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.2010 Feb 19:11:11.
doi: 10.1186/1471-2091-11-11.

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

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Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

Thomas Schmidt et al. BMC Biochem..

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.

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Figures

Figure 1
Figure 1
Morphology of rubber particles fromTaraxacum koksaghyz. Transmission electron micrographs at low a) and high b) magnification. c) Rubber particle size distribution measured by photon correlation spectroscopy in latex of 4- and 12-month-old plants. Magnifications in a) and b) are indicated by bar size.
Figure 2
Figure 2
13C NMR spectrum of rubber particles isolated fromTaraxacum koksaghyz. The signal arising from the deuterated sample is indicated. The inset displays the structure and the carbon numbering for poly(cis-1,4-isoprene).
Figure 3
Figure 3
Incorporation of IPP into rubber particles. a) Time course-dependent and b) rubber particle protein dependent incorporation of [1-14C]IPP. c)13C NMR signal of C-2 of poly(cis-1,4-isoprene) from the labeling experiment with [1,2,4-13C3]IPP. The filled circles indicate13C atoms from [1, 2, 4-13C3]IPP. Values in a) and b) represent mean (± standard deviation) from three biological repetitions.
Figure 4
Figure 4
Molecular mass distribution of [1-14C]IPP-labeled rubber synthesizedin vitro. a) SEC profile of labeled material. b)formula imagew andformula imagen of the labeled polymer. IPP incorporation assay was performed and stopped after the time points shown. Extracted polymer material was fractionated by SEC and the radioactivity of the resulting fractions was determined by scintillation. Proteinase K (ProtK)-treated particles controlled for IPP trapped on the rubber particles through non-enzymatic mechanisms. Progression lines represent the mean of two measurements.
Figure 5
Figure 5
Detection ofT. koksaghyzCPT in the rubber phase. Latex was harvested and divided into pellet (lane 1), C-serum (lane 2) and rubber phase (lane 3). Proteins were separated by SDS-PAGE and either stained with Coomassie Brilliant Blue a) or transferred to a membrane for western blot analysis using antibodies against TkCPTs b).
Figure 6
Figure 6
Plasmids for heterologous expression ofTkCPT1-3. For heterologous expression ofTkCPT1-3inS. cerevisiaeand protoplasts ofN. tabacumthe corresponding cDNAs were cloned into the plasmids pYEX-BX a) and pCAMBIA-1305.1 b), respectively.PCUP1,S. cerevisiae CUP1promoter; P(A)1, cauliflower mosaic virus (CaMV) 35S polyadenylation signal sequence;URA3,S. cerevisiaeURA3 locus; 35S-P, CaMV 35S promoter;GUS, beta-glucuronidase synthetic construct including catalase intron (I) (GenBank: AAK29426); P(A)2,Agrobacterium tumefaciensD-nopaline synthase polyadenylation signal sequence.
Figure 7
Figure 7
Functional complementation of the temperature-sensitive dedol-PP synthase rer2 yeast mutant SNH23-D7 by TkCPTs. a) Yeast strain SNH23-D7 transformed with plasmid pYEX-BX, pYEX-TkCPT1, pYEX-TkCPT2 and pYEX-TkCPT3 were dropped on SD-URA plates and incubated for 2 days at the temperatures shown. b) Western blots were performed with antibodies against TkCPTs to detect TkCPT in yeast SNH23-D7 (rer2) transformants pYEX-BX (vector control) (1), pYEX-TkCPT1 (2), pYEX-TkCPT2 (3) and pYEX-TkCPT3 (4).
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