Progress 05/11/05 to 05/31/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This is a Bridge CRIS project that resulted from splitting CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). In the last year a CRIS Project Plan following on from this bridge project has been written and completed OSQR review. The new CRIS was initiated in June 2006 and follows on in large part from this bridge project. Fruits and vegetables are major components of the human diet contributing a large portion of vitamins, minerals, antioxidants, and fiber. While plant-based food nutritional composition has clear and profound potential for positive human benefit, it has also proven to be a difficult set of traits to modify (via either traditional breeding or transgenic approaches) due to underlying complex
biosynthetic and regulatory pathways. Synthesis and accumulation of these compounds is the result of coordinated activity of many genes that may also impact additional aspects of plant growth, development and environmental response making metabolic engineering especially challenging. Effective manipulation of crop nutrient traits for human benefit will therefore require greater knowledge of the pathways involved in their synthesis and the regulatory systems that control them. Under this project we utilized emerging genomics technologies and strategies to define the genetic basis of carotenoid accumulation and metabolic flux and we initiated efforts toward creation of germplasm resources for evaluation of carotenoid accumulation, dietary availability and nutritive value. Carotenoids are a major focus of our effort as this class of chemicals includes compounds necessary for human health and well being (e.g. precursors to vitamin A) in addition to strong anti-oxidants associated with
positive nutritive effects. Genes and germplasm resources developed under this project will be used to establish mechanisms regulating nutrient metabolism and accumulation as well as to demonstrate the availability and nutritional value of different carotenoids as constituents of the human diet. Antioxidants including carotenoids have received considerable attention from plant and nutritional researchers in recent years due to reported links to prevention of cancer and other degenerative diseases. Cancer is the second leading cause of death in the USA with over 500,000 deaths annually and it cost the USA $125.3 billion in medical expense in 2003 (America Cancer Society). Diet plays an important role in the onset of cancer and it accounts for about one third of all cancer-related deaths. Thus, plant foods designed to reduce cancer risk in have great potential as value-added commodities for producers and consumers. Moreover, reducing cancer risk through food-based strategies would
likely result in large savings in healthcare costs and improve human health and well-being in a cost-effective and sustainable way. The research activities defined under this project will help address scientifically the value of antioxidants in disease prevention and additional aspects of human nutrition. Carotenoids were selected as a focus class of compounds as they have both known nutritional value (e.g. as vitamin A precursors) and have been frequently associated with additional health benefits whose merits require further testing. To avoid an overly narrow focus we will devote some effort to additional compounds of known or suspected nutritional and/or health-promoting value in the human diet. These will include small or pilot studies on the genetic regulation of synthesis and/or accumulation of ascorbate, folic acid and selenium in edible crop tissues. Research proposed on identifying molecular strategies for increasing nutritional content of crops has been identified as an
important aspect of two components of National Program 302. These are Component 1 - Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement (Problem Statement 1B: Applying Genomics to Crop Improvement) and Component II - Biological Processes that Improve Crop Productivity and Quality (Problem Statement IIC: High Value Products). 2. List by year the currently approved milestones (indicators of research progress) This CRIS project is a bridge CRIS that has led to development of a new CRIS that successfully underwent OSQR earlier this year. Consequently, only the FY2006 milestones are listed here. Milestones for FY 06 1. Grow, harvest and store at -80C normal and mutant tomato fruit tissues for microarray expression analysis. 2. Grow, harvest and store at -80C tomato and wild-species introgression line fruit tissues for microarray expression analysis. 3. Analyze carotenoid levels in tomato carotenoid mutant and variable lines as a first step in
