Location: Bioproducts Research
2023 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 in Fiscal Year (FY) 2023 for project 2030-21240-022-000D, titled, “Domestic Production of Natural Rubber and Resins”.
In FY23, progress was made under 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". ARS researchers in Albany, California, hypothesized that the minimum components of the putative rubber transferase complex are three proteins known as cisprenyl transferase (CPT), CPT-binding protein (CBP), and small rubber particle protein (SRPP). The ARS researchers transformed tobacco plants with a plant transformation vector carrying these three guayule proteins as a proof-of-concept they form the rubber biosynthesis enzymatic complex. Through collaboration with researchers at the University of Nevada at Reno, multiple transgenic Arabidopsis lines expressing these three genes have been generated. Preliminary analysis of these transgenic tobacco and Arabidopsis lines indicates some appear to synthesize a polyisoprene, a rubber-like molecule, and the molecular weight is being investigated.
In related work, two of the above three proteins (CPT and CBP) were recombinantly produced in bacteria (Escherichia coli), affinity chromatography purified, and found to be active in solution. The same two proteins from the Hevea rubber tree were recombinantly produced and purified by research collaborators at Reno, Nevada, and found to be active as well. ARS researchers are testing these proteins under various conditions to optimize activity, including in the presence of liposomes produced by ARS researchers at Peoria, Illinois. The ex vivo activity confirms CPT and CBP are two minimum proteins needed for rubber synthesis, conditional to the presence of reaction additives necessary for synthesis of high-quality rubber (i.e. high molecular weight rubber). These experiments are helping define the fundamental mechanisms by which plants produce rubber.
Plants produce and store natural rubber in rubber particles (RPs). These tiny structures are analogous to lipid droplets (LDs); both are produced by plants from the cell’s endoplasmic reticulum, although the major components enclosed in the phospholipid monolayer structure of LDs are triacylglycerols (TAG) oils, not polyisoprene. Both RPs and LDs evolved to store insoluble plant products safely inside plant cells. Both RPs and LDs share many common membrane proteins which play important roles in controlling the size and stabilizing RP or LD. One important such protein is SEIPIN which determines the size of LDs. Surprisingly, ARS researchers found that guayule genetically modified for high expression of SEIPIN1 had smaller rubber particle size. These results helped them understand the fundamentals of how rubber particles are formed.
ARS researchers also made progress on Sub-objective 1B: "Increase natural rubber yield in guayule through controlled expression of transcription factors related to plant stress response". Rubber synthesis in guayule is highly upregulated by cold, drought, wounding, and other stresses in its natural environment or under controlled conditions. 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), regulate many stress responsive genes, and a guayule DREB1D (PaDREB1D) was highly induced in cold-treated stem tissue where active rubber synthesis and accumulation occurred. ARS scientists have characterized PaDREB1D in guayule and transgenic Arabidopsis. Spatial gene expression profiling of PaDREB1D revealed that guayule stems had the highest expression level among different organs examined, suggesting an important role of PaDREB1D in stem tissues, where rubber is made. Under cold or freezing temperatures, PaDREB-1D significantly increased its expression in stems, and other tissues: peduncle, and root, followed by leaf and flower. Sequence analysis revealed that PaDRED1D contains DNA-binding domains responsible for regulating cold-responsive (COR) gene transcription. ARS researchers studied the function of PaDREB1D by transferring PaDREB1D into a model plant, Arabidopsis. Transgenic Arabidopsis constitutively expressing PaDREB1D turned on expression of a set of Arabidopsis COR genes under both room temperature (24 degrees C) and cold (4 degrees C), whereas wild-type Arabidopsis expressed these COR genes only upon cold treatment. The transgenic plants also exhibited enhanced freezing tolerance under freezing temperature at -5 degrees C, showing a survival rate of 88–98 percent compared with that of wild-type at 0 percent. ARS researchers demonstrated that PaDREB1D is a functional member of the guayule DREB gene family and plays a critical role not only in stem function, but also in cold and freeze tolerance in whole guayule plants.
Additional insights on how stress affects rubber production in guayule were published. When guayule plants were wounded and/or heat-treated under controlled laboratory conditions, the highest rubber and resin production was found for plants with both cold + wounding stress. Some stress hormones responded to cold, some to stress, but only a few to the combination. Combining these results with the gene expression studies above will help to connect molecular signaling pathways in guayule. Separately, the first results from a new field study, in collaboration with ARS scientists in Parlier, California, detected higher rubber and resin production in guayule cultivated under poor soil and water quality stress. Guayule has potential to be grown in irrigation sediment soils such as those found in the western San Joaquin Valley of California and may product higher concentration of rubber under these stressful cultivation conditions.
In related research, through a Community Science Project (CSP) collaboration with the Joint Genome Institute (JGI) in Berkeley, California, Boyce Thompson Institute, USDA, and Bridgestone Americas, first results from genome sequencing of a trio of important guayule lines have been completed. A chromosome-level sequence assembly had been prepared for the guayule diploid line Cal-3.
In Sub-objective 1C: "Apply CRISPR/Cas9 technology to improve rubber yield in guayule", 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. In FY23, progress was made for specific molecular design of the necessary components.
In 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", ARS researchers evaluated heat-treated rice hulls as rubber compound fillers. The high silica content of rice hulls can create challenges for bioproducts, but in rubber compounds was an advantage. Chemical coupling between heat-treated rice hulls and tire rubber reduced compound hysteresis, and important indicator for fuel economy when used in tread compounds. Results have been published.
In Sub-objective 3C: "Develop bio-based antioxidants for stabilization of natural rubber and resin", ARS researchers completed evaluation and analysis of transgenic guayule plants overexpressing tocopherol (vitamin E, a potent antioxidant molecule). Most transgenic lines produce more tocopherols compared to the non-transformed guayule, however under the conditions tested the protective effect on rubber quality in plants stems was marginal. Surprisingly, natural rubber and resin production in transgenic lines was significantly lower than non-transformed guayule controls. This suggests that the carbon metabolizing isoprenoid pathway in plastids utilized for tocopherols biosynthesis may also be involved in rubber biosynthesis. Rubber is produced from carbon in the plant cytosol (not plastids); this is the first evidence of a role for carbon from the plastids, that will inform future metabolic engineering efforts.
In Sub-objective 3C: “Develop bio-based antioxidants for stabilization of natural rubber and resin", stakeholder interest in biobased and biodegradable rubber compounds continued to grow in FY23. ARS researchers made progress in identifying safer additives to prevent ozone degradation of rubber compounds. ARS scientists also published computational models to elucidate the mechanism for ozone attack on rubber, and devised strategies for biobased or biodegradable compounds that are safer for the environment. These compounds may impart ozone resistance to tire compounds. Candidate compounds have been prepared and are under evaluation.
Accomplishments
1. Multi-gene transformation demonstrated in guayule. Genetic engineering of guayule is one important tool for crop improvement. Introduction of a new trait, or modification of an existing one, may require insertion or alteration of several genes or genetic elements. ARS researchers in Albany, California, constructed a DNA cassette for guayule Agrobacteria-mediated genomic transformation consisting of four tocopherol biosynthesis genes, each driven by a unique promoter sequence necessary for in vivo expression. All four genes were inserted in the genome and properly expressed. This is the first evidence a large cassette of DNA can successfully be inserted in guayule, a species known to be challenging for transformation. This critical technical achievement is important for (1) possible insertion of an entire metabolic pathway, and (2) for CRISPR/Cas9 editing efforts in guayule, to produce edited plants free of foreign DNA.
2. Alternative rubber compound additives. Tire manufacturers are increasingly pursuing biobased materials including natural rubber, biobased fillers, and safer chemical additives. ARS scientists in Albany, California, have responded to environmental concerns for one specific additive, a p-phenylene diamine (PPD) derivative that forms a toxic quinone (PPDQ) lethal to aquatic species. Computational studies were performed and published, with the University of California, Berkeley, to elucidate the mechanism for ozone reactions. Next, green strategies were devised for alternative compounds that are safer for the environment. Candidate compounds have been prepared and are under evaluation.
Review Publications
Torres, L.F., McCaffrey, Z., Williams, T.G., Wood, D.F., Orts, W.J., McMahan, C.M. 2023. Evidence of silane coupling in torrefied agro-industrial residue-filled poly(styrene-co-butadiene) rubber compounds. Journal of Applied Polymer Science. 140(12). Article e53646. https://doi.org/10.1002/app.53646.
Chen, G.Q., Ponciano, G.P., Dong, C., Dong, N., Johnson, K., Bolton, T.T., Williams, T.G., Wood, D.F., Placido, D.F., McMahan, C.M., Dyer, J.M. 2023. Overexpressing an Arabidopsis SEIPIN1 reduces rubber particle size in guayule. Industrial Crops and Products. 195. Article 116410. https://doi.org/10.1016/j.indcrop.2023.116410.
Placido, D., McMahan, C.M., Lee, C.C. 2022. Wounding and cold stress increase resin and rubber production of Parthenium argentatum cultivar G711. Industrial Crops and Products. 193. Article 116174. https://doi.org/10.1016/j.indcrop.2022.116174.
Rossomme, E.C., Hart-Cooper, W.M., Orts, W.J., McMahan, C.M., Head-Gordon, M. 2023. Computational studies of rubber ozonation explain the effectiveness of 6PPD as an antidegradant and the mechanism of its quinone formation. Environmental Science and Technology. 57(13):5216-5230. https://doi.org/10.1021/acs.est.2c08717.
Banuelos, G.S., Placido, D.F., Zhu, H., Centofanti, T., Zambrano, M., Heinitz, C.C., Lone, T.A., McMahan, C.M. 2022. Guayule as an alternative crop for natural rubber production grown in B- and Se-laden soil in Central California. Industrial Crops and Products. 189. Article 115799. https://doi.org/10.1016/j.indcrop.2022.115799.
