Location: Bioproducts Research
2020 Annual Report
Objectives
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
Subobjective 1A: Genetically modify guayule for improved rubber yields- we will engineer guayule for over-expression of isoprenoid genes and/or down-regulation of carbon-competing pathways to increase rubber content. Independently transformed lines and controls will be analyzed: gene expression, rubber/resin content, rubber transferase activity, inulin, squalene, lipids and TAGs. We will apply the knowledge with that developed in 1) regulation of biochemical pathways 2) storage of hydrocarbons in plants 3) guayule genomics tools.
Sub-objective 1B: Identify biochemical regulation of enzymes- we will use yeast, S. cerevisiae, a single-celled eukaryote that responds to IPP by producing ergosterol, as a model to study HMGR, IDI, and FPP synthase impact on ergosterol. Results will be translated to tobacco to evaluate post-translational modifications in a model plant.
Sub-objective 1C. Develop genetic transformation of Kazak dandelion- a robust transformation system will be developed by 1) screening diploid seedlings to identify highly regenerating lines 2) optimizing culture conditions 3) evaluating explant sources (hypocotyl, stem, leaf petiole), and 4) assessing seed production. Self-compatible lines will facilitate genetic studies on relationships among transgene dosage, gene expression level, and rubber content.
Subobjective 2A- We will attempt to elucidate the roles of naturally-occurring proteins in Hevea rubber particles. This knowledge will inform modification of the chemical, physical, and/or biological properties of guayule and Kazak dandelion rubbers to meet industrial requirements. We will study interactions of proteins, amino acids, and lipids with rubber, then employ biobased post-harvest treatments. If unsuccessful, we will apply chemical treatments.
Subobjective 2B: To elucidate the roles of lipid in rubber biosynthesis- the molecular species of various lipid classes in rubber particles of guayule, Kazak dandelion and Hevea will be quantified using HPLC and MS. Lipid profiles from the native guayule and Kazak dandelion will be compared to those from genetically modified plants.
Subobjective 2C: Develop novel processes to fractionate crude guayule - we will evaluate a series of processes including 1) re-precipitation, 2) microfiltration, 3) liquid-liquid extraction, and 4) microfiltration/ultrafiltration to de-rubberize and fractionate guayule resin into major components.
Sub-objective 3A. Develop knowledge of hydroxy fatty acid (HFA) synthesis in lesquerella- we will engineer key genes to increase HFA levels in lesquerella seed oil. We will apply Agrobacterium-mediated transformation, and identify stable transgenic lines by germinating T1 seeds in selection medium. Plants/seeds will be characterized (transgene copy number for T1 using qPCR, fatty acid and TAG composition in using T2 seeds). If the total HFA content does not reach the target 70% in transgenic lesquerella, alternative promoters will be studied. Sub-objective 3B. Develop HFA-producing camelina- knowledge gained from engineering lesquerella for increased HFA content will inform strategies to raise HFA-production in the domestic oilseed crops camelina.
Progress Report
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
1. 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).
Review 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. https://doi.org/10.1016/j.bcab.2020.101617.
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. https://doi.org/10.1016/j.indcrop.2020.112245.
