Location: Bioproducts Research
2020 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
This report documents progress for project 2030-21410-022-00D, "Domestic Production of Natural Rubber and Resin," which started July 2020 and continues research from project 2030-21410-021-00D, "Domestic Production of Natural Rubber and Industrial Seed Oils."
Research has been conducted under Sub-objective 1A,‘Over-express enzymes and proteins involved in natural rubber synthesis and accumulation in guayule, including components of the rubber transferase complex’, and Sub-objective 1B, 'Increase natural rubber yield in guayule through controlled expression of transcription factors related to plant stress response', under Objective 1, 'Genetically modify guayule for improved commercial rubber yields.'
Genetic modification of guayule requires different strong promoters to drive the expression of multiple genes involved in natural rubber (NR) synthesis. Hevea produces NR in laticifer, a specialized tissue for synthesis and storage of high concentration of NR without disturbing plant growth. Two transformation vectors carrying laticifer specific promoters of rubber elongation factor (REF) gene and protease inhibitor-like protein (PI) genes from Hevea fused to a visual reporter gene, (pND6-REFP and pND6-PIP), have been transferred into Agrobacterium strain AGL1 and EHA101 for genetic transformation of guayule. Preliminary results indicated that there was no significant difference in efficiency of callus production between using AGL1 and EHA101. Since this is the first time that AGL1Q is being tested in guayule, it provides an additional tool for genetic engineering of guayule.
For natural rubber bioengineering, the structure of the potentially most impactful enzyme, the Rubber Transferase, has not been established. Single gene modifications have been met with mixed results, and the Rubber transferase is most likely an enzyme complex. A stacked 3-gene plant transformation construct has been prepared containing hypothesized components of the rubber transferase enzyme complex, for transformation into guayule and model plants.
Progress has also been made in Sub-objective 1B, ‘Increase natural rubber yield in guayule through controlled expression of transcription factors related to plant stress response’. A new strategy, flowering reduction in guayule, is the subject of associated agreement 2030-21410-021-15R, “Sustainable Bioeconomy for Arid Regions (SBAR)”. Prior field studies suggest reducing flowers might increase rubber content, and a bioengineering approach using four target genes (APETALA1, SEPATTALA3, FLOWERING TERMINUS, LEAFY) all transcription control factors related to flowering, is underway. In all cases we seek to downregulate the control factors to reduce the extent of flowering. Several thousand transformation attempts have yielded putative transformed plants incorporating the SEPETALLA3 and LEAFY genes. This project has provided the opportunity to improve the transformation efficiency for guayule, and factors such as timing, types of leaf cuts, and light intensity have been studied.
For Sub-objective 2A, ‘Enrich the high-value terpene fraction of guayule resin via environmentally-friendly extraction and filtration processes’, a series of green solvents have been used for liquid-solid extraction of guayule resin. While all solvents tested removed some components, alcohol solvents removed different components than other solvents, suggesting they may be useful for serial separations.
Progress was also made in Sub-objective 3A, ‘Evaluate the use of heat-treated agricultural residues as bio-based reinforcing fillers in natural rubber compounds.' A series of natural rubber compounds were prepared using torrefied (heat treated) rice hulls or almond shells as a reinforcing filler. The torrefied filler was used in place of part or all of the conventional filler, carbon black for concentrations from 0 to 100 weight percent. The effect of torrefied fillers on the compound processability, the curing process, dynamic properties, and mechanical properties was investigated. A manuscript has been submitted.
For Sub-objective 3C, the team is evaluating whether Vitamin E (tocopherols) could be an effective biobased antioxidant in rubber compounds. Initial rubber mixing studies suggest alpha-tocopherol is five times more effective as an antioxidant than the petroleum-based additives used. Another approach is to take advantage of natural antioxidants already produced by plants like guayule. Production of higher levels of natural antioxidants could improve the quality of guayule natural rubber by improving post-harvest stability. In collaboration with the University of Nevada, Reno, guayule plants overexpressing 4 genes responsible for tocopherol (Vitamin E) synthesis were created. Initial results suggest higher rubber content in cold-treated plants, perhaps due to improved oxidative stability.
Accomplishments
1. RNA database developed to support guayule development. Guayule, a perennial desert shrub native to the southwestern United States and northern Mexico, is under development as a domestic source of natural rubber, a critical agricultural material. Rubber yield in guayule increases under various environmental stresses, including drought stress. At the molecular level, drought stress results in differential expression of genes regulating various metabolic pathways. ARS scientists in Albany, California, and Maricopa, Arizona, in collaboration with scientists from the University of Arizona (Tuscon, Arizona) and Cornell University (Ithaca, New York), generated a guayule transcriptome (RNASeq) database from field-grown guayule subjected to water-deficit (drought) and well-watered (control) irrigation treatments. RNA transcripts associated with plant drought stress response as well as water homeostasis were highly abundant, but not those for rubber biosynthesis. The data suggest rubber biosynthesis point of control is not at the gene transcriptional level but possibly at the post-transcriptional or translational (protein synthesis) levels. The RNASeq database offers valuable data for future guayule genomic analysis related to plant development and metabolism regulation.
Review Publications
Nelson, A.D., Ponciano, G.P., McMahan, C.M., Ilut, D.C., Pugh, N.A., Elshikha, D.E., Hunsaker, D.J., Pauli, D. 2019. Transcriptomic and evolutionary analysis of the mechanisms by which P. argentatum, a rubber producing perennial, responds to drought. Biomed Central (BMC) Plant Biology. 19:494. https://doi.org/10.1186/s12870-019-2106-2.
Placido, D.F., Dierig, D.A., Cruz, Von, M.V., Ponciano, G.P., Dong, C., Dong, N., Huynh, T.T., Williams, T.G., Cahoon, R.E., Wall, G.W., Wood, D.F., Mcmahan, C.M. 2020. Downregulation of an allene oxide synthase gene improves photosynthetic rate and alters phytohormone homeostasis in field-grown guayule. Industrial Crops and Products. 153:112341. https://doi.org/10.1016/j.indcrop.2020.112341.
