New aspects of rubber biosynthesis

New aspects of rubber biosynthesis. Following a review of the site of rubber biosynthesis in Hevea brasiliensis and Parthenium argentalum, evidence is given for the initiation of polyisoprene molecules from (ranMerpenoid precursors including geranylgeranyl pyrophosphate. All franj-¹⁴C-geranylgeraniol has been isolated from incubations of H. brasiliensis latex serum with ¹⁴C-isopentenyl pyrophosphate. Gel-filtration chromatography of the serum yields very small rubber particles of high biosynthetic activity, and two proteinaceous fractions. One of these increases the biosynthesis of rubber and may contain the enzyme, isopentenyldiphosphate δ-isomerase, whilst the other appears to inhibit rubber formation. The nature and molecular weight of the rubber formed in vitro is discussed and a mechanism for the de novo formation of rubber particles is suggested.     

Characterization of natural rubber biosynthesisin Ficus benghalensis

Natural rubber was identified for the first time in the latex of Ficus benghalensis, and the rubber biosynthetic activity in latex and rubber particles was investigated. ¹³C NMR analysis of samples prepared by successive extractions with acetone and benzene confirmed that the benzene-soluble residues were natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of F. benghalensis was approximately 17 %. Gel permeation chromatography revealed that the molecular mass of the natural rubber from F. benghalensis was approximately 1 500 kDa. The high rubber content and large molecular size suggest that F. benghalensis is a good candidate for an alternative rubber source. Examination of latex serum from F. benghalensis by SDS-polyacrylamide gel electrophoresis revealed a small number of proteins with major proteins of 31 and 55 kDa in size. The 31-kDa protein was predominant in catalytically-active rubber particles. Determination of metal ion concentration in latex and a comparison of the effect of ethylenediamine-tetraacetic acid on in vitro rubber biosynthesis in F. benghalensis, F. carica and Hevea brasiliensis suggest that the divalent metal ion present in latex serum is an important physiological factor controlling the rubber biosynthetic activities in these plant species. Microscopic examination revealed that the rubber in F. benghalensis occurred in a series of laticifer cells located in concentric zones in the inner bark of stems and branches.     

Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.     

巴西橡胶树胶乳中DUF1262结构域蛋白结构与基因表达分析

为了揭示天然橡胶生物合成酶互作蛋白结构及其在天然橡胶生物合成过程中的功能。本研究以橡胶树胶乳橡胶粒子总蛋白为研究对象,采用免疫共沉淀实验技术以天然橡胶合成关键酶顺式-异戊二烯基转移酶（CPT）抗体从胶乳中捕获了1个含DUF1262结构域的未知功能蛋白。生物信息学分析表明橡胶树基因组中包含50个编码含DUF1262结构域蛋白的基因序列;蛋白质相互作用网络分析表明DUF1262结构域蛋白可能参与调节信号转导或转录调控等过程;荧光定量PCR结果表明编码该蛋白基因的转录本在根、叶、花、枝和胶乳等组织中广泛分布,但在胶乳中表达较低,在树皮表达较高;水杨酸、脱落酸、过氧化氢及干旱处理可增强该基因在叶片中的转录水平。本研究证明DUF1262参与橡胶树逆境反应等生理过程,为揭示胶乳生物合成调控机制提供新线索。     

Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis

We have purified "rubber transferase" from latex of the commercial rubber tree Hevea brasiliensis and find that it is a dimer with a monomeric molecular mass of 38,000 Da, requires Mg2+, and is stabilized by thiols in agreement with studies of a partially purified preparation previously described (Archer, B. L., and Cockbain, E. G. (1969) Methods Enzymol. 15, 476-480). Greater than 90% of the [1-14C]isopentenyl pyrophosphate which is incorporated into deproteinated rubber particles by the purified prenyltransferase is added to high molecular mass polyisoprene (greater than 20,000 Da). Purified prenyltransferase and deproteinated rubber particles reconstitute 40-60% of the biosynthetic activity of whole latex in samples matched for rubber content. Incorporation is linear with added rubber particles up to at least 10 mg/ml rubber or 20 microM rubber molecules (based on a number average molecular mass of 500,000 Da). Prenyltransferase concentrations estimated in whole latex (0.37% or 160 nM) are sufficient to saturate all elongation sites in whole latex, and addition of purified prenyltransferase does not increase [1-14C]isopentenyl pyrophosphate incorporation. Deproteinated rubber particles can be titrated with the pure enzyme (Kd = 9 nM) demonstrating that the fraction of rubber molecules available for addition is low (approximately 0.01%). An estimated 7,000 isoprene units are added per complex at a rate of 1/s in a typical assay. Hevea prenyltransferase catalyzes the formation of cis-isoprene in the presence of rubber particles. However, in the absence of rubber particles and in the presence of dimethylallyl pyrophosphate, the purified prenyltransferase catalyzes the formation of geranyl pyrophosphate and all trans-farnesyl pyrophosphate as demonstrated by thin layer chromatography, gas chromatography, and molecular exclusion chromatography.     

The biosynthesis of rubber: Incorporation of isopentenyl pyrophosphate into purified rubber particles by a soluble latex serum enzyme

1. The rubber particles in Hevea brasiliensis latex have been partially purified by `washing' with buffer solution, and separated into active fractions of different particle size. 2. The enzyme responsible for incorporating isopentenyl pyrophosphate into rubber is distributed between the surface of the rubber particles and the aqueous serum phase of the latex. The enzyme at the surface can be removed or inactivated if the rubber particles are washed sufficiently with buffer solution. Enzyme in the serum phase can be concentrated by fractional precipitation with ammonium sulphate. 3. To incorporate isopentenyl pyrophosphate into rubber in vitro, active rubber particles are required as well as enzyme and soluble cofactors. The activity of the rubber particles per unit surface area increases with diminishing particle size.     

Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis)

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.     

Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica

The rubber-producing tree, Ficus elastica (the Indian rubber tree), requires the same substrates for rubber production as other rubber-producing plants, such as Hevea brasiliensis (the Brazilian or Para rubber tree), the major source of commercial natural rubber in the world, and Parthenium argentatum (guayule), a widely studied alternative for natural rubber production currently under commercial development. Rubber biosynthesis can be studied, in vitro, using purified, enzymatically active rubber particles, an initiator such as FPP, IPP as the source of monomer, and a metal cofactor such as Mg2+. However, unlike H. brasiliensis and P. argentatum, we show that enzymatically active rubber particles purified from F. elastica are able to synthesize rubber, in vitro, in the absence of added initiator. In this paper, we characterize, for the first time, the kinetic differences between initiator-dependent rubber biosynthesis, and initiator-independent rubber biosynthesis, and the effect of cofactor concentration on both of these processes.     

Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis

Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis‐prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF‐silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF‐silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production.     

Chromosome-level wild Hevea brasiliensis genome provides new tools for genomic-assisted breeding and valuable loci to elevate rubber yield

The rubber tree (Hevea brasiliensis) is grown in tropical regions and is the major source of natural rubber. Using traditional breeding approaches, the latex yield has increased by sixfold in the last century. However, the underlying genetic basis of rubber yield improvement is largely unknown. Here, we present a high-quality, chromosome-level genome sequence of the wild rubber tree, the first report on selection signatures and a genome-wide association study (GWAS) of its yield traits. Population genomic analysis revealed a moderate population divergence between the Wickham clones and wild accessions. Interestingly, it is suggestive that H. brasiliensis and six relatives of the Hevea genus might belong to the same species. The selective sweep analysis found 361 obvious signatures in the domesticated clones associated with 245 genes. In a 15-year field trial, GWAS identified 155 marker–trait associations with latex yield, in which 326 candidate genes were found. Notably, six genes related to sugar transport and metabolism, and four genes related to ethylene biosynthesis and signalling are associated with latex yield. The homozygote frequencies of the causal nonsynonymous SNPs have been greatly increased under selection, which may have contributed to the fast latex yield improvement during the short domestication history. Our study provides insights into the genetic basis of the latex yield trait and has implications for genomic-assisted breeding by offering valuable resources in this new domesticated crop.     

Laser diffraction: A new tool for identification and studies of physiological effectors involved in aggregation-coagulation of the rubber particles from Hevea latex

Latex coagulation is the main limiting factor of rubber yield in Hevea brasiliensis (rubber tree). Using laser diffraction, we set up and optimized a new method for monitoring the kinetics of rubber particle (RP) aggregation, a prerequisite for latex coagulation. In contrast to any previous method used, laser diffraction allows continuous monitoring changes in size of RP aggregates, thereby allowing characterization and quantification of the processes involved in latex coagulation. Using this technique, we confirm that RP aggregating factors are proteins compartmentalized within latex cell vacuoles (lutoids), which, especially at relatively acidic physiological pH, can induce formation of RP aggregates large enough to induce plugging of severed latex vessel extremities. Conversely, latex cytosol was found to harbor anti-aggregating proteins. Further, we were able to titrate the RP-aggregating efficiency of the intralutoidic serum and the anti-aggregating efficiency of the cytosol. Preliminary assays showed that these two parameters were correlated with the yield potential of the tested rubber clones. This method will allow identification and characterization of proteins involved in latex coagulation, hence in rubber yield. We suggest that laser diffraction could be used to monitor the kinetics and characterize the physiological processes involved in aggregation of any particles, organelles or cells.     

Laticifer-specific cis-prenyltransferase silencing affects the rubber, triterpene, and inulin content of Taraxacum brevicorniculatum

Certain Taraxacum species, such as Taraxacum koksaghyz and Taraxacum brevicorniculatum, produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; EC 2.5.1.20) is a cis-prenyltransferase (CPT). Here, we present a comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently isolated CPTs known to be associated with rubber particles (TbCPT1 to -3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by nuclear magnetic resonance, size-exclusion chromatography, and gas chromatography-mass spectrometry indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-coenzyme A reductase, the key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool. To our knowledge, this is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.     

Micro‐organisms in latex and natural rubber coagula of Hevea brasiliensis and their impact on rubber composition,structure and properties

Natural rubber, produced by coagulation of the latex from the tree Hevea brasiliensis, is an important biopolymer used in many applications for its outstanding properties. Besides polyisoprene, latex is rich in many nonisoprene components such as carbohydrates, proteins and lipids and thereby constitutes a favourable medium for the development of micro‐organisms. The fresh rubber coagula obtained by latex coagulation are not immediately processed, allowing the development of various microbial communities. The time period between tree tapping and coagula processing is called maturation, during which an evolution of the properties of the corresponding dry natural rubber occurs. This evolution is partly related to the activity of micro‐organisms and to the modification of the biochemical composition. This review synthesizes the current knowledge on microbial populations in latex and natural rubber coagula of H. brasiliensis and the changes they induce on the biochemistry and technical properties of natural rubber during maturation.     

橡胶蛋白——种与胶乳凝固有关的具有抗真菌活性的植物凝集素

 橡胶蛋白(Hevein)是一种存在于巴西橡胶树(Hevea brasiliensis)乳管细胞特化的液泡——黄色体(lutoid)内、与几丁质结合的小分子(4.7kD)单链蛋白质,在橡胶粒子的凝絮和胶乳的凝固过程中表现出极为重要的作用,是一类具有抗真菌活性的植物凝集素。     

Structural characterization of rubber from jackfruit and euphorbia as a model of natural rubber

A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from Hevea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from Hevea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The alpha-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal =CH-CH2OP group.     

Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex

Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy–immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.     

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree)

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches—resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9–194.2 kDa and in isoelectric point values of 4.0–11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.