2009 Annual Report 1a.Objectives (from AD-416) 1. Determine if unintended effects are produced in transgenic crops, using fruit ripening in tomato as a model system. 1A. Determine if unintended effects are produced in transgenic crops, using gene expression analysis as a monitoring tool. 1B. Determine if unintended effects are produced in the fruit of transgenic crops that affect fruit quality or composition, through metabolomic and proteomic profiling and an examination of agronomic trait performance. 2. Determine if unintended effects are reduced in transgenic plants through the use of promoters with tissue-specific expression. 1b.Approach (from AD-416) 1) Utilize genomic, metabolomic, proteomic and agronomic approaches to evaluate phenotypic difference between tomatoes. 1A) Utilize natural diversity between tomato cultivars, together with conventional breeding techniques, to capture a reasonable phenotypic range from diverse tomato germplasm. 1B) Utilize RNAi and artificial microRNA gene silencing technologies to adjust RIN gene expression levels and alter fruit ripening. Compare phenotypic effects of transgenes to the range observed with conventional cultivars.. 2)Leverage research on fruit specific or ripening stage specific promoter sequences to further tailor the modulation of RIN gene expression in the target tissue. Assess the efficacy of tailored gene modulation on reducing unintended effects via genomic, metabolomic, proteomic and agronomic monitoring. 3.Progress Report Transgenic modification of crop plants to improve product quality or agronomic performance is a commonly utilized tool for modern agriculture. However, consumer concerns regarding transgenic crops have restricted the use of plant transformation from many sectors of agricultural production. Part of the consumer aversion to transgenic plants is connected to fears of unintended effects to plant composition or food quality. ARS can play a role in biotechnology risk assessment by developing information to help guide decision-making by regulators, producers and consumers. One expects to see highly significant differences in food composition between cultivars due to differences in quality that have been the targets of genetic selection (ie. breeding). One may also see significant differences in food composition due to genetic drift between varieties or environmental or random factors. The significance of compositional differences between varieties can therefore only be fully appreciated when these multiple sources of variation are considered. We are estimating the boundaries of stakeholder acceptable phenotypic diversity in tomato. This will allow us to benchmark our work in biotechnology risk assessment and estimate the significance of variation observed within transgenic tomatoes relative to that between conventional cultivars. We are using a collection of diverse heirloom tomato varieties and breeding lines to make this initial estimate via non-targeted metabolomic profiling. The metabolomic profiling utilizes both liquid chromatograph/mass spectroscopy (LC/MS; in collaboration with scientists at Ithaca NY) and nuclear magnetic resonance (NMR; in collaboration with scientists at Wyndmoor PA) to detect as many different compounds as possible, without preconception of what will be important. In parallel, we have constructed 40 new transgenic tomato varieties that reduce the expression of the ripening inhibitor (rin) gene, a major regulator of fruit ripening in tomato. Commercial tomato breeders worldwide use a conventional mutant form of the ripening inhibitor gene to prolong shelf life and reduce production-associated costs. Our first estimate on phenotypic diversity came from greenhouse grown tomatoes. This small-scale study featured rin mutants in two different genetic backgrounds, plus normal relatives and a single transgenic event that had a modest degree of gene silencing. Samples were analyzed using LC-MS and NMR and the data assessed using multivariate statistics. This experiment was expanded in the summer 2008 field season from the perspective of stakeholder acceptable phenotypic variation: 8 breeding lines or commercially released varieties; 8 heirloom varieties, several with altered fruit color; 7 ripening mutants. Metabolomic and molecular characterization of these samples are underway. The summer 2009 field season has an even greater expansion, combining heirloom varieties, breeding lines and genetic mutants, with field-based evaluation of 25 different transgenic events that exhibit a range of gene silencing of the rin gene. Samples will be collected from the summer 2009 field and analyzed in early to mid FY2010. 4.Accomplishments 1. Defined the compositional differences between heirloom and modern tomato varieties. There is little systematic knowledge about tomato composition. We identified numerous compounds and differences between heirloom and modern tomato varieties. We found simple sugars such as glucose and fructose and antioxidants such as kaempferol, narignenin and quercetin were higher in the heirloom variety Ailsa Craig. Breeding lines and commercialized varieties from North Carolina State University had higher levels of coumaric acid (an antioxidant) and phenylalanine (an amino acid). This information enables tomato breeders to enhance nutritional quality more rapidly and assist food scientists to define key components of tomato quality. 6.Technology Transfer None Review Publications Kochian, L.V., Hoekenga, O., Magalhaes, J., Pineros, M. 2009. Maize aluminum tolerance. In: Bennetzen, J.L., Hale, E.S.C., editors. Handbook of Maize: It's Biology. New York, NY: Springer. p. 367-380. Hoekenga, O. 2008. Using metabolomics to estimate unintended effects in transgenic crop plants: problems, promises and opportunities. Journal of Biomolecular Techniques. (3):159-166.