Structural analysis of cassiicolin, a host-selective protein toxin from Corynespora cassiicola

Cassiicolin is a host-selective toxin (HST) produced by the fungus Corynespora cassiicola (strain CCP). It is responsible for the Corynespora leaf fall (CLF) disease, which is among the main pathologies affecting rubber tree (Hevea brasiliensis). Working on purified cassiicolin and using electron microscopy, we have demonstrated that this 27-residue O-glycosylated protein is able to induce cellular damages identical to those induced by the fungus on rubber tree leaves and displays the same host selectivity. The solution structure and disulfide pairing of cassiicolin have been determined using NMR spectroscopy and simulated annealing calculations. Cassiicolin appears to have an original structure with a prolate ellipsoid shape. It adopts an over-all fold consisting of three strands arranged in a right-handed twisted, antiparallel beta-sheet knitted by three disulfide bonds. Its conformation resembles that found in small trypsine-like inhibitors isolated from the brain, the fat body and the hemolymph of locust grasshoppers. But cassiicolin has no sequence homology with these protease inhibitors, and lacks their characteristic substrate-binding loop. Probably, this motif represents one of the few highly stabilized "minimal" scaffolds, with a high sequence permissiveness, that nature has selected to evolve over different phyla and to support different functions. The knowledge of the 3D structure opens the way to the delineation of the mechanism of action of the toxin using site-directed mutagenesis.     

Identification of natural rubber and characterization of rubber biosynthetic activity in fig tree

Natural rubber was extracted from the fig tree (Ficus carica) cultivated in Korea as part of a survey of rubber producing plants. Fourier transform infrared and (13)C nuclear magnetic resonance analysis of samples prepared by successive extraction with acetone and benzene confirmed that the benzene-soluble residues are natural rubber, cis-1,4-polyisoprene. The rubber content in the latex of fig tree was about 4%, whereas the rubber content in the bark, leaf, and fruit was 0.3%, 0.1%, and 0.1%, respectively. Gel-permeation chromatography revealed that the molecular size of the natural rubber from fig tree is about 190 kD. Similar to rubber tree (Hevea brasiliensis) and guayule (Parthenium argentatum Gray), rubber biosynthesis in fig tree is tightly associated with rubber particles. The rubber transferase in rubber particles exhibited a higher affinity for farnesyl pyrophosphate than for isopentenyl pyrophosphate, with apparent K(m) values of 2.8 and 228 microM, respectively. Examination of latex serum from fig tree by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major proteins of 25 and 48 kD in size, and several proteins with molecular mass below 20 and above 100 kD. Partial N-terminal amino acid sequencing and immunochemical analyses revealed that the 25- and 48-kD proteins were novel and not related to any other suggested rubber transferases. The effect of EDTA and Mg(2+) ion on in vitro rubber biosynthesis in fig tree and rubber tree suggested that divalent metal ion present in the latex serum is an important factor in determining the different rubber biosynthetic activities in fig tree and rubber tree.     

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.     

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.     

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。     

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。     

SNP Identification from RNA Sequencing and Linkage Map Construction of Rubber Tree for Anchoring the Draft Genome

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly.     

Characterization of the complete chloroplast genome of Hevea brasiliensis reveals genome rearrangement, RNA editing sites and phylogenetic relationships

Rubber tree (Hevea brasiliensis) is an economical plant and widely grown for natural rubber production. However, genomic research of rubber tree has lagged behind other species in the Euphorbiaceae family. We report the complete chloroplast genome sequence of rubber tree as being 161,191 bp in length including a pair of inverted repeats of 26,810 bp separated by a small single copy region of 18,362 bp and a large single copy region of 89,209 bp. The chloroplast genome contains 112 unique genes, 16 of which are duplicated in the inverted repeat. Of the 112 unique genes, 78 are predicted protein-coding genes, 4 are ribosomal RNA genes and 30 are tRNA genes. Relative to other plant chloroplast genomes, we observed a unique rearrangement in the rubber tree chloroplast genome: a 30-kb inversion between the trnE(UUC)-trnS(GCU) and the trnT(GGU)-trnR(UCU). A comparison between the rubber tree chloroplast genes and cDNA sequences revealed 51 RNA editing sites in which most (48 sites) were located in 26 protein coding genes and the other 3 sites were in introns. Phylogenetic analysis based on chloroplast genes demonstrated a close relationship between Hevea and Manihot in Euphorbiaceae and provided a strong support for a monophyletic group of the eurosid I.     

