"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

The multifaceted roles of R2R3 transcription factor HlMYB7 in the regulation of flavonoid and bitter acids biosynthesis, development and biotic stress tolerance in hop (Humulus lupulus L.).

Hop (Humulus lupulus) biosynthesizes the highly economically valuable secondary metabolites, which include flavonoids, bitter acids, polyphenols and essential oils. These compounds have important pharmacological properties and are widely implicated in the brewing industry owing to bittering flavor, floral aroma and preservative activity. Our previous studies documented that ternary MYB-bHLH-WD40 (MBW) and binary WRKY1-WD40 (WW) protein complexes transcriptionally regulate the accumulation of bitter acid (BA) and prenylflavonoids (PF). In the present study, we investigated the regulatory functions of the R2R3-MYB repressor HlMYB7 transcription factor, which contains a conserved N-terminal domain along with the repressive motif EAR, in regulating the PF- and BA-biosynthetic pathway and their accumulation in hop. Constitutive expression of HlMYB7 resulted in transcriptional repression of structural genes involved in the terminal steps of biosynthesis of PF and BA, as well as stunted growth, delayed flowering, and reduced tolerance to viroid infection in hop. Furthermore, yeast two-hybrid and transient reporter assays revealed that HlMYB7 targets both PF and BA pathway genes and suppresses MBW and WW protein complexes. Heterologous expression of HlMYB7 leads to down-regulation of structural genes of flavonoid pathway in Arabidopsis thaliana, including a decrease in anthocyanin content in Nicotiana tabacum. The combined results from functional and transcriptomic analyses highlight the important role of HlMYB7 in fine-tuning and balancing the accumulation of secondary metabolites at the transcriptional level, thus offer a plausible target for metabolic engineering in hop.

Growth regulators and their reflection on different hop genotypes cultivated under in vitro conditions / Reguladores de crescimento e seu reflexo em diferentes genótipos de lúpulo cultivados in vitro

Hops is a new culture in Brazil. Tissue culture can be an important technique for rapid hop propagation. This paper aims to characterize responses from different genotypes under different growth regulators through the interrelationship of response variables important to hop in vitro growth. Three genotypes were cultivated in six culture media with different combinations of growth regulators, BAP (6-benzylaminopurine), IAA (3-indolacetic acid) and GA3 (gibberellic acid). The means were compared by orthogonal contrasts and the interrelationship of the response variables was performed by path analysis. American genotypes showed favorable root development under the BAP + IAA combination, while the use of IAA improved shoot development. The origin of genotypes was important for defining the best protocol for in vitro cultivation. The path coefficient showed that the variable number of shoots has stronger direct effect on the number of nodal segments. Additionally, in tissue culture assays, the use of a covariable and proper error distribution significantly increased experimental accuracy.

O lúpulo é uma nova cultura no Brasil. A cultura de tecidos pode ser uma técnica importante para a propagação rápida do lúpulo. Este artigo tem como objetivo caracterizar respostas de diferentes genótipos sob diferentes reguladores de crescimento através da inter-relação de variáveis de resposta importantes para o crescimento in vitro. Três genótipos foram cultivados em seis meios de cultura com diferentes combinações de reguladores de crescimento, BAP (6-benzilaminopurina), AIA (ácido 3-indolacético) e GA3 (ácido giberélico). As médias foram comparadas por contrastes ortogonais e a inter-relação das variáveis de resposta foi realizada por análise de trilha. Os genótipos americanos apresentaram desenvolvimento radicular favorável sob a combinação BAP + AIA, enquanto o uso do AIA melhorou o desenvolvimento da parte aérea. A origem dos genótipos foi importante para definir o melhor protocolo para o cultivo in vitro. O coeficiente de trilha mostrou que a variável número de brotos tem um efeito direto mais forte no número de segmentos nodais. Adicionalmente, em experimentos com cultura de tecidos, o uso de uma covariável e distribuição de erro adequada aumentou significativamente a precisão experimental.

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.

Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants.

The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five viroid species (Apple fruit crinkle viroid (AFCVd), Citrus bark cracking viroid (CBCVd), Hop latent viroid (HLVd), Hop stunt viroid (HSVd), and Potato spindle tuber viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different viroid species, suggesting that the MED expression is viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.

Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae.

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

Enabling Heterologous Synthesis of Lupulones in the Yeast Saccharomyces cerevisiae.

