Journal Article

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.)

Patzak, Josef, Kocábek, Tomáš, Procházková, Jitka, Füssy, Zoltán, Heyerick, Arne, Matoušek, Jaroslav

2012

Overview

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 Hl Myb3 (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, Hl Myb7, 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 P chs _H1 and P chs 4 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.

• Publisher: BioMed Central,Springer Nature B.V
• Subject: Agriculture / Binding sites / Biochemistry and physiology / Biomedical and Life Sciences / Biosynthesis / Cloning / Enzymes / Flavonoids - biosynthesis / Genes / Humulus - genetics / Humulus - metabolism / Humulus lupulus / Life Sciences / Metabolites / Plant Proteins - genetics / Plant Proteins - metabolism / Plant Sciences / Regulation / Research Article / Software / Transcription Factors - genetics / Transcription Factors - metabolism / Tree Biology