Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Overexpression of HbGRF4 or HbGRF4-HbGIF1 Chimera Improves the Efficiency of Somatic Embryogenesis in Hevea brasiliensis.

Transgenic technology is a crucial tool for gene functional analysis and targeted genetic modification in the para rubber tree (Hevea brasiliensis). However, low efficiency of plant regeneration via somatic embryogenesis remains a bottleneck of successful genetic transformation in H. brasiliensis. Enhancing expression of GROWTH-REGULATING FACTOR 4 (GRF4)-GRF-INTERACTING FACTOR 1 (GIF1) has been reported to significantly improve shoot and embryo regeneration in multiple crops. Here, we identified endogenous HbGRF4 and HbGIF1 from the rubber clone Reyan7-33-97, the expressions of which dramatically increased along with somatic embryo (SE) production. Intriguingly, overexpression of HbGRF4 or HbGRF4-HbGIF1 markedly enhanced the efficiency of embryogenesis in two H. brasiliensis callus lines with contrasting rates of SE production. Transcriptional profiling revealed that the genes involved in jasmonic acid response were up-regulated, whereas those in ethylene biosynthesis and response as well as the S-adenosylmethionine-dependent methyltransferase activity were down-regulated in HbGRF4- and HbGRF4-HbGIF1-overexpressing H. brasiliensis embryos. These findings open up a new avenue for improving SE production in rubber tree, and help to unravel the underlying mechanisms of HbGRF4-enhanced somatic embryogenesis.

Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup.

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

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.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

BACKGROUND: Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. METHODS: Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. RESULTS: Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. CONCLUSIONS: These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

Magnetic activated carbon synthesized using rubber fig tree leaves for adsorptive removal of tetracycline from aqueous solutions.

The current study emphasizes the activated carbon fabrication from rubber fig leaves, the establishment of its composite with iron oxide nanoparticles (RFAC@Fe2O3), and its relevance in the adsorptive elimination of tetracycline. The physical and functional properties of RFAC@Fe2O3 nanocomposite were uncovered by multiple approaches. Elemental analysis portrayed the existence of carbon, oxygen, and iron, while FESEM analysis revealed that Fe2O3 nanoparticle agglomerates were entrenched in the activated carbon matrix rendering it a rough abrasive texture. FT-IR analysis reported the presence of functional groups attributing to CC, -OH, crystalline iron oxide, and Fe-O stretching vibrations, and XRD corroborated graphitic crystalline structure, oxygenated functional groups attached to carbon accompanied by crystalline plane corresponding to Fe2O3 nanoparticles. XPS spectra depicted signature peaks for C, O, and Fe, while VSM studies designated its superparamagnetic nature. The high surface area (662.73 m2/g), pore size (3.12 nm), and mesoporous nature of RFAC@Fe2O3 make it apt for the adsorption of pollutants from contaminated samples. The adsorption of tetracycline (50 ppm) by RFAC@Fe2O3 was maximum at pH 4.0. As the nanocomposite dosage and stirring speed increased to 2.0 g/L and 150 rpm, maximum adsorption was observed due to more active binding sites and improved mixing. Freundlich isotherm along with pseudo-second-order model well described adsorption process divulging that tetracycline was adsorbed onto RFAC@Fe2O3 composite in multi-layers by chemisorption. Thermodynamic analysis signified negative values for ΔG°, while positive values for ΔH° and ΔS were obtained, indicating spontaneous feasible endothermic adsorption.

Antioxidant and photoprotective role of latex C-serum from Hevea brasiliensis during 15-week UVB irradiation in male hairless SKH-1 mice.

