This work benefited from the networking activities coordinated within the EU-funded COST ACTION FA1106 "An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine". This work was supported by the 'Completamento del Centro di Genomica Funzionale Vegetale' project funded by the CARIVERONA Bank Foundation and by the 'Valorizzazione dei Principali Vitigni Autoctoni Italiani e dei loro Terroir (Vigneto)' project funded by the Italian Ministry of Agricultural and Forestry Policies. SDS was financed by the Italian Ministry of University and Research FIRB RBFR13GHC5 project "The Epigenomic Plasticity of Grapevine in Genotype per Environment Interactions".

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B., Zamboni, A., Zenoni, S., Delledonne, M., Pezzotti, M., Ger&#243;s, H., Chaves, M., Delrot, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> The Biochemestry of the Grape Berry. 11, (2012).</li><li>Anesi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+scientific+interpretation+of+the+terroir+concept:+plasticity+of+the+grape+berry+metabolome.">Towards a scientific interpretation of the terroir concept: plasticity of the grape berry metabolome.</a> BMC Plant Biol. 15, 1-17 (2015).</li><li>Berdeja, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+limitation+and+rootstock+genotype+interact+to+alter+grape+berry+metabolism+through+transcriptome+reprogramming.">Water limitation and rootstock genotype interact to alter grape berry metabolism through transcriptome reprogramming.</a> Hort Res. 2, 1-13 (2015).</li><li>Carbonell-Bejerano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solar+ultraviolet+radiation+is+necessary+to+enhance+grapevine+fruit+ripening+transcriptional+and+phenolic+responses.">Solar ultraviolet radiation is necessary to enhance grapevine fruit ripening transcriptional and phenolic responses.</a> BMC Plant Biol. 14, 1-16 (2014).</li><li>Carbonell-Bejerano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+sampling+bias+in+molecular+studies+of+grapevine+fruit+ripening:+transcriptomic+assessment+of+the+density+sorting+method.">Reducing sampling bias in molecular studies of grapevine fruit ripening: transcriptomic assessment of the density sorting method.</a> Theor Exp Plant Phys. 28 (1), 109-129 (2016).</li><li>Carbonell-Bejerano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+oscillatory+transcriptional+programs+in+grapevine+ripening+fruits.">Circadian oscillatory transcriptional programs in grapevine ripening fruits.</a> BMC Plant Biol. 14, 1-15 (2014).</li><li>Cavallini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversification+of+grapevine+MYB5a+and+MYB5b+in+the+control+of+flavonoid+biosynthesis+in+a+petunia+anthocyanin+regulatory+mutant.">Functional diversification of grapevine MYB5a and MYB5b in the control of flavonoid biosynthesis in a petunia anthocyanin regulatory mutant.</a> Plant &amp; Cell Physiol. 55 (3), 517-534 (2014).</li><li>Cramer, G. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Grapevine+Anthocyanin+Acyltransferase,+Transcriptionally+Regulated+by+VvMYBA,+Can+Produce+Most+Acylated+Anthocyanins+Present+in+Grape+Skins.">A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins.</a> Plant Physiol. 169 (3), 1897-1916 (2015).</li><li>Royo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental,+transcriptome,+and+genetic+alterations+associated+with+parthenocarpy+in+the+grapevine+seedless+somatic+variant+Corinto+bianco.">Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco.</a> J Exp Bot. , 259-273 (2015).</li><li>Venturini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+transcriptome+characterization+of+Vitis+vinifera+cv.+Corvina+unveils+varietal+diversity.">De novo transcriptome characterization of Vitis vinifera cv. 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The authors have nothing to disclose.

This article describes the metabolomics, transcriptomics and statistical analysis protocols used to interpret the grape berry terroir concept. Metabolomics analysis by HPLC-ESI-MS is sensitive enough to detect large numbers of metabolites simultaneously, but relative quantitation is affected by the matrix effect and ion suppression/enhancement. However, a similar approach has already been used to describe the ripening and post-harvest withering of Corvina berries, and the correction of matrix effects had a limited impact...

