Progress 04/13/15 to 04/12/20
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the final report for project 2030-21410-021-00D ⿿Domestic Production of Natural Rubber and Industrial Seed Oils, which has been replaced by new project 2030-21410-022-00D, ⿿Domestic Production of Natural Rubber and Resins." For additional information, reference the new project report. For Objective 1, ⿿Develop varieties and commercially-viable post-harvest practices that maximize the market value of U.S. produced guayule and Kazak dandelion,"economic sustainability for the guayule crop in the southwestern United States might be secured with increased rubber yield. In support of Sub-objective 1A, ⿿Genetically modify guayule for improved rubber yields⿿, crop improvement included the following approaches. First, increasing gene expression in the primary biosynthesis pathway increased rubber content up to 30% using two key pathway genes, 3-hydroxy-3- methylglutaryl-CoA reductase (HMGR) and farnesyl pyrophosphate synthase (FPPs), especially under a cold-inducible promoter. Results have been published, two patents awarded, and a third patent filed. The second approach was to divert carbon away from other products to increase rubber content. Genetic downregulation of the first step to carbohydrate synthesis (sucrose: sucrose-1-fructosyl transferase) reduced the carbohydrate content, but with only a small increase in rubber. In a similar approach, production of the hydrocarbon squalene was reduced when squalene synthase was downregulated. Some, but not all, diverted carbon was used by the plant to produce more rubber. In both cases, lab studies revealed more rubber production under cold treatment, as expected, but also, surprisingly, in roots, probably due to crowding stress. Additional studies in guayule⿿s response to stress, such as wounding, are underway. ARS scientists discovered that guayule plants with a single gene modification, to reduce allene oxide synthase, the main rubber particle protein, resulted in a major increase in rubber content. In addition, the plants were significantly larger. Increased rubber was related to higher photosynthetic rates and to levels of the plant stress response hormones, an effect which could be duplicated by soil treatment. Some, but not all the improvements, were found in a 2-year field trial of engineered plants in Eloy, Arizona. Results have been published and two patents filed. Using the tools of biotechnology to increase yield in rubber-producing crops would be enhanced with more basic knowledge. In support of Sub- objective 1B, ⿿Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber⿿, studies focused on DNA and RNA analysis. ARS scientists at Albany, California, along with collaborators at Cornell University (Ithaca, New York) completed a major research milestone in publication of the first guayule genome sequence and assembly. The data can be used by breeders to improve traits like rubber yield and disease resistance in the crop and are now available for researchers worldwide. It is known that rubber biosynthesis in guayule increases during cold stress, but a field study conducted by ARS researchers (Albany, California and Maricopa, Arizona) confirmed drought-stressed plants also had higher rubber content. Comparison of the expressed genes of control and drought-stressed plants brought new insight. One discovery was that the rubber pathway gene, HMGR, has at least five forms. In parallel, analysis of HMGR expression in Kazakh dandelion revealed that TkHMGR1 (out of 11 forms) is the most highly expressed, and more so in roots than in leaves. These findings pave the way for further molecular and genetic studies of HMGR. Using lettuce as a model, another enzyme, the isopentenyl diphosphate-dimethylalyl diphosphate isomerase, may serve a possible gatekeeper role for production of isoprenoids. A third enzyme, germacrene A synthase, may also be important. Kazakh dandelion (Taraxacum kok-saghyz, Tk) produces natural rubber with industrial value. For Sub-objective 1C, ⿿Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion⿿, ARS scientists screened 76 Tk seedling lines, identified nine individual Tk plants with highly efficient shoot regeneration, and established a protocol with optimal hormone concentration. The use of an antibiotic, Kanamycin (25 mg/ l) was effective for eliminating non-transgenic plants. Self-compatibility is an important trait. Among the nine Tks, Tk#12 produced selfed seeds by hand-pollination. Both Hevea and Tk produce rubber in laticifer cells. New transformation vectors have been made that contain laticifer-specific promoters of rubber elongation factor (REF) and protease inhibitor-like protein (PIP) genes from Hevea. Continued efforts on transformation are underway through collaboration with the Ohio State University (Wooster, Ohio). Rubber particles from different species vary in proteins and lipids. Hevea (rubber tree) latex particles⿿ non-rubber constituents contribute to the outstanding properties of its rubber. Progress was made to better understand why in Sub-objective 2A, ⿿Elucidate the roles of naturally- occurring proteins in Hevea rubber particles⿿ and Sub-objective 2B, ⿿Elucidate the roles of lipid in the biosynthesis of rubber and on the mechanical properties of dry rubber⿿. In a series of studies, proteins, amino acids, and lipids were evaluated