Location: Plant, Soil and Nutrition Research2018 Annual Report
1: Analyze the genome, transcriptome, metabolite and protein components that drive fruit and vegetable development, maturation, ripening, nutrient content and quality traits. 1A: Develop comprehensive systems-based gene expression, proteomic and metabolic data for fruit ripening and development from a range of fruit tissues and sub-tissues. 1B: Determine the regulatory control of chromoplast development and carotenoid accumulation using the Or gene as a model. 1C: Identify genes and proteins affecting Or-regulated chromoplast development and carotenoid accumulation. 1D: Continue efforts toward an improved tomato reference genome sequence. 2: Identify the genes and quantitative trait loci (QTL) that underlie variation in crop traits associated with vegetable and fruit biology, including processes that determine nutrient content, quality and shelf life. 2A: Analyze tomato genetic and phenotypic variation and associated gene expression changes resulting from defined introgressions of wild species DNA so as to identify loci and genes underlying fruit quality and nutrient content. 2.B: Elucidate how the epigenome contributes to regulating tomato fruit ripening and quality. 3: Determine the molecular function and utility of genes that contribute to target fruit and vegetable traits. 4: Evaluate the translatability of validated nutrient quality gene sequence activities in additional crop species.
A number of general themes will be followed to secure progress toward all four primary objectives and associated sub-objectives described below. 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 loci and corresponding genes underlying fruit and vegetable quality and nutritional content loci. Candidate genes will be isolated and 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. For example, while chromoplasts can be generated from fully developed chloroplasts, as is most commonly observed during fruit ripening of, for example, tomato and pepper, chromoplasts can also be derived from proplastids or other non-colored plastids as is the case in melon, watermelon, and carrot (Burger et al., 2008; Kim et al., 2010; Tadmor et al., 2005). Better understanding of such 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.
This is the final report for project 8062-21000-037-00D, which terminated on May 21, 2018. All project milestones were met or substantially met and a number were exceeded in that results stemming from milestones were in some cases pursued via additional research activities. In summary, three basic strategies were employed to identify and functionally characterize genes associated with crop nutrient quality. The first is identification of genes known through mutation to influence nutrient or other quality parameters. These activities address NP301 component 301.3.C.2010 and 301.4.2010 as DNA markers were developed that are being used by seed companies to accelerate their breeding programs for nutrient, ripening, and shelf-life traits. Second, gene targets and/or interacting proteins of these regulators have been identified and analyzed in addition to homologs of these genes in other species indicating the potential to use an easy to work with model crop such as tomato for biological discovery on high value crops that are less experimentally tractable. We have completed whole genome characterization of DNA (promoter) interactions between the RIN, NOR and EIN3 transcription factors and their respective target genes. Exceeding the goals of the project we have developed DNA constructs and initiated development of transgenic plants that will allow whole genome analysis of additional transcription factors shown to be important contributors to ripening control under this project. We further demonstrated the critical role of DNA methylation dynamics in ripening control. Our recent data on melon fruit ripening suggests a similar methylation dynamics phenomena and further indicates a role for ethylene in mediating these changes. Working with collaborators at the University of Florida we also showed that the epigenome can contribute to fruit quality alterations resulting from stress, in particular chilling, a widely deployed tool during postharvest storage. The role of the epigenome in ripening control and its influence of fruit quality characteristics will be a significant aspect of the ensuing project. In addition to primary regulation of plant processes critical to food and nutritional security, we also focused on specific metabolic pathways important to crop nutrient content. Substantial progress has been made specifically regarding carotenoid pathway regulation and manipulation. Carotenoids possess strong anti-oxidant activities with some compounds such as beta-carotene serving as vitamin A precursors in the human body. Other pathway outputs contribute to fruit aromas and serve as plant hormones. Under this project, newly identified interacting proteins (e.g. with Or) provide us new genetic tools and new directions for in-depth elucidation of the complex regulatory networks controlling carotenoids and nutritional quality of food crops. We identified an unanticipated target for enhancement of carotenoid content, phytoene desaturase, which we showed becomes limiting when phytoene synthase is elevated (for example during ripening) and is the target for multiple points of endogenous regulatory control. We also demonstrated that expression of a heterologous protein in tomato bypassed endogenous regulatory circuitry yielding substantially elevated carotenoid content. These findings represent increased basic understanding of an important biochemical pathway influencing crop performance, quality and nutrient value while also providing genetic insights and tools facilitating improved crop quality and nutrition via breeding or targeted genetic intervention. Nutrient pathways including the carotenoid pathway will continue to be targeted in the ensuing project. Through the course of this project the tomato genome sequence continued to be leveraged and improved. In the last year, new sequence assembly and annotation versions have been developed through integration of long read single molecule sequencing data which has resulted in placement of nearly all previously unassigned sequence (referred to as Chromosome 0) to the 12 tomato chromosomes. Updated sequences are released though our collaborators at the Boyce Thompson Institute via the solgenomics.net website. Continued domestic and international collaborative efforts during the course of this project have resulted in a de novo genome sequence of the tomato wild species S. pennellii and S. lycopersicoides as a collaborative effort with labs in the Cold Spring Harbor Institute, Belgium and the Boyce Thompson Institute with ongoing activity to develop an improved de novo sequence of S. pimpinellifolium. This first is a wild relative of tomato harboring many natural disease resistance genes, the second is a wild tomato relative adapted to cold and drought environments and the third is the progenitor of the domesticated tomato. All have enormous and largely untapped breeding potential that will be facilitated by these genome sequences. These efforts were beyond the scope of the original project. Finally, nutrient-associated gene identification through comparative gene and/or protein expression analysis on a range of germplasm from cauliflower and tomato varieties with altered nutrient composition was undertaken in an effort to understand regulation of such pathways important for both human nutrition and crop quality. The scientific outcomes of these efforts are noted above. Central to their success was development of novel protocols for precise chemical determination and quantitation. Several protocols facilitating analysis of carotenoids and additional metabolites were developed and published during the course of the project. With regard to data dissemination we 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. Tomatoes are among the most widely consumed fruit in the U.S. and are central components of many home gardens. 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. ARS researchers in Ithaca, New York, demonstrated that a protein from tomato and originally identified in orange colored cauliflower provides a crucial regulatory step in limiting the synthesis of carotenoids in tomatoes. Expression of this protein during early fruit development resulted in accumulation of carotenoids (lycopene and beta-carotene) prior to ripening initiation resulting in higher ripe fruit carotenoid levels. No changes in other ripening parameters were observed. These results suggest that the gene encoding this protein can be manipulated via traditional breeding or targeted genetic engineering to improve nutrient and visual quality in tomato and additional fruit crops.
2. 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 consumed fresh, as processed products (sauces and paste) and are also central components of many home vegetable gardens. Breeders for decades have attempted to minimize the proportion of the fruit represented by the seeds and watery locule and increase the fleshy pericarp or outer portion of the fruit. ARS researchers in Ithaca, New York, demonstrated that a single tomato gene is largely responsible for the conversion of tissue more like the pericarp to the watery locule during ripening. Repression of this gene resulted in firm fleshy fruit with little to no watery locule and also resulted in identification of a natural mutation in the same gene. These results provide genetic insights and tools to breed for more fleshy tomato fruit with the potential to translate such characteristics to other fruit crops of interest via identification of equivalent genes in their genomes.
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Zhang, B., Tieman, D., Chen, J., Xu, Y., Chen, K., Fei, Z., Giovannoni, J.J., Klee, H. 2016. Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation. Proceedings of the National Academy of Sciences. 113:12580-12585.
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