2010 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 In the last twelve months, we made progress on several projects within the research plan and took steps to refocus our attention on a new model system. We are using diverse, conventional varieties to estimate the boundaries of stakeholder acceptable phenotypic variation so as to understand the significance of changes observed in transgenic tomato fruit. This information should help to assess and improve genetic engineering technology and also potentially allay consumer concerns regarding transgenic crop improvement, by allowing a statistically rigorous treatment of the concept of “substantial equivalence”. In the summer of 2008, we grew 23 diverse, conventional tomato varieties under field conditions and began to characterize them using mass spectrometry (MS), nuclear magnetic resonance (NMR) and gene expression analyses. In the summer of 2009, we grew a 16 variety subset of the larger collection and 25 transgenic tomato varieties on a research farm. We also grew accessions from 15 other Solanaceous species with berry-type fruit, with both edible (e.g. tomatillo) and inedible (e.g. potato) accessions. The Solanaceae diversity experiment expanded the boundaries for stakeholder acceptable variation, generated the first food composition data for several specialty fruit crops (e.g. husk cherry), and allowed us to ask questions regarding the evolution of novel fruit characteristics. While our original research plan proposed the use of multiple technologies, we have concentrated our efforts on the high throughput characterization of chemical composition (metabolomics) of our fruit samples. We spent part of the year examining patterns of gene expression (transcriptomics) using quantitative PCR; however, we could find no obvious patterns within the genes we chose as representative of ripening-regulated genes and redoubled our efforts on MS-based metabolomics. We also proposed examining the fruit proteome, which is the collection of proteins in fruit, but reached the opinion that there were not adequate resources to test the substantial equivalence of transgenic tomatoes from every perspective. Taking advantage of recent developments in statistics, we applied both well-known and novel methods to analyze our metabolomics data. We identified patterns within the data so as to easily distinguish tomato varieties from one another; these data also suggest which compounds may underlie particular quality traits and should provide valuable new information to tomato breeders and food scientists. Tomato has been an excellent model system to study unintended effects to food quality in transgenic crops. However, asking similar questions using corn as a model may have greater relevance and immediacy to address stakeholder concerns. In anticipation of a bridging research project to begin in FY2011, we redirected our attention to asking corn-based questions. Our summer 2010 research field is filled with diverse, conventional corn varieties to allow us to estimate the boundaries of stakeholder acceptable phenotypic diversity. We are also adjusting our focus from modifying fruit ripening in tomato to examining iron nutritional quality in corn. 4.Accomplishments 1. Identified an heirloom tomato variety with novel chemical properties. Alpha-tomatine is a glycoalkaloid with multiple pharmacological properties, including reducing LDL cholesterols, inhibiting lung cancer and fungal cell growth. Alpha-tomatine is found in unripe, green tomatoes but is normally broken down during fruit ripening, such that ripe tomatoes have little or none. ARS scientists in Ithaca, NY identified an heirloom variety of tomato that contains high levels of alpha-tomatine in red ripe fruit during a survey of chemical diversity in heirloom and modern tomato varieties. This variety may provide a convenient and palatable source for this important compound. Review Publications Buescher, E., Achberger, T., Amusan, I., Giannini, A., Ochsenfeld, C., Rus, A., Lahner, B., Hoekenga, O., Yakubova, E., Harper, J., Guerinot, M., Zhang, M., Salt, D.E., Baxter, I.R. 2010. Natural Genetic Variation in Selected Populations of Arabidopsis thaliana is Associated with Ionomic Differences. PLoS One. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011081 Krill, A.M., Kirst, M., Kochian, L.V., Buckler Iv, E.S., Hoekenga, O. 2010. Association and linkage analysis of aluminum tolerance genes in maize. PLoS One. 5(4):e9958. DOI: 10.1371/journal.pone.0009958. Dubois, P., Olsefski, G.T., Hoekenga, O., Flint Garcia, S.A., Brutnell, T. 2010. Physiological and genetic characterization of end-of-day far-red light response in maize seedlings. Plant Physiology. 10.1104/pp.110.159830.