Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz
- Thomas Schmidt†1,
- Malte Lenders†1,
- Andrea Hillebrand1,
- Nicole van Deenen1,
- Oliver Munt1,
- Rudolf Reichelt2,
- Wolfgang Eisenreich3,
- Rainer Fischer4,
- Dirk Prüfer1, 4 and
- Christian Schulze Gronover4Email author
© Schmidt et al; licensee BioMed Central Ltd. 2010
Received: 15 October 2009
Accepted: 19 February 2010
Published: 19 February 2010
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.
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 ( w) 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 w 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.
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.
Natural rubber poly(cis-1,4-isoprene) with a molecular mass of 10-10,000 kDa is one of the most important industrial raw materials in the world, and its sole commercial source is currently the para rubber tree Hevea brasiliensis . Other sources, such as Russian dandelion (Taraxacum koksaghyz Rodin) and Guayule (Parthenium argentatum), could be useful in the event of supply shortages as well as providing a suitable alternative for people with allergies to hevein, a major allergen present in H. brasiliensis latex [2, 3].
The biosynthesis of natural rubber takes place in the latex of laticifers or specialized parenchyma cells in the bark , where it is stored in rubber particles as an end product. Ultrastructural analysis of rubber particles from different species [4–6] revealed an almost identical globular structure that contains a homogeneous hydrophobic rubber core surrounded by an intact monolayer membrane. The monolayer membrane includes a mixture of lipids, proteins and other molecules with the hydrophilic portions of the phospholipids and glycosylated particle-bound proteins facing the cytoplasm [6–10]. The size of rubber particles ranges from 0.08-2 μm in H. brasiliensis, 0.2-6.5 μm in Ficus species and 1-2 μm in P. argentatum [6, 11].
Natural rubber is synthesized by adding activated 2-methyl-1,3-butadiene (isopentenyl diphosphate, IPP) to the growing chain [12, 13]. This reaction is catalyzed by specific long-chain cis-prenyltransferases (CPTs, EC 22.214.171.124), which are probably located on the surface of rubber particles. According to their function, CPTs are classified as short-, medium- or long-chain polymerizing enzymes and can be distinguished from trans-prenyltransferases (TPTs) by the presence of five conserved protein motifs . They are found in bacteria , yeast , animals including humans  and plants [18–22].
Recently, two CPTs (RER2 and SRT1) were isolated from Saccharomyces cerevisiae and were shown to be responsible for the biosynthesis of dolichol, a long-chain polyprenol with a saturated alpha-isoprene unit, which serves as a glycosyl carrier for protein glycosylation in the endoplasmic reticulum . The first plant CPT was identified in Arabidopsis thaliana (ACPT), and appears to be required for normal growth and development . The latex of H. brasiliensis contains at least two CPTs, designated HRT1 and HRT2 (for Hevea rubber transferase). The addition of recombinant HRT2 to washed latex particles supplemented with radioactively-labeled IPP resulted in the significant production of a high-molecular-weight labeled rubber product, whereas recombinant HRT1 showed no significant activity . In vitro, initiation of rubber biosynthesis by HRT requires intact particles, isopentenyl diphosphate (IPP), allylic diphosphates such as farnesyl diphosphate (FPP) and divalent metal cations (Mg2+ or Mn2+) as a co-factor [26–28]. However, all attempts to purify a functional rubber transferase from rubber particles have failed, suggesting that the native enzyme needs additional factors for its activity .
In this study, we report the comprehensive analysis of rubber particles from T. koksaghyz. The rubber particles contained very pure poly(cis-1,4-isoprene) and retained their capacity to produce natural rubber ex vivo. Immunological analysis revealed that CPTs associated with these particles remain fully functional when expressed as recombinant proteins either in Saccharomyces cerevisiae or tobacco protoplasts.
Results and Discussion
Physical characterization of purified T. koksaghyz rubber particles
Concentration and chemical properties of T. koksaghyz rubber
NMR data of poly(cis-1,4-isoprene) from T. koksaghyz.
Observed correlations in
Published 13 C-NMR chemical shifts**
5, 1, 4
In vitro biosynthetic activity of rubber particles
The rubber biosynthetic activity of T. koksaghyz rubber particles was characterized in vitro by assaying the incorporation of the radiolabeled precursor IPP, which should become incorporated into the polymer and thus trapped in the rubber particles [21, 33]. The IPP incorporation assay was carried out using intact isolated rubber particles as well as particles pre-treated with proteinase K to destroy particle-associated enzymes and other proteins. The treated and untreated particles were tested by SDS-PAGE and Coomassie Brilliant Blue staining, showing that the protein bands normally found in the particles were eliminated by proteinase K treatment (data not shown). This control allowed us to distinguish between physical and enzymatic incorporation of IPP into the particle, but had no influence either on particle integrity or stability. Earlier investigations into the in vitro activity of rubber particles used boiled particles as a control , which was not suitable for T. koksaghyz particles because of their rapid temperature-dependent agglomeration which changes the surface area to which IPP could be attached.
