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.     

乙烯利刺激橡胶树增产及其分子生物学基础

本文介绍近年来乙烯利刺激橡胶树增产的分子生物学研究进展,讨论乙烯利刺激橡胶树胶乳增产的可能原因,并对这一领域今后研究的发展趋势进行了展望。     

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     

Sucrose Synthetase in the Latex of Hevea brasiliensis Muell. Arg

Sucrose synthetase (EC 2.4.1.13[EC]) was found in the latex of therubber tree but the activity of sucrose phosphate synthetase(EC 2.4.1.14[EC]) was not detected. The enzyme was purified andsome properties have been investigated. Examination of the kineticsof sucrose synthesis revealed K_m of 0.56 mM for uridine diphosphoglucoseand 3.85 mM for fructose. Mg²⁺ and cyanide activated sucrosesynthesis but reduced the cleavage reaction. Increased pH hadthe same effect, the synthetic activity being higher than theactivity of sucrose breakdown within the physiological levelsof latex pH. In the latex of regularly tapped trees, the total enzyme activityin the direction of synthesis was about 10% or less of the totalinvertase activity at pH 7.0. Because of the strong limitationof invertase under natural conditions, the proportion of actualsynthetase activity is, however, much higher and evidence ispresented that in the latex of regularly tapped trees this activitysignificantly reduces carbohydrate breakdown. Some indications have been obtained that this involvement ofsucrose synthetase is weakened by application of Ethrel to thebark. A reduction of its synthetic activity, accompanied byan acceleration of sucrose utilization in latex cytoplasm andby an increase of latex yield, could be observed before thetreatment-induced rise of pH enhancing inver.     

一种快速、高效的橡胶树胶乳总RNA提取方法

胶乳是橡胶树（Hevea brasiliensis）乳管中特殊的细胞质,主要由橡胶粒子、黄色体、F-W粒子和普通细胞质成分构成,其中橡胶粒子占20％-40％,蛋白含量高达1％-2％。由于高比例橡胶粒子和蛋白质的干扰,目前使用的胶乳RNA提取方法都具有步骤繁琐、胶乳需求量大、操作技巧性强不易掌握等缺点。为快速、高效地获取高质量的胶乳RNA,我们在现有方法的基础上摸索出一套步骤简单、容易操作、快速、高效提取橡胶树胶乳总RNA的简易方法,获得了较好的实验效果。紫外分光光度计、RT-PCR和RACE分析结果表明,使用该方法提取的胶乳RNA质量完全能够满足相应的分子操作,但所需时间仅为目前常用方法的50％,RNA获得率提高了2-3倍,操作难度大大降低。     

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.     

The Latex of Hevea brasiliensis Contains High Levels of Both Chitinases and Chitinases/Lysozymes

The latex of the commercial rubber tree, Hevea brasiliensis, was fractionated by ultracentrifugation as described by G. F. J. Moir ([1959] Nature 184: 1626-1628) into a top layer of rubber particles, a cleared cytoplasm, and a pellet that contains primarily specialized vacuoles known as lutoids. The proteins in each fraction were resolved by two-dimensional gel electrophoresis. Both the pellet fraction and cleared cytoplasm contained large amounts of relatively few proteins, suggesting that laticifers serve a very specialized function in the plant. More than 75% of the total soluble protein in latex was found in the pellet fraction. Twenty-five percent of the protein in the pellet was identified as chitinases/lysozymes, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of bacterial cell walls. Both the chitinase and lysozyme activities were localized exclusively in the pellet or lutoid fraction. The chitinases/lysozymes were resolved into acidic and basic classes of proteins and further purified. An acidic protein (molecular mass 25.5 kD) represented 20% of the chitinase activity in latex; this protein lacked the low level of lysozyme activity that is associated with many plant chitinases. Six basic proteins, having both chitinase and lysozyme activities in various ratios and molecular mass of 27.5 or 26 kD, were resolved. Two of the basic proteins had very high lysozyme specific activities which were comparable to the specific activities reported for animal lysozymes. Like animal lysozymes, but unlike previously characterized plant chitinases/lysozymes, these basic chitinases/lysozymes were also capable of completely lysing or clearing suspensions of bacterial cell walls. These results suggest that laticifers may serve a defensive role in the plant.     

Involvement of HbPIP2;1 and HbTIP1;1 Aquaporins in Ethylene Stimulation of Latex Yield through Regulation of Water Exchanges between Inner Liber and Latex Cells in Hevea brasiliensis

Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl₂. In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.Hevea brasiliensis (rubber tree) is the only commercial source of natural rubber. Rubber (cis-1,4-polyisoprene) is synthesized as rubber particles in highly specialized cells, which, when mature, form concentric mantels of articulated “laticifer” networks in the inner liber of the rubber tree (Hébant and de Faÿ, 1980; Hébant, 1981). Upon bark tapping, the laticifers are severed and their fluid cytoplasm (“latex”) flows out until coagulation processes lead to the plugging of their extremity (d''Auzac, 1989a; Yeang, 2005). Gooding (1952) was the first to show that tapping induced, during the latex flow, progressive dilution of the latex coming from the remote areas to the tapping cut. Thus, tapping the rubber tree induces not only latex flow but also complex water circulation at the bottom of the trunk from the inner liber tissues (Lustinec et al., 1968; d''Auzac, 1989b) and from the xylem through the vascular rays, which have been reported to be numerous in H. brasiliensis (Hébant and de Faÿ, 1980).The rubber particles account for up to 55% to less than 30% of the collected latex volume depending on the season, the tapping hour, the tree age, the rubber clone, and the exploitation system. The latex flow rate and duration are the first intrinsic factors known to limit rubber yield: the faster and the longer the latex flow, the higher the yield (d''Auzac, 1989a). These two factors are under the control of the turgor pressure in the liber tissues (Gooding, 1952; Buttery and Boatman, 1964), of the latex dry rubber content (DRC) or total solid content (TSC), and finally of the latex coagulation efficiency (Kongsawadworakul and Chrestin, 2003). In addition, DRC is positively linked to latex viscosity and thus inversely linked to latex fluidity and yield (Van Gils, 1951).Treatment of the rubber tree bark with Ethrel, an ethylene releaser, markedly increases the production of latex (d''Auzac and Ribaillier, 1969) owing to transient partial removing of the yield-limiting factors. In particular, stimulation of rubber tree yield, either as previously with synthetic auxins or as now with Ethrel, has so far been reported to (1) decrease latex DRC, TSC, and osmolarity (Baptist and de Jonge, 1955; Tjasadihardja and Kardjono, 1974; Coupé and Chrestin, 1989), indicating latex dilution; (2) extend the bark drainage area (Boatman, 1966; Lustinec et al., 1967); and (3) decrease the laticifer-plugging index (Yip and Gomez, 1980). Furthermore, it has been reported that the rubber clones with the lowest latex DRC corresponded to those yielding the highest latex volume and dry rubber production but displayed only slight response to stimulation, and inversely (Tjasadihardja and Kardjono, 1974; Lee and Tan, 1979; Gohet et al., 2003). For example, without stimulation, PB217, a rubber clone with a high yield potential, is characterized by relatively high TSC, short latex flow, and low metabolic activity (Obouayeba et al., 1996). However, with stimulation, PB217 fully expresses its yield potential, due to prolonged latex flow and higher metabolic activity in response to ethylene. Therefore, the PB217 rubber clone is a good model to study and understand the effects of ethylene stimulation on latex yield.The circulation of water between the different liber tissues, as well as the latex water content, are of major importance in the processes of latex flow. Water coming from the phloem and the xylem can use two complementary routes to circulate between and within tissues: (1) symplastic pathways (Varney et al., 1993), where water and solutes can move from cell to cell through the plasmodesmata (Blackman and Overall, 2001); and (2) intercellular spaces, or apoplastic pathways (Canny, 1995). The apoplastic water cannot easily cross biological membranes, but this process can be facilitated by water channels called “aquaporins.” Unlike the other cells that surround them (parenchyma cells, vascular ray cells, sieve tubes companion cells, etc.), mature latex vessels are devoid of plasmodesmata (de Faÿ et al., 1989). Thus, water fluxes across the laticifer plasmalemma are probably mainly controlled by aquaporins.Aquaporins belong to a ubiquitous large family of major intrinsic proteins (MIPs) known to facilitate water and/or small neutral solute fluxes across cell membranes (Chrispeels and Maurel, 1994; Maurel et al., 2008). Plant aquaporins have been classified in four subfamilies, including the two major ones: PIPs (for plasma membrane intrinsic proteins) and TIPs (for tonoplast intrinsic proteins). The PIP family has been clustered into two major groups as PIP1 and PIP2. PIP2s have been shown to be far more efficient than PIP1 in mediating water transport (Baiges et al., 2002). In addition, although phosphorylation, pH, Ca²⁺, and osmotic gradients can affect water channel activity (Johansson et al., 1998; Chaumont et al., 2005; Maurel et al., 2008), the MIP gene expression level has been shown to play a major role in controlling the membrane water permeability. Expression of MIP genes is regulated during development and by different environmental factors, such as light (Cochard et al., 2007) and various stresses. They have been reported to be either down-regulated (Aharon et al., 2003; Quist et al., 2004; Alexandersson et al., 2005) or up-regulated (Guerrero et al., 1990) in response to water stress or to freezing-thawing events (Sakr et al., 2003).As mentioned above, yield stimulation with Ethrel was reported by several authors to induce dilution of latex. To address the possible role of aquaporins in this process, we constructed a full-length cDNA library from Hevea inner bark RNA of control and Ethrel-stimulated trees. Among about 4,000 ESTs sequenced, three aquaporins encoding PIP1, PIP2, and TIP isoforms were found. The full-length HbPIP2;1 and HbTIP1;1 cDNAs were cloned. They were further characterized with respect to the kinetics of ethylene effects on their expression, in relation to latex dilution and production, of mature virgin rubber trees of the PB217 clone. The function of both HbPIP2;1 and HbTIP1;1 proteins was verified, and their respective promoters were analyzed in silico. Aquaporin gene expression and function in response to ethylene treatments are proposed to favor water circulation within the inner bark tissues of rubber trees, thereby helping ease and prolong latex flow, hence increasing rubber yield.     

The Role of Lipids and Proteins in the Mechanism of Latex Vessel Plugging in Hevea brasiliensis

The phospholipid content of the bottom fraction of latex as well as the neutral lipids of rubber particles were determined in thirty-one clonal mother trees of RRII clones of Hevea brasiliensis Muell. Arg. Ratios between cationic proteins and anionic proteins in the B-sera, which is the sera contained in the lutoid particles in latex, obtained from clones RRIM-501, PB 6/9, RRII-105 and Tjir-l were determined by electrophoresis. The influence of these factors on plugging index (a measure of the magnitude of latex vessel plugging) was investigated. Lutoid instability, as indicated by bursting index, is negatively ocrrelated with the phospholipid content of the bottom fraction of latex. The neutral lipid content of rubber particles is positively correlated with the colloidal stability of latex. The latex vessel plugging during latex flow is found to be negatively correlated with both the lutoid stability and the neutral lipids in the rubber particle. A high ratio of cationic and anionic proteins in B-serum may also enhance the process of plugging.     

Inhibition of Ethylene Production by Fatty Acids in Fruit and Vegetable Tissues

Fatty acids of chain length from C₄ to C₁₂ inhibited ethyleneproduction in wounded albedo tissue of Hassaku (Citrus hassakuHort. ex Tanaka) fruit. Of the fatty acids tested, caprylicacid (C₈) and capric acid (C₁₀) were the most effective. Lauricacid (C₁₂) was less effective, and caproic acid (C₆) and butyricacid (C₄) were the least effective. Caprylic acid at 5 mM markedlyinhibited ethylene production in not only wounded albedo tissueof citrus fruit but also apple (Malus sylvestris Mill.) cortex,tomato (Lycopersicon esculentum Mill.) pericarp, cucumber (Cucumissativus L.) cortex, banana (Musa AAA group Cavendish subgroup)pulp, broccoli (Brassica oleracea L.) floret, spinach (Spinaciaoleracea L.) leaf, lettuce (Lactuca sativa L.) leaf and mungbean (Vigna radiata [L.] Wilczek) hypocotyl. Caprylic acid inhibitedethylene production at the step of conversion of l-aminocyclopropane-l-carboxylicacid to ethylene. The inhibition could be partially relievedby transferring the tissue to caprylic acid-free medium. (Received June 15, 1982; Accepted August 13, 1982)     

Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells

The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ΔpH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H⁺-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.     

A Comparison of Abscission of Rubber (Hevea brasiliensis) Leaves Infected with Microcyclus ulei with Leaf Abscission Induced by Ethylene Treatment, Deblading and Senescence

Studies on the histology and on effects of growth substancesand phenols as well as changes in activities of pectinmethylesterase indicated that the mechanism of abscission of Hevealeaflets infected with Microcyclus ulei differed from the mechanismof abscission of debladed, ethylene treated and senescent leaves.An abscission layer which was formed during abscission of debladed,ethylene-treated and senescent leaves was absent during abscissionof heavily diseased leaves. The ratio of pectinmethyl esteraseactivities in tissues distal to the abscission zone to activitiesin tissues proximal to the zone decreased in debladed and ethylenetreated leaves but such decreases were not detected during abscissionof Hevea leaves infected with M. ulei. Hevea brasiliensis Muell. Arg., rubber, leaf abscission, Microcyclus ulei, ethylene, indol-3-ylacetic acid, kinetin     

Range of Hevea brasiliensis Pollen Dispersal Estimated by Esterase Isozyme Markers

A study was carried out to estimate the distance Hevea brasiliensispollen could be dispersed under natural conditions. Seeds werecollected at varying distances from the boundary between twoadjacent fields that were each planted with a pure stand ofa genetic clone. Esterase isozyme markers were used to determineif the seeds had been derived from self- or cross-pollination.The incidence of cross-pollination was then examined in relationto the distance from the inter-clonal boundary. A logarithmicmodel gave the best fit (r²=0.864) and suggested that pollencould travel distances in the order of 0.3 to 1.1 km. Copyright1999 Annals of Botany Company Hevea brasiliensis (rubber tree), pollen dispersal, cross-pollination, self-pollination, esterase isozymes.     

基因枪法获得GAI转基因橡胶树植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg²⁺ diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg²⁺. These results suggest the presence of a Mg²⁺/H⁺ antiporter. The maximum Mg²⁺/H⁺ exchange rate was observed at pH 8.5. The K_m values for Mg²⁺ (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K⁺ and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg²⁺/H⁺ exchange was electroneutral. Mg²⁺/H⁺ exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K_m for Mg²⁺ are similar to those reported for the Mg²⁺/2Na⁺ antiporter in animals cell. These data are consistent with the existence of a Mg²⁺/2H⁺ antiporter in a plant tonoplast.