Journal of Experimental Botany, Vol. 54, No. 384, pp. 985-992,
March 1, 2003
© 2003 Oxford University Press
The micromorphology and protein characterization of rubber particles in Ficus carica, Ficus benghalensis and Hevea brasiliensis
Received 17 September 2002; Accepted 29 November 2002
1 Department of Forest Products and Technology, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju, 500-757 Korea
2 Division of Applied Plant Science and Agricultural Plant Stress Research Center, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju, 500-757 Korea
3 Kumho Life and Environmental Science Laboratory, 1 Oryong-dong, Buk-gu, Gwangju, 500-712 Korea
4 To whom correspondence should be addressed at Chonnam National University: Fax: +82 62 530 2047. E-mail: hskang{at}chonnam.chonnam.ac.kr
Abbreviations: CPT, cis-prenyltransferase; CPD, critical point drying; IDP, isopentenyl diphosphate; SEM, scanning electron microscopy; SRPP, small rubber particle protein; TEM, transmission electron microscopy.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.
Key words: Electron microscopy, Ficus benghalensis, Ficus carica, Hevea brasiliensis, immunolocalization, micromorphology, rubber particle, rubber particle protein.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The surface of rubber particles contains the enzymes and/or factors necessary for rubber biosynthesis, and is the place where rubber biosynthesis occurs (Benedict et al., 1990; Cornish and Backhaus, 1990; Cornish, 2001). Rubber particles are globular particles in which the hydro phobic rubber polymers are surrounded by a monolayer membrane containing various kinds of lipids, proteins, and other components (Hasma and Subramaniam, 1986; Hasma, 1991; Siler et al., 1997; Cornish et al., 1999; Wood and Cornish, 2000). Since rubber transferase, the key enzyme for rubber biosynthesis, is one of the proteins associated with the monolayer membrane, it is considered a membrane protein. It loses its activity once it is isolated from rubber particles. Therefore, in an effort to identify the rubber transferase and/or additional factors important for rubber biosynthesis, many research groups have investigated rubber particles and the proteins associated with them in Hevea brasiliensis (Attanyaka et al., 1991; Cornish, 1993; Dennis and Light, 1989; Goyvaerts et al., 1991; Light and Dennis, 1989; Oh et al., 1999), Parthenium argentatum Gray (guayule) (Backhaus et al., 1991; Benedict et al., 1990; Cornish and Backhaus, 1990; Cornish et al., 1994; Pan et al., 1995; Siler and Cornish, 1993), Ficus elastica (Cornish and Siler, 1996; Siler and Cornish, 1994), Ficus carica (Kang et al., 2000a), and Ficus benghalensis (Kang et al., 2000b).
Despite continued efforts to identify rubber transferase, the nature of this extremely interesting enzyme has not yet been verified. In H. brasiliensis a 14 kDa protein tightly associated with the large rubber particles was suggested to be a rubber elongation factor (Dennis and Light, 1989), and its gene has been cloned (Attanyaka et al., 1991; Goyvaerts et al., 1991). However, the direct role of this protein in rubber biosynthesis has not been proven. More recently, Oh et al. (1999) have isolated a cDNA from H. brasiliensis which encodes a major rubber particle protein of 24 kDa, tightly associated with small rubber particles (designated SRPP). They demonstrated that the SRPP plays a positive role in isopentenyl diphosphate (IDP) incorporation into the rubber polymer. In guayule, the most abundant rubber particle protein of 52 kDa was sequenced to be a P450 allen oxide synthase (Pan et al., 1995). Based on a series of cross-specific immunoinhibition analyses, a 375 kDa protein was suggested to be a rubber transferase in F. elastica (Cornish et al., 1994; Siler and Cornish, 1993, 1994). Proteins present in the latex and rubber particles of F. benghalensis have been analysed, and the protein of 31 kDa was found to be the most abundant in catalytically-active rubber particles (Kang et al., 2000b).