Development, characterization, genetic diversity and cross-species/genera transferability of ILP markers in rubber tree (Hevea brasiliensis)

To construct a linkage map enriched with tapping panel dryness (TPD)-related markers, we firstly utilized rubber tree ESTs associated with TPD to develop intron length polymorphism (ILP) markers. In this study, 52 new ILP markers were further developed. Together with the ILP markers previously reported, 102 ILP markers developed from TPD-related ESTs were analyzed within 39 Hevea germplasm in detail. The PCR success rate and polymorphism rate of ILP markers was 97.06 and 61.62 %, respectively. The results based on PCR amplification and sequence analyses provided the evidences on cross-species/genera transferability of rubber tree ILP markers. The average polymorphic information content (PIC) values of 39 Hevea germplasm were about 0.1719, indicating that the genetic base of Hevea germplasm selected in this study was very narrow. Among 39 Hevea germplasm, the PIC value of wild rubber tree accessions was the highest, followed by Hevea species and cultivated rubber tree clones. Based on the similarity coefficient of ILP markers, 39 Hevea germplasm were divided into three groups including cultivated clones, wild accessions and Hevea species, suggesting that the classification was generally related to the characterization of Hevea germplasm. The ILP markers developed in this study further enriches the number of molecular marker in rubber tree, and the ILP markers will have a wide application in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc. Moreover, the ILP markers transferred cross-euphorbiaceae family might be utilized in cassava, castor bean and physic nut.     

A Study on Manganese Content in Soils and Leaves of Rubber Trees in Rubber Plantation Areas of China

The present paper deals with the status of manganese (Mn) in rubber trees on 110 rubber plantation at 46 counties in the rubber tree growing regions of China. The content of Mn in rubber tree leaves ranges from 31 to 2167 ppm, averaging 390 ppm which is higher than that in the leaves of normal rubber trees (150–300 ppm). Thus it can be concluded that the present status of Mn in China's rubber trees is higher. Nevertheless, the content of Mn of soil of rubber plantations in China varies significantly according to different soil types, fertilization and management. The difference between the highest and the lowest contents of total Mn could be as high as 140 times and an even greater difference of 200–1240-fold between the highest and the lowest contents of replaceable and reducible Mn has been observed. The storage of reducible Mn is generally insufficient in the soil. Therefore, the symptom of leaf yellowing due to deficiency of Mn has developed in rubber trees in certain parts of rubber tree growing regions in China. The essential oure for leaf yellowing of rubber trees is to raise Mn content in the tree by proper application of fertilizer. The procedures, dosage and timing of the fertilizer for leaf yellowing treatments are suggested.     

巴西橡胶树的分子生物学研究进展

巴西橡胶树是一种重要的热带经济作物,由于橡胶树体内橡胶含量多,且容易采收,所以橡胶树一直是天然橡胶的商业来源。相比于模式植物和粮食等经济作物来说,分子生物学研究显滞后。,而且橡胶树是多年生乔木,经济性状多集中于胶乳,因此研究难度大,研究也不多,本就巴西橡胶的分子生物学方面的研究进行综述。     

植物焦磷酸酶(PPase)的研究进展

植物焦磷酸酶(PPase)可分为存在于细胞质中可溶性的无机焦磷酸酶和与膜结合的不可溶性焦磷酸酶.后者不仅能水解焦磷酸,同时还具有质子泵的功能.橡胶树乳管中与黄色体膜结合的不可溶性焦磷酸酶,是调控橡胶生物合成的一个必不可少的酶.对植物焦磷酸酶的结构及其功能和分子生物学研究的进展进行了综合论述,并着重阐述了焦磷酸酶在橡胶树橡胶生物合成中的作用.     

