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.

The effect of supplementation with rubber seed kernel pellet on in vitro rumen fermentation characteristics and fatty acid profiles in swamp buffalo.

BACKGROUND: Rubber seed kernel is a by-product derived from rubber tree plantations. It is rich in C18 unsaturated fatty acids (UFA) and has the potential to be used as a protein source for ruminant diets. This investigation has been conducted to determine the influence of rubber seed kernel pellet (RUSKEP) supplementation on in vitro rumen fermentation characteristics and fatty acid profiles in swamp buffalo. Using a completely randomized design (CRD) and supplementation of RUSKEP at 0, 2, 4, 6, 8, and 10% dry matter (DM) of substrate. RESULTS: The supplementation with RUSKEP had no effect on gas kinetics, cumulative gas production, or degradability. Ruminal pH decreased linearly (P < 0.01) and ammonia-nitrogen (NH3-N) concentration decreased quadratically (P < 0.01) by RUSKEP supplementation. The proportion of acetate (C2) decreased linearly (P < 0.01), but propionate (C3) and butyrate (C4) increased linearly (P < 0.01), resulting in a decrease in the acetate to propionate ratio (C2:C3) (P < 0.01) by RUSKEP supplementation. With an increasing level of dietary RUSKEP, there was a slight increase in UFA in the rumen by increasing the oleic acid (OA; C18:1 cis-9 + trans-9), linoleic acid (LA; C18:2 cis-9,12 + trans-9,12), and α-linolenic acid (ALA; C18:3 cis-9,12,15) concentrations (P < 0.01). CONCLUSIONS: Adding up to 10% of RUSKEP could improve in vitro rumen fermentation and C18 unsaturated fatty acids, especially ALA, in swamp buffalo.

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.

Differential Analysis of Mycelial Proteins and Metabolites From Rigidoporus Microporus During In Vitro Interaction With Hevea Brasiliensis.

Rigidoporus microporus is the fungus accountable for the white root rot disease that is detrimental to the rubber tree, Hevea brasiliensis. The pathogenicity mechanism of R. microporus and the identity of the fungal proteins and metabolites involved during the infection process remain unclear. In this study, the protein and metabolite profiles of two R. microporus isolates, Segamat (SEG) and Ayer Molek (AM), were investigated during an in vitro interaction with H. brasiliensis. The isolates were used to inoculate H. brasiliensis clone RRIM 2025, and mycelia adhering to the roots of the plant were collected for analysis. Transmission electron microscope (TEM) images acquired confirms the hyphae attachment and colonization of the mycelia on the root of the H. brasiliensis clones after 4 days of inoculation. The protein samples were subjected to 2-DE analysis and analyzed using MALDI-ToF MS/MS, while the metabolites were extracted using methanol and analyzed using LC/MS-QTOF. Based on the differential analyses, upregulation of proteins that are essential for fungal evolution such as malate dehydrogenase, fructose 1,6-biphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase hints an indirect role in fungal pathogenicity, while metabolomic analysis suggests an increase in acidic compounds which may lead to increased cell wall degrading enzyme activity. Bioinformatics analyses revealed that the carbohydrate and amino acid metabolisms were prominently affected in response to the fungal pathogenicity. In addition to that, other pathways that were significantly affected include "Protein Ubiquitination Pathway," Unfolded Protein Response," "HIFα Signaling," and "Sirtuin Signaling Pathway." The identification of responsive proteins and metabolites from this study promotes a better understanding of mechanisms underlying R. microporus pathogenesis and provides a list of potential biological markers for early recognition of the white root rot disease.

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.

Isolation and Identification of Secondary Metabolites Produced by Phytopathogenic Fungus Corynespora cassiicola from Hevea brasiliensis.