Due to the presence of the enzymes for rubber biosynthesis in rubber particles, it is important to investigate the structure of rubber particles and to characterize the rubber particle-associated proteins in various plant species in order to identify the rubber transferase and/or additional important factors for rubber biosynthesis and also to understand the relationship between the size or composition of rubber particles and the commercial value of rubbers. The microstructure and particle size distribution of rubber particles from H. brasiliensis, P. argentatum, F. elastica, and Euphorbia lactiflua have been characterized by various electron microscopic techniques (Gomez and Hamzah, 1989; Yeang et al., 1995; Cornish et al., 1999; Wood and Cornish, 2000). However, studies involving immunolocalization of the membrane proteins are limited. Bahri and Hamzah (1996) used an immunogold labelling technique in conjunction with electron microscopy to confirm the presence of the 14 kDa protein and SRPP on the surface of H. brasiliensis rubber particles.
In this study, the micromorphology of rubber particles was examined from F. carica and F. benghalensis, the Ficus species recently identified as rubber-producing plants by this group (Kang et al., 2000a, b), and compared it with that from H. brasiliensis. In addition, in order to understand whether rubber particles of these three plant species contain similar proteins, the presence of those rubber particle proteins deemed important was investigated by western analysis and transmission electron microscopy (TEM)–immunogold cytochemistry. One of the proteins investigated in this study was the SRPP. This major rubber particle protein in H. brasiliensis has been suggested to be involved in rubber biosynthesis (Oh et al., 1999). The other protein was the cis-prenyltransferase (CPT) that has a catalytic activity similar to rubber transferase. It catalyses a sequential condensation of IDP with allylic diphosphate, but produces the shorter chain length polyprenyl diphosphates ranging in carbon number from C50120 (Ogura and Koyama, 1998). It was, therefore, important to investigate the presence of these proteins on the surface of rubber particles in H. brasiliensis, F. carica, and F. benghalensis, and biochemical and TEM-immunogold techniques were used to obtain the information.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
The latex samples of F. carica and F. benghalensis were obtained from the plants grown in a greenhouse maintained at 30 °C under constant light. H. brasiliensis latex was collected from regularly tapped rubber trees (H. brasiliensis RRIM600) at the Rubber Research Institute of Malaysia, and the latex samples on ice were shipped to this laboratory. The latex was collected directly into the ice-cold buffer containing 100 mM TRIS-HCl, pH 7.5, 5 mM MgSO4, and 5 mM dithiothreitol.
Preparation of washed rubber particles
The latex of H. brasiliensis was centrifuged at 20 000 g for 30 min at 4 °C, and the latex of F. carica and F. benghalensis was centrifuged at 10 000 g for 5–10 min at 4 °C. The top creamy fraction of rubber particles was collected, resuspended in the same buffer, and recentrifuged. The rubber particles of F. carica and F. benghalensis sedimented during the washing cycle, whereas the Hevea rubber particles remained afloat. The non-rubber fractions were discarded, and this washing procedure was repeated three times as described previously (Cornish and Backhaus, 1990; Siler and Cornish, 1993; Kang et al., 2000a). The washed rubber particles were used directly for western blot and immunogold label analyses.