Lupulones, naturally produced by glandular trichomes of hop (Humulus lupulus), are prenylated phloroglucinol derivatives that contribute the bitter flavor of beer and demonstrate antimicrobial and anticancer activities. It is appealing to develop microbial cell factories such that lupulones may be produced via fermentation technology in lieu of extraction from limited plant resources. In this study, the yeast Saccharomyces cerevisiae transformants harboring a synthetic lupulone pathway that consisted of five genes from hop were constructed. The transformants accumulated several precursors but failed to accumulate lupulones. Overexpression of 3-hydroxy-3-methyl glutaryl co-enzyme A reductase, the key enzyme in precursor formation in the mevalonate pathway, also failed to achieve a detectable level of lupulones. To decrease the consumption of the precursors, the ergosterol biosynthesis pathway was chemically downregulated by a small molecule ketoconazole, leading to successful production of lupulones. Our study demonstrated a combination of molecular biology and chemical biology to regulate the metabolism for heterologous production of lupulones. The strategy may be valuable for future engineering microbial process for other prenylated natural products.

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages.

Aroma compounds provide attractiveness and variety to alcoholic beverages. We discuss the molecular biology of a major subset of beer aroma volatiles, fruity and floral compounds, originating from raw materials (malt and hops), or formed by yeast during fermentation. We introduce aroma perception, describe the most aroma-active, fruity and floral compounds in fruits and their presence and origin in beer. They are classified into categories based on their functional groups and biosynthesis pathways: (1) higher alcohols and esters, (2) polyfunctional thiols, (3) lactones and furanones, and (4) terpenoids. Yeast and hops are the main sources of fruity and flowery aroma compounds in beer. For yeast, the focus is on higher alcohols and esters, and particularly the complex regulation of the alcohol acetyl transferase ATF1 gene. We discuss the release of polyfunctional thiols and monoterpenoids from cysteine- and glutathione-S-conjugated compounds and glucosides, respectively, the primary biological functions of the yeast enzymes involved, their mode of action and mechanisms of regulation that control aroma compound production. Furthermore, we discuss biochemistry and genetics of terpenoid production and formation of non-volatile precursors in Humulus lupulus (hops). Insight in these pathways provides a toolbox for creating innovative products with a diversity of pleasant aromas.

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.

Chemotyping of new hop (Humulus lupulus L.) genotypes using comprehensive two-dimensional gas chromatography with quadrupole accurate mass time-of-flight mass spectrometry.

Comprehensive two-dimensional gas chromatography with quadrupole accurate mass time-of-flight mass spectrometry (GC×GC-Q-TOFMS) is employed to profile Humulus lupulus L. (hop) essential oils. Comparison of characterised essential oils allows discrimination among chemotypes. Experimental and commercial hop genotypes displayed distinguishable chemotypic patterns among the volatile secondary metabolites making up their essential oils. In total, 210-306 unique compounds were detected (depending on specific genotype), with 99 of these compounds either positively or tentatively identified. Identified volatile secondary metabolites were grouped into esters, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes and ketones. Terpenoids were the dominant chemical families across all hop genotypes analysed, representing between 67% and 90% of the total ion count. The multidimensional chromatographic profiles of hop essential oils are extremely information-rich, making GC×GC-Q-TOFMS useful for fast screening of new hybrid hop genotypes, and therefore informing breeding strategies to derive new commercial hop cultivars for the development of distinctive and desirable beers.

Propagation and some physiological effects of Citrus bark cracking viroid and Apple fruit crinkle viroid in multiple infected hop (Humulus lupulus L.).

The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.

The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.).

Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

Cloning and molecular analysis of HlbZip1 and HlbZip2 transcription factors putatively involved in the regulation of the lupulin metabolome in hop (Humulus lupulus L.).

Hop (Humulus lupulus L.), the essential source of beer flavor is of interest from a medicinal perspective in view of its high content in health-beneficial terpenophenolics including prenylflavonoids. The dissection of biosynthetic pathway(s) of these compounds in lupulin glands, as well as its regulation by transcription factors (TFs), is important for efficient biotechnological manipulation of the hop metabolome. TFs of the bZIP class were preselected from the hop transcriptome using a cDNA-AFLP approach and cloned from a cDNA library based on glandular tissue-enriched hop cones. The cloned TFs HlbZIP1A and HlbZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively, and both are similar to the group A3 bZIP TFs according to the composition of characteristic domains. While HlbZIP1A is rather neutral (pI 6.42), HlbZIP2 is strongly basic (pI 8.51). A truncated variant of HlbZIP1 (HlbZIP1B), which is strongly basic but lacks the leucine zipper domain, has also been cloned from hop. Similar to the previously cloned HlMyb3 from hop, both bZIP TFs show a highly specific expression in lupulin glands, although low expression was observed also in other tissues including roots and immature pollen. Comparative functional analyses of HlbZip1A, HlbZip2, and subvariants of HlMyb3 were performed in a transient expression system using Nicotiana benthamiana leaf coinfiltration with Agrobacterium tumefaciens strains bearing hop TFs and selected promoters fused to the GUS reference gene. Both hop bZIP TFs and HlMyb3 mainly activated the promoters of chalcone synthase chs_H1 and the newly cloned O-methyl transferase 1 genes, while the response of the valerophenone synthase promoter to the cloned hop TFs was very low. These analyses also showed that the cloned bZIP TFs are not strictly G-box-specific. HPLC analysis of secondary metabolites in infiltrated Petunia hybrida showed that both hop bZIP TFs interfere with the accumulation and the composition of flavonol glycosides, phenolic acids, and anthocyanins, suggesting the possibility of coregulating flavonoid biosynthetic pathways in hop glandular tissue.

