Project description:Transcriptional profiling of hop samples throughout in vitro culture on medium with sucrose and hormones IAA and BAP Study of gene expression involved in stress response during in vitro culture and in organogenic nodule formation Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. To investigate the spatio-temporal sequence of events that underlies competence acquisition for organogenesis three main stages were selected: (i) internodes at the time of excision from the parent plant (T0); (ii) internodes grown for 15 days on culture medium in which several prenodular structures are formed inside the calli (T15d); and (iii) nodule forming tissue after 28 days of culture (T28d). Additionally a control was included corresponding to 28 days of culture without hormones, lacking nodule formation and plantlet regeneration abilities, in order to identify genes specifically involved in morphogenesis (T28dWH). Organogenic nodule formation is a morphogenic process that shares features with somatic embryogenesis though in the former no shoot/root pole is established and plantlet regeneration can occur from different peripheral regions of nodules. Previous studies have shown that the organogenesis-determining period (when cells are determined to form nodules) occurs in between 15 and 25 days of culture corresponding to prenodular and first nodular stages. When nodules are fully developed they are surrounded by layers of elongated, highly vacuolated cells that may degenerate when nodules start differentiating plantlets. Plantlet regeneration from organogenic nodules can be observed after 45 days of culture. However, organogenic nodules after 28 days of culture are already determined to undergo plantlet regeneration, since they no longer require exogenous supply of hormones. Explants cultured in medium without hormones can develop incipient prenodular structures with vascular tissue but not nodules, thus lacking morphogenic potential. Keywords: Plant development, Time course

Project description:Transcriptional profiling of hop samples throughout in vitro culture on medium with sucrose and hormones IAA and BAP Study of gene expression involved in stress response during in vitro culture and in organogenic nodule formation Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. To investigate the spatio-temporal sequence of events that underlies competence acquisition for organogenesis three main stages were selected: (i) internodes at the time of excision from the parent plant (T0); (ii) internodes grown for 15 days on culture medium in which several prenodular structures are formed inside the calli (T15d); and (iii) nodule forming tissue after 28 days of culture (T28d). Additionally a control was included corresponding to 28 days of culture without hormones, lacking nodule formation and plantlet regeneration abilities, in order to identify genes specifically involved in morphogenesis (T28dWH). Organogenic nodule formation is a morphogenic process that shares features with somatic embryogenesis though in the former no shoot/root pole is established and plantlet regeneration can occur from different peripheral regions of nodules. Previous studies have shown that the organogenesis-determining period (when cells are determined to form nodules) occurs in between 15 and 25 days of culture corresponding to prenodular and first nodular stages. When nodules are fully developed they are surrounded by layers of elongated, highly vacuolated cells that may degenerate when nodules start differentiating plantlets. Plantlet regeneration from organogenic nodules can be observed after 45 days of culture. However, organogenic nodules after 28 days of culture are already determined to undergo plantlet regeneration, since they no longer require exogenous supply of hormones. Explants cultured in medium without hormones can develop incipient prenodular structures with vascular tissue but not nodules, thus lacking morphogenic potential. Keywords: Plant development, Time course 3 time points: 3 Biological replicates and 1 technical replicate (1 dye-swap). 1 control at beginning of culture and 1 control wihout hormones. One replicate per array. Each clone printed twice in the same sub-grid

Project description:BACKGROUND: Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high ?-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. RESULTS: Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial) clades. CONCLUSIONS: Our analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. This study provides evidence for the lupulin gland-specific biosynthesis of BCAAs and prenyl diphosphates to provide precursors for the production of bitter acids. The biosynthetic pathway leading to BCAAs in lupulin glands involves the plastidial enzyme, HlBCAT2. The mitochondrial enzyme HlBCAT1 degrades BCAAs as the first step in the catabolic pathway leading to branched chain-acyl-CoAs.

Project description:Menopause is a critical stage in every woman's life. It can cause a distressing time for women by creating various vasomotor symptoms (VMS). Phytoestrogens can potentially exert various favourable effects and alleviate VMS in postmenopausal women. The hop (Humulus lupulus L.) contains 8-prenylnaringenin (8-PN), the most potent phytoestrogen known to date. The hop is eight times stronger than any other herbal oestrogens. This study aims to conduct a comprehensive systematic review and a meta-analysis survey of the effects of hop in the management of VMS in postmenopausal women.Only randomised controlled clinical trials, with cluster randomisation and crossover, blinded and non-blinded designs, conducted between 2000 and 2015, will be included in this review. Quasi-experimental and observational studies as well as case reports will be excluded. The studies will be selected if their participants were aged 40-60 years, had elevated follicle-stimulating hormone (FSH) levels and/or menstrual irregularities, and experienced discomforting VMS (at least hot flashes or night sweats). The primary outcome will be the rate of response to treatment, such as changes in frequency and intensity of symptoms in the intervention and placebo groups. 'Hop', 'Humulus', 'menopause', 'vasomotor', 'hot flashes', 'phytoestrogen' and 'night sweats' will be used as search key words. Prior to their inclusion in the review, the selected papers will be assessed by two independent reviewers for methodological validity. Any disagreements will be resolved through a third reviewer. The risk of bias will be independently determined using the Cochrane Risk of Bias Tool. The quality of the papers will be assessed based on the CONSORT checklist.Results will be disseminated through traditional academic literature. Dissemination of results will occur by peer-reviewed publications. The results of our project can help reproductive health researchers when evaluating the discomforts of research procedures described in study protocols or when designing a study. Information on experiences of menopausal women involved in previous studies may also help in future research. The expected dissemination actions are effective treatment in designing strategies that aim to develop women's health and healthcare providers when offering treatment for women with vasomotor symptoms.

Project description:Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of standardization of botanical extracts to multiple chemical markers for both safety and desired bioactivity.

Dataset Information

Organogenic nodule development in hop (Humulus lupulus L.): transcript and metabolic responses.

Publications

Organogenic nodule development in hop (Humulus lupulus L.): transcript and metabolic responses.

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