Molecular Studies of the Protein Complexes Involving Cis-Prenyltransferase in Guayule (Parthenium argentatum), an Alternative Rubber-Producing Plant

Abstract

Guayule (<i>Parthenium argentatum</i>) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic <i>cis</i>-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for <i>cis</i>-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving <i>cis</i>-prenyltransferases (CPTs) and CPT-Binding Proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here, we identified four guayule genes - three <i>PaCPTs</i> (<i>PaCPT1-3</i>) and one <i>PaCBP</i>, whose protein products organize PaCPT/PaCBP complexes. Co-expression of both <i>PaCBP</i> and each of the <i>PaCPTs</i> could complemented the dolichol (a short <i>cis</i>-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the <i>PaCPT/PaCBP</i>-expressing yeast efficiently incorporated ¹⁴C-isopentenyl diphosphate into dehydrodolichyl diphosphates; however, NR with high molecular weight could not be synthesized in <i>in vitro</i> assays. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three <i>PaCPT</i>s, guayule transcriptomics analysis indicated that the <i>PaCPT3</i> is predominantly expressed in stem and induced by cold-stress, suggesting its involvement in NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule.