Chalcone synthase homologues from Humulus lupulus: some enzymatic properties and expression

The enzymatic properties of four chalcone synthase homologues CHS_H1, VPS, CHS 2 and CHS 4 from Humulus lupulus L. were investigated after heterologous expression in Escherichia coli. It was found that both VPS and CHS_H1 can utilize isovaleryl-CoA and isobutyryl-CoA as substrates producing compounds with positions in thin layer chromatography characteristic for phloroisovalerophenone and phloroisobutyrophenone. These reactions are accompanied by the formation of associated byproducts. The formation of naringenin chalcone can be catalyzed primarily by CHS_H1. Comparatively the ability of VPS to perform chalcone synthase reaction is very limited. Since only CHS_H1 has true chalcone synthase activity, this enzyme can be considered a key enzyme in prenylflavonoid biosynthesis. Both CHS 2 and CHS 4 utilize isovaleryl-CoA and isobutyryl-CoA as substrates, but the reactions were prematurely terminated. In comparison with VPS and CHS_H1, the optimum pH of CHS 2 was shifted to lower value. High expression of chalcone synthase-like genes were found in maturating hop cones of cultivars with high bitter acid content (Agnus, Magnum, Target) by Northern and Western blotting using probes specific for vps, chs_H1, chs 4 and polyspecific serum risen against recombinant protein CHS4, respectively. It was also found that these cultivars maintained expression of CHS homologues for a longer period of time during cone development in contrast to time-limited expression of CHS homologues in cultivars with low bitter acids content.     

Isolation and characterization of polymorphic microsatellites for assessment of genetic variation of hops (Humulus lupulus L.)

Hop (Humulus lupulus L.) cultivars are vegetatively propagated and it is difficult to differentiate them during the process of propagation. Fingerprinting with molecular markers based on DNA could be a useful means of identifying different cultivars. Simple sequence repeats, or microsatellite markers, are the most suitable marker for genetic fingerprinting because they are multi‐allelic and co‐dominant. For this purpose, we have developed primers for 10 new polymorphic microsatellite loci that are suitable for genetic fingerprinting in hop.     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

啤酒花节间外植体再生体系的建立

在国内首次建立了两个啤酒花主栽品种（麒麟丰禄,青岛大花）的节间外植体再生体系。实验表明：啤酒花无菌苗的节间为适于不定芽再生韵外植体;适宜的再生培养基为Ms基本培养基＋B5维生素＋葡萄糖20g／L,附加IAA0．25mg／L＋TDZ2mg／L（麒麟丰禄）或IAA0．125mg／L＋TDZ1．5mg／L（青岛大花）,以7g／L琼脂固化,pH5．8;不定芽再生频率可达57％（麒麟丰禄）或16％（青岛大花）。啤酒花再生体系的建立,为利用植物基因工程技术培育抗病虫害啤酒花新品系奠定了基础。     

Anti-inflammatory and anti-tumor-promoting effects of 5-deprenyllupulonol C and other compounds from Hop (Humulus lupulus L.)

A new phloroglucinol derivative, 5‐deprenyllupulonol C ( 1 ), along with four other phloroglucinol derivatives, 2 – 5 , five chalcones, 6 – 10 , four flavanones, 11 – 14 , two flavonol glycosides, 15 and 16 , and five triterpenoids, 17 – 21 , were isolated from the female inflorescence pellet extracts of hop (Humulus lupulus L.). Upon evaluation of these compounds against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, twelve compounds, i.e., 1 – 4, 11 – 14, 17 – 19 , and 21 , showed potent inhibitory effects on EBV‐EA induction, with IC₅₀ values in the range of 215–393 mol ratio/32 pmol TPA. In addition, eleven compounds, i.e., 1 – 4, 6, 11, 12, 14, 17, 18 , and 20 , were found to inhibit TPA‐induced inflammation (1 μg/ear) in mice, with ID₅₀ values in the range of 0.13–1.06 μmol per ear. Further, lupulone C ( 2 ) and 6‐prenylnaringenin ( 14 ) exhibited inhibitory effects on skin‐tumor promotion in an in vivo two‐stage mouse‐skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter.     

New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L

One hundred and thirty-five microsatellite markers were developed for hop Humulus lupulus L. from di- and trinucleotide-enriched libraries. Seventy-eight primers showed amplification in two tested genotypes. Twenty-four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop.     