It is known that UVB radiation induces several adverse skin alterations starting from simple photoaging to skin cancer. In addition, it was demonstrated that reactive oxygen species (ROS) were found to be related to cancer development and progression. The aim of study was to examine whether male hairless (SKH-1) mice (Mus musculus) that were subchronically exposed to UVB radiation presented with actinic keratosis (AK) and squamous cell carcinoma lesions, and that treatment with latex C-serum cream significantly prevented abnormal skin development. Data demonstrated for the first time the photoprotective activity of latex C-serum extracted from the rubber tree Hevea brasiliensis var. subconcolor Ducke. Latex C-serum prevented the progression of AK to squamous cell carcinoma in SKH-1 mice, indicating that mice topically treated with latex C-serum presented only AK lesions and treatment with the highest concentration (10%) significantly reduced epidermal thickness, suggesting diminished cell proliferation. Latex C-serum protected the skin of mice against oxidative stress damage, increasing catalase (CAT) activity, regenerating glutathione (GSH) levels, lowering thiobarbituric acid-reactive species (TBARS) production and regenerating the total antioxidant capacity (TAC) of the skin. Evidence that UV radiation in skin induced systemic alterations and erythrocytic analysis indicated that latex C-serum increased CAT activity and GSH levels. Taken together these data indicate that latex C-serum plays an important antioxidant and photoprotective role, preventing serious damage to the skin following exposure to UVB radiation.

Rubber latex yield is affected by interactions between antecedent temperature, rubber phenology, and powdery mildew disease.

Rubber (Hevea brasiliensis) latex production is crucial to the local economy, yet Xishuangbanna's climate is considered sub-optimal for rubber cultivation. The prevalence of the powdery mildew disease (Oidium heveae) in this region has decreased the annual latex yield by 20%. Rubber latex yield is influenced by several factors, including temperature, disease, other biotic conditions, and plantation management. However, the interrelationships and potential influencing networks between rubber latex yield and these factors are rarely quantitatively assessed, and understanding their impacts on latex yield could inform better management practices. To address this gap, we investigated the effects of temperature, phenology, and powdery mildew disease on rubber latex yield in March using observational data on daily rubber latex yield combined with detailed phenology, powdery mildew, and temperature data from 2004 to 2010 in a state farm in the Xishuangbanna, Yunnan, China. We found that the critical influencing periods of daily temperature difference (or diurnal temperature difference) on the rubber latex yield were during Nov 27-Jan 19 and Jan 21-Mar 17. Partial least square regression analysis and variance partitioning analysis were conducted on the 35 phenological variables, eight powdery mildew-related variables, and two climatic variables. The most influential factors were identified as the factors of the daily temperature differences during Jan-Mar, the duration of leaf flushing phenology, and mean and maximum percentage of leaves infected by powdery mildew. Subsequent canonical correlation analysis and linear regression found that temperature difference directly affected the rubber latex yield and indirectly affected the yield through phenology and powdery mildew disease. Raised daily temperature differences from Jan to Mar had the greatest impact, leading to a higher rubber latex yield. Our comprehensive quantitative assessment revealed the relative importance of antecedent daily temperature differences, phenology, and powdery mildew disease as well as their complex interconnections in influencing rubber latex yield. Our findings are essential to future studies on both powdery mildew disease and rubber latex yield, and also develop rubber latex models.

Brassinosteroids Regulate the Water Deficit and Latex Yield of Rubber Trees.

Brassinolide (BR) is an important plant hormone that regulates the growth and development of plants and the formation of yield. The yield and quality of latex from Hevea brasiliensis are regulated by phytohormones. The understanding of gene network regulation mechanism of latex formation in rubber trees is still very limited. In this research, the rubber tree variety CATAS73397 was selected to analyze the relationship between BR, water deficit resistance, and latex yield. The results showed that BR improves the vitality of rubber trees under water deficit by increasing the rate of photosynthesis, reducing the seepage of osmotic regulatory substances, increasing the synthesis of energy substances, and improving the antioxidant system. Furthermore, BR increased the yield and quality of latex by reducing the plugging index and elevating the lutoid bursting index without decreasing mercaptan, sucrose, and inorganic phosphorus. This was confirmed by an increased expression of genes related to latex flow. RNA-seq analysis further indicated that DEG encoded proteins were enriched in the MAPK signaling pathway, plant hormone signal transduction and sucrose metabolism. Phytohormone content displayed significant differences, in that trans-Zeatin, ethylene, salicylic acid, kinetin, and cytokinin were induced by BR, whereas auxin, abscisic acid, and gibberellin were not. In summary, the current research lays a foundation for comprehending the molecular mechanism of latex formation in rubber trees and explores the potential candidate genes involved in natural rubber biosynthesis to provide useful information for further research in relevant areas.

Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree.

Plant PP2C genes are crucial for various biological processes. To elucidate the potential functions of these genes in rubber tree (Hevea brasiliensis), we conducted a comprehensive analysis of these genes using bioinformatics methods. The 60 members of the PP2C family in rubber tree were identified and categorized into 13 subfamilies. The PP2C proteins were conserved across different plant species. The results revealed that the HbPP2C genes contained multiple elements responsive to phytohormones and stresses in their promoters, suggesting their involvement in these pathways. Expression analysis indicated that 40 HbPP2C genes exhibited the highest expression levels in branches and the lowest expression in latex. Additionally, the expression of A subfamily members significantly increased in response to abscisic acid, drought, and glyphosate treatments, whereas the expression of A, B, D, and F1 subfamily members notably increased under temperature stress conditions. Furthermore, the expression of A and F1 subfamily members was significantly upregulated upon powdery mildew infection, with the expression of the HbPP2C6 gene displaying a remarkable 33-fold increase. These findings suggest that different HbPP2C subgroups may have distinct roles in the regulation of phytohormones and the response to abiotic and biotic stresses in rubber tree. This study provides a valuable reference for further investigations into the functions of the HbPP2C gene family in rubber tree.

A putative effector of the rubber-tree powdery mildew fungus has elicitor activity that can trigger plant immunity.

MAIN CONCLUSION: A putative powdery mildew effector can elicit defense responses including reactive oxygen species and callose accumulations in model plants Nicotiana benthamiana and Arabidopsis thaliana and host plant Hevea brasiliensis. Powdery mildew fungi cause severe diseases in many agricultural plants, such as the mildew fungus Erysiphe quercicola infecting the rubber tree (Hevea brasiliensis), causing latex yield losses. However, effectors of E. quercicola were rarely functionally characterized. In this study, we identified a highly specific candidate-secreted effector protein, EqCSEP04187, from E. quercicola. This putative effector is expressed at the late stage but not the early stage during infection. The constitutive expression of EqCSEP04187 in model plants Nicotiana benthamiana and Arabidopsis thaliana elicited defense responses, as did transient expression of EqCSEP04187 in protoplasts of H. brasiliensis. Introducing EqCSEP04187 into another H. brasiliensis-associated fungal pathogen, Colletotrichum gloeosporioides, inhibited H. brasiliensis infection, and infection by E. quercicola was decreased in the A. thaliana eds1 mutant expressing EqCSEP04187. Further analysis suggests that these reductions in infection were the consequences of EqCSEP04187 eliciting defense responses. Our study suggests that this putative effector has elicitor activity that can improve plant resistance.

Unraveling the Host-Selective Toxic Interaction of Cassiicolin with Lipid Membranes and Its Cytotoxicity.

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for Corynespora leaf fall disease in susceptible rubber trees. Currently, the molecular mechanisms of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more sensitive to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Mature Cas1 and Cas2 play an essential role in causing membrane disruption. Cytotoxicity tests on rubber leaves of Rubber Research Institute of Vietnam (RRIV) 1, RRIV 4, and Prang Besar (PB) 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryogenic scanning electron microscopy analyses of necrotic leaf tissues treated with Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and to disrupt specific membranes.

Simultaneous quantification of volatile fatty acids and nonvolatile organic acids in Hevea brasiliensis latex.

