2011 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.
The project objective is to elucidate mechanisms of nutrient accumulation in crop plant tissues so that the underlying genes can be used as breeding markers or transgenes to develop more healthy and nutritious crops. This work relates directly to National Program 301 objectives as indicated below. Three basic strategies continue to be employed to identify genes associated with crop nutrient quality. The first is identification of genes known through mutation to influence nutrient or other quality parameters. Examples include the cauliflower Orange (Or) mutation resulting in elevated beta-carotene, the tomato ripening-inhibitor (rin) mutation which results in impaired ripening, and the high-pigment (hp) mutation which leads to elevated tomato carotenoids. Proteins which interact with Or continue to be characterized and provide new candidates for determining how the OR protein facilitates elevated carotenoid levels in plant tissues. 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 these 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 (suggesting potential for broad applicability to diverse crops species). For example, we have found that the tomato rin gene may be a master regulator of ripening. This suggests it would be an optimal target for manipulating the complete fruit ripening process to optimize both production traits (i.e., uniform ripening and shelf-life) and consumer traits (flavor, texture and nutritional quality). The release of the tomato genome sequence in December 2009 which ARS scientists at the Robert Holley Center have led has been a powerful resource for the identification of key genes regulating nutritional content, ripening and shelf-life. This represents a significant enabling tool for the plant scientific community as the availability of the tomato genome sequence has facilitated our comparisons to genomes of related vegetable crops (e.g. potato) and identification of gene candidates for similar functions of interest to those defined in tomato (e.g. fruit maturation regulators in pepper and eggplant). The third track toward nutrient-associated gene identification is through comparative gene and/or protein expression analysis on a range of germplasm representing traditional and wild tomato species . Proteomic and gene expression profiling have been completed on a series of developing tomato fruit that were also characterized for nutrient quality to assist in defining where and when ripening processes occur and when. Finally, our public database has been consolidated and updated to house and disseminate all of the information reported here in addition to integrated data recovered from the public domain. These activities and especially the database address NP301 component 301.4.B.2010 by supplying data in the public domain that help build and test hypotheses pertaining to “biological processes to improve crop productivity and quality” especially as related to fruit.
Characterized a tomato gene which serves as a natural regulator of uniform tomato fruit ripening. The development and ripening of fleshy fruits is imperative to seed dispersal for many plants and impacts the food and nutrition needs of humans and other species. ARS researchers at the Robert W. Holley Center for Agriculture and Health at Ithaca, NY have identified a tomato gene whose natural function is to alter pigmentation in the top versus the bottom of tomato fruit. The gene designated “uniform” or “u” has been long known due to the existence of a mutant version that has been used in tomato breeding for decades to develop lines with fruit of uniform ripening and coloring. The gene underlying this mutation has now been identified and DNA sequence used to develop a molecular tool both to assist tomato breeding for uniform color and ripening but also a potential tool to identify similar genes in additional fruit species.
Identified two banana genes that regulate banana ripening and extend shelf-life. Banana is an important fruit crop for many developed countries including the US and a staple in parts of Africa and Asia. Short shelf-life results in considerable crop loss, incurs additional expense for storage and handling and impacts food security especially in countries where banana and its relatives (plantains) are dietary staples. ARS researchers at the Robert W. Holley Center at Ithaca, NY in collaboration with colleagues at the Boyce Thompson Institute and ARO Israel have identified two banana genes, MaMADS1 and MaMADS2, that regulate ripening. Banana plants altered via transgenic approaches in expression of either of these genes produce fruit with 1 – 2 weeks of additional shelf life. Such banana plants hold the promise of extending fruit shelf life and availability with minimal cost, thus promoting both food security and improved economy of banana storage and distribution.