啤酒花茎段培养及快速繁殖技术研究

以啤酒花的幼嫩茎段作为外植体,研究出理想的一次性成苗培养基：ＭＳ＋６－ＢＡ０．０１ｍｇ／Ｌ＋ＩＡＡ０．１ｍｇ／Ｌ＋琼脂６．５ｇ／Ｌ＋葡萄糖２％。试管苗月增殖率４．２,生根率１００％,成苗率９０％。采用试管苗微扦插的方式进行快速繁殖,经锻炼后直接移入经改造后的土壤中,移栽成活率达９３．８％。     

Development of a shoot multiplication system for hop (Humulus lupulus L.)

Summary Nodal explants from hop were exposed to plant growth regulators to determine suitable media for initiation from axillary buds and subsequent micropropagation. Efficient culture establishment (96.6% of explants) was achieved on Murashige and Skoog (MS) medium (modified to contain 1 mg l⁻¹ thiamine hydrochloride) supplemented with 0.57 μM indoleacetic acid (IAA) and 2.22 μM 6-benzylaminopurine (BA). Subsequent transfer of explants to treatments containing an auxin ([1-naphthaleneacetic acid], NAA or IAA) and BA, 6-[γ,γ-dimethylallylamino]purine (2iP), kinetin (KIN) or thidiazuron (N-phenyl-N′-1,2,3-thidiazol-5-ylurea [TDZ]) resulted in significantly different amounts of multiplication. Optimal TDZ-supplemented media elicited a greater than threefold increase in the number of shoots and nodes generated per explant compared to optimal media containing BA, 2iP and KIN. Shoots were successfully rooted using half-strength MS supplemented with 5.71 μM IAA and 4.9 μM indolebutyric acid (IBA), and regenerated plants were successfully transferred to soi. An overall micropropagation schedule, which can be implemented into hop commercialization programs, includes: (i) establishment in MS with 0.57 μM IAA and 2.22 μM BA; (ii) multiplication in MS with 0.57 μM IAA and 2.27 μM TDZ; (iii) elongation in MS; and (iv) rooting in half-strength MS with 5.71 μM IAA and 4.9 μM IBA.     

Modulation of breast cancer cell survival by aromatase inhibiting hop (Humulus lupulus L.) flavonoids

Hop flavonoids are being regarded as attractive molecules to prevent or treat certain forms of cancer. Studies have focused mainly on xanthohumol, the most abundant prenylated chalcone existing in hops extract. However, during the production of beer, or after its ingestion, xanthohumol originates different metabolites, among which isoxanthohumol and 8-prenylnaringenin. The aim of this work was to study the effect of the prenylflavonoids xanthohumol, isoxanthohumol and 8-prenylnaringenin on the breast cancer Sk-Br-3 cell line proliferation, apoptosis and activity of the enzyme aromatase (estrogen synthase). Aromatase activity was determined by a tritiated water assay, cell proliferation was assessed by [³H]thymidine incorporation, sulforhodamine B protein measurement and Ki-67 immunostaining and apoptosis was determined by TUNEL. Our results show that all tested prenylflavonoids were able to inhibit aromatase activity and thus, estrogen formation. Additionally, breast cancer cell line proliferation was decreased and apoptosis induced by all three compounds. The presence of 17β-estradiol in treatment medium was able to revert the effect of the prenylflavonoids on cellular proliferation. These observations strengthen the idea that hop flavonoids may have anti-breast cancer effects and shed new light on a possible mechanism of action by which these effects occur, namely through their ability to decrease estrogen synthesis.     

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.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

Histologial Studies on the Root-Knot Nematodes Nonresistant and Resistant Root Apex of Humulus lupulus L.

The nematodes (Meloidogy incognita Chitwood.) invade near the root apex of the hop (Humulus lupulus L.), and usually lie near the meristematic zone. Fallowing their entrance, there are the formation of the giant cells and the consequent continuous divisions and activities of the flat meristematic cells, which result in formation of the nematode galls. Their sizes are also related to the number of nematodes. String beads nematode galls are often formed at the nematode-non resistant root part of the "Comet". The process of nematodes entering and later changing of the cells and tissue is almost the same as the formation of the nematode gall in other plants. Fewer nematodes can also enter the root tip of the highly resistant "Qingdao Dahua", but the subsequent special differentiation of the cells and tissues in the root have greatly restrained the activities of nematodes and ultimately to their death.     

Factors Controlling Flowering in the Hop (Humulus lupulus L.)

