Early Activation by Ethylene of the Tonoplast H-Pumping ATPase in the Latex from Hevea brasiliensis

The treatment of Hevea brasiliensis (rubber tree) bark by chloro-2-ethyl phosphonic acid (ethrel), an ethylene-producing compound, induces a significant increase in the tonoplast H⁺-translocating ATPase activity in the latex during the first 24 hours after the application of the stimulating agent. Moreover, the tonoplast-bound ATPase is highly activated when vacuoles (lutoids) are resuspended in ultrafiltrated cytosol. This effect is amplified during ethrel stimulation. Preliminary assays to characterize the endogenous effector(s) suggest that the activator(s) could be a heat-resistant compound with a low molecular weight, most likely an anion. The activation of the tonoplast-bound ATPase and the associated activation of the protons translocation across the lutoid membrane, could explain the cytosolic alkalinization observed in latex following the ethrel treatment of Hevea bark, which results in an enhanced rubber production.     

Hevains: Serine-centred proteases from the latex of Hevea brasiliensis

Hevains b and l, isolated respectively from the serum and lutoids of freeze-dried latex from Hevea brasiliensis, were purified to homogeneity and compared with hevain a from commercial, ammonia-treated latex. The M_rs of hevains a and b are 69 000 and 58 000, respectively, and both exist in several charged forms. The amino acid compositions of the two enzymes differ significantly, but the reactivities to a variety of ester and protein substrates are similar, as are the pH optima. Hevain l is a distinct protease of M_r 80 000 and unique amino acid composition. It displays esterolytic activity and will digest insulin B chain, but is not proteolytic to azocollagen, azocasein, bovine serum albumen or haemoglobin. The activities of all three enzymes are dependent on the presence of serine and histidine residues.     

Absorption des acides amines par les lutoïdes du latex d'Hevea brasiliensis

Absorption of Amino Acids by Lutoids from the Latex of Hevea brasiliensis. The latex of Hevea brasiliensis is a fluid protoplasm containing organelles with a single membrane that are called lutoids. They are microvacuoles of a lysosomal character. The intact lutoids can easily be isolated in an isotonic medium by the usual procedures of cellular fractionation. Previous studies have shown the existence of a compartmentation of the free amino acids in latex. While acidic components dominate in the cytoplasmic serum, the lutoid serum contains mainly basic components. These findings, as well as the part played by the lutoids in the latex flow and in the process of latex coagulation, led the authors to study the penetration of acidic, neutral and basic amino acids into the lutoids. Labelled amino acids were used for the purpose. Special interest was given to the uptake of lysine, which is transported into the lutoids against a concentration gradient in the absence of a metabolic energy source. The process is linear for at least 30 min and follows Michaelis–Menten kinetics as a function of the lysine concentration. The uptake is strictly temperature and pH dependent. It increases in the presence of ATP. Lysine uptake by lutoids is also increased by application of Ethrel, ethylene generator, to the bark of tapped trees. 2,4-Dinitrophenol (2,4-DNP), ammonium chloride and N-ethylmaleimide (NEM) inhibit the influx. Neutral l -amino acids do not affect the uptake of l -lysine, while l arginine inhibits lysine transport competitively. d -lysine is without effect on l -lysine uptake by lutoids. Under conditions used in this study no lysine efflux is observed. A mechanism governing the penetration of basic amino acids against a concentration gradient is discussed, and the possible role of a proton gradient is suggested.     

Electron transport in the membrane of lutoids from the latex ofHevea brasiliensis

1. An antimycin-insensitive NADH-cytochromec oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex ofHevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH.2. Twob-type cytochromes are present in the membranes. Cytochromeb₅₆₃ is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 °K. A second cytochrome, cytochromeb₅₆₁, seems to be reducible by hydrosulfite only.3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochromeP-450 could not be demonstrated.4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Ethylene stimulation of latex production in Hevea brasiliensis

Rubber tree (Hevea brasiliensis) is an important industrial crop for natural rubber production. Ethylene, as a stimulant of latex production in H. brasiliensis, has been widely used in commercial latex production. However, the mechanism of ethylene action are not completely elucidated, especially in molecular aspect. Here, we focus on the molecular biological progression of ethylene stimulation of latex production. Our data and all previous information showed ethylene had little direct effect on accelerating rubber biosynthesis. The prolonged latex flow and acceleration of sucrose metabolism by ethylene may be the main reasons for the stimulation of latex yield by ethylene.Key words: Hevea brasiliensis, ethylene, rubber production, gene, sucrose     

Modification of pH of latex cytoplasm by ethylene

In the absence of glycolytic activity the pH of isolated latex cytoplasm of Hevea brasiliensis increases progressively during the first hours of incubation. This increase is greatly enhanced after bark treatment with 2-chloroethylphosphonic acid. The process of alkalization is not enzymic and is oxygen dependent. This observation is discussed in relation to the increase in latex pH in situ under hormone action.     

ATPase membranaire de vacuoles lysosomales: Les lutoides du latex d'Hevea brasiliensis

Membranes of the lutoids present in the latex of Hevea brasiliensis possess an ATPase which is separable from adsorbed residual acid phosphatase. The pH optimum of the ATPase is 7.75 in Tris—HCl and is displaced to 6.5 in K-phosphate buffer. A divalent cation is obligatory (Mg  Mn > Ca). ATP-Mg is the natural substrate of the enzyme. Monovalent cations have practically no action on the enzyme. It is, however, activated by anions, both inorganic (Cl^?, HCO₃^?) and organic (malate, aspartate, tartrate). The enzyme has a higher specificity for ATP than for GTP, CTP or UTP and is non-competitively inhibited by ADP. The enzyme is temperature sensitive and a break in the Arrhenius plot occurs at about 20°, characteristic of membrane-bound enzymes. SH-group poisons inhibit enzyme activity as do classical uncouplers at high concentrations (about 10^?3 M). A hypothesis is formulated whereby the membrane-bound lutoid ATPase functions as a proton pump in order to maintain the acid pH of a vacuolar and lysosomal compartment.     

