Most cited article - PubMed ID 27392499
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.)
Hop (Humulus lupulus L.) is an emblematic industrial crop in the French North East region that developed at the same time as the brewing activity. Presently, this sector, especially microbreweries, are interested in endemic wild hops, which give beer production a local signature. In this study, we investigated the genetic and metabolic diversity of thirty-six wild hops sampled in various ecological environments. These wild accessions were propagated aeroponically and cultivated under uniform conditions (the same soil and the same environmental factors). Our phytochemical approach based on UHPLC-ESI-MS/MS analysis led to the identification of three metabolic clusters based on leaf content and characterized by variations in the contents of twelve specialized metabolites that were identified (including xanthohumol, bitter acids, and their oxidized derivatives). Furthermore, molecular characterization was carried out using sixteen EST-SSR microsatellites, allowing a genetic affiliation of our wild hops with hop varieties cultivated worldwide and wild hops genotyped to date using this method. Genetic proximity was observed for both European wild and hop varieties, especially for Strisselspalt, the historical variety of our region. Finally, our findings collectively assessed the impact of the hop genotype on the chemical phenotype through multivariate regression tree (MRT) analysis. Our results highlighted the 'WRKY 224' allele as a key discriminator between high- and low-metabolite producers. Moreover, the model based on genetic information explained 40% of the variance in the metabolic data. However, despite this strong association, the model lacked predictive power, suggesting that its applicability may be confined to the datasets analyzed.
- MeSH
- Phenotype MeSH
- Genetic Variation * MeSH
- Genotype MeSH
- Humulus * genetics metabolism classification chemistry MeSH
- Plant Leaves genetics metabolism chemistry MeSH
- Microsatellite Repeats genetics MeSH
- Tandem Mass Spectrometry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
Viroids are small, non-coding, pathogenic RNAs with a significant ability of adaptation to several basic cellular processes in plants. TFIIIA-7ZF, a splicing variant of transcription factor IIIA, is involved in replication of nuclear-replicating viroids by DNA-dependent polymerase II. We overexpressed NbTFIIIA-7ZF from Nicotiana benthamiana in tobacco (Nicotiana tabacum) where it caused morphological and physiological deviations like plant stunting, splitting of leaf petioles, pistils or apexes, irregular branching of shoots, formation of double-blade leaves, deformation of main stems, and modification of glandular trichomes. Plant aging and senescence was dramatically delayed in transgenic lines. Factors potentially involved in viroid degradation and elimination in pollen were transiently depressed in transgenic leaves. This depressed "degradome" in young plants involved NtTudor S-like nuclease, dicers, argonoute 5, and pollen extracellular nuclease I showing expression in tobacco anthers and leaves. Analysis of the "degradome" in tobacco leaves transformed with either of two hop viroids confirmed modifications of the "degradome" and TFIIIA expression. Thus, the regulatory network connected to TFIIIA-7ZF could be involved in plant pathogenesis as well as in viroid adaptation to avoid its degradation. These results support the hypothesis on a significant impact of limited TFIIIA-7ZF on viroid elimination in pollen.
