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.     

Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562 aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.     

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.     

Cloning and analysis of valerophenone synthase gene expressed specifically in lupulin gland of hop (Humulus lupulus L.)

Resin and essential oil derived from hop (Humulus lupulus L.) cones are very important compounds for beer brewing, and they specifically accumulate in the lupulin gland of hop cones. In order to identify the genes responsible for the biosynthetic pathway of these compounds and use the identified genes for hop breeding using Marker Assisted Selection and transformation techniques, genes expressed specifically in the lupulin gland were cloned and sequenced. One of them was suggested to be similar to the chalcone synthase gene from the DNA sequence. The translation product of the gene had the activity of valerophenone synthase, which catalyzes a part of the synthesis reaction of alpha-acid and beta-acid. Northern analysis showed that the valerophenone synthase gene seemed to be expressed specifically in the lupulin gland.     

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.     

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.     

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.     

Glandular trichomes of Humulus lupulus var. Brewer's Gold: Ontogeny and histochemical characterization of the secretion

Leaves of Humulus lupulus possess two types of glandular trichomes: - peltate (lupulin) and bulbous.
Peltate trichomes are formed from a protodermal cell by two anticlinal divisions in perpendicular planes, followed by two periclinal ones that give rise to the initials of the head cells, the basal and the stalk cells. Head cells divide successively in radial and irregular planes. Fully developed peltate trichomes are built of a glandular head consisting of 30 to 72 cells, four stalk cells and four basal cells.
Bulbous trichomes are also formed from a protodermal cell by an anticlinal division followed by two periclinal ones that produce the initials of the glandular head cells, and the basal and stalk cells. Fully developed bulbous trichomes consist of four (occasionally eight) head glandular cells, two stalk cells and two basal cells.
The density of peltate trichomes decreases with the expansion of the leaves.
Both peltate and bulbous trichomes secrete essential oils. Peltate trichomes are the preferential site for the synthesis of bitter resins. Tannic acids could not be detected histochemically either in peltate or in bulbous trichomes. Both types of trichomes produce secretion that accumulates in the subcuticular space, being released, in the case of bulbous trichomes, by rupture of the cuticle.     

An investigation of the storage and biosynthesis of phenylpropenes in sweet basil

Plants that contain high concentrations of the defense compounds of the phenylpropene class (eugenol, chavicol, and their derivatives) have been recognized since antiquity as important spices for human consumption (e.g. cloves) and have high economic value. Our understanding of the biosynthetic pathway that produces these compounds in the plant, however, has remained incomplete. Several lines of basil (Ocimum basilicum) produce volatile oils that contain essentially only one or two specific phenylpropene compounds. Like other members of the Lamiaceae, basil leaves possess on their surface two types of glandular trichomes, termed peltate and capitate glands. We demonstrate here that the volatile oil constituents eugenol and methylchavicol accumulate, respectively, in the peltate glands of basil lines SW (which produces essentially only eugenol) and EMX-1 (which produces essentially only methylchavicol). Assays for putative enzymes in the biosynthetic pathway leading to these phenylpropenes localized many of the corresponding enzyme activities almost exclusively to the peltate glands in leaves actively producing volatile oil. An analysis of an expressed sequence tag database from leaf peltate glands revealed that known genes for the phenylpropanoid pathway are expressed at very high levels in these structures, accounting for 13% of the total expressed sequence tags. An additional 14% of cDNAs encoded enzymes for the biosynthesis of S-adenosyl-methionine, an important substrate in the synthesis of many phenylpropenes. Thus, the peltate glands of basil appear to be highly specialized structures for the synthesis and storage of phenylpropenes, and serve as an excellent model system to study phenylpropene biosynthesis.     

Natural and non-natural prenylated chalcones: synthesis, cytotoxicity and anti-oxidative activity

A general strategy for the synthesis of 3'-prenylated chalcones was established and a series of prenylated hydroxychalcones, including the hop (Humulus lupulus L.) secondary metabolites xanthohumol (1), desmethylxanthohumol (2), xanthogalenol (3), and 4-methylxanthohumol (4) were synthesized. The influence of the A-ring hydroxylation pattern on the cytotoxic activity of the prenylated chalcones was investigated in a HeLa cell line and revealed that non-natural prenylated chalcones, like 2',3,4',5-tetrahydroxy-6'-methoxy-3'-prenylchalcone (9, IC(50) 3.2+/-0.4microM) as well as the phase 1 metabolite of xanthohumol (1), 3-hydroxyxanthohumol (8, IC(50) 2.5+/-0.5microM), were more active in comparison to 1 (IC(50) 9.4+/-1.4microM). A comparison of the cytotoxic activity of xanthohumol (1) and 3-hydroxyxanthohumol (8) with the non-prenylated analogs helichrysetin (12, IC(50) 5.2+/-0.8) and 3-hydroxyhelichrysetin (13, IC(50) 14.8+/-2.1) showed that the prenyl side chain at C-3' has an influence on the cytotoxicity against HeLa cells only for the dihydroxylated derivative. This offers interesting synthetic possibilities for the development of more potent compounds. The ORAC activity of the synthesized compounds was also investigated and revealed the highest activity for compounds 12, 4'-methylxanthohumol (4), and desmethylxanthohumol (2), with 4.4+/-0.6, 3.8+/-0.4, and 3.8+/-0.5 Trolox equivalents, respectively.     

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.     

Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and β-acids in transgenic plants compared to wild-type plants.     

The leaf epidermome of Catharanthus roseus reveals its biochemical specialization

Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis-enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed.     

Prenylflavonoid variation in Humulus lupulus: distribution and taxonomic significance of xanthogalenol and 4'-O-methylxanthohumol

The resins produced by either lupulin or leaf glands of over 120 plants of Humulus lupulus and one plant of H. japonicus (Cannabinaceae) were analyzed for the presence of prenylated flavonoids. The H. lupulus taxa investigated were H. lupulus var. lupulus from Europe, H. lupulus var. cordifolius from Japan, and H. lupulus from North America. Fifty-two of the plants examined were cultivars of European, American, and Japanese origin. Twenty-two flavonoids were detected in the glandular exudates of H. lupulus by HPLC-MS MS. Xanthohumol (3'-prenyl-6'-O-methylchalconaringenin) was the principal prenylflavonoid in all H. lupulus plants and was accompanied by 11 structurally similar chalcones. Ten flavonoids were identified as the flavanone isomers of these chalcones. Three other prenylchalcones were isolated from H. lupulus cv. 'Galena', one of which was identified as 3'-prenyl-4'-O-methylchalconaringenin (named 'xanthogalenol'). The distribution of three 4'-O-methylchalcones, i.e. xanthogalenol, 4'-O-methylxanthohumol, and 4',6'-di-O-methylchalconaringenin, was found to be limited to wild American plants from the Missouri-Mississippi river basin, H. lupulus var. cordifolius, and most of their descendents. These 4'-O-methylchalcones were absent from cultivars of European origin, and from wild hops from Europe and southwestern USA. The flavonoid dichotomy (presence versus absence of 4'-O-methylchalcones) indicates that there are at least two evolutionary lineages within H. lupulus (European and Japanese American), which is in agreement with morphological, molecular, and phytogeographical evidence. Leaf glands of H. japonicus from eastern Asia did not produce the H. lupulus prenylflavonoids.