The secondary metabolites of the phytopathogenic fungus Corynespora cassiicola CC01 from Hevea brasiliensis were investigated. As a result, two new compounds, 5-acetyl-7-hydroxy-6- methoxybenzofuran-2(3H)-one (1) and (S)-2-(2,3-dihydrofuro [3,2-c]pyridin-2-yl)propan-2-ol (2), together with seven known compounds, 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (3), 3,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (4), curvulin acid (5), 2-methyl-5-carboxymethyl- 7-hydroxychromone (6), tyrosol (7), p-hydroxybenzoic acid (8) and cerevisterol (9), were isolated from the fermentation extract by comprehensive silica gel, reverse phase silica gel, Sephadex-LH20 column chromatography and high-performance liquid chromatography (HPLC). The structures of these compounds were identified by using high-resolution electrospray mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy (NMR), optical rotation, ultraviolet and infrared spectroscopy techniques and a comparison of NMR data with those reported in the literature. Compounds 1 and 2 were new compounds, and compounds 3-9 were discovered from this phytopathogenic fungus for the first time. Compounds 1-9 were tested for phytotoxicity against the fresh tender leaf of Hevea brasiliensis, and the results show that none of them were phytotoxic. Additionally, these compounds were subjected to an antimicrobial assay against three bacteria (E. coli, methicillin-resistant Staphylococcus aureus and Micrococcus luteus), but they showed no activity.

Lipid Composition of Latex and Rubber Particles in Hevea brasiliensis and Taraxacum kok-saghyz.

Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Pará rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85-99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (< 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.

Structural Analysis of the Terminal Groups in Commercial Hevea Natural Rubber by 2D-NMR with DOSY Filters and Multiple-WET Methods Using Ultrahigh-Field NMR.

The terminal groups of natural rubber (NR) are widely believed to play a crucial role in defining the excellent mechanical and other physical properties of processed NR products. Despite their presumed importance, the chemical structures of the terminal groups are elusive in widely used NR species with a high degree of polymerization, such as Hevea natural rubber (H-NR). In previous studies, structural analysis by solution NMR has been carried out on the terminal units of NR after chemical treatment involving chemical alterations, such as deproteinization with enzymes and other chemicals. However, there is concern that such chemical treatments may alter the properties of the terminal units. In this study, we established an NMR-based approach to analyze the structures of the terminal units in commercial H-NR without any chemical treatments, or with only a mild treatment of some samples, such as acetone extraction for removing the impurities. To suppress the signals of low-molecular-weight impurities, we have developed methods combining DOSY-based diffusion filters with multiple-WET (MWET) 2D-NMR, which we introduced previously to suppress strong signals from main-chain of polymer and solvents (Tanaka et al. Macromolecules, 2016, 49, 5750-5754). Using the new method and MWET 2D-NMR methods with high-field NMR at a 1H frequency of 900 MHz, we observed NMR signals of the terminal units of chemically untreated commercial H-NR for the first time. The NMR results for eight commercial H-NR samples consistently demonstrated the presence of at least five kinds of terminating-end (α-terminus) units of the H-NR polymer chain in addition to NMR signals for the initiating-end (ω-terminus) units. Our NMR analyses revealed for the first time that none of the α-terminal groups form a phosphate ester.

Unrestrictive identification of post-translational modifications in Hevea brasiliensis latex.

The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in modern society. Post-translational modifications (PTMs) of the latex proteins are important for the stability and functionality of the proteins. In this study, latex proteins were acquired from the C-serum, lutoids, and rubber particle layers of latex without using prior enrichment steps; they were fragmented using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation (ETD) activation methods. PEAKS 7 were used to search for unspecified PTMs, followed by analysis through PTM prediction tools to crosscheck both results. There were 73 peptides in 47 proteins from H. brasiliensis protein sequences derived from UniProtKB were identified and predicted to be post-translationally modified. The peptides with PTMs identified include phosphorylation, lysine acetylation, N-terminal acetylation, hydroxylation, and ubiquitination. Most of the PTMs discovered have yet to be reported in UniProt, which would provide great assistance in the research of the functional properties of H. brasiliensis latex proteins, as well as being useful biomarkers. The data are available via the MassIVE repository with identifier MSV000082419.

The 14-3-3 protein HbGF14a interacts with a RING zinc finger protein to regulate expression of the rubber transferase gene in Hevea brasiliensis.

Hevea brasiliensis is a key commercial source of natural rubber (cis 1,4-polyisoprene). In H. brasiliensis, rubber transferase is responsible for cis-1,4-polymerization of isoprene units from isopentenyl diphosphate and thus affects the yield of rubber. Little is known about the regulatory mechanisms of the rubber transferase gene at a molecular level. In this study we show that the 5'UTR intron of the promoter of the rubber transferase gene (HRT2) suppresses the expression of HRT2. A H. brasiliensis RING zinc finger protein (designated as HbRZFP1) was able to interact specifically with the HRT2 promoter to down-regulate its transcription in vivo. A 14-3-3 protein (named as HbGF14a) was identified as interacting with HbRZFP1, both in yeast and in planta. Transient co-expression of HbGF14a and HbRZFP1-encoding cDNAs resulted in HbRZFP1-mediated HRT2 transcription inhibition being relieved. HbGF14a repressed the protein-DNA binding of HbRZFP1 with the HRT2 promoter in yeast. We propose a regulatory mechanism by which the binding of HbGF14a to HbRZFP1 interferes with the interaction of HbRZFP1 with the HRT2 promoter, thereby repressing the protein-DNA binding between them. This study provides new insights into the role of HbGF14a in mediating expression of the rubber transferase gene in Hevea brasiliensis.

