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 LupulusL.)

Abstract

AbstractBackgroundLupulin 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 TFsHlMyb2(M2),HlbHLH2(B2) andHlWDR1(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) ofchs_H1 in ternary combinations of B2, W1 and either M2 or the previously characterizedHlMyb3 (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 nativechs_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 ofchs_H1-activating TFs. Comparative analyses of hop andA. thalianaTFs 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 thechs_H1 gene that depends on variable activation by combinations of R2R3Myb, bHLH and WDR TF homologues and inhibition by a Myb repressor.