comparative metabolite/gene expression analysis. 4. Develop new data correlation tools for the Tomato Expression Database (TED) to facilitate comparative expression and metabolite analysis and put them online. The purpose of this tool creation activity will be to provide online access to all public researchers simple, fast and reliable methods for identifying genes related to fruit development and whose expression patterns correlate with changes in levels of measured nutrient compounds. Correlation of nutrient and metabolite levels will allow us and others to select candidates for regulation of target nutrients. 5. Develop tomato COP10 RNAi gene repression construct and transform into A. tumefaciens. Tomato COP10 is a gene whose Arabidopsis counterpart has been shown to regulate light responses. Light is a regulator of carotenoid accumulation and thus a regulator of light response would be predicted to impact carotenoid levels. 6. Create repression and over-expression
constructs for cauliflower Or gene and transform into A. tumefaciens. 7. Isolate cauliflower Or protein complex from mutant high-carotenoid tissues. 8. Isolate organo-Se related genes via microarray analysis. 9. Develop DNA constructs for over-expression and repression of organo- Se genes in broccoli. 4a List the single most significant research accomplishment during FY 2006. Carotenoid and Transcriptional Profiling in Tomato: Carotenoid and transcriptional profiling data were developed for a collection of tomato carotenoid mutants. Correlative analysis of this data revealed that transcription of most genes in the carotenoid synthesis pathway is under feedback regulation. These results have been reported at a number of international conferences and have led to research in several labs (and our own) focused on defining the underlying molecular mechanisms of this regulatory system. These results have significant implications relevant to attempts to modify carotenoid accumulation
in crop plants as they suggest on the one hand that endogenous monitoring and regulatory systems may counter some strategies for engineering crop carotenoid accumulation. Alternatively, they also imply that manipulation of carotenoid levels may be achieved more simply and effectively if appropriate regulators are identified and targeted for manipulation. 4b List other significant research accomplishment(s), if any. 1. Use of cauliflower OR gene in creation of high carotenoid crops. The cauliflower OR gene regulates levels of carotenoids in cauliflower curds though mechanisms that remain unknown but seem to influence storage capability rather than synthesis. We tested the ability to influence carotenoid levels in other crops by expressing the OR gene in potato tubers. Resulting transgenic lines resulted in several fold elevation of tuber carotenoids including high levels of beta-carotene (precursor to vitamin A). This result demonstrates the utility of OR in heterologous systems
and for influencing carotenoid levels in non-floral tissues. 2. Use of the tomato RIN gene to regulate ripening in additional fruit species. In collaboration with researchers at Texas A&M and Warwick University (UK), we have shown that homologs of the tomato RIN gene are effective regulators of ripening in melon and strawberry, respectively. Specifically, the tomato RIN gene was used to identify homologous melon and strawberry sequences which were normally expressed in ripening fruit. These sequences were then used to create gene repression constructs (RNAi) which were transferred to the melon and strawberry genomes. Resulting transgenic plants showed delayed ripening of one to several weeks. These lines are being characterized further for effects on shelf- life, fruit quality and crop performance. The implications of this work are that a wide range of species and representing at least two different ripening physiologies share a common ripening regulator in the RIN transcription
factor. 3. Identification of a regulatory network influencing fruit ripening and carotenoid accumulation. Using available mutants and recently developed transgenic lines we tested the regulatory relationships of three transcription factors which influence ripening. Tests of epistasis indicated that the most comprehensive regulator of ripening available is the gene encoded at the tomato NOR locus, which in turn is necessary for expression of the RIN MADs-box transcription factor, which in turn regulates induction of the TDR4 MADS box transcription factor during ripening. This regulatory cascade of NOR - RIN - TDR4 is the earliest component of ripening regulation defined to date and represents a set of genes for targeting ripening regulation and downstream control of carotenoid accumulation in fruit species. 4. Altering expression of selenocysteine methytransferase in broccoli. Selenocysteine Se-methyltransferase is the key enzyme involved in the biosynthesis of
Se-methylselenocysteine, the bioactive form of selenium with carcinogenic activity. We have generated transgenic broccoli plants overexpressing and down-regulating expression of selenocysteine Se- methyltransferase. We have analyzed these transformants and shown that the BoSMT RNAi lines contain low levels of BoSMT mRNA with reduced amounts of Se-methylselenocysteine accumulation. Surprisingly, the overexpression of BoSMT in broccoli did not result in significantly elevated levels of Se-methylselenocysteine accumulation, indicating that Se-methylselenocysteine may be further metabolized into other Se compounds, which is under current investigation. 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Boyce Thompson Institute for Plant Research (BTI) entitled "The molecular basis of nutritional characteristics in edible fruit tissues" 1907-21000-019-01S. This project focused on development of tools for tomato
functional genomics in collaboration with the group of Dr. Greg Martin of the BTI. Specific activities have included ongoing construction of four databases that facilitate dissemination and examination of processed gene expression and metabolite accumulation data related to nutritional genomics experiments in tomato fruit. All four databases can be reached through the umbrella Tomato Expression Database (TED) home page at http://ted.bti.cornell.edu/. Tomato fruit tissues representing a continuum of development from 7 days post anthesis through advanced stages of ripening were harvested from normal and nearly isogenic mutant varieties in addition to fruit of the related species pepper. Expression profiling of Nr mutant fruit tissues has been completed as has pepper and the tomato rin mutant. We continued to maintain and distribute all tomato ESTs developed in prior years of this project to the world-wide research community and have recently included distribution of tomato BAC clones
to countries participating in the international tomato genome sequencing effort. We are currently testing a second-generation public tomato array based on an oligonucleotide (as opposed to cDNA) platform. This project terminated 7/31/2005 and has been replaced by a new SCA. 5. Describe the major accomplishments to date and their predicted or actual impact. This was a short-term bridge project in place until activation of 21000- 025-00D in June, 2006. The major accomplishments include the insights into feedback regulation of the carotenoid synthesis pathway described in 4a. These results impact scientists focused on metabolic engineering of carotenoid pathways in edible plant tissues and are fully reside under National Program 302, component 1 (Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-
user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The major accomplishments under this bridge project impact scientists and researchers focused on understanding the underlying genetic regulation of nutritional pathways in plants (specifically carotenoids). Data has been reported at several scientific conferences and will be incorporated into future scientific publications. The remainder of activities under this CRIS have resulted in preliminary data in support of our new CRIS project 21000-025-00D.
Impacts
(N/A)
Publications
- Giovannoni, J.J., El-Rakshy, S. 2005. Genetic regulation of tomato fruit ripening and development and implementation of associated genomics tools. Acta Horticulturae. 682:63-72.
- Giovannoni, J.J. 2006. Breeding new life into plant metabolism. Nature Biotechnology. 24:418-419.
- Barry, C., Giovannoni, J.J. 2006. Ripening in the tomato green-ripe mutant is inhibited by ectopic expression of a novel protein that disrupts ethylene signaling. Proceedings of the National Academy of Sciences. 103:7923-7928.
- Mueller, L., Tanksley, S., Giovannoni, J.J., Van Eck, J. 2005. The tomato sequencing project, the first cornerstone of the international solanaceae project (sol). Comparative and Functional Genomics. 6:153-158.
- Fei, Z., Tang, X., Alba, R., Giovannoni, J.J. 2006. Tomato expression database (ted): a suite of data presentation and analysis tools. Nucleic Acids Research. 34:D766-D770.
- Barry, C., Thompson, A., Seymour, G., Grierson, D., Giovannoni, J.J. 2005. Ethylene insensitivity conferred by the green-ripe (gr) and never-ripe 2 (nr-2) ripening mutants of tomato. Plant Physiology. 138:267-275.
- Moore, S., Payton, P.R., Wright, M., Tanksley, S., Giovannoni, J.J. 2005. Utilization of tomato microarrays for comparative gene expression analysis in the solanceae. Journal of Experimental Botany. 56:2885-2895.
- Alba, R., Payton, P.R., Fei, Z., Debbie, P., Martin, G., Tanksley, S., Giovannoni, J.J. 2005. Transcriptome and selected metabolite analysis reveal multiple points of ethylene control during tomato fruit development. The Plant Cell. 17:2954-2965.
- Thannhauser, T.W., Yang, Y., Zhang, S., Damasceno, C., Sherwood, R., Rose, J. 2006. Itraq based protein quantitative analysis of the cell wall proteome of pathogen-infected tomato leaves. Journal of Biomolecular Techniques. 17:40.
