Effect of Non-Rubber Constituents on Guayule and Hevea Rubber Intrinsic Properties

To meet the increasing demand for natural rubber (NR), currently sourced from the tropical rubber tree Hevea brasiliensis, and address price volatility and steadily increasing labor costs, alternate rubber-producing species are in commercial development. One of these, guayule (Parthenium argentatum), has emerged on the market as a commercial source of high quality rubber. Non-rubber constituents play an important role in the physical properties of NR products. The intrinsic composition of the two NR materials differs and these differences may be a principal cause of the performance differences between them.We have compared the effect of non-rubber constituents, such as protein, lipids, resin and rubber particle membranes. Firstly, a film casting method was developed to obtain rubber films with a uniform thickness. Secondly, the glass transition temperature of different rubbers was determined by dynamic mechanical analysis, and tensile properties were tested for uncompounded materials. Guayule natural rubber (GNR), from which most of the membranes were removed while in latex form (MRGNR) was found to have higher intrinsic strength than GNR or gel-free NR (FNR). An acetone extraction was performed to quantify the resin and free lipids in the rubber samples

Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

<p>Abstract</p> <p>Background</p> <p><it>Parthenium argentatum </it>(guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of <it>P. argentatum </it>was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, <it>Lactuca sativa, Guitozia abyssinica </it>and <it>Helianthus annuus </it>revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of <it>Parthenium </it>species and lines.</p> <p>Results</p> <p>The complete plastid genome of <it>P. argentatum </it>is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in <it>L. sativa, G. abyssinica </it>and <it>H. annuus</it>, we demonstrate that the <it>P. argentatum </it>chloroplast genome sequence is most closely related to that of <it>H. annuus</it>. Similar to chloroplast genomes in <it>G. abyssinica, L. sativa </it>and <it>H. annuus</it>, the plastid genome of <it>P. argentatum </it>has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the <it>mat</it>K and <it>psb</it>A-<it>trn</it>H spacer chloroplast DNA barcodes, three of the four <it>Parthenium </it>species tested, <it>P. tomentosum</it>, <it>P. hysterophorus </it>and P. <it>schottii</it>, can be differentiated from <it>P. argentatum</it>. In addition, we identified lines within <it>P. argentatum</it>.</p> <p>Conclusion</p> <p>The genome sequence of the <it>P. argentatum </it>chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of <it>P</it>. <it>argentatum </it>that are important for producing latex.</p

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes

Abstract 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 (N50 =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 (Satellite Targeted) 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. Keywords: Natural rubber, Genome, Assembly, Annotation, Class II transposable element, Non-autonomous, Transposo

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in \u3ci\u3eParthenium argentatum\u3c/i\u3e

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

Unraveling the Mystery of Natural Rubber Biosynthesis

Monitoring the growth of in vitro natural rubber was accomplished by high resolution size exclusion chromatography, SEC. Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphate monomer in the presence of farnesyl pyrophosphate initiator. The high-resolution SEC was able to detect the formation of new rubber. Changes in the low molecular weight fraction were also detected. Gravimetric analysis revealed ∼30% mass gain after the in vitro synthesis. The overall gel content was found to be reduced, which further supported the formation of new rubber. This is the first report that utilizes high-resolution SEC to monitor the in vitro NR growth without the use of radiolabeling

Computational studies of rubber ozonation explain the effectiveness of 6PPD as an antidegradant and the mechanism of its quinone formation

The discovery that the commercial rubber antidegradant 6PPD reacts with ozone (\ce{O3}) to produce a highly toxic quinone (6PPDQ) spurred a significant research effort into non-toxic alternatives. Identification of non-toxic alternatives has been hampered by lack of a detailed understanding of the mechanism of protection that 6PPD affords rubber compounds against ozone. Herein, we report high-level density functional theory studies into early steps of rubber and PPD ($p$-phenylenediamine) ozonation, identifying key steps that contribute to the antiozonant activity of PPDs. In this, we establish that our density functional theory approach can achieve chemical accuracy for many ozonation reactions, which are notoriously difficult to model. Using adiabatic energy decomposition analysis, we examine and dispel the notion that one-electron charge transfer initiates ozonation in these systems, as is sometimes argued. Instead, we find direct interaction between \ce{O3} and PPD carbon atoms is kinetically accessible, and that this motif is more significant than interactions with PPD nitrogens. The former pathway results in a hydroxylated PPD intermediate, which reacts further with \ce{O3} to afford 6PPD hydroquinone and, ultimately, 6PPDQ. This mechanism directly links the toxicity of 6PPDQ to the antiozonant function of 6PPD. These results have significant implications for development of alternative antiozonants, which are discussed

Computational Studies of Rubber Ozonation Explain the Effectiveness of 6PPD as an Antidegradant and the Mechanism of Its Quinone Formation

The discovery that the commercial rubber antidegradant 6PPD reacts with ozone (O3) to produce a highly toxic quinone (6PPDQ) spurred a significant research effort into nontoxic alternatives. This work has been hampered by lack of a detailed understanding of the mechanism of protection that 6PPD affords rubber compounds against ozone. Herein, we report high-level density functional theory studies into early steps of rubber and PPD (p-phenylenediamine) ozonation, identifying key steps that contribute to the antiozonant activity of PPDs. In this, we establish that our density functional theory approach can achieve chemical accuracy for many ozonation reactions, which are notoriously difficult to model. Using adiabatic energy decomposition analysis, we examine and dispel the notion that one-electron charge transfer initiates ozonation in these systems, as is sometimes argued. Instead, we find direct interaction between O3 and the PPD aromatic ring is kinetically accessible and that this motif is more significant than interactions with PPD nitrogens. The former pathway results in a hydroxylated PPD intermediate, which reacts further with O3 to afford 6PPD hydroquinone and, ultimately, 6PPDQ. This mechanism directly links the toxicity of 6PPDQ to the antiozonant function of 6PPD. These results have significant implications for development of alternative antiozonants, which are discussed

Transcriptome and gene expression analysis in cold-acclimated guayule (\u3ci\u3eParthenium argentatum\u3c/i\u3e) rubber-producing tissue

Natural rubber biosynthesis in guayule (Parthenium argentatum Gray) is associated with moderately cold night temperatures. To begin to dissect the molecular events triggered by cold temperatures that govern rubber synthesis induction in guayule, the transcriptome of bark tissue, where rubber is produced, was investigated. A total of 11,748 quality expressed sequence tags (ESTs) were obtained. The vast majority of ESTs encoded proteins that are similar to stress-related proteins, whereas those encoding rubber biosynthesis- related proteins comprised just over one percent of the ESTs. Sequence information derived from the ESTs was used to design primers for quantitative analysis of the expression of genes that encode selected enzymes and proteins with potential impact on rubber biosynthesis in field-grown guayule plants, including 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, small rubber particle protein, allene oxide synthase, and cis-prenyl transferase. Gene expression was studied for field-grown plants during the normal course of seasonal variation in temperature (monthly average maximum 41.7 °C to minimum 0 °C, from November 2005 through March 2007) and rubber transferase enzymatic activity was also evaluated. Levels of gene expression did not correlate with air temperatures nor with rubber transferase activity. Interestingly, a sudden increase in night temperature 10 days before harvest took place in advance of the highest CPT gene expression level

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in \u3ci\u3eParthenium argentatum\u3c/i\u3e

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

Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule.

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