Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz.
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.
Schmidt
Thomas
T
Fraunhofer Institut für Molekularbiologie und Angewandte Okologie, Forckenbeckstr, 6, 52074 Aachen, Germany.
Lenders
Malte
M
Hillebrand
Andrea
A
van Deenen
Nicole
N
Munt
Oliver
O
Reichelt
Rudolf
R
Eisenreich
Wolfgang
W
Fischer
Rainer
R
Prüfer
Dirk
D
Gronover
Christian Schulze
CS
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
D045782:Hemiterpenes; D007840:Latex; C455100:1,4-polyisoprene; D012408:Rubber; D014166:Transferases; C106821:cis-prenyl transferase; D005966:Glucuronidase