Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues.

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.

Field to Greenhouse: How Stable Is the Soil Microbiome after Removal from the Field?

Plant-soil feedback (PSF) processes impact plant productivity and ecosystem function, but they are poorly understood because PSFs vary significantly with plant and soil type, plant growth stage, and environmental conditions. Controlled greenhouse studies are essential to unravel the mechanisms associating PSFs with plant productivity; however, successful implementation of these controlled experiments is constrained by our understanding of the persistence of the soil microbiome during the transition from field to greenhouse. This study evaluates the preservation potential of a field soil microbiome when stored in the laboratory under field temperature and moisture levels. Soil microbial diversity, taxonomic composition, and functional potential were evaluated via amplicon sequencing at the start of storage (W0), week 3 (W3), week 6 (W6), and week 9 (W9) to determine the effect of storage time on soil microbiome integrity. Though microbial richness remained stable, Shannon diversity indices decreased significantly at W6 for bacteria/archaea and W3 for fungi. Bacterial/archaeal community composition also remained stable, whereas the fungal community changed significantly during the first 3 weeks. Functional predictions revealed increased capacity for chemoheterotrophy for bacteria/archaea and decreased relative proportions of arbuscular mycorrhizal and ectomycorrhizal fungi. We show that preservation of the field soil microbiome must be a fundamental component of experimental design. Either greenhouse experiments should be initiated within 3 weeks of field soil collection, or a preliminary incubation study should be conducted to determine the time and storage conditions required to sustain the integrity of the specific field soil microbiome being studied.

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.

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.

RNASeq analysis of drought-stressed guayule reveals the role of gene transcription for modulating rubber, resin, and carbohydrate synthesis.

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.

Transcriptomic and evolutionary analysis of the mechanisms by which P. argentatum, a rubber producing perennial, responds to drought.

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.

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in Parthenium argentatum.

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.

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

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.

Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism.

Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer.

Transcriptome and gene expression analysis in cold-acclimated guayule (Parthenium argentatum) 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.

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.

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

BACKGROUND: Parthenium 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. RESULTS: The 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. CONCLUSION: The 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.

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species.

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Heveabrasiliensis and Parthenium argentatum. Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz-DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz-DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.

A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases.

A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross-linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as X-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, rubber elongation factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate.

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray.

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.