2012 Annual Report 1a.Objectives (from AD-416): The overall objective of the proposed research is to develop improved plant germplasm and advanced process technology enabling domestic production of two strategic industrial raw materials – domestic natural rubber and castor oil, along with their valuable co-products. The crops for industrial production of rubber are Parthenium argentatum (guayule), Taraxacum kok-saghyz (Russian dandelion) and Hevea brasiliensis (Hevea), and for castor oil are Ricinus communis (castor) and Lesquerella fendleri (Lesquerella). Domestic production of both castor and rubber has taken place in the past, yet critical issues denied sustainability of these important industrial crops. We propose to address these via the following objectives: Objective 1: Develop germplasm that enables domestic commercial production of natural rubber and ricinoleate by metabolic engineering and conventional breeding. Objective 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications. Identify chemical processes and/or modifications to develop superior performance properties of natural rubber from domestic sources. Identify processing methods to enhance the value of castor oil byproducts. 1b.Approach (from AD-416): Improvements in natural rubber yield will be sought through genetic manipulation of guayule and Russian dandelion plants. The biosynthesis of ricinoleate in Lesquerella will be metabolically engineered. Transformation methods will be developed and optimized; efficacious high expression and tissue-specific promoters will be identified. Genetically altered lines will be phenotypically characterized for alterations in enzymatic activity and production of target metabolites. Promising lines will be tested in controlled environment greenhouse and/or field trials on site or at collaborator locations. Improved germplasm will also be developed/identified through characterization of NPGS castor and Russian dandelion lines. Mutagenesis will be used to develop high yield, low toxin and allergen castor lines. More specific knowledge of the biosynthetic pathways for production of natural rubber and hydroxy fatty acids will be achieved through physiological, cell biology, and biochemical studies. Knowledge of the detailed physical and chemical characteristics of natural rubber from alternative crops will be developed to elucidate the mechanisms for differences from incumbent materials and to provide strategies for technological equivalence. The processing of castor seed meal by bioconversion to reduce toxin and allergen content of byproducts will be researched. 3.Progress Report: Progress was made on both main objectives and their sub-objectives, all of which fall under National Program 306, Component 3: Biobased Products, including 3A: Agricultural Feedstocks and Byproducts, and 3B: Develop Biobased Products and Sustainable Technologies/Processes. Progress on this report falls under Problem 3A: Many Agricultural Feedstocks and Byproducts are Underutilized, and Problem 3B: Biobased Products and Sustainable Technologies/Processes are Desired. Under Objective 1: Develop germplasm that enables domestic commercial production of natural rubber and ricinoleate by metabolic engineering and conventional breeding, significant progress was made in acquisition and utilization of genomic sequence information for guayule (published in 2012) and Lesquerella. Knowledge of genomic sequences is the first step to more effective engineering of plant biochemical pathways for improved quantity and quality of natural products of interest. Genetic modification of the oil-producing plant Lesquerella has progressed, using a seed-specific promoter to focus only on plant tissues of interest, those where oil is stored. A new protocol under development allows a significant improvement in the regeneration rate for guayule in vitro culture (tissue culture). The new protocol may enable creation of transgenic plants 30% faster than the previous methods. Genetically-modified guayule showed a significantly higher recovery rate following pollarding (cutting at the base) compared to controls in field evaluations. Field evaluations of castor continued in 2012, to determine whether improved yield seen in greenhouse testing translates to field conditions. In cooperation with NPGS, evaluation of germplasm of natural-rubber producing Russian Dandelion lines, collected in Kazakhstan in 2008, entered its third year. Under Objective 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications, a process for solvent extraction of guayule natural rubber from ground plant tissue was scaled to pilot plant (25L) reactor, based on laboratory studies, to successfully recover kg quantities of solid guayule natural rubber. In ongoing studies, progress has been made to differentiate the impact of specific amino acids and proteins on physical and chemical performance of natural rubber. In castor (ricinoleate) research, new analytical methods identified previously-unknown components of castor seed oil featuring similar chemical functions (hydroxy fatty acids) as conventional castor oil. Specifically, diacylglycerol and triacylglycerol containing polyhydroxy fatty acids were found in castor oil. The ratios of regioisomers of triacylglycerols and tetraacylglycerols containing mono- di- and tri-hydroxy fatty acids in castor oil were estimated using the new method. These oils may have value in industrial applications. In addition, a simple seed treatment has been identified improves the germination rate of castor seed, reducing the problem of irregular stands and lowered yield. 