In experiments on the effects of daylength on the growth andflowering of the perennial hop it was shown that Humulus lupulusis a short-day plant. The absolute length of the short day isimportant since very short days (8h) induce dormancy beforeflowering can occur. Light-break treatment may therefore promoteor inhibit flowering according to the associated main photoperiod.A minimum node number must have been differentiated before thehop can be induced to initiate flowers, an effect analogousto the juvenile condition. Minimum leaf number and criticaldaylength for induction depend on variety. At low temperature,induction is possible with longer photoperiods. Promotion offlowers by growth retardants (B9 and CCC) in unfavourable daylengths,and delay of initiation by gibberellic acid treatment were alsoobserved.     

Response of Neoseiulus fallacis Garmen and Galendromus occidentalis Nesbitt (Acari: Phytoseiidae) to Tetranychus urticae Koch (Acari: Tetranychidae)-Damaged Hop Humulus lupulus (L.) (Urticales: Cannabaceae)

Abstract 1 The response of Neoseiulus fallacis Garmen and Galendromus occidentalis (Acari: Phytoseiidae) to Tetranychus urticae Koch (Acari: Tetranychidae)‐damaged and undamaged hop, Humulus lupulus (L.), plants was tested using a Y‐tube olfactometer. 2 Neoseiulus fallacis but not G. occidentalis was attracted to volatiles from T. urticae‐damaged hop plants when paired with undamaged plants. 3 The response of N. fallacis to these volatiles was stronger for plants severed at the soil surface than for intact plants. 4 There was no difference in the response of N. fallacis to severed or intact hop plants that had no spider mite damage, indicating that artificial wounding by severing alone does not elicit the production of attractive volatiles detectable to N. fallacis. These results are consistent with the existence of cross‐talk between signalling pathways initiated by feeding damage and artificial wounding that result in elevated levels of predator‐attracting volatiles.     

NMR assignment of iso-alpha-acids from isomerised extracts of Humulus lupulus L. cones

Iso-alpha-acids are known to contribute to the characteristic bitter taste of beer. Six iso-alpha-acids were isolated from a carbon dioxide extract of the cones of Humulus lupulus L. by centrifugal partition chromatography followed by separation through beta-cyclodextrin binding. This method overcame the low yield issue of most isolation procedures that results from the low stability of these compounds to light and oxygen. Their full identification was performed using one- and two-dimensional NMR spectrometry, including (1)H- and (13)C-NMR, (1)H-(1)H COSY, HMQC and HMBC and electrospray ionisation mass spectrometry. The results confirmed the structures of the isolated compounds as trans-isocohumulone, cis-isocohumulone, trans-isohumulone, cis-isohumulone, trans-isoadhumulone and cis-isoadhumulone. Epimers can be easily distinguished by observing the chemical shift differences of the H-5, H-1', H-2' and C-5 signals and the different splitting pattern of H-5 and H-2'.     

High throughput isolation of microsatellites in hop (Humulus lupulus L.)

Procedures to generate usable microsatellite marker sequences should be optimized for cost-effectiveness in each species. For hop, we have used a cocktail of several restriction enzymes to digest the genomic DNA. This is followed by capture of microsatellite-containing sequences with long microsatellite probes attached to a membrane. The enrichment level for GA and GT libraries was 37% and 35%, respectively, and 100% of the clones contained microsatellite sequences. Libraries can be generated from genomic DNA in approximately 10 d.     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

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.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

Preparation of green colorimetric pH sensor using Humulus lupulus L. (common hop) biomolecular extract for sweat monitoring

Novel smart cotton diagnostic assay was developed toward onsite sensing of sweat pH variations for possible medical applications such as drug test and healthcare purposes. Humulus lupulus L. extract was obtained according to previously reported procedure. As reported by high-performance liquid chromatography (HPLC), the extract demonstrated the presence of hop acids, prenylchalcones, and prenylflavanones, which is responsible for the colorimetric changes. The extract was applied to cellulose fibers employing potassium aluminum sulfate as mordant. This was observed by the formation of mordant/xanthohumol nanoparticles onto cotton surface. The absorption spectra and CIE (Commission Internationale de l'Eclairage) Lab screening of the prepared cotton assay showed colorimetric changes in association with hypsochromic shift from 600 nm to 433 nm upon exposure to sweat simulant fluid (pH < 7). The biochromic activity of the xanthohumol-finished cotton depends mainly on the halochromic performance of the xanthohumol chromophore to show a colorimetric switch from yellow to white owing to intramolecular charge transfer in the xanthohumol molecule. No substantial defects were detected in gas-permeability and stiffness of the treated fabrics. Satisfactory fastness was approved for the xanthohumol-dyed diagnostic cotton assay.