2012 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 overall 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. 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. 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 is a master regulator of ripening at the level of both transgene effects on multiple crop species and via analysis of RIN binding to numerous fruit ripening gene promoters. These results confirm RIN is 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). In the last year we have also developed and characterized transgenic plants altered in expression of the tomato UNIFORM RIPENING (u) gene and showed that this gene influences fruit chloroplast development and photosynthesis, and thus directly impacts sugar accumulation, appearance and quality of ripe fruit. The tomato genome sequence has been completed by an international consortium of plant scientists and led by ARS scientists at the Robert Holley Center resulting in publication in 2012 and is a powerful resource for the identification of key genes regulating nutritional content, ripening and shelf-life. In the last year this project has also continued work on refinement of the tomato genome sequence to bring it to the highest quality standard possible with available resources. The third track toward nutrient-associated gene identification is through comparative gene and/or protein expression analysis on a range of germplasm from cauliflower and tomato varieties with altered nutrient composition in an effort to understand regulation of these pathways which are important in human nutrition and crop quality. Proteomic and gene expression profiling in the last year has focused on identification of genes and proteins influenced by the cauliflower OR and tomato RIN and U proteins.
Finally, our public database has been 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
Defined the regulatory mechanisms in flavonoid biosynthesis. ARS researchers at Ithaca, New York have investigated the general regulatory mechanism of anthocyanin biosynthesis in red cabbage. Through a combination of candidate analysis and fine-mapping, we have successfully isolated the gene responsible for purple cauliflower formation and demonstrated the importance of the involvement of transposable elements in gene transcription for phenotypic changes in plants. The isolated gene can also serve as an important genetic tool for the breeding of Brassica vegetables with enhanced health-promoting properties and visual appeal.
Identification of a key regulator of tomato fruit pigmentation and ripening. Efforts by ARS researchers at Ithaca, New York under the prior project resulted in positional cloning of the tomato uniform ripening (u) gene which impacts fruit color. Research efforts under the prior project demonstrated that the underlying gene is a mutated transcription factor and wide selection of this trait in cultivated germplasm contributes to poor quality of mass produced tomato fruit.
Completion of the tomato genome sequence. The 950 Mb tomato genome sequence was accomplished under the prior project with the ARS researchers at Ithaca, New York leading the US contribution toward a large multinational effort. The tomato genome sequence is of exceptional quality and was reported in Nature with extensive world-wide media attention. 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.
Klee, H., Giovannoni, J.J. 2011. Genetics and control of tomato fruit ripening and quality attributes. Annual Review of Genetics. 45:41-59. DOI: 10.1146/annurev-genet-110410-132507
Elitzur, T., Vrebalov, J., Giovannoni, J.J., Ocampo, E., Goldschmidt, E., Friedman, H. 2010. The regulation of MADS-box gene expression during ripening of banana and their regulatory interation with ethylene. Journal of Experimental Botany. 61:1523-1535.
Goyal, R.K., Kumar, V., Shukla, V., Mattoo, R., Yongsheng, L., Chung, S.H., Giovannoni, J.J., Mattoo, A.K. 2012. Features of a unique intronless cluster of class I small heat shock protein genes in tandem with box C/D snoRNA genes on chromosome 6 in tomato (Solanum lycopersicum). Planta. 235(3):453-471.
Zhong, S., Joung, J., Zheng, Y., Liu, B., Shao, Y., Xiang, J., Zhangjun, F., Giovannoni, J.J. 2011. High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harbor Protocols. 8:940-949. DOI: 10.1101/pdb.prot5652.
Lopez-Casado, G., Covey, P.A., Bedlinger, P.A., Mueller, L.A., Thannhauser, T.W., Zhang, S., Fei, Z., Giovannoni, J.J., Rose, J. 2012. The proteome of tomato pollen as a test case. Proteomics. 12(6):761-774. DOI: 10.1002/pmic.201100164.
Lee, J., Joung, J., Mcquinn, R., Chung, M., Fei, Z., Tieman, D., Klee, H., Giovannoni, J.J. 2012. Combined transcriptome, genetic diversity and metabolite profiling in tomato fruit reveals the ethylene response factor SlERF6 to play an important role in ripening and carotenoid accumulation. Plant Journal. 70:191-204.
Powell, A., Nguyen, C., Hill, T., Aktas, H., Figueroa-Balderas, R., Barry, C., Liu, Y., Chetelat, R., Granell, A., Van Deynze, A., Giovannoni, J.J., Bennett, A. 2012. Uniform ripening (U) encodes a Golden 2-like transcription factor regulating tomato fruit chloroplast development. Science. 336(6089):1711-1715. DOI: 10.1126/Science.1222218.
Din, V., Tieman, D.M., Tohge, T., Mcquinn, R.P., De Vos, R.C., Osorio, S., Schmelz, E.A., Taylor, M.G., Smits-Kroon, M.T., Schuurink, R.C., Haring, M.A., Giovannoni, J.J., Fernie, A.R., Klee, H.J. 2011. Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit. The Plant Cell. 23:2738-2753.
Brown, A., Paterson, A., Li, L. 2012. Genomics and breeding in food crops. In: Benkeblia, N., editor. OMICs technologies: tools for food science. Boca Raton, FL: CRC Press. p. 141-162.
Yang, Y., Qiang, X., Owsiany, K., Zhang, S., Thannhauser, T.W., Li, L. 2011. Evaluation of different multidimensional LC-MS/MS pipelines for iTRAQ-based proteomic analysis of potato tubers in response to cold storage. Journal of Proteome Research. 10(10):4349-4886.
Ramos, S., Rutzke, M., Hayes, R.J., Faquin, V., Guilherme, L.R., Li, L. 2010. Selenium accumulation in lettuce germplasm. Planta. 233:649-660.
Chiu, L., Zhou, X., Burke, S., Wu, X., Prior, R.L., Li, L. 2010. The purple cauliflower arises from activation of a MYB transcription factor. Plant Physiology. 154:1470-1480.
Cilia, M., Howe, K.J., Fish, T., Smith, D., Mahoney, J., Tamborindeguy, C., Burd, J.D., Thannhauser, T.W., Gray, S.M. 2011. Biomarker discovery from the top down: protein biomarkers for efficient virus transmission by insects (Homoptera: Aphididae) discovered by coupling genetics and 2-D DIGE. Proteomics. 11:2440-2458.
Cilia, M., Thannhauser, T.W., Gray, S.M. 2010. Tangible benefits of the pea aphid genome sequencing in proteomics research: enhancements in protein identification, data incorporation, and evaluation criteria. Journal of Insect Physiology. Available: http://www.ncbi.nlm.nih.gov/pubmed/21070785.
Cilia, M., Tamborindeguy, C., Fish, T., Howe, K.J., Thannhauser, T.W., Gray, S.M. 2011. Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein isoform expression to polerovirus transmission. Journal of Virology. 85(5):2148-2166.