Sequence Analysis of a “True” Chalcone Synthase (chs_H1) Oligofamily from hop (Humulus lupulus L.) and PAP1 Activation of chs_H1 in Heterologous Systems JAROSLAV MATOUS ˇ EK, †,‡ LUKA Ä S ˇ VRBA, † JOSEF S ˇ KOPEK, † LIDMILA ORCTOVA Ä , † KAREL PES ˇ INA, † ARNE HEYERICK, # DAVID BAULCOMBE, § AND DENIS DE KEUKELEIRE* ,# Biological Centre AS CR, Institute of Plant Molecular Biology, Branis ˇovska ´ 31, 37005 C ˇ eske ´ Bude ˇjovice, Czech Republic, Faculty of Biological Sciences, University of South Bohemia, Branis ˇovska ´ 31, 37005 C ˇ eske ´ Bude ˇjovice, Czech Republic, Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University (UGent), Harelbekestraat 72, B-9000 Ghent, Belgium, and The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom Screening of a cDNA library of the hop cv. Osvald’s 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for “true” chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the “native” chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome. KEYWORDS: Chalcone synthases; transcriptional factors; secondary metabolites; Humulus lupulus; hop cDNA library; Petunia hybrida; Nicotiana benthamiana; plant transformation INTRODUCTION Recent developments in plant biotechnology are based on the knowledge of functional genomics that employ transcriptomics, proteomics, and developing metabolomics to understand the function and regulation of plant genes (for reviews, see 1, 2). The glandular trichomes (lupulin glands) in hop (Humulus lupulus L.) form a specific part of the hop metabolome having a relatively stable biochemical composition that can be useful for characterization of hop cultivars and genotypes (3). This stability suggests the possibility of efficiently manipulating the lupulin metabolome by changing structural and regulatory genes using molecular genetic techniques and “Myb” biotechnology approaches using Myb transcription factors as tools for meta- bolic engineering in plants (4). Several compounds in the lupulin metabolome are of par- ticular interest in view of their highly interesting medicinal properties (5, 6). In this respect, current research is focused on prenylated flavonoids that constitute a subclass of polyphenols. * To whom correspondence should be addressed. Tel: +3292648055. Fax: +3292648192. E-mail: Denis.DeKeukeleire@UGent.be. † Institute of Plant Molecular Biology. ‡ University of South Bohemia. # Ghent University (UGent). § John Innes Centre. 7606 J. Agric. Food Chem. 2006, 54, 7606-7615 10.1021/jf061785g CCC: $33.50 © 2006 American Chemical Society Published on Web 09/06/2006