- Keywords
- Nicotiana tabacum, nucleolytic enzymes, plant aging, plant morphology changes, plant transformation, transcription factors, viroid,
- MeSH
- RNA, Small Untranslated * MeSH
- Pollen genetics MeSH
- Nicotiana genetics MeSH
- Transcription Factor TFIIIA MeSH
- Tobacco Use MeSH
- Viroids * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- RNA, Small Untranslated * MeSH
- Transcription Factor TFIIIA MeSH
BACKGROUND: Hop (Humulus lupulus L.) bitter acids are valuable metabolites for the brewing industry. They are biosynthesized and accumulate in glandular trichomes of the female inflorescence (hop cone). The content of alpha bitter acids, such as humulones, in hop cones can differentiate aromatic from bitter hop cultivars. These contents are subject to genetic and environmental control but significantly correlate with the number and size of glandular trichomes (lupulin glands). RESULTS: We evaluated the expression levels of 37 genes involved in bitter acid biosynthesis and morphological and developmental differentiation of glandular trichomes to identify key regulatory factors involved in bitter acid content differences. For bitter acid biosynthesis genes, upregulation of humulone synthase genes, which are important for the biosynthesis of alpha bitter acids in lupulin glands, could explain the higher accumulation of alpha bitter acids in bitter hops. Several transcription factors, including HlETC1, HlMYB61 and HlMYB5 from the MYB family, as well as HlGLABRA2, HlCYCB2-4, HlZFP8 and HlYABBY1, were also more highly expressed in the bitter hop cultivars; therefore, these factors may be important for the higher density of lupulin glands also seen in the bitter hop cultivars. CONCLUSIONS: Gene expression analyses enabled us to investigate the differences between aromatic and bitter hops. This study confirmed that the bitter acid content in glandular trichomes (lupulin glands) is dependent on the last step of alpha bitter acid biosynthesis and glandular trichome density.
- Keywords
- Bitter acids, Differential gene expression, Glandular trichome development, Hop, Humulus lupulus, Lupulin gland,
- MeSH
- Humulus metabolism MeSH
- Transcription Factors metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Transcription Factors MeSH
Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that "forcing" overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.
Viroids are small infectious pathogens, composed of a short single-stranded circular RNA. Hop (Humulus lupulus L.) plants are hosts to four viroids from the family Pospiviroidae. Hop latent viroid (HLVd) is spread worldwide in all hop-growing regions without any visible symptoms on infected hop plants. In this study, we evaluated the influence of HLVd infection on the content and the composition of secondary metabolites in maturated hop cones, together with gene expression analyses of involved biosynthesis and regulation genes for Saaz, Sládek, Premiant and Agnus cultivars. We confirmed that the contents of alpha bitter acids were significantly reduced in the range from 8.8% to 34% by viroid infection. New, we found that viroid infection significantly reduced the contents of xanthohumol in the range from 3.9% to 23.5%. In essential oils of Saaz cultivar, the contents of monoterpenes, terpene epoxides and terpene alcohols were increased, but the contents of sesquiterpenes and terpene ketones were decreased. Secondary metabolites changes were supported by gene expression analyses, except essential oils. Last-step biosynthesis enzyme genes, namely humulone synthase 1 (HS1) and 2 (HS2) for alpha bitter acids and O-methytransferase 1 (OMT1) for xanthohumol, were down-regulated by viroid infection. We found that the expression of ribosomal protein L5 (RPL5) RPL5 and the splicing of transcription factor IIIA-7ZF were affected by viroid infection and a disbalance in proteosynthesis can influence transcriptions of biosynthesis and regulatory genes involved in of secondary metabolites biosynthesis. We suppose that RPL5/TFIIIA-7ZF regulatory cascade can be involved in HLVd replication as for other viroids of the family Pospiviroidae.
- Keywords
- HLVd, Humulus lupulus, bitter acids content, differential gene expression, essential oils, hop, hop latent viroid, xanthohumol,
- Publication type
- Journal Article MeSH
Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.
