Location: Bioproducts Research
2022 Annual Report
Objectives
Objective 1: Genetically modify guayule for improved commercial rubber yields.
Sub-objective 1A: Over-express enzymes and proteins involved in natural rubber synthesis and accumulation in guayule, including components of the rubber transferase complex.
Sub-objective 1B: Increase natural rubber yield in guayule through controlled expression of transcription factors related to plant stress response.
Sub-objective 1C. Apply CRISPR/Cas9 technology to improve rubber yield in guayule.
Objective 2: Develop environmentally sustainable, commercially viable processes for fractionation and modification of guayule resin co-product into higher value products.
Sub-objective 2A: Enrich the high-value terpene fraction of guayule resin via environmentally-friendly extraction and filtration processes.
Sub-objective 2B: Chemically modify guayule resin components to enhance their market value.
Objective 3: Enable marketable natural rubber composites incorporating food waste and byproducts.
Sub-objective 3A: Evaluate the use of heat-treated agricultural residues as bio-based reinforcing fillers in natural rubber compounds.
Sub-objective 3B: Assess the feasibility of using meat-processing byproducts and other agricultural residues as bio-based rubber compound additives.
Sub-objective 3C: Develop bio-based antioxidants for stabilization of natural rubber and resin.
Approach
Sub-objective 1A: Over-express enzymes and proteins involved in natural rubber synthesis –Co-expression of genes associated with the Rubber Transferase and the MVA pathway will provide targets for preparation of a vector using GAANTRY technology that can insert multiple transgenes simultaneously into plants. We will generate at least 10 independent transformed guayule lines using Agrobacterium-mediated transformation. Transgene insertion will be confirmed, and genotype and phenotype analysis performed.
Sub-objective 1B: Controlled expression of transcription factors – We will construct transformation vectors, overexpressing transcription factors. Guayule transformation, transgene confirmation, transgene expression, and other phenotypes including rubber content will be determined.
Sub-objective 1C. Apply CRISPR/Cas9 technology to improve rubber yield. – Using plant codon optimized synthetic Cas9 nuclease, to reduce off-target effects, Agrobacterium-mediated transformation of guayule will be performed to target the reporter gene GUS (ß-glucuronidase). Once the Cas9 is proven to be functional in guayule, we will target the AOS1 for gene editing. Transgenic lines will be further evaluated by standard methods.
Sub-objective 2A: Enrich the high-value terpene fraction of guayule resin –The utility of filtration technology for fractionation of guayule resins will be evaluated, with a focus on green solvents and low temperature processing.
Sub-objective 2B: Chemically modify guayule resin components –
We will determine if saponification and methanolysis of complex guayule resin mixtures can/should be applied as a fractionation strategy, as a means to valuable products.
Sub-objective 3A: Evaluate the use of heat-treated agricultural residues –
We will conduct torrefaction of the guayule bagasse and other crop residues. Natural rubber composites will be prepared and characterized using with the torrefied biobased fillers compared to conventionally used fillers.
Sub-objective 3B: Assess the feasibility of using agricultural residues –
We will focus on protein sources from agricultural operations, initially meat by-products. Materials will be characterized for chemical and physical properties, and protein stability. Model natural rubber compounds will be formulated in which commercial meat by-products will be added to, or used in place of, synthetic anti-degradants and vulcanization aids, and the impact on compound performance assessed.
Sub-objective 3C: Develop bio-based antioxidants –
We will determine the efficacy of in vivo stabilization of guayule rubber by tocopherols, and ex vivo use of biobased antioxidants for guayule extraction processing and compounding.
Progress Report
Progress was made under Sub-objective 1A. In plants, natural rubber is synthesized by a complex of at least two proteins, 1) a cis-prenyl transferase (CPT) and 2) a cis-prenyl transferase binding-protein (CBP). A third protein, the small rubber particle protein (SRPP) may also play a role. ARS researchers in Albany, California, hypothesize that overexpression of the genes encoding these proteins might have a significant effect on rubber production in plants. ARS scientists constructed a transformation vector carrying all three guayule genes, CPT3, CBP, and SRPP. The effect of these gene on rubber production in plants is being investigated. Through an associated agreement with the University of Nevada, Reno, multiple transgenic plant lines have been generated for a model plan (Arabidopsis), now under testing.
In subordinate project 2030-21410-022-001R, Sustainable Bioeconomy for Arid Regions, ARS researchers transformed guayule plants to downregulate genes that promote flowering. In some, but not all cases, less flowering was observed. ARS researchers are gaining valuable insight on which genes control flowering in guayule.
In related work, rubber particles (RPs) are analogous to lipid droplets (LDs), in that both are generated from the endoplasmic reticulum (ER) although the major components enclosed in the phospholipid monolayer structure of LDs are triacylglycerols (TAG), not polyisoprene. RP and LD share many common membrane proteins that play important roles in controlling the size and stabilizing RP or LD. One important protein is SEIPIN, which determines the size of LDs. ARS researchers are testing the function of the SEIPIN1 gene in affecting RP size and rubber accumulation in guayule.
Progress was also made in Sub-objective 1B. Transcription factors (TFs) are regulatory proteins induced by signals such as environmental stresses. A well-studied TF gene family, dehydration responsive element binding proteins (DREBs) regulates many stress-responsive genes, and a guayule DREB1D (PaDREB1D) showed high ribonucleic acid (RNA) expression in cold-treated stem tissue where active rubber synthesis and accumulation occurred. ARS scientists have successfully cloned PaDREB1D from guayule and prepared a transformation vector featuring PaDREB1D fused to a green fluorescent protein that allows visual studies of the function of PaDREB1D. To investigate the potential biological function of PaDREB1D, ARS scientists generated over 20 independent transgenic plant lines for a model plant (Arabidopsis), now under testing. The cold signal transduction through PaDREB1D is being studied, including characterizing PaDREB1D-regulated gene expression and freezing tolerance of these transgenic Arabidopsis under non-acclimated conditions.
In Sub-objective 1C, transformation vectors for gene editing of guayule have been designed and are being constructed. We anticipate completing these by end of 2022, then start gene editing transformation for guayule.
In related research, through a Community Science Project (CSP) collaboration with the Joint Genome Institute (JGI; Berkeley, California), ARS scientists co-collected tissue samples from three varieties of guayule growing in Salinas, California, Parlier, California, and Eloy, Arizona. Tissues were sampled at four times points, processed, and rubber biosynthesis rate measured. The biosynthesis rate was higher for guayule tissues collected during colder months as expected. JGI scientists are performing RNA sequencing on the same plants, moving toward a molecular understanding of how cold stress induces rubber production in guayule. In another part of the CSP, ARS scientists led material preparation for three important guayule germplasms. Guayule germplasm was screened to identify a line, AZ2-D, which has a high capacity of shoot proliferation under tissue culture conditions. Through collaboration with the Arizona Genomics Institute, shoot tip materials yielded high molecular weight DNA in superior quality. The DNA samples are being utilized for whole genome sequencing and data analysis.
In support of Sub-objective 3A, ARS researchers in Albany, California, prepared natural and synthetic rubber composites using torrefied biobased fillers as full or partial replacements for carbon black or silica usually used in rubber compounds. The process of torrefication changes the properties of biomass to make it work better as a fuel source. In most cases, low levels of replacements did not compromise compound properties. In a new development, experiments by ARS scientists demonstrated that torrefied rice hulls could be chemically coupled to tire-grade rubber, reducing heat build-up under dynamic stress. Results suggest the material could be used in tire tread compounds without compromising vehicle fuel economy.
For Sub-objective 3C, the project has generated mature guayule plants that are genetically modified to overexpresses tocopherols, i.e., Vitamin E, a potent antioxidant. A new test protocol was developed to measure the quality and quantity of rubber, before and after heat aging of plant stems, to simulate field drying conditions. If successful, guayule plants with higher Vitamin E in tissues will have improved post-harvest stability.
Tire industry stakeholders are keenly interested in safer additives to prevent ozone degradation of tires. ARS scientists have 1) developed computational models to elucidate the mechanism for ozone attack on rubber, and 2) devised strategies for biobased or biodegradable compounds that are safer for the environment and might provide oxygen and ozone resistance in tire compounds. Candidate compounds have been prepared and are under evaluation.