Evaluation of peptides release using a natural rubber latex biomembrane as a carrier.

The biomembrane natural (NRL-Natural Rubber Latex), manipulated from the latex obtained from the rubber tree Hevea brasiliensis, has shown great potential for application in biomedicine and biomaterials. Reflecting the biocompatibility and low bounce rate of this material, NRL has been used as a physical barrier to infectious agents and for the controlled release of drugs and extracts. The aim of the present study was to evaluate the incorporation and release of peptides using a latex biomembrane carrier. After incorporation, the release of material from the membrane was observed using spectrophotometry. Analyses using HPLC and mass spectroscopy did not confirm the release of the antimicrobial peptide [W6]Hylin a1 after 24 h. In addition, analysis of the release solution showed new compounds, indicating the degradation of the peptide by enzymes contained in the latex. Additionally, the release of a peptide with a shorter sequence (Ac-WAAAA) was evaluated, and degradation was not observed. These results showed that the use of NRL as solid matrices as delivery systems of peptide are sequence dependent and could to be evaluated for each sequence.

Microbial communities in natural rubber coagula during maturation: impacts on technological properties of dry natural rubber.

AIM: To characterize microbial communities present in natural rubber (NR) coagula from Hevea brasiliensis latex during maturation and identify microbial taxa (bacteria and fungi) having an impact on dry NR properties. METHODS AND RESULTS: Microbial community dynamics in NR coagula maturated under controlled conditions were compared and related with the evolution of dry NR properties. The pyrosequencing of 16S (119 837 effective reads) and 18S (131 879 effective reads) rRNA gene regions was performed on 21 samples covering different maturation times and two aeration conditions. Results showed a relatively high bacterial richness (Chao1 estimates of 200-1000) associated with significant bacterial dynamics. Lactic acid bacteria (LAB) were dominant in the first days of maturation. Then, in aerobic conditions, development of Actinobacteria represented by the family Microbacteriaceae was associated with alkalinization of the samples and a higher sensitivity of NR to thermo-oxidation as evaluated by its plasticity retention index (PRI). In anaerobiosis, the reduced development of bacteria, mostly LAB present, was associated with improved NR properties (higher initial plasticity P0 and PRI). CONCLUSIONS: The involvement of micro-organisms in the evolution of dry NR properties during the maturation of NR coagula was confirmed. The importance of the structure and dynamics of microbial communities is specifically highlighted. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural rubber is a key elastomer for the tyre industry and for a variety of other applications. The majority of raw NR is obtained by natural coagulation of H. brasiliensis latex under the activity of micro-organisms. An improved understanding of the microbial communities involved in the maturation of NR coagula may lead to an improvement in the production process of raw NR to provide a better consistency in NR quality.

Proteomics analysis of latex from Hevea brasiliensis (clone RRIM 600).

The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in today's modern society. Following ultracentrifugation, the latex can be separated into 3 layers: C-serum, lutoids, and rubber particles. Previous studies have shown that a large number of proteins are present in these 3 layers. However, a complete proteome for this important plant is still unavailable. Protein sequences have been recently translated from the completed draft genome database of H. brasiliensis, leading to the creation of annotated protein databases of the following H. brasiliensis biosynthetic pathways: photosynthesis, latex allergens, rubberwood formation, latex biosynthesis, and disease resistance. This research was conducted to identify the proteins contained within the latex by way of de novo sequencing from mass spectral data obtained from the 3 layers of the latex. Peptides from these proteins were fragmented using collision-induced dissociation, higher-energy collisional dissociation, and electron-transfer dissociation activation methods. A large percentage of proteins from the biosynthetic pathways (63% to 100%) were successfully identified. In addition, a total of 1839 unique proteins were identified from the whole translated draft genome database (AnnHBM).

