Increase in cone biomass and terpenophenolics in hops ( Humulus lupulus L.) by treatment with prohexadione-calcium

Humulus lupulus L. (hop), a specialty crop bred for flavor characteristics of the inflorescence, is an essential ingredient in beer. Hop inflorescences, commonly known as hop cones, contain terpenophenolic compounds, which are important for beer flavoring and of interest in biomedical research. Hop breeders focus their efforts on increasing cone biomass and terpenophenolic content. As an alternative to traditional breeding, hops were treated with prohexadione-calcium (Pro-Ca), a growth inhibitor previously shown to have positive agronomic effects in several crops. Application of Pro-Ca to hop plants during cone maturation induced increases in cone biomass production by 1.5-19.6% and increased terpenophenolic content by 9.1-87.3%; however, some treatments also induced significant decreases in terpenophenolic content. Induced changes in cone biomass production and terpenophenolic accumulation were most dependent on cultivar and the developmental stage at which plants were treated.     

Formation and accumulation of alpha-acids,beta-acids,desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.)

Important secondary metabolites, present in hops (Humulus lupulus L.), include alpha-acids and beta-acids, which are essential for the brewing of beer, as well as the prenylated chalcones, desmethylxanthohumol, and xanthohumol, which exhibit interesting bioactive properties. Their formation and accumulation in five selected hop varieties, Wye Challenger, Wye Target, Golding, Admiral, and Whitbread Golding Variety, were quantitatively monitored by high-performance liquid chromatography using UV detection. All target compounds were present from the onset of flowering, not only in female hop cones but also in male inflorescences, albeit in low concentrations. During development from female inflorescences to cones, levels of alpha-acids, beta-acids, desmethylxanthohumol, and xanthohumol gradually increased, while each hop variety exhibited individual accumulation rates. Furthermore, these compounds were present in leaves of fully grown hops as well. The study demonstrated that key compounds for flavor and potential beneficial health effects associated with beer not only reside in the glandular lupulin structures but also are distributed over various parts of the hop plant.     

Phytochemical and morphological characterization of hop (Humulus lupulus L.) cones over five developmental stages using high performance liquid chromatography coupled to time-of-flight mass spectrometry, ultrahigh performance liquid chromatography photodiode array detection, and light microscopy techniques

Hop (Humulus lupulus L.) inflorescences, commonly known as "hop cones", are prized for their terpenophenolic contents, used in beer production and, more recently, in biomedical applications. In this study we investigated morphological and phytochemical characteristics of hop cones over five developmental stages, using liquid chromatography coupled to time-of-flight mass spectrometry (LC-TOF-MS), and ultrahigh performance liquid chromatography photodiode array detection (UHPLC-PDA) methods to quantitate 21 polyphenolics and seven terpenophenolics. Additionally, we used light microscopy to correlate phytochemical quantities with changes in the morphology of the cones. Significant increases in terpenophenolics, concomitant with glandular trichome development and associated gross morphological changes, were mapped over development to fluctuations in contents of polyphenolic constituents and their metabolic precursor compounds. The methods reported here can be used for targeted metabolic profiling of flavonoids, phenolic acids, and terpenophenolics in hops, and are applicable to quantitation in other crops.     

Constitutive expression of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its derivatives in substantial quantities

Resveratrol, a well-known phytoalexin and antioxidant, is produced by the action of stilbene synthase (STS) in some plant species. Hop (Humulus lupulus L.) plants of the Tettnang variety were transformed with a gene encoding for STS from grapevine. Under the control of the constitutive 35S cauliflower mosaic virus promoter, expression of the transgene resulted in accumulation of resveratrol and high levels of its glycosylated derivatives in leaves and inflorescences. Piceid, the predominant derivative, reached a concentration of up to 560 microg/g of fresh weight (f.w.) in hop cones, whereas no stilbenes were detected in nontransformed controls (wild-type). In transgenic plants the amounts of alpha- and beta-acids, naringenin chalcone, and prenylated flavonoids did not change significantly when compared with nontransformed plants. Transgenic plants showed normal morphology and flower development as did the nontransformed controls. The results clearly show that in hop constitutive expression of sts interferes neither with plant development nor with the biosynthesis of secondary metabolites relevant for the brewing industry. Since resveratrol is a well-known phytoalexin and antioxidant, sts transgenic hop plants could display enhanced pathogen resistance against microbial pathogens, exhibit new beneficial properties for health, and open new venues for metabolic engineering.     

