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.     

Relationship between latex yield of Hevea brasiliensis and antecedent environmental parameters

A study on the relationship between latex yield and antecedent environmental data was undertaken for five clones (RRII203, RRII118, RRIM600, RRII105 and GT1) of Hevea brasiliensis (rubber) in Agartala, northeast India, a region in which rubber is not traditionally cultivated. The explained variance for the regression equations based on parameters determined on the day of tapping and up to 3 days prior to it, varied from 72% to 37% during the NWT period and 94-83% during the WT period. Soil moisture storage, 1 and 3 days prior to tapping, was found to be the primary parameter affecting yield for the NWT and WT periods, respectively. It was observed that the clone RRII105, with a comparatively lower yield to that of RRIM600, was more susceptible to daily WD conditions during the non-winter season. RRIM600 and RRII105 being high-yielding clones were also found to be fairly dependent on the AT of the day prior to tapping. The mean lag period correlation of this parameter with yield was also found to be higher during the WT period than during the NWT period. As a whole, the mean lag period based on prior measurements of environmental variables showed optimum correlation with yield at 15-20 days prior to the day of tapping. The study also confirms that varied responses of yield with environmental factors in this non-traditional region of rubber cultivation depend on clonal character.     

Expression of the sucrose transporter 3 (<Emphasis Type="Italic">HbSUT3</Emphasis>) in rubber tree and its relation to latex yield

Sucrose is not only a precursor of rubber biosynthesis, but it also participates in other latex regeneration between repeated tapping. Sucrose transporter (HbSUT) genes play key roles in sinks and sources of the rubber biosynthesis pathway. HbSUT3 was dominant in expression in latex among the SUT member genes. Therefore, the expression level of HbSUT3 in latex is a potential indicator distinguishing between high- and low-yielding clones. The aim of this research was to assess the potential of this gene in selective breeding to improve latex yield, from the correlation of HbSUT3 expression with latex yield. Four high-yielding clones were sampled in this study and compared with the common RRIM 600 clone as the baseline, paired by the field. Among the putative full-length cDNAs of Hevea sucrose transporters available in the NCBI database, only HbSUT3 was detected. The HbSUT3 gene was overexpressed in the four rubber clones relative to the control, and the NK1 clone had the highest expression level. The expression level of HbSUT3 correlated positively with latex yield but negatively with the sucrose content of latex. Gene expression analysis of rubber seedlings indicated that the bark had higher expression of this gene than the leaves, and the levels correlated with latex yields of these clones. These data provided new candidate selection criteria for use in the early selection of high-yielding rubber clones, necessary for rapid cycle breeding programs.     

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.     

A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation

An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M(r) analyzed by SDS-PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca(2+). Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogenous latex lectin-like protein in the sequential process of latex coagulation.     

Pythium vexans causing patch canker of rubber trees on Hainan Island,China

This is the first report of patch canker disease of rubber trees (clone RRIM600) in China. It is characterized by discrete irregular patches of rotted, discolored bark and wood, accompanied by a decrease in latex flow. A total of seven isolates of Pythium vexans were obtained from the diseased bark of the trunks and roots of rubber trees. Inoculating these isolates into healthy, mature rubber trees resulted in symptoms similar to patch canker and the same fungal species was re-isolated from the diseased tissues. This is also the first record of Py. vexans in Hainan.     

巴西橡胶树胶乳黄色体蛋白提取及双向电泳方法初探

巴西橡胶树（Hevea brasiliensis）的黄色体在胶乳凝固和保护植株过程中有重要作用。本文比较使用三氯醋酸/丙酮（TCA/ ACE）、Tris缓冲液、磷酸缓冲液提取橡胶树胶乳黄色体总蛋白的双向电泳效果。确定一种适合双向电泳的蛋白提取方法。结果表明Tris缓冲液提取法得到的双向电泳图谱可以达到300个,尤其是低丰度蛋白呈现性较好,适合提取黄色体蛋白以进行双向电泳。     

云南保存橡胶树魏克汉种质资源亲子系谱分析

对“农业部景洪橡胶树种质资源圃”（云南景洪）保存的290份魏克汉种质资源进行了亲子系谱分析。结果表明,无性系GT1、PR107、PB86、RRIM600、云研277-5、PB5/51、IAN873的后代为210份,占72.41%。从表型看,魏克汉种质资源群体变异丰富,实际上遗传基础很窄。本文以GT1、PR107、PB86、RRIM600、云研277-5、PB5/51、IAN873为主线建立了亲子系谱图,并对部分种质资源的性状进行了分析,获得一些性状的传递遗传规律,供育种工作者参考。     

The Regulation of Invertase Activity in the Latex of Hevea brasiliensis Muell. Arg: THE EFFECTS OF GROWTH REGULATORS, BARK WOUNDING, AND LATEX TAPPING

