Colleen M. McMahan

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.

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.

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.

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.

Development of Crops to Produce Industrially Useful Natural Rubber

Natural rubber, cis-1,4-polyisoprene, is an essential industrial commodity that most developed countries have to import. Hevea brasiliensis (Hevea), grown in tropical and subtropical areas, is the primary source of natural rubber. The high quality and quantity of the rubber cause us to focus on understanding rubber production in Hevea and two temperate plant species, guayule (Parthenium argentatum) and Russian dandelion (Taraxacum kok-saghyz). We review the cell biology, physiology, and biochemistry of rubber production in these three species. Rubber is synthesized on subcellular vesicles called rubber particles. Purified rubber particles alone contain all necessary factors for rubber production. We have used genomic approaches to identify expressed genes associated with rubber-producing tissues and proteomics to identify proteins associated with rubber particles. The protein and EST identifications guided our analysis of key proteins in rubber production, including cis-prenyltransferase, rubber elongation factor, small rubber particle protein, allene oxide synthase, HMG-CoA reductase, and allylic diphosphate synthases. We discuss biotechnological approaches to improve rubber production.

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.

A bicistronic transgene system for genetic modification of Parthenium argentatum

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.

Standard Operating Protocol for Growing Transgenic Sunflower Plants in Contained Environments

Biotechnology provides the tools to develop industrial products for modern society that would not be possible in any other way (3). A biotechnological approach to improve sunflower was initiated in 2001 in a collaborative research project that involved genetic transformation of sunflower. Development of transgenic sunflowers creates concern about the possible flow of transgenes into wild sunflower populations, where these populations might be genetically altered in a detrimental way (2,8,10,12). Thus, pollen from transgenic sunflowers must be contained to prevent the hybridization of transgenic sunflower with wild sunflower. Concerns about biotechnology and gene flow from transgenic crops into their wild relatives have been studied and discussed by numerous researchers and organizations including Council for Agricultural Science and Technology (4), Daniell (5), Ellstrand et al. (7), Ellstand (6), and Wolfenbarger (13). The objective of this article is to present the standard operating protocol (SOP) we developed and have used for several years for growing transgenic sunflower plants in controlled, contained environments. This SOP may be of assistance to other researchers who work with transgenic plants when preparing their own SOP. A resource we found useful in developing our SOP was Adair et al. (1). Our SOP described in this article has been modified to include additional background information and descriptions of our operation to tailor it to the readership of this publication.