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United States Department of Agriculture

Agricultural Research Service

2010 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
The overall project objective is to elucidate mechanisms of nutrient (esp. carotenoid) 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 have been 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 and the tomato ripening-inhibitor (rin) mutation which results in impaired ripening and the high-pigment (hp) mutation which leads to elevated tomato carotenoids. In the last year, proteins which interact with Or have continued to be characterized and provide new candidates for determining how the OR protein facilitates elevated carotenoid levels in plant tissues. In a second research track, 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). In the case of the gene encoded by the rin locus, we have identified numerous gene targets which interact with this key ripening regulator. In summary, these results suggest that rin regulates genes in most known pathways and processes associated with ripening, suggesting this gene is indeed a master regulator of ripening. This feature of rin suggests it would be an optimal target for manipulating the complete fruit ripening process so as to optimize both production traits such as uniform ripening and shelf-life in addition to consumer traits such as flavor, texture and nutritional quality. The release of the tomato genome sequence in December 2009 has allowed more accurate proteomics analysis which in turn has enabled more comprehensive systems approaches toward identification of key genes regulating nutritional content, ripening and shelf-life. The third track toward nutrient-associated gene identification and characterization is through comparative gene and/or protein expression analysis on a range of germplasm representing traditional and wild species genetic diversity. Proteomics and gene expression profiling has been completed on a series of developing tomato fruit that were also characterized for nutrient quality in addition to specific fruit tissues to assist in defining where and when ripening processes are initiated and manifested. For example, data recovered in the last year demonstrates that the fruit epidermis is the primary site of synthesis and deposition of phenolic pigments while the pericarp (the tomato fruit wall) is largely devoid of such compounds (and associated gene expression) but high in carotenoid synthesis and accumulation. New transgenic plants have been developed in the last year and new genes clearly impacting fruit ripening and nutrient content have been identified. Our public database has been consolidated and updated to house and disseminate this information in addition to integrated data recovered from the public domain.

1. Characterization of a broccoli gene that impacts nutritional content in vegetable crops. Broccoli accumulates high level of bioactive forms of selenium. Both biosynthesis and volatilization of selenium compounds affect the accumulation of the bioactive forms of selenium. To reduce selenium volatilization for eventually producing healthy crops, we have isolated a broccoli methyltransferase gene whose product mediates selenium volatilization in both bacteria and plants. This methyltransferase represents the first plant enzyme that is not directly involved in sulfur/selenium metabolism yet mediates selenium volatilization. In the last year, researchers at the USDA-ARS Robert W. Holley Center in Ithaca, NY have validated and refined the characterization of this broccoli methytransferase gene confirming its potential utility in enhancing the nutritional value of crops via reduced nutrient loss via natural volatilization. The discovery opens up new avenues toward increasing the accumulation of bioactive compounds in plants.

2. Characterization of a tomato gene which serves as a natural repressor of 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. Researchers at the Robert W. Holley Center for Agriculture and Health in Ithaca, NY have identified a tomato gene whose natural function is to slow down the ripening process. The gene designated AP2a due to its similarity to a gene shown to regulate flower development in the weed Arabidopsis was shown to be elevated in expression during ripening. Removal of gene activity in genetically modified tomato plants resulted in increased production of the hormone ethylene that naturally ripens fruits and accelerated ripening. These results reveal a new player participating in the regulation of fruit ripening and a new target for manipulation (via breeding or transgene manipulation) of ripening and associated characteristics including fruit texture, color, and shelf-life with downstream implications for food security and crop nutritional quality.

5.Significant Activities that Support Special Target Populations
As in past years, during FY10 we hosted a number of international graduate students, postdocs and professors for training in tomato genome tool development and use. Faculty members from Italy, Israel and China were hosted for sabbaticals ranging from 3–9 months. Postdocs from Spain and Germany and graduate students from China, Italy and The Netherlands visited our lab for periods of 1–6 months to receive training in development and use of tomato genomics resources and infrastructure. All of these individuals were supported by fellowships from their respective governments or from international NGOs.

We also hosted three women/minority summer interns. All three summer interns were hosted in the labs of this project through a program administered through the Boyce Thompson Institute and with National Science Foundation support. The program is intended to support interest among under-represented minorities for the sciences in general and plant science in particular. All students generated written research proposals, completed 9-week research projects and presented 15 min. oral or poster presentations to an evaluation committee of Cornell professors sponsored by BTI. Specific project activities included genetic analysis of a tomato carotenoid mutation, characterization of gene candidates for fruit-specific promoters to be used in driving genes of interest in fruit tissues and, characterization of transgenic tomato plants altered in carotenoid content.

Review Publications
Matayas, A., Gapper, N., Chung, M., Giovannoni, J.J., Rose, J. 2009. Biology and genetic engineering of fruit maturation for enhanced quality and shelf-life. Current Opinion in Biotechnology. 20:197-203.

Slocombe, S., Schauvinhold, I., Azziz, N., Larson, T., Giovannoni, J.J., Dixon, R., Broun, P. 2009. Genomic analysis of branched chain fatty acid and acyl sugar production in Solanum pennellii and Nicotiana benthamiana. Plant Physiology. 148:1830-1846.

Waller, J., Akhtar, T., Lara-Nunez, A., Gregory, J., Giovannoni, J.J., Hanson, A. 2010. Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit. Molecular Plant. 3:66-77.

Vrebalov, J., Pan, I., Arroyo, A., Chung, M., Poole, M., Rose, J., Seymour, G., Giovannoni, J.J., Irish, V. 2009. Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene, TAGL1. The Plant Cell. 21:3041:3062.

Li, L., Lu, S. 2008. Carotenoid Metabolism: the Biosynthesis, Regulation, and Beyond. Journal of Integrative Plant Biology. 50:778-785.

Zhou, X., Li, L. 2010. Think outside of the box: selenium volatilization altered by a broccoli gene in the ubiquinone biosynthetic pathway. Plant Signaling and Behavior. 5:74-75.

Zhou, X., Van Eck, J., Li, L. 2008. Use of the cauliflower OR gene for improving crop nutritional quality. Biotechnology Annual Review. 14(6):171-190.

Salas Fernandez, M.G., Hamblin, M., Li, L., Rooney, W.L., Tuinstra, M.R., Kresovich, S. 2008. Quantitative trait loci analysis of endosperm color and carotenoid content in sorghum grain. Crop Science. 48:1732-1743.

Yuan, Y., Chiu, L., Li, L. 2009. Transcriptional regulation of anthocyanin biosynthesis in red cabbage. Planta. 230:1141-1153.

Xiangjun, Z., Cooke, P.H., Li, L. 2009. Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development. Journal of Experimental Botany. 61:357-367.

Van Eck, J., Zhou, X., Lu, S., Li, L. 2010. Modulation of carotenoid accumulation in transgenic potato by inducing chromoplast formation with enhanced sink strength. In: Fett-Neto, A., editor. Metabolic Engineering of Plant Secondary Pathways with Methods in Molecular Biology Book Series. Chapter 6. New York, NY: Springer. p. 77-93.

Stack, S., Royer, S., Shearer, L., Chang, S., Giovannoni, J.J., Westfall, D., Anderson, L. 2009. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome. Cytogenetics and Genome Research. 124:339-350.

Last Modified: 9/1/2015
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