Western blot analysis
Rubber particle proteins were solubilized by incubating the rubber particles in a detergent solution containing 0.1% (w/v) Triton X-100 and 1% (w/v) SDS. The detergent-treated suspension was centrifuged at 13 000 rpm for 10 min, and the supernatant fraction was separated by SDS–12% PAGE. The gels were subsequently used for western blotting where the proteins in the gel were transferred to a polyvinylidene difluoride membrane. For the detection of the SRPP, the membrane was incubated with the buffer containing the polyclonal rabbit antibody raised against the SRPP from H. brasiliensis as described earlier (Kang et al., 2000a). For the detec tion of the cis-prenyltransferase, the polyclonal rat antibody was raised against the synthetic peptide (DLMIRTSGEQRISNF) in which the sequence was designed based on the conserved amino acid sequence of CPTs from plants and micro-organisms, which corresponds to the positions from 246 to 260 of Arabidopsis CPT (Oh et al., 2000). After three cycles of washing with TBS-T buffer or PBS-milk, respectively, the membrane was incubated for 1 h with anti-IgG antiserum conjugated to horseradish peroxidase (Amersham Pharmarcia Biotech). After a further three cycles of washing with the same buffer, the proteins on the membrane were detected by the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
Electron microscopy
The shape and diameter of rubber particles were determined by scanning electron microscopy (SEM). Initial attempt to characterize particle shape and diameter using critical point drying (CPD), a widely used method for studying biological materials, proved unsuccessful because this method caused the severe collapse of particles. A novel air-drying method that was developed specifically for this work, and which proved most suitable, is described here. The purified rubber fraction from freshly collected latex was fixed in 3% glutaraldehyde in 50 mM sodium cacodylate buffer (containing 1% tannic acid) for 1 h at room temperature. After washing in several changes of buffer, the samples were post-fixed in 1% aqueous osmium tetroxide also for 1 h at room temperature, and subsequently washed in several changes of distilled water. A small drop of the suspension (appropriately diluted) was air-dried on a piece of cover glass. The cover glass was then placed on a stub applied with a double-sided tape, sputter-coated with gold and examined with a Hitachi S-2400 SEM.
Immunocytochemistry
The initial attempts to process samples using the fixation procedures employed routinely for immunocytochemical work were not successful. Neither the paraformaldehyde–glutaraldehyde (2.5%: 0.5%) combination nor glutaraldehyde (3%) alone could stabilize the spherical form of rubber particles. The glutaraldehyde–tannic acid combination, as used for SEM work, was effective in keeping the natural form of rubber particles: however, gold-labelling was poor and non-specific. The method that worked best was fixation with osmium tetroxide.
The purified rubber fraction from H. brasiliensis, F. carica, and F. benghalensis was fixed with 1% osmium tetroxide (in 50 mM sodium cacodylate buffer) for 1 h at room temperature. After washing in buffer, a small drop of the rubber was placed on formvar-coated nickel grids and air-dried. Procedure for immunogold labelling was similar to that described by Kim et al. (2002). Prior to treatment with the primary antibody, grids were sequentially floated on drops of glycine (in PBS buffer), distilled water, PBS buffer, and normal goat serum (NGS)–PBS buffer. Grids from all samples were treated with the SRPP antibody (1:200 dilution) and CPT antibody (1:100 dilution) for 90 min at 37 °C. After sequential washes in PBS–non-fat milk–Tween 20, PBS, and TRIS-HCl, samples were treated with the following gold conjugated (10 nm) secondary antibodies (1:20 dilution) for 2 h at room temperature. Samples exposed to the SRPP antibody were treated with anti-rat IgG and those exposed to the CPT antibody were treated with anti-rabbit IgG. Samples were then washed sequentially with TBS-tween 20, TBS, TRIS-HCl, and distilled water. For the control, samples were processed as above, except that they were not incubated with the primary antibodies. Grids were air-dried and examined with a JEOL JEM1010 TEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Comparison of the shape and size of rubber particles
Rubber particles of all three species, H. brasiliensis, F. carica, and F. benghalensis, were predominantly spherical in shape (Fig. 1). Only in F. benghalensis were some particles also elongated, but this appears to have been induced by centrifugation and thus is not a normal shape. Although several size populations of particles were observed for all species examined, the range was greatest for Hevea, which also had the smallest size particles (Figs 1A, 2). Only marginal differences were observed between F. carica and F. benghalensis size populations as well as in the size of individual particles (Figs 1B, C, 2). For Hevea, it was possible to categorize particles into three size groups: largest particles with a diameter range of 0.4–0.75 µm, intermediate size particles with a diameter range of 0.25–0.35 µm, and the smallest particles with a diameter range of 0.08–0.2 µm. The particles of F. carica and F. benghalensis could be categorized into two size groups, although the size range for the largest particles of F. benghalensis was somewhat greater compared with F. carica. Also, the largest particles of F. carica were slightly larger than those of F. benghalensis. Smaller sized particles were less abundant than larger sized particles for both species. The largest particles of F. carica measured 3.7–6.5 µm in diameter, and those of F. benghalensis measured 3.0–6.0 µm. The smaller particles of F. carica measured 1.6–3.0 µm and those of F. benghalensis measured 1.6–2.3 µm.
