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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

2018 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 sub-objectives, 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, research was conducted on guayule and Kazakh dandelion (for rubber production) and lesquerella and camelina (for oil production). Sub-objective 1A: To genetically modify guayule for improved rubber yields, ARS researchers in Albany, California, developed new tools for more effective genetic modifications. The first, use of a short peptide linker, allows 2 genes to be introduced at the same time and same place for efficient transformation. The second, a cold-inducible promoter (CBF2P), controls the effects of the introduced genes to only when the temperature is cold (5-10 degrees Celsius or 40-50 degrees Fahrenheit). Since guayule mostly produces rubber in the winter, CBF2P helps increase rubber yields, in synergy with the plants’ natural cold response. Up to 30 percent increase in rubber content was observed when the two approaches were combined. Separately, increased rubber accumulation was also found for guayule plants modified to produce lower levels of squalene, a terpene that may compete with rubber for fixed carbon. Full plant characterization is underway. In associated agreement, 2030-21410-021-04C, “Genetic Modification of Guayule”, a 2-year field trial was completed for guayule plants genetically modified to control the levels of allene oxide synthase (AOS), the major rubber particle protein for guayule natural rubber. Field plants showed different levels of gene expression even after 2 years in the challenging semi-arid (Eloy, Arizona) environment. Higher rates of photosynthesis for modified plants were found in the field, as was seen in the laboratory. Mechanistic studies suggest at least part of the mode of action is related to changes in the levels of plant hormones and their stress response in guayule. In associated agreement, 2030-21410-021-06S, “Quality improvement of Guayule Natural Rubber”, ARS researchers in Albany, California, created guayule plants over-expressing 4 genes responsible for tocopherol (Vitamin E) synthesis with the hypothesis that Vitamin E preserves rubber quality after harvest. Initial results suggest higher rubber content in the plants, perhaps because of improved oxidative stability. ARS scientists in Albany, California, completed a major research milestone in publication of the first guayule genome sequence and assembly. The DNA reads are now publicly available. Several genomic assemblies were prepared and have been transferred to industrial and academic labs. Results are being used in genotype fingerprinting of guayule cultivars and in applied bioengineering research. Under associated agreement, 2030-21410-021-15R, “Sustainable Bioeconomy for Arid Regions (SBAR)”, the initial steps toward downregulation of flowering were completed. Guayule flowers in spring, but also at other times during the year (indeterminately), and field studies suggest reducing flowers might increase rubber content. The new guayule genomic information from the sequencing project was used to identify candidate genes. The top two genes have been cloned and initial transformations are underway. Sub-objective 1B: Significant progress was made to identify biochemical regulation of enzymes in the plant pathways that will lead to increased yield of rubber. It is well-known that rubber biosynthesis in guayule increases during cold stress, but a recent field study conducted by ARS researchers confirmed drought-stressed plants also had higher rubber content. A comparison of the expressed genes of control and drought-stressed plants brought new insight as to how drought stress affects rubber production. Analysis of these genes resulted in a list of candidates related to rubber synthesis that are targets for improving rubber yield. An important discovery, that a critical rubber pathway gene, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), has at least two forms, HMGR1 and HMGR2, provided new insight into the genetic and molecular basis of rubber production in guayule. Lettuce (Lactuca sativa L.) is a potentially useful model plant for elucidating rubber biosynthesis. It is known to make a natural rubber of molecular weight (MW)>1 million kilo Dalton (kD). ARS researchers in Albany, California, showed that rubber content of latex from different tissues of the bolting plant had the same MW. Because the generation time is 3-4 months and it is easily transformed, lettuce has great potential for elucidating rubber biosynthesis and regulation. The Isopentenyl diphosphate-Dimethylallyl diphosphate Isomerase (IDI) is a potential gatekeeper enzyme in isoprenoid biosynthesis. Analysis of the lettuce genome database will identify additional gene sequences for enzymes that utilize isopentenyl pyrophosphate (IPP) with the goal of inactivating them to enhance availability of IPP for rubber biosynthesis. Sub-objective 1C: To develop an effective protocol for highly efficient genetic transformation of Kazak dandelion (Tk), transformation experiments were performed using an Agrobacterium strain which contains a rubber elongation factor promoter (REFP) from Hevea fused to a visual detectable reporter gene. Identification of REFP activity in Tk provides a tool for targeted expression of genes involved in rubber synthesis in Tk. Investigations are underway to establish a protocol for healthy shoot regeneration. Sub-objective 2A: Rubber particles from different species have different levels and types of proteins. Hevea (rubber tree) latex proteins are quite different from other species and contribute to the outstanding properties of its rubber. Progress was made to better understand the role of these proteins. Different types of proteins and extracted Hevea protein (REF) were added to guayule latex to change the composition. Some properties, such as thermal stability and rate of cure, were enhanced, but the material strength was only slightly affected. To test whether protein-polymer linkages are formed in vivo, guayule plants were transformed with Hevea REF protein. The genes were highly expressed but high amounts of protein did not accumulate. Latex has been extracted from the plants for further testing. Sub-objective 2B: Rubber particle lipids may also have an important role in the high material strength of Hevea rubber. ARS researchers in Albany, California, compared the structure and function of rubber particle lipids in guayule and Hevea. The intact molecular species of the acylglycerols (AG) in the lipids were compared using high performance liquid chromatography-mass spectrometry (HPLC-MS). Results show the fatty acids found in guayule rubber particles are mainly linear, including mostly unsaturated structures. A significant percentage was composed of linolenic or linoleic acids. Interestingly, chemically functional hydroxy fatty acids were found in both guayule and Hevea particles; however, most of the fatty acids associated with Hevea rubber particles were unusual furan type structures, including a newly discovered lipid. Results suggest that differences in lipids may explain the differences in physical properties observed. Sub-objective 2C: The economics of guayule would be enhanced by higher value use of coproducts, including guayule resin. ARS researchers isolated small amounts of sesquiterpene via basic resin hydrolysis. The next step is scale-up to acquire larger quantities in order to evaluate chemical modifications that convert it to a potentially useful chemical compound. The use of guayule resin components as additives in renewable biodiesel is also under investigation. Sub-objective 3A: Using high-performance analysis technologies, detailed changes of seed oil molecules, triacylglycerols (TAGs), and their structures were investigated in transgenic lesquerella lines that express a specific castor gene. This change increased hydroxy fatty acid (HFA) ricinoleic acid from 2 percent to 17 percent. A total of 36 different TAG structures were detected and their contents were quantified. Results revealed that the castor gene increased HFA content by particularly enriching ricinoleic acid in tri-HFA-TAGs in lesquerella, a new mechanism for underlying HFA biosynthesis in seeds. Sub-objective 3B: ARS researchers in Albany, California, sequenced and analyzed transcriptomes from developing seeds, leaf, and flower bud of a lesquerella relative, Physaria lindheimeri (PI). Pl produces 80 percent HFA in seed oil. Genes involved in HFA biosynthesis in Pl were all identified. A multi-gene stacker transformation vector which is important to enhance HFA in Camelina, is under construction.

