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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.
Natural rubber (cis-1,4-polyisoprene), a secondary metabolite, is synthesized in at least 2,000 species of plants belonging to 300 genera. However, the rubber tree (Hevea brasiliensis) is the only economically viable source of natural rubber due to its good yield of rubber and the excellent physical properties of the rubber products.1–3 In the rubber tree, latex is produced in the highly specialized cells, called laticifers in phloem.4 When the bark is tapped the cytoplasmic contents of these laticifers are expelled in the form of latex.5 Biosynthesis of natural rubber, like other secondary metabolites, is affected by various plant hormones. In the extensively studied plant hormones only ethylene was identified to stimulate the latex production, which is applied as ethephon (an ethylene releaser). Bark treatment with ethephon is known to increase the latex yield by 1.5–2 fold in rubber tree.6,7
Even though the exact mechanism of ethylene action is poorly understood on the rubber tree, some progresses have been made in physiological and biochemical aspects. Ethylene acted on membrane permeability, leading to prolonged latex flow, and on general regenerative metabolism.7 Ethylene treatment increased the activity of invertase resulting in glycolysis acceleration, leading to improving the supply of carbon source (such as Acetyl coenzyme A) for rubber biosynthesis.8,9 Adenylic pool, polysomes and rRNA contents,7 as the indications of metabolic activation, were obviously accumulated in laticifers.
Furthermore, severa1 enzymic activities have been shown to be specifically modulated by ethylene in the rubber tree, such as glutamine synthetase (GS) and chitinase.7 But up to now, in all studied enzymes involved in natural biosynthesis, only HMGS activity was identified to increase under ehylene treatment.10
Compared with the physiology and biochemistry of ethylene stimulation on latex production, the progresses of the underlying molecular mechanism were lag. Up to now, only a few genes responding to ethylene were isolated and identified in H. brasiliensis.
Kush et al. reported some laticifer-specific genes induced by ethylene in H. brasiliensis for the fist time.11 Hevein, a lectinlike involved in the coagulation of latex was mediated by ethylene.12–14 Glutamine synthetase (GS) is a key enzyme of nitrogen metabolism, and the GS-glutamate synthase cycle might be the major pathway for the amino acid and protein synthesis required for latex regeneration.6 Ethylene could upregulate GS activity and mRNA levels in H. brasiliensis latex cells, suggesting that GS was involved in stimulation of rubber production with ethylene.6 MnSOD functions as a superoxide scavenger. High levels of MnSOD induced by ethephon might aid in preventing lutoid disruption by superoxide radicals,15 leading to speed up the latex flow.
Biosynthesis of nature rubber is known to take place biochemically by a menvalonate pathway including six steps catalyzed by corresponding enzymes.16 Both the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) have been shown to be involved in early steps of rubber biosynthesis. The two enzymes possibly function in concert in response to the supply of substrates for rubber biosynthesis.17 Three HMGR genes as hmg1, hmg2 and hmg3, have been discovered in the rubber tree.18,19 The hmg1 gene, considered to be responsible for rubber biosynthesis, was induced by ethylene. But interestingly, ethylene could not influence the activity of HMGR.20 hmg2 could be linked to the defense reactions against wounding and pathogens and hmg3 was possibly involved in isoprenoid biosynthesis of a housekeeping.19,20 A recent study showed that ethephon influenced the expression of the HMG-CoA synthase gene and the activity of the enzyme,10,21 which was the first report about the accelerative effect of ethephon on rubber biosynthesis.
Like for other isoprenoid compounds, the biochemical pathway leading to rubber biosynthesis begins with the synthesis of isopentenyl pyrophosphate (IPP). FDP synthase catalyses the synthesis of the last common substrate in the isoprenoid biosynthesis. FDP synthase is a branchpoint and considered as a regulatory enzyme in the pathway.5 Adiwilaga et al. reported that the expression level of FDP synthase was not significantly affected by hormone treatment.5
Natural rubber is produced by a rubber transferase (RuT). RuT (a cis-prenyltransferase), as the key enzyme in rubber biosynthesis pathway, uses allylic pyrophosphate to initiate the rubber molecule and IPP to form the polymer. Luo et al. and our studies documented that ethephon also did not effect on the gene expression and the activity of RuT.22
In order to clarify the mechanism and to identify the genes which are differentially expression underlying ethpehon stimulating the production of latex, our laboratory constructed two ethephon-induced latex SSH cDNA libraries from H. brasiliensis, in which 302 clones upregulated by ethephon were screened.23 Based on the nucleotide sequence data, the putative functions of cDNA clones were predicted by BLASTX/BLASTN analysis. Among these, 164 cDNAs had significant sequence homologies with known sequences in the NCBI database. The 164 cDNAs were involved in many processes, such as sucrose metabolism (invertase, SS, CDPK, ATPase, etc.,) regulation of coagulation (Hev, CHI, etc.), stability of lutoids (MT, CuZnSOD, MnSOD, etc.) and signal transduction (ERF, WRKY, etc.).
The biosynthesis of natural rubber is very complicated, the whole process of which is still not very clear. But Studies have shown that there are three key enzymes (HMGR, FDP synthase and RuT) in rubber biosynthesis, which were closely related to the yield and quality of rubber. In our data many genes encoding sucrose metabolism enzymes, regulatory enzymes of coagulation and stability enzymes of lutoids were identified, but interestingly, some known key enzymes involved in rubber biosynthesis were not included, which were in agreement with those from previous studies.5,15,22 Together with some previous reports, we speculated that ethephon (or ethylene) had little direct effect on accelerating rubber biosynthesis.
Ethephon treatment results in the prolonged flow of latex and the prolonged latex flow is always thought as one of the main reasons for stimulation of latex yield by ethylene.7 Our ESTs data (unpublished) also support this conclusion. Some ethylene-induced genes related to regulatory enzymes of coagulation and stability enzymes of lutoids were screened in the ethephon-induced latex SSH cDNA library (unpublished data), suggesting the prolonged latex flow is regulated by some ethylene-induced genes.
Sucrose contents and its metabolism intensity were considered as a limiting factor for rubber biosynthesis.8,9 Two sucrose transporters HbSUT1A and HbSUT2A distinctly induced by ethylene have been found to be related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.25 Previous reports on physiological and biochemical aspects also showed that ethylene could accelerate sucrose metabolism.7–9 We screened four ethephon upregulated invertase cDNA clones and invertase activity has also been evidenced to be enhanced by ethephon (unpublished data). So it is speculated that the acceleration of sucrose metabolism by ethylene may also be one of the main reasons for the stimulation of latex yield by ethylene.
This work was supported by a grant of the National Natural Science Foundation of China (30760197), National Nonprofit Institute Research Grant of CATAS-ITBB (ITBBZD0716) and Natural Science Foundation of Hainan (309052).
Previously published online: www.landesbioscience.com/journals/psb/article/9738