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.     

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.