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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Research Project #428635

Research Project: Domestic Production of Natural Rubber and Industrial Seed Oils

Location: Bioproducts Research

2017 Annual Report

Objective 1: Develop varieties and commercially-viable post-harvest practices that maximize the market value of U.S.-produced guayule and Kazak dandelion. Sub-objective 1A: Genetically modify guayule for improved rubber yields. Sub-objective 1B: Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber. Sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion. Objective 2: Enable new commercially-viable processes for expanding the manufacture of industrial products based on guayule and Kazak dandelion. Subobjective 2A: Modify the protein components of guayule rubber to increase its market value. Subobjective 2B: Elucidate the roles of lipid in the biosynthesis of rubber and on the mechanical properties of dry rubber. Subobjective 2C: Develop novel processes to fractionate crude guayule resin into value-added components. Objective 3: Enable the commercial production of hydroxy fatty acids from oilseed crops already grown in the U.S. Sub-objective 3A. Develop knowledge of HFA synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Sub-objective 3B. Develop HFA-producing camelina.

Subobjective 1A: Genetically modify guayule for improved rubber yields- we will engineer guayule for over-expression of isoprenoid genes and/or down-regulation of carbon-competing pathways to increase rubber content. Independently transformed lines and controls will be analyzed: gene expression, rubber/resin content, rubber transferase activity, inulin, squalene, lipids and TAGs. We will apply the knowledge with that developed in 1) regulation of biochemical pathways 2) storage of hydrocarbons in plants 3) guayule genomics tools. Sub-objective 1B: Identify biochemical regulation of enzymes- we will use yeast, S. cerevisiae, a single-celled eukaryote that responds to IPP by producing ergosterol, as a model to study HMGR, IDI, and FPP synthase impact on ergosterol. Results will be translated to tobacco to evaluate post-translational modifications in a model plant. Sub-objective 1C. Develop genetic transformation of Kazak dandelion- a robust transformation system will be developed by 1) screening diploid seedlings to identify highly regenerating lines 2) optimizing culture conditions 3) evaluating explant sources (hypocotyl, stem, leaf petiole), and 4) assessing seed production. Self-compatible lines will facilitate genetic studies on relationships among transgene dosage, gene expression level, and rubber content. Subobjective 2A- We will attempt to elucidate the roles of naturally-occurring proteins in Hevea rubber particles. This knowledge will inform modification of the chemical, physical, and/or biological properties of guayule and Kazak dandelion rubbers to meet industrial requirements. We will study interactions of proteins, amino acids, and lipids with rubber, then employ biobased post-harvest treatments. If unsuccessful, we will apply chemical treatments. Subobjective 2B: To elucidate the roles of lipid in rubber biosynthesis- the molecular species of various lipid classes in rubber particles of guayule, Kazak dandelion and Hevea will be quantified using HPLC and MS. Lipid profiles from the native guayule and Kazak dandelion will be compared to those from genetically modified plants. Subobjective 2C: Develop novel processes to fractionate crude guayule - we will evaluate a series of processes including 1) re-precipitation, 2) microfiltration, 3) liquid-liquid extraction, and 4) microfiltration/ultrafiltration to de-rubberize and fractionate guayule resin into major components. Sub-objective 3A. Develop knowledge of hydroxy fatty acid (HFA) synthesis in lesquerella- we will engineer key genes to increase HFA levels in lesquerella seed oil. We will apply Agrobacterium-mediated transformation, and identify stable transgenic lines by germinating T1 seeds in selection medium. Plants/seeds will be characterized (transgene copy number for T1 using qPCR, fatty acid and TAG composition in using T2 seeds). If the total HFA content does not reach the target 70% in transgenic lesquerella, alternative promoters will be studied. Sub-objective 3B. Develop HFA-producing camelina- knowledge gained from engineering lesquerella for increased HFA content will inform strategies to raise HFA-production in the domestic oilseed crops camelina.

