Abstract
BackgroundLupulin 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.ResultsHomologues 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.ConclusionsThis 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.
Highlights
Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities
This gene is coding for a crucial hop “true” chalcone synthase endowed with the ability to produce naringenin chalcone with a high catalytical rate [16] and, it is likely playing an important role in the biosynthetic pathway leading to the accumulation of prenylated chalcones in lupulin glands, including xanthohumol and desmethylxanthohumol [14,16]
In the present study new transcription factor (TF) homologues corresponding to the plant R2R3Myb, bHLH and WDR families were cloned using a cDNA library derived from hop (Humulus lupulus L.) lupulin gland tissue
Summary
Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities. Hop has been known for a long time specific target tissues such as the liver [8] Another lupulinderived prenylflavonoid, 8-prenylnaringenin (8-PN), is one of the most potent phytoestrogens known to date [9,10]. The biosynthesis of naringenin chalcone in hop cones as a prenylflavonoid precursor is attributed to the gene encoding the CHS_H1 protein having a so-called “true” chalcone synthase (EC 2.3.1.74) activity, by which it efficiently catalyzes the production of naringenin chalcone by condensation of three malonyl-CoA units and p-coumaroyl-CoA [14,15]. The VPS-like homologue CHS4 shows high expression in lupulin glands, the enzyme does not catalyze the formation of naringenin chalcone and its function is currently still unknown [15,18]
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