Large-scale data analysis based on the genomes, transcriptomes, proteomes and metabolomes of plants provides unprecedented insight into the behavior of complex systems, such as the terroir characteristics of wine which reflect the interactions between grapevine plants and their environment. Because the terroir of a wine can be distinct even when identical grapevine clones are grown in different vineyards, genomics analysis is of little use because the clonal genomes are identical. Instead it is necessary to look at correlations between gene expression and the metabolic properties of the berries, which determine the quality traits of wine. The analysis of gene expression at the level of the transcriptome benefits from the similar chemical properties of all transcripts, which facilitates quantitative analysis by exploiting universal characteristics such as hybridization to immobilized probes on microarrays. In contrast, universal analytical methods in proteomics and metabolomics are more challenging because of the huge physical and chemical diversity of individual proteins and metabolites. In the case of metabolomics this diversity is even more extreme because individual metabolites differ vastly in size, polarity, abundance and volatility, so no single extraction process or analytical method offers a holistic approach.
Among the analytical platforms suitable for non-volatile metabolites, those based on high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) are much more sensitive than alternatives such as HPLC with ultraviolet or diode array detectors (HPLC-UV, HPLC-DAD) or nuclear magnetic resonance (NMR) spectroscopy, but quantitative analysis by HPLC-MS can be influenced by phenomena such as the matrix effect and ion suppression/enhancement1-3. The investigation of such effects during the analysis of Corvina grape berries by HPLC-MS using an electrospray ionization source (HPLC-ESI-MS), showed that sugars and other molecules with the lowest retention times were strongly underreported, probably also reflecting the large number of molecules in this zone, and that the abundance of other molecules could be underestimated, overestimated or unaffected by the matrix effect, but the data normalization for the matrix effect seemed to have limited impact on the overall results4,5. The method described herein is optimized for the analysis of medium-polarity metabolites that accumulate at high levels in grape berries during ripening, and which are significantly impacted by the terroir. They include anthocyanins, flavonols, flavan-3-ols, procyanidins, other flavonoids, resveratrol, stilbenes, hydroxycinnamic acids and hydroxybenzoic acids, which together determine the color, taste and health-related properties of wines. Other metabolites, such as sugars and aliphatic organic acids, are ignored because quantitation by HPLC-MS is unreliable due to the matrix effect and ion suppression phenomena5. Within the polarity range selected by this method, the approach is untargeted in that it aims to detect as many different metabolites as possible6.
Transcriptomics methods that allow thousands of grapevine transcripts to be monitored simultaneously are facilitated by the availability of the complete grapevine genome sequence7,8. Early transcriptomics methods based on high-throughput cDNA sequencing have evolved with the advent of next-generation sequencing into a collection of procedures collectively described as RNA-Seq technology. This approach is rapidly becoming the method of choice for transcriptomics studies. However, a large body of literature based on microarray, which allow thousands of transcripts to be quantified in parallel by hybridization, has accumulated for grapevine. Indeed, before RNA-Seq became a mainstream technology, many dedicated commercial microarray platforms had been developed allowing grapevine transcriptome to be inspected in great detail. Among the vast variety of platforms, only two allowed genome-wide transcriptome analysis9. The most evolved array allowed the hybridization of up to 12 independent samples on a single device, thus reducing the costs of each experiment. The 12 sub-arrays each comprised 135,000 60-mer probes representing 29,549 grapevine transcripts. This device has been used in a large number of studies10-24. These two platforms have now been discontinued but a new custom microarray has recently been designed and represents a more recent development as it contains an even greater number of probes representing additional newly discovered grapevine genes25.
The large-sale datasets produced by transcriptomics and metabolomics analysis require suitable statistical methods for data analysis, including multivariate techniques to determine correlations between different forms of data. The most widely used multivariate techniques are those based on projection, and these can be unsupervised, such as principal component analysis (PCA), or supervised, such as bidirectional orthogonal projection to latent structures discriminant analysis (O2PLS-DA)26. The protocol presented in this article utilizes PCA for exploratory data analysis and O2PLS-DA to identify differences between groups of samples.

