Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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 (ÿ-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. 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. Artificial Intelligence (AI)/Machine Learning (ML) Machine learning was used to advance research in both 1- the plant genomics and 2- rubber additive chemistry aspects of the project in FY24. In plant genomics, artificial intelligence, and machine learning methods were used in this project to analyze guayule genome and transcriptome data, facilitating mechanistic studies of regulatory control of rubber biosynthesis and advance guayule genomic resources. Methods used include both unsupervised ML for dimensionality reduction, such as principal component analysis for sample clustering and deep learning system, such as AlphaFold 3 to predict structure models of the rubber transferase components/complex. Various AI tools deployed on SCINET high-performance computing clusters benefit the project by allowing high-throughput computation of the high dimensional and complex genomic sequence analysis. In rubber additive chemistry, classical machine learning models were used to relate chemometric data (orbital energies, ionization potentials, etc.) to experimental reactivities of molecules toward ozone. Chemometric data were computed using SCINet (Ceres), while models were trained using local hardware. This work is expected to enable high-throughput screening of novel structures for use as rubber antidegradants. ACCOMPLISHMENTS 01 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.
Impacts
(N/A)
Publications
- 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.
- 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.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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 (ÿ-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. 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. Artificial Intelligence (AI)/Machine Learning (ML) Artificial intelligence and machine learning methods were used for this project to aid development of sustainable and non-toxic rubber anti- degradants to replace existing commercial products. Methods used include multiple linear and partial least squares regression techniques. Open- source Python programming libraries such as Pandas and Scikit-Learn were used in conjunction with the Jupyter notebook environment, deployed on ARS workstations as well as the Ceres supercomputing cluster. These methods have benefited the project by allowing for high-throughput computational screening of proposed antidegradants, allowing for more efficient experimental processes. ACCOMPLISHMENTS 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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 (ÿ-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 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 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/20 to 09/30/21
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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 (�-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. In support of Sub-objective 1A, natural rubber is synthesized by at least a cis-prenyl transferase (CPT) and a cis-prenyl transferase binding- protein (CBP). A (guayule homologue of) the small rubber particle (RP) protein (SRPP) may function in stabilization and coagulation of rubber molecules in RP. We constructed a conventional transformation vector carrying three guayule genes, CPT3, CBP and SRPP to evaluate their effect for engineering rubber production in guayule. Progress was also made in Sub-objective 1B. 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, DREBs (dehydration responsive element binding proteins) 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 in Albany, California, have successfully cloned and constructed PaDREB1D in a binary vector for studying the function of PaDREB1D. The success of this project allows the ability of transgenic guayule lines overexpressing the PaDREB1D to be engineered for increased rubber synthesis in the absence of a cold signal. In support of Sub-objective 1C, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was applied to improve rubber yield in guayule, and progress was made toward designing constructs for gene editing of guayule. In collaboration with the Joint Genome Institute, a draft genomic DNA assembly was prepared, providing some of the critical plant DNA sequences needed for precise editing. In subordinate project 2030-21410-022-01R, Sustainable Bioeconomy for Arid Regions, guayule plants were transformed to downregulate genes that promote flowering. It is possible that plants with fewer flowers may produce more rubber, or have other desirable traits. Evaluation of the plants is continuing, Progress was also made on Sub-objective 3A. Natural rubber composites were prepared using torrefied biobased fillers as full or partial replacements for carbon black, the conventional petroleum-based filler. Moderate levels of biobased fillers could be used without compromising the material performance. To address Sub-objective 3B, a series of experimental compounds were prepared. The antioxidant performance of meat by-product extracts was evaluated. Record of Any Impact of Maximized Teleworking Requirement: The continued maximized telework/limited laboratory access requirements had more impact in fiscal year (FY) 2021 than in FY20. During FY20 some publications were prepared and a lot of training took place. But by FY21 the impact of training, especially for laboratory-related activities crucial to support staff, was less effective since there were little to no opportunities to put the training into practice. For our CRIS, the research must take place at the bench, and the lack of access and continuity had a negative impact on progress toward Project Objectives and Milestones. Impactful research is performed in cross-disciplinary teams. While existing collaborations can be fostered, for a while, with maximized telework, new collaborations cannot be readily established. Mentorship can take place under maximized telework only when both parties are committed. We now have zero interns for the first time in 20+ years, which negatively affects the morale of staff in place and the future of ARS. ACCOMPLISHMENTS 01 Genomic resources for guayule. Modern breeding techniques for the natural rubber-producing crop, Parthenium argentatum (guayule), require background information characterizing the plant DNA. In collaboration with the Joint Genome Institute, a draft guayule DNA assembly was successfully developed from a vigorous hybrid line used in commercial crop development. In addition, ARS researchers in Albany, California, measured the differences in gene expression between high and low rubber- producing field plants by RNA sequencing. The resulting RNA assembly, or transcriptome, highlights which genes are expressed driving production of rubber, resins, and carbohydrates in guayule. The two new databases featuring the DNA and RNA assembled sequences are now available to researchers and commercial developers worldwide, linked and hosted by ARS GrainGenes servers.
Impacts
(N/A)
Publications
- Torres, L.F., McCaffrey, Z., Washington, W., Williams, T.G., Wood, D.F., Orts, W.J., McMahan, C.M. 2021. Torrefied agro-industrial residue as filler in natural rubber compounds. Journal of Applied Polymer Science. 138(28). Article e50684. https://doi.org/10.1002/app.50684.
- Cornish, K., Dacosta, B., McMahan, C.M. 2020. Temporal analysis of natural rubber transferases reveals intrinsic distinctions for in vitro synthesis in two rubber-producing species. Current Topics in Biochemical Research. 21:45-58.
- Chen, G.Q., Kim, W., Johnson, K., Park, M., Lee, K., Kim, H. 2021. Transcriptome analysis and identification of lipid genes in Physaria lindheimeri, a genetic resource for hydroxy fatty acids in seed oil. International Journal of Molecular Sciences. 22(2). Article 514. https:// doi.org/10.3390/ijms22020514.
- Chen, G.Q., Johnson, K., Nazarenus, T.J., Ponciano, G.P., Morales, E., Cahoon, E.B. 2021. Genetic engineering of lesquerella with increased ricinoleic acid content in seed oil. Plants. 10(6). Article 1093. https:// doi.org/10.3390/plants10061093.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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 (�-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. 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 01 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.
Impacts
(N/A)
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.
- 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.
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