Fate of resveratrol and piceid through different hop processings and storage times

Trans-Piceid and trans-resveratrol contents of hop cones, hop pellets, CO2 extracts, and spent hop from American varieties (harvest 2004) were determined by reverse-phase high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry [RP-HPLC-APCI(+)-MS/MS]. Pelletization induced strong stilbene degradation in some cultivars. Similarly, 1 year of storage at 4 degrees C led to a huge loss of trans-piceid, especially in the case of hop cones (much faster than in model media, although well protected from light and oxygen). Therefore, after 8 months of storage, the overall stilbene content was in the same range whatever the conditioned form. Absent in fresh hop cones or pellets, cis-resveratrol was released from cis-piceid in all stored samples. On the other hand, no delta-viniferin was detected despite it is present in light-protected model media spiked with trans-piceid. Because supercritical carbon dioxide proved inefficient for recovering resveratrol and piceid from pellets, spent hop emerged as the most interesting material for subsequent specific stilbene extraction.     

The impact of climate change on the yield and quality of Saaz hops in the Czech Republic

The impact of climate change on the production and quality of hops Humulus lupulus will depend on future weather conditions in the growing season. Our simulations suggest that hops will be particularly vulnerable to a change in climate. Even with the modest warming so far experienced yields have stagnated and quality declined. Recorded observations show an increase in air temperature which is associated with an earlier onset of hop phenological phases and a shortening of the vegetation period. Simulations using future climate predict a decline in both yields, of up to 7–10%, and α-acid content, of up to 13–32%, the latter a major determinant of quality. The concentration of hop cultivation in a comparatively small region in the Czech Republic makes it more vulnerable than if the crop were grown in more areas with different climates. Thus climate change may gradually lead to changes in the regionalization of hop production. Policy assistance may be necessary for the adaptation of the Czech hop growing industry to changed climatic conditions.     

Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades

The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.     

Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.     

Occurrence of odorant polyfunctional thiols in the Super Alpha Tomahawk hop cultivar. Comparison with the thiol-rich Nelson Sauvin bitter variety

Tomahawk hop (Humulus lupulus) is a recently developed Super Alpha cultivar (14-18% α-acids w/w), already widely used by brewers to impart bitterness and a citrus-like aroma to beer. By comparison with two bitter varieties (Nelson Sauvin and Nugget) and two aromatic ones (Cascade and Saaz), the Tomahawk cultivar showed a very particular terpenoid profile, rich in both α- and β-selinenes (>600 mg/kg IST equiv in total), methyl geranate (>40 mg/kg IST equiv), and geraniol (>200 mg/kg). Tomahawk also proved to contain a wide variety of odorant polyfunctional thiols. The major β-sulfanyl acetate, 3-sulfanyl-2-ethylpropyl acetate, newly identified here, was found at similar levels in the famous Sauvignon-like Nelson Sauvin and Tomahawk varieties (15-44 μg/kg IST equiv). On the other hand, lower levels of total β-sulfanyl alcohols were measured in Tomahawk, although 3-sulfanylhexan-1-ol was found at a similar level and the 3-sulfanyl-4-methylpentan-1-ol previously claimed to be specific to the Nelson Sauvin variety was also evidenced in the Super Alpha cultivar (9-13 μg/kg IST equiv). As revealed by boiling and fermentation, Tomahawk hop also contains very interesting bound polyfunctional thiols that should be investigated for better use by brewers.     

Microsatellite DNA Analysis of Wild Hops, Humulus lupulus L.

To study the relationships and genetic diversity among wild hops, Humulus lupulus, we analyzed 133 samples of wild hops collected from Europe, Asia and North America using polymorphism on 11 microsatellite loci. Although only three primers showed bands in Japanese hops, all other samples showed polymorphic bands at most loci. There were no duplicate genotypes among samples of European, Chinese and North American hops, and each individual hop could be distinguished completely. The phylogenetic tree constructed from DA distance with the UPGMA method showed a large cluster comprised of European hops, although Russian hops from the Caucasus and Altai regions were separate from the European cluster. Chinese and North American samples gave distinct clusters suggesting genetic differentiation. This study has indicated that hop microsatellite DNA is differentiated, and is dependent upon the origin in regions of Europe, Asia and North America.     