|
|
Western analysis of the proteins on the surface of rubber particles
The presence of the two proteins, the SRPP and CPT that are suggested to be involved in rubber biosynthesis, was investigated by western analyses. Specifically, it was necessary to probe whether the rubber particles of F. carica, Hevea rubber tree, and F. benghalensis contain the two proteins on their surfaces. As shown in Fig. 3A, the SRPP and the 14 kDa protein on Hevea rubber particles cross-reacted with the SRPP antibody. The cross-reactivity of both the SRPP and the 14 kDa protein with the polyclonal antibody raised against the SRPP was well established, since the SRPP and 14 kDa protein share a high degree of sequence homology in their amino acid sequences (Oh et al., 1999; Kang et al., 2000a). By contrast, neither the F. carica proteins nor the F. benghalesis proteins reacted with the SRPP antibody. These results indicate that the SRPP, abundantly present on the rubber particles of Hevea rubber trees, is not a common protein necessary for rubber biosynthesis in rubber-producing plants.
|
For the detection of the CPT, the polyclonal rat antibody was raised against the synthetic peptide (DLMIRTSGE QRISNF) in which the sequence was designed based on the conserved amino acid sequence of CPTs from plant and micro-organisms, and corresponds to the positions from 246 to 260 of Arabidopsis CPT (Oh et al., 2000). Figure 3B shows that the CPT antibody did not react with any of the proteins isolated from the rubber particles of F. carica, H. brasiliensis, and F. benghalensis. Since the CPT is ubiquitous in all living organisms, the protein extract isolated from tobacco leaves was used as a positive control. The tobacco leaf protein of about 32 kDa in size was clearly reacted with the CPT antibody. The absence of cross-reactivity of the proteins isolated from F. carica, H. brasiliensis, and F. benghalensis with the CPT antibody indicates that cis-prenyltransferase does not reside on the surface of rubber particles, and is not a key determinant necessary for rubber biosynthesis in plants.
Immunogold labelling of the proteins on the surface of rubber particles
The TEM examination of rubber samples, which had been incubated with the SRPP antibody and subsequently treated with gold conjugated secondary antibody, provided evidence for intense gold labelling of H. brasiliensis rubber particles, and the absence of labelling of F. carica and F. benghalensis rubber particles. Several H. brasiliensis rubber particles are shown at low magnification in Fig. 4A. At this magnification gold particles were just detected as small, electron-dense granules associated exclusively with rubber particles, and there were no indications of any background labelling. Gold particles were attached to the margins of all rubber particles and, in the case of the smaller particles, which are less dense, gold particles also appeared to be distributed all over the surface. Gold particle labelling is more clearly visible in the high magnification view in Fig. 4B. The size of rubber particles is variable in the population shown in this figure. All rubber particles were intensely labelled, and numerous gold particles were associated with the margins of the particles. Where resolvable, gold particles could also be seen all over the surface of rubber particles. There appeared to be no difference between large and small rubber particles of H. brasiliensis with regard to labelling intensity. However, confirmation of this point will require quantification, which was not undertaken in this work. The complete absence of gold labelling of F. carica and F. benghalensis rubber particles is shown in Fig. 5. TEM examination of rubber samples, which had been incubated with the CPT antibody and subsequently treated with gold conjugated secondary antibody, did not show any gold labelling of rubber particles in all the species examined (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Observations of the size, form and architecture of rubber particles, and characterization of the proteins on the surface of rubber particles are of great importance in understanding the mechanism of rubber chain elongation and chain length determination, and also in relation to the properties of the final rubber products. It was stated at the outset that microscopic characterization of rubber particles is difficult (Condon and Fineran, 1989) probably because the membrane which encloses rubber material within individual particles is highly sensitive to the chemical treatments used in conventional microscopy preparations. Aldehydes destabilize latex particles, rendering them susceptible to fusion, and dehydrating agents, such as acetone and ethanol, cause extraction of internal contents (Condon and Fineran, 1989). It is therefore not surprising that the initial attempts to process rubber samples by conventional preparation methods for SEM and TEM observations were unsuccessful. Tannic acid is known to stabilize some tissue components against extraction (Simionescu and Simionescu, 1976), which was used as an additional fixative mixed with aldehyde. Tannic acid has also been used to increase osmification of tissue culture cells (Katsumoto et al., 1981). In the SEM work, initial fixation was with a combination of glutaraldehyde and tannic acid, and post-fixation with osmium tetroxide. It appears that a combination of these treatments stabilized rubber particles adequately for them to withstand the forces of surface tension generated during air-drying. Interestingly, the same fixation procedure used in conjunction with CPD failed to stabilize particles, large populations of which were severely collapsed. It is likely that the bounding membrane was not completely stabilized and became leaky during the ethanol-dehydration step of CPD, resulting in the extraction of the internal contents of rubber particles, and their consequent collapse. This highlights the importance of developing a suitable method for preserving rubber particles in their natural size and form, because these, together with compositional characteristics, are likely to be important factors in determining the properties of rubber in industrial processing.