Effects of replacing fish meal with rubber seed meal on growth, nutrient utilization, and cholesterol metabolism of tilapia (Oreochromis niloticus × O. aureus).

A feeding trial was conducted to evaluate the effects of replacing fish meal with rubber seed meal (RSM) on growth, nutrient utilization, and cholesterol metabolism of tilapia (Oreochromis niloticus × Oreochromis aureus). Five experimental diets were formulated with 0, 150, 300, 450, and 600 g kg-1 RSM replacing graded levels of fish meal, respectively. Each diet was randomly assigned to triplicate groups of 25 fish (initial average weight 65.3 g) per aquarium in a rearing system maintained at 29 ± 1 °C for 8 weeks. Dietary 150 g kg-1 RSM inclusion did not affect the weight gain and daily growth coefficient, whereas these were depressed by a further inclusion. Additionally, feed efficiency ratio and protein efficiency ratio were not affected by dietary RSM inclusion regardless of inclusion level. However, the inclusion of 450 and 600 g kg-1 RSM decreased the mid-intestinal trypsin, lipase, and amylase activities; the hepatic acyl-CoA/cholesterol acyl transferase; low-density lipoprotein receptor; and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities. Similarly, dietary 600 g kg-1 RSM inclusion inhibited the plasma catalase and hepatic glutathione peroxidase activities. These results indicated that 150 g kg-1 RSM can be included in tilapia diets, whereas higher inclusion of RSM inhibited the growth rate, digestive enzyme activity, antioxidant capacity, and cholesterol metabolism.

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.

Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues.

Isolation of a furan fatty acid from Hevea brasiliensis latex employing the combined use of pH-zone-refining and conventional countercurrent chromatography.

Furan fatty acids are valuable and bioactive minor fatty acids that usually contribute <0.1% to the fatty acid content of food samples. Their biological role still remains unclear as authentic furan fatty acid standards are not readily available and thorough experimental studies verifying the relevance of furan fatty acids are thus virtually impossible. An efficient protocol for the isolation of the furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid from hydrolyzed and centrifuged latex of Hevea brasiliensis was developed using countercurrent chromatography. A first run using pH-zone-refining countercurrent chromatography provided 48.4 mg of 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid from 210 mg latex extract in a purity of 95%. The purity was increased to 99% by means of one second run in conventional countercurrent chromatography mode. The Structure and purity of 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid were determined by gas chromatography coupled to mass spectrometry and (1)H and (13)C NMR spectroscopy.

Identification, Functional Study, and Promoter Analysis of HbMFT1, a Homolog of MFT from Rubber Tree (Hevea brasiliensis).

A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical ß-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.

Effects of resistant and susceptible rubber germplasms on development, reproduction and protective enzyme activities of Eotetranychus sexmaculatus (Acari: Tetranychidae).

Systematic research or technical support regarding rubber germplasm resistance against mites was not performed yet. To develop a preliminary understanding of the mite-resistance mechanisms of rubber germplasms, stably resistant rubber germplasms were obtained, the development and reproduction of Eotetranychus sexmaculatus that fed on leaves of resistant and susceptible rubber germplasms were examined in the laboratory, and the activities of protective enzymes in this mite species were also compared. The results indicated that: (1) among the 23 rubber core germplasms identified, five (IRCI12, Reyan87-6-5, IAN717, RRIM600 and RRIC52) steadily developed resistance to E. sexmaculatus; (2) E. sexmaculatus that fed on the highly resistant germplasm IRCI12 did not complete development and reproduction-the female adults laid only 4.90 eggs on average, and none of these eggs hatched; (3) the resistant germplasms extended the duration of each developmental stage, reduced the fecundity, egg hatchability, and female offspring percentage, and significantly decreased the offspring survival rate compared with the susceptible germplasms; and (4) during each developmental stage of the mites that fed on resistant rubber germplasms, decreased activities (by 0.25-fold to 0.63-fold times) of the protective enzymes peroxidase, ascorbate peroxidase, polyphenol oxidase, superoxide dismutase and catalase were observed compared with those in the mites that fed on susceptible rubber germplasms (P < 0.05). These findings may explain why E. sexmaculatus did not complete their development and reproduction on the resistant rubber germplasms. This study lays a foundation for elucidation of the mechanism of rubber resistance to mites and provides experimental material and technical support for the breeding of mite-resistant rubber plants.

Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles.