Progress 08/01/02 to 06/03/04
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? The major problem is to develop a tomato that is rich in lycopene and other nutritious, health-promoting carotenoids. Tomato is a major agricultural commodity in the United States and the major source of lycopene in Western diets. Epidemiological studies show a strong correlation between consumption of tomatoes and tomato products and a lowered risk of both prostate and gastrointestinal cancers and cardiovascular disease. This protective effect is attributed to the antioxidant properties of lycopene, the compound responsible for the red color of tomato fruit. Lycopene is the most powerful biological antioxidant and is able to quench singlet oxygen from free radical formation resulting from oxidative reactions in tissues, thereby protecting organs from damage. Other carotenoids such as beta-carotene,
lutein, and zeaxanthin also have strong antioxidant activity and are believed to protect against other debilitating diseases such as macular degeneration and cataracts. Research objectives: (1) To identify genes that increase the biosynthesis of lycopene by a cool-temperature-induced mechanism that enhances ripening in VFNT Cherry tomato fruit and calyces cultured in vitro. (2) To identify factors involved in triggering this mechanism top aid in enhancing the ripening process. (3) To screen a group of bioregulators for their ability to increase total content of lycopene and/or the cis- lycopene (and other carotenoids) in tomato fruit and search for key control points in biosynthesis. Cancers of the prostate gland and digestive tract are now major malignancies throughout the world. In fact, prostate cancer is one of the most common malignancies among men in affluent nations. Lycopene and other carotenoids are highly desirable to tomato growers and processors and the general public,
enhancing both appeal and potential health benefits of the tomato. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 1998) 1. Screen bioregulators [2-(4-chlorophenyl-thio) triethylamine (CPTA), 2- (3,5,-dichorophenoxy)triethylamine(DCPTA), 2-(4-methylphenoxy) trimethylamine (MPTA), NBMA, and NBIA for their effects on lycopene- isomer production. (The last two compounds are patented, and their use was under license.) 2. Purchase necessary equipment, determine appropriate method, and adapt procedure for carotenoid extraction and lycopene-isomer identification, separation, and quantification to our new HPLC system. Year 2 (FY 1999) 1. Analyze carotenoid isomers in tomatoes treated with bioregulators to identify bioregulators that are effective in producing cis-lycopene isomers and measure any other changes in carotenoid composition resulting from treatment. 2. Document finds from in vitro-bioregulator studies on tomato in journal publication. 3.
Begin tests on greenhouse tomato plants of any of the bioregulators that result in the production of increased amounts of cis-lycopene isomers and/or other beneficial carotenoids in in-vitro-grown tomatoes. 4. Isolate RNA from appropriate tomato tissue for screening to determine activated genes in high lycopene production of cis- and trans- lycopene isomer production. 5. Set up gene expression screens to detect genes involved in activated lycopene synthesis in our cool-temperature-induced tomato system and genes involved in the synthesis of cis vs. trans isomers of lycopene. Year 3 (FY 2000) 1. Identify genes involved in FY 2000, using RNA from isolated tomato tissues. 2. Document results of field tests of bioregulators on altered lycopene content in tomato fruit. 3. Develop strategies to transfer lycopene-activation process (improved methods to insert genes into specific retions of the plant genomes) to the field. Year 4 (FY 2001) 1. Document results on (a) genes activated in
cool-temperature-induced tomato system that are involved in enhanced lycopene biosynthesis and (b) genes activated in the biosynthesis of cis-lycopene isomers. 2. Test strategies developed in FY 2000 (to engineer plants) to produce tomato fruit that contain high lycopene and high cis-lycopene isomers. 3. Document results on factors produced during cool temperature-induced activation of lycopene production (FY 2000, No. 3). 4. Screen for genes involved in the activation of the synthesis of other beneficial carotenoids (e.g., flavor volatiles, zeaxanthin). Year 5 (FY 2002) 1. Grow fruit using strategies developed in FY 2001 (no. 2). 2. When fruit in FY 2001 (no. 2) are produced and analyzed, begin collaborative bioavailability studies in human subjects, using high- lycopene tomatoes (high all-trans-lycopene isomer, high cis-lycopene and low lycopene) and analyze carotenoids in human serum samples. 3. Document results of bioengineering high-lycopene/high-carotenoid tomato plants