- Keywords
- AFCVd and CBCVd propagation and eradication, Nicotiana tabacum, TUDOR S-nuclease, male gametophyte, recombinant AGO, small RNA, strand-specific viroid RT-qPCR, viroid degradation, viroid replication,
- MeSH
- Phenotype MeSH
- Host-Pathogen Interactions MeSH
- Nucleic Acid Conformation MeSH
- Plant Diseases virology MeSH
- Pollen virology MeSH
- Virus Replication MeSH
- RNA, Viral MeSH
- Nicotiana virology MeSH
- Viroids * MeSH
- Viral Load MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- RNA, Viral MeSH
The hop plant (Humulus lupulus L.) produces several valuable secondary metabolites, such as prenylflavonoid, bitter acids, and essential oils. These compounds are biosynthesized in glandular trichomes (lupulin glands) endowed with pharmacological properties and widely implicated in the beer brewing industry. The present study is an attempt to generate exhaustive information of transcriptome dynamics and gene regulatory mechanisms involved in biosynthesis and regulation of these compounds, developmental changes including trichome development at three development stages, namely leaf, bract, and mature lupulin glands. Using high-throughput RNA-Seq technology, a total of 61.13, 50.01, and 20.18 Mb clean reads in the leaf, bract, and lupulin gland libraries, respectively, were obtained and assembled into 43,550 unigenes. The putative functions were assigned to 30,996 transcripts (71.17%) based on basic local alignment search tool similarity searches against public sequence databases, including GO, KEGG, NR, and COG families, which indicated that genes are principally involved in fundamental cellular and molecular functions, and biosynthesis of secondary metabolites. The expression levels of all unigenes were analyzed in leaf, bract, and lupulin glands tissues of hop. The expression profile of transcript encoding enzymes of BCAA metabolism, MEP, and shikimate pathway was most up-regulated in lupulin glands compared with leaves and bracts. Similarly, the expression levels of the transcription factors and structural genes that directly encode enzymes involved in xanthohumol, bitter acids, and terpenoids biosynthesis pathway were found to be significantly enhanced in lupulin glands, suggesting that production of these metabolites increases after the leaf development. In addition, numerous genes involved in primary metabolism, lipid metabolism, photosynthesis, generation of precursor metabolites/energy, protein modification, transporter activity, and cell wall component biogenesis were differentially regulated in three developmental stages, suggesting their involvement in the dynamics of the lupulin gland development. The identification of differentially regulated trichome-related genes provided a new foundation for molecular research on trichome development and differentiation in hop. In conclusion, the reported results provide directions for future functional genomics studies for genetic engineering or molecular breeding for augmentation of secondary metabolite content in hop.
- Keywords
- Humulus lupulus, RNA sequencing, bitter acids, lupulin glands, prenylflavonoids, terpenoids, trichome,
- MeSH
- Flavonoids biosynthesis chemistry metabolism MeSH
- Gene Ontology MeSH
- Humulus chemistry metabolism MeSH
- Plant Leaves genetics metabolism MeSH
- Propiophenones chemistry metabolism MeSH
- Gene Expression Regulation, Plant MeSH
- Plant Proteins genetics metabolism MeSH
- RNA-Seq MeSH
- Terpenes chemistry metabolism MeSH
- Transcription Factors metabolism MeSH
- Transcriptome genetics MeSH
- Trichomes genetics metabolism ultrastructure MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Flavonoids MeSH
- Propiophenones MeSH
- Plant Proteins MeSH
- Terpenes MeSH
- Transcription Factors MeSH
- xanthohumol MeSH Browser
Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.
- Keywords
- Citrus bark cracking viroid, differentially expressed genes, hop, pathogen, transcriptome analysis, viroids,
- MeSH
- Humulus genetics metabolism virology MeSH
- Plant Leaves genetics metabolism virology MeSH
- Plant Diseases genetics virology MeSH
- Gene Expression Regulation, Plant MeSH
- Plant Proteins genetics metabolism MeSH
- Plant Viruses genetics isolation & purification physiology MeSH
- Gene Expression Profiling MeSH
- Viroids classification genetics isolation & purification physiology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Plant Proteins MeSH
BACKGROUND: The hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression. RESULTS: The transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines. CONCLUSIONS: Taken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.
- Keywords
- Bitter acids, Flavonoids, Genetic transformation, Humulus lupulus, Secondary metabolite, Transcription factors, Transcriptome analysis,
- MeSH
- Molecular Sequence Annotation MeSH
- Gene Expression MeSH
- Plants, Genetically Modified MeSH
- Genomics * MeSH
- Humulus genetics MeSH
- Plant Proteins genetics MeSH
- Gene Expression Profiling * MeSH
- Transcription Factors genetics MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Plant Proteins MeSH
- Transcription Factors MeSH