Treatments which increase latex yield, e. g. bark scraping,latex tapping, and bark application of 2, 4-D or 2-chloroethylphosphonicacid (CEPA) were found to enhance the activity of latex invertase.In previously untapped trees, both the introduction of tappingand the application of 2, 4-D brought about an increase in thelevel of invertase. In regularly tapped trees, the amount oflatex invertase is several times higher than in untapped treesand evidence was obtained that its activity is regulated bythe variation of latex pH. The pH of latex of the clone investigated(PR 107) was shown to vary between 6.3 and 7.1 whereas the activityof invertase, as assayed directly in the latex, has a sharpoptimum at pH 7.5 and falls rapidly with the shift of pH tothe acid side. There was no increase in the content of latexinvertase when trees adapted to regular tapping were treatedwith 2, 4-D. The effect of auxin on actual invertase activitywas essentially mediated through related increase of latex pH.The CEPA and bark scraping were also shown to increase latexpH in tapped trees. The treatment of the bark of tapped trees with CEPA increasedthe level of latex sucrose, as did auxins. Bark scraping alsohad a slight stimulatory effect. The K_m of latex invertase asa function of pH was found to change in the same way as V_max,being highest at pH optimum.     

Genetic and epigenetic uniformity of polyembryony derived multiple seedlings of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

Hevea brasiliensis Muell. Arg (Para rubber tree) is a tropical tree species of Amazonian origin widely cultivated in several parts of the world for natural rubber, a highly priced commodity inevitable for the world rubber industry. Large, tree to tree variation in growth and latex yield among individual plants of high yielding Hevea clones is a common phenomenon observed in mature rubber plantations. The genetic heterogeneity of the seedlings which are used as rootstocks for propagation through budgrafting is considered as a major factor  responsible for this variation. In order to minimize this variation, attempts were made to develop highly uniform rootstock material via an in vitro technique by inducing zygotic polyembryony in Hevea. Immature open pollinated fruits of a high yielding clone RRII 105 were cultured by half ovulo embryo culture technique. Multiple embryos were induced from the 8–10-week-old zygote with a novel combination of gibberellic acid (GA₃), kinetin, and zeatin. Plantlets were successfully generated from the multiple embryos and raised in the field post hardening. Screening using genetic and epigenetic molecular markers revealed that the multiple seedlings developed are highly uniform and are of single zygotic origin. Development of plants having genetic and epigenetic uniformity suggests that this technique is ideal for raising uniform rootstock material in Hevea which may significantly reduce intraclonal variations. Moreover, these plants could serve as ideal material for physiological and molecular investigations towards the understanding of  stock–scion interaction process in rubber.     

Callus friability and somatic embryogenesis inHevea brasiliensis

The influence of plant growth regulators, sucrose, calcium and various macronutrient media on callus friability and somatic embryogenesis was investigated inHevea brasiliensis Müll. Arg. Friable and embryogenic calli were spontaneously formed in two rubber tree clones (PR 107 and RRIM 600) on the Medium for Hevea (MH), with 3,4-dichlorophenoxyacetic acid (3,4-d), kinetin and sucrose, while compact embryogenic calli were enhanced in three other clones (PB 260, PB 235 and GT1). Callus friability was enhanced in clone PB 260 when the concentration of one growth factor (3,4-d or kinetin) was reduced from 4.5 μLM to 0.45 μM during the first culture, or when high sucrose or calcium levels 351 mM and 12 mM, respectively) were maintained during subcultures. The different macronutrient media did not alter callus texture but only use of MH and Murashige and Skoog (MS) media led to somatic embryogenesis. Friable calli obtained by modifying the auxin/cytokinin balance lost their embryogenic potential. In contrast, those obtained on media with high sucrose or calcium concentrations were mainly composed of embryogenic cells embedded in a mucilaginous matrix. Such calli could be of potential interest for establishing embryogenic cell suspension cultures.     

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.     

巴两橡胶树胶乳黄色体蛋白提取及双向电泳方法初探

巴西橡胶树(Hevea brasiliensis)的黄色体在胶乳凝固和保护植株过程中有重要作用。本文比较使用三氯醋酸/丙酮(TCA/ ACE)、Tris缓冲液、磷酸缓冲液提取橡胶树胶乳黄色体总蛋白的双向电泳效果。确定一种适合双向电泳的蛋白提取方法。结果表明Tris缓冲液提取法得到的双向电泳图谱可以达到300个,尤其是低丰度蛋白呈现性较好,适合提取黄色体蛋白以进行双向电泳。     

Relationship between cyanogenesis and latex stability on tapping panel dryness in rubber trunk girth

