Plants produce natural rubber in specialized organelles, termed rubber particles (RP). The structure of the RP consists of a polyisoprene (rubber) core surrounded by a shell layer composed of a lipid monolayer and proteins. Among the prominent RP-associated proteins is small rubber particle protein (SRPP), the second most abundant protein on the Hevea brasiliensis rubber particle monolayer membrane. In this study, a combination of protein modeling, biophysical analysis, biochemistry, and transmission electron microscopy have been utilized to further characterize Hevea SRPP1 (HbSRPP1) membrane architectural rearrangement capabilities. Results obtained reveal that HbSRPP1 possesses characteristics reminiscent of apolipoproteins, including the ability to reorganize aqueous phospholipid dispersions into stable, disk-shaped nanoparticles, termed nanodisks. This research provides the groundwork for further studies into the mechanism of SRPP's interaction with the RP membrane surface which likely involves conformational changes in RP topology.

Rubber dandelion Taraxacum kok-saghyz (Tk) has been developed as an alternative crop to diversify natural rubber production. Germplasm utilization and biotechnology are potential approaches to developing commercially viable Tk crops. We surveyed a Tk germplasm (accession W6-35169, plant name KAZ08-014, L. E. Rodin) and found high variations in its plant morphology, in vitro callus and shoot induction, and self-incompatibility. We identified plant #12, which was competent in regenerating shoots and producing selfed seeds. This individual is particularly useful in basic and applied research to improve a Tk crop.

Guayule (Parthenium argentatum) is a rubber producing plant. Genetic engineering of guayule to improve natural rubber content requires the use of promoters functional in stem tissues where most of guayule natural rubber is produced. We isolated a promoter region from a gene coding the Small Rubber Particle Protein 1. Transgenic guayule lines expressing the Small Rubber Particle Protein 1 promoter fused to β-glucuronidase reporter gene were developed. The promoter, active in leaf, stem and root tissues drives significant levels of transgene expression, especially in the stem tissue. The isolated Small Rubber Particle Protein 1 promoter is a new molecular element in the toolbox available for guayule improvement through genetic engineering strategies.

The drought-adapted shrub guayule (Parthenium argentatum) produces rubber, a natural product of major commercial importance, and two co-products with potential industrial use: terpene resin and the carbohydrate fructan. The rubber content of guayule plants subjected to water stress is higher compared to that of well-irrigated plants, a fact consistently reported in guayule field evaluations. To better understand how drought influences rubber biosynthesis at the molecular level, a comprehensive transcriptome database was built from drought-stressed guayule stem tissues using de novo RNA-seq and genome-guided assembly, followed by annotation and expression analysis. Despite having higher rubber content, most rubber biosynthesis related genes were down-regulated in drought-stressed guayule, compared to well-irrigated plants, suggesting post-transcriptional effects may regulate drought-induced rubber accumulation. On the other hand, terpene resin biosynthesis genes were unevenly affected by water stress, implying unique environmental influences over transcriptional control of different terpene compounds or classes. Finally, drought induced expression of fructan catabolism genes in guayule and significantly suppressed these fructan biosynthesis genes. It appears then, that in guayule cultivation, irrigation levels might be calibrated in such a regime to enable tunable accumulation of rubber, resin and fructan.

Guayule (Parthenium argentatum A. Gray) is a promising alternative for the sustainable production of natural rubber. In addition to rubber, guayule produces a wide range of secondary metabolites, such as resin, essential oils, and phenolic compounds, with a variety of potential applications in fields as diverse as biopesticides, biomedicine, cosmetics, and other industries. However, the dynamics of these metabolites in different accessions of guayule are not known in detail. Therefore, in the present study, a comprehensive multivariate analysis was performed with the data obtained by the research group over five years (82 metabolites in 27 accessions grown under standardized conditions) to evaluate their genetic and metabolic variability. Advanced statistical tools such as Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), and Spearman correlation were used. These analyses revealed that resin and rubber content alone are insufficient for accession differentiation. In contrast, sesquiterpenes, particularly guayulins, appeared to be strong biomarkers for genetic classification, correlating strongly with rubber and resin biosynthetic pathways. In addition, guayulin A and guayulin C seem to have a high correlation with rubber. With respect to guayulins, this analysis also seems to indicate that their biosynthesis occurs in the leaves, starting with guayulin C and D, and that they are then translocated to the stem. These results suggest that a comprehensive metabolite profiling approach, focusing on specific compound groups, can improve guayule breeding strategies aimed at maximizing rubber yield and resin utilization.

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis.

A conventional source of natural rubber (NR) is from Hevea (Hevea brasiliensis). NR production from Hevea is limited by its narrow growth region and susceptibility to leaf blight disease. In response to these limitations, and to address NR supply and security, guayule (Parthenium argentatum Gray) has been developed as an alternative crop. NR consists mainly of cis-1,4-polyisoprene synthesized in/on rubber particles (RPs). RPs resemble lipid droplets (LDs) in that both are generated from endoplasmic reticulum (ER) and consist of hydrophobic cores and phospholipid monolayers. A prominent ER-related protein seipin plays an essential role in controlling LD biogenesis from the ER surface. Prior expression of AtSEIPIN1 in Arabidopsis increased LD size and consequently increased its core constituent triacylglycerol content in seed. To investigate if AtSEIPIN1 can similarly impact RPs and production of cis-1,4-polyisoprene, we generated three independent transgenic guayule lines overexpressing AtSEIPIN1 (SEIoe). Compared with wild-type, fewer RPs were observed, and RP size was significantly reduced in all SEIoe lines. Although not always statistically significant, NR quantity and quality were also reduced in SEIoe lines. Resin contents were reduced in mature stems, but not in young plantlets of SEIoe lines. The mechanisms of how AtSEIPIN1 influences RP biogenesis and NR production in guayule are discussed.

Production of natural rubber by Parthenium argentaum (guayule) requires increased yield for economic sustainability. An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by downregulation of squalene synthase (SQS), such that the pool of farnesyl diphosphate (FPP) substrate might instead be available to initiate natural rubber synthesis. Downregulation of SQS resulted in significantly reduced squalene and slightly increased rubber, but not in the same tissues nor to the same extent, partially due to an apparent negative feedback regulatory mechanism that downregulated mevalonate pathway isoprenoid production, presumably associated with excess geranyl pyrophosphate levels. A detailed metabolomics analysis of isoprenoid production in guayule revealed significant differences in metabolism in different tissues, including in active mevalonate and methylerythritol phosphate pathways in stem tissue, where rubber and squalene accumulate. New insights and strategies for engineering isoprenoid production in guayule were identified.

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants.

Guayule (Parthenium argentatum) is under development in the southwestern United States as a source of domestic natural rubber; increased rubber yield was found for greenhouse-grown guayule with downregulated allene oxide synthase (AOS). The objective of this study was to evaluate natural rubber production in guayule plants with varying levels of AOS gene expression grown in a field environment. Four plant genotypes: wildtype plants, vector controls, and transgenic plants with overexpressed and downregulated allene oxide synthase (total of 960 plants) were grown at the Bridgestone Guayule Research Farm in Eloy, Arizona between May 2016 to May 2018. Plant phenotypes were evaluated at 6-, 12-, 18-, and 24-months. Downregulated allene oxide synthase (AOSi) genotypes showed remarkable phenotypes including higher photosynthetic activity (net assimilation rate), total number of branches per plant, and plant stembark thickness compared with wildtype, vector control, and overexpressed AOS (AOSoe) lines. Moreover, the rubber particles were smaller and had higher activity (radiolabeled isoprenyl pyrophosphate (IPP) incorporation per g of rubber) in the AOSi genotypes than control and AOSoe lines. The downregulation of AOS also led to significant changes in phytohormone levels, which may have influenced the plants' morphology and physiology. However, in contrast to laboratory and greenhouse studies, natural rubber concentration and yield were not higher in AOSi plants.

We report functional genomics studies of a CYP74 rubber particle protein from Parthenium argentatum, commonly called guayule. Previously identified as an allene oxide synthase (AOS), this CYP74 constitutes the most abundant protein found in guayule rubber particles. Transgenic guayule lines with AOS gene expression down-regulated by RNAi (AOSi) exhibited strong phenotypes that included agricultural traits conducive to enhancing rubber yield. AOSi lines had higher leaf and stem biomass, thicker stembark tissues, increased stem branching and improved net photosynthetic rate. Importantly, the rubber content was significantly increased in AOSi lines compared to the wild-type (WT), vector control and AOS overexpressing (AOSoe) lines, when grown in controlled environments both in tissue-culture media and in greenhouse/growth chambers. Rubber particles from AOSi plants consistently had less AOS particle-associated protein, and lower activity (for conversion of 13-HPOT to allene oxide). Yet plants with downregulated AOS showed higher rubber transferase enzyme activity. The increase in biomass in AOSi lines was associated with not only increases in the rate of photosynthesis and non-photochemical quenching (NPQ), in the cold, but also in the content of the phytohormone SA, along with a decrease in JA, GAs, and ABA. The increase in biosynthetic activity and rubber content could further result from the negative regulation of AOS expression by high levels of salicylic acid in AOSi lines and when introduced exogenously. It is apparent that AOS in guayule plays a pivotal role in rubber production and plant growth.

Background Guayule (Parthenium argentatum Gray) is a drought tolerant, rubber producing perennial shrub native to northern Mexico and the US Southwest. Hevea brasiliensis, currently the world's only source of natural rubber, is grown as a monoculture, leaving it vulnerable to both biotic and abiotic stressors. Isolation of rubber from guayule occurs by mechanical harvesting of the entire plant. It has been reported that environmental conditions leading up to harvest have a profound impact on rubber yield. The link between rubber biosynthesis and drought, a common environmental condition in guayule's native habitat, is currently unclear. Results We took a transcriptomic and comparative genomic approach to determine how drought impacts rubber biosynthesis in guayule. We compared transcriptional profiles of stem tissue, the location of guayule rubber biosynthesis, collected from field-grown plants subjected to water-deficit (drought) and well-watered (control) conditions. Plants subjected to the imposed drought conditions displayed an increase in production of transcripts associated with defense responses and water homeostasis, and a decrease in transcripts associated with rubber biosynthesis. An evolutionary and comparative analysis of stress-response transcripts suggests that more anciently duplicated transcripts shared among the Asteraceae, rather than recently derived duplicates, are contributing to the drought response observed in guayule. In addition, we identified several deeply conserved long non-coding RNAs (lncRNAs) containing microRNA binding motifs. One lncRNA in particular, with origins at the base of Asteraceae, may be regulating the vegetative to reproductive transition observed in water-stressed guayule by acting as a miRNA sponge for miR166. Conclusions These data represent the first genomic analyses of how guayule responds to drought like conditions in agricultural production settings. We identified an inverse relationship between stress-responsive transcripts and those associated with precursor pathways to rubber biosynthesis suggesting a physiological trade-off between maintaining homeostasis and plant productivity. We also identify a number of regulators of abiotic responses, including transcription factors and lncRNAs, that are strong candidates for future projects aimed at modulating rubber biosynthesis under water-limiting conditions common to guayules' native production environment.

Parthenium argentatum (guayule) was transformed with a bicistronic transgene containing a viral 2A cleavage sequence. The transgene includes the coding sequences of two key enzymes of the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and farnesyl pyrophosphate synthase (FPPS), involved in rubber biosynthesis. The viral 2A peptide sequence located between the two transgenes allowed for their co-expression via the Arabidopsis CBF2 (C-Binding repeat Factor 2) cold-inducible promoter. We identified three independent transgenic lines expressing the bicistronic transgenes upon cold treatment and examined the rubber content in the in vitro guayule plants.

Background Guayule ( Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. Results The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N 50 = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar ( Sa tellite Tar geted) elements, which are non-autonomous Mu- like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. Conclusion The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

The natural rubber producing plant guayule (Parthenium argentatum Gray) stores carbohydrates mainly in the form of fructans, synthesized and stored in the same tissues at the same time as the rubber polymer, and a potential source of carbon for rubber biosynthesis. The first committed step to fructan synthesis is catalyzed by sucrose:sucrose-1-fructosyltransferase (1-SST), which was downregulated to test whether reduction of carbohydrate synthesis would divert carbon instead to rubber biosynthesis. Guayule leaf strips were transformed by Agrobacterium-mediated technology, and plants with downregulated 1-SST were evaluated in the laboratory and greenhouse. The plant tissue fructan concentration was reduced significantly, and sucrose concentration increased, especially in root tissues of greenhouse-grown plants. However, increased natural rubber production did not result.

Natural rubber biosynthesized via the isoprenoid pathway by domestic plant sources, such as guayule (Parthenium argentatum) may be more economically sustainable with improved yields, through breeding or targeted metabolic engineering of the isoprenoid pathway. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is considered a key regulatory enzyme of isoprenoid carbon flux in mammals, in microbial systems, and possibly in plants. The objective of our study was to modify isoprenoid production in guayule through overexpression of the HMGR gene.A transformation plasmid pND4-HMGR(tAN) was constructed with a modified binary vector and the HMGR gene from Aspergillus nidulans containing only the 465 amino acid catalytic domain driven by a constitutive promoter. Five independent transgenic lines were obtained via leaf disc Agrobacterium-mediated transformation. In the laboratory, the NR content of 2-month-old in vitro plantlets showed a 65% increase in rubber over the vector control for one line (HMGR6), and lower resin production for another (HMGR2). In field evaluations, the genetically modified HMGR6 line was differentiated from control lines in size, biomass, and plant morphology descriptors, but not in rubber or resin content. Remarkably, the survival rate of all HMGR-modified plants following pollarding harvest, was better than controls, with the highest survival rate for line HMGR6.In summary, we report the first genetic modification of guayule to overexpress the isoprenoid pathway enzyme HMGR. Survival during regrowth was significantly improved for HMGR overexpressing plants, suggesting enhanced carbon flux to important secondary isoprenoid metabolites, such as growth phytohormones.