Progress Report
Progress was made on all three objectives and their subobjectives, all of which fall under National Program 306, Quality and Utilization of Agricultural Products. To address Problem Statement 2C, i.e., develop new cultivars to improve the quality and productivity of non-food biobased products, crops improvement research was conducted on guayule and Kazakh dandelion (for rubber production) and lesquerella and camelina (for oil production). Subobjective 1A: Genetically modify guayule for improved rubber yields. A series of engineered plants were created to increase yield and also to better understand the biochemical pathways in guayule. Plants with reduced allene oxide synthase (AOS), the main rubber particle protein in guayule, have been created. Reducing the amount of AOS protein in guayule rubber particles caused a major increase in rubber content, plus the plants were significantly larger. A provisional patent was filed on the technology, as these plants are being evaluated in a designed field trial, in collaboration with an industrial Cooperative Research and Development Agreement (CRADA) partner. Separately, reduction of carbohydrate (fructan) levels in modified plants was successful, but those guayule plants did not divert the excess carbon to higher rubber production. In associated agreement 2030-21410-021-06S, Quality Improvement of Guayule Natural Rubber with the University of Nevada, Reno, guayule plants over-expressing 4 genes responsible for tocopherol (Vitamin E) synthesis have been created. It is thought that Vitamin E may preserve rubber quality after harvest. Finally, guayule plants have been built containing a series of novel gene expression control elements. Evaluation is underway. ARS researchers continue to increase the rubber yield in Kazakh dandelion, a rubber-producing crop that is more suited to northern states and cooler climates. In Sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion, a series of lab experiments explored the best conditions for transformation of the plant. Kazak dandelion (Tk) transformation experiments were performed using an Agrobacterium strain carrying a specific reporter gene known as pCambia2301. A shoot regeneration rate of 57 percent was observed for control root explants, compared with 30 percent for Agrobacterium-treated explants. Various stress phenotypes such as light green shoots, leaf thinning, and arrested development were observed among regenerated shoots. We tried addition of chemicals known to help plant development: polyamines (putrescine, spermidine, and spermine) or polyamine precursors (arginine and ornithine). However, these compounds did not promote healthy shoot development in Tk. Further investigations are underway to establish a protocol for healthy shoot regeneration. Using the tools of biotechnology to increase yield in rubber-producing crops would certainly be more efficient if we had more knowledge about what controls rubber production in plants. In Sub-objective 1B: Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber, the latest tools of biotechnology were applied to lettuce, again, a rubber-producing crop that can be readily transformed to understand the rubber-producing (isoprenoid) pathway. Specifically, the enzyme Isopentenyl diphosphate-Dimethylallyl diphosphate Isomerase (IDI) was studied because it is a potential gatekeeper enzyme in rubber biosynthesis and is the first common enzyme for multiple pathways seen in lettuce. The antibody for the IDI from lettuce was chosen, as lettuce is a rubber producer that can act as a model crop for genetic transformation. The biochemical pathways for oil (fatty acid) production in plants are better known, which aids our efforts to increase the amount of high value hydroxyl fatty acid (HFA) production in lesquerella and camelina. These plants have the potential to be used for U.S. production of oils like castor oil, long valued for its chemical and physical properties. Significant progress was made in Sub-objective 3A., Develop knowledge of HFA synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Transgenic lesquerella plants expressing a castor gene (RcLPAT2) increased the production of castor oil-like fatty acids (oils) from 2 to 17 percent. But transgenic expression of a different castor gene (RcLPAT299) did not change oil composition, thus indicating that this second gene (RcLPAT299) did not participate in fatty acid synthesis in lesquerella oil. These results direct us to new investigations targeting other genes that might further improve fatty acid synthesis in lesquerella oil. To apply the successful genes to camelina, we need an efficient transformation system. Sub-objective 3B is to Develop HFA-producing camelina. However, transformation of camelina with a promising gene (hydroxylase gene (FAH12) from Physaria lindheimeri (Pl), PlFAH12), had limited success. A potential obstacle could be that the selection gene used in the vector to transform the transgenic camelina production is too weak. New vectors with better, more-broadly applicable selection genes are under construction. Progress was also made to address NP306 Problem Statement 2B: Enable technologies for expanding market applications of existing biobased products. Research was conducted to expand the market applications for guayule rubber. In Subobjective 2A, Modify the protein components of guayule rubber to increase its market value, progress was made using two approaches. First, commercial proteins and protein extracts from Hevea (rubber tree) plants were blended with latex from guayule. The physical properties of the guayule latex blends were improved in many cases. Importantly, the study illustrated the broader potential for proteins as bio-based additives in tire compounds. A publication from this work was selected for a “Frontiers” issue of Rubber Chemistry and Technology, by editors representing the American Chemical Society Rubber Division, which is considered an honor for such papers. Secondly, guayule plants were transformed to express the same rubber-particle protein found in Hevea latex. Very high levels of gene expression were found, but the amount of new protein was lower than expected. We continue to evaluate the full impact of the modification. Our project is uniquely suited to examine the roles of both proteins and lipids on the quality and quantity of natural rubber produced in plants. Research was conducted toward Subobjective 2B, Elucidate the roles of lipid in the biosynthesis of rubber. Various molecular species of lipid affect the biosynthesis of rubber and the mechanical properties of dry rubber. For example, molecular species of lipid containing unusual furan fatty acids, hydroxy fatty acids and normal fatty acids in rubber particles of latex from guayule were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS). We will now focus on the most prominent types of lipids to study the roles on the biosynthesis of rubber and mechanical properties of dry rubber. Separately, lipids were extracted from Hevea (rubber tree) plants, and were blended with latex from guayule. The result was an increase in the gel (insoluble) content, especially when Hevea proteins were also added. Gel in natural rubber provides material strength advantages. Finally, research was conducted under NP306 Problem Statement 2B towards producing new marketable non-food biobased products derived from agricultural products and byproducts. Establishment of new markets for the organic resin co-product from guayule cultivation could impact the economic sustainability of this ‘new’ crop. In Subobjective 2C, Develop novel processes to fractionate crude guayule resin into value-added components, methods are under development for isolating guayulins and terpenoids by solvent extraction or distillation. Once isolated, we are evaluating modification of these components by bioconversion. A series of samples of guayule resin provided by industrial partners has been tested to determine the molecular weight, size and compositions of the molecules using Gas Chromatography-Mass Spectrometry (GC-MS). Composition varied by source and extraction solvent. However, in experiments to date, while organic solvents with varying polarity affected the quantity of the compounds, the overall metabolic profile; i.e., the types of compounds was similar, regardless of solvent. The method used, however, was insensitive to lower molecular weight terpenes (like pinene resin) so will be refined. Finally, the antimicrobial activity for selected fractions of guayule resin was evaluated.