The current method used in latex industries to determine the volatile fatty acids contents of Hevea brasiliensis latex is steam distillation. However, the accuracy of the method has been debated for some time. We assessed the accuracy of the method and developed a new, more reliable high-performance liquid chromatographic method of determining acids in latex. The volatile fatty acids (formic, acetic, propionic, butyric, and valeric acids) and nonvolatile organic acids (oxalic, malic, lactic, citric, and succinic acids) in latex are directly determined simultaneously for the first time with high sensitivity and without losses during sample preparation. To avoid errors from derivatization, an acid-resistant Prevail HPLC column and a gradient mobile phase of 25 mM potassium dihydrogen phosphate (pH 2.5) and acetonitrile were employed. Under optimum conditions, the calibrations of both types of acids demonstrated satisfactory correlation coefficients of  ≥0.990, with limits of detection ranging from 0.02 to 395 mM. The developed method demonstrated the profiles of acids in field and concentrated latex of the same batch. Moreover, the evolution of the profiles of all studied acids in both types of latex during a 3-month period was also revealed.

Development of Novel Markers for Yield in Hevea brasiliensis Muell. Arg. Based on Candidate Genes from Biosynthetic Pathways Associated with Latex Production.

Scarcity of functional genetic markers associated with candidate genes (CGs) is a serious constraint for marker-assisted selection in the natural rubber producing tree, Hevea brasiliensis. In order to develop markers associated with rubber yield, five CGs involved in latex biosynthesis were characterized from 16 popular Hevea varieties. Novel SNPs and indels were identified and developed into markers using simple genotyping techniques like allele-specific PCR, CAPS, etc. A progeny population was genotyped using these markers to validate them, to understand their segregation pattern and to map them to a genetic linkage map. Parent-specific maps were constructed using pseudo-test cross strategy with the help of additional markers. The sequence structure information generated will be useful for future studies on gene mapping, functional relevance of coding SNPs and evolution of rubber biosynthesis genes in Hevea. Concurrently, the markers developed may serve as powerful tools for yield-based selection and for genetic diversity and pedigree studies in Hevea. Above all, the marker assays designed for genotyping could be economically carried out in any laboratory having basic molecular biology infrastructure and expertise.

Germination and in vitro development of mature zygotic embryos and protein profile of seedlings of wild and cultivated Hevea brasiliensis.

The main factors governing Hevea brasiliensis germination and seedling establishment remains unclear. We examined the effect of growth regulators Indole 3-Acetic Acid (IAA) and 6-Benzylaminopurine (BAP), and their interactions on germination and the development of mature zygotic embryos (MZE) and protein profile of Hevea brasiliensis seedlings from wild and cultivated (clone PB 250) genotypes. Embryonic axes excised from seeds (wild and clone PB 250) were inoculated in Murashige and Skoog medium (control) and supplemented with IAA (3 µM) and BAP (6 µM) individually and their combination (3 µM IAA + 6 µM BAP). For both genotypes, the mature embryos displayed a high percentage of germination and establishment, and the seedlings were characterized by protein bands ranging from 7 to 30 kDa. Notably, the wild genotype showed proteins in the 14 kDa range, which may be associated with one of the major rubber elongation factors (REF). The wild and clone genotypes presented different behavior and strategies in relation to the protein profile in the presence of different growth regulators. Although the latex biosynthetic pathway and its mechanisms of regulation still remain largely unknown, our results aid in our understanding of the dynamics of proteins in different rubber tree clones in vitro.

Ectopic Expression of HbRPW8-a from Hevea brasiliensis Improves Arabidopsis thaliana Resistance to Powdery Mildew Fungi (Erysiphe cichoracearum UCSC1).

The RPW8s (Resistance to Powdery Mildew 8) are atypical broad-spectrum resistance genes that provide resistance to the powdery mildew fungi. Powdery mildew of rubber tree is one of the serious fungal diseases that affect tree growth and latex production. However, the RPW8 homologs in rubber tree and their role of resistance to powdery mildew remain unclear. In this study, four RPW8 genes, HbRPW8-a, b, c, d, were identified in rubber tree, and phylogenetic analysis showed that HbRPW8-a was clustered with AtRPW8.1 and AtRPW8.2 of Arabidopsis. The HbRPW8-a protein was localized on the plasma membrane and its expression in rubber tree was significantly induced upon powdery mildew infection. Transient expression of HbRPW8-a in tobacco leaves induced plant immune responses, including the accumulation of reactive oxygen species and the deposition of callose in plant cells, which was similar to that induced by AtRPW8.2. Consistently, overexpression of HbRPW8-a in Arabidopsis thaliana enhanced plant resistance to Erysiphe cichoracearum UCSC1 and Pseudomonas syringae pv. tomato DC30000 (PstDC3000). Moreover, such HbRPW8-a mediated resistance to powdery mildew was in a salicylic acid (SA) dependent manner. Taken together, we demonstrated a new RPW8 member in rubber tree, HbRPW8-a, which could potentially contribute the resistance to powdery mildew.

Comparative analysis of latex transcriptomes reveals the potential mechanisms underlying rubber molecular weight variations between the Hevea brasiliensis clones RRIM600 and Reyan7-33-97.

BACKGROUND: The processabilities and mechanical properties of natural rubber depend greatly on its molecular weight (MW) and molecular weight distribution (MWD). However, the mechanisms underlying the regulation of molecular weight during rubber biosynthesis remain unclear. RESULTS: In the present study, we determined the MW and particle size of latex from 1-year-old virgin trees and 30-year-old regularly tapped trees of the Hevea clones Reyan7-33-97 and RRIM600. The results showed that both the MW and the particle size of latex varied between these two clones and increased with tree age. Latex from RRIM600 trees had a smaller average particle size than that from Reyan7-33-97 trees of the same age. In 1-year-old trees, the Reyan7-33-97 latex displayed a slightly higher MW than that of RRIM600, whereas in 30-year-old trees, the RRIM600 latex had a significantly higher MW than the Reyan7-33-97 latex. Comparative analysis of the transcriptome profiles indicated that the average rubber particle size is negatively correlated with the expression levels of rubber particle associated proteins, and that the high-MW traits of latex are closely correlated with the enhanced expression of isopentenyl pyrophosphate (IPP) monomer-generating pathway genes and downstream allylic diphosphate (APP) initiator-consuming non-rubber pathways. By bioinformatics analysis, we further identified a group of transcription factors that potentially regulate the biosynthesis of IPP. CONCLUSIONS: Altogether, our results revealed the potential regulatory mechanisms involving gene expression variations in IPP-generating pathways and the non-rubber isoprenoid pathways, which affect the ratios and contents of IPP and APP initiators, resulting in significant rubber MW variations among same-aged trees of the Hevea clones Reyan7-33-97 and RRIM600. Our findings provide a better understanding of rubber biosynthesis and lay the foundation for genetic improvement of rubber quality in H. brasiliensis.

Transcriptome analysis of Pará rubber tree (H. brasiliensis) seedlings under ethylene stimulation.

BACKGROUND: Natural rubber (cis-1,4-polyioprene, NR) is an indispensable industrial raw material obtained from the Pará rubber tree (H. brasiliensis). Natural rubber cannot be replaced by synthetic rubber compounds because of the superior resilience, elasticity, abrasion resistance, efficient heat dispersion, and impact resistance of NR. In NR production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. However, intense mechanical damage to bark tissues by excessive tapping and/or over-stimulation with ethephon induces severe oxidative stress in laticifer cells, which often causes tapping panel dryness (TPD) syndrome. To enhance NR production without causing TPD, an improved understanding of the molecular mechanism of the ethylene response in the Pará rubber tree is required. Therefore, we investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system. RESULTS: After ethephon treatment, 3270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel. CONCLUSIONS: Dynamic changes in gene expression occur specifically in response to ethephon treatment. Certain genes identified may potentially contribute to latex production or TPD suppression. These data provide valuable information to understand the mechanism of ethylene stimulation, and will contribute to improved management practices and/or molecular breeding to attain higher yields of latex from Pará rubber trees.

Hevea brasiliensis latex proteomics: a review of analytical methods and the way forward.

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation.