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Logo of bmcpsBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Plant Biology
 
BMC Plant Biol. 2012; 12: 27.
Published online Feb 20, 2012. doi:  10.1186/1471-2229-12-27
PMCID: PMC3340318
Combinatorial analysis of lupulin gland transcription factors from R2R3Myb, bHLH and WDR families indicates a complex regulation of chs_H1 genes essential for prenylflavonoid biosynthesis in hop (Humulus Lupulus L.)
Jaroslav Matoušek,corresponding author1,3 Tomáš Kocábek,1 Josef Patzak,2 Zoltán Füssy,1,3 Jitka Procházková,1 and Arne Heyerick4
1Biology Centre ASCR v.v.i, Institute of Plant Molecular Biology, Branišovská 31, 370 05 České Budějovice, Czech Republic
2Hop Research Institute, Co. Ltd, Kadaňská 2525, 438 46 Žatec, Czech Republic
3Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic
4Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
corresponding authorCorresponding author.
Jaroslav Matoušek: jmat/at/umbr.cas.cz; Tomáš Kocábek: kocabek/at/umbr.cas.cz; Josef Patzak: j.patzak/at/telecom.cz; Zoltán Füssy: zoltan.fussy/at/gmail.com; Jitka Procházková: prochazkova/at/umbr.cas.cz; Arne Heyerick: Arne.Heyerick/at/UGent.be
Received July 21, 2011; Accepted February 20, 2012.
Abstract
Background
Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities. The detailed analysis of the transcription factor (TF)-mediated regulation of the oligofamily of one of the key enzymes, i.e., chalcone synthase CHS_H1 that efficiently catalyzes the production of naringenin chalcone, a direct precursor of prenylflavonoids in hop, constitutes an important part of the dissection of the biosynthetic pathways leading to the accumulation of these compounds.
Results
Homologues of flavonoid-regulating TFs HlMyb2 (M2), HlbHLH2 (B2) and HlWDR1 (W1) from hop were cloned using a lupulin gland-specific cDNA library from the hop variety Osvald's 72. Using a "combinatorial" transient GUS expression system it was shown that these unique lupulin-gland-associated TFs significantly activated the promoter (P) of chs_H1 in ternary combinations of B2, W1 and either M2 or the previously characterized HlMyb3 (M3). The promoter activation was strongly dependent on the Myb-P binding box TCCTACC having a core sequence CCWACC positioned on its 5' end region and it seems that the complexity of the promoter plays an important role. M2B2W1-mediated activation significantly exceeded the strength of expression of native chs_H1 gene driven by the 35S promoter of CaMV, while M3B2W1 resulted in 30% of the 35S:chs_H1 expression level, as quantified by real-time PCR. Another newly cloned hop TF, HlMyb7, containing a transcriptional repressor-like motif pdLNLD/ELxiG/S (PDLNLELRIS), was identified as an efficient inhibitor of chs_H1-activating TFs. Comparative analyses of hop and A. thaliana TFs revealed a complex activation of Pchs_H1 and Pchs4 in combinatorial or independent manners.
Conclusions
This study on the sequences and functions of various lupulin gland-specific transcription factors provides insight into the complex character of the regulation of the chs_H1 gene that depends on variable activation by combinations of R2R3Myb, bHLH and WDR TF homologues and inhibition by a Myb repressor.
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