(FY 2000 no. 4). 4. Identify genes found that activate the biosynthesis of other beneficial carotenoid (under FY 2001, no. 4) and (b) develop strategies for their transfer to plants for field studies. 5. Document results on engineering tomato plants that produce high- lycopene and/ or high cis-lycopene-containing tomato fruit (see FY 2001, no. 2). Year 6 (FY 2003) 1. Continue bioavailability studies with trials testing the effect of oil on bioavailability and analyze human serum samples as in FY 2002 (no. 2) for carotenoid composition. 2. Document bioavailability studies (FY 2002, no. 2 and FY 2003, no. 1). 3. Test strategies developed to transfer genes leading to tomato fruit. 3. Milestones: A. Milestones addressed and met in FY 2004. Additional accomplishment of milestone FY 1998, #1: Analyzed results of multiple in vitro and greenhouse experiments on the effect of NBMA [see (D) under Other Significant Accomplishments]. FY 2000 Milestone #1: This work was delayed until FY
2003 and 2004 because the tomato oligo chip for microarray analyses was not available to our CRADA partner until that time [see (B) under Other significant accomplishments]. Accomplishment of milestones that depend upon this one consequently have been delayed. Plans are in place to extend the work and complete the project under a new CRIS Project No. 5325-41440-004-00D. FY 2002 Milestone #2: This milestone was delayed because human studies at the cooperating ARS site had not been completed and human serum samples were not available until this year [see ((e) under Other significant accomplishments]. Those studies that depend upon the accomplishment of this milestone will follow under our new CRIS project. All milestones have been completed with the exception of the following: FY 2000, Milestone #2: Field tests of bioregulators on altered lycopene content of tomato fruit are being postponed until an interested CRADA partner is found. FY 2001, Milestone #2: This work is in progress
and those depending upon the results will be continued in the future under our new CRIS project. B. Milestones to address over next three years. This project is terminating. Future research will be merged into a new CRIS Project 5325-41440-004-00D. 4. What were the most significant accomplishments this past year? A. Carotenoids such as lycopene have been shown to have numerous health benefits. Researchers in the Processed Foods Research Unit at WRRC, Albany, CA analyzed the carotenoid and fatty acid composition of gac fruit (Momordica cochinchinesis), a fruit that is grown in Southeast Asia. Gac contained 30-50 times more lycopene and 50-150 times more beta-carotene than tomato. It also contained high concentrations of unsaturated fatty acids which increase absorption of lycopene and beta-carotene in humans. This research identified an excellent source of health promoting carotenoids and fatty acids, providing a means to supply these essential nutrients to populations suffering
from degenerative diseases such as blindness, cancer and cardiovascular disease. B. Tomato catsup is an excellent source of lycopene, carotenoids, and antioxidant compounds. Researchers in the Processed Foods Research Unit at WRRC, Albany, CA analyzed 13 commercial catsups for carotenoid content, antioxidant activity, and percentage solids. Organic catsups had the highest lycopene, carotenoid and antioxidant levels. Solids content was not an indicator of nutritional value. This research was important in demonstrating that nutritional value cannot be assessed merely by appearance or advertising appeal of various food products. Research on ways to increase the lycopene content in tomatoes is needed to improve the healthfulness of tomatoes for consumers and the food industry. Increased understanding of how the tomato system produces lycopene is needed to meet this goal. Researchers in the Processed Foods Research Unit at WRRC, Albany, CA collaborated with an industrial partner
