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.

Host defence in a developmental context.

SUMMARY Disease resistance takes place within the context of the host developmental programme. The cellular and molecular basis of the developmental control of resistance is virtually unknown. It is clear from mutant studies that developmental processes are impacted when defence factors are altered and it is equally clear that alteration of developmental factors impacts defence functions. A review of current knowledge regarding the interplay of resistance and development is presented. Stage-specific limitations on defence represent an important target for crop improvement.