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.     

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.     

Ethephon Increases Rubber Tree Latex Yield by Regulating Aquaporins and Alleviating the Tapping-Induced Local Increase in Latex Total Solid Content

The concentration of phloem solute generally falls from leaves to roots. However, a local increase in latex total solid content (LILTSC) was identified near the tapping cut of rubber trees. To understand the mechanism of ethephon-stimulated latex yield, the formation and ethephon (an ethylene releaser) alleviation of the LILTSC near the tapping cut were examined. It was found that the LILTSC near the tapping cut of a tapped rubber tree was caused by the tapping-accelerated rubber biosynthesis which began following the first tapping and became significant after the fourth tapping. Ethephon stimulation markedly reduced the LILTSC. The latex yield change pattern upon ethephon stimulation was associated with the kinetic change of LILTSC and the decomposition dynamic of ethephon into ethylene. Once the LILTSC was reduced by ethylene release upon ethephon stimulation, the latex yield increased; however, when the ethylene release upon ethephon stimulation receded, the LILTSC was restored and the effect of ethephon stimulation dissipated. The reduction of LILTSC by ethephon stimulation could be ascribed to the translocation property of ethylene in plants and its regulation of aquaporins. Because maximum ethylene release upon tapping-cut-ethephon-application occured close to the tapping cut, the aquaporins were more up-regulated in this region, leading to a reduction of the LILTSC and an increase in latex yield. All these results suggest that the LILTSC near the tapping cut was caused by tapping; the ethephon-induced aquaporin up-regulation and LILTSC reduction are involved in the mechanism of ethephon-promoted latex yield.     

Stimulatory effects of 2,4-dichlorophenoxyacetic acid and of 1-naphthylacetic acid on sucrose level,invertase activity and sucrose utilization in the latex ofHevea brasiliensis

Summary Treatment of the bark ofHevea brasiliensis with 2,4-dichlorophenoxyacetic acid (2,4-D) or l-naphthylacetic acid (NAA) greatly increases sucrose level, invertase activity and sucrose utilization in the latex; the efficacy of 2,4-D is considerably greater than that of NAA. The greater sucrose utilization is the consequence of increased invertase activity. The changes occur as soon as the first tapping following bark treatment. It is suggested that the rise in both sucrose level and utilization in the latex serum mediate the effect of auxins on latex production. This is most likely related to a faciliation of latex outflow resulting from an increase in the osmotic and turgor pressure in the laticiferous tissue, as well as to enhanced regeneration of latex.The latex invertase has been found to be of a weakly alkaline type, with a sharp pH optimum at 7.15–7.20 in citrate-phosphate buffer. Its activity falls of rapidly on the acid side, being almost zero at pH 6.4. Since the natural pH of latex generally varies between pH 6.5 and 7.0, it is suggested that pH is an important factor in the regulation of invertase activity in the latex, and that the limiting nature of invertase-mediated sucrose hydrolysis in latex serum is caused by unfavourable conditions for invertase activity rather than by a scarcity of this enzyme.Expert of the International Atomic Energy Agency.     

Effects of Tapping, Wounding, and Growth Regulators on Turgor Pressure in Hevea brasiliensis Muell. Arg.

Various forms of wounding result in increases in hydrostaticpressure in the laticiferous phloem tissue of Hevea brasiliensis;regular tapping, a form of controlled wounding, probably causesa similar response. When a tree is ring-barked, there is a transientfall in turgor immediately above and below the ring, presumablyowing to loss of latex during cutting. This is followed by apressure increase which is particularly marked above the ring,suggesting the accumulation of metabolites. Isolation of an‘island’ of tissue, by cuts down to the wood, resultsin a steep fall in turgor within it, although a relatively slowrecovery may follow. Partially isolated panels show smallerfalls and a more rapid rise. It appears that a functional phloemconnexion permits a more rapid recovery of osmotic and turgorpressures following latex losses, both in the isolation experimentsand in normal tapping. Growth regulators such as 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) increase latex yields by prolonging latex flowafter tapping. In untapped trees treatment with 2,4,5-T resultsin a slow and fairly small increase in turgor pressure, butthis effect may not be apparent if trees are regularly tapped.When the tapping cut is opened, there is a rapid fall in pressureimmediately under the cut. This loss in turgor spreads throughthe latex-vessel system as latex flows towards the cut, butrecovery is apparent near the cut even before flow ceases. Pressuregradients indicate a rapid formation of a localized resistanceto flow at or near the cut surface. This process appears toplay a major part in restricting flow. The increased yieldswhich result both from regular tapping and after 2,4,5-T treatmentappear to result from a delay in this sealing process. The mechanismsby which the barrier to flow is built up and delayed by 2,4,5-Ttreatment are not clearly understood.     

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.     

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.     

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.     

Control of carbohydrate metabolism by ethylene in latex vessels ofHevea brasiliensis MUEL. ARC in relation to rubber production

The treatment of the bark of regularly tapped trees with 2-ohloroethylphosphonio acid (CEPA) in the long run brings down sucrose level in the latex even when latex production is not increased. The sucrose deficit thus caused persists for a number of months after the treatment probably due to the depletion of carbohydrate reserves by the stimulation. The loss of carbohydrates not related to an increase of rubber produotion grows as the concentrations of applied CEPA are raised. The treatment increases the activity of phosphoenolpyruvate (PEP) carboxylase and decreases pyruvate deoarboxylation in mitochondria-free oytoplasmic latex serum. The pyruvate deoarboxylase has a sharp optimum at pH 6.5 and is almost inactive at pH 7.5 while PEP oarboxylase, like invertase, has the optimum at weakly alcaline pH and is only slightly active at pH 6.5. The CEPA-induced changes in the activity of these enzymes are related to an increase of latex pH after the treatment. Consistently with this the evolution of CO2 from sugars catabolised in the serum was found to decrease with increasing pH. It is concluded that the observed ethylene-induced changes in enzyme aotivities bring about in latex vessels a deviation of the glycolysis-fed metabolio flux from synthetic pathways to mitochondrial oxidations and consequently a decrease in the efficiency of sucrose conversion to rubber.     

The leaf, inner bark and latex cyanide potential of Hevea brasiliensis: Evidence for involvement of cyanogenic glucosides in rubber yield

The latex of Hevea brasiliensis, expelled upon bark tapping, is the cytoplasm of anastomosed latex cells in the inner bark of the rubber tree. Latex regeneration between two tappings is one of the major limiting factors of rubber yield. Hevea species contain high amounts of cyanogenic glucosides from which cyanide is released when the plant is damaged providing an efficient defense mechanism against herbivores. In H. brasiliensis, the cyanogenic glucosides mainly consist of the monoglucoside linamarin (synthesized in the leaves), and its diglucoside transport-form, linustatin. Variations in leaf cyanide potential (CNp) were studied using various parameters. Results showed that the younger the leaf, the higher the CNp. Leaf CNp greatly decreased when leaves were directly exposed to sunlight. These results allowed us to determine the best leaf sampling conditions for the comparison of leaf CNp. Under these conditions, leaf CNp was found to vary from less than 25 mM to more than 60 mM. The rubber clones containing the highest leaf CNp were those with the highest yield potential. In mature virgin trees, the CNp of the trunk inner bark was shown to be proportional to leaf CNp and to decrease on tapping. However, the latex itself exhibited very low (if any) CNp, while harboring all the enzymes (β-d-diglucosidase, linamarase and β-cyanoalanine synthase) necessary to metabolize cyanogenic glucosides to generate non-cyanogenic compounds, such as asparagine. This suggests that in the rubber tree bark, cyanogenic glucosides may be a source of buffering nitrogen and glucose, thereby contributing to latex regeneration/production.     

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.     

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     

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,MAPs）基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE（Rapid Amplification ofcDNA Ends）进行差异片段的5＇和3＇端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412.序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因.半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理（伤害及乙烯处理）使其表达上调.     

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.     

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.     

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.     

Trunk contraction ofHevea brasiliensis due to tapping

Determination of bark contraction due to tapping made simultaneously at different positions on the trunk ofHevea trees (young PR 107, S/2 ↓) provided information about the flow area and the speed of contration spreading. The relation between truck contraction at the given distance from the cut and the amount of latex tapped was experessed in mathematical terms. A quantitative relation was also established between the yields of latex and the linear dimension of flow areas in longitudinal direction below the middle of tapping cut. From the contraction curves it is deduced that the lateral flow of latex becomes relatively more important with increasing yields.     

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.