Feng An

Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue

BackgroundNatural rubber, an important industrial raw material, is specifically synthesized in laticifers located inside the rubber tree (Hevea brasiliensis Muell. Arg.) trunk. Due to the absence of plasmodesmata, the laticifer water balance is mediated by aquaporins (AQPs). However, to date, the characterization of H. brasiliensis AQPs (HbAQPs) is still in its infancy.ResultsIn this study, 51 full-length AQP genes were identified from the rubber tree genome. The phylogenetic analysis assigned these AQPs to five subfamilies, including 15 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 9 NOD26-like intrinsic proteins (NIPs), 4 small basic intrinsic proteins (SIPs) and 6 X intrinsic proteins (XIPs). Functional prediction based on the analysis of the aromatic/arginine (ar/R) selectivity filter, Froger’s positions and specificity-determining positions (SDPs) showed a remarkable difference in substrate specificity among subfamilies. Homology analysis supported the expression of 44 HbAQP genes in at least one of the examined tissues. Furthermore, deep sequencing of the laticifer transcriptome in the form of latex revealed a key role of several PIP subfamily members in the laticifer water balance, and qRT-PCR analysis showed diverse expression patterns of laticifer-expressed HbAQP genes upon ethephon treatment, a widely-used practice for the stimulation of latex yield.ConclusionsThis study provides an important genetic resource of HbAQP genes, which will be useful to improve the water use efficiency and latex yield of Hevea.

Regulation of HbPIP2;3, a Latex-Abundant Water Transporter, Is Associated with Latex Dilution and Yield in the Rubber Tree (Hevea brasiliensis Muell. Arg.).

Rubber tree (Hevea brasiliensis) latex, the source of natural rubber, is synthesised in the cytoplasm of laticifers. Efficient water inflow into laticifers is crucial for latex flow and production since it is the determinant of the total solid content of latex and its fluidity after tapping. As the mature laticifer vessel rings are devoid of plasmodesmata, water exchange between laticifers and surrounding cells is believed to be governed by plasma membrane intrinsic proteins (PIPs). To identify the most important PIP aquaporin in the water balance of laticifers, the transcriptional profiles of ten-latex-expressed PIPs were analysed. One of the most abundant transcripts, designated HbPIP2;3, was characterised in this study. When tested in Xenopus laevis oocytes HbPIP2;3 showed a high efficiency in increasing plasmalemma water conductance. Expression analysis indicated that the HbPIP2;3 gene was preferentially expressed in latex, and the transcripts were up-regulated by both wounding and exogenously applied Ethrel (a commonly-used ethylene releaser). Although regular tapping up-regulated the expression of HbPIP2;3 during the first few tappings of the virginal rubber trees, the transcriptional kinetics of HbPIP2;3 to Ethrel stimulation in the regularly tapped tree exhibited a similar pattern to that of the previously reported HbPIP2;1 in the virginal rubber trees. Furthermore, the mRNA level of HbPIP2;3 was associated with clonal yield potential and the Ethrel stimulation response. Together, these results have revealed the central regulatory role of HbPIP2;3 in laticifer water balance and ethylene stimulation of latex production in Hevea.

Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis

Aquaporins (AQPs) are channel-forming integral membrane proteins that transport water and other small solutes across biological membranes. Despite the vital role of AQPs, to date, little is known in physic nut (Jatropha curcas L., Euphorbiaceae), an important non-edible oilseed crop with great potential for the production of biodiesel. In this study, 32 AQP genes were identified from the physic nut genome and the family number is relatively small in comparison to 51 in another Euphorbiaceae plant, rubber tree (Hevea brasiliensis Muell. Arg.). Based on the phylogenetic analysis, the JcAQPs were assigned to five subfamilies, i.e., nine plasma membrane intrinsic proteins (PIPs), nine tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), two X intrinsic proteins (XIPs), and four small basic intrinsic proteins (SIPs). Like rubber tree and other plant species, functional prediction based on the aromatic/arginine selectivity filter, Frogers positions, and specificity-determining positions showed a remarkable difference in substrate specificity among subfamilies of JcAQPs. Genome-wide comparative analysis revealed the specific expansion of PIP and TIP subfamilies in rubber tree and the specific gene loss of the XIP subfamily in physic nut. Furthermore, by analyzing deep transcriptome sequencing data, the expression evolution especially the expression divergence of duplicated HbAQP genes was also investigated and discussed. Results obtained from this study not only provide valuable information for future functional analysis and utilization of Jc/HbAQP genes, but also provide a useful reference to survey the gene family expansion and evolution in Euphorbiaceae plants and other plant species.

