2007 Annual Report 1a.Objectives (from AD-416) The primary objective of this research project is to identify genes and define molecular mechanisms that regulate nutritional content, quality and availability of plant-based foods with a primary emphasis on carotenoids. The value of such research will be in expanding the knowledge base of molecular biology related to crop nutrient quality and more specifically, to understanding of primary and secondary biochemical pathways and associated genetic regulatory systems that influence nutritional characteristics of plant-derived foods. Discoveries resulting from activities pursued through this project will lead to molecular tools for testing biochemical and molecular regulatory hypotheses and eventually for manipulating crop nutrient profiles and\or content. Implementation of said discoveries will be through both creation of genetically modified crops plants and indirect genetic manipulation via DNA markers associated with target nutrient traits. Resulting genetically modified plants will further be useful in testing not only nutrient levels but also availability to humans through diet within the context of a given crop tissue or derived food. Specific broad objectives of this project include: Objective 1: Define genetic regulatory mechanisms that control endogenously regulated and environmentally influenced synthesis and accumulation of carotenoids in plant-based foods. Objective 2: Develop and characterize genetic and biochemical plant attributes contributing to regulation of accumulation of carotenoids with exploratory efforts toward additional plant-derived nutrients. 1b.Approach (from AD-416) Efforts will focus on the use of tomato and cauliflower for identification and characterization of nutrient-related genes with primary emphasis on carotenoids. We propose expansion of the tomato model to include analysis of genome-wide expression patterns during fruit development and ripening. We will perform comparative expression profiling of pre-ripening and ripening fruit from normal, ancestral and mutant varieties, combined with HPLC analysis of carotenoid accumulation to identify candidate transcription factors impacting fruit carotenoid accumulation. Additional insights into transcriptional control of carotenoid accumulation will be developed through analysis of protein accumulation profiles in addition to (and in some cases in support of) transcription data. A major goal of this project is to identify novel genes involved in or regulating a specific metabolite pathway using correlation analysis between genotype, metabolite and gene expression data. We will develop both biology-driven and statistics-driven integration tools that will be presented to the research community and broader public via the world wide web. The secondary model for our activities will be cauliflower as both a source of unique genetic variation related to carotenoids and a member of the Brassicaceae which includes many important vegetable species. Previously, we have demonstrated that expression of the Or genomic DNA allele in transgenic cauliflower induced carotenoid accumulation. To begin to shed light on the nature of the Or mutation and endogenous OR protein function, we propose to generate both "knockout" and over-expression lines in cauliflower. We will employ a range of proteomics approaches including 2-hybrid, gel filtration and mass spectrometry to identify proteins that interact with Or. Finally, as a relatively minor activity and in an effort to identify future areas of promise, we will begin to improve our understanding of Se metabolism in plants for enhancing the biosynthesis of functional forms of organoselenium compounds. We will employ molecular and genomics approaches to identify, isolate and characterize important genes controlling Se metabolism. 3.Progress Report This project was initiated in June 2006 with the general goal of developing basic insights that would support the development of food crops with enhanced nutrient quality. Specific objectives center on identification of genes that might serve as tools via breeding or targeted genetic engineering for enhanced crop quality. All yearly milestones for the project were met or exceeded and the project is operating within budget. Three strategies have been employed to identify genes associated with nutrient quality. First is identification of genes known by mutation to influence nutrient or other quality parameters. Examples include the cauliflower Orange (Or) mutation resulting in elevated beta-carotene accumulation and the tomato ripening-inhibitor (rin) mutation that results in impaired fruit ripening (isolated under the prior CRIS). The Or gene was isolated and shown via transgenic manipulation to regulate antioxidant carotenoid accumulation in cauliflower curd. It was additionally shown to be capable of facilitating high-level beta-carotene accumulation in potato tubers while rin homologs were shown to regulate ripening and antioxidants in strawberry and melon. In these latter regards, proof-of-concept for