as additives for guayule latex. Usually, addition of commercial proteins and amino acids reduced bulk viscosity and improved thermo-oxidative stability. Similar results were found when using protein extracts from Hevea plants. Analysis of rubber particle lipid extracts from guayule and Hevea revealed that fatty acids in guayule rubber particles are mainly linolenic or linoleic acids. Hevea particle lipids were unusual furan type structures, including a newly discovered form. Furan lipids likely serve as radical scavengers during tapping (wounding stress) or to quench radicals formed during storage. This may explain the superior stability of Hevea rubber compared to guayule rubber. While high levels of natural rubber strain-induced crystallization, a goal of the project, were not achieved, improvement in other properties suggests these biobased materials may provide safer and more environmentally benign alternatives to traditionally used additives. Research also progressed for all Objective 3, 'Enable the commercial production of hydroxy fatty acids from oilseed crops already grown in the U.S.', sub-objectives. Castor oil is the conventional source of hydroxy fatty acid (HFA) containing 90% ricinoleic acid (18:1OH) with many industrial uses; howerver, since castor contains toxic substances, it is desirable to produce oils with high castor oil substitute content in other plants. For Sub-objective 3A, ⿿Develop knowledge of hydroxy fatty acid (HFA) synthesis in lesquerella to accelerate development of HFA- producing domestic oilseed crops⿿. Lesquerella does not have biologically toxic compounds and contains a major HFA, lesquerolic acid (20:1OH), at 55-60% of seed oil. Therefore, lesquerella is being developed as a new industrial oilseed crop in the United States. Genetic improvement of lesquerella could be an effective approach, and the impact of several genes on HFA content was studied. HFAs in lesquerella are located only at sn-1 and sn-3 positions of triacylglycerols (TAG), by introducing castor lysophosphatidic acid acyltransferase 2 gene (RcLPAT2) into lesquerella, the 18:1OH content at the sn-2 position of TAG increased from 2% to 17%, and consequently, seeds accumulated more castor oil-like TAGs. We revealed a new mechanism of RcLPAT2 in increasing castor oil-like TAGs in lesquerella by detailed analysis of the oil structure. In lesquerella, 20:1OH is synthesized through elongation of 18:1OH, and the step is regulated by an elongase, PfKCS18. Lesquerella also produces small amount of densipolic acid 18:2OH. A lesquerella PfFAD3 gene is responsible for the conversion of 18:1OH to 18:2OH. By silencing PfKCS18 and PfFAD3, we generated transgenic lesquerella that dramatically increased 18:1OH content from ~3% to ~27%. This is a major step toward the development of castor oil-producing lesquerella. Physaria lindheimeri (Pl) is a wild species closely related to lesquerella but contains over 85% 20:1OH in its seed oil. Key genes involved in HFA synthesis in Pl were identified. P. lindheimeri genes should be readily adapted to the molecular machinery of gene expression in lesquerella as both belong to Physaria genera. This strategy holds promise to boost the HFA level in lesquerella. Camelina is an existing industrial oilseed crop and serves as a platform for novel oil production. In support of Sub-objective 3B, ⿿Develop hydroxy fatty acid (HFA)-producing camelina⿿, essential genes from HFA- producing plants (castor, lesquerella, and P. lindheimeri) have been transferred to Camelina. Camelina expressing the key gene encoding 12 hydroxylase from Physaria lindheimeri (PlFAH12) produced about 15% HFA in seed oil. Co-expressing PlFAH12 and a lesquerella diacylglycerol acyltranstransferase (PfDGAT) which is a key gene for oil synthesis in Camelina increased the HFA content to 30% of seed oil. A transformation vector stacking multiple key genes responsible for HFA synthesis in P. lindheimeri was constructed, including PlFAH12, lysophosphatidic acid acyltransferase 2 (PlLPAT2), diacylglycerol acyltranstransferase (PlDGAT), phospholipid:DAG acyltransferase (PlPDAT), and PC:DAG phosphocholine transferase (PlPDCT). Camelina expressing these genes provides a new safe source for commercial production of HFA. Accomplishments 01 Discovery of new castor genes for genetic engineering of castor oil- producing lesquerella. Castor oil contains hydroxy fatty acid (HFA) with 90% of total fatty acids is ricinoleate (18:1OH) used for industrial applications. The production of castor oil is hampered by the presence of the toxin ricin in its seed. Lesquerella accumulates 60% HFAs in seed oil and is free of ricin. Developing 18:1OH-producing lesquerella would provide a safe source of HFAs readily usable by existing industrial technologies. In addition to a previous demonstrated castor lysophosphatidyl acyltransferase 2 gene (RcLPAT2) which increases 18:1OH in lesquerella, ARS scientists in Albany, California, discovered two new isoforms, RcLPATB and RcLPAT3B, capable of increasing HFA production in Arabidopsis. The newly identified genes provide targets for further enhancement of HFA in lesquerella or other commercial oilseeds. This research was part of a collaborative effort among the USDA, Sejong University (South Korea) and National Institute of Agricultural Science, Rural Development Administration (South Korean government).
Impacts
(N/A)
Publications
- Chen, G.Q., Lin, J.T., Van Erp, H., Johnson, K., Lu, C. 2020. Regiobiochemical analysis reveals the role of castor LPAT2 in the accumulation of hydroxy fatty acids in transgenic lesquerella seeds. Biocatalysis and Biotransformation. 25:10167.
- Kim, H., Park, M., Lee, K., Suh, M., Chen, G.Q. 2020. Variant castor lysophosphatidic acid acyltransferases acylate ricinoleic acid in seed oil. Industrial Crops and Products. 150:112245.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. 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 ÿ-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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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 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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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 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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This report covers the project, Domestic Production of Natural Rubber and Industrial Seed Oils, which began in April of 2015, and is part of National Program 306 Quality and Utilization of Agricultural Products (Component 2: Non-Food). Progress was made advancing all objectives during FY16. In domestic natural rubber research, under sub-objective 1A: Genetically modify guayule for improved rubber yields, previously modified lines were characterized to better understand the effect of the genetic modification on the plants� properties, especially natural rubber yield. Modification of guayule to reduce the amount of stored carbohydrate might divert carbon to natural rubber production. In laboratory studies, guayule plants modified to downregulate the first step to carbohydrate synthesis, the sucrose-1-fructosyltransferase gene 1- SST, increased rubber and resin yield for laboratory grown (in vitro) plants, while fructan carbohydrate production was lowered, suggesting deviated carbon flux from carbohydrate to natural rubber production. However, results for greenhouse plants showed more impact in root tissue, where more of the fructan is stored. In other studies, increased rubber content was found for modified plants overexpressing two key natural rubber biosynthesis pathway genes, HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and FPPs (farnesyl pyrophosphate synthase), especially when using a cold-inducible promoter. A patent has been filed. Under sub-objective 1B: Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber, an understanding of how plants control natural rubber accumulation will aid strategies to higher yield. Studies have been initiated to understand biochemical regulation of two key pathway genes, HMGR and IPPi (the isopentenyl pyrophosphate-dimethylallyl pyrophosphate isomerase). The HMGR protein has been previously expressed in the yeast, S. cerevisiae. Antibody development for both enzymes is underway. Further natural rubber research took place under sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion. Plants that grow well, and produce new plants by shoot regeneration, under laboratory conditions are needed for an effective protocol. A total number of 76 individual Kazak dandelion seedlings were screened for their shoot regeneration efficiency from root explants. Individuals with high efficiency of shoot regeneration from root explants have been identified. Another important aspect is the ability to distinguish modified plants from controls. The use of an antibiotic, Kanamycin (at concentration at 25 mg/l) has been demonstrated to be effective to eliminate non- transgenic shoots. In subobjective 2A: Modify the protein components of guayule rubber to increase its market value, a series of commercial proteins (gelatin, soy, albumin, casein, zein, gliadin and gluten) were added to guayule natural rubber as a latex blend. In general, protein addition reduced bulk viscosity and improved thermo-oxidative stability. Gel and green strength of the polymer-protein blends were increased, with the exception of gliadin, but not to levels observed for Hevea. Effects on vulcanization and mechanical properties in compounds were surprisingly influenced by the antioxidants used. Our results demonstrate the potential of proteins as bio-based rubber compounding additives. Results have been submitted for publication. In subobjective 2B: Elucidate the roles of lipid in the biosynthesis of rubber and on the mechanical properties of dry rubber we also seek to enhance the properties, and thereby the market value, of domestic latex and rubber. The outstanding