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     

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.     

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     

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.     

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.     

Chromosome-level wild Hevea brasiliensis genome provides new tools for genomic-assisted breeding and valuable loci to elevate rubber yield

The rubber tree (Hevea brasiliensis) is grown in tropical regions and is the major source of natural rubber. Using traditional breeding approaches, the latex yield has increased by sixfold in the last century. However, the underlying genetic basis of rubber yield improvement is largely unknown. Here, we present a high-quality, chromosome-level genome sequence of the wild rubber tree, the first report on selection signatures and a genome-wide association study (GWAS) of its yield traits. Population genomic analysis revealed a moderate population divergence between the Wickham clones and wild accessions. Interestingly, it is suggestive that H. brasiliensis and six relatives of the Hevea genus might belong to the same species. The selective sweep analysis found 361 obvious signatures in the domesticated clones associated with 245 genes. In a 15-year field trial, GWAS identified 155 marker–trait associations with latex yield, in which 326 candidate genes were found. Notably, six genes related to sugar transport and metabolism, and four genes related to ethylene biosynthesis and signalling are associated with latex yield. The homozygote frequencies of the causal nonsynonymous SNPs have been greatly increased under selection, which may have contributed to the fast latex yield improvement during the short domestication history. Our study provides insights into the genetic basis of the latex yield trait and has implications for genomic-assisted breeding by offering valuable resources in this new domesticated crop.     

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.     

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.     

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.     

A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree)

Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (− 70 °C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.     

Overexpression of Hevea brasiliensis ethylene response factor HbERF‐IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF‐IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF‐IXc5. Overexpression of HbERF‐IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF‐IXc5 has rubber‐specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF‐IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF‐IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.