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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

2019 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
In FY19, significant progress was made for all objectives in the project: Domestic Production of Natural Rubber and Industrial Seed Oils, which seeks to develop new cultivars to improve the quality and productivity of non-food biobased products. Research was conducted on guayule and Kazakh dandelion (for rubber production) and lesquerella and camelina (for oil production). In support of Sub-objective 1A, guayule was genetically modified in an attempt to improve rubber yields through the downregulation of a guayule rubber particle protein (allene oxide synthase AOS), which increased rubber content up to 4-fold in lab studies and was published in FY19. The increased rubber was related to the levels of the plant’s stress response hormones, an effect which could be duplicated by soil treatments. Additional studies in guayule’s response to stress, such as wounding, are underway. Additionally, ARS scientists discovered that over-expressing four genes responsible for tocopherol (Vitamin E) synthesis in guayule plants resulted in increased rubber content under cold treatment. It is thought that Vitamin E may preserve rubber quality after harvest. Plants have been moved to the greenhouse, and when they are large enough, the oxidative stability of the latex rubber will be evaluated. As well, progress continued toward downregulation of flowering. Guayule flowers in spring, but also at other times during the year (indeterminately), and field studies suggest reducing flowers might increase rubber content. Three transformation vectors, representing three transcription factors related to flowering, have been constructed. Leaf disc transformations are underway. Separately, ARS and university researchers have collected and characterized a set of field plants, with associated soil rhizosphere samples, to investigate the relationship between rubber biosynthesis and the soil microbiome/soil chemistry. In support of Sub-objective 1B, the guayule genome completed last year has enabled new progress for the strategy and tactics of guayule bioengineering. Evaluation of gene expression for field plants exposed to drought (and high rubber content) compared to controls has identified single genes and transcription control factors that may be related to rubber biosynthesis, therefore, provide attractive targets for modification. The original strategy to work with tobacco as a model species was changed to focus on lettuce as a model system for rubber production. Rubber production by the wild lettuce Lactuca virosa has been evaluated. The plant is known as a prodigious producer of latex which contains lacticin and lactucopicrin. These compounds have analgesic and sedative effects and have been used in the past as a replacement for the natural opioids from poppy. The latex is present in leaves during growth and in the stems of flowering plants. The dried latex is 98 percent resin and two percent rubber. While the rubber content of the latex is disappointing, the plant represents a near null for rubber production, potentially useful in identifying key components involved in rubber production by comparison to guayule. The focus of research under this Sub-objective was on two enzymes in lettuce that are involved in isoprenoid production. One is the Germacrene A synthase that metabolizes farnesyl diphosphate (FDP) to the sesquiterpene compound Germacrene A, which is further metabolized to a series of sesquiterpenoid compounds. Since such compounds form the resin fraction of latex, interference of the synthesis of germacrene A could block formation of one component of resin and possibly enhance rubber production by making more FDP available to initiate rubber biosynthesis. Molecular tools including antibodies have been generated to evaluate the role of this enzyme in lettuce development. A second enzyme of interest is the isopentenyl diphosphate(IDP)-dimethylallydiphosphate(DMADP) isomerase (IDI) which catalyzes the interconversion of IDP and DMADP, the key enzyme providing substrates for the synthesis of FDP. As such, it is positioned at the termination of IDP and DMADP synthesis and the beginning of polyisoprenoid biosynthesis. It is known to be a regulatory enzyme in other systems and based on sequence contain numerous sites for phosphorylation and possible nuclear localization. It is not yet characterized in development, but antibodies and primers have been generated for molecular characterization of IDI in lettuce development. Kazak dandelion Taraxacum kok-saghyz (TK) is another species of interest for industrial rubber production in temperate climates. In support of Sub-objective 1C, transformation experiments are underway through collaboration with The Ohio State University. Transformation vectors were constructed that contain laticifer-specific promoters of rubber elongation factor gene and protease inhibitor-like protein gene from Hevea fused to a visual detectable ß-glucuronidase (GUS) reporter gene. These promoters should restrict gene expression to the tissues of interest, the laticifers, where rubber is stored. Identification of GUS expression in TK would confirm laticifer-tissue specific expression and provide new tools for targeted expression of genes involved in rubber synthesis in TK. In support of Sub-objective 2A, laboratory studies previously showed addition of commercial proteins and amino acids could improve rubber compound properties. A similar approach is underway evaluating meat processing by-products (example for chicken feathers) as rubber compound additives. In support of Sub-objective 2B, research published last year showed that rubber particle lipids from guayule versus Hevea natural rubber are very different in chemical structure. In FY19, research showed that addition of two-three percent Hevea lipid (and lipid + protein) extracts to guayule latex made modest improvements. However, addition of a synthetic furan fatty acid ester, (methyl 10-oxo-10-(5-pentylfuran-2-yl) decanoate) improved guayule rubber compound performance. Furan lipids likely serve as radical scavengers during tapping (wounding stress) or to quench radicals formed from rubber degradation during storage. The economics of guayule would be enhanced by higher value use of coproducts, including guayule resin. Guayule resin is a complex mixture of sesquiterpenoid compounds, acylglycerols, free fatty acids, sterols, steroidal compounds and low molecular weight rubber. Separation of these compounds is key to enhancing the value of the resin. The resin as isolated during the rubber extraction process is difficult to work with, as a tar-like substance not readily poured or solubilized. Yet, it contains substances of potential value, including sesquiterpenes that have been shown to display anti-termite activity, aromatic compounds with anti-oxidant, perfume and other applications, low molecular weight rubber for possible use in adhesives, triacylglycerols that can be converted to biodiesel or solvent and a sterol-triterpenoid fraction of unknown use. In Sub-objective 2C, attempts to decolorize the resin were unsuccessful due to the viscous nature of the resin and use of silica-based chromatography was of limited use in separating components. Methanolysis of guayule resin made it easier to work with the resin, reducing the viscosity considerably. It also reduces the vapor pressure of any carboxylates present, making them more amenable to fractionation by distillation. We are proceeding to characterize processes to identify commercially viable means to fractionate the derivatized resin. In order to enable the commercial production of hydroxy fatty acids from oilseed crops already grown in the U.S., we started with Sub-objective 3A, by developing knowledge about hydroxy fatty acid (HFA) synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Using a transgenic approach, two important lesquerella fatty acid synthesis genes (desaturase 3 (FAD3) genes, PfFAD3-1 and PfFAD3-2), have been fully characterized. We discovered that PfFAD3-1 is a functional gene, but not PfFAD3-2. Sequence analysis reveals putative variations in PfFAD3-2, which could cause the loss of its function. PfFAD3-1 is the key in generating linolenic acid (18:3) and densipolic acid in lesquerella. This study not only enhances our understanding of molecular mechanisms underlying FAD3 activity in lesquerella, but also provides critical information for implementing genetic approaches to develop new crops. Over-expression of PfFAD3-1 in crops could increase 18:3 which enable crops to resist cold at freezing temperature. Alternatively, silencing PfFAD3-1 in lesquerella would eliminate 18:3 in seed oil which is favorable for low-oxidative features of oil desirable biofuel application. Finally, progress was also made on Sub-objective 3B, in developing hydroxy fatty acid (HFA) producing camelina. Physaria lindheimeri (Pl) produces 80 percent HFA in seed oil. A transformation vector carrying multi-genes from PI is constructed and transformed into Camelina. HFA production in Camelina is under investigation.

