2008 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.
This project was initiated in June of 2006 taking several research strategies toward the goal of developing basic biological insights that would support the development of food crops with enhanced nutrient quantity and/or quality. Specific objectives center on identification of genes and corresponding proteins which influence crop nutrient quality and which might serve as tools for enhanced crop quality. All yearly milestones for the project were met or exceeded and the project is operating within the allocated budget. 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. Gene targets of the RIN protein have also been identified. Significant progress has 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. This is particularly true with the LC-MS/MS data generated on our Model 4700 mass spectrometer. This instrument has low throughput for LC-MS/MS applications and the data generated on this instrument has a high false discovery rate (> 10%). To address the throughput issue we are replacing the 4700 with a new high throughput instrument (SYNAPT HDMS, Waters) which will be available to us in the fall of 2008. We have developed a peptide isoelectric focusing method to replace the strong cation exchange step of our 2D LC separation scheme which allows us to use peptide pI as a filter to eliminate false positives. This, coupled with new methods developed to test putative identification hypotheses will allow us to identify true false positives on a peptide by peptide basis, eliminating the need to rely on statistical approaches.
The third track toward nutrient-associated gene identification and characterization is through comparative gene and/or protein expression analysis. Proteomics and gene expression profiling has been performed on developing tomato fruit that were also characterized for nutrient quality in an initial effort toward this end. New transgenic plants have been developed in the last year and several genes impacting fruit carotenoid content have been identified. During the last year we have continued to asses the utility of wild-species tomato introgression lines for comparative expression profiling and nutrient metabolite analysis of the same lines. Several genes are now being analyzed in transgenic plants to test whether they indeed impact nutrient profiles. Our public database has been updated to house and disseminate this information. In the last year we have consolidated our four prior databases into one easier use tool with enhanced functionality. NP 302, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2C.
Characterization of a transcription factor regulating ripening; While a number of ripening-associated genes have been isolated from tomato and other species, few “master regulator” genes have been identified to date. One such gene previously identified by our group is the RIPENING-INHIBITOR (RIN) transcription factor. In an effort to expand our understanding of fruit ripening control and to develop new gene targets for breeding and genetic engineering, we have recently identified a new regulator which apparently acts in the control cascade of RIN and which impacts downstream ripening traits including accumulation of nutritionally important carotenoids and important quality attributes such as softening and ascorbate (vitamin C) accumulation. Regulatory analysis of this gene suggests that it represents a control mechanism operating independent of the ripening hormone ethylene control and thus may represent a regulatory target common to fruiting species which do not require ethylene for ripening (e.g. strawberry, grape, citrus). Ongoing analysis will reveal whether or not this gene may prove useful as a tool in regulating post-harvest shelf-life, texture and nutrient quality in fruit crop species.
Consolidation and expansion of 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 of this and the preceding project we have developed four 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. The database can be accessed at www.ted.bti.cornell.edu. 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).
These activities will facilitate the achievement of various components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement in that it represents an improved Proteomic technology that can extend genomic understanding to the level of gene products.
Functional characterization of a gene influencing accumulation of carotenoids in vegetable foods. Carotenoids are important plant derived nutritional compounds that when deficient in the human diet can lead to severe disease consequences. In prior years we characterized the cauliflower OR (ORANGE) protein that facilitates high level accumulation of carotenoids in cauliflower curds (a clearly beneficial characteristic) yet also results in altered plant growth and morphology with potentially negative effects on yield and appearance. To better understand these phenomena, we isolated a putative regulatory protein that interacts with OR. We show that the protein affects the plant’s response to plant hormones via short roots and delayed flowering. This provides a new functional role of this isolated protein, helps explain the effect of OR on plant growth and presents a target for manipulation toward the objective of increased carotenoid levels but absent negative side effects on plant growth and field performance and productivity.
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 Argentina and graduate students from Taiwan 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 two minority summer interns. Three undergraduate summer interns were hosted in the labs of this CRIS 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 three 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 isolation of fruit-specific promoters and microarray analysis of tomato introgression lines for identification of candidate genes associated with fruit nutrient quality.
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Lopez, A., Yang, Y., Thannhauser, T.W., Li, L. 2008. Phytoene desaturase is present in a large protein complex in the plastid membrane. Physiologia Plantarum. 133:190-198.
Lopez, A., Van Eck, J., Conlin, B., Paolillo, D., O'Neill, J., Li, L. 2008. Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers. Journal of Experimental Botany. 59:213-223.
Giovannoni, J.J. 2007. Fruit ripening mutants yield insight into ripening control. Current Opinion in Plant Biology. 10:283-284.
Barry, C., Giovannoni, J.J. 2007. Ethylene and Fruit Ripening. Journal of Plant Growth Regulation. 26:143-159.
Beatriz, C., Giovannoni, J.J. 2008. Molecular Biology of ethylene during tomato fruit development and maturation. Plant Science. 175:106-113.
Datema, E., Mueller, L., Buels, R., Giovannoni, J.J., Visser, R., Stiekema, W., Vanham, R. 2008. Comparative BAC end sequence analysis of tomato and potato reveals overrepresentation of specific gene families in potato. Biomed Central (BMC) Plant Biology. 8:34.
Keller, C., Readman, C., Shin, H., Bosdet, I., Wilkin, J., Vrebalov, J., Giovannoni, J.J., Douglas, C. 2007. A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation. Plant Journal. 50:1063-1078.