Completed and released the whole tomato genome sequence. ARS researchers at the Robert W. Holley Center for Agriculture and Health at Ithaca, NY, are primary leaders of an international effort to sequence the tomato genome. The tomato genome sequence is now in the public domain (http://solgenomics.net) and is among the highest quality of plant genome sequences in terms of accuracy and coverage in large part due to the sequences, tools and resources developed over the last 10 years by Robert W. Holley laboratory researchers working with collaborators at Cornell University (and the affiliated Boyce Thompson Institute), the University of Oklahoma and Colorado State University. The international effort includes participants from ten primary countries with additional contributors representing many additional nations. The tomato genome sequence will drive future crop improvement activities for tomato and serve as a reference sequence to facilitate crop improvement for many related and important crops including pepper, potato, eggplant and coffee.
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Giovannoni, J.J. 2010. The apple genome: ripe for harvest. Nature Genetics. 42:822-823.
Pan, I., Giovannoni, J.J., Irisch, V. 2010. Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development. Journal of Experimental Botany. 61:1795-1806.
Rugkong, A., Mcquinn, R., Giovannoni, J.J., Rose, J., Watkins, C. 2011. Expression of ripening-related genes in cold stored tomato fruit. Postharvest Biology and Technology. 61:1-14.
Enfissi, E., Barneche, F., Ahmed, I., Lichtle, C., Giovannoni, J.J., Bowler, C., Bramley, P., Fraser, P. 2010. Integrative transcript and metabolite analysis of DE-ETIOLATED1 down-regulated tomato fruit reveals the underlying metabolic and cellular events associated with their nutritionally enhanced chemotype. The Plant Cell. 22:1190-1215.
Joung, J., Corbett, A., Fellman, S., Tieman, D., Klee, H., Giovannoni, J.J., Fei, Z. 2009. Plant MetGenMAP: an integrative analysis system for plant systems biology. Plant Physiology. 151:1758-1768.
Costa, F., Alba, R., Schouten, H., Sansavini, S., Fei, Z., Giovannoni, J.J. 2010. Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening. Biomed Central (BMC) Plant Biology. 10:229.
Gonda, I., Bar, E., Portnoy, V., Schaffer, A., Tadmor, Y., Gepstein, S., Giovannoni, J.J., Katzir, N., Lewinsohn, E. 2010. Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit. Journal of Experimental Botany. 61:1111-1123.
Zhou, X., Mcquinn, R., Fei, Z., Wolters, A., Van Eck, J., Brown, C.R., Giovannoni, J.J., Li, L. 2011. Regulatory control of high levels of carotenoid accumulation in potato tubers. Plant, Cell & Environment. 34:1020-1030.
Harel-Beja, R., Portnoy, V., Fei, Z., Giovannoni, J.J., Ori, N., Lewinsohn, E., Tadmor, Y., Schaffer, A., Katzir, N. 2010. A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes. Theoretical and Applied Genetics. 121:511-533.
Chung, M., Vrebalov, J., Alba, R., Lee, J., Mcquinn, R., Klein, P., Giovannoni, J.J. 2010. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant Journal. 64:936-947.
Kamenetzky, L., Asis, R., Bassi, S., De Godoy, F., Bermudez, L., Fernie, A., Vrebalov, J., Giovannoni, J.J., Rossi, M., Carrari, F. 2010. Genomic analysis of wild tomato introgressions determining metabolism- and yield-associated traits. Plant Physiology. 152:1772-1786.
Seymour, G., Ryder, C., Cevik, V., Hammond, J., Popovich, A., King, G., Vrebalov, J., Giovannoni, J.J., Manning, K. 2011. A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria x ananassa Duch.) fruit, a non-climacteric tissue. Journal of Experimental Botany. 62:1179-1188.
Fei, Z., Joung, J., Tang, X., Zheng, Y., Huang, M., Lee, J., Mcquinn, R., Tieman, D., Klee, H., Giovannoni, J.J. 2010. Tomato functional genomics database (TFGD): a comprehensive collection and analysis package for tomato functional genomics. Nucleic Acids Research. 39:D1156-D1163.