title: Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz.
abstract: BACKGROUND
  Natural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as Hevea brasiliensis, Ficus spec. and the desert shrub Parthenium argentatum, there have been no in planta functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion Taraxacum koksaghyz, which has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. However, there is very little molecular data available for either the rubber polymer itself or its biosynthesis in T. koksaghyz.
  
  
  RESULTS
  We established a method for the purification of rubber particles--the active sites of rubber biosynthesis--from T. koksaghyz latex. Photon correlation spectroscopy and transmission electron microscopy revealed an average particle size of 320 nm, and 13C nuclear magnetic resonance (NMR) spectroscopy confirmed that isolated rubber particles contain poly(cis-1,4-isoprene) with a purity > 95%. Size exclusion chromatography indicated that the weight average molecular mass (Mw) of T. koksaghyz natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min(-1) mg(-1). Ex vivo rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-14C]isopentenyl pyrophosphate (IPP) incorporation at a M of 2,500 kDa. Characterization of recently isolated cis-prenyltransferases (CPTs) from T. koksaghyz revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast.
  
  
  CONCLUSIONS
  T. koksaghyz rubber particles are similar to those described for H. brasiliensis. They contain very pure, high molecular mass poly(cis-1,4-isoprene) and the chain elongation process can be studied ex vivo. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described T. koksaghyz CPTs are the major rubber chain elongating enzymes in this species. T. koksaghyz is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.
authors.affiliation: Fraunhofer Institut für Molekularbiologie und Angewandte Okologie, Forckenbeckstr, 6, 52074 Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmidt
authors.forename: Malte
authors.initials: M
authors.lastname: Lenders
authors.forename: Andrea
authors.initials: A
authors.lastname: Hillebrand
authors.forename: Nicole
authors.initials: N
authors.lastname: van Deenen
authors.forename: Oliver
authors.initials: O
authors.lastname: Munt
authors.forename: Rudolf
authors.initials: R
authors.lastname: Reichelt
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eisenreich
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Dirk
authors.initials: D
authors.lastname: Prüfer
authors.forename: Christian Schulze
authors.initials: CS
authors.lastname: Gronover
mesh_terms: D002850:Chromatography, Gel; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D045782:Hemiterpenes / Q000737:chemistry; D007840:Latex / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010316:Particle Size; D012408:Rubber / Q000737:chemistry* / Q000378:metabolism; D031205:Taraxacum / Q000737:chemistry* / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D007840:Latex; C455100:1,4-polyisoprene; D012408:Rubber; D014166:Transferases; C106821:cis-prenyl transferase; D005966:Glucuronidase