Location: Bioproducts Research
2015 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 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
1. 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.
2. 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.