- Thannhauser, T.W., Yang, Y., Zhang, S. 2006. Comparison of protein identification by maldi-tof/tof and lc electrospray ionization tandem ms for 2d gel separated cauliflower samples. Journal of Biomolecular Techniques. 17:48.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This is a new Bridge CRIS project that has arisen from CRIS project 1907- 21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species) that terminated in FY2005 and has been split into two new CRIS projects; this project and a second CRIS on improving transport and detoxification of selected mineral elements in crop plants. A draft CRIS Project Plan has been written and will undergo OSQR review in the fall of 2005. Fruits and vegetables are major components of the human diet contributing a large portion of vitamins, minerals, antioxidants, and fiber. While plant-based food nutritional composition has clear and profound potential for positive human benefit, it has also proven to be a difficult set of traits to modify (via either traditional breeding or
transgenic approaches) due to underlying complex biosynthetic and regulatory pathways. Synthesis and accumulation of these compounds is the result of coordinated activity of many genes that may also impact additional aspects of plant growth, development and environmental response making metabolic engineering especially challenging. Effective manipulation of crop nutrient traits for human benefit will therefore require greater knowledge of the pathways involved in their synthesis and the regulatory systems that control them. We will utilize emerging genomics technologies and strategies (defined here broadly as transcriptomics, proteomics, metabolomics, and other omics approaches for characterizing large-scale genome regulated activities) to define the genetic basis of carotenoid accumulation and metabolic flux and we will additionally create germplasm resources for evaluation of carotenoid accumulation, dietary availability and nutritive value. Carotenoids will be a major focus of
our effort as this class of chemicals includes compounds necessary for human health and well being (e.g. precursors to vitamin A) in addition to strong anti-oxidants associated with positive nutritive effects. Genes and germplasm resources developed under this project will be used to establish mechanisms regulating nutrient metabolism and accumulation as well as to demonstrate the availability and nutritional value of different carotenoids as constituents of the human diet. These same tools are likely to have broad effects that may open doors to study additional nutrient compounds sharing similar control mechanisms (e.g. flavonoids). Antioxidants including carotenoids have received considerable attention from plant and nutritional researchers in recent years due to reported links to prevention of cancer and other degenerative diseases. Cancer is the second leading cause of death in the USA with over 500,000 deaths annually and it cost the USA $125.3 billion in medical expense in
2003 (America Cancer Society). Diet plays an important role in the onset of cancer and it accounts for about one third of all cancer-related deaths. Thus, plant foods designed to reduce cancer risk in have great potential as value-added commodities for producers and consumers. Moreover, reducing cancer risk through food-based strategies would likely result in large savings in healthcare costs and improve human health and well-being in a cost-effective and sustainable way. The research activities defined under this project will help address scientifically the value of antioxidants in disease prevention and additional aspects of human nutrition. Carotenoids were selected as a focus class of compounds as they have both known nutritional value (e.g. as vitamin A precursors) and have been frequently associated with additional health benefits whose merits require further testing. To avoid an overly narrow focus we will devote some effort to additional compounds of known or suspected
nutritional and/or health-promoting value in the human diet. These will include small or pilot studies on the genetic regulation of synthesis and/or accumulation of ascorbate, folic acid and selenium in edible crop tissues. Research proposed on identifying molecular strategies for increasing nutritional content of crops has been identified as an important aspect of two components of National Program 302. These are Component 1 - Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement (Problem Statement 1B: Applying Genomics to Crop Improvement) and Component II - Biological Processes that Improve Crop Productivity and Quality (Problem Statement IIC: High Value Products). 2. List the milestones (indicators of progress) from your Project Plan. This CRIS project is a bridge CRIS that is scheduled to undergo OSQR review later this year. Hence, only the FY2006 milestones are listed here as future year milestones will be influenced by the outcome of the
OSQR review. Milestones for FY 06 Grow, harvest and store at 80C normal and mutant tomato fruit tissues for microarray expression analysis. Grow, harvest and store at 80C tomato and wild-species introgression line fruit tissues for microarray expression analysis. Analyze carotenoid levels in tomato carotenoid mutant and variable lines as a first step in comparative metabolite/gene expression analysis. Develop new data correlation tools for the Tomato Expression Database (TED) to facilitate comparative expression and metabolite analysis and put them online. The purpose of this tool creation activity will be to provide online access to all public researchers simple, fast and reliable methods for identifying genes related to fruit development and whose expression patterns correlate with changes in levels of measured nutrient compounds. Correlation of nutrient and metabolite levels will allow us and others to select candidates for regulation of target nutrients. Develop tomato COP10
RNAi gene repression construct and transform into A. tumefaciens. Tomato COP10 is a gene whose Arabidopsis counterpart has been shown to regulate light responses. Light is a regulator of carotenoid accumulation and thus a regulator of light response would be predicted to impact carotenoid levels. Create repression and over-expression constructs for cauliflower Or gene and transform into A. tumefaciens. Isolate cauliflower Or protein complex from mutant high-carotenoid tissues. Isolate organo-Se related genes via microarray analysis. Develop DNA constructs for over-expression and repression of organo-Se genes in broccoli. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. See AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). This is a bridge CRIS.
Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This is a Bridge project and this question will be addressed in the new CRIS report (approval anticipated in early 2006). 4a What was the single most significant accomplishment this past year? See AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). This is a Bridge CRIS. 4b List other significant accomplishments, if any. Because this is a bridge CRIS that is scheduled to undergo OSQR review later this year, please see the AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species) for a description of the other significant accomplishment during FY 2004. 4d Progress report. Because
this is a bridge CRIS that is scheduled to undergo OSQR review later this year, the Progress Reports for ongoing subordinate projects are listed here. Please see the AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species) for the Progress Reports for subordinate projects that have terminated. 1907-21000-019-01S This report serves to document research conducted under grant agreement between ARS and the Boyce Thompson Institute for Plant Research (BTI). Additional details of this research can be found in the report for the parent project 1907-21000-012-00D. This project focused on development of tools for tomato functional genomics in collaboration with the group of Dr. Greg Martin of the BTI. Specific activities have included ongoing construction of four databases that facilitate dissemination and examination of processed gene expression and metabolite accumulation data related to
nutritional genomics experiments in tomato fruit. All four databases can be reached through the umbrella Tomato Expression Database (TED) home page at http://ted.bti.cornell.edu/. Tomato fruit tissues representing a continuum of development from 7 days post anthesis through advanced stages of ripening were harvested from normal and nearly isogenic mutant varieties in addition to fruit of the related species pepper. Expression profiling of Nr mutant fruit tissues has been completed as has pepper and the tomato rin mutant. We continued to maintain and distribute all tomato ESTs developed in prior years of this project to the world-wide research community and have recently included distribution of tomato BAC clones to countries participating in the international tomato genome sequencing effort. We are currently testing a second-generation public tomato array based on an oligonucleotide (as opposed to cDNA) platform. 1907-21000-019-02R This report serves to document research conducted
under grant agreement between ARS and BARD. Additional details of this research can be found in the report for the parent project 1907-21000-012-00D. This is the final year of a three-year project with the objective of developing tools for melon genomics and their utilization for analysis of fruit quality. Specific objectives for the ARS lab included sequencing of approximately 1500 melon fruit ESTs (clones provided by the Israeli co-PI) and creation of a small melon array. Fruit samples derived from lines of varying quality will be profiled by our Israeli collaborators (Katzir lab) for expression using the small melon array to be developed under this project. To date the ARS lab has sequenced approximately 3500 ESTs over twice the number promised due to cost savings. 2200 melon unigenes were re- arrayed and DNA template prepared for creation of microarrays. Test arrays have been created and protocols are near completion for release of the first public melon array. We have also
established collaborations with melon researchers in Spain that will result in inclusion of an additional 1000 ESTs on our array in the future. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. See AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). This is a Bridge CRIS in place until the new CRIS focused on nutritional genomics of crop species is in place. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? See AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). This is a bridge CRIS. 7.
List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). See AD-421 for the terminated CRIS project 1907-21000-012-00D (Using genomic/proteomic technologies to improve quality and stress tolerance of crop species). This is a Bridge CRIS.
Impacts
(N/A)
Publications
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