2006 Annual 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 project is aligned with the National Program 306, Quality and Utilization of Agricultural Products. Mankind has used plant extracts for thousands of years for the prevention of disease, the treatment of disease symptoms, as insecticides, to control microbe growth, for weed control, and many more functions. Many of these activities are due to the highly diverse secondary metabolites (the phyto-chemicals) produced by plants to deal with their environmental stresses. While many, if not most, of these phytochemicals have already been identified chemically, the exact chemical and biological role of their effect, both alone or in concert with the other phytochemicals in a specific plant species, on other plants, against insects, microbes, and in the animals that ingest them, is still not very well characterized. One of the main hurdles to promote this area of research is the lack of significant quantities of purified phytochemicals for use as analytical standards and for biological evaluation. Some of these phyto-chemicals may be shown to have multiple roles in various situations effective as a specific pesticide, usable as an intermediate in chemical synthesis and a functional role in human health. This project research team specializes in the identification, isolation and analysis of phytochemicals from plants and plant products, and assessing their functional activity in biological systems, as well as evaluated methodology to enhance the accumulation of these compounds in the plant tissues. The current project is focusing on the analysis of phytochemicals with either pest control activity or health promoting activity in humans and animals. It also finds new uses with added monetary value for low-value co-products from crops, such as soybeans, corn, herbs, alternative oil seed crops, crucifer species, alternative uses for weed species, and others. Knowledge of natural compounds present in plants with agricultural and health benefits would allow plant breeders and agricultural processors to optimize these compounds. The development of rapid and accurate analytical methodology, and the preparation of purified analytical and experimental standards for the analysis of plant phyto-chemicals is a key part of the new research efforts aimed at unlocking the mode of action of phytochemicals in both plant and animal systems. Often, these compounds are found in relatively low concentrations in the plants themselves. They may be accumulated only under certain conditions, such as the growth of reproductive structures or under specific types of stress. Hence, it is often expensive and time consuming to purify these phytochemicals from the plants for use as standards and in biological activity studies. The evaluation of external factors, such as nutrient composition, atmospheric gas composition and temperature control, etc., in culture conditions, may lead to enhanced accumulation of specific phyto-chemicals in plants. Accurate analytical methodology, in cooperation with rigorous biological studies, is required to determine the effect of these compounds on health and to determine the doses and timing required to obtain maximized positive biological, pesticide and health effects. 2.List by year the currently approved milestones (indicators of research progress) Objective 1: Develop or optimize protocols for the identification, quantitation and elucidation of phytochemicals from plant tissues, products and by-products that have effective biological activity against agricultural pests, demonstrate biological activity in cell culture or animal nutritional studies, or have chemical functionality in industrial processes. FY 2005: Finish isolation of A group saponin standards, develop method(s) for total saponin analysis in soy and develop methods for isolation of intact glucosinolates from various seed sources. Prepare large-scale pure B group saponins. With industrial collaborators, investigate legume processing streams for phenolic and saponin content. Continue fractionation of antioxidants from corn distillers solubles. Isolate and identify anthoquinones from sicklepod. FY 2006: Develop methods for large-scale isolation of A group saponins from soy; scale-up isolation of pure key glucosinolates. Continue assessment of crucifer species for glucosinolates; determine methods to isolate indole glucosinolates in a stable form. Isolate and identify phenolics and saponins from industrial processing streams and develop analytical procedures. Characterize antioxidants from corn distillers solubles. Continue anthoquinone scale-up isolation. FY 2007: Based on feedback from bioassay and nutritional research collaborators, develop methods for scale-up purification of phytochemicals of interest from industrial processing streams, especially from alternative legume and crucifer crops. Begin assessing other new crop processing streams for phytochemical composition. FY 2008 & 2009: Based on literature, phytochemical assessment and feedback from collaborators, continue assessing other new crop processing streams for phyto-chemical composition, such as the phytosterols. Objective 2: Develop or optimize protocols to enhance production of key phyto-chemicals by - (a) optimizing conditions for accumulation in plant tissues, or (b) from specific processed agricultural materials for the production of purified phytochemicals in quantities sufficient for use as standards, and in further biological studies to fully characterize their metabolism and function. Utilize these standards and research results to characterize enriched whole plant tissues and processed fractions for use as alternative pesticides and in nutritional studies FY 2005: Develop methodology for the optimized production of specific glucosinolate hydrolysis products. Continue work on the identification of soy saponin metabolites in mammalian cell culture and feeding studies. Perform in-house bioassay studies on glucosinolate degradation product activities against weeds, insects and fungi. Prepare stable glucosinoate containing products from plant materials. Continue studies on model plant culture systems to optimize production of known phytochemicals using various culture parameters. Continue investigation of high value phytochemical-producing plants to adapt to plant culture systems FY 2006: Scale-up production of specific glucosinolate hydrolysis products and, with collaborators, perform pilot scale production of thiourea antioxidants. With collaborators, perform large scale field study assay for activity of glucosinolate-containing plant products. Optimize culture/crop conditions, using data from the model systems, for plants producing high-value plant phytochemicals, and assess levels produced by analysis. FY 2007: Transfer thiourea antioxidant production to customers. Assess field study glucosinolate trials and determine levels of production of active glucosinolates. Determine methodology to scale-up plant production systems on a pilot scale for optimal mass production with minimal costs. FY2008 & 2009: Transfer alternative glucosinolate pest control methodology to customers. Begin examination of alternative phytochemicals, such as the anthoquinones, for potential biological activities. Transfer technology to customers and continue to assess other plant species for use in culture systems and production of secondary metabolites. 4a.List the single most significant research accomplishment during FY 2006. Complete Quantification of Group A and Group B Soyasaponins in Soybeans: This work addresses the problem statement -- New technologies to convert commodities and processing byproducts into important value-added products will fill demonstrated needs in the National Program 306, Quality and Utilization of Agricultural Products. A combination of high-pressure extraction and preparative High Pressure Liquid Chromatography (HPLC) was used to purify the group A and group B soyasaponins from soy germ for use as analytical standards and in biological assays. A standardized sample preparation and extraction method was developed for the analysis of the phytochemicals found in soy and processed soy products, which is reproducible in other laboratories. Complete saponin analysis of the extracts prepared from soy germ (hypo cots), hulls and cotyledons show that a significant portion of the saponins is concentrated in the germ. The germ contains nearly all the group A soyasaponins, while the group B soyasaponins are nearly equally distributed between the germ and cotyledons. The work was done in-house and resulted in a publication. This work has resulted in a reproducible method to identify and quantitate all the saponin forms (A & B) in soybeans and soy products. The impact of dietary saponins in cell culture models and whole animal models is just now being evaluated, and this work will provide research material to determine if the biological activity of the two types of saponins found in soy is similar or different. 4b.List other significant research accomplishment(s), if any. Tissue Cultures Optimization Improves Secondary Metabolite Production: Development of optimal whole plant culture conditions to optimize the production of secondary metabolites in plant tissues. This work addresses the problem statement -- New technologies to convert commodities and processing byproducts into important value-added products will fill demonstrated needs in the National Program 306, Quality and Utilization of Agricultural Products. In order to obtain high yields of secondary products from plants in vitro, we studied the influence of the physical environment with and without supplemental carbon dioxide additions. The in vitro environment being sterile necessitates the occurrence of high chamber relative humidity (~100 %). These high humidity levels significantly retard normal growth and foliage development which correspondingly reduces secondary metabolism. By intermediate air applications, normal foliage growth and development as well as higher secondary metabolism occurs. We are currently exploiting these findings to improve secondary metabolism from in vitro grown plantlets. 4c.List significant activities that support special target populations. None. 4d.Progress report. Significant progress has been made on the development of methods to purify gram quantities of specific soy isoflavones and soy saponins. This material is being provided to collaborators at Iowa State University and the University of Illinois for evaluation of their biological effects. A number of papers and funded grants have resulted from this interaction. The analytical methodology has been applied to measuring levels of saponins and isoflavones in a variety of soy products. Significant progress has been made on the development of methods to purify gram quantities of a variety of glucosinolates from crucifer species as well. This material is being used as analytical standards and as a source to prepare degradation products for evaluation in pest control, industrial antioxidant production and for evaluation in human disease prevention. This material is being provided to collaborators at Bradley University and the University of Illinois Medical School Research Facility in Peoria. We are expanding our research efforts in the evaluation of the chemical and biological activity of compounds found in dry corn distillers grains. These materials have been shown to contain potent anti-oxidant activity, and preliminary laboratory and limited field work has shown that this material contains potent pesticidal activity against weedy species as well as providing nutrient enrichment to the soil. Research work on this will continue into FY 2007 and 2008. The analytical methodology available in this research project has been used to identify and quantitate phytochemicals in several other collaborative projects, including furan metabolites in yeast cultures (NCAUR), anthoquinones in sicklepod seeds (sicklepod workshop), fractionation of oviposition stimulants for stinkbugs from soy (EMBRAPA Brazil, which is the Brazilian Agricultural Research Service), anthocyanins from corn, petunia and gentium (NCAUR, Bradley University). Biologically active compounds (antioxidants) are being isolated and characterized from corn distillers solubles/solids in collaboration with researchers at the University of Illinois. It is generally believed that occurrence of high biomass (primary metabolism) coupled with high phytochemicals production are mutually exclusive. Plants require carbon obtained from atmospheric photosynthesis for both primary and secondary metabolism. Within the tissue culture environment, exogenous sugar provides the carbon for these processes. We have found a closer correlation between secondary metabolism and primary metabolism to sugar content in the media. We are continuing our studies to exploit these observations to increase secondary metabolism coupled to high biomass production in vitro. In order to obtain high yields of secondary products from plant foliage in vitro, we studied the influence of the physical environment with and without supplemental carbon dioxide additions. The in vitro environment being sterile necessitates the occurrence of high chamber relative humidity (~100%). These high humidity levels significantly retard normal growth and foliage development which correspondingly reduces secondary metabolism. By intermediate air applications, normal foliage growth and development as well as higher secondary metabolism occurs. We are currently exploiting these findings to improve secondary metabolism from in vitro grown plantlets. 5.Describe the major accomplishments to date and their predicted or actual impact. This project is part of the National Program 306, Quality and Utilization of Agricultural Products (100%) program components, New Processes, New Uses and Value-Added Foods and Biobased Products (e.g., phytonutrients, phytochemicals). There have been a number of observations linking diets high in levels of certain phytochemicals with the prevention of certain chronic disease; the specific details are still largely unknown. The specific mode of action of these phyto-chemicals in the body, whether they work alone or in concert with other dietary and bodily components, how much is needed in the diet to produce the optimal effect, and when and how long they need to be ingested have yet to be ascertained. It is vital for this nutritional research to go forward to characterize the exact chemical composition of plant materials which have "health-promoting activities," that is, to determine the chemical structure and amounts of phytochemicals in these plants, to determine which ones have the key biological activities and then provide quantities of pure phytochemicals for whole animal and human nutritional studies. Phytochemicals isolated from other plants have been shown to be active in the prevention of plant growth, insect feeding and fungal growth in agricultural systems. Chemical analysis and purification from this lab is aimed at providing these important research tools for the purification and evaluation of plant phytochemicals. We are coordinating our research with a number of other research groups in Peoria and around the nation on these problems. Purified phytochemicals from soy produced by this lab and in cooperative research with private companies have produced quantities sufficient for a number of bioassay studies on whole plants, fungal cultures, insects, and mammalian cell culture systems. Additional phytochemicals, which have been implicated as having health-promoting activities, are being purified from several sources for evaluation in collaborating biological research labs. Purified phytochemicals (glucosinolates) from crucifer species are needed to evaluate and quantitate their biological mode of action against plant pests and in the prevention of certain chronic diseases. They can also be used in the industrial production of chemical antioxidants. Efficient methodology has been developed to produce quantities of pure glucosinolates and a number of their degradation products. The glucosinolate levels in the seeds of several commercially grown plant species have been determined. Purified phytochemicals (the saponins and isoflavones) from soy processing products have been transferred to a number of researchers in the nutrition and agriculture production field at the University of Illinois, the University of Missouri, Archer Daniels Midland Company, and to EMBRAPA research facilities in Brazil. This has resulted in the production of several papers on the evaluation of the biological role these phytochemicals play in the prevention of several animal diseases. The variability in accumulation of these compounds in soy cultivars was investigated in collaboration with a visiting scientist from EMBRAPA Brazil. We quantitated the isoflavones and saponin levels in 500 soy samples of a number of cultivars grown in different climatic conditions in Brazil. The collaborator is currently correlating this information. Significant progress has been made on the development of methods to analyze the volatile emissions given off by tissue cultured and soil potted plants. Thus far, we have conducted extensive research with electronic noses. Electronic noses are employed because they obtain instantaneous results. Two electronic noses, i.e. the z-Nose and Cyranose, are employed to evaluate volatile emissions, in a time-course fashion. External applications applied to test plants reveal rapid emissions of volatiles (essential oils). GC-MS coupled with a computerized library of chemical identifications has aided in the identification of volatile essential oils emitted by a large variety of plant spp. Large quantities of carbon dioxide are released into the atmosphere as a by-product by processors producing industrial ethanol, which could be utilized to grow plants faster or to make them produce larger amounts of desirable chemicals. We have found that production of secondary chemicals of economic importance, by plants, increases in high carbon dioxide atmospheres. Optimal levels of carbon dioxide for the production of these desirable chemicals have been identified. This may lead to procedures to obtain high production of specific phytochemicals that are important for health in cultured plant systems. Tissue cultured plants are being used as model systems to determine methodologies to enhance the production of phytochemicals in plants. 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? Technical information on basic research on the analytical methodology for biological phytochemicals from soybeans and other plant species is currently being transferred between scientists at several university partnerships, an industrial partner and, in addition, to ARS research groups here in Peoria, Prosser, WA, and in Charleston, SC. Technical information was transferred to four other soy processing companies on the evaluation of processed materials from soy for both isoflavone content and saponin content. Additional information on the determination of phytochemical identification and quantitation was provided to several labs around the world. It is hoped that this research will lead to a better understanding of what chemicals are required to (a) be used as alternative pest control in organic farming systems, (b) produce alternative phytochemically-derived ingredients for industrial chemical synthesis, enhance the accumulation of important pharmaceutically active compounds in plants, determine the dose and time frame needed to use functional foods for the prevention of certain diseases, such as cancer, and how much is required. This information, when confirmed, can be made available to the general public. Technical information on the nature of chemicals found in plants which affect the juvenility of certain insects is being transferred to ARS scientists at Columbia, MO. Technical information on the levels and identity of chemicals known to be involved in the resistance of corn to infestation by earworms and borers is being transferred to ARS scientists here in Peoria to aid them in the evaluation of their breeding programs. New chemicals from corn are being identified with the help of these research programs. The Organization for Economic and Cooperative Development (OECD), (Paris, France, a part of the EUC) granted Mark Berhow a research fellowship for May 16 to June 2, 2006, at Institute of Entomology, Czech Academy of Sciences, Branisovska, Czech Republic, to work with Dr. Magdalena Hodkova. The technology transferred included methodology for the coordination of the extraction, purification and character-ization of uncharacterized juvenile hormones in Pyrrhocoris apterus. The results of this research will contribute towards developing more efficient insect predator mass rearing protocols, which will allow for the development of a biological pest control program using beneficial insects to supplement or replace chemical pest control. In addition, because of the potential similarity between this juvenile hormone and that of other Heteroptera insects, which are pests of major crops, this information may contribute to the development of new biological-based methods for insect pest management. This will contribute towards the implementation of more innovative and biologically compatible insect pest manage-ment plans. This was an extension of work on the characterization of plant natural products by Liquid Chromatography (LC)-mass spectroscopy. Mark Berhow and Steven Vaughn have provided analytical standards for flower and bean anthocyanins, soy saponins, soy isoflavones, citrus flavonoids, and crucifer glucosinolates, as well as a description for their analytical methodology to researchers from other ARS locations, universities and private industry. To validate this transfer of methodology, several samples have been analyzed to confirm analytical results. Brent Tisserat has collaborated with the University of Nebraska-Lincoln, on conducting field tests with autoclaved fungal biomass (AFB) as a semi-biocontrol agent to combat Wheat scab, a major wheat disease. To date, AFB has shown potential in the greenhouse environment to significantly retard Wheat scab. He also consulted with private industry representatives to utilize carbon dioxide to enhance aging of soybeans in order to accelerate their breeding program. 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). Vaughn, S.F. (invited speaker). Organic seedmeals with potential use in turf. Midwest Turf Growers field day, June 1, 2006, Summit Seed, Inc., Manteno, IL. Panizzi, A.R., Berhow, M.A. and Bartelt, R.J. Oviposition of the southern green stink bug, Nezara viridula (L.) on artificial substrates conditioned by chemical extracts from soybean plants. XX Brazilian Congress of Entomology, Gramado, RS, Brazil, September 05-10, 2004, Program and Abstracts, p. 104. Silva, F.A.C., Panizzi, A.R., Berhow, M.A., and Marques, F.A. Oviposition behavior of pentatomids on artificial substrate mediated by chemical extracts from soybean. IV Brazilian Meeting on Chemical Ecology, Piracicaba, SP, Brazil, November 29 to December 02, 2005. Johnson, E.T., Berhow, M.A. and Dowd, P.F. P1-Mediated transgenic secondary metabolite production in corn silks moderately enhances insect resistance. Annual Meeting of the Phytochemical Society of North America, July 8-12, 2006. Jochum, C.C., Yuen, G.Y. and Tisserat, B. Effects of induced systemic resistance-activating agents on Fusarium head blight. P. 211-214. In: Proceedings of the 2005 National Fusarium Head Blight Forum, Milwaukee, WI, Dec. 11-13, 2005. U.S. Wheat and Barley Scab Initiative, Michigan State Univ., East Lansing, MI. Review Publications Berhow, M.A., Kong, Suk Bin, Vermillion, K.E., Duval, S.M. 2006. Complete quantification of group A and group B soyasaponins in soybeans. Journal of Agricultural and Food Chemistry. 54:2035-2044. Vaughn, S.F., Palmquist, D.E., Duval, S.M., Berhow, M.A. 2006. Herbicidal activity of glucosinolate-containing seedmeals. Weed Science. 54(4):743-748. Berhow, M.A., Johnson, E.T., Dowd, P.F., Duval, S.M. 2006. Extraction and characterization of anthocyanins from pigmented flowers and black seed hulls [abstract]. Phytochemical Society of North America Meeting and Newsletter. p. 22 Tisserat, B. 2005. Establishing tissue-cultured sweetgum plants in soil. HortTechnology. 15:5-9. Ellington, A.A., Berhow, M.A., Singletary, K.W. 2006. Inhibition of Akt signaling and enhanced erk1/2 activity are involved in induction of macroautophagy by triterpenoid b-group soyasaponins in colon cancer cells. Carcinogenesis. 27(2):298-306. Wu, Y., Payne Wahl, K.L., Vaughn, S.F. 2006. Corn gluten meal odorants and volatiles after treatments to improve flavor. Cereal Chemistry. 83:228-234. Mao, W., Berhow, M.A., Zangerl, A.R., Mcgovern, J., Berenbaum, M.R. 2006. Cytochrome p450-mediated metabolism of xanthotoxin by Papilio multicaudatus. Journal of Chemical Ecology. 32:523-536.