through a trust agreement, 5325-41440-004-01T, to obtain a genetic map of a particular tomato sepal that produces large amounts of lycopene at cool temperatures. Changes in gene expression during ripening were evaluated to determine which carotenoid biosynthesis genes were induced or suppressed during this process. This research provided information necessary to increase the healthfulness of tomatoes. Lycopene, the principle carotenoid in tomatoes, is both an excellent natural colorant and a cancer preventative agent. Researchers in the Processed Foods Research Unit at WRRC, Albany, CA developed a highly efficient, environmentally friendly process to extract food-grade lycopene from tomato waste. This process and product was invented through a collaboration with a small business and a patent is being filed on the discovery. This research, when commercialized, will lower the cost of lycopene production significantly, allowing its health promoting and coloring properties to be
better utilized by the food industry and providing a value added outlet for tomato waste. Carotenoids in tomatoes, such as lycopene, are effective in preventing cancer; however, the mechanisms for their effects are poorly understood. Researchers in the Processed Foods Research Unit at WRRC, Albany, CA collaborated with researchers in the Western Human Nutrition Research Center, Davis, CA to compare the bioavailability of tomatoes high in trans-lycopene versus those high in cis-lycopene. Increased understanding of the bioavailability of trans- versus cis- isomers will enable us to improve the healthfulness of food products through both processing and formulation. C. Significant activities that support special target populations. None. D. Progress Report. None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Carotenoid enhancement. Tomatoes naturally contain a health-promoting, red-colored compound lycopene, which
contributes significantly to the health of the consuming public. ARS researchers developed a high- lycopene-producing tomato and are analyzing genes that are activated or suppressed to produce this effect. ARS confirmed that treating tomato seeds with low concentrations (that cannot be detected in the plant) of a bioregulator compound increases lycopene production in the fruit. If these effects are reproduced in field trials, the procedure will provide an alternative method to produce high antioxidant-containing tomatoes. Tomatoes contain mostly the trans-isomer of lycopene, while human plasma contains much higher concentrations of cis-lycopene isomers. The cis isomers are more bioavailable than trans-. ARS researchers developed a simple, rapid method to separate and analyze lycopene isomers, which are useful in studies to answer question on the relative nutritional benefits of these isomers. ARS researchers found that gac fruit (Momordica cochinchinensis) are a very rich
potential source of lycopene, beta-carotene, and unsaturated fatty acids. Carotenoids and fatty acid composition in fruit and seeds were analyzed, and, in collaboration with scientists at Ohio State University, the bioaccessibility of lycopene from the aril was found to be high. Therefore, this source of carotenoids has the potential to alleviate dietary deficiencies leading to blindness in much of the underdeveloped countries in the world. 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? ARS developed a method of extracting lycopene from tomato waste, using an environmentally friendly solvent. A stabilizer that protects oxidation and degradation of lycopene during extraction was also discovered. An invention disclosure has been filed and a patent
application is being prepared. These results should make possible U.S. competition for the food-grade lycopene-supplementation market. Discussions have begun on the development of a CRADA with a commercial partner interested in manufacturing this product. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Presentations: Bartley, Glenn E. and Ishida, Betty K. Transcription factor gene expression during tomato fruit ripening. Presented at Plant Genomics 2003, American Society of Plant Biology, Western Section meeting, Davis, CA, October 10-11, 2003. Log No. 155376. Ishida, Betty K., Turner, Charlotta, Chapman, Mary H., and McKeon, Thomas A. Carotenoids and fatty acids in gac (Momordica cochinchinensis Spreng) fruit. Gordon Research Conference on Carotenoids. Ventura, CA, January 4-9, 2004. Poster presentation. Log No. 155378. Bartley, Glenn E., Ishida, Betty K., Zhu, Tong, and Ono, Makoto. Expression
of carotenoid biosynthesis genes during cool temperature sepal morphogenesis. Gordon Research Conference on Carotenoids. Ventura, CA, January 4-9, 2004. Poster presentation. Log No. 168144.
Impacts
(N/A)
Publications
- Ishida, B.K., Turner, C., Chapman, M.H., McKeon, T.A. 2004. Fatty acids and carotenoid composition in gac (momordica cochinchinensis spreng) fruit. Journal of Agricultural and Food Chemistry. Vol 52, p. 274-279.
- Bartley, G.E., Ishida, B.K. 2003. Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis. BMC Plant Biology, Vol 3, #4, p. 1-11.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The major problem is to develop a tomato that is rich in lycopene and other valuable nutritional and health-promoting carotenoids. Research objectives: (1) To identify genes that increase the biosynthesis of lycopene by a cool-temperature-induced mechanism that enhances ripening in VFNT Cherry tomato fruit and calyces cultured in vitro. (2) Identify factors that are involved in triggering this mechanism so that we can enhance the ripening process. We are also screening a group of bioregulators for their ability to increase total content of lycopene and/or the cis-lycopene (and other carotenoids) in tomato fruit and will search for key control points in their biosynthesis. 2. How serious is the problem? Why does it matter? The tomato is a major agricultural commodity in the United States. However, economic and nutritional benefits remain unfulfilled because of emphasis on tomato
durability in transport instead of flavor, nutrition, and color. Recently, epidemiological studies showed a strong correlation between the consumption of tomatoes and tomato products and a lowered risk of both cardiovascular disease and prostate and gastrointestinal cancers. This protective effect has implicated lycopene, the compound responsible for the red color of tomato fruit. Prostate cancer and cancers of the digestive tract are now major malignancies throughout the world. In fact, prostate cancer is one of the most common malignancies among men in affluent nations. Lycopene has been shown to be more effective against proliferation of human endometrial, mammary, and lung cancer cells than either alpha- or beta-carotene. Lycopene is believed to protect organs from damage due to free radicals because of its antioxidant properties, quenching singlet oxygen and reacting with oxygen- centered radicals. The trans-isomer of lycopene predominates in tomato fruit, but human plasma
contains a larger percentage of cis-isomers. Cis- isomers of lycopene are more bioavailable than the trans-isomer. Other carotenoids in tomato that are also valuable to human health and nutrition are volatile flavor compounds, zeaxanthin, and lutein. The last two compounds are present in all photosynthetic plants. These compounds are highly desirable to tomato growers and processors and the general public, enhancing both appeal and potential health benefits of the tomato. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research is assigned to National Program 301 (40%) emphasizing tomato genetic improvement to achieve quality and nutritional improvement as well as the development of new technological approaches to effect genetic improvement. This research is also assigned to National Program 306 (60%) emphasizing efforts to characterize, understand, and improve the quality and market value of tomatoes through
enhancement of potentially important phytonutrients. (NP 301 Plant Microbial and Insect genetic Resources Genomics and Genetic Improvement; NP 306 New Uses, Quality and Marketability of Plants Animal Products). 4. What were the most significant accomplishments this past year? A. Tomatoes naturally contain health-promoting and red-colored compound known as lycopene that, if present in greater concentration, could contribute significantly to the health of the consuming public. ARS researchers at the Western Regional Research Center used a special plant- free method of growing and ripening tomatoes that have unusually high lycopene levels to identify genetic control of lycopene formation. Regulation of putative developmental genes, possibly involved in ripening and development, and their relative abundance were examined. Differences were found in gene expression that might be important to tomato cultivar variation and therefore to developing a field-grown tomato having greater health
benefits. B. 1) Sources of lycopene and other valuable carotenoids are of interest because of their nutritional and commercial value. ARS researchers examined the composition of Gac fruit and found the aril (oily pulp surrounding seeds) to be very high in lycopene, beta-carotene, and lutein and in fatty acids (oleic, linoleic, and palmitic acids). Gac aril in Southeast Asia is mixed with rice and served at festive occasions. Gac aril could provide an important source of protective phytonutrients, and its high unsaturated fatty acid content would serve to enhance bioavailability of carotenoids in this food. Bioaccessibility of lycopene and carotenes is essential to delivery and effectiveness of these carotenoids at their target sites of protective action. ARS scientists on the Processed Foods Research unit undertook collaborative studies with researchers at the Ohio State University Human Nutrition Department on in-vitro simulation of gastric and intestinal phases of digestion and
incubation of cultured Caco-2 human intestinal cells with a rice mixture containing Gac aril and Gac oil. These studies showed that cis isomers of lycopene and alpha- and beta-carotenes are accumulated and retained to a significantly greater extent than trans- lycopene. These results indicate that Gac aril and oil are excellent sources of bioaccessible carotenes and lycopene. C. See B above. Target population: General, but particularly important to children during infancy and puberty. D. 1) Using molecular biological methods, ARS researchers examined putative developmental genes that are possibly involved in tomato fruit development and ripening and determined the relative abundance of a number of transcripts of genes that might be involved in regulation of these processes. Results confirmed some previous database-mining results and showed differences in gene expression that might be important to differences among tomato cultivars. This information will be valuable in developing
a more nutritious field-grown tomato (Bartley, G.E. and Ishida, B.K. "Developmental gene regulation during tomato fruit ripening and in- vitro sepal Morphogenesis," awaiting final acceptance in Plant Biology). 2) In collaboration with the Western Human Nutrition Research Center, bioavailability studies of cis- and trans- lycopene from tomatoes in humans were completed, and data are awaiting analyses. 3) Enhancement of lycopene production in tomatoes by bioregulators was confirmed. Data analysis is being completed. 4) Studies on lycopene bioavailability in hamsters were completed. Data analysis is underway. 5) At the request of a potential industrial CRADA partner, exploratory experiments began on effectiveness of several environmentally friendly solvents in a new procedure to extract lycopene from tomato-processing waste. 6) Research under a CRADA between ARS and an industrial research partner (Project No. 5325-21430-009-01T Activated Lycopene Biosynthesis in In- Vitro-Grown Cherry
Tomato and Calyces) determined the best times to sample tissues that are ripening in vitro at 18 and 28 degrees C for microarray analysis. RNA samples at selected times were sent to ARS' CRADA partner for analyses. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A) Carotenoid enhancement. Tomatoes naturally contain a health-promoting and red-colored compound known as lycopene, which contributes significantly to the health of the consuming public. ARS researchers developed a high-lycopene-producing tomato and are analyzing genes that are activated to produce this effect. B) ARS has confirmed that treating tomato seeds with small concentrations (which cannot be detected in the plant) of a bioregulator compound increases lycopene production in the fruit. If these studies are substantiated by field trials, the procedure will provide an alternative method for producing high antioxidant-containing tomatoes. C) Tomatoes
contain primarily the trans-isomer of lycopene, while human plasma contains much higher concentrations of cis-lycopene isomers than is present in food. Cis-lycopene isomers are more bioavailable than trans. ARS researchers developed a simple, rapid method to separate and analyze trans- and cis-isomers of lycopene, which will be useful in studies to answer questions on nutritional benefits of lycopene isomers. D) Gac fruit (Momordica cochinchinensis) was found to be a very rich potential source of lycopene, as well as beta-carotene and lutein. Fruit and seed composition were analyzed, and, in collaboration with scientists in the Human Nutrition Department at Ohio State University, the bioaccessibility of lycopene from the aril was found to be high. 6. What do you expect to accomplish, year by year, over the next 3 years? 2004: When data from microarray analyses of tomato fruit development experiments are received from our CRADA partner, results will be assessed to determine which
activated genes are the keys to fruit ripening regulation. Results of earlier studies on catsup, Gac fruit composition, and bioaccessibility of lycopene and carotenes from gac oil and aril will be published. 2005 and 2006: Collaborations with researchers at Ohio State University Human Nutrition Department will continue on bioassessibility of lycopene and carotenes in gac oil and aril. Data analysis will be completed on the effect of bioregulator(s)on lycopene production, on bioavailability of cis-and trans-lycopene from tomato in humans and on bioavailability of lycopene from tomato in hamsters. 7. 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? ARS has a CRADA to analyze differential gene expression in tomato fruit and calyces (sepals) cultured at
16 and 28 degrees C, using microarray analysis at a research facility that has the necessary expertise and equipment for this task. This work is in progress. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Vuong, L.T., Chitchumroonchokchai, C., Chapman, M., Ishida, B.K., King, J. , Failla, M.L. High bioaccessibility of carotenes and lycopenes in gac oil and gac fruit aril. FASEB Journal. 2003. v. 17. Abstract 758-759. Log No. 147129.
Impacts
(N/A)
Publications
- Bartley, G.E. Oxidative Dehydrogenation, Horvath, I.T. editor. J. Wiley and Sons, New York, N.Y. Encyclopedia of Catalysis, 2003. v. 5. p. 424-449.
- Ishida, B.K., Bartley, G.E., Chapman, M.H. A Search for a High Lycopene Tomato. Proceedings from Tomato Breeders Round Table. 2003. p. 30-31.
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