the utility of these genes in altering anti-oxidant levels of plant-based foods was achieved. The second strategy for nutrient-associated gene isolation entailed identification of genes physically associated with those defined by mutation. An Or-interacting protein was identified and isolated. The third track employs comparative gene/protein/nutrient analysis. A number of activities take this approach in which experimental systems are developed to identify genes/proteins whose expression tracks changes in nutrients during development or among genotypes. During the last year we have also begun to assess the utility of wild-species tomato introgression lines (each having a small and unique segment of DNA from a wild relative integrated via breeding) for comparative expression profiling using a new tomato gene chip (developed in large part by our group) and nutrient analysis of the same lines. Our public database has been updated to disseminate this information through a user interface that assists hypothesis development and testing. Research is also conducted under the following Agreements: 1907-21000-025-03S Identification and Functional Genomics of Genes Impacting Phytonutrient Levels and Metal Tolerance in Food Crop Species, specific cooperative agreement with Boyce Thompson Institute (BTI); 1907-21000-025-04N Functional Genomics Analysis of Fruit Flavor and Nutrition Pathways, non-funded agreement with BTI; 1907-21000-025-05R Control of Carotenoid Biosynthesis by a Novel Regulatory Gene Identified in Cauliflower, NRI reimbursable agreement; 1907-21000-025-06S Enhancing Carotenoid Accumulation in Staple Crops, specific cooperative agreement with Cornell; and 1907-21000-025-07N, non-funded agreement with Cornell. Additional details can be found in the specific reports for these agreements. The Specific Cooperative Agreement is monitored via regular meetings with cooperators. 4.Accomplishments 1. Title: Isolation and characterization of OR and associated proteins; We have successfully isolated the cauliflower Or gene and demonstrated that it can be used as a novel genetic tool to increase carotenoid content in an important staple crop. The Or gene encodes a novel protein in controlling carotenoid accumulation. We have also isolated a protein associating with the OR protein. Functional characterization of the OR-interacting protein will aid in our efforts to unravel the molecular mechanism of the Or gene for further enhancing carotenoid content in food crops to enhance their nutritional value. 2. Title: Enhancing the concentration of bioactive compounds in Broccoli; A number of organoselenium compounds have been shown to be bioactive molecules linked to anti-carcinogenic activity. We have successfully isolated a methyltransferase gene from broccoli and shown that it stimulates the production of volatile selenium compounds in bacteria. This gene is being examined to see if it can be reduced in expression (via gene silencing) to reduce the net conversion (and loss) of these bioactive compounds into volatile ones. Repression of this methyltransferase would be predicted to yield a net increase in the concentrations of these potentially helpful compounds naturally found in broccoli. 3. Title: Isolation of a novel regulator of fruit development and ripening from tomato; While a number of ripening-associated genes have been isolated from tomato and other species, few “master regulator” genes have been identified to date. One such gene previously identified by our group is the RIPENING-INHIBITOR (RIN) MADS-box transcription factor. In characterizing other members of this gene family in tomato we have identified a new MADS-box gene which acts prior to RIN in development and is necessary not only for ripening to occur but also in achieving full fruit size and mass. Ongoing analysis will reveal whether or not this gene may prove useful as a tool in regulating post-harvest shelf-life, nutrient quality (as it also clearly impacts carotenoid accumulation) and yield in fruit crop species. 4. Title: Development of a public tool for tomato gene expression profiling. Tomato serves as a model system for many biological inquiries including those related to fruit development, quality and pathogen response. In collaboration with several international partners we have developed an oligonucleotide-based gene chip (microarray) that allows simultaneous characterization of 12,000 independent tomato genes (approximately one third of the total genes encoded in the tomato genome) from any tissue of interest. The TOM2 tomato gene chip has been quality tested and was released to the scientific community in the last year. Public and private research labs the world over are using this tool for large-scale gene expression analyses in tomato and related crop species (such as pepper and eggplant). 