article must therefore be hereby marked "advertisement" inI tTo whom reprint requests should be sent at present address: Depart- accordance with 18 U.S.C. §1734 solely to indicate this fact. ment of Biology, New York University, New York, NY 10003. 9033 Downloaded by guest on October 1, 2021 9034 Evolution: Helariutta et al. Proc. Natl. Acad. Sci. USA 93 (1996) (15), and their sequences were used for classification and a linear gradient of methanol (0-60% in 40 min) in H20. The designation of the subcloning strategy. Both strands of the eluent was monitored at 220 nm using a Waters 990 + diode GCHS17 and GCHS26 clones were determined using the array detector. For spectral information, data from 200 to 400 deletion strategy both manually and with an automated se- nm was collected at a resolution of 1.4 nm. Furthermore, the quence determination system (ALF; Pharmacia; ref. 18). radioactivity of each fraction was measured by scintillation Sequence alignments were done using the CLUSTAL program of counter, and the peak fractions were analyzed by TLC. the PC/gene package (IntelliGenetics). Amplification of GCHS2-Like Sequences from Dahlia, Leib- RESULTS nitzia, Onoseris, and Taraxacum. Partially degenerate (inosine containing) primers including restriction enzyme sites were Isolation and Characterization of GCHS17 and GCHS26 used to amplify fragments from various asteraceous species. Clones. Among 3.4 million plaque-forming units screened, 5'-TGCACTCGAGTGA(A/C/G/T)AA(A/G)ACAGC(A/ eight A clones hybridizing to GCHS1-3 cDNA probes were C/G/T)ATAAA(A/G)AA-3' and 5'-ACTGGGATCCACC- isolated. According to the classification based on the ampli- (A/C/G/T)GGGTG(A/C/G/T)ACCATCCA(A/G)AA-3' fication of a 181-bp fragment from a conserved region (15), the corresponding to peptides C(D/E)KTAIKK and FWMVH- clones were deduced to represent four different sequences. PGG were used for specific amplification of GCHS2-like Two novel genes, GCHS17 and GCHS26, having a continuous genes. For amplification, plant DNA was extracted by the reading frame (except for an intron) showed similarity to the method of Dellaporta et al. (19) and purified in isopycnic CsCl GCHS2 gene. The exon/intron boundaries as well as the start gradients (18). PCR was performed using a "touch-down" and stop codons of the reading frames were deduced based on the strategy: 10 times (940 75 s; 500 5 min adding -1° per cycle, general similarity of CHS enzymes at the amino acid sequence slope +220, 10 per 10 s; 720 5 min) followed by 31 times (940 level and by comparison to the GCHS2 cDNA sequence. 75 s; 530 2 min; 720 5 min). To verify that the amplification GCHS17 is a truncated clone missing approximately the first 50 products did not contain any chimeric artifacts due to recom- codons. It has a 28-bp first exon, a 1638-bp intron, and a 1016-bp bination among related gene family members (20), a second, second exon. GCHS26 harbors the entire reading frame: a 196-bp independent amplification was performed. After PCR, frag- first exon, a 483-bp intron, and a 1016-bp second exon. ments were separated from primers by gel electrophoresis, We compared the deduced amino acid sequences of purified from agarose and digested with the corresponding GCHS17 and GCHS26 to each other and to the other astera- restriction enzyme pair for cloning into plasmid pOK12 (21) ceous CHS superfamily sequences. The sequences form two for sequence analysis of both strands. subgroups: usual CHS-like sequences (with 88-93% intra- Phylogenetic Analysis of CHS-Like Gene Sequences. Nucle- group identity) and GCHS2-like sequences (83-84% intra- otide sequences representing 20 sequences (corresponding to group identity). The identity between the two groups is the amino acid sequence from Rll to S389 in GCHS1) were 73-77%.