In additional research related to Sub-objective 3C, lesquerella produces industrially useful hydroxy fatty acids (HFA). In collaboration with Washington State University, ARS scientists, in Albany, California, created over ten independent transgenic lesquerella lines with suppressed expression of diacylglycerol transferase 1 gene. These materials are being analyzed to elucidate a novel triacylglycerol synthesis pathway for accumulation of HFA in lesquerella.
Accomplishments
1. Guayule tolerates marginal soils and irrigation waters. Guayule is a natural rubber-producing industrial crop that is tolerant of drought, marginal soils, and poor-quality irrigation waters, such as those found in the westside of the San Joaquin Valley, California. In FY22, ARS scientists in Albany, California, and Parlier, California, published results of a greenhouse and field study, where six guayule accessions were grown in poor quality soils and irrigated with water high in salt, boron, and selenium. Three of the accessions (N566, R1037, and R1093) showed low to medium toxicity under most conditions, and remarkably, salt treatment resulted in higher natural rubber concentration in three guayule accessions (N566, 11635, 11604). The findings warrant future investigations of guayule cultivation under field conditions typical of the western San Joaquin Valley of California.
2. Carbon metabolism in guayule. The arid-adapted guayule plant is under development as a climate smart crop in the southwestern United States. It naturally sequesters remarkable quantities of carbon, which are converted perennially into biomass as well as secondary metabolites of economic importance, most significantly natural rubber. Carbon metabolism in guayule is complex, especially so since rubber production is linked to abiotic stress such as cold, under conditions which also trigger changes in photosynthetic rates and in carbohydrate metabolism. ARS scientists in Albany, California, in collaboration with researchers at the Joint Bioenergy Institute (Emeryville, California) have applied metabolomics tools to reveal a better understanding of carbon metabolism in guayule. In growth chamber plants, rubber synthesis pathway metabolites, including the upstream precursor mevalonate, were more concentrated in guayule stem tissues compared with leaf and root tissues, yet leaf tissue presented the highest concentrations of the rubber biosynthesis initiator farnesyl pyrophosphate (FPP), and monomer, isopentenyl pyrophosphate (IPP). Results will inform future strategies for crop yield improvement.
Review Publications
Placido, D.F., Heinitz, C.C., McMahan, C.M., Banuelos, G.S. 2021. Guayule is an industrial crop that can be grown for its natural rubber production and phytoremediation capability in the Western San Joaquin Valley, California. Current Plant Biology. 28. Article 100223. https://doi.org/10.1016/j.cpb.2021.100223.
Dong, C., Ponciano, G.P., Huo, N., Gu, Y.Q., Ilut, D., McMahan, C.M. 2021. RNASeq analysis of drought-stressed guayule reveals the role of gene transcription for modulating rubber, resin, and carbohydrate synthesis. Scientific Reports. 11. Article 21610. https://doi.org/10.1038/s41598-021-01026-7.
Ramirez Cavidad, D., Hathwaik, U.I., Cornish, K., McMahan, C.M., Michel Jr., F. 2022. Alkaline pretreatment of Taraxacum kok-saghyz (TK) roots for the extraction of natural rubber (NR). Biochemical Engineering Journal. 181. Article 108376. https://doi.org/10.1016/j.bej.2022.108376.
Santim, R., Sanchez, A., da Silva, M., McMahan, C.M., Malmonge, J. 2022. Electrically conductive nanocomposites produced by in situ polymerization of pyrrole in a natural rubber latex medium. Polymer Composites. 43(5):2972-2979. https://doi.org/10.1002/pc.26591.
Placido, D., Dong, N., Amer, B., Dong, C., Ponciano, G.P., Kahlon, T.S., Whalen, M., Baidoo, E., McMahan, C.M. 2022. Downregulation of squalene synthase broadly impacts isoprenoid biosynthesis in guayule. Metabolites. 12(4). Article 303. https://doi.org/10.3390/metabo12040303.
Park, M., Lee, K., Chen, G.Q., Kim, H. 2022. Enhanced production of hydroxy fatty acids in arabidopsis seed through modification of multiple gene expression. Biotechnology for Biofuels. 15. Article 66. https://doi.org/10.1186/s13068-022-02167-1.