<table><tbody><tr><td>Mill Grinder</td><td>IKA</td><td>IKA A11 basic</td><td></td></tr><tr><td>HPLC Autosampler</td><td>Beckman Coulter</td><td>&amp;nbsp;-</td><td>System Gold 508 Autosampler</td></tr><tr><td>HPLC System</td><td>Beckman Coulter</td><td>&amp;nbsp;-</td><td>System Gold 127 Solvent Module HPLC</td></tr><tr><td>C18 Guard Column</td><td>Grace</td><td>&amp;nbsp;-</td><td>Alltima HP C18 (7.5 mm x 2.1 mm; 5 &amp;mu;m) Guard Column</td></tr><tr><td>C18 Column</td><td>Grace</td><td>&amp;nbsp;-</td><td>Alltima HP C18 (150 mm x 2.1 mm; 3 &amp;mu;m) Column</td></tr><tr><td>Mass Spectometer</td><td>Bruker Daltonics</td><td>&amp;nbsp;-</td><td>Bruker Esquire 6000; The mass spectometer was equipped with an ESI source and the analyzer was an ion trap.</td></tr><tr><td>Extraction solvents and HPLC buffers</td><td>Sigma</td><td>34966</td><td>Methanol LC-MS grade</td></tr><tr><td>Sigma</td><td>94318</td><td>Formic acid LC-MS grade</td><td></td></tr><tr><td>Sigma</td><td>34967</td><td>Acetonitrile LC-MS grade</td><td></td></tr><tr><td>Sigma</td><td>39253</td><td>Water&amp;nbsp; LC-MS grade</td><td></td></tr><tr><td>Minisart RC 4 Syringe filters (0.2 &amp;mu;m)</td><td>Sartorius</td><td>17764</td><td></td></tr><tr><td>Softwares for data collection (a) and processing (b)</td><td>Bruker Daltonics</td><td>-</td><td>Bruker Daltonics Esquire 5.2 Control (a); Esquire 3.2 Data Analysis and MzMine 2.2 softwares (b)</td></tr><tr><td>Spectrum Plant Total RNA kit</td><td>Sigma-Aldrich</td><td>STRN250-1KT</td><td>For total RNA extractino from grape pericarps</td></tr><tr><td>Nanodrop 1000</td><td>Thermo Scientific</td><td>1000</td><td></td></tr><tr><td>BioAnalyzer 2100</td><td>Agilent Technologies</td><td>G2939A</td><td></td></tr><tr><td>RNA 6000 Nano Reagents</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>RNA Chips</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>Agilent Gene Expression Wash Buffer 1</td><td>Agilent Technologies</td><td>5188-5325</td><td></td></tr><tr><td>Agilent Gene Expression Wash Buffer 2</td><td>Agilent Technologies</td><td>5188-5326</td><td></td></tr><tr><td>LowInput QuickAmp Labeling kit One-Color</td><td>Agilent Technologies</td><td>5190-2305</td><td></td></tr><tr><td>Kit RNA Spike In - One-Color</td><td>Agilent Technologies</td><td>5188-5282</td><td></td></tr><tr><td>Gene Expression Hybridization Kit</td><td>Agilent Technologies</td><td>5188-5242</td><td></td></tr><tr><td>RNeasy Mini Kit (50)</td><td>Qiagen</td><td>74104</td><td>For cRNA Purification</td></tr><tr><td>Agilent SurePrint HD 4X44K 60-mer Microarray</td><td>Agilent Technologies</td><td>G2514F-048771&amp;nbsp;</td><td></td></tr><tr><td>eArray</td><td>Agilent Technologies</td><td>-</td><td>&lt;a href=&quot;https://earray.chem.agilent.com/earray/&quot;&gt;https://earray.chem.agilent.com/earray/&lt;/a&gt;</td></tr><tr><td>Gasket slides</td><td>Agilent Technologies</td><td>G2534-60012</td><td>Enable Agilent SurePrint Microarray 4-array Hybridization</td></tr><tr><td>Thermostatic bath</td><td>Julabo</td><td>-</td><td></td></tr><tr><td>Hybridization Chamber</td><td>Agilent Technologies</td><td>G2534-60001</td><td></td></tr><tr><td>Microarray Hybridization Oven</td><td>Agilent Technologies</td><td>G2545A</td><td></td></tr><tr><td>Hybridization Oven Rotator Rack</td><td>Agilent Technologies</td><td>G2530-60029</td><td></td></tr><tr><td>Rotator Rack Conversion Rod</td><td>Agilent Technologies</td><td>G2530-60030</td><td></td></tr><tr><td>Staining kit</td><td>Bio-Optica</td><td>10-2000</td><td>Slide-staining dish and Slide rack</td></tr><tr><td>Magnetic stirrer device</td><td>AREX Heating Magnetic Stirrer</td><td>F20540163&amp;nbsp;</td><td></td></tr><tr><td>Thermostatic Oven</td><td>Thermo Scientific</td><td>Heraeus - 6030</td><td></td></tr><tr><td>Agilent Microarray Scanner</td><td>Agilent Technologies</td><td>G2565CA</td><td></td></tr><tr><td>Scanner Carousel, 48-position</td><td>Agilent Technologies</td><td>G2505-60502</td><td></td></tr><tr><td>Slide Holders</td><td>Agilent Technologies</td><td>G2505-60525</td><td></td></tr><tr><td>Feature extraction software v11.5</td><td>Agilent Technologies</td><td>-</td><td>inside the Agilent Microarray Scanner G2565CA</td></tr><tr><td>SIMCA + V13 Software</td><td>Umetrics</td><td></td><td></td></tr></tbody></table>

the terroir concept interpreted through grape berry metabolomics and transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

