Construction of gene expression system in hop (Humulus lupulus) lupulin gland using valerophenone synthase promoter

The promoter region of the valerophenone synthase (VPS) gene was isolated from hop (Humulus lupulus). VPS, a member of the chalcone synthase (CHS) super-family, catalyzes the biosynthesis reaction of the hop resin that significantly accumulates in the cone's secretory gland called the "lupulin gland". The typical H-box and G-box sequences, which exist in many plants' CHS promoters and act as cis-elements for tissue specificity, UV-light induction, etc., were not found in the isolated VPS promoter, although the H-box-like sequence (CCTTACC, CCTAACC) and the core sequence (ACGT) of the G-box were observed. The transformation experiment using the VPS promoter-UIDA gene fusion revealed that the promoter acts not only in the lupulin gland but also in the glands of leaf and stem. On the other hand, the VPS promoter activity was not induced by UV-irradiation.     

Developmental Morphology of Hop Stunt Viroid-infected Hop Plants and Analysis of Their Cone Yield1)

Infection of hop plants with hop stunt viroid (HSV) results in the retardation of the growth rate except for the rate of leaf emergence and the disappearance of the fold-like structure over the epidermal cell. Mature cones from HSV-infected hop plants remained small-sized and the content of α-acid was half to one third of that of HSV–free hop cones. In HSV-infected hop cones, the lupulin glands are distributed most abundantly on the bracteoles and the perianths and their numbers are reduced by at least 60% of that in the HSV-free control. Scanning electron micrographs confirm that most of the lupulin glands on bracteoles from HSV-infected hop cones shrivel severely, but not those from HSV-free hop cones. They also reveal that the lupulin glands on the perianths from both, HSV-free and HSV–infected hop cones become withered. Moreover, spherical granules (1.2 to 1.9μm in diameter) were not observed on the surface of the lupulin glands from HSV-infected hop cones.     

Enzymatic reactions by five chalcone synthase homologs from hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol

The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.     

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg(2+) as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.     

Xanthohumol and related prenylflavonoids from hops and beer: to your good health!

Xanthohumol (3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone) is the principal prenylated flavonoid of the female inflorescences of the hop plant ('hops'), an ingredient of beer. Human exposure to xanthohumol and related prenylflavonoids, such as 8-prenylnaringenin and isoxanthohumol, is primarily through beer consumption. Xanthohumol has been characterized a 'broad-spectrum' cancer chemopreventive agent in in vitro studies, while 8-prenylnaringenin enjoys fame as the most potent phytoestrogen known to date. These biological activities suggest that prenylflavonoids from hops have potential for application in cancer prevention programs and in prevention or treatment of (post-)menopausal 'hot flashes' and osteoporosis. Xanthohumol and 8-prenylnaringenin are metabolized into many flavonoid derivatives with modified 3,3-dimethyl allyl (prenyl) moieties. Xanthohumol is formed in lupulin glands by a specialized branch of flavonoid biosynthesis that involves prenylation and O-methylation of the polyketide intermediate chalconaringenin. Although a lupulin gland-specific chalcone synthase is known, the aromatic prenyltransferase and O-methyltransferase participating in xanthohumol have not been identified. The prenylflavonoid pathway is a possible target for breeding or biotechnological modification of hops with the aim of increasing xanthohumol levels for beer brewing and 8-prenylnaringenin levels for pharmaceutical production.     

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.     

Cell type-specific gene expression in the Drosophila melanogaster male accessory gland.

The accessory gland of the male Drosophila melanogaster plays a vital role in reproduction. This secretory organ synthesizes products that are transferred to the female and are necessary to elicit the proper physiological and behavioral responses in the female. The accessory gland is composed of two morphologically distinct secretory cell types, the main cells and the secondary cells. Previous studies identified some genes expressed in main cells or in all accessory gland cells. In this paper we use P-element mediated enhancer traps to examine gene expression in the accessory gland. We show that, in addition to genes expressed in main cells only or in all accessory gland secretory cells, there are genes expressed specifically in secondary cells. Each cell type is uniform in the expression of its genes. Our results demonstrate that the two cell types are not only morphologically distinct but also biochemically distinct. We also show that the two cell types differ in their regulation of gene expression in response to mating activity.     

Terpene biosynthesis in glandular trichomes of hop

Hop (Humulus lupulus L. Cannabaceae) is an economically important crop for the brewing industry, where it is used to impart flavor and aroma to beer, and has also drawn attention in recent years due to its potential pharmaceutical applications. Essential oils (mono- and sesquiterpenes), bitter acids (prenylated polyketides), and prenylflavonoids are the primary phytochemical components that account for these traits, and all accumulate at high concentrations in glandular trichomes of hop cones. To understand the molecular basis for terpene accumulation in hop trichomes, a trichome cDNA library was constructed and 9,816 cleansed expressed sequence tag (EST) sequences were obtained from random sequencing of 16,152 cDNA clones. The ESTs were assembled into 3,619 unigenes (1,101 contigs and 2,518 singletons). Putative functions were assigned to the unigenes based on their homology to annotated sequences in the GenBank database. Two mono- and two sesquiterpene synthases identified from the EST collection were expressed in Escherichia coli. Hop MONOTERPENE SYNTHASE2 formed the linear monterpene myrcene from geranyl pyrophosphate, whereas hop SESQUITERPENE SYNTHASE1 (HlSTS1) formed both caryophyllene and humulene from farnesyl pyrophosphate. Together, these enzymes account for the production of the major terpene constituents of the hop trichomes. HlSTS2 formed the minor sesquiterpene constituent germacrene A, which was converted to β-elemene on chromatography at elevated temperature. We discuss potential functions for other genes expressed at high levels in developing hop trichomes.