5.Significant Activities that Support Special Target Populations We have hosted a Scientist from the Institute of Agricultural and Environmental Research, Tennessee State University (TSU), an 1890 University. The purpose of the visit (6/28/07-8/8/07) was to provide training in advanced proteomics techniques, access to state of the art instrumentation and software and guidance concerning the design, planning and execution of proteomics experiments. The work carried out by this scientist while at Ithaca will be used to generate preliminary data to support a planned grant proposal to the National Research Initiative in the fall of 2007. This effort was undertaken with a view to improve both the educational and research opportunities available at an institution that has traditionally served socially disadvantaged and historically underserved. 6.Technology Transfer Number of new CRADAs and MTAs 1 Number of active CRADAs and MTAs 1 Number of patent granted 1 Number of web sites managed 5 Number of non-peer reviewed presentations and proceedings 9 Number of newspaper articles and other presentations for non-science audiences 6 Review Publications Li, L., Lu, S., Van Eck, J., O'Halloran, D., Zhou, X., Lopez, A.B., Cosman, K., Conlin, B., Paolillo, D., Garvin, D.F., Vrebalov, J., Kochian, L.V., Kupper, H., Earle, E., Cao, J. 2006. The cauliflower or gene encodes a cysteine-rich zinc finger domain-containing protein that induces high-level of B-carotene accumulation. The Plant Cell. 18:3594-3605. Giovannoni, J.J. 2007. Fruit ripening and its manipulation. In: Gan, S., editor. Senescence Process of Plants. Oxford, UK: Blackwell Publishing Ltd. p.278-295. Giovannoni, J.J. 2004. Genetic regulation of fruit development and ripening. The Plant Cell. 16:S170-180. Giovannoni, J.J., El-Rakshy, S. 2005. Genetic regulation of tomato fruit ripening and development and implementation of associated genomics tools. Acta Horticulturae. 682:63-72. Martel, C., Giovannoni, J.J. 2007. Fruit ripening. In: Roberts, J., Gonzalez-Carranzas, Z., editors. Plant Cell Separation and Adhesion. Oxford, UK: Blackwell Publishing Ltd. p. 164-176. Barry, C., Giovannoni, J.J. 2006. Ripening in the tomato green-ripe mutant is inhibited by ectopic expression of a novel protein that disrupts ethylene signaling. Proceedings of the National Academy of Sciences. 103:7923-7928. Rose, J.K., Bashir, S., Giovannoni, J.J., Jahn, M.M., Saravanan, R.S. 2004. Tackling the plant proteome: practical approaches, hurdles and experimental tools. Plant Journal. 39:715-733. Adams-Phillips, L., Barry, C., Giovannoni, J.J. 2004. Signal transduction systems regulating fruit ripening. Trends in Plant Science. 9:331-338. Mueller, L., Tanksley, S., Giovannoni, J.J., Van Eck, J. 2005. The tomato sequencing project, the first cornerstone of the international solanaceae project (sol). Comparative and Functional Genomics. 6:153-158. Ponce-Valadez, M., Watkins, C., Moore, S., Giovannoni, J.J. 2005. Differential gene expression analysis of strawberry cultivar responses to elevated co2 concentrations during storage using a tomato cdna microarray. Acta Horticulturae. 682:255-262. Fei, Z., Tang, X., Alba, R., Giovannoni, J.J. 2006. Tomato expression database (ted): a suite of data presentation and analysis tools. Nucleic Acids Research. 34:D766-D770. Basset, G., Ravanel, S., Quinlivan, E., White, R.A., Giovannoni, J.J., Rebeille, F., Nichols, B., Shinozaki, K., Gregory, J., Hanson, A. 2004. Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant Journal. 40:453-461. Barry, C., Thompson, A., Seymour, G., Grierson, D., Giovannoni, J.J. 2005. Ethylene insensitivity conferred by the green-ripe (gr) and never-ripe 2 (nr-2) ripening mutants of tomato. Plant Physiology. 138:267-275. Moore, S., Payton, P.R., Wright, M., Tanksley, S., Giovannoni, J.J. 2005. Utilization of tomato microarrays for comparative gene expression analysis in the solanceae. Journal of Experimental Botany. 56:2885-2895. Wang, Y., Tang, X., Cheng, Z., Mueller, L., Giovannoni, J.J., Tanksley, S. 2006. Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome. Genetics. 172:2529-2540. Alba, R., Payton, P.R., Fei, Z., Debbie, P., Martin, G., Tanksley, S., Giovannoni, J.J. 2005. Transcriptome and selected metabolite analysis reveal multiple points of ethylene control during tomato fruit development. The Plant Cell. 17:2954-2965. Manning, K., Tor, M., Poole, M., Hong, Y., Thompson, A., King, G., Giovannoni, J.J., Seymour, G. 2006. A naturally occurring epigenetic mutation in an sbp-box gene inhibits tomato fruit ripening. Nature Genetics. 38:948-952. Liu, Y., Roof, S., Ye, Z., Barry, C., Van Tuinen, A., Vrebalov, J., Bowler, C., Giovannoni, J.J. 2004. Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proceedings of the National Academy of Sciences. 26:9897-9902. Lia, C., Liua, G., Schilmillera, A., In Leea, G., Jayantya, S., Sagemana, C., Vrebalov, J., Giovannoni, J.J., Howe, G. 2005. Role of - oxidation in jasmonate biosynthesis and systemic wound signaling in tomato. The Plant Cell. 17:971-986. Alba, R., Fei, Z., Payton, P.R., Liu, Y., Debbie, P., Rose, J., Martin, G., Tanksley, S., Jahn, M., Giovannoni, J.J. 2004. Ests, cdna microarrays, and gene expression profiling: tools for dissecting plant physiology and development. Plant Journal. 36:697-714.