Location: Bioproducts Research
2024 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 2030-21410-022-000D, titled, "Domestic Production of Natural Rubber and Resins", which started in April 2020.
In support of Sub-objective 1A, a protein of interest, the Small Rubber Particle Protein (SRPP) common to all rubber producing plants, was studied. ARS researchers in Albany, California, isolated a promoter region of the guayule homolog (Guayule Homolog to SRPP, GHS), translationally fused it to a reporter gene (GUS gene) and developed transgenic guayule lines for molecular characterization. Results show the isolated PaGHS promoter region constitutively expresses GUS in the stem tissues in four independent lines. Ongoing identification of the PaGHS promoter motifs responsive to environmental stresses will be informative to understand the cold/drought-induction mechanism of rubber biosynthesis in guayule (Sub-objective 1B). This promoter is also a new tool for genetic engineering approaches to improve rubber biosynthesis in guayule (Sub-objective 1C).
Natural rubber is synthesized by at least a cis-prenyl transferase (CPT) and a cis-prenyl transferase binding-protein (CBP). ARS researchers constructed a conventional transformation vector carrying three guayule genes, CPT3, CBP, and SRPP. In collaboration with the University of Nevada at Reno, multiple transgenic Arabidopsis lines expressing these genes have been generated. Plant analysis including stable integration of these transgenes into genome, spatial and temporal gene expression, and functional characterization of the transgenic plants are being investigated.
Progress was also made for Sub-objective 1B. Natural Rubber (NR) synthesis in guayule is upregulated by cold. Transcription factors (TFs) are regulatory proteins induced by signals such as cold. ARS researchers in Albany, California, identified a guayule transcription factor that is a promising candidate for overexpression in guayule, potentially boosting NR production without the need for cold induction. In related research, through a Community Science Project (CSP) collaboration with the Joint Genome Institute in Berkeley, California, and the Boyce Thompson Institute in Ithaca, New York, progress continued for genome sequencing of a trio of important guayule lines. The genomes of two of these guayule lines (CAL-3 and AZ-2) have been assembled. ARS scientists are using these newly chromosome-level assembled and functionally annotated genomes to advance guayule genetic transformation and facilitate mechanistic studies of regulatory control of rubber biosynthesis in guayule. Specifically, 1) A genome guided transcriptome field study has successfully identified a few regulatory elements related to temperature-based control of rubber biosynthesis in guayule, and 2) Precise application of gene editing with accurate CRISPR vector designs are now enabled. Analysis of the genomic and transcriptomic data showed interesting results in FY24. Rubber particle biosynthesis genes were found to be generally down regulated when guayule was under cold stress. This counter intuitive finding, concluded from comparative and functional genomic analysis of a multi-location field experiment, with various temperature profile supported an earlier hypothesis made by ARS researchers in Albany, California, from a drought field study, that guayule rubber biosynthesis control point is at the post-transcriptional level and beyond.
In support of Sub-objective 1C, two different Cas9 enzyme sequences were identified (out of many possible choices) as candidates for guayule genome editing. One is the classic Streptococcus pyogenes Type II ‘SpCas9’ (plant codon-optimized), and the other is ‘BP-Cas9’ with a dual synthetic bipartite nuclear localization signal to increase editing efficiency as demonstrated in Arabidopsis. Both Cas9s are currently being synthesized for incorporation into guayule CRISPR editing constructs. The best performing Cas9 will be used for editing guayule rubber biosynthesis genes to increase rubber yields.
Also in support of Sub-objective 1C, ARS researchers adopted a new cloning method for simplified and efficient construction of guayule genome editing vectors. DNA sequencing data from the above CSP project is being used to move forward in CRISPR/Cas9 editing of guayule. The new Golden Gate cloning method allows building a library of guayule molecular components such as promoters, terminators and selection markers, that can easily be combined for custom assembly and editing of any gene of interest.
ARS research for Objective 2 has pivoted to focus on characterization of how genotype and growth conditions impact resin production in guayule. Rubber production in guayule is much lower in the greenhouse compared to the field, but interestingly, resin production is about the same for greenhouse and field plants. In collaboration with ARS scientists from the National Plant Germplasm System, Parlier, California, a greenhouse study was performed to compare phenotypes, including resin production, for 10 guayule lines, including one historical line from Manzanar germplasm. Phenotype data were collected after one year of growth for six plants per accession. The Manzanar plants showed the lowest biomass, and smallest stem thickness, among the accessions studied. Rubber content was intermediate but, interestingly, resin content lowest, compared to the other lines. By design, the accessions included guayule diploid, tetraploid, and hybrid plants, which were readily differentiated by several descriptors, especially leaf morphology. Leaf area, width, and perimeter, especially, differentiated diploid lines, as expected. Other plant descriptors, particularly those related to flower morphology, were remarkably similar across dissimilar genotypes. One exception was the Manzanar line with a lower disc flower count compared to most other lines in this study. A comparison of greenhouse phenotypes to field phenotypes is underway.
In a series of greenhouse and field experiments, various guayule genotypes were grown in soil and irrigation water conditions similar to those found in the westside of California’s San Joaquin Valley. Selected guayule ecotypes showed tolerance to saline soil and poor-quality irrigation water. Interestingly, in some cases rubber and resin production were increased, by as much as two-fold (% dry weight basis) when plants were grown under chemical stressors. This trend also varied by accession. In an ongoing field lysimeter trial, the highest chemical stresses showed the highest rubber and resin accumulation after 14 months of growth. This study is continuing in collaboration with ARS scientists in Parlier, California.
In support of Sub-objective 3A, ARS researchers evaluated a new type of biochar, i.e., pyrolyzed waste biosolids, as full or partial replacement for carbon black in rubber compounds. Partial replacement resulted in little effects on compound properties, suggesting a possible application for the material. The current investigation is focusing on the effect of particle size on rubber performance.
For Sub-objective 3B, benchmark testing was performed to quantify the biobased content of currently used natural rubber balloon films. Rubber balloons are composed of natural rubber and have few additives. Testing by ASTM D6866 determined the biobased content to be close to 100%. The industrial collaborator is pursuing certification for the USDA BioPreferred Program. ARS scientists also tested biodegradability of rubber balloon films, using a method adapted from the ASTM D5338, Aerobic Biodegradation standard. Initial results suggest faster biodegradation for natural rubber compared to petroleum-based thermoplastics, suggesting natural rubber particles might present different environmental impacts than microplastics in the environment.
In support of Sub-objective 3C, ARS researchers published peer-reviewed results of transgenic guayule plants overexpressing tocopherol (a potent antioxidant molecule). Increasing in vivo tocopherol content in stem tissues of guayule via transgenesis provided some thermo-oxidative protection against rubber degradation, however overproduction of tocopherols resulted in a disruption of the isoprenoid pathways that synthesize rubber, resin, and the triterpenoid argentatins resulting in significantly lower contents in the transgenic lines. Importantly, these research results confirmed a role for the plastid isoprenoid pathway (methyl-erythtiol-4-phosphate) in rubber biosynthesis, highlighting the importance of this pathway.
Stakeholder interest in biobased and biodegradable rubber compounds, especially for safer antiozonants, continued to grow in FY24. Development of new materials is hampered by the complex and costly in-rubber testing required. In response, ARS researchers developed a screening method consisting of polymer solutions with and without candidate antiozonants, exposed to (bubbling) ozone, which causes dramatic reductions of solution viscosity in minutes. More than 100 additives have been characterized, identifying promising antiozonants capable of protecting the rubber from ozonation in solution. In addition, novel antiozonant molecules have been synthesized and tested using the screening method. The most promising candidates have been provided to an industrial partner for in-rubber evaluation.
Accomplishments
1. Guayule genomes sequenced. Guayule research has long been impeded by poor genomic resources. ARS Researchers in Albany, California, through a Community Science Project (CSP) collaboration with the Joint Genome Institute in Berkeley, California, and the Boyce Thompson Institute in Ithaca, New York, completed genome sequencing for two important guayule lines (CAL-3 and AZ-2). ARS scientists are using these newly chromosome-level assembled and functionally annotated genomes to advance guayule genetic transformation and facilitate mechanistic studies of regulatory control of rubber biosynthesis in guayule. Specifically, the information has resulted in 1) identification of regulatory elements related to temperature-based control of rubber biosynthesis in guayule and 2) precise application of gene editing with accurate CRISPR vector designs.
Review Publications
Kushwara, P., Soto Velázquez, A.L., McMahan, C.M., Neilson, J. 2024. Field to greenhouse: How stable is the soil microbiome after removal from the field? Microorganisms. 12(1). Article 110. https://doi.org/10.3390/microorganisms12010110.
King-Smith, N., Molnar, K., Blakeslee, J., McMahan, C.M., Pillai, A., Mutalkhanov, M., Puskas, J., Cornish, K. 2023. Extractable latex yield from Taraxacum kok-saghyz roots is enhanced by increasing rubber particle buoyancy. Industrial Crops and Products. 206. Article 117698. https://doi.org/10.1016/j.indcrop.2023.117698.
Ponciano, G.P., Dong, N., Dong, C., Breksa III, A.P., Vilches, A.M., Abutokaikah, M.T., McMahan, C.M., Holguin, F.O. 2024. Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues. Phytochemistry. 222. Article 114060. https://doi.org/10.1016/j.phytochem.2024.114060.
Sousa, E.A., Sanches, A., Vilches, J., da Silva, M., de Paula, F.R., McMahan, C.M., Malmonge, J. 2024. Ribbon-like microfiber of vulcanized and non-vulcanized natural rubber obtained by the solution blow spinning. Polymers for Advanced Technologies. 35(2). Article e6306. https://doi.org/10.1002/pat.6306.
Chen, G.Q., Dong, N., Johnson, K., Dong, C., Scheller, H.V., Williams, T.G., Wood, D.F. 2024. A guayule C-repeat binding factor is highly activated in guayule under freezing temperature and enhances freezing tolerance when expressed in Arabidopsis thaliana. Industrial Crops and Products. 212. Article 118303. https://doi.org/10.1016/j.indcrop.2024.118303.