Effect of Mg2+ ion on [1-14C]IPP incorporation of T. koksaghyz rubber particles.
MgCl 2 [mM]
[1- 14 C]IPP incorporation
[Bq μg -1 protein]
0.57 (± 0.023)
0.83 (± 0.037)
0.89 (± 0.039)
0.91 (± 0.040)
0.52 (± 0.023)
0.04 (± 0.002)
Molecular mass and polydispersity of labeled and unlabeled material from ex vivo IPP incorporation.
w (10 5 Da)
n (10 5 Da)
w n -1
unlabeled 5-120 min
51.7 (± 1.34)
24.6 (± 1.42)
labeled 5-20 min
26.5 (± 1.01)
7.1 (± 0.45)
labeled 40-120 min
22.5 (± 2.27)
4.1 (± 0.30)
Particle associated cis-1,4-polyprenylcistransferases
Functional analysis of TkCPT1-3
Heterologous expression of TkCPTs in Nicotiana tabacum protoplasts.
GUS activity (μ mol min-1μ g-1)
CPT activity (pmol min-1mg-1)
1.78 (± 0.10)
1.34 (± 0.16)
3.48 (± 0.38)
2.73 (± 0.50)
0.97 (± 0.15)
2.05 (± 0.37)
2.28 (± 0.70)
1.22 (± 0.21)
2.90 (± 0.64)
2.50 (± 0.16)
We have investigated the physical properties and developmental profile of rubber particles from T. koksaghyz latex and have shown that they possess intrinsic cis-1,4-polyprenylcistransferase (rubber transferase) activity of 0.2 pmol min-1 mg-1 which can be partially reconstituted in vitro without further co-factors. We are the first to demonstrate conclusively that CPTs are an intrinsic part of the rubber particle and we have demonstrated a correspondence between the CPT activity in isolated rubber particles and the recently identified CPT genes TkCPT1-3 in T. koksaghyz, which are able to complement a yeast strain deficient in CPT activity and maintain their activity in tobacco protoplasts. In vitro rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-14C]isopentenyl pyrophosphate (IPP) incorporation at a weight average molecular mass of 2,500 kDa. Our data indicate that TkCPT1-3 are responsible for the rubber chain elongation that occurs in T. koksaghyz rubber particles and that their roles in this regard may be redundant. Our experiments provide crucial background information that will allow the development of T. koksaghyz as a potential alternative commercial source of rubber.
Plant material and cultivation conditions
Taraxacum koksaghyz plants were obtained from the Botanical Gardens Karlsruhe (Karlsruhe, Germany) and cultivated at 18°C with a 16-h photoperiod (20 klx) in controlled growth chambers or in the greenhouse. Plants were cultivated in a prefertilized 1:1 mixture of standard soil (ED73 Einheitserde, Fröndenberg, Germany) and garden mold (Botanical Garden Münster, Germany) and fertilized every 4 weeks with a commercial fertilizer according to the manufacturer's recommendations (Hakaphos Plus, Compo GmbH, Münster, Germany).
Rubber preparation and determination
For the isolation of native and functional rubber particles we followed the general procedure described for H. brasiliensis [27, 43] with the following modifications. Latex was harvested from petioles or roots of 20 week old T. koksaghyz plants, if not stated otherwise, by dissecting the tissue with a razor blade and transferring the expelling latex into an equal volume of ice-cold rubber extraction buffer (100 mM Tris.Cl (pH 7.8), 350 mM sorbitol, 10 mM NaCl, 5 mM MgCl2, 5 mM DTT) and centrifuging (12,000 × g, 20 min, 4°C). The latex separated into three fractions (pellet, C-serum and rubber phase) the latter two of which were transferred to a new tube and centrifuged as above. The rubber phase containing the rubber particles was transferred to a fresh tube and washed with 800 μl rubber extraction buffer and then dissolved in rubber extraction buffer and stored briefly at 4°C. To determine the dry rubber content, 20 μl of latex was transferred to a fresh tube and gently overlaid with 20 μl glacial acetic acid to coagulate the rubber particles. Afterwards, the coagulum was air-dried for 24 h.
Photon correlation spectroscopy
Freshly prepared rubber particles were dispersed by brief ultrasonication and then filtered with a 5 μm syringe filter prior to dynamic light scattering with a Zetasizer Nano ZS (Malvern Instruments GmbH, Herrenberg, Germany) containing a He-Ne laser (4.0 mW at 633 nm) and an Avalanche photodiode detector with a Q.E. >50% at 633 nm. Measurements were performed according to the manufacturer's instructions.
Transmission electron microscopy
Freshly prepared T. koksaghyz latex rubber phase was diluted 1:3 with rubber extraction buffer containing 1 mM DTT, and a small droplet was placed for 1 min on a freshly glow-discharged carbon layer (thickness ~ 10 nm) onto a Pioloform film supported by a commercial Cu-mesh grid. After removing excess liquid, the grid was washed with double-distilled water and stained with 2% aqueous uranyl acetate for 30 s before air drying. Transmission electron microscopy (TEM) was carried out using a Philips EM 410 (acceleration voltage 80 kV) in the bright-field mode, and micrographs were recorded on Imaging Plates (Ditabis, Pforzheim, Germany).
To measure the incorporation of IPP into rubber, the latex rubber phase was mixed with assay buffer to a final concentration of 100 mM Tris-HCl (pH 7.5), 2.5 mM CaCl2, 10 mM DTT, 1 mM sodium azide, 0.05% Triton X-100, 5 mM MgCl2, 2.8 μM E,E- farnesyl diphosphate (Sigma-Aldrich) and 7.2 μM [1-14C]- or [1, 2, 4-13C3]isopentenyl pyrophosphate (IPP) (GE Healthcare). The reaction mixture was incubated at 30°C for 2 h and then stopped by heating to 95°C for 5 min. The reaction products were hydrolyzed to corresponding alcohols using 2 μl of potato acid phosphatase (1 U μl-1 dissolved in double-distilled water) and 398 μl phosphatase buffer (50 mM sodium acetate (pH 4.7), 0.1% Triton X-100, 60% (v/v) methanol) for 2 h at 37°C as described . The products were extracted by shaking with 600 μl n-hexane for 1 h, air dried and resuspended in 300 μl n-hexane. Radioactivity was measured using a scintillation counter (Beckman Scintillation Counter LS6500) after mixing 50 μl of the extracts with 4 ml Rotiszint® eco plus (Roth, Karlsruhe, Germany). Extracts were air dried and resolved in tetrahydrofuran. Size-exclusion chromatography to determine the molecular size distribution of natural rubber and rubber produced in radioactive assays was carried out as previously described .
One-dimensional 1H and 13C NMR spectra were measured at 500 and 125 MHz, respectively, using a DRX500 or AVANCE 500 spectrometer (Bruker, Rheinstetten, Germany). Two-dimensional HMQC, HMBC and COSY spectra were measured with the AVANCE 500 spectrometer using an inverse probe-head and standard parameter sets implemented in TOPSPIN 1.1. The solvent was deuterated dichloromethane and the temperature was 27°C. The experimental time for 13C NMR spectra was typically 15 h (corresponding to more than 10,000 scans). Data were processed using TOPSPIN 1.1 or MestReNova.
Construction of expression vectors for heterologous expression
RNA from T. koksaghyz latex was isolated as described previously  and cDNA synthesis was carried out using the SuperScript II™ Reverse Transcriptase Kit (Invitrogen, Karlsruhe, Germany) with an oligo(dT) primer. For all three TkCPT s (TkCPT1, TkCPT2 and TkCPT3) cDNA was generated using the primer combination cpt-TK_EcoRI (5'-AAA GAA TTC ATG CAA GTG AAT CCA ATC ATT ACT AC-3') and cpt-TK-rev_SalI (5'-AAA GTC GAC TTA TGC CTG CTT CTT CTT CTT CTC C-3'). The products were inserted into the pCRII-TOPO® vector (Invitrogen, Karlsruhe, Germany), sequenced and then transferred using Eco RI and Sal I restriction sites into the expression vector pGEX4-T1 (GE Healthcare Europe GmbH, Freiburg, Germany). This allowed the expression of TkCPTs as N-terminal fusions to glutathione-S-transferase (GST) for heterologous expression in Escherichia coli BL21-cells and downstream antibody generation.
For heterologous expression of TkCPT1-3 in Nicotiana tabacum var. SR1 protoplasts, the corresponding cDNAs were amplified with the primers TkCPT_PciI (5'-AAA ACA TGT TAC AAG TGA ATC CAA TCA TTA CTA C-3') and TkCPT-rev_XbaI (5'-AAA ACA TGT TAC AAG TGA ATC CAA TCA TTA CTA C-3') and transferred using the Nco I and Xba I sites into the pUC18-based pAM vector containing the CaMV 35S promoter and polyadenylation sequences from pRT104 . The cassette was then transferred using the Kpn I and Eco RI sites into pCAMBIA-1305.1 (GenBank: AF354045), which already contains the gus A reporter gene including a catalase intron under the control of the CaMV 35S promoter.
For expression in yeast, the TkCPT cDNAs were released from pAM using Xho I and Eco RI and introduced into vector pYEXBX (Clontech Laboratories Inc., Saint-Germain-en-Laye, France), which had been digested with Sal I and Eco RI.
Generation of antibodies against TkCPTs
The three TkCPT clones in pGEX4-T1 were overexpressed in 300-ml cultures of E. coli strain BL21, induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). Purified TkCPT1 was sequenced by MALDI-MS and administered to rabbits by EUROGENTEC (Seraing, Belgium). The pre-immune and antibody sera were tested for specificity by western blot against the recombinant proteins TkCPT1, TkCPT2 and TKCPT3.
SDS-PAGE and western blots
SDS-PAGE was carried out using 15 μg protein per lane from the pellet, C-serum and rubber phases of fresh T. koksaghyz latex. Protein concentrations were determined using the Bradford method . Proteins were separated on SDS-PAGE gels and either stained with Coomassie Brilliant Blue or transferred to nitrocellulose membranes as described . The membranes were incubated with the primary antibody (1:500 dilution) for 1 h at room temperature, washed and then incubated with a mouse anti-rabbit IgG conjugated to horseradish peroxidase (Sigma, Munich, Germany) according to the manufacturer's instructions. Membranes with HRP-coupled secondary antibodies were imaged on X-ray films by chemiluminescence detection.
Expression of TkCPTs in yeast
Yeast strain SNH23-D7 (MATa rer2-2 mfa1::ADE2 mfa2::TRP1 bar1::HIS3 ade2 trp1 his3 leu2 ura3 lys2)  was cultivated in standard YPD medium for 48 h at 20°C, then transformed  with pYEXBX-TkCPT1-3, and the pYEXBX base vector as a control. After regeneration, cells were plated on SD-URA agar, colonies for each construct were transferred to 0.9% NaCl and adjusted to different optical densities (1, 0.1, 0.01 and 0.001) and 5-μ l droplets were spotted onto SD-URA plates. After 48 h incubation in SD-URA liquid medium, denatured protein extracts were prepared from 20 OD600 units  and used for SDS-PAGE and western blot analysis.
Expression in protoplasts and GUS assay
Protoplasts were isolated from Nicotiana tabacum var. SR1 and Ca(NO3)2 polyethylene glycol-mediated DNA transfer was performed as described  using 3.3 × 105 protoplasts and 10 μ g of pCambia1305.1-TkCPT1-3 DNA per transfection. Frozen protoplasts were sheared in IPP assay buffer, briefly centrifuged and the supernatants pooled from six replicate transfections. Protein concentration was determined using the Bradford method  and bovine serum albumin as a standard. Approximately 100 μg of protein extract was used in the IPP incorporation assay and 1 μg in the glucuronidase activity assay with 4-methylumbelliferylglucuronide (4-MUG) as the substrate .
This work was supported by a grant from the Ministry of Science and Education of Germany (grant no. FKZ 0313712), by EVONIK Industries AG, by the Hans-Fischer Gesellschaft Munich (to WE), by the Deutsche Bundesstiftung Umwelt (to ML) and the federal state North-Rhine Westphalia co-financed by the European Union.
Arabidopsis thaliana cis-prenyltransferase
heteronuclear multiple bond correlation
heteronuclear multiple quantum coherence
Hevea brasiliensis rubber transferase
nuclear magnetic resonance
Saccharomyces cerevisiae dehydrodolichyl diphosphate synthase
size exclusion chromatography
Saccharomyces cerevisiae cis-1,4-polyprenylcistransferases
transmission electron microscopy
Taraxacum koksaghyz cis-1,4-polyprenylcistransferases
The authors would like to thank Ursula Malkus (Institut für Medizinische Physik und Biophysik, Münster) for carrying out the TEM analysis of rubber particles. The technical assistance of Sandra Ponanta (Institut für Biochemie und Biotechnologie der Pflanzen, Münster), Raphael Soeur (Fraunhofer Institut für Molekularbiologie und Angewandte Ökologie, Aachen) and Karin Wacker (Fraunhofer Institut für Chemische Technologie, Pfinztal) is gratefully acknowledged. We would like to thank Prof. Dr. Thomas Hirth (Fraunhofer Institut für Grenzflächen und Bioverfahrenstechnik, Stuttgart) and Dr. Ulrich Fehrenbacher (Fraunhofer Institut für Chemische Technologie, Pfinztal) for providing SEC and PCS facilities. We are grateful to Dr Akihiko Nakano and Dr Ken Sato (RIKEN, Japan) for kindly providing the yeast strain SNH23-D7.
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