4.Accomplishments 1. Enhancing oil content in Lesquerella. Genetic improvement of Lesquerella, an oilseed producing crop, could provide a domestic source of hydroxy fatty acids, high value biobased raw materials. First, genetic modification of Lesquerella has been demonstrated, using a seed-specific promoter to focus only on plant tissues of interest, those where oil is stored. Next, transgenic lines were successfully modified to incorporate a key gene from castor responsible for hydroxy fatty acid (HFA) synthesis. Fifteen transgenic lines have been made, including 4 lines with increased HFA production. Transgenic Lf lines with enhanced HFA and oil content could be developed for commercial production. 2. Genomic sequence information developed for guayule. Knowledge of genetic sequences is the first step to more effectively engineer biochemical pathways for production of natural rubber. ARS researchers (Albany, CA) published the first genomic sequence information for guayule, a natural rubber-producing crop under developed in the southwestern US. A transcriptome (highly expressed gene sequences) from cold-treated bark tissue high in rubber production revealed sequences for all known rubber-biosynthesis genes, many stress-related genes, and genes of unknown function. The information is a significant step forward in determination of the molecular basis of rubber. Metabolic engineering for increased yield in guayule is a key to sustainability of this important domestic natural rubber-producing crop. 3. Key technology gaps identified for development of vehicle tires from guayule. Expansion of cultivation of guayule, a natural rubber-producing crop under development in the US southwest, would be significantly enhanced by demonstration of the suitability of use of guayule rubber in modern tires. ARS Scientists (Albany, CA, and Maricopa, AZ), in collaboration with multiple partners, developed a detailed program to close key technology gaps in feedstock development, agronomics, extraction process development, and design, construction, and testing of a commercial passenger tire based on guayule rubber. Extramural funding was successfully approved for the program starting June 1, 2012. The project includes comprehensive sustainability analysis for guayule cultivation, processing, tire construction, and use. This program will fully assess the opportunity to achieve sustainable natural rubber supply security and the benefits of rural development in semi-arid southwestern US agriculture. The knowledge gained will inform future public and private domestic rubber research and development toward a more sustainable path. Review Publications Severino, L.S., Auld, D.L., Baldanzi, M., Chen, G.Q., He, X., Crosby, W., Machado, O.L., Morris, J.B., Zieler, H. 2012. A review on the challenges for increassed production of castor. Agronomy Journal. 104(4):853-880. Ponciano, G.P., Mcmahan, C.M., Wengshuang, X., Lazo, G.R., Coffelt, T.A., Collins-Silva, J., Nural-Taban, A., Golley, M., Shintani, D.K., Whalen, M.C. (2012). Transcriptome and gene expression analysis in cold-acclimated guayule (Parthenium argentatum)rubber-producing tissue. Phytochemistry. 79:57-66. Kumar, S., Hahn, F.M., Baidoo, E., Kahlon, T.S., Wood, D.F., Mcmahan, C.M., Cornish, K., Keasling, J., Daniell, H., Whalen, M.C. 2011. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Metabolic Engineering. 14(1):19-28. Collins Silva, J., Nural, A.T., Scott, D.J., Hathwaik, U., Woosley, R., Schegg, K., Mcmahan, C.M., Whalen, M.C., Cornish, K., Shintani, D.K. 2012. The role of the small rubber particle protein in determining rubber yields and polymer length in Russian dandelion. Phytochemistry. 79:46-56. Chundawat, S.P.S., Chang, L., Gunawan, C., Balan, V., Mcmahan, C., Dale, B.E. 2012.Guayule as a feedstock for lignocellulosic biorefineries using ammonia fiber expansion (AFEX) pretreatment. Industrial Crops and Products. 37: 486-492. Chiang, K.C., Xie, W., Mcmahan, C.M., Puskas, J. 2011. Unraveling the mystery of natural rubber biosythesis part I: investigation of the composition and growth of in vitro natural rubber using high resolution size exclusion chromatography. Rubber Chemistry and Technology. 84(2):166-177. Lin, J.T., Chen, G.Q. 2012. Identification of minor acylglycerols less polar than triricinolein in castor oil by mass spectrometry. American Chemistry Society Abstracts. dx.doi.org/10.1007/s11746-012-2082-2. Chen, G.Q., Thilmony, R.L., Lin, J.T. 2012. Transformation of Lesquerella fendleri with the new binary vector pGPro4-35S. Online Journal of Biological Sciences. 11(3)90-95. DOI: 10.3844/ojbsci.2011.90.95. Lin, J.T., Chen, G.Q. 2012. Ratios of regioisomers of minor acylglycerols less polar than triricinolein in castor oil estimated by mass spectrometry. Journal of the American Oil Chemists' Society. dx.doi.org/10.1007/s11746-012-2083-1.