白菜NAC转录因子BrcCUC3基因克隆及其对叶缘和主枝发育的影响

植物NAC (矮牵牛NAM基因、拟南芥ATAF1/2和CUC2基因)转录因子CUP-SHAPED COTYLEDON(CUC)亚家族成员在植物茎尖分生组织形态建成、器官边界分离、叶发育方面发挥着重要作用.采用基因同源克隆的方法获得了白菜(Brassica rapa ssp.chinensis)基因BrcCUC3,并转化拟南芥(Arabidopsis thaliana)做了初步的功能鉴定.研究结果表明,白菜Brc CUC3的编码区长1 008 bp,编码335个氨基酸,基因结构分析显示BrcCUC3包含3个外显子、2个内含子,内含子剪接位点符合GT-AG规则.氨基酸序列分析表明,BrcCUC3蛋白具有典型的植物NAC domain结构域.BrcCUC3编码区氨基酸序列与其它植物的CUC3蛋白有很高的一致性,尤其与甘蓝(Brasica oleracea)、萝卜(Raphanus sativus)和拟南芥CUC3蛋白高度一致,一致性分别达到98％,97％和83％.在不同物种CUC3的系统进化树上,BrcCUC3归属于双子叶植物分支的十字花科亚组,由不同植物20条CUC3编码区氨基酸序列所建立的系统进化树与真实的植物进化基本一致.荧光定量PCR分析结果表明,BrcCUC3在白菜叶深裂株系叶片中的表达量比叶全缘叶片中的高.利用根癌农杆菌(A grobacterium tumefaciens)浸花法转化拟南芥,获得了转BrcCUC3基因的拟南芥植株.过表达BrcCUC3的转基因拟南芥呈现叶缘出现裂刻、主枝增加的新表型.初步说明该基因参与叶形和主枝的发育调控,为揭示白菜叶形发育分子调控机制和通过基因工程创制植物叶形新种质提供分子依据.     

Germplasm sources of protective glandular leaf trichomes of hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis>)

We report here that small but numerous foliar glands are present near the base of the abaxial leaf blades of hop (Humulus lupulus L.) at about 2.6 times the density found in the more distal regions, and this concentration of glands appear to protect the basal region from damage by insects. Distinctive wild taxa of H. lupulus are native to distinctive regions of the north temperate world. The much higher presence of protective glands in the three native North American Humulus taxonomic varieties in comparison with the varieties native to Europe and Asia suggests specialized leaf feeders in North America have been responsible for the evolution of greater gland density. Commercial domesticated hop cultivars trace most of their germplasm to the European hop (H. lupulus var. lupulus), which possesses a much lower concentration of leaf glands than the native hops of all other regions of the world, and therefore is likely relatively susceptible to damage. We suggest that breeding for increased density of protective foliage glands may be a non-chemical way of alleviating foliar feeding damage in cultivated hops.     

Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques

Application of aroma extract dilution analysis on the volatiles obtained from dried cones of Spalter Select hops grown in the German hop-growing area of Hallertau revealed 23 odorants in the flavor dilution (FD) factor range of 16-4096, 20 of which could be identified. On the basis of high FD factors, trans-4, 5-epoxy-(E)-2-decenal, linalool, and myrcene were identified as the most potent odorants, followed by ethyl 2-methylpropanoate, methyl 2-methylbutanoate, (Z)-1,5-octadien-3-one, nonanal, (E,Z)-1,3, 5-undecatriene, 1,3(E),5(Z),9-undecatetraene, propyl 2-methylbutanoate, 4-ethenyl-2-methoxyphenol, and 1-octen-3-one. Ten of the high-impact hop aroma compounds had previously not been identified as hop constituents and, in particular, 1,3(E),5(Z), 9-undecatetraene has not yet been reported as a food odorant. In an extract obtained from fresh hops, in addition to the odorants found in dry hops, (Z)-3-hexenal was characterized as a further key odorant rendering an additional green aroma note to the fresh material.     

拟南芥甘油二酯激酶(AtDGK7)基因抗逆的生理作用

构建表达载体PBI121-AtDGK7,转化拟南芥（Arabidopsis thaliana）获得过表达转基因植株。通过抗逆生理指标测定、ABA、NaCl和甘露醇抗性试验, 以及半定量RT-PCR方法,对拟南芥甘油二酯激酶(AtDGK7)基因的抗逆生理功能进行初步研究。结果表明,过表达AtDGK7转基因拟南芥对高浓度ABA的敏感性比野生型要低,能更好地抵御盐胁迫。通过测定脯氨酸(Pro)和丙二醛(MDA)含量以及超氧物歧化酶(SOD)活性,进一步证实过表达AtDGK7转基因拟南芥植株的耐低温能力增强。     

Identification and genetic structure of wild Italian <Emphasis Type="Italic">Humulus lupulus</Emphasis> L. and comparison with European and American hop cultivars using nuclear microsatellite markers

Nine genic SSR loci were used to evaluate the genetic diversity and identify accessions in wild Italian Humulus lupulus L., in comparison with widely cultivated European and U.S. commercial cultivars. A collection of 80 wild hop samples from Italy and 43 hop cultivars from Europe and U.S., were characterized. Allelic frequency analysis revealed 65 distinct Italian genotypes and differentiated all the commercial cultivars; moreover, specific alleles were observed for wild and cultivated hops. The number of alleles identified in the wild population were 104 and 123 within all the accessions. The maximum polymorphic information content was evidenced for locus HlGA23 in the Italian wild population and in the whole set of accessions (0.905 and 0.902 respectively). The dendrogram constructed from Euclidean distance with the UPGMA method showed two main clusters, one including commercial American and European accessions and one mostly composed by wild Italian accessions. Model-based clustering (Bayesian method) placed the accessions into five germplasm groups, one of which was characterized by Italian genotypes only. The study showed for the first time the great biodiversity present in Italy, and the remarkable differences with European and American hops. It was also found that within the population of north-central Italy a large genetic variability is present, suited to be studied and exploited; this genetic wealth could be used in future breeding programs in order to develop new hop varieties carrying characteristics useful for brewers.     

The effect of nickel and copper deposition from a mining and smelting complex on coniferous regeneration in the boreal forest of northern Manitoba

Airborne pollutants in the form of SO₂ and heavy metals have been dispersed over the boreal forest in the Thompson area of northern Manitoba since 1961. Metal deposition has been found in soils and plant material to a distance of 35 km from the Inco nickel smelter. Studies were initiated in 1979 to determine effects of Ni and Cu on coniferous regeneration of jack pine (Pinus banksiana Lamb.) and black spruce (Picea mariana (Mill) BSP.).Studies included metal accumulation in cones, effects on seed quality, seed germination and seedling response in metal contaminated soils. Nickel and Cu accumulation in cones of both species declined rapidly with distance from the smelter. Seed quality and germination potential was not related to metals in cones or air pollutant influence in general. Nickel and Cu accumulation in surface organic soils was inversely related to distance from the smelter. A significant inverse correlation was determined between seedling growth and Cu/Ni concentrations in surface organic soils. Metal accumulation in soils within 5 km of the nickel smelter inhibited root growth of conifer seedlings.     

Effects of beer and hop on ionotropic gamma-aminobutyric acid receptors

Beer induced the response of the ionotropic gamma-aminobutyric acid receptors (GABA(A) receptors) expressed in Xenopus oocytes, indicating the presence of gamma-aminobutyric acid (GABA)-like activity. Furthermore, the pentane extract of the beer, hop (Humulus lupulus L.) oil, and myrcenol potentiated the GABA(A) receptor response elicited by GABA. The GABA(A) receptor responses were also potentiated by the addition of aliphatic esters, most of which are reported to be present in beer flavor. Aliphatic esters showed the tendency to decrease in the potentiation of the GABA(A) receptor response with an increase in their carbon chain length. When myrcenol was injected to mice prior to intraperitoneal administration of pentobarbital, the pentobarbital-induced sleeping time of mice increased additionally. Therefore, the beer contained not only GABA-like activity but also the modulator(s) of the GABA(A) receptor response.     

Inheritance of alloplasmic fertility restoration in eggplant (Solanum melongena L.)

Cytoplasmic male sterility (CMS) system has been exploited worldwide in field and vegetable crops. In eggplant, alloplasmic CMS lines were developed through interspecific hybridization between Solanum aethiopicum L.?×?S. melongena L., while the restorer (R) lines were isolated from the reciprocal cross. The knowledge about inheritance of Rf gene is must for its further use in breeding and molecular studies. Therefore, four sets of CMS (D-CMS 291A, D-CMS 99A, D-CMS 5A and D-CMS 72A) and restorer (R 2-1, R 3-4, R 6-2 and R 2596-2) lines were used to develop F₁, F₂ and backcross progenies, to understand the inheritance mechanism. Phenotyping of all the populations and test of goodness of fit revealed involvement of a single dominant gene (Rf) for fertility restoration. The visual scoring of flowers for male sterility and fertility was further validated with the tests on pollen stainability, germination and index. Among others, media containing 0.5% agar?+?300 ppm calcium nitrate?+?5% sucrose?+?50 mg/l boric acid?+?400 mg/l PEG 4000 furnished the best results for in vitro pollen germination. Differences between and within male sterile and restorer lines were observed for pistil and stamen length and girth, pollen stainability and germination. Stable expression of CGMS and restorer lines in all the generation progenies confirmed their utility in future eggplant breeding programs.