1. Discovery of a major lesquerella gene for oilseed biosynthesis. The arid-land plant, lesquerella, produces Hydroxy Fatty Acid (HFA) in its seed oil. HFA is a valuable raw material for the chemical industry and can be used as an excellent lubricity enhancer in diesel and ultralow sulfur diesel fuels, replacing sulfur-containing petroleum-based additives. ARS scientists in Albany, California, revealed two isoforms of a key HFA biosynthesis pathway gene, PfFAD3-1 and PdFAD3-2, through deep data mining of a lesquerella seed RNA data set (transcriptome). In collaboration with Sejong University and National Institute of Agricultural Science, Rural Development Administration, South Korea, they discovered that PfFAD3-1 is a functional gene, but not PfFAD3-2. Therefore, controlling PfFAD3-1 expression in lesquerella could reduce linolenic acid (18:3) in lesquerella which is favorable for low-oxidative biofuels, or increase 18:3 to create a more cold-resistant plant. This study enhances the understanding of molecular mechanisms underlying FAD3 activity in lesquerella and provides critical information for implementing genetic approaches to develop new crops.

2. Isolation of a rubber dandelion plant ideally suitable for breeding purposes. Kazakh dandelion, also known as rubber dandelion, can be grown in the northern U.S. for the high-quality natural rubber produced in its roots. However, not all varieties of rubber dandelion are suited to application of bioengineering technology for crop improvement. ARS scientists in Albany, California, identified a specific line, Tk#12, for which all plants are diploid, in vitro regenerable and are self-compatible by hand-pollination. These combined characteristics make Tk#12 ideal for application of bioengineering technology for crop improvement, and useful for conventional breeding as well. A set of plants has been transferred to the Ohio State University for studying stable inheritance of traits including transgenes.

Review Publications
Chen, G.Q., Johnson, K., Morale, E. 2018. Recurrent selection for improved oil content in castor bean. In: Kole, C., Robinowicz, P. editors. The Castor Bean Genome. Compendium of Plant Genomes. Cham, Switzerland: Springer Nature. p.67-75.
Hathwaik, U.I., Lin, J.T., McMahan, C.M. 2018. Molecular species of triacylglycerols in the rubber particles of Parthenium argentatum and Hevea brasiliensis. Biocatalysis and Agricultural Biotechnology. 16:107-114.
Lee, K., Kim, E., Jeon, I., Lee, Y., Chen, G.Q., Kim, H. 2019. Lesquerella FAD3-1 gene is responsible for the biosynthesis of trienoic acid and dienoic hydroxy fatty acids in seed oil. Industrial Crops and Products. 134:257–264.
Placido, D.F., Dong, N., Dong, C., Cruz, V., Dierg, D., Cahoon, R.E., Kang, B., Huynh, T.T., Whalen, M.C., Ponciano, G.P., McMahan, C.M. 2019. Downregulation of a CYP74 rubber particle protein increases natural rubber production in Parthenium argentatum . Frontiers in Plant Science. 10:760.
Ramirez-Cadavid, D., Cornish, K., Hathwaik, U.I., Kozak, R., McMahan, C.M., Michel, F. 2019. Development of novel processes for aqueous extraction of natural rubber from Taraxacum kok-saghyz (TK). Journal of Chemical Technology & Biotechnology. 94(8):2452-2464.