The case study described in this article yielded a final data matrix comprising 552 signals (m/z features) including molecular ions plus their isotopes, adducts and some fragments, relatively quantified among 189 samples (7 vineyards x 3 ripening stages x 3 growing seasons x 3 biological replicates). The total number for data points was therefore 104,328. Fragmentation tree analysis resulted in the annotation of 282 m/z features, corresponding to metabolites plus adducts...

Watch this Scientific Journal Video about The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics at JoVE.com

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

the-terroir-concept-interpreted-through-grape-berry-metabolomics

Universidad San Sebastian

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JoVE Journal

Genetics

10.3K Views.  University of Verona. This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

Video: The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

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In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative transcriptomics. It also serves as a primer for biologists to develop data scientist computational skills, e.g. executing bash commands, visualization and management of large data sets. All command line code and further explanations of each command or step can be found on the wiki (<a href="https://wiki.cyverse.org/wiki/x/dgGtAQ">https://wiki.cyverse.org/wiki/x/dgGtAQ</a>). The Discovery Environment and Atmosphere platforms are connected together through the CyVerse Data Store. As such, once the initial raw sequencing data has been uploaded there is no more need to transfer large data files over an Internet connection, minimizing the amount of time needed to conduct analyses. This protocol is designed to analyze only two experimental treatments or conditions. Differential gene expression analysis is conducted through pairwise comparisons, and will not be suitable to test multiple factors. This workflow is also designed to be manual rather than automated. Each step must be executed and investigated by the user, yielding a better understanding of data and analytical outputs, and therefore better results for the user. Once complete, this protocol will yield de novo assembled transcriptome(s) for underserved (non-model) organisms without the need to map to previously assembled reference genomes (which are usually not available in underserved organism). These de novo transcriptomes are further used in pairwise differential gene expression analysis to investigate genes differing between two experimental conditions. Differentially expressed genes are then functionally annotated to understand the genetic response organisms have to experimental conditions. In total, the data derived from this protocol is used to test hypotheses about biological responses of underserved organisms.

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved Non-model Organisms

This protocol outlines a comparative de novo transcriptome assembly and annotation workflow for novice bioinformaticians. The workflow is available for free entirely through CyVerse and connected by the Data Store. Command line and graphical user interfaces are used, but all code needed is available to copy and paste.

This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Biochemistry

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as knockout mutants, wild accessions and advanced breeding populations, there is a rising burden for gene functional annotation. In this protocol, annotation of plant gene function using combined co-expression gene analysis, metabolomics and informatics is provided (Figure 1). This approach is based on the theory of using target genes of known function to allow the identification of non-annotated genes likely to be involved in a certain metabolic process, with the identification of target compounds via metabolomics. Strategies are put forward for applying this information on populations generated by both forward and reverse genetics approaches in spite of none of these are effortless. By corollary this approach can also be used as an approach to characterise unknown peaks representing new or specific secondary metabolites in the limited tissues, plant species or stress treatment, which is currently the important trial to understanding plant metabolism.

Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.

Given the ever expanding number of model plant species for which complete genome sequences are available and the abundance of bio-resources such as ...

Biology

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Profiling the Bacterial Community in Grapes Using Amplicon Metagenomics

Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/&#181;L to 87 ng/&#181;L. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

Author Spotlight: Exploring the Fermentation Microbiome Through Next-Generation Sequencing

Here, we present a protocol to describe amplicon metagenomic for determining the bacterial community of Traminette grapes, fermenting grapes, and final wine.

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

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Chapters in this video

In Vitro Transcription Assays and Their Application in Drug Discovery

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

Leveraging CyVerse Resources for De Novo Comparative Transcriptomics of Underserved (Non-model) Organisms

Controllable Ion Channel Expression through Inducible Transient Transfection

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

Annotation of Plant Gene Function via Combined Genomics, Metabolomics and Informatics

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing