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Background: Hop (Humulus lupulus L.) bitter acids are valuable metabolites for the brewing industry. They are biosynthesized and accumulate in glandular trichomes of the female inflorescence (hop cone). The content of alpha bitter acids, such as humulones, in hop cones can differentiate aromatic from bitter hop cultivars. These contents are subject to genetic and environmental control but significantly correlate with the number and size of glandular trichomes (lupulin glands)., Results: We evaluated the expression levels of 37 genes involved in bitter acid biosynthesis and morphological and developmental differentiation of glandular trichomes to identify key regulatory factors involved in bitter acid content differences. For bitter acid biosynthesis genes, upregulation of humulone synthase genes, which are important for the biosynthesis of alpha bitter acids in lupulin glands, could explain the higher accumulation of alpha bitter acids in bitter hops. Several transcription factors, including HlETC1, HlMYB61 and HlMYB5 from the MYB family, as well as HlGLABRA2, HlCYCB2-4, HlZFP8 and HlYABBY1, were also more highly expressed in the bitter hop cultivars; therefore, these factors may be important for the higher density of lupulin glands also seen in the bitter hop cultivars., Conclusions: Gene expression analyses enabled us to investigate the differences between aromatic and bitter hops. This study confirmed that the bitter acid content in glandular trichomes (lupulin glands) is dependent on the last step of alpha bitter acid biosynthesis and glandular trichome density., (© 2021. The Author(s).)

Background: The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression., Results: The transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines., Conclusions: Taken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.

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 prenylflavonoids (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 modulation 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

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.

Hop (Humulus lupulus L.), the essential source of beer flavor is of interest from a medicinal perspective in view of its high content in health-beneficial terpenophenolics including prenylflavonoids. The dissection of biosynthetic pathway(s) of these compounds in lupulin glands, as well as its regulation by transcription factors (TFs), is important for efficient biotechnological manipulation of the hop metabolome. TFs of the bZIP class were preselected from the hop transcriptome using a cDNA-AFLP approach and cloned from a cDNA library based on glandular tissue-enriched hop cones. The cloned TFs HlbZIP1A and HlbZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively, and both are similar to the group A3 bZIP TFs according to the composition of characteristic domains. While HlbZIP1A is rather neutral (pI 6.42), HlbZIP2 is strongly basic (pI 8.51). A truncated variant of HlbZIP1 (HlbZIP1B), which is strongly basic but lacks the leucine zipper domain, has also been cloned from hop. Similar to the previously cloned HlMyb3 from hop, both bZIP TFs show a highly specific expression in lupulin glands, although low expression was observed also in other tissues including roots and immature pollen. Comparative functional analyses of HlbZip1A, HlbZip2, and subvariants of HlMyb3 were performed in a transient expression system using Nicotiana benthamiana leaf coinfiltration with Agrobacterium tumefaciens strains bearing hop TFs and selected promoters fused to the GUS reference gene. Both hop bZIP TFs and HlMyb3 mainly activated the promoters of chalcone synthase chs_H1 and the newly cloned O-methyl transferase 1 genes, while the response of the valerophenone synthase promoter to the cloned hop TFs was very low. These analyses also showed that the cloned bZIP TFs are not strictly G-box-specific. HPLC analysis of secondary metabolites in infiltrated Petunia hybrida showed that both hop bZIP TFs interfere with the accumulation and the composition of flavonol glycosides, phenolic acids, and anthocyanins, suggesting the possibility of coregulating flavonoid biosynthetic pathways in hop glandular tissue.

A hop-specific cDNA library from glandular tissue-enriched hop cones was screened for Myb transcription factors. cDNA encoding for R2R3 Myb, designated HlMyb3, was cloned and characterized. According to the amino acid (aa) sequence, HlMyb3 shows the highest homology to GhMyb5 from cotton and is unrelated to the previously characterized HlMyb1 from the hop. Southern blot analyses indicated that HlMyb3 is a unique gene, which was detected in various Humulus lupulus cultivars, but not in Humulus japonicus. Reverse transcription and real-time PCR revealed the highest levels of HlMyb3 mRNA in hop cones at a late stage of maturation and in colored petiole epidermis, while the lowest levels were observed in hop flowers. Two alternative open reading frames starting in the N-terminal domain of HlMyb3, encoding for proteins having 269 and 265 amino acids with apparent molecular masses of 30.3 and 29.9 kDa, respectively, were analyzed as transgenes that were overexpressed in Arabidopsis thaliana, Nicotiana benthamiana, and Petunia hybrida plants. Transformation with the longer 269 aa variant designated l-HlMyb3 led to a flowering delay and to a strong inhibition of seed germination in A. thaliana. Nearly complete flower sterility, dwarfing, and leaf curling of P. hybrida and N. benthamiana l-HlMyb3 transgenotes were noted. On the contrary, the shorter 265-aa-encoding s-HlMyb3 transgene led in A. thaliana to the stimulation of initial seed germination, to fast initiation of the lateral roots, and to quite specific branching phenotypes with many long lateral stems formed at angles near 90 degrees . Limited plant sterility but growth stimulation and rather branched phenotypes were evident for s-HlMyb3 transgenotes of P. hybrida and N. benthamiana. It was found that both HlMyb3 transgenes interfere in the accumulation and composition of flavonol glycosides and phenolic acids in transformed plants. These effects on heterologous transgenotes suggest that the HlMyb3 gene may influence hop morphogenesis, as well as metabolome composition during lupulin gland maturation.