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.     

Natural rubber biosynthesis in plants,the rubber transferase complex,and metabolic engineering progress and prospects

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.     

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.     

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.     

First report of Bipolaris bicolor causing a leaf spot disease on rubber tree

The rubber tree (Hevea brasiliensis) is the only resource for commercial natural rubber production and thus has economic importance in Southeast Asia. A spot disease on the leaves of a rubber tree was first discovered in 2017 in Hainan, China. In this study, the fungal isolate MA1 from the infected tissues was determined to be a pathogen of the spot disease by satisfying Koch's postulates. The isolate MA1 was identified as Bipolaris bicolor based on the morphological characteristics and multigene phylogenetic analysis. Among fungicides, prochloraz, iprodione and pyraclostrobin significantly inhibited hyphal growth of B. bicolor under in vitro conditions. This study constitutes the first report on the association of B. bicolor with leaf spot disease of rubber trees worldwide.     

Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis

Hydroxy‐methylglutaryl coenzyme‐A synthase (HMGS) is a rate‐limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non‐coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three‐dimensional structural studies highlighting the structural positioning of non‐synonymous SNPs from different HMGS haplotypes revealed that the ligand‐binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.     

Ultrastructure of Laticifers Modifications in Hevea brasiliensis Infected with Root Rot Fungi

This paper presents some aspects of latex coagulation inside latice vessels of roots of Hevea brasiliensis infected by two fungi: Rigidoporus lignosus and Phellinus noxius. Three stages are described in latex coagulation: the phase of latex destabilisation characterized by the bursting of vacuoles and lysosomes membranes; the phase of latex coagulation characterized by the fusion of rubber particles and the disorganization of the cytoplasm; the formation of shots of rubber clumps indicating the final stage of coagulation.     

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.     

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.     

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.     

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1–5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1–5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3–5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.     

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.     

Alternative sources of natural rubber

Rubber (cis-1,4-polyisoprene) is one of the most important polymers naturally produced by plants because it is a strategic raw material used in more than 40,000 products, including more than 400 medical devices. The sole commercial source, at present, is natural rubber harvested from the Brazilian rubber tree, Hevea brasiliensis. Primarily due to its molecular structure and high molecular weight (>1 million daltons) this rubber has high performance properties that cannot easily be mimicked by artificially produced polymers, such as those derived from, e.g., bacterial poly-hydroxy-alkanoates (PHAs). These high performance properties include resilience, elasticity, abrasion resistance, efficient heat dispersion (minimizing heat build-up under friction), and impact resistance. Medical rubber gloves need to fit well, be break-resistant, allow the wearer to retain fine tactile sensation, and provide an effective barrier against pathogens. The sum of all these characteristics cannot yet be achieved using synthetic gloves. The lack of biodiversity in natural rubber production renders continuity of supply insecure, because of the risk of crop failure, diminishing acreage, and other disadvantages outlined below. A search for alternative sources of natural rubber production has already resulted in a large number of interesting plants and prospects for immediate industrial exploitation of guayule (Parthenium argentatum) as a source of high quality latex. Metabolic engineering will permit the production of new crops designed to accumulate new types of valued isoprenoid metabolites, such as rubber and carotenoids, and new combinations extractable from the same crop. Currently, experiments are underway to genetically improve guayule rubber production strains in both quantitative and qualitative respects. Since the choice for gene activities to be introduced or changed is under debate, we have set up a complementary approach to guayule with yeast species, which may more quickly show the applicability and relevance of genes selected. Although economic considerations may prevent commercial exploitation of new rubber-producing microorganisms, transgenic yeasts and bacteria may yield intermediate or alternative (poly-)isoprenes suitable for specific applications. Received: 9 August 1999 / Received revision: 15 October 1999 / Accepted: 16 October 1999     

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.     

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.     

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     

Regulation of Natural Rubber Biosynthesis by Proteins Associated with Rubber Particles

Natural rubber, cis-1,4-polyisoprene, is an essential raw material used in thousands of products, many of which are absolutely necessary for medical purposes. Natural rubber is obtained from latex, an aqueous emulsion present in the laticiferous vessels of the natural rubber-producing plants. Hevea brasiliensis (the Brazilian rubber tree) currently is the only commercially important source of natural rubber. H. brasiliensis crops have very little genetic variability, leaving rubber plantations at risk of serious pathogenic attacks. In addition, repeated exposure to residual proteins in latex products derived from H. brasiliensis have led to serious and widespread allergic (type I) hypersensitivity. Therefore, identification of alternative sources of natural rubber is a very important biotechnological task. Potentially, Russian dandelion (Taraxacum kok-saghyz) may be such an alternative because significant amounts of natural rubber are produced in its root system. However, H. brasiliensis is a more efficient producer of natural rubber than T. kok-saghyz. Thus, improvement of rubber biosynthesis in plants is a first-priority problem of modern biotechnology. In this review, we describe proteins that may increase the concentration of natural rubber in laticiferous vessels of T. kok-saghyz and its close relative Taraxacum brevicorniculatum, when overexpressed in the plants. These proteins, cis-prenyltransferases, rubber transferase activator, and small rubber particle proteins, are directly involved in synthesis of the polyisoprene chain. We also analyze the effects of their expression levels on the production of natural rubber in vivo.     

Climate and natural production of rubber (Hevea brasiliensis) in Xishuangbanna,southern part of Yunnan province,China

According to the author's and his collaborators' investigations, the climate influences the growth of rubber trees (Hevea brasiliensis) in Xishuangbanna, the southern part of Yunnan Province, China, in at least four aspects: (1) The yield of latex per tapping and the final yield of dry rubber per tree per year or per unit area per year; (2) the growth rate, as expressed by increment of girth in cm; (3) the survival during the over-wintering period; (4) the initiation or suppression of certain diseases; In this paper the author would like to describe the influence of climatic elements on yield of latex and on survival during the over-wintering period. As for the other two aspects, only general comments are given.     

Age-dependent and jasmonic acid-induced laticifer-cell differentiation in anther callus cultures of rubber tree

Main conclusion

Callus cultures of rubber tree may serve as an efficient model to screen and study environmental factors and phytohormones that stimulate laticifer cell differentiation and improve latex yield. The number of laticifer cells in bark is one of the most important factors determining the biosynthesis and economic value of rubber trees (Hevea brasiliensis). The differentiation of laticifer cells in planta has been characterized, whereas laticifer-cell differentiation in callus cultures in vitro is largely unknown. In this study, we present molecular and physiological evidences for laticifer-cell differentiation in calli derived from rubber tree anthers. RT-PCR analysis showed that three key genes rubber elongation factor (REF), small rubber particle protein (SRPP), and cis-prenyl transferase (CPT) that are essential in latex biosynthesis in rubber tree bark also were transcribed in anther calli. Laticifer cell development in callus cultures was age-dependent; the cells began to appear at 58 days after initiation of culture, and the percentage of laticifer cells increased steadily with increasing callus age. Addition of 0–2 mg/L jasmonic acid (JA) to the media significantly promoted the differentiation of laticifer cells in callus cultures. However, JA concentrations higher than 3 mg/L were not optimum for laticifer cells differentiation; this result was not observed in previous in planta studies. Laticifer cells differentiated on media with pH 5.8–7.0, with an optimum of pH 6.2, whereas a higher pH inhibited differentiation. These results indicate that the anther-derived rubber tree callus may serve as a new and more efficient model to study environmental factors that influence laticifer cell differentiation, and may be useful for research on new technologies to improve latex yield, and to screen for commercially useful phytohormones.