1. Guayule genome. In 2018, ARS scientists at Albany, California, along with collaborators at Cornell University (Ithaca, New York) and Cooper Tire (Findlay, Ohio), completed a major research milestone in publication of the first Parthenium argentatum (guayule) genome sequence and assembly. The results provide fundamental DNA data which can be used by breeders to improve traits like natural rubber yield and disease resistance in the crop. The data are now publically available for researchers worldwide. In addition, organizations of the DNA data- known as assemblies- were constructed to provide more insight into the most important genes. Results are being used by industry and universities to fingerprint guayule varieties and develop crop improvement strategies.

2. Guayule field trials of bioengineered plants with higher photosynthesis rates completed. ARS scientists in Albany, California, along with Bridgestone Americas researchers, completed a 2-year field trial of bioengineered plants at the Bridgestone Research Farm in Eloy, Arizona. Experimental plants had higher photosynthesis rates and as a result grew larger, compared to control plants. The bioengineered plants have the potential to increase natural rubber yields, critical to economic sustainability of the crop. Additional laboratory studies are in process to understand the basic biology behind the improvements. Two U.S. Patents and two international patents on the technology were filed in 2018.

Review Publications
Lin, J.T. Chen, G.Q. 2018. Castor and Lesquerella oils: production, composition, and uses. In: Chen, G.Q., editor. Quantification if the Molecular Species of Acylglycerols Containing Hydroxy Fatty Acids in Lesquerella Oil Using High-performance Liquid Chromatography and Mass Spectrometry. New York, NY: Nova Science Publisher Inc. p. 19-34.
Lin, J.T. 2018. Castor and Lesquerella oils: production, composition, and uses. In: Chen, G.Q., editor. Composition of Acylglycerols in Castor Oil and their Biosynthetic Pathway. New York, NY: Nova Science Publisher Inc. p. 1-18.
Chen, G.Q., Johnson, K., Morales, E., Ibanez, A.M., Lin, J.T. 2017. A high-oil castor cultivar developed through recurrent selection. Industrial Crops and Products. 111:8-10.
Zhu, Y., Xie, L., Chen, G.Q., Lee, M., Loque, D., Scheller, H.V. 2018. A transgene design for enhancing oil content in Arabidopsis and Camelina seeds. Biotechnology for Biofuels. 11:46.
Franco, J.V., Wang, Y., Huo, N., Ponciano, G.P., Colvin, H.A., McMahan, C.M., Gu, Y.Q., Belknap, W.R. 2018. Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes. BMC Genomics. 19:271.
He, X., Patfield, S.A., Cheng, L.W., Stanker, L.H., Rasooly, R., McKeon, T.A., Zhang, Y., Brandon, D.L. 2017. Detection of abrin holotoxin using novel monoclonal antibodies. Toxins. 9(12):386.
Ponciano, G.P., Dong, N., Chen, G.Q., McMahan, C.M. 2018. A bicistronic transgene system for genetic modification of Parthenium argentatum. Plant Biotechnology Reports. 12(2):149-155.
Sabaini, P.S., Boateng, A.A., Schaffer, M.A., Mullen, C.A., Elkasabi, Y.M., McMahan, C.M., Macken, N. 2018. Techno-economic analysis of guayule (parthenium argentatum) pyrolysis biorefining: production of biofuels from guayule bagasse via tail-gas reactive pyrolysis. Industrial Crops and Products. 112:82-89.