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

Abstract

Guayule ( Parthenium argentatum ) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis -1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis -polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis -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 PaCPTs ( PaCPT1-3 ) and one PaCBP , whose protein products organize PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis -polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP -expressing yeast efficiently incorporated 14 C-isopentenyl diphosphate into dehydrodolichyl diphosphates; however, NR with high molecular weight could not be synthesized in in vitro assays. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPT s, guayule transcriptomics analysis indicated that the PaCPT3 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.

Natural rubber biosynthesis in plants, the rubber transferase complex, and metabolic engineering progress and prospects

“…The fourth nonenzymatic protein (RTA) also is named CBP (CPT‐binding protein), and, for clarity, this is the abbreviation we use below as we combine these reports. Thus, CBP directly interacts with enzymatically active CPTs and CPT/CBP complexes appear to be involved in both dolichol and NR biosynthesis (Brasher et al ., ; Epping et al ., ; Qu et al ., ; Lakusta et al ., ; Table ).…”

“…CPT genes were reported to be required for rubber biosynthesis in T. kok‐saghyz, L. sativa and H. brasiliensis (Epping et al ., ; Qu et al ., ; Yamashita et al ., ; Table ), but their ability to produce allylic pyrophosphate initiators (Cornish, , 201a,b) can make interpretation of such experiments difficult. Four CPT ‐like sequences were identified in a P. argentatum cold‐acclimated EST collection (Ponciano et al ., ), and recently, three CPT genes ( PaCPT1‐3 ) and one CBP ( PaCBP ) also were identified in this species (Lakusta et al ., ). When PaCBP was co‐expressed with each PaCPT in turn they formed a complex which could synthesize dehydrodolichyl‐PP in microsomes (Lakusta et al ., ).…”

“…Four CPT ‐like sequences were identified in a P. argentatum cold‐acclimated EST collection (Ponciano et al ., ), and recently, three CPT genes ( PaCPT1‐3 ) and one CBP ( PaCBP ) also were identified in this species (Lakusta et al ., ). When PaCBP was co‐expressed with each PaCPT in turn they formed a complex which could synthesize dehydrodolichyl‐PP in microsomes (Lakusta et al ., ). Although no rubber was made, PaCPT3 was both highly expressed in rubber‐producing stem tissue and was further induced by cold stress, as was PaCBP .…”

Strategies for the production of biochemicals in bioenergy crops

“…The polymerization process of the rubber chain is initiated primarily from FPP and a CPT complex, consisting of a rubber elongation factor (REF), a small rubber particle protein (SRPP), and CPT-binding proteins (CPTBP). Finally, the termination has been proposed to be controlled by ubiquitinproteasome proteolysis [308,309].…”

Long-Chain Polyisoprenoids Are Synthesized by AtCPT1 in Arabidopsis thaliana

“…The prenyl diphosphate biosynthetic pathway in eukaryotic cells requires the interaction of CPT with an accessory CPT-binding protein for determination of polyprenol chain length and examples of such complexes are human (hCIT/NgBR) [11], yeast (Rer2/Srt1/NUS1) [12] and some plants, including tomato (SlCPT3/SlCPTBP) [13], lettuce (CPT3/CPTL2) [14], Arabidopsis (CPT3/LEW1) [15], ginseng (CPT1/PgCPLT2) [16] and guayule (PaCPT1-3/PaCBP) [17].…”