Katrina Cornish

Alternative sources of natural rubber

Abstract Rubber (cis-1,4-polyisoprene) is one of the most important polymers naturally produced by plants because it is a strategic raw material used in more than 40,000 products, including more than 400 medical devices. The sole commercial source, at present, is natural rubber harvested from the Brazilian rubber tree, Hevea brasiliensis. Primarily due to its molecular structure and high molecular weight (>1 million daltons) this rubber has high performance properties that cannot easily be mimicked by artificially produced polymers, such as those derived from, e.g., bacterial poly-hydroxy-alkanoates (PHAs). These high performance properties include resilience, elasticity, abrasion resistance, efficient heat dispersion (minimizing heat build-up under friction), and impact resistance. Medical rubber gloves need to fit well, be break-resistant, allow the wearer to retain fine tactile sensation, and provide an effective barrier against pathogens. The sum of all these characteristics cannot yet be achieved using synthetic gloves. The lack of biodiversity in natural rubber production renders continuity of supply insecure, because of the risk of crop failure, diminishing acreage, and other disadvantages outlined below. A search for alternative sources of natural rubber production has already resulted in a large number of interesting plants and prospects for immediate industrial exploitation of guayule (Parthenium argentatum) as a source of high quality latex. Metabolic engineering will permit the production of new crops designed to accumulate new types of valued isoprenoid metabolites, such as rubber and carotenoids, and new combinations extractable from the same crop. Currently, experiments are underway to genetically improve guayule rubber production strains in both quantitative and qualitative respects. Since the choice for gene activities to be introduced or changed is under debate, we have set up a complementary approach to guayule with yeast species, which may more quickly show the applicability and relevance of genes selected. Although economic considerations may prevent commercial exploitation of new rubber-producing microorganisms, transgenic yeasts and bacteria may yield intermediate or alternative (poly-)isoprenes suitable for specific applications.

Similarities and differences in rubber biochemistry among plant species.

This report reviews aspects of the biochemical regulation of rubber yield and rubber quality in three contrasting rubber-producing species, Hevea brasiliensis, Parthenium argentatum and Ficus elastica. Although many similarities are revealed, considerable differences also exist in enzymatic mechanisms regulating biosynthetic rate and the molecular weight of the rubber biopolymers produced. In all three species, rubber molecule initiation, biosynthetic rate and molecular weight, in vitro, are dependent upon substrate concentration and the ratio of isopentenyl pyrophosphate (IPP, the elongation substrate, or monomer) and farnesyl pyrophosphate (FPP, an initiator), but these parameters are affected by intrinsic properties of the rubber transferases as well. All three rubber transferases are capable of producing a wide range of rubber molecular weight, depending upon substrate concentration, clearly demonstrating that the transferases are not the prime determinants of product size in vivo. However, despite these commonalities, considerable differences exist between the species with respect to cosubstrate effects, binding constants, effective concentration ranges, and the role of negative cooperativity in vitro. The P. argentatum rubber transferase appears to exert more control over the molecular weight it produces than the other two species and may, therefore, provide the best prospect for the source of genes for transformation of annual crop species. The kinetic data, from the three contrasting rubber-producing species, also were used to develop a model of the rubber transferase active site in which, in addition to separate IPP and allylic-PP binding sites, there exists a hydrophobic region that interacts with the linear portion of allylic-PP initiator proximal to the pyrophosphate. Substrate affinity increases until the active site is traversed and the rubber interior of the rubber particle is reached. The kinetic data suggest that the hydrophobic region in H. brasiliensis and F. elastica is about 1.8 nm long but only 1.3 nm in P. argentatum. The estimates are supported by measurements of the rubber particle monolayer membrane using electron paramagnetic resonance spectroscopy.