1. Guayule rubber tire. Guayule (Parthenium argentatum) is under development in the southwestern U.S. as a source of domestic natural rubber, organic resins, and biofuel feedstock. Tires use almost 80 percent of imported natural rubber, so an understanding of the technical fit of guayule rubber for use in modern tires is critical to developing that market. Research under a consortium project funded by the Biomass Research and Development Initiative reached a successful conclusion when passenger tires built with 100 percent guayule rubber, in place of imported and petroleum-based rubber, passed Department of Transportation specified testing. Consortium members included ARS researchers at Albany, California, and Maricopa Arizona, university partners, and rubber and tire industry leaders. Tire industry representatives also reported that the tires passed more stringent internal testing, and that a 75 percent guayule rubber version was suitable for sale immediately, pending material availability. A modern day technical benchmark for the use of guayule natural rubber has now been established for a commodity application.

2. Guayule yield improvement patents. Economic sustainability of the developing guayule crop in the Southernwestern U.S. might be secured with increased natural rubber yield. ARS scientists in Albany, California, have successfully developed tools and techniques to engineer guayule plants with higher rubber yield. Two U.S. patents were issued this fiscal year, both of which enable production of higher levels of isoprene pyrophosphate, the monomer used by plants to synthesize natural rubber polymers. Patent U.S. 14209,255 (December 20, 2016) is a method to transform the chloroplasts of guayule. Patent 9,574,203 (February 21, 2017) covers guayule plants transformed to overexpress a critical enzyme in the isoprenoid pathway. These technologies are now available to guayule breeders and growers for developing high rubber content lines.

