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.     

Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X‐100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30–35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M_w/M_n of 2.9. ¹H NMR, ¹³C NMR, and FTIR analysis revealed the characteristic of the cis‐1,4‐polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 589–594, 2012.     

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.     

The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree)

Efficient sucrose loading in rubber‐producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K_m value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H ⁺ symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber‐containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue‐specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield‐stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.     

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz – role of cis‐prenyltransferase‐like 1 protein

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.     

Chitinase and β-1,3-glucanase in the lutoid-body fraction of Hevea latex

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a β-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charge neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.     

Organic acids and protein compounds causing the photoluminescence properties of natural rubber membranes and the quenching phenomena from Au nanoparticle incorporation

Natural rubber membranes were fabricated using latex from Hevea brasiliensis trees (clone RRIM 600) by casting, and controlling the time and temperature of thermal treatment. Three temperatures were used: 65, 80 and 120 °C and the corresponding annealing times of 6, 8, 10 and 12 h. The centrifugation of the latex produces the constituent phases: solid rubber (F1), serum or protein components (F2) and bottom fraction (F3). The photoluminescence properties could be correlated with organic acid components of latex. Natural rubber membranes were used as the active substrate (reducing agent) for the incorporation of colloidal Au nanoparticles synthesized by in situ reduction at different times. The intensity of photoluminescence bands assigned to the natural rubber decreases with the increase in amount of nanoparticles present on the membrane surface. It can be assumed that Au nanoparticles may be formed by reduction of the Au cation reacting with functional groups that are directly related to photoluminescence properties. However, the quenching of fluorescence may be attributed to the formation of a large amount of metal nanostructures on the natural rubber surface. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation

An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M(r) analyzed by SDS-PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca(2+). Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogenous latex lectin-like protein in the sequential process of latex coagulation.     

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.     

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.     

Biological coagulation of skim latex using Acinetobacter sp. isolated from natural rubber latex centrifugation effluent

An Acinetobacter sp, isolated from latex centrifugation effluent, effectively coagulated skim rubber from skim latex. After coagulation for 48 h without the addition of any nutrients, at an optimum dilution of 1:10(v/v) and with an inoculum concentration of 6.4 mg dry cell /ml, the yield of the skim rubber was 8 % (w/v) and the COD of the residual solution was only 0.4 g/l. chemical coagulation at the same dilution resulted in 7 % (w/v) yield of dry rubber content and 2.2 g COD /l.     

Guayule and Russian Dandelion as Alternative Sources of Natural Rubber

ABSTRACT

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.     

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.     

Homologous Hevea brasiliensis REF (Hevb1) and SRPP (Hevb3) present different auto-assembling

HbREF and HbSRPP are two Hevea brasiliensis proteins present on rubber particles, and probably involved in the coagulation of latex. Their function is unclear, but we previously discovered that REF had amyloid properties, which could be of particular interest during the coagulation process. First, we confirmed that REF and SRPP, homologous and principal proteins in hevea latex, are not glycoproteins. In this work, we investigated various aspects of protein interactions: aggregation, auto-assembling, yeast and erythrocyte agglutination, co-interactions by various biochemical (PAGE, spectroscopy, microscopy), biophysical (DLS, ellipsometry) and structural (TEM, ATR-FTIR, PM-IRRAS) approaches. We demonstrated that both proteins are auto-assembling into different aggregative states: REF polymerizes as an amyloid rich in β-sheets and forms quickly large aggregates (> μm), whereas SRPP auto-assembles in solution into stable nanomultimers of a more globular nature. Both proteins are however able to interact together, and SRPP may inhibit the amyloidogenesis of REF. REF is also able to interact with the membranes of yeasts and erythrocytes, leading to their agglutination. In addition, we also showed that both REF and SRPP did not have antimicrobial activity, whereas their activity on membranes has been clearly evidenced. We may suspect that these aggregative properties, even though they are clearly different, may occur during coagulation, when the membrane is destabilized. The interaction of proteins with membranes could help in the colloidal stability of latex, whereas the protein–protein interactions would contribute to the coagulation process, by bringing rubber particles together or eventually disrupting the particle monomembranes.     

Ethylene stimulation of latex production in Hevea brasiliensis

Rubber tree (Hevea brasiliensis) is an important industrial crop for natural rubber production. Ethylene, as a stimulant of latex production in H. brasiliensis, has been widely used in commercial latex production. However, the mechanism of ethylene action are not completely elucidated, especially in molecular aspect. Here, we focus on the molecular biological progression of ethylene stimulation of latex production. Our data and all previous information showed ethylene had little direct effect on accelerating rubber biosynthesis. The prolonged latex flow and acceleration of sucrose metabolism by ethylene may be the main reasons for the stimulation of latex yield by ethylene.Key words: Hevea brasiliensis, ethylene, rubber production, gene, sucrose     

Hevea latex lectin binding protein in C-serum as an anti-latex coagulating factor and its role in a proposed new model for latex coagulation

A distinct protein specifically recognized by its strong interaction with Hevea latex lectin (HLL) was detected in the aqueous C-serum fraction of centrifuged fresh latex. This C-serum lectin binding protein (CS-HLLBP) exhibited strong inhibition of HLL-induced hemagglutination. The CS-HLLBP was purified to homogeneity by a protocol that included ammonium sulfate fractionation, size exclusion and ion exchange chromatography. The purified CS-HLLBP had a specific HI titer of 0.23microg ml(-1). Its M(r)s analyzed by SDS-PAGE was ca. 40kDa and that by gel filtration was ca. 204kDa. It has a pI value of 4.7, an optimum activity between pH 6 and10 and was heat stable up to 50 degrees C. The HI activity of CS-HLLBP was abolished upon treatment with chitinase. The CS-HLLBP inhibited HLL-induced rubber particle aggregation in a dose dependent manner. A highly positive correlation between CS-HLLBP activity and rubber yield per tapping was found. The correlations for fresh latex (r=0.98, P<0.01) and dry rubber (r=0.95, P<0.01) were both highly significant. This indicated that the CS-HLLBP might be used as a reliable marker for the mass screening of young seedlings to identify and select clones with potential to be superior producers of rubber. A latex anti-coagulating role of the CS-HLLBP is proposed. The findings described in this 3 paper series have been used to propose a new model of rubber latex coagulation that logically describes roles for the newly characterized latex lectin and the two lectin binding proteins.     

A method for protein extraction from different subcellular fractions of laticifer latex in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> compatible with 2-DE and MS

Background  

Proteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.