未来气候变化对海南橡胶树春季物候期的影响

为研究未来气候变化对海南岛橡胶树春季物候期(第一蓬叶展叶期和春花期)的影响,以国内外关于橡胶树物候期量化研究和橡胶树观测试验数据为基础,结合作物生长钟模型,建立橡胶树春季物候期模型,开发成计算机软件RubberSP并进行适宜性评价。在此基础上,通过贝叶斯模型平均法(BMA)结合耦合模式比较计划第五阶段(CMIP5)多模式数据集中的5个大气环流模式(GCMs),分别在RCP2.6、RCP4.5和RCP8.5气候情景下,以1986—2017年为基准时段,预估2020—2099年气候变化对橡胶树春季物候期的可能影响。结果表明:RubberSP的模拟精度较高,模拟值与实测值的决定系数R²为0.73～0.87,均方根误差RMSE为3.26～4.15 d,归一化均方根误差NRMSE为3.4%～7.4%。BMA方法可以有效地处理单一GCMs带来的不确定性问题,较好地反映出气温变化趋势;预估RCP2.6、RCP4.5和RCP8.5情景下,海南岛至21世纪末较基准时段分别升温超过0.3、1.0和2.5 ℃。在未来气候情景下,春季物候期出现日序提前,产胶量提高的可能性变大。日序等值线均向西北方向移动,海南岛橡胶树种植最适宜区有向西北方向扩大的可能。第一蓬叶展叶期的空间差异性变大,春花期则略微变小。橡胶树春季物候期在未来3种情景下提前或推迟的变化幅度随RCP情景下升温的幅度而变化,RCP2.6最平缓,RCP8.5最剧烈。     

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).     

The rubber tree (Hevea brasiliensis Muell. Arg.) homologue of the LEAFY/FLORICAULA gene is preferentially expressed in both male and female floral meristems

The rubber tree (Hevea brasiliensis Muell. Arg.) is an important source of natural rubber in tropical regions and, as with many woody species, shows a long juvenile phase. To understand the genetic and molecular mechanisms underlying the reproductive process in rubber trees, H. brasiliensis RRIM600 flower and inflorescence development have been characterized, the rubber tree FLORICAULA/LEAFY (FLO/LFY) orthologue, HbLFY, cloned, and its expression patterns were analysed during vegetative and reproductive development. The rubber tree, similar to other Euphorbiaceae species, produces lateral inflorescences containing male, female, and bisexual flowers. HbLFY is expressed in lateral meristems that give rise to inflorescences and in all flower meristems, consistent with a role in reproductive development. Complementation studies using Arabidopsis lfy mutants indicated that the biological function of LFY might be conserved among Brassicaceae and Euphorbiaceae species.     

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.     

Synchronous flowering of the rubber tree (Hevea brasiliensis) induced by high solar radiation intensity

How tropical trees flower synchronously near the equator in the absence of significant day length variation or other meteorological cues has long been a puzzle. The rubber tree (Hevea brasiliensis) is used as a model to investigate this phenomenon. The annual cycle of solar radiation intensity is shown to correspond closely with the flowering of the rubber tree planted near the equator and in the subtropics. Unlike in temperate regions, where incoming solar radiation (insolation) is dependent on both day length and radiation intensity, insolation at the equator is due entirely to the latter. Insolation at the upper atmosphere peaks twice a year during the spring and autumn equinoxes, but the actual solar radiation that reaches the ground is attenuated to varying extents in different localities. The rubber tree shows one or two flowering seasons a year (with major and minor seasons in the latter) in accordance with the solar radiation intensity received. High solar radiation intensity, and in particular bright sunshine (as distinct from prolonged diffuse radiation), induces synchronous anthesis and blooming in Hevea around the time of the equinoxes. The same mechanism may be operational in other tropical tree species.     

Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries,promoter sequence and gene expression analysis

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.     

Hev b 9, an enolase and a new cross-reactive allergen from hevea latex and molds. Purification, characterization, cloning and expression.

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.