Lupulin Glands Research Articles

IDENTIFICAÇÃO E QUANTIFICAÇÃO DOS TEORES DE α E β-ÁCIDOS DOS LÚPULOS PLANTADOS NO ESPÍRITO SANTO POR CROMATOGRAFIA LÍQUIDA DE ALTA EFICIÊNCIA (HPLC)

Brazil is the third largest beer producer in the world, behind only to China and the United States. However, one of the essential raw materials to produce the drink, hops (Humulus Lupulus L.), still faces significant dependence on imports. Inside the lupulin glands located in female hop flowers are compounds that provide bitterness, aroma and important biological activities. It is the molecules known as α and β-acids (Humulones and Lupulones), together with their essential oils that contribute to this. The quantification of these α and β-acids is vital for brewers and researchers, since they improve the sensorial and functional characteristics of beers. In the last decade, through the certification of seedlings of this flower in Brazil, some Brazilian states have been presenting varieties well adapted to the climate. The states of the South and Southeast, at the forefront of this cultivation, have been playing a crucial role in this process of popularizing Hops. Thus, the objective of this work was the quantification, by High Performance Liquid Chromatography (HPLC), of the α and β-acids present in samples from Espírito Santo and other states in the country. The results indicated a high amount of β-acids in the Teamaker variety (12.51 g 100 g-1, m/m), while α-acids were predominant in varieties such as Zeus (14.89 g 100 g-1, m/m) and Comet (14.69 g 100 g-1, m/m). The study also demonstrated that the flower processing stage (harvesting, drying and storage) still requires greater care, since some varieties studied (Polaris and East Rent Golding) present a significant amount of compounds already oxidized due to poor conservation.

Updating the Knowledge on the Secretory Machinery of Hops (Humulus lupulus L., Cannabaceae).

Cannabaceae species garner attention in plant research due to their diverse secretory structures and pharmacological potential associated with the production of secondary metabolites. This study aims to update our understanding of the secretory system in Hops (Humulus lupulus L.), an economically important species especially known for its usage in beer production. For that, stems, leaves, roots, and inflorescences were collected and processed for external morphology, anatomical, histochemical, ultrastructural and cytochemical analyses of the secretory sites. Our findings reveal three types of secretory structures comprising the secretory machinery of Hops: laticifer, phenolic idioblasts and glandular trichomes. The laticifer system is articulated, anastomosing and unbranched, traversing all plant organs, except the roots. Phenolic idioblasts are widely dispersed throughout the leaves, roots and floral parts of the species. Glandular trichomes appear as two distinct morphological types: capitate (spherical head) and peltate (radial head) and are found mainly in foliar and floral parts. The often-mixed chemical composition in the secretory sites serves to shield the plant from excessive UVB radiation, elevated temperatures, and damage inflicted by herbivorous animals or pathogenic microorganisms. Besides the exudate from peltate glandular trichomes (lupulin glands), latex and idioblast content are also likely contributors to the pharmacological properties of different Hop varieties, given their extensive presence in the plant body.

CRISPR/Cas9-mediated mutagenesis of the mediator complex subunits MED5a and MED5b genes impaired secondary metabolite accumulation in hop (Humulus lupulus)

Hop (Humulus lupulus L.) is an important commercial crop known for the biosynthesis of valuable specialized secondary metabolites in glandular trichomes (lupulin glands), which are used for the brewing industry. To achieve burgeoning market demands is the essentiality of comprehensive understanding of the mechanisms of biosynthesis of secondary metabolites in hop. Over the past year, several studies using structural biology and functional genomics approaches have shown that Mediator (MED) serves as an integrative hub for RNAP II-mediated transcriptional regulation of various physiological and cellular processes, including involvement of MED5a and MED5b in hyperaccumulation of phenylpropanoid in A. thaliana. In the present work, an unprecedented attempt was made to generate Hlmed5a/med5b double loci mutant lines in hop using a CRISPR/Cas9-based genome editing system. The Hlmed5a/med5b double loci mutant lines showed reduced expression of structural genes of the flavonoid, humulone, and terpenoid biosynthetic pathways, which was more pronounced in the lupulin gland compared to leaf tissue and was consistent with their reduced accumulation. Phenotypic and anatomical observations revealed that Hlmed5a/med5b double loci mutant line exhibited robust growth, earlier flowering, earlier cone maturity, reduced cone size, variations in floral structure patterns, and distorted lupulin glands without any remarkable changes in leaf morphology, intensity of leaf color, and chlorophyll content. Comparative transcriptome analysis of leaf and lupulin gland tissues indicates that the expression of enzymatic genes related to secondary metabolite biosynthesis, phytohormone biosynthesis, floral organs, flowering time, and trichome development, including other genes related to starch and sucrose metabolism and defense mechanisms, were differentially modulated in the Hlmed5a/med5b lines. The combined results from functional and transcriptomic analyses illuminates the pivotal function of HlMED5a and HlMED5b in homeostasis of secondary meatbolites accumulation in hop.