Rubber transferase activity in rubber particles of guayule

Abstract Rubber transferase (RuT) activity, measured as incorporation of radiolabelled isoprene from [ 14 C]isopentenyl pyrophosphate (IPP) into rubber, was assayed in suspensions of washed rubber particles (WRPs) purified from stembark tissue of Parthenium argentatum . Isolated WRPs had high RuT activity which was not diminished even after repeated washing, demonstrating the firm association of the enzyme with the particles. The activity of RuT was characterized with respect to substrate and WRP concentration. The rate of IPP-incorporation was dependent upon the concentration of two substrates, IPP and the allylic pyrophosphate starter molecule E , E -farnesyl pyrophosphate (FPP). The enzyme present in 6 × 10 10 WRPs per ml was saturated by 1 mM IPP and 20 μM FPP. Under saturating cosubstrate concentrations the apparent K m of RuT was ca 300 μM IPP and 3 μM FPP. Analysis of WRPs by SDS-PAGE revealed a simple protein profile characteristic of guayule rubber particles. A successful and facile assay for IPP-polymerization by isolated rubber particles is described.

Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous, monolayer biomembrane

Abstract. The physical characteristics of rubber particles from the four rubber (cis-1,4-polyisoprene) producing species Euphorbia lactiflua Phil., Ficus elastica Roxb., Hevea brasiliensis Müll. Arg., and Parthenium argentatum Gray, were investigated using transmission electron microscopy (TEM) and electron-paramagnetic-resonance (EPR) spin labeling spectroscopy. Transmission electron microscopy showed the rubber particles to be composed of a spherical, homogeneous, core of rubber enclosed by a contiguous, electron-dense, single-track surface layer. The biochemical composition of the surface layer and its single-track TEM suggested that a monolayer biomembrane was the surface structure most compatible with the hydrophobic rubber core. The EPR spectra for a series of positional isomers of doxyl stearic acid, used to label the surface layer of the rubber particles, exhibited flexibility gradients and evidence for lipid-protein interactions for all four rubber particle types that is consistent with a biomembrane-like surface. The EPR spectra confirmed that the surface biomembrane is a monolayer. Thus, rubber particles appear similar to oil bodies in their basic architecture. The EPR spectra also provided information on protein location and degree of biomembrane penetration that correlated with the known properties of the rubber-particle-bound proteins. The monolayer biomembrane serves as an interface between the hydrophobic rubber interior and the aqueous cytosol and prevents aggregation of the particles. An unexpected observation for the probes in pure polyisoprene was evidence of an intrinsic flexibility gradient associated with the stearic acid molecule itself.

Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts.

Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into the tobacco chloroplast genome. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.

Hypoallergenicity of guayule rubber particle proteins compared to Hevea latex proteins

Abstract Hevea brasiliensis Muell. Arg. is currently the sole commercial source of natural rubber. However, products made from Hevea latex are responsible for causing allergies affecting an increasing number of people world-wide. In this paper we test the hypothesis that latex allergy can be circumvented by using rubber of low hypoallergenicity from other species. We also report studies to determine the feasibility of ameliorating latex allergy in Hevea. We show that antibodies raised against proteins extracted from films of ammoniated Hevea latex did not recognize latex proteins from two other rubber producing plant species, guayule (Parthenium argentatum Gray) and Ficus elastica Roxb., indicating that Hevea latex allergy can be circumvented using rubber from other species. The hypoallergenicity of guayule and F. elastica latex was confirmed in preliminary clinical trials (reported elsewhere). F. elastica rubber is mostly of low molecular weight and short chain, whereas guayule rubber is comparable in quality to that of Hevea. Guayule latex should be suitable for the manufacture of high-quality, hypoallergenic natural rubber products for the Hevea-hyperallergic individual. The Hevea latex protein antibodies recognized not only proteins from raw Hevea latex, but also rubber particle preparations from which the soluble latex proteins were removed. Hence extensive removal of rubber particle-bound proteins, in addition to the soluble latex proteins, would be required to produce a safe Hevea latex product. Latex samples from different commercial lines of H. brasiliensis reacted similarly to the antibodies, indicating that clonal selection is unlikely to be helpful in eliminating latex antigens.

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

BackgroundParthenium argentatum (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 P. argentatum 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, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines.ResultsThe complete plastid genome of P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum.ConclusionThe genome sequence of the P. argentatum 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 P. argentatum that are important for producing latex.

A protein from Ficus elastica rubber particles is related to proteins from Hevea brasiliensis and Parthenium argentatum

Abstract Rubber particles, having high rubber transferase (RuT) activity, were isolated from Ficus elastica latex. The predominant protein in these rubber particles is a large hydrophobic glycoprotein (termed LPR for large protein from rubber particles), with an apparent monomeric M r of 376 000 on SDS-PAGE. A M r of 750 000 was determined with native PAGE, indicating that LPR probably exists as a dimer. Antibodies raised to purified LPR recognized blotted intact F. elastica rubber particles, as well as LPR, demonstrating that LPR is situated at or near the surface of the particles. Anti-LPR antibodies also recognized enzymatically active rubber particles and rubber particle proteins from two other rubber-producing plants, Hevea brasiliensis and Parthenium argentatum . This indicates that the rubber particles of all three rubber-producing species contain proteins with common antigenic determinants. Large proteins, similar in size to LPR, were found in preparations of rubber particles from H. brasiliensis and P. argentatum . These results are the first demonstration that rubber particles from different species contain similar proteins that may share common functions in rubber biosynthesis and/or rubber particle structure.

Microstructure of Purified Rubber Particles

Purified rubber particles from Hevea brasiliensis (Brazilian rubber tree), Parthenium argentatum (guayule), Ficus elastica (Indian rubber tree), and Euphorbia lactiflua were examined and compared using conventional scanning electron microscopy (SEM), field‐emission SEM, cryo‐SEM, and transmission electron microscopy (TEM). Rubber particles of all four species were spherical; they varied in size and had a uniform homogeneous material, the rubber core, surrounded by a contiguous monolayer (half‐unit) membrane. Frozen‐hydrated and/or untreated particles from H. brasiliensis and P. argentatum deformed and fused readily, whereas those from F. elastica and E. lactiflua retained their spherical shapes. These results indicate that the surface components of the H. brasiliensis and P. argentatum particles are more fluid than those of F. elastica or E. lactiflua. When fixed in aldehyde, F. elastica particles retained their spherical exterior shapes but had hollow centers, whereas H. brasiliensis and P. argentatum particles completely collapsed. In aldehyde–osmium tetroxide–fixed material, the rubber core of F. elastica was poorly preserved in some particles in which only a small amount of the rubber core remained adhering to the monolayer membrane, leaving a hollow center. Euphorbia lactiflua particles were well preserved in terms of retaining the rubber core; however, the membrane was not as easily discernible as it was in the other three species. Both H. brasiliensis and P. argentatum were well preserved following fixation; their cores remained filled with rubber, and their monolayer membranes were defined. The addition of potassium permanganate to the fixation‐staining regime resulted in higher‐contrast micrographs and more well defined monolayer membranes.

Metabolism of Abscisic Acid

The involvement of abscisic acid (ABA) in many physiological processes has been suggested [1, 36], but in most cases the role of ABA has not been established unequivocally. The main reason for this uncertainty is that it is not possible at present to grow plants devoid of ABA, since no ABA-less mutants are available and no specific inhibitors of ABA biosynthesis have been found. Nevertheless, it is clear that ABA plays a role in mediating responses to water stress. For example, application of ABA to turgid leaves causes rapid closure of stomata, and levels of ABA in leaves of mesophytic plants increase many times under conditions of water stress. In ABA-deficient mutants (wilty or droopy) applied ABA causes stomatal closure, but the ABA content of these mutants is not increased by water stress. Another phenomenon involving ABA is seed maturation. Observations made on ABA-deficient mutants of various species indicate that precocious germination of developing embryos (vivipary) is prevented by ABA [16, 18].