Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci The R2R3 transcription factor HlMYB8 and its role in flavonoid biosynthesis in hop (Humulus lupulus L.) Tomáš Kocábek a, ⁎ , Ajay Kumar Mishra a , Jaroslav Matoušek a , Josef Patzak b , Anna Lomnická a,c , Mudra Khare a , Karel Krofta b a Biology Centre of the Czech Academy of Sciences v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05 České Budějovice, Czech Republic b Hop Research Institute Co. Ltd., Kadaňská 2525, 438 46 Žatec, Czech Republic c University of South Bohemia in České Budějovice, Faculty of Science, Branišovská 1760, 370 05 České Budějovice, Czech Republic ARTICLE INFO Keywords: Bitter acids Chalcone synthase Humulus lupulus Flavonoids R2R3 myb RNA sequencing ABSTRACT Hop is an important source of medicinally valuable secondary metabolites including bioactive prenylated chalcones. To gain in-depth knowledge of the regulatory mechanisms of hop flavonoids biosynthesis, full-length cDNA of HlMyb8 transcription factor gene was isolated from lupulin glands. The deduced amino acid sequence of HlMyb8 showed high similarity to a flavonol-specific regulator of phenylpropanoid biosynthesis AtMYB12 from Arabidopsis thaliana. Transient expression studies and qRT-PCR analysis of transgenic hop plants overexpressing HlMyb8 revealed that HlMYB8 activates expression of chalcone synthase HlCHS_H1 as well as other structural genes from the flavonoid pathway branch leading to the production of flavonols (F3H, F’3H, FLS) but not pre- nylflavonoids (PT1, OMT1) or bitter acids (VPS, PT1). HlMyb8 could cross-activate Arabidopsis flavonol-specific genes but to a much lesser extent than AtMyb12. Reciprocally, AtMyb12 could cross-activate hop flavonol- specific genes. Transcriptome sequence analysis of hop leaf tissue overexpressing HlMyb8 confirmed the mod- ulation of several other genes related to flavonoid biosynthesis pathways (PAL, 4CL, ANR, DFR, LDOX). Analysis of metabolites in hop female cones confirmed that overexpression of HlMyb8 does not increase prenylflavonoid or bitter acids content in lupulin glands. It follows from our results that HlMYB8 plays role in a competition between flavonol and prenylflavonoid or bitter acid pathways by diverting the flux of CHS_H1 gene product and thus, may influence the level of these metabolites in hop lupulin. 1. Introduction Female hop (Humulus lupulus L.) cones are widely used in brewing industry to provide the beer bitterness and aroma and accredited to have potential pharmaceutical or biomedical applications. The com- mercial value of cones lies in lupulin glands, which contain several biologically active components, including essential oils, bitter acids, prenylflavonoids, having many different beneficial effects on human health [1–3]. Xanthohumol (XN) is the most abundant prenylflavonoid with its overall concentration in the cone dry mass varying from 0.2 and 1.1% depending on the genotype and the environmental conditions [4]. The other flavonoids, isoxanthohumol (IX) and 8-prenylnaringenin (8- PN) are present in ten to hundred-fold lower concentrations in comparison to XN level [2]. One of the recent efforts of hop breeders is to gain new hop cultivars with elevated levels of prenylflavonoids but met with limited success [5,6]. Flavonoids are generally synthesized via phenylpropanoid pathway which constitutes most detailed studied biosynthetic route among sec- ondary metabolism in plants [7–9]. The phenylalanine serves as the precursor molecule for flavonoid synthesis, which is converted to cin- namic acid by phenylalanine ammonia lyase (PAL) (Fig. 1). One mo- lecule of CoA-ester of cinnamic acid and three molecules of malonyl- CoA are condensed into the naringenin chalcone. This reaction is cat- alyzed by the enzyme chalcone synthase (CHS). The chalcone is iso- merised to a flavanone by chalcone flavanone isomerase enzyme (CHI). From these central intermediates, the pathway splits into different https://doi.org/10.1016/j.plantsci.2018.01.004 Received 3 November 2017; Received in revised form 12 January 2018; Accepted 14 January 2018 ⁎ Corresponding author. E-mail address: kocabek@umbr.cas.cz (T. Kocábek). Abbreviations: 8PN, 8-prenylnaringenin; ANR, anthocyanin reductase; BA, bitter acids; CHI, chalcone isomerase; CHS, chalcone synthase; DEG, differentially expressed genes; DFR, dihydroflavonol reductase; F3′H, flavonoid 3-hydroxylase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, gene on- tology; GUS, β-glucuronidase; IX, isoxanthohumol; LDOX, leucoanthocyanidin dioxygenase; MBW, a general MYB-bHLH-WDR transcription factor complex; MU, 4-methylumbelliferone; PA, proanthocyanidin; PAL, phenylalanine ammonia lyase; RNA-seq, RNA sequencing utilizing high-throughput deep sequencing of cDNA; qRT-PCR, quantitative real-time polymerase chain reaction; XN, Xanthohumol Plant Science 269 (2018) 32–46 0168-9452/ © 2018 Elsevier B.V. All rights reserved. T