Photosynthate allocation and productivity of latex vessels inHevea brasiliensis

Manipulations of production systems in rubber tree which were intended to improve sucrose translocation in tapped bark resulted in an increase of latex sucrose and of latex production and reduced the incidence of nonyielding laticiferous tissue. This was achieved by shortening the tapping cut from full to half spiral, by changing the descending direction into an ascending mode of tapping or by annual change-over of tapping panel allowing for a longer time the regeneration of bark removed above the location of the cut. The increase of latex yield did not result in a significant decrease in the growth of trees over a period of three years. Clonal differences in nonyielding bark appeared to be related to differences in sucrose depletion by tapping. In clone PB 235 which exhibited low latex sucrose, a reduction of tapping frequency resulted in an increase in sucrose level and in a decrease of bark “dryness” tending to an increase in total yield. The tapping manipulations examined did not affect latex flow characteristics such as the plugging index of latex vessels and the bursting index of lutoids. The results stress the importance of photosynthate allocation for the physiology of laticiferous system productivity and indicate the possibilities of improving assimilate economy in rubber trees. On mission as export of the International Atomic Energy Agency.     

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.     

Some aspects of sucrose transport and utilization in latex producing bark ofHevea brasiliensis Muel. Arg

The radiotracer technique is described for transport and metabolic studies in latex producing bark. This technique provided further evidence that carbohydrates are supplied to latex vessels as sucrose and that sucrose is readily used for rubber synthesis. Bark treatment with ethylene resulting in latex flow stimulation is shown to activate within a few hours sucrose and water transport into the latex vessels. The available knowledge suggests an implication of an energy-dependent process of proton excretion which could explain the earlier observed ethylene induced alkalization of latex cytosol enhancing invertase and sucrose utilization. An increase of tapping intensity decreases the level of latex sucrose and changes its distribution pattern in drained bark. There is a clonal difference in the early response to exploitation. Bark excision by conventional tapping inhibits downward sucrose translocation, the inhibition being proportional to the surface of consumed bark above the tapping cut. Seasonal variation in the level of latex sucrose is related to variations in sunshine duration and sucrose concentration rises during the process of refoliation. The results stress the importance of sucrose supply to latex vessels for latex producing potentialities and indicate an association of low sucrose availability in latex vessels with premature latex vessel degeneration and bark dryness.     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.     

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.     

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     

巴西橡胶树一个AP2／EREBP转录因子的克隆及表达分析

本文采用RACE技术从巴西橡胶树中鉴定出一个AP2／EREBP转录因子。该转录因子全长cDNANl225bp,最长开放阅读框699bp,预测编码蛋白包含232个氨基酸;序列比对分析发现,该转录因子具有一段保守的AP2／EREBP结构域,与拟南芥Soloist（At4g13040）具有较高的相似性,将该基因命名HbSoloist。半定量胙PcR分析结果表明,HbSoloist在巴西橡胶树的胶乳、叶片、树皮和花中均有表达。与健康橡胶树相比,死皮树胶乳HbSoloist表达量明显下降。研究发HbSoloist表达受乙烯利、茉莉酸和低温处理调控,表明HbSoloist基因可能在巴西橡胶树乙烯、茉莉酸和低温反应中发挥作用。     

使用微丝骨架解聚剂对橡胶树进行刺激采胶的研究

初步研究了不同浓度的(1%,5%,10%)KI、饱和浓度的大环内酯作为微丝骨架解聚剂对橡胶树的刺激排胶效果.2%的甲基纤维素处理的橡胶树作为空白.测定了各处理橡胶树的胶乳产量及胶乳的6种生理参数,即干胶含量、总固形物含量、蔗糖含量、无机磷含量、硫醇含量以及镁离子含量.结果表明施用1%KI和饱和浓度大环内酯的橡胶树胶乳产量大量增加,其增产幅度与作为天然橡胶常用刺激采胶剂--0.3%的乙烯利的增产幅度大致相当.比较通过1%KI和饱和浓度大环内酯刺激采胶的胶乳和0.3%的乙烯利刺激采胶的胶乳的各生理参数发现,3种处理得来的胶乳干胶含量和总固形物含量并没有明显的差别,但各处理的其它4个生理参数却差别明显,这意味着KI和饱和浓度大环内酯使橡胶树胶乳增产机制可能与乙烯利的机制不同.值得一提的是,高浓度的KI对橡胶树有明显的副作用,长时间的使用会引起橡胶树死皮病的发生.     

A SIMPLE APPARATUS FOR THE COLLECTION OF HEVEA LATEX UNDER ASEPTIC CONDITIONS

SUMMARY: A simple apparatus, designed to eliminate the bacterial infection of Hevea latex which occurs on tapping, is described. It produces two materials, ultraviolet irradiated latex and non-irradiated latex. An attempt has been made to maintain normal tapping conditions on a half-spiral cut. Under these conditions the latex flow time and the dry rubber content were both increased.     

Localization and Behaviour of Actin Microfilaments in Laticiferous Cells of Hevea brasiliensis in Latex Exploitation

Actin microfilaments of laticiferous cells and bark wounds in Hevea brasiliensis were studied using TRITC-phalloidin fluorescent microscopy. Actin in latex from mature rubber trees was also investigated using SDS-PAGE and western-boltting. TRITC-fluorescent substance plugged the end of laticifers when latex flow stopped. Actin was detected only in C serum among the four latex fractions. Higher actin content was found in the latex collected at the beginning of tapping than in that collected just before latex flow stopped. Lower actin content was detected in the latex from rubber trees with more intensive exploitation. The present study indicated that actin microfilaments might play an important role in regulation of latex flow and plugging of the laticifers wounds.     

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.     

橡胶树PR107微割排胶影响面的膨压测定及分析

对中国热带农业科学研究院试验场7队割制为割阴刀、1/8树围四天一刀(1/8s↑d/4)的橡胶树品种PR107胶树不同高度树干进行膨压测定。结果表明,割胶前橡胶树树干膨压分布不均匀,割胶后膨压下降较少。说明胶树茎干膨压一直维持在较高水平是气刺微割排胶时间较长的原因之一。     

Ethylene Production by Hevea brasiliensis Tissues Treated with Latex Yield-stirnulatory Compounds

Leaflets and pieces of immature stem of Hevea brasiliensis weretreated with a range of compounds, and the subsequent ratesof release of ethylene compared with the reported efficacy ofthe individual substances as stimulants of latex yield. Themost effective ethylene inducers were also the best stimulants.Amongst the 53 compounds tested, no compound which was not ayield stimulant induced the formation of significant amountsof ethylene. Of all the known stimulants tested, only acetylenewas neither converted to, nor induced the formation of ethylene.     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。