features of the incumbent Hevea natural rubber have been attributed to the presence of non-rubber constituents, mainly proteins and lipids, which contribute to strain- induced crystallization. Guayule latex particles have similar lipid content, but quite different lipid composition compared to Hevea, which may be responsible for the disparity in properties. Hevea lipids were isolated from latex particles; the primary components were confirmed by LC-MS as furanoid type phospholipids, in agreement with literature reports. Blends of guayule latex with Hevea lipids and with a model furanoid lipid have been prepared. Use of guayule crops co-products could significantly impact its economic sustainability. In subobjective 2C: Develop novel processes to fractionate crude guayule resin into value- added components, chemical, solvent and enzymatic treatments to isolate components of guayule resin have been evaluated. In collaboration with an industrial partner, guayule resin in 3 forms, crude extract, purified, and leaf extract, have been evaluated. The industrial purification process reduced residual rubber to below 2%. An initial protocol for resin fractions� extraction and GC-MS characterization has been developed. In addition, we have discussed collaboration with SRRC on application of some resin components in developing wood products resistant to termites. Oilseeds research progressed under sub-objective 3A: Develop knowledge of hydroxyl fatty acids (HFA) synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Castor oil is composed of triacylglycerols (TAGs) containing about 90% of hydroxy fatty acids and has many industrial uses because of the structure. However, since castor contains toxic substances, it is desirable to produce TAGs with high hydroxy fatty acid (castor oil substitute) content in other oilseed plants. Lesquerella is an arid-land crop that could be used to produce hydroxyl fatty acids (HFA) similar to valuable castor oil. Genetic improvement of lesquerella could be an effective approach, and the effect of several genes on HFA content was studied. A key gene is HFA biosynthesis in plants is castor lysophosphatidic acid acyltransferase (RcLPAT2). The RcLPAT2 gene from castor plant was successfully transferred to lesquerella. The resulting lesquerella showed increased production of HFA at the sn-2 position, similar to castor. The increase was significant, from 2% to 17%, demonstrating that RcLPAT2 can be used to engineer a new HFA-producing crop, such as lesquerella, Camelina and canola. Another key enzymatic step (elongase) on the biosynthetic pathway of triacylglycerols in lesquerella has been identified and can be blocked to increase the content of hydroxy fatty acid of triacylglycerols in lesquerella. Genetic transformations of lesquerella with another castor LPAT gene, RcLPAT299 were also performed. However, RcLPA299 did not increase HFA content at the sn-2 position of seed TAGs. The results provide important information for re-directing future research. In addition, fatty acid composition was changed in 16 transgenic lesquerella lines with lowered expression for two other genes: 3-Ketoacyl-Coenzyme A Synthase (KCS3RNAi) and omega-3 fatty acid desaturase (FAD3RNAi). As expected, castor oil-like HFA was increased in positive lines. In summary, research identified key enzymatic steps in lesquerella can be used to produce castor oil for industrial uses. Camelina is already established as an industrial oilseed producing crop in the western United States. It is possible that it can also be used to produce valuable hydroxyl fatty acids (HFA)s. Under Sub-objective 3B: Develop HFA-producing camelina, a modification strategy was designed that required the use of the lipid biosynthesis gene PIFAH12 (oleate 12 hydroxylase). This gene was isolated and cloned from Physaria lindheimeri (Lindheimer�s bladderpod), a cruciferous flowering plant. A transformation vector carrying the PlFAH12 gene has been designed and constructed. The transformation vector has been sent to a Montana State University collaborator, for testing the effect of PlFAH12 in Camelina. Separately, data mining of a lesquerella seed transcriptome and expression profiling of lesquerella diacylglycerol acyltranstransferase (PfDGAT), Phospholipid:DAG acyltransferase (PfPDAT), and PC:DAG phosphocholine transferase (PfPDCT) gene family members have been performed. The results have been published. Accomplishments 01 Field evaluation of improved guayule lines. Increasing yield of natural rubber in guayule plants is the main goal of current genetic improvement studies. ARS scientists at Albany, California, discovered a single gene modification that can increase rubber content by up to four fold in the laboratory. During 2016, greenhouse evaluations confirmed the increase in rubber content, also discovered the same gene results in larger, greener plants. This remarkable combination could increase grower yields through both a higher percent of rubber and biomass. In collaboration with an industrial partner, a field trial was initiated. More than 1,500 tissue cultured plants were transported from ARS in Albany, California, to the field location, transitioned to soil and planted out in the Arizona field. 02 Identification of a specific gene to enhance production of castor oil in lesquerella. Castor seed oil is a conventional source of valuable hydroxy fatty acid (HFA) which has numerous industrial applications. Lesquerella seed oil also contains HFA, but unlike like castor seed which contain toxin ricin, lesquerella seeds represent a safe source of HFA. If lesquerella oil can be engineered to resemble castor oil, it would provide an alternative source of castor oil that is safe, cost- competitive, and readily adaptable by existing industrial technologies. ARS scientists discovered a key castor gene as a target for genetic engineering a castor oil-producing lesquerella crop. This gene, lysophosphatidic acid acyltransferase 2 (RcLPAT2), was used to create transgenic lesquerella plants in collaboration with Washington State University at Pullman and Rothamsted Research at United Kingdom. The result is the first demonstration that RcLPAT2 can be used to increase HFA, a valuable property for the engineering of a new castor oil- producing crop, such as lesquerella, Camelina, and canola.
Impacts
(N/A)
Publications
- Chen, G.Q., Van Erp, H., Martin-Moreno, J., Johnson, K., Morales, J.S., Browse, J., Eastmond, P.J., Lin, J.T. 2016. Expression of castor LPAT2 enhances ricinoleic acid content at the sn-2 position of triacylglycerols in lesquerella seed. International Journal of Molecular Sciences. 17(4) :507. doi: 10.3390/ijms17040507.
- McKeon, T.A., Brandon, D.L., He, X. 2015. Improved method for extraction of castor seed for toxin determination. Biocatalysis and Agricultural Biotechnology. 5:56-57. doi: 10.1016/j.bcab.2015.12.007.
- Hou, C.T., Lin, J.T., Ray, K. 2015. Identification of molecular species of polyol oils produced from soybean oil by Pseudomonas aeruginosa E03-12 NRRL B-59991. Biocatalysis and Agricultural Biotechnology. 4(4):500-505. doi: 10.1016/j.bcab.2015.08.017.
- Orts, W.J., McMahan, C.M. 2016. Biorefinery developments for advanced biofuels from a widening array of biomass feedstocks. BioEnergy Research. 9(2):430-446. doi: 10.1007/s12155-016-9732-4.
- Torres, L., Mcmahan, C.M., Ramadan, L.E., Holtman, K.M., Tonoli, G.H., Flynn, A., Orts, W.J. 2015. Effect of multi-branched PDLA additives on the mechanical and thermomechanical properties of blends with PLLA. Journal of Applied Polymer Science. doi: 10.1002/app.42858.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This new project which began in April of 2015, continues research from �Improvement and Utilization of Natural Rubber-and Castor Oil-producing Industrial Crops�, 2030-21410-020-00D. Please see the report for this project for additional information. In domestic natural rubber (NR) crop research under Sub-objective 1A: Genetically modify guayule for improved rubber yields, guayule was genetically modified to reduce the amount of carbohydrate produced, by down regulation of 1-SST (sucrose: sucrose-1-fructosyl transferase). Initial lab in vitro cultured plants had higher rubber levels, suggesting the carbon was diverted to rubber production. Greenhouse plants did not confirm initial results, although lower levels of sugars and carbohydrates were found. Separately, overexpression of a proprietary gene led to higher NR accumulation in young guayule plants; greenhouse studies are in process. In collaboration with the University of Nevada Reno baseline levels of natural antioxidants (AO) in guayule tissue culture, greenhouse, and field plants were measured under various conditions. Target genes for increasing AO production in guayule were identified, cloned from Arabidopsis, and vectors prepared. Transformation activities will start in the next few months. Research in Sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion, also showed progress. Protocols for shoot regeneration were tested on leaf and root explants. Individuals with high efficiency of shoot regeneration from leaf explants have been identified. Kanamycin as a selection tool is being evaluated on root explants. The protocols will be optimized following these studies. To address Subobjective 2A: Modify the protein components of guayule rubber to increase its market value, a series of commercial proteins were blended with guayule latex to determine if protein-polymer interactions, created in vitro, could lead to more Hevea-like rubber. While high levels of strain- induced crystallization, a goal of the project, were not achieved, other physical and chemical properties did show surprising improvement. This suggests that commercial proteins could serve as biobased additives for industrial rubber compounds. Research continues in collaboration with Cooper Tire. To address Subobjective 2C: Develop novel processes to fractionate crude guayule: Guayule resin has potential industrial uses both in adhesives and in solvent preparations, replacing solvents and adhesives generated from petroleum with those derived from renewable resources. Methods for analyzing the resin fraction have been identified and research will proceed on evaluating guayule cultivars for resin production and components. Oilseeds research progressed under Sub-objective 3A: Develop knowledge of hydroxyl fatty acids (HFA) synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Homozygous lesquerella lines expressing a castor lysophosphatidic acid acyltransferase (RcLPAT2) have been characterized. The resulted lesquerella showed increased production of HFA at the sn-2 position of seed triacylglycerols, which is expected. The data are being prepared for a manuscript for publication. Genetic transformations of lesquerella with another castor LPAT gene, RcLPAT299, are being performed. The results will be used for a future design of gene stacking experiments to enhance HFA accumulation in lesquerella. Homozygous transgenic lesquerella lines expression silencing constructs of 3-Ketoacyl-Coenzyme A Synthase (KCS3RNAi) and omega-3 fatty acid desaturase (FAD3RNAi) are being evaluated. The positive lines are expected to produce more uniform HFA. Finally, the structures of the molecular species of tetraacylglycerols in lesquerella oil were characterized by mass spectrometry. These structural characterizations inform proposed biosynthetic pathways and potentially, production of tetraacylglycerols for industrial uses. Research in camelina, new to the project team, is also progressing well for Sub-objective 3B: Develop HFA-producing camelina. A gene encoding oleate 12 hydroxylase from Physaria lindheimeri (PlFAH12) has been isolated by PCR and cloned. A transformation vector carrying the PlFAH12 gene has been designed and constructed. The transformation vector has been sent to a Montana State University collaborator, for testing the effect of PlFAH12 in Camelina. Data mining of a lesquerella seed transcriptome and expression profiling of lesquerella diacylglycerol acyltranstransferase (PfDGAT), Phospholipid:DAG acyltransferase (PfPDAT), and PC:DAG phosphocholine transferase (PfPDCT) gene family members have been performed. Candidate genes are being evaluated for their efficacies on enhancing HFA in Camelina. Accomplishments 01 Genetic modification of guayule for higher rubber yield. Increasing yield of natural rubber in guayule plants is the main goal of genetic improvement studies for ARS scientists in Albany, California. A single gene modification that can increase rubber content by up to four fold was discovered in laboratory and greenhouse studies. Gene expression was well correlated to the level of rubber measured. Moreover, modified plants are larger and greener than controls, suggesting carbon fixation plays a role in how these improved plants work. 02 Tetraacylglycerols in lesquerella oil. Structural differentiation of the molecular species of chemical components of lesquerella oil has been performed by ARS scientists in Albany, California. These oils have physical properties different from those of traditionally used oils, but can be used in industry similar to those, e. g., as viscosity and pour point improvers for lubricants. The newly-identified molecular species contained one or two normal fatty acids with the remainder being lequerolic acid, a hydroxyl-functional fatty acid valued for its ability to be used as a starting point for production of biobased plastics, etc.
Impacts
(N/A)
Publications
- Boateng, A.A., Mullen, C.A., Elkasabi, Y.M., Mcmahan, C.M. 2015. Guayule (parthenium argentatum) pyrolysis biorefining: production of hydrocarbon compatible bio-oils from guayule bagasse via tail-gas reactive pyrolysis. Fuel. 158:948-956.
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