Guayule and Russian dandelion as alternative sources of natural rubber

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.     

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Natural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink‐to‐source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum and functionally characterized the key enzyme fructan 1‐exohydrolase (1‐FEH), which catalyses the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1‐FEH almost doubled the rubber content in the roots of two dandelion species without any trade‐offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber‐enriched varieties for industrial rubber production.     

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.     

Establishment of new crops for the production of natural rubber

Natural rubber is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic alternatives. The rubber tree Hevea brasiliensis is the almost exclusive commercial source of natural rubber currently and alternative crops should be developed for several reasons, including: a disease risk to the rubber tree that could potentially decimate current production, a predicted shortage of natural rubber supply, increasing allergic reactions to rubber obtained from the Brazilian rubber tree and a general shift towards renewables. This review summarizes our knowledge of plants that can serve as alternative sources of natural rubber, of rubber biosynthesis and the scientific gaps that must be filled to bring the alternative crops into production.     

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.     

Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein,TkSRPP3, result in qualitative and quantitative changes in rubber metabolism

Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer.     

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     

Rubber-The Primary Sources For American Production

Despite extensive war-time investigations of other rubber-containing latex plants, the Pará rubber tree of Brazil remains, by any measure, the world's foremost source, and only guayule and the Russian dandelion display any possibility of becoming secondary sources of commercial importance.     

Antioxidant Sources from Leaves of Russian Dandelion

Taraxacum kok‐saghyz (TKS) is a dandelion species native to Kazakhstan, Uzbekistan and north‐west China, considered as a promising alternative source of natural rubber from its roots. The aim of this study was to investigate the possible exploitation of TKS leaves, a rubber byproduct, as a source of phenolic compounds with antioxidant properties for potential applications in forage, nutraceutical and pharmacological fields. Two accessions (TKS016, TKS018) grown under Mediterranean conditions of Sardinia were evaluated at vegetative and flowering stages. The leaves of TKS018 had the highest antioxidant capacity (19.6 mmol trolox equivalent antioxidant capacity 100 g^?1), total phenolic (106.4 g gallic acid equivalent kg^?1), tannic phenolics (58.5 g gallic acid equivalent kg^?1) and total flavonoid contents (22.9 g catechin equivalent kg^?1). At both phenological stages, TKS016 showed significantly lower values than TKS018 in 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), total phenolic and tannic phenolics. Six individual molecules were identified, namely chlorogenic, cryptochlorogenic, caffeic, sinapic, chicoric and 3,4‐dimethoxycinnamic acids. Chicoric (8.53–10.68 g kg^?1 DW) and chlorogenic acids (4.18–7.04 g kg^?1 DW) were the most abundant. TKS leaves represent a valuable source of chicoric acid with potential application as antioxidant to be used as herbal medicine and nutrition for production of healthy food/feed.     

Population Genetics of the Rubber-Producing Russian Dandelion (Taraxacum kok-saghyz)

The Russian dandelion, Taraxacum kok-saghyz (TKS), is a perennial species native to Central Asia that produces high quality, natural rubber. Despite its potential to help maintain a stable worldwide rubber supply, little is known about genetic variation in this species. To facilitate future germplasm improvement efforts, we developed simple-sequence repeat (SSR) markers from available expressed-sequence tag (EST) data and used them to investigate patterns of population genetic diversity in this nascent crop species. We identified numerous SSRs (1,510 total) in 1,248 unigenes from a larger set of 6,960 unigenes (derived from 16,441 ESTs) and designed PCR primers targeting 767 of these loci. Screening of a subset of 192 of these primer pairs resulted in the identification of 48 pairs that appeared to produce single-locus polymorphisms. We then used the most reliable 17 of these primer pairs to genotype 176 individuals from 17 natural TKS populations. We observed an average of 4.8 alleles per locus with population-level expected heterozygosities ranging from 0.28 to 0.50. An average pairwise F_ST of 0.11 indicated moderate but statistically significant levels of genetic differentiation, though there was no clear geographic patterning to this differentiation. We also tested these 17 primer pairs in the widespread common dandelion, T. officinale, and a majority successfully produced apparently single-locus amplicons. This result demonstrates the potential utility of these markers for genetic analyses in other species in the genus.     

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.     

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.     

Characterization of polymorphic microsatellite markers for Microcyclus ulei,causal agent of South American leaf blight of rubber trees

South American leaf blight caused by the ascomycete Microcyclus ulei is the most harmful disease of the rubber tree in Latin America and a potential threat to Asiatic and African natural rubber production. Until now, the variability of this fungus was assessed through observation of pathogenicity of isolates on a range of rubber tree clones with known resistance reactions. The present study describes the process used to design 11 microsatellite markers and evaluates their usefulness in detecting genetic polymorphism. Nine of these markers were polymorphic among six isolates from Brazil (with two to three alleles per locus) and five markers were polymorphic among four isolates from French Guiana (with two to four alleles per locus).     

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.     

Biotechnological production of astaxanthin with <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>/<Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

The oxygenated β-carotene derivative astaxanthin exhibits outstanding colouring, antioxidative and health-promoting properties and is mainly found in the marine environment. To satisfy the growing demand for this ketocarotenoid in the feed, food and cosmetics industries, there are strong efforts to develop economically viable bioprocesses alternative to the current chemical synthesis. However, up to now, natural astaxanthin from Haematococcus pluvialis, Phaffia rhodozyma or Paracoccus carotinifaciens has not been cost competitive with chemically synthesized astaxanthin, thus only serving niche applications. This review illuminates recent advances made in elucidating astaxanthin biosynthesis in P. rhodozyma. It intensely focuses on strategies to increase astaxanthin titers in the heterobasidiomycetous yeast by genetic engineering of the astaxanthin pathway, random mutagenesis and optimization of fermentation processes. This review emphasizes the potential of P. rhodozyma for the biotechnological production of astaxanthin in comparison to other natural sources such as the microalga H. pluvialis, other fungi and transgenic plants and to chemical synthesis.     

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.     

The potential of small exclosures in assisting regeneration of coffee shade trees in South‐Western Ethiopian coffee forests

Ethiopian Afromontane moist forests where coffee grows as understorey shrub are traditionally managed by the local communities for coffee production through thinning of the shade tree canopy and slashing of competing undergrowth. This management practice has a negative impact on the coffee shrubs, because the removal of shade tree saplings and seedlings reduces the succession potential of the shade tree canopy, which threatens the very existence of the shade coffee production system. We assessed the functionality of small exclosures to initiate coffee shade tree canopy restoration through natural regeneration. Our results show that small exclosures have a strong restoration potential for the coffee shade trees preferred by farmers (Albizia schimperiana, A. gummifera and Millettia ferruginea), as evidenced from their seedling abundance, survival and growth. The regeneration of late‐successional tree species of the moist Afromontane forest was not successful in the small exclosures, most probably due to the low abundance or absence of adult trees as seed sources for regeneration. Therefore, temporary establishment of small exclosures in degraded coffee forest fragments where shade trees are getting old or dying is recommended for sustainable shade coffee production.     

Heterologous expression of chloroplast‐localized geranylgeranyl pyrophosphate synthase confers fast plant growth,early flowering and increased seed yield

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast‐targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS‐transgenic lines (control) or wild‐type plants. The gibberellin levels in HaGGPS‐transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS‐transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS‐expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.     

橡胶草90年来主要研究成果及最新研究进展

天然橡胶是重要的国防战略物质,巴西橡胶树（Hevea brasiliensis（Willd.ex A.Juss.） Muell.Arg）是天然橡胶的唯一来源,天然橡胶商业化形式极为单一,潜在的供给不足问题亟待解决。因此,寻找可替代巴西橡胶树的产胶植物一直受到全世界高度重视。蒲公英属橡胶草（Taraxacum kok-saghyz Rodin）根部含有与橡胶树橡胶类似的天然橡胶分子,该植物主要分布在温带和寒带地区,具有易于机械化收获、生长周期短、遗传转化相对容易等特点,是最具开发潜力的产胶植物。本文对橡胶草90年（1931-2018）来的研究历史和主要成果进行了概括,对近10年取得的最新成果进行了深度分析,并预测橡胶草在未来天然橡胶产业中的作用,期望为开展橡胶草商业化生产和橡胶生物合成相关基础研究提供一定的参考。