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United States Department of Agriculture

Agricultural Research Service

2009 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
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 crop 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. In the last year proteins which interact with Or were isolated and characterized and provide new candidates for elucidation the function of the OR protein. In a second research track, gene targets and/or interacting proteins of these regulators have also been identified and analyzed in addition to homologs of these genes in other species (suggesting potential for broad applicability to diverse crops species). Significant progress has also been made in the development of new methodologies to minimize or eliminate experimental problems that have led to improved confidence in the data we generate. Proteomics infrastructure and analysis has been upgraded to facilitate more accurate proteomics analysis which in turn enables 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 performed on developing tomato fruit that were also characterized for nutrient quality. 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. Identified a broccoli gene that impacts levels of bioactive selenium 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. The discovery opens up new aspects in increasing the accumulation of bioactive compounds by reducing their volatilization in plants.

2. Identified a transcription factor linking fleshy fruit development and subsequent 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. Fossil records suggest the emergence of fleshy-fruited species from progenitors bearing dry and dehiscent fruit with examples of conversion between fruit types over evolutionary time. The existence of closely related species with dry and fleshy fruits, as in the Solanaceae where tomato and pepper produce fleshy fruits while petunia and tobacco produce dry capsules, would suggest that the molecular basis of such differences are not necessarily complicated. The association of fleshy fruit development with ripening further suggests that these processes may be related. We have previously mined tomato microarray and digital expression profiling data from wild species introgression lines which serve as a reservoir of genetic diversity to identify genes associated with ripening and recovered the AGAMOUS-like gene TAGL1 whose expression persists from early carpel development through maturation and is up-regulated in concert with ripening. RNAi repression of TAGL1 in tomato resulted in reduced pericarp thickness and ripening inhibition suggesting a single molecular bridge linking fleshy pericarp development and subsequent fruit ripening.

3. Consolidated and expanded the Tomato Functional Genomics Database. Tomato serves as a model system for many biological inquiries including those related to fruit development, quality and pathogen response. In prior years we have developed and updated several online public databases that housed and served data from microarray expression profiling experiments, metabolite profiles, QTL (quantitative trait locus) mapping and genetic and clone resources. In the last year we have consolidated these databases into a single tomato gene expression database that includes tools for analyzing and querying the incorporated data and we have dramatically increased the data content by including many additional public sources of data that researchers can query and analyze online. The database can be accessed at Public and private research labs the world over are using this tool for large-scale gene expression analyses in tomato and related crop species (such as pepper and eggplant). A second database devoted to cucurbit genomics data has also been developed using the tomato format and infrastructure (

5.Significant Activities that Support Special Target Populations
We hosted several international graduate students, postdocs and professors for training in tomato genome tool development and use. A faculty member from Italy, postdocs from Spain, Italy, and Germany and graduate students from China and Italy visited our lab for periods of 1 – 9 months to receive training in development and use of tomato genomics resources and infrastructure. All of these people were supported by fellowships from their respective governments.

We hosted four women/minority summer interns. All four undergraduate 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 four 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. One of these interns won the award for best poster. Specific project activities included genetic analysis of a new ripening mutation, isolation of fruit-specific promoters, characterization of transgenic tomato plants altered in carotenoid content and microarray analysis of tomato introgression lines for identification of candidate genes associated with fruit nutrient quality.

6.Technology Transfer

Number of Invention Disclosures Submitted1
Number of Web Sites Managed3

Review Publications
Portnoy, V., Benyamini, Y., Giovannoni, J.J., Schaffer, A., Tadmor, Y., Lewinshon, E., Katzir, N. 2008. The Molecular and Biochemical Basis for Varietal Variation in Sesquiterpene Content in Melon (Cucumis melo L.) Rinds. Plant Molecular Biology. 66:647-661.

Mueller, L., Tanksley, S., Giovannoni, J.J., Vaneck, J., Stack, S., Buels, R. 2009. A snapshot of the emerging tomato genome sequence. The Plant Genome. 2:78-92.

Akhtar, T., Naponelli, V., Gregory, J., Giovannoni, J.J., Hanson, A. 2008. Tomato y-glutamilhydrolases: expression, characterization, and evidence for heterodimer formation. Plant Physiology. 148:775-784.

Wang, S., Liu, J., Feng, Y., Niu, X., Giovannoni, J.J., Liu, Y. 2008. Altered Plastid Levels and Potential for Improved Fruit Nutrient Content via Down-regulation of the Tomato DDB1 Interacting Protein CUL4. Plant Journal. 55:89-103.

Wechter, W.P., Levi, A., Harris-Shultz, K.R., Davis, A.R., Fei, Z.J., Katzir, N., Giovannoni, J.J., Salman-Minkov, A., Hernandez, A., Thimmapuram, J., Tadmor, Y., Portnoy, V., Trebitsh, T. 2008. Gene expression in developing watermelon fruit. Biomed Central (BMC) Genomics. 9:275-282.

Ioannidi, E., Kalamaki, M., Engineer, C., Petaraki, I., Alexandrou, D., Giovannoni, J.J., Kanellis, A. 2009. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. Journal of Experimental Botany. 60:663-678.

Ponce-Valdez, M., Moore-Fellman, S., Giovannoni, J.J., Gan, S., Watkins, C. 2009. Differential fruit gene expression in two strawberry cultivars in response to elevated CO2 during storage revealed by a heterologous fruit microarray approach. Postharvest Biology and Technology. 51:131-140.

Barry, C., Mcquinn, R.P., Chung, M., Besuden, A., Giovannoni, J.J. 2008. Amino acid substitutions in homologs of the STAY-GREEN (SGR) protein are responsible for the green-flesh and chlorophyll retainer mutations of tomato and pepper. Plant Physiology. 147:179-187.

Last Modified: 8/27/2015
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