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.

HlMyb3, a putative regulatory factor in hop (Humulus lupulus L.), shows diverse biological effects in heterologous transgenotes.

A hop-specific cDNA library from glandular tissue-enriched hop cones was screened for Myb transcription factors. cDNA encoding for R2R3 Myb, designated HlMyb3, was cloned and characterized. According to the amino acid (aa) sequence, HlMyb3 shows the highest homology to GhMyb5 from cotton and is unrelated to the previously characterized HlMyb1 from the hop. Southern blot analyses indicated that HlMyb3 is a unique gene, which was detected in various Humulus lupulus cultivars, but not in Humulus japonicus. Reverse transcription and real-time PCR revealed the highest levels of HlMyb3 mRNA in hop cones at a late stage of maturation and in colored petiole epidermis, while the lowest levels were observed in hop flowers. Two alternative open reading frames starting in the N-terminal domain of HlMyb3, encoding for proteins having 269 and 265 amino acids with apparent molecular masses of 30.3 and 29.9 kDa, respectively, were analyzed as transgenes that were overexpressed in Arabidopsis thaliana, Nicotiana benthamiana, and Petunia hybrida plants. Transformation with the longer 269 aa variant designated l-HlMyb3 led to a flowering delay and to a strong inhibition of seed germination in A. thaliana. Nearly complete flower sterility, dwarfing, and leaf curling of P. hybrida and N. benthamiana l-HlMyb3 transgenotes were noted. On the contrary, the shorter 265-aa-encoding s-HlMyb3 transgene led in A. thaliana to the stimulation of initial seed germination, to fast initiation of the lateral roots, and to quite specific branching phenotypes with many long lateral stems formed at angles near 90 degrees . Limited plant sterility but growth stimulation and rather branched phenotypes were evident for s-HlMyb3 transgenotes of P. hybrida and N. benthamiana. It was found that both HlMyb3 transgenes interfere in the accumulation and composition of flavonol glycosides and phenolic acids in transformed plants. These effects on heterologous transgenotes suggest that the HlMyb3 gene may influence hop morphogenesis, as well as metabolome composition during lupulin gland maturation.

Sequence analysis of a "true" chalcone synthase (chs_H1) oligofamily from hop (Humulus lupulus L.) and PAP1 activation of chs_H1 in heterologous systems.

Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.

Comparative chemical attributes of native North American hop, Humulus lupulus var. lupuloides E. Small.

The genetic diversity of 159 representative genotypes of native hop (Humulus lupulus var. lupuloides E. Small, Cannabaceae) from 34 selected populations was assessed by relative magnitudes and ranges of alpha acids (AA), beta acids (BA), and the cohumulone (CoH) component of alpha acids, with reference to temporal changes between 1989-1990 and 2001, and to the same attributes in American and European hop cultivars, principally H. lupulus var. lupulus L. Chemical profiles of these genotypes were generated by high pressure liquid chromatography (HPLC) of methanol extracts from their processed samples (cones). The alpha ratio (AR, alpha acids / alpha+beta acids) measured the degree to which alpha acids predominated in cone extracts. Synchronous ranges of AR and CoH were also selected for graphic portrayals of native hop genotypic diversity. Cones sampled and analyzed from eight populations that were accessible in both 1989 and 2001 were distinct in chemical attributes, indicating a succession of genotypes, and suggesting temporal cycling of H. lupulus var.lupuloides germplasm. The principal distinctions between the two sub-species were a markedly higher proportion of CoH (38-88% vs. 19-41%) in alpha acids of H. l. var. lupuloides, and generally higher concentrations of AA in cultivars of both American and European commercial hop cultivars, predominantly H. lupulus var. lupulus. All of the 159 native hop genotypes also contained detectable levels of xanthohumol and xanthogalenol, prenylflavonoids recently reported to have mammalian anti-cancer activity. Some native genotypes had previously exhibited natural repellence of insect and mite pests; thus H. lupulus var. lupuloides germplasm offers a diverse resource of underutilized and yet undefined biochemicals.

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.