The presence of high cyanogenic glycoside concentrations may predispose plant to the tapping panel dryness (TPD). This study aimed to verify the involvement of cyanogenesis in the reduction of latex stability and in the establishment of TPD. The following parameters were evaluated in rubber tree trunk bark: concentration of cyanogenic glycosides with determination of cyanogenic potential (HCNp) and latex stability with lutoid bursting index (LBI). The study of the relationship between cyanogenesis and TPD was performed by semiquantitative comparison of hydrogen cyanide (HCN) gas released from the trunk bark under the following conditions: without (0%) and with (100%) TPD. The positive correlations between HCNp values and LBI indicate that cyanogenic glycosides present in the bark reduce latex stability, resulting in low yield due to the short duration of flow during tapping. The largest amount of HCN released by trunk bark tissues when the plant exhibits TPD symptoms strengthens the evidence of the involvement of this compound in the establishment of this condition.     

A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation

In the first of this three paper series, an in vitro latex coagulation was shown to arise from aggregation of rubber particles (RP) and lutoid membranes. RP aggregation was shown to be induced by a specific Hevea latex lectin-like protein (HLL) present on the lutoid membrane. In this second paper, a binding protein (BP) ligand counterpart for HLL was identified. This RP-HLLBP, having a specific interaction, with HLL was isolated from RP and characterized. The protein was extracted from the small RP in the presence of a surfactant (0.2% Triton-X-100) and further purified to homogeneity. Purification steps included acetone precipitation, heat-treatment, and column chromatography. The presence of RP-HLLBP was monitored by its ability to compete with erythrocytes in the hemagglutination inhibition (HI) assay. The purified RP-HLLBP had an HI titre of 1.37 microgml(-1), a pI value of 5.4, optimum activity at pH 5-8 and was thermostable up to 60 degrees C. On SDS-PAGE a single glycoprotein with M(r) of 24 kDa was detected while on native PAGE the major Mr was about 120 kDa. The purified RP-HLLBP was shown to inhibit latex coagulation. Chitinase, but no other glycosidase tested, abolished its HI action and inhibited HLL-induced RP aggregation in a competitive dose dependent manner. This indicated the presence of, and role for, N-acetylglucosamine residues in the binding recognition. The Hevea latex lectin-like protein can thus be referred to as a Hevea latex lectin. Based on protein identification by peptide mass fingerprinting, the RP-HLLBP was confirmed to be the small rubber particle protein (SRPP). This work has unambiguously determined the role of an intrinsic RP glycoprotein (RP-HLLBP or SRPP) as a key component in formation of the rubber latex coagulum.     

Seasonal suitability of three rubber tree clones to Calacarus heveae (Acari, Eriophyidae)

The suitability of rubber tree clones to Calacarus heveae was inferred from the life cycle, reproduction and survivorship of this mite. The assays were performed under controlled conditions with leaflets detached from 6-year-old plants. The development of 20 C. heveae individuals on each of the clones GT 1, PB 235 and RRIM 600 was analysed. This experiment was performed four times during periods when C. heveae was abundant in the field: (P1) November–December 2005, (P2) January–February, (P3) March–April and (P4) May–June 2006. Accordingly, the leaflets used in each assay represented the physiological condition of the host plant during each period. This approach allowed us to evaluate the seasonal suitability of rubber tree clones to C. heveae. We observed seasonal differences in the suitability of rubber tree clones to mite attack. The mites reared on the PB235 had a shorter development period, the highest egg production and highest survivorship. This evidence showed that the PB 235 was the most suitable of those tested. We also observed that the leaflets used in the assays during periods P2 and P3 were the most favourable for the development of C. heveae. This finding emphasises the seasonal suitability of rubber tree leaflets. On the other hand, GT 1 showed higher resistance against C. heveae than did RRIM 600 and PB 235, primarily during the period from November to February. This result indicated that use of the GT 1 clone to control the mite might represent an alternative for growers.     

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.     

Susceptibility of rubber trees to Loranthaceae in Gabon

Abstract

In Gabon, two major species of Loranthaceae, Phragmanthera capitata (Sprengel) Balle and Globimetula braunii (Engl.) Danser to a lesser extent, parasitize Hevea brasiliensis (Müll.) Arg. The behavior of different clones of rubber trees facing the Loranthaceae parasitism has been studied in the large industrial plantation of Mitzic and in three neighboring smallholding plantations. The clones PR 261, RRIM 600 and PB 260, although never totally resistant, are far less susceptible to Loranthaceae than the clones AVROS 2037, PB 217, PB 235 and GT 1. The parasite development kinetics was more rapid on the most susceptible clones. Phellem thickness, cortical parenchyma thickness and tannin cell density were independent of susceptibility. The number of tree ramifications was significantly different between the clones. Interestingly, on the least susceptible trees, being also the most ramified, the parasite showed the slowest development.