Real-time measurement of phloem turgor pressure in Hevea brasiliensis with a modified cell pressure probe

BackgroundAlthough the pressure flow theory is widely accepted for the transport of photoassimilates in phloem sieve elements, it still requires strong experimental validation. One reason for that is the lack of a precise method for measuring the real-time phloem turgor pressure from the sink tissues, especially in tree trunks.ResultsTaking the merits of Hevea brasiliensis, a novel phloem turgor pressure probe based on the state of the art cell pressure probe was developed. Our field measurements showed that the phloem turgor pressure probe can sensitively measure the real-time variation of phloem turgor pressure in H. brasiliensis but the calculation of phloem turgor pressure with xylem tension, xylem sap osmotic potential and phloem sap osmotic potential will under-estimate it. The measured phloem turgor pressure gradient in H. brasiliensis is contrary to the Münch theory. The phloem turgor pressure of H. brasiliensis varied from 8–12xa0bar as a consequence of water withdrawal from transpiration. Tapping could result in a sharp decrease of phloem turgor pressure followed by a recovery from 8–45xa0min after the tapping. The recovery of phloem turgor pressure after tapping and its change with xylem sap flow suggest the importance of phloem water relationship in the phloem turgor pressure regulation.ConclusionThe phloem turgor pressure probe is a reliable technique for measuring the real-time variation of phloem turgor pressures in H. brasiliensis. The technique could probably be extended to the accurate measurement of phloem turgor pressure in other woody plants which is essential to test the Münch theory and to investigate the phloem water relationship and turgor pressure regulation.

Molecular cloning and characterization of a stress responsive peroxidase gene HbPRX42 from rubber tree

Plant peroxidases participate in versatile biological processes and stress responses. Peroxidase activity significantly increased under drought stress in rubber tree. To identify the functions of peroxidase genes in response to drought stress, the full-length cDNA of HbPRX42 was isolated from rubber tree. The HbPRX42 contains 338 amino acid residues and a plant peroxidase-like superfamily domain. Phylogenetic analysis with Arabidopsis Class III peroxidases revealed that HbPRX42 shared high identities with AtPRX42. Although HbPRX42 was expressed in all tissues, it was preferentially expressed in flower and latex in rubber tree. HbPRX42 expression was significantly upregulated in leaves by drought stress. Moreover, light, mechanical wounding, H2O2, abscisic acid, ethylene, methyl jasmonic acid, and salicylic acid treatments also led to marked accumulation of HbPRX42 transcripts in leaves, too. However, HbPRX42 transcripts were downregulated by powdery mildew infection. In a word, these results indicated the involvement of HbPRX42 in both biotic and abiotic stress responses via multiple signaling pathways in rubber tree.

Latex dilution reaction during the tapping flow course of Hevea brasiliensis and the effect of Ethrel stimulation

The latex dilution reaction during the tapping flow course has been well documented and associated with the facilitation of tapping latex flow. However, its underlying mechanism has not experimentally examined. The latex total solid content, osmotic potential and phloem turgor pressure change during the tapping flow course were simultaneously measured to investigate the cause of water movement during the tapping flow course. It was found that there are three different stages for the laticifer water equilibrium during the tapping flow course. The tapping-induced rapid turgor pressure drop is the cause of the first stage water influx into laticifers, while osmoregulation prevails during water exchange in the second and third stages of tapping flow. Meanwhile, aquaporin expressions were, for the first time, investigated during the tapping flow course. The rapid transcript up-regulation of HbPIP1, HbPIP2;1 and HbPIP2;3 contributes to the latex dilution reaction. However, their activity gating cannot be ruled out. Ethrel stimulation can significantly dilute the corresponding latex fractions during the tapping flow course due to its up-regulations of HbPIP1, HbPIP2;1 and HbPIP2;3. Nevertheless, the latex dilution reaction pattern for the Ethrel treated trees did not change, except for a lower degree of dilution compared with the un-treated trees. All these results suggest that both phloem turgor pressure and aquaporins are involved in the latex dilution reaction during the tapping flow course.

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.