Location: Plant, Soil and Nutrition Research2019 Annual Report
Objective 1: Identify loci and functionally characterize underlying genes that contribute to fruit and vegetable shelf-life, appearance, flavor, texture, and nutritional quality by characterizing cultivated and wild species diversity so as to develop a better understanding of corresponding trait biology and to develop new molecular tools for breeding. (See uploaded postplan for sub-objectives) Objective 2: Generate genome-scale DNA sequence data, gene expression profiles, proteomic and metabolite data of fruit and vegetable crops for facilitating trait discovery and trait improvement. (See uploaded postplan for sub-objectives) Objective 3: Develop and test models for the regulation of fruit and vegetable development and quality traits at the genome level that incorporate epigenome dynamics and epigenetic regulators. (See uploaded postplan for sub-objectives) Objective 4: Develop and utilize new advanced analytical approaches to characterize fruit and vegetable proteins and chemical metabolites, their modifications and interactions, via targeted and genome-scale methodologies. (See uploaded postplan for sub-objectives) Objective 5: Develop, test, and thoroughly analyze at the whole genome level gene editing technologies in tomato for use in enhancing nutrient levels and shelf-life, and in selected high value crops for use in breeding and research. (See uploaded postplan for sub-objectives)
The overall approach of this project will be use of molecular, genetic and genomics approaches to address our objectives centered on advancing our understanding of fruit and vegetable quality and deploying said knowledge toward crop improvement. We will take advantage of existing germplasm in the form of mutant/variant lines and segregating populations and/or wild species introgression lines to identify genes underlying fruit and vegetable quality and nutritional content. Candidate genes will be isolated, sequenced, and characterized for gene expression attributes in addition to allelic variation that will be correlated with trait and/or metabolic outputs. Functional analyses will be undertaking for candidate quality and nutrition impacting genes through identification and development, respectively, of chemical/natural or transgenic mutations. In some instances, we will test potential for translation of insights from model and crop systems studies to additional crop and stable crop species. Better understanding of processes underlying fruit and vegetable quality will facilitate design of molecular strategies to improve crop quality attributes in both primary experimental crop systems and targets of translational biology. Through these undertakings, we will develop transgenic and gene edited lines to address gene function. We will further utilize said lines and additional lines developed as controls to assess the nature and degree of genome changes resulting from transformation or gene editing and the extent of possible biotechnological risk, if any.
This is the first annual report for this project. A significant component of the project involves assessment of tomato genetic diversity toward understanding fruit development, ripening and nutritional quality while simultaneously using resulting materials for biotechnology risk assessment with an emphasis on emerging gene editing approaches. In the first year, researchers grew tomato populations resulting from crosses of cultivated and wild species. These populations are both recombinant inbred (RIL) and introgression line (IL) populations – a key feature being that they are inbred/true breeding, allowing low resolution mapping of observed traits and recovery of identical lines for future experiments. A current population of interest is derived from a cross between tomato and its most diverged sexually compatible relative, Solanum lycopersicoides, which is adapted to extreme cold and drought in the Andes mountain range. ARS researchers in Ithaca, New York, completed phenotyping under drought conditions and are currently analyzing gene expression, fruit quality, photosynthetic and transpiration data that will help identify genes underlying these traits. In collaboration with researchers at the Boyce Thompson Institute (BTI) and in Belgium, Ithaca researchers have recently completed de novo sequencing of the S. lycopersicoides genome. Using genome sequence and gene expression data (including the sequence derived from RNA-seq analysis) researchers have created high resolution molecular maps of the cross-over events defining individual population members, facilitating rapid definition of candidate genes for traits mapped to specific chromosomal regions. Ithaca researchers have begun using these populations and their transcriptome data to explore the degree to which whole genome transcriptome data can be influenced by genetic variation when using a single genome as reference. Efforts to characterize transcription factors regulating tomato fruit ripening have resulted in recent identification and functional characterization of regulators influencing specific ripening processes. One such gene, SlLOB1, is primarily responsible for influencing cell wall remodeling during fruit maturation as it regulates genes involved in cell wall synthesis, breakdown and associated textural features. A second gene functions primarily in breakdown of the locule tissue surrounding the seed prior to ripening initiation. ARS researchers in Ithaca, New York, have also begun to assess the activities of fruit and ripening-related transcription factors via various approaches that reveal sites of DNA interaction. DNA constructs for these efforts have been completed in the last year. A number of additional fruit transcription factor gene targets have been identified for gene editing and corresponding DNA constructs have been developed for plant transformation. Resulting gene edited lines will be used both to assess functional attributes of these genes in the fruit maturation process and will also serve as resources to address efficiency, off-targeting, and the generation of unanticipated DNA modifications resulting from gene editing in an effort to provide information on biotechnology risk of this fast emerging approach to stable genome modification. Working with collaborators from BTI, Cornell, the U. of Florida, U. of Georgia and China, researchers developed a tomato pan-genome based on the original Heinz1706 reference, hundreds of re-sequenced tomato accession in the public domain and approximately 150 newly sequenced accessions. This effort resulted in identification of approximately 5000 new genes or gene variants absent from the reference genome. Researchers fully explored one gene that was prevalent in accessions of the wild tomato progenitor S. pimpinnellifolium but largely absent in modern cultivated tomatoes. Examination of the most recent varieties revealed this gene, a lipoxygenase involved in fatty acid metabolism, has been making a resurgence in modern breeding indicating active selection. The gene influences production of fatty acid volatiles associated with tomato aroma and flavor. Researchers demonstrated that it also influenced