Molecular characterization of cis-prenyltransferase complexes in guayule (Parthenium argentatum)
Date
2017-12-17
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Abstract
Natural Rubber (NR) is a biopolymer with an irreplaceable role in the fields of medicine, manufacturing, and transport. Despite its necessity, the Brazilian Rubber Tree (Hevea brasiliensis) remains virtually the sole source of NR. As the majority of NR plantations are in Southeast Asia, its production is threatened by climate and disease. While NR supply remains insecure, biochemical studies have identified two essential proteins: cis-prenyltransferases (CPTs) and cis-prenyltransferase Binding Proteins (CBPs). With recent studies elucidating the biochemical properties of plant CPTs and CBPs, a potential mechanism for NR biosynthesis has been proposed. As the bulk of NR research has so far focused on two phylogenetically close relatives, lettuce and dandelion, further evidence is required from a more genetically distant plant species. This research is focused on guayule (Parthenium argentatum, Asteraceae family) – an alternative NR-producing plant long-diverged from lettuce and dandelion. CBP and CPT homologs, PaCBP and PaCPT1-3, were identified through publicly available transcriptomics databases and various biochemical assays were undertaken. In vivo split-ubiquitin yeast two hybrid and co-immunoprecipitation assays have indicated strong levels of interaction between the PaCPTs and PaCBP. Interestingly, our findings also show that guayule CPTs, together with guayule’s CBP, are responsible for the production of dolichol-like polymers in vitro rather than NR. This was shown by using harvested microsomes from yeast that expressed heterologous guayule CBP and CPTs in a biochemical assay. The biochemical assay was performed using [14C]-radiolabeled isopentenyl pyrophosphate as substrate. The polymer sizes were determined through reverse-phase thin layer chromatography. Another method used to determine the function of CPT and CBP was to rescue the lethal phenotype of dolichol synthase-deficient yeast by co-expressing CPT and CBP. Furthermore, phylogenetic and expression analyses of PaCPT1-3 revealed PaCPT3 as both likely involved in NR biosynthesis and highly expressed throughout the plant. PaCPT3 may therefore prove to be suitable for future NR studies and biotechnological applications. Taken together, our data provide further evidence of a NR biosynthetic mechanism that is conserved across the Asteraceae family, while also increasing guayule’s viability as both a production alternative and model for the study of NR.
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Keywords
Biochemistry, cis-prenyltransferase, cis-prenyltransferase binding protein, natural rubber, parthenium argentatum, guayule
Citation
Lakusta, A. M. (2017). Molecular characterization of cis-prenyltransferase complexes in guayule (Parthenium argentatum) (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.