Humulus lupulus L. Strobilus Photosynthetic Capacity and Carbon Assimilation.

The economic value of Humulus lupulus L. (hop) is recognized, but the primary metabolism of the hop strobilus has not been quantified in response to elevated CO2. The photosynthetic contribution of hop strobili to reproductive effort may be important for growth and crop yield. This component could be useful in hop breeding for enhanced performance in response to environmental signals. The objective of this study was to assess strobilus gas exchange, specifically the response to CO2 and light. Hop strobili were measured under controlled environment conditions to assess the organ's contribution to carbon assimilation and lupulin gland filling during the maturation phase. Leaf defoliation and bract photosynthetic inhibition were deployed to investigate the glandular trichome lupulin carbon source. Strobilus-level physiological response parameters were extrapolated to estimate strobilus-specific carbon budgets under current and future atmospheric CO2 conditions. Under ambient atmospheric CO2, the strobilus carbon balance was 92% autonomous. Estimated strobilus carbon uptake increased by 21% from 415 to 600 µmol mol-1 CO2, 14% from 600 to 900 µmol mol-1, and another 8%, 4%, and 3% from 900 to 1200, 1500, and 1800 µmol mol-1, respectively. We show that photosynthetically active bracts are a major source of carbon assimilation and that leaf defoliation had no effect on lupulin production or strobilus photosynthesis, whereas individual bract photosynthesis was linked to lupulin production. In conclusion, hop strobili can self-generate enough carbon assimilation under elevated CO2 conditions to function autonomously, and strobilus bracts are the primary carbon source for lupulin biosynthesis.

Impact of pre-freezing and microwaves on drying behavior and terpenes in hops (Humulus lupulus)

Hop buds (Humulus lupulus) are paramount to beer bittering, flavoring, and microbiological stability. To optimize post-harvest processing, fresh and pre-frozen hops were subjected to freeze-drying, hot air and microwave-assisted hot air drying. Pre-freezing occurred at − 80°C, prior to drying at 35°C, 50°C, and 65°C, with different microwave power (0 W, 100 W and 200 W, where 0 W represented conventional hot air drying). Results show that hops drying kinetics can be described using the predictive Page and Logarithmic mathematical models. Obtained R2, SSE, and RMSE values ranged between 0.999 and 0.982, 0.035–0.001, and 0.058–0.004, respectively. Irrespective of the drying condition, pre-freezing reduced drying time by 0.17–85.9 %. Pre-freezing hop buds increases the effective moisture diffusion coefficient, and it increases with higher drying temperature and microwave power, ranging between 5.91 × 10−10 m2 s−1 and 2.43 × 10−7 m2 s−1. SEM analyses indicate that pre-freezing causes structural damage to lupulin glands. The average concentration of myrcene, limonene, caryophyllene, and humulene for fresh hops were 15.08 mg g−1, 0.27 mg g−1, 3.09 mg g−1, and 6.52 mg g−1 respectively. For the dried samples under the various conditions, the concentration ranged from 12.20 mg g−1 to 0.53 mg g−1 (myrcene), 0.26 mg g−1 to 0.12 mg g−1 (limonene), 1.49 mg g−1 to 0.31 mg g−1 (caryophyllene), and 2.69 mg g−1 to 0.52 mg g−1 (humulene). Results affirm that pre-freezing plant material prior to drying can shorten postharvest processing times, and this method can potentially be applied to other industrial crops. This study highlights the importance of controlled postharvest processing to ensure industrial crop quality.

Genome-Wide Analyses of MADS-Box Genes in Humulus lupulus L. Reveal Potential Participation in Plant Development, Floral Architecture, and Lupulin Gland Metabolism.

MADS-box transcription factors (TFs) are involved in multiple plant development processes and are most known during the reproductive transition and floral organ development. Very few genes have been characterized in the genome of Humulus lupulus L. (Cannabaceae), an important crop for the pharmaceutical and beverage industries. The MADS-box family has not been studied in this species yet. We identified 65 MADS-box genes in the hop genome, of which 29 encode type-II TFs (27 of subgroup MIKCC and 2 MIKC*) and 36 type-I proteins (26 α, 9 β, and 1 γ). Type-II MADS-box genes evolved more complex architectures than type-I genes. Interestingly, we did not find FLOWERING LOCUS C (FLC) homologs, a transcription factor that acts as a floral repressor and is negatively regulated by cold. This result provides a molecular explanation for a previous work showing that vernalization is not a requirement for hop flowering, which has implications for its cultivation in the tropics. Analysis of gene ontology and expression profiling revealed genes potentially involved in the development of male and female floral structures based on the differential expression of ABC homeotic genes in each whorl of the flower. We identified a gene exclusively expressed in lupulin glands, suggesting a role in specialized metabolism in these structures. In toto, this work contributes to understanding the evolutionary history of MADS-box genes in hop, and provides perspectives on functional genetic studies, biotechnology, and crop breeding.

Developmental regulation of lupulin gland-associated genes in aromatic and bitter hops (Humulus lupulus L.)

BackgroundHop (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).ResultsWe 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.ConclusionsGene 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.

The use of biotechnology in hop (Humulus lupulus L.) improvement

Hop (Humulus lupulus L.) is a clonally propagated, dioecious, perennial, climbing plant used commercially for their secondary metabolites. The resins containing α- and β-acids, and essential oils produced by the lupulin glands, present on the female flowers are used to add bitterness, aroma and flavour to beer. Recently, flavonoids, including chalcones and flavanones, of hops have been shown to exert a variety of biological activities, including oestrogenic and anticancerogenic characteristics. In this review, we provide a overview of the techniques and opportunities presented by the integration of plant biotechnology into hop improvement. The use of tissue culture techniques such as micropropagation, meristem culture, in vitro storage, adventitious shoot induction, callus culture and cell suspension culture in hops are briefly reviewed. The usefullness of genetic transformation technology to introduce novel traits into hop is also discussed.

Gene Expression of Agronomically Important Secondary Metabolites in cv. ‘USDA Cascade’ Hop (Humulus lupulus L.) Cones during Critical Developmental Stages

The transcriptome from lupulin glands and associated bracts from cone tissue of hop (Humulus lupulus) c.v. ‘Cascade’, during three stages of development: early, mid, and late or near-harvest, was sequenced. Significant increases were found in expression patterns of many genes involved in the biosynthesis of bitter acids, xanthohumol, and volatile secondary metabolites or “hop oils” during the middle stage of cone development. The biosynthesis of thiol precursors responsible for popular “tropical fruit” flavors in beer is not well known, but homologs of genes hypothesized to be involved in this process tend to be up-regulated during the late stage in hop cones. More research needs to be performed to describe the pathway of thiol precursor biosynthesis in hops. Hierarchical clustering revealed overlap of samples taken from each developmental stage, likely due to non-uniform ripening of cones on the plant. It is proposed that the mid-stage of cone development is critical for the development of important flavor-producing secondary metabolites in hops, and this is supported by previous research describing concentrations of secondary metabolites. It is hypothesized that abiotic stress during the mid-stage of cone development may be quite detrimental to the bitter acid concentrations ultimately found in hops.

Performance of Tetranychus urticae (Acari: Tetranychidae) on three hop cultivars (Humulus lupulus).

Tetranychus urticae Koch (Acari: Tetranychidae) is a major pest species in numerous crops including hop (Humulus lupulus L.). Substantial T. urticae infestation was observed to occur in this recently introduced crop in Brazil. The adoption of less suitable cultivars to the pest species is highly desirable for integrated pest management. We used free-choice trials and two-sex life table analysis to determine the preference and population growth of T. urticae under laboratory conditions using three of hop cultivars currently expanding in Brazil (Mantiqueira, Victoria, and Yakima Gold). We also estimated the density of non-glandular trichomes and lupulin glands found on the abaxial leaf surface of these cultivars and correlated them with performance parameters of T. urticae. Mantiqueira appeared to be the least preferred by adult females for attractiveness and oviposition suggesting existence of antixenosis on this cultivar. Female immature stages developed slower on Yakima Gold and Mantiqueira, but no difference was observed between the latter and Victoria. Fecundity and longevity were significantly lower on Mantiqueira than on Victoria and Yakima Gold. No significant differences were observed among cultivars for intrinsic rate of increase (r), finite rate of increase (λ), and net rate of reproduction (R0), suggesting the absence of antibiosis. Although, lupilin gland densities were higher on Mantiqueira and Yakima Gold than on Victoria, no significant correlations were observed between these defensive traits and performance parameters of T. urticae. However, 30-day population simulations of T. urticae suggest that Yakima Gold is the least susceptible, Mantiqueira is moderately susceptible, and Victoria is highly susceptible.

Gene expression for secondary metabolite biosynthesis in hop (Humulus lupulus L.) leaf lupulin glands exposed to heat and low-water stress

Hops are valued for their secondary metabolites, including bitter acids, flavonoids, oils, and polyphenols, that impart flavor in beer. Previous studies have shown that hop yield and bitter acid content decline with increased temperatures and low-water stress. We looked at physiological traits and differential gene expression in leaf, stem, and root tissue from hop (Humulus lupulus) cv. USDA Cascade in plants exposed to high temperature stress, low-water stress, and a compound treatment of both high temperature and low-water stress for six weeks. The stress conditions imposed in these experiments caused substantial changes to the transcriptome, with significant reductions in the expression of numerous genes involved in secondary metabolite biosynthesis. Of the genes involved in bitter acid production, the critical gene valerophenone synthase (VPS) experienced significant reductions in expression levels across stress treatments, suggesting stress-induced lability in this gene and/or its regulatory elements may be at least partially responsible for previously reported declines in bitter acid content. We also identified a number of transcripts with homology to genes shown to affect abiotic stress tolerance in other plants that may be useful as markers for breeding improved abiotic stress tolerance in hop. Lastly, we provide the first transcriptome from hop root tissue.

Flavonoids as Phytoestrogenic Components of Hops and Beer.

The value of hops (Humulus lupulus L.) in beer production has been undisputed for centuries. Hops is rich in humulones and lupulones which gives the characteristic aroma and bitter taste, and preserves this golden drink against growing bacteria and molds. Besides α- and β-acids, the lupulin glands of hop cones excrete prenylated flavonoids, which exhibit a broad spectrum of biological activities and therefore has therapeutic potential in humans. Recently, interest in hops was raised due to hop prenylated flavanones which show extraordinary estrogen activities. The strongest known phytoestrogen so far is 8-prenylnaringenin (8-PN), which along with 6-prenylanaringenin (6-PN), 6,8-diprenylnaringenin (6,8-DPN) and 8-geranylnaringenin (8-GN) are fundamental for the potent estrogen activity of hops. This review provides insight into the unusual hop phytoestrogens and shows numerous health benefits associated with their wide spectrum of biological activities including estrogenic, anticancer, neuropreventive, antinflamatory, and antimicrobial properties, which were intensively studied, and potential applications of these compounds such as, as an alternative to hormone replacement therapy (HRT).

Dissection of Dynamic Transcriptome Landscape of Leaf, Bract, and Lupulin Gland in Hop (Humulus lupulus L.).

The hop plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.

Phenological phases of flowering in hop (Humulus lupulus L.) and their correspondence with microsporogenesis and microgametogenesis

Hop (Humulus lupulus L.) suffered, as many other crops, a shrinkage of its intraspecific agrobiodiversity. Biotechnological methods of breeding would offer new opportunities to produce improved varieties with interesting phytochemical profiles and adaptable to the challenging conditions of climate change. Doubled haploid (DH) technology could be a useful tool to increase hop agrobiodiversity but, unfortunately, there is a complete lack of information about hop flower biology. For this reason, the main aim of this work is the study of the different phenological phases of flowering in hop and the corresponding developmental stages of microspores/pollen grains contained therein. The results obtained allowed the identification of morphological markers (anther and flower bud length), easy and fast to measure, that would speed up the selection of flower buds containing the highest percentage of vacuolated microspores and young pollen, the stages considered in most species as the most responsive to androgenesis. A further result, derived from the flower bud and anther microscopical observation, evidenced the increase of lupulin glands on bud and anther surface as the bud proceeds in development from microsporogenesis to microgametogenesis.

Phenolic composition and antioxidant, antimicrobial and cytotoxic properties of hop (Humulus lupulus L.) Seeds

Hop (Humulus lupulus L.) is a perennial climbing plant cultivated for commercial use, mainly in brewing industry, since the female inflorescences (cones) contain lupulin glands that confer the characteristic bitterness, aroma and flavour to beer. However, the bioactive potential of the seeds found in these cones remains unexplored. In this study, the phenolic composition of hop seeds was analysed by LC-DAD-ESI/MSn and its antioxidant, cytotoxic and antimicrobial activities were screened against oxidizable substrates, human tumour and non-tumour cell lines, and different bacteria and fungi, respectively. (+)-Catechin and (-)-epicatechin were identified in the seeds, totalizing 17.6 ± 0.7 mg/g extract. The seed extract showed antioxidant activity mainly through the formation inhibition of thiobarbituric acid reactive substances (TBARS), and had cytotoxic effects against MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma), and HepG2 (hepatocellular carcinoma) tumour cells, but no toxicity towards the normal PLP2 cell line was observed. The extract showed remarkable antimicrobial properties, being better than the positive controls against almost all tested bacteria and fungi of the genus Penicillium. These results highlight the potential of hop seeds as a source of functional ingredients with applications in the food and pharmaceutical sectors.

Discrimination of geographical origin of hop (Humulus lupulus L.) using geochemical elements combined with statistical analysis.

Beer is a popular alcoholic beverage worldwide, traditionally made from water, barley and hop (Humulus lupulus L.) strobili. The strobili contain lupulin glands whose components (mostly bitter acids and polyphenols) confer unique and locally different flavours to beer types. It is therefore relevant for brewers and consumers to precisely know the geographical origin of hop plants used for high-quality beer. Hop plants belonging to the variety Hallertau Perle, grown in two locations, Cavalese and Imèr, of the Trentino Region (Italy) were analysed to establish a direct relationship between the chemical elements detected in soil and in plant parts. Chemical elements were determined by X-ray fluorescence and inductively coupled plasma mass spectrometry in soil, leaf and strobili samples from Cavalese and Imèr. The data from the two areas were compared by a nonparametric test (Mann-Whitney) and multivariate statistics (principal component analysis and partial least squares discriminant analysis). The geochemical characterization and the statistical analyses showed different concentrations of major and trace elements in soil and plant parts from the two areas. A reliable correlation could be established between some elements in soil and strobili samples, that is Nb, Fe, Rb and Zr for Cavalese and Mg, Ni, Zn and Zr for Imèr. These elements could therefore be used as geochemical fingerprints to identify the geographical origin of strobili from the two study areas, an approach useful to verify the origin of hop plants for the production of high-quality beer.

Registration of High Alpha‐Acid Male Hop Germplasm USDA 21267M

USDA 21267M (Reg. No. GP‐37, PI 559093) male hop (Humulus lupulus L. var. lupulus) germplasm was released to the public 6 Dec. 2011. This germplasm has been used by the USDA–ARS for hop cultivar development for several decades. Nonagency interest in this germplasm was expressed, and release of this material is considered beneficial for the US public. USDA 21267M arose from a cross, made in 1973 involving the hop cultivar Comet and the USDA–ARS hop male germplasm 21110M. The defining characteristics of USDA 21267M are its high alpha acid levels observed in lupulin glands as well as good breeding value for yield, alpha and beta acid levels, and high xanthohumol levels. It is expected that hop breeders will use this germplasm as a parent for high‐yielding, high‐alpha hop breeding material. The release of USDA 21267M will give both public and private breeders new germplasm for varietal development.

The R2R3 transcription factor HlMYB8 and its role in flavonoid biosynthesis in hop (Humulus lupulus L.)

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.

A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid‐related compounds and possible transport proteins

Female flowers of hop (Humulus lupulus) are an essential source of terpenoid-related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono- and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D-LC-MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism-related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid-derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra- and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes.

Fast Sampling, Analyses and Chemometrics for Plant Breeding: Bitter Acids, Xanthohumol and Terpenes in Lupulin Glands of Hops (Humulus lupulus).

The valuable secondary metabolites in hops (bitter acids, xanthohumol, volatile monoterpenes and sesquiterpenes) are sequestered in lupulin glands (extracellular trichomes) which can be collected and analysed with little or no sample preparation. To determine whether high throughput screening of lupulin glands composition, by fast analyses and chemometrics, could be used for breeder selection of hops with key flavour attributes. Lupulin glands from 139 plants (39 cultivars/advanced selections) were analysed by Raman and 1 H NMR spectroscopy, and head-space solid-phase microextraction (HS-SPME) GC-FID. The digital X,Y-data were subjected to principal component analysis (PCA) and the results compared with conventional analyses of extracts of whole hops from the same plants. Quantitative 1 H NMR analyses were also done for the bitter acids. Raman spectroscopy rapidly identified hops cultivars with high xanthohumol concentrations and high α:β bitter acid ratios. 1 H NMR spectroscopy was slower, requiring a solvent extraction, but distinguished cultivars by cohumulone content as well as α:β acid ratios. HS-SPME-GC rapidly distinguished aroma hops with high myrcene and farnesene contents, and pinpointed a novel selection with unusual sesquiterpenes. The quantitative NMR analyses showed correlations between bitter acid concentrations related to biosynthetic pathways. Analysis of lupulin glands gave reliable results for the main quality indicators used by hops breeders, potentially avoiding harvesting, drying and solvent extracting whole hops. PCA of digital X,Y-data rapidly discriminated different hops chemotypes, and highlighted plants with potential for new flavourcultivars. Copyright © 2016 John Wiley & Sons, Ltd.