levels of carotenoid derived apocarotenoid volatiles associated with flavor and consumer liking. The increased prevalence of this rare gene in modern breeding coincides with renewed interest by breeders in selecting for flavor in response to consumer complaints that many production tomatoes are lacking in this regard. In additional efforts to understand the molecular basis of fruit and vegetable quality and nutritional composition, progress continued toward dissecting OR gene function as related to phytoene synthase (PSY) activity in mediating carotenoid biosynthesis and associated nutrient composition in crops. Researchers demonstrated that in addition to regulating carotenoid biosynthesis and chromoplast formation, OR also influences chromoplast number to affect total carotenoid accumulation. Researchers showed that OR specifically interacts with the central chloroplast division factor ARC3 to influence chromoplast division and manipulation of plastid division factors can greatly affect carotenoid levels in edible tissues, thus providing a new strategy for crop carotenoid enrichment. Researchers additionally found significant activity differences between PSY isozymes, the rate-limiting enzyme for carotenoid biosynthesis. Researchers identified key amino acid residues underlying these differences, highlighting the potential for using synthetic biology for engineering or breeding of carotenoid-enriched crops. The later work was recently published in Plant Physiology and the former work is under review. During this year, progress was also made in performing RNA-seq and QTL-seq analysis of both melon and cauliflower to identify additional genes that regulate carotenoid and nutrition traits. The QTL-seq data was used to identify nutrient QTLs while analysis of the expression data is just beginning. A significant portion of project efforts are devoted to developing and improving methods of molecular analysis. Researchers made improvements in the basic protocols for carotenoid analysis with respect to sampling, extraction and time required. Researchers initiated a non-targeted analysis of flavonoids from tomato peels. This work involves the identification of known flavonoids but also the identification and structural characterization of novel flavonoids via mass spectrometry, UV/Vis and NMR spectroscopy. Structural characterization requires isolation of significant quantities of the targeted compounds and thus this study has developed preparative scale chromatographic methods to fill this need. Researchers have evaluated pressure cycling as a means to carry out micro-extractions of metabolites from plant tissues. Qualitative results are promising; however obtaining reliable quantitative results remains a challenge. ARS researchers in Ithaca, New York believe that the problems are not related to any aspect of the pressure cycling strategy but rather issues relating to our ability to accurately estimate the amount of material in our micro-samples. This, makes it difficult to properly normalize. Researchers believe this is a tractable problem and remains an active area of investigation. ARS researchers continue to evaluate convergence chromatography for use in metabolite profiling. To date most applications have been targeted to carotenoids and flavonoids. Applications involving carotenoids have been largely successful. Applications involving simple, unmodified flavonoids (e.g., kaempferol, naringenin, genistein etc.) have also been successful. However, flavonoids that have been modified by the addition of polar adducts (i.e., flavonoid glycosides) represent more of a challenge. Fortunately this latter class of compounds can be analyzed by orthogonal methods including reverse phase UPLC. Our convergence chromatograph has been coupled to a single quadrupole mass spectrometer which provides unit mass resolution. While sufficient for the analysis of targeted compounds it is not suitable for untargeted analyses where higher mass accuracy is required to ensure proper identification. This past year the Center acquired a high resolution mass spectrometer (Model 6600 Triple TOF from AB Sciex) and researchers are developing an interface with a view towards coupling this with the convergence chromatograph. In addition to improving the mass resolution from +/- 1 Da to +/_ 1 ppm, this configuration will also add another orthogonal dimension of separation as the new instrument also incorporates an ion mobility feature. In short, researchers continue to develop new and optimized protocols to support the molecular characterization that underlies the research objectives of this project. With regard to data dissemination, ARS researchers help support and release our data through the following public databases operated by colleagues at the Boyce Thompson Institute and Cornell University including the Tomato Functional Genomics Database http://ted.bti.cotnell.edu; Tomato Epigenome Database http://ted.bti.cornell.edu/epigenome/; Tomato Expression Atlas http://tea.solgenomics.net and the Solgenomics Database http://solgenomics.net.
1. Characterization of a protein influencing fruit and vegetable nutritional content. Carotenoids are important dietary antioxidants and some serve as precursors to vitamin A, a necessary nutrient. People do not synthesize carotenoids and must acquire them through their diet though diets of many Americans are deficient in carotenoids. Plant plastids are specialized structures that synthesize and store large amounts of carotenoids and as such, plastid numbers are directly related to the nutritional quality of edible crops. Researchers at the USDA in Ithaca, New York, identified a protein named ORANGE from orange cauliflower and demonstrated that it works with other “division factors” in regulating carotenoid accumulation by regulating plastid numbers. Unraveling this function of ORANGE provides a novel target for manipulating plastid division in many crops and thus enhance nutritional quality and uptake for consumers.
2. Identification of gene from wild tomatoes lost in domestication – an opportunity to improve bland tomato flavor though breeding. Tomatoes are the most valuable fruit crop world-wide and are among the most widely consumed fruit or vegetables in the U.S. at 70 lbs. per capita annually. They are important sources of dietary carotenoids including the antioxidant lycopene (which give tomatoes their characteristic red color) and beta-carotene, which our bodies convert to the necessary nutrient vitamin A, yet many consumers complain that store-bought tomatoes lack flavor. ARS researchers in Ithaca, New York, identified a rare version of a gene called TOMLoxC that is prevalent in wild ancestors of tomato but lost from most cultivated lines. They showed this gene contributes to flavor through an aroma compound that taste panels show consumers like. They further demonstrated that this gene, while still rare, is seeing a resurgence in some modern tomato varieties in parallel with a renewed interest in flavor by breeders attempting to address consumer demands. This work was published in the journal Nature Genetics and received wide media coverage.
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