Comparative Study on Plant Latex Particles and Latex Coagulation in Ficus benjamina,Campanula glomerata and Three Euphorbia species

Among latex-producing plants, mainly the latex of Hevea brasiliensis has been studied in detail so far, while comprehensive comparative studies of latex coagulation mechanisms among the more than 20,000 latex-bearing plant species are lacking. In order to give new insights into the potential variety of coagulation mechanisms, the untreated natural latices of five latex-bearing plants from the families Euphorbiaceae, Moraceae and Campanulaceae were visualised using Cryo-SEM and their particle size compared using the laser diffraction method. Additionally, the laticifers of these plants species were examined in planta via Cryo-SEM. Similar latex particle sizes and shape were found in Ficus benjamina and Hevea brasiliensis. Hence, and due to other similarities, we hypothesize comparable, mainly chemical, coagulation mechanisms in these two species, whereas a physical coagulation mechanism is proposed for the latex of Euphorbia spp. The latter mechanism is based on the huge amount of densely packed particles that after evaporation of water build a large surface area, which accelerates the coagulation procedure.     

Cinetique d'action de l'acide 2-chloro ethylphosphonique sur les polyribosomes du latex d'Hevea brasiliensis

Effects of 2-chloroethylphosphonic acid on the latex of Hevea brasiliensis were studied during 7 days after its application on the tapping panel of the tree. Ribosome polymerisation shows a dramatic rise within 12 hr after treatment. This shift is overcome at first without rRNA synthesis. Increase of latex production and latex pH exhibit similar features and start 24 hr after application of the, stimulant. After 4 days, rRNA concentration and the stability of the lutoïds (microvacuoles with lysosomal characteristics) change significantly.     

Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production

Background and Aims

The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel^®) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described.

Methods

Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel.

Key Results

Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application.

Conclusion

The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.Key words: Hevea brasiliensis, laticifers, latex production, ethylene, sucrose transporters     

Hevamine: A crystalline basic protein from Hevea brasiliensis latex

The isolation and purification of a basic protein from the bottom fraction of Hevea brasiliensis latex is described. Two protein fractions were obtained by chromatography on carboxymethyl cellulose which also differed in their electrophoretic mobility in polyacrylamide gel, but the similarity of their other properties precludes their classification as two protein entities at this stage.     

A neutral cytoplasmic phosphatase from the latex of Hevea brasiliensis

The cytoplasmic serum prepared from the latex of Hevea brasiliensis contains an Mg²⁺ -dependent phosphatase named ‘neutral cytoplasmic phosphatase’ (NCP) which is found at the first tapping of the tree. NCP has been purified and some of its properties have been studied.     

Glycolipid composition of Hevea brasiliensis latex

Glycolipids of fresh latex from three clones of Hevea brasiliensis were characterized and quantified by HPLC/ESI-MS. Their fatty acyl and sterol components were further confirmed by GC/MS after saponification. The four detected glycolipid classes were steryl glucosides (SG), esterified steryl glucosides (ESG), monogalactosyl diacylglycerols (MGDG) and digalactosyl diacylglycerols (DGDG). Sterols in SG, ESG and total latex unsaponifiable were stigmasterol, β-sitosterol and Δ⁵-avenasterol. The latter was found instead of fucosterol formerly described. Galactolipids were mainly DGDG and had a fatty acid composition different from that of plant leaves as they contained less than 5% C18:3. Glycolipids, which represented 27–37% of total lipids, displayed important clonal variations in the proportions of the different fatty acids. ESG, MGDG and DGDG from clone PB235 differed notably by their higher content in furan fatty acid, which accounted for more than 40% of total fatty acids. Clonal variation was also observed in the relative proportions of glycolipid classes except MGDG (8%), with 43–51% DGDG, 30–34% SG and 7–19% ESG. When compared with other plant cell content, the unusual glycolipid composition of H. brasiliensis latex may be linked to the peculiar nature of this specialized cytoplasm expelled from laticiferous system, especially in terms of functional and structural properties.     

Purification and characterization of hevain,a serine protease from Hevea brasiliensis

A serine protease of MW 69000 has been isolated, in homogeneous form, from the latex of Hevea brasiliensis. The enzyme, named hevain, has only limited esterolytic and proteolytic abilities, a maximum activity in the pH range 6.5–7.5, and a pI of 4.3. Hevain has a notably high content of acidic amino acids, while the aromatic residues are present in relatively minor amounts.     

Composes phenoliques du latex d'Hevea brasiliensis: aglycones

The latex of Hevea brasiliensis; clone PR 107, contains from 160 to 1100 μg total phenolics per ml. This wide variability is associated with season, tapping system and application or not of a stimulant (Ethrel). The following aglycones have been identified in hydrolysed extracts: vanillic, salicyclic, syringic, gentisic, p- and m-hydroxybenzoic and protocatechuic acids; scopoletin, aesculetin and coumarin; ferulic, sinapic, caffeic, o- and p-coumaric acids; quercetin and kaempferol; tyrosine and dihydroxyphenylalanine. Flavans and condensed tannins are also present in latex.