The shape and size distribution of the rubber particles detected in this study compare well with the previously reported results (Gomez and Hamzah, 1989; Cornish et al., 1993; Wood and Cornish, 2000). Among the three plant species examined, H. brasiliensis, producing high molecular weight rubber, had the smallest rubber particles and F. carica, producing low molecular weight rubber, had the largest rubber particles. It has been suggested in the previous report that higher molecular weights were associated with the smaller rubber particles (Yeang et al., 1995). However, more extensive analysis is required for a better understanding of the correlation between rubber particle size and the molecular weight of rubber in plants.
Conventionally, a mixture of paraformaldehyde and glutaraldehyde has been used to fix biological materials in immuno-TEM studies. In the fixation medium, these components are used in such a proportion that a proper balance between adequate fixation of cell components and antigenicity is achieved. However, in this immuno-TEM work, glutaraldehyde-based fixation resulted in poor stabilization of rubber particles and non-specific gold labelling. The fixation of the rubber fraction directly in osmium tetroxide proved to be the most satisfactory method. Although for immunocytochemistry this is not a fixative of choice because of concerns that it may adversely affect tissue antigenicity, the high labelling intensity of H. brasiliensis rubber particles, which had been incubated in the SRPP antibody, suggests that immunogenic properties of the bounding membrane of rubber particles were not affected by osmium fixation, and this is a suitable fixation method for rubber material.
The role of the SRPP in rubber biosynthesis has not been clearly verified. The study has provided additional information by investigating whether the SRPP found on the surface of Hevea rubber particles is also present on the rubber particles of F. carica and F. benghalensis. The observations presented here clearly indicate that the rubber particles of F. carica and F. benghalensis do not contain the SRPP found in H. brasiliensis (Figs 4, 5). The absence of the SRPP in F. carica and F. benghalensis implies that the SRPP, the most abundant protein in rubber tree, is not a universal protein required for rubber biosynthesis in rubber-producing plant species. It has been suggested that the SRPP plays a positive role in rubber biosynthesis (Oh et al., 1999). If this protein is a rubber transferase or forms a rubber transferase complex, a similar protein should be present on the rubber particles of every rubber-producing plant species. No cross-reactivity was found of the rubber particle proteins isolated from F. carica and F. benghalensis with the SRPP antibody, which implies that the SRPP is not a rubber transferase required for rubber chain elongation.
Rubber transferase belongs to a family of CPT that catalyses the synthesis of linear prenyl diphosphates involved in the biosynthesis of various isoprenoid compounds including natural rubber. The plant CPT gene has recently been cloned from Arabidopsis, and the analysis has shown that the Arabidopsis CPT catalyses the formation of polyprenyl diphosphates with the predominant carbon number C120 (Oh et al., 2000). Since no report confirming the function of CPT genes from rubber-producing plants has yet been published, it is of importance to investigate if the CPTs are present on the surface of rubber particles. The western and immunogold label analyses showed that the rubber particles of all three plant species tested, F. carica, H. brasiliensis, and F. benghalensis, do not contain the CPT. This implies that CPT itself is not a part of the rubber transferase complex. Although the three rubber-producing plants do not contain CPTs on their rubber particles, it would be of interest to isolate the CPT from rubber-producing plant species, and to test if the CPT is involved in catalysing the formation of higher molecular weight polymers similar to natural rubber.
|
Acknowledgements |
|---|
This work was supported in part by a grant (No. R11-2001-09201004-0) from the Korea Science and Engineering Foundation (KOSEF) to the Agricultural Plant Stress Research Center, and by a grant (No. 297066-5) from the Agricultural Research Promotion Center, Korea Ministry of Agriculture. We thank Mr Sug-Ju Ko and the Jeonnam Agricultural Research and Extension Service for use of their TEM facilities. Adya Singh is grateful to KOSEF for the Brain Pool Scientist Award.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Attanyaka DPSTG, Kekwick RGO, Franklin FCH. 1991. Molecular cloning and nucleotide sequencing of the rubber elongation factor gene from Hevea brasiliensis. Plant Molecular Biology 16, 1079–1081.[CrossRef][ISI][Medline]
Backhaus RA, Cornish K, Chen SF, Huang, DS, Bess VH. 1991. Purification and characterization of an abundant rubber particle protein from guayule. Phytochemistry 30, 2493–2497.[CrossRef][ISI]
Bahri ARS, Hamzah S. 1996. Immunocytochemical localization of rubber membrane protein in Hevea latex. Journal of Natural Rubber Research 11, 88–95.
Benedict CR, Madhavan S, Greenblatt GA, Venkatachalam KV, Foster MA. 1990. The enzymatic synthesis of rubber polymer in Parthenium argentatum Gray. Plant Physiology 92, 816–821.
Condon JM, Fineran BA. 1989. The effect of chemical fixation and dehydration on the preservation of latex in Calystegia silvatica (Convolvulaceae): examination of exudate and latex in situ by light and scanning electron microscopy. Journal of Experimental Botany 40, 925–939.
Cornish K. 1993. The separate roles of plant cis and trans prenyl transferase in cis-1,4-polyisoprene biosynthesis. European Journal of Biochemistry 218, 267–271.[ISI][Medline]
Cornish K. 2001. Similarities and differences in rubber biochemistry among plant species. Phytochemistry 57, 1123–1134.[CrossRef][ISI][Medline]
Cornish K, Backhaus RA. 1990. Rubber transferase activity in rubber particles of guayule. Phytochemistry 29, 3809–3813.[CrossRef][ISI]
Cornish K, Siler DJ. 1996. Characterization of cis-prenyl transferase activity localized in a buoyant fraction of rubber particles from Ficus elastica latex. Plant Physiology and Biochemistry 34, 377–384.
Cornish K, Siler DJ, Grosjean OK. 1994. Immunoinhibition of rubber particle-bound cis-prenyltransferases in Ficus elastica and Parthenium argentatum. Phytochemistry 35, 1425–1428.[CrossRef]
Cornish K, Siler DJ, Grosjean OK, Goodman N. 1993. Fundamental similarities in rubber particle architecture and function in three evolutionarily divergent plant species. Journal of Natural Rubber Research 8, 275–285.
Cornish K, Wood DF, Windle JJ. 1999. Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogenous rubber core enclosed by a contiguous, monolayer biomembrane. Planta 210, 85–96.[CrossRef][ISI][Medline]
Dennis MS, Light DR. 1989. Rubber elongation factor from Hevea brasiliensis: identification, characterization, and role in rubber biosynthesis. Journal of Biological Chemistry 264, 18608–18617.
Gomez JB, Hamzah S. 1989. Particle size distribution in Hevea latex-some observations on the electron microscopic method. Journal of Natural Rubber Research 4, 204–211.
Goyvaerts E, Dennis MS, Light DR, Chua NH. 1991. Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis. Plant Physiology 97, 317–321.
Hasma H. 1991. Lipids associated with rubber particles and their possible role in mechanical stability of latex concentrates. Journal of Natural Rubber Research 6, 105–114.
Hasma H, Subramaniam A. 1986. Composition of lipids in latex of Hevea brasiliensis clone RRIM 501. Journal of Natural Rubber Research 1, 30–40.
Kang H, Kang MY, Han KH. 2000a. Identification of natural rubber and characterization of rubber biosynthetic activity in fig trees. Plant Physiology 123, 1133–1142.
Kang H, Kim YS, Chung GC. 2000b. Characterization of natural rubber biosynthesis in Ficus benghalensis. Plant Physiology and Biochemistry 38, 979–987.[CrossRef][ISI]
Katsumoto T, Naguro T, Tino A, Takagi A. 1981. The effect of tannic acid on the preservation of tissue culture cells for scanning electron microscopy. Journal of Electron Microscopy 30, 177–182.
Kim YS, Wi SG, Grinwald C, Schmitt U. 2002. Immuno electron microscopic localization of peroxidases in the differentiating xylem of Populus spp. Holzforschung 56, 355–359.[CrossRef]
Light DR, Dennis MS. 1989. Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis. Journal of Biological Chemistry 264, 18589–18597.
Ogura K, Koyama T. 1998. Enzymatic aspects of isoprenoid chain elongation. Chemical Reviews 98, 1263–1276.[CrossRef][ISI][Medline]
Oh SK, Han KH, Ryu SB, Kang H. 2000. Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from Arabidopsis thaliana: implication in rubber biosynthesis. Journal of Biological Chemistry 275, 18482–18488.
Oh SK, Kang H, Shin DH, Yang J, Chow KS, Yeang HY, Wagner B, Breiteneder H, Han KH. 1999. Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis. Journal of Biological Chemistry 274, 17132–17138.
Pan Z, Durst F, Werck-Reinchhart D, Gardner HW, Camara B, Cornish K, Backhaus RA. 1995. The major protein of guayule rubber particle is a cytochrome P450: characterization based on cDNA cloning and spectroscopic analysis of the solubilized enzyme and its reaction products. Journal of Biological Chemistry 270, 8487–8494.
Siler DJ, Cornish K. 1993. A protein from Ficus elastica rubber particles is related to proteins from Hevea brasiliensis and Parthenium argentatum. Phytochemistry 32, 1097–1102.[CrossRef]
Siler DJ, Cornish K. 1994. Identification of Parthenium argentatum rubber particle proteins immunoprecipitated by an antibody that specifically inhibits rubber transferase activity. Phytochemistry 36, 623–627.[CrossRef]
Siler DJ, Goodrich-Tanrikulu M, Cornish K, Stafford AE, Mckeon TA. 1997. Composition of rubber particles of Hevea brasiliensis, Parthenium argentatum, Ficus elastica, and Euphorbia lactiflua indicates unconventional surface structure. Plant Physiology and Biochemistry 35, 881–889.
Simionescu N, Simionescu M. 1976. Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. Journal of Cell Biology 70, 608–621.
Wood DF, Cornish K. 2000. Microstructure of purified rubber particles. International Journal of Plant Science 161, 435–445.[CrossRef][ISI][Medline]
Yeang HY, Yip E, Hamzah S. 1995. Characterization of zone 1 and zone 2 rubber particles in Hevea brasiliensis latex. Journal of Natural Rubber Research 10, 108–123.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  K.-S. Chow, K.-L. Wan, Mohd. N. M. Isa, A. Bahari, S.-H. Tan, K Harikrishna, and H.-Y. Yeang Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex J. Exp. Bot., July 1, 2007; 58(10): 2429 - 2440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Kim, S. B. Ryu, Y. S. Kwak, and H. Kang A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro* J. Exp. Bot., February 1, 2004; 55(396): 377 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Singh, S. G. Wi, H. Kang, G. C. Chung, and Y. S. Kim Simple and Rapid Methods for SEM Observation and TEM Immunolabeling of Rubber Particles J. Histochem. Cytochem., August 1, 2003; 51(8): 1105 - 1108. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||