Review Publications
Chen, G.Q., Johnson, K., Morales, E., Mackey, B.E., Lin, J.T. 2016. Rapid development of a castor cultivar with increased oil content. Industrial Crops and Products. 94:586-588. doi:10.1016.j.indcrop.2016.09.020.[Corrigendum: Industrial Crops and Products: 2017, p.101:103.]
Dong, N., Dong, C., Ponciano, G.P., Holtman, K.M., Placido, D.F., Coffelt, T.A., Whalen, M.C., McMahan, C.M. 2017. Fructan reduction by downregulation of 1-SST in guayule. Industrial Crops and Products. doi: 10.1016/j.indcrop.2017.04.034.
Hou, C.T., Lin, J.T., Dulay, R.R., Ray, K.J. 2017. Identification of molecular species of acylglycerols of Philippine wild edible mushroom, Ganoderma lucidum. Biocatalysis and Agricultural Biotechnology. 9:19-27.
Kang, S., McKeon, T.A. 2016. Lipase catalyzed methanolysis of tri-(12-hydroxy stearoyl)-glycerol in organic solvents. Advances in Enzyme Research. 4(4):152-157. doi: 10.4236/aer.2016.44014.
Kim, H., Lee, K., Donghwan, S., Lee, J., Chen, G.Q., Hwang, S. 2016. Transcriptome analysis and identification of genes associated with omega-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens. Biomed Central (BMC) Genomics. 17:474. doi: 10.1186/s12864-016-2805-0.
Lhamo, D., McMahan, C.M. 2017. Study of protein addition on properties of guayule natural rubber. Rubber Chemistry and Technology. doi: 10.5254/rct.17.83746.
Lin, J.T., Chen, G.Q. 2017. Structural characteristics of the molecular species of tetraacylglycerols in lesquerella (Physaria fendleri) oil elucidated by mass spectrometry. Biocatalysis and Agricultural Biotechnology. 10(10):167-173. doi: 10.1016/j.bcab.2017.03.005.
McKeon, T.A. 2016. Fatty acid composition of seed oil from Fremontodendron californicum. American Journal of Plant Sciences. 7(15):2107-2111. doi: 10.4326/ajps.2013.715188.
McKeon, T.A. 2016. Castor (Ricinus communis L.). In: McKeon, T.A, Hayes, D.G., Hildebrand, D.F.,Weselake, R.J., editors. Industrial Oil Crops. Waltham, MA: Elsevier Academic Press and Urbana, IL: AOCS Press. p. 75-112.
McKeon, T.A., Hayes, D.G., Hildebrand, D.H., Weselake, R.J. 2016. Introduction to industrial crops. In: McKeon, T.A., Hayes, D.G., Hildebrand, D.F., Weselake, R.J., editors. Industrial Oil Crops. Waltham, MA: Elsevier Academic Press and Urbana, IL: AOCS Press. p. 1-13.
Monadjemi, S., McMahan, C.M., Cornish, K. 2016. Effect of non-rubber constituents on Guayule and Hevea rubber intrinsic properties. Journal of Research Updates in Polymer Science. 5(5):87-96. doi: 10.6000/1929-5995.2016.05.03.1.
Sikandar, S., Ujor, V.C., Ezeji, T.C., Rossington, J.L., Michel, F.C., Mcmahan, C.M., Ali, N., Cornish, K. 2017. Thermomyces lanuginosus STm: a source of thermostable hydrolytic enzymes for novel application in extraction of high-quality natural rubber from Taraxacum kok-saghyz (rubber dandelion). Industrial Crops and Products. 103(2017):161-168. doi:10.1016/j.indcrop.2017.03.044.
Lin, J.T., Fagerquist, C.K., Chen, G.Q. 2016. Ratios of regioisomers of the molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil estimated by mass spectrometry. Journal of the American Oil Chemists' Society. 93(2):183-191. doi: 10.1007/s11746-015-2769-2.
Chen, G.Q., Riiff, T.J., Johnson, K., Morales, J.S., Kim, H.U., Lee, K., Lin, J.T. 2017. Seed development and hydroxy fatty acid biosynthesis in physaria lindheimeri. Industrial Crops and Products. 108(108):410-415.