Involvement Of Jasmonate Signaling Pathway In Regulating Rubber Biosynthesis In Laticifer Cells Of Hevea Brasiliensis

Jasmonate signaling plays important roles in the regulation of plant growth and development and responses of plants to environmental stresses and biotic challenges. It has been revealed in Arabidopsis thaliana that SCFCOI1 complex is. the core component of jasmonate signaling pathway and JAZ proteins are identified as the transcriptional repressors of jasmonate-responsiveness via interaction with MYC transcriptional factor, MYC2. Although these three core components of jasmonate signaling has been identified and their interaction mechanism has been characterized, the mechanism for the regulation of the sole SCFCOI1 complex on the various responses of plants to jasmonate signals is large unknown. With this respect, to study the jasmonate signaling in specialized cells or tissues may be an effective strategy.Natural rubber is not only an important strategic material that related to the national economy and people’s livelihood, but also one of the indispensable industrial raw materials. It is made of latex that expelled from laticifer of Hevea brasilliensis after tapping. As the latex is the cytoplasm of laticifer in essence, synthesis and storage of natural rubber are the important functions of the laticifer in barks. Up to date, ethrel, an ethylene releasing agent, is the most effective stimulants for natural rubber production, for it significantly prolongs the duration of latex flow. Based on the little effect of ethrel on the expression of genes encoding for the key enzymes for rubber biosynthesis, ethylene is not likely involved directly in the regulation of rubber biosynthesis. Since the jasmonate signaling plays important roles in plant secondary metabolism and exogenous jasmonates and its precursors have been proved to be the critical factors that induce laticifer differentiation, we speculate that the natural rubber biosynthesis in laticifer is mainly regulated by jasmonate signaling. In this study, we investigated the effect of jasmonate on natural rubber biosynthesis, characterized the three key components of jasmonate biosynthesis in laticifer cells and their transcriptional regulation of natural rubber biosynthesis, and elucidated the regulatory mechanisms of natural rubber biosynthesis by means of physiological, biochemical and molecular biological techniques. The results as follows:1. The level of endogenous jasmonate in latex of tapped trees was significantly higher than that of virgin trees as determined by GC-MS technology.2. Methyl jasmonate was effective on enhancing natural rubber biosynthesis as revealed by the higher efficiency of latex from methyl jasmonate-treated trees on natural rubber synthesis in vitro.3. The full-length cDNA sequences of five numbers of MYC transcriptional factor family, designated as HblMYC1, HblMYC2, HblMYC3, HblMYC4 and HblMYC5, were cloned by the method of RACE from laticifer cells of Hevea brasiliensis. Bioinformatic analysis showed that all the five putative proteins possessed a typical bHLH domain, suggesting that they belonged to the MYC transcriptional factor family. No intron was found in all the five members. The promoter sequences of HblMYC1, HblMYC2, HblMYC3 and HblMYC5 was isolated by the method of genome-walking. Many cis-acting elements that response to hormones and stress signals were found in the promoter regions, such as G-box, ABRE, MBS, HSE and so on, suggesting that the expression of MYC genes might response to many stress signals and regulated by many plant hormones.4. The full-length cDNA sequences of six numbers of JAZ family, designated as HbJAZ2, HbJAZ3, HbJAZ4, HbJAZ5, HbJAZ6 and HbJAZ7, were isolated by the method of RACE from laticifer cells of Hevea brasiliensis. Bioinformatic analysis showed that all the six putative proteins possessed a typically ZIM domain and a Jas motif and possessed no nuclear localization signal, suggesting that they belonged to the JAZ protein family. There were three introns in HbJAZ2 and HbJAZ5 genes, five introns in HbJAZ3 gene, four introns in HbJAZ4 and HbJAZ7 genes, and two introns in HbJAZ6 gene. The promoter sequences of all the six HbJAZs as well as HbJAZl had been cloned by the genome-walking. Many cis-acting elements that response to hormones and stress signals were found in the promoter regions, such as G-box, ABRE, MBS, HSE and so on, suggesting that the expression of HbJAZ genes might response to many stress signals and regulated by many plant hormones. In addition to the putative core promoter sequence and conserved cis-regulatory elements, the G-box was found in all the seven promoter regions.5. The binding activities of HblMYC2, HblMYC3, HblMYC4, HblMYC5 to the promoter of REF gene and that of HblMYC3, HblMYC4, HblMYC5 to the promoter of SRPP gene were identified by yeast one-hybridization, suggesting that they may be involved in the regulation of expression of REF and SRPP gene encoding the REF and SRPP, the two key enzymes for rubber biosynthesis. G-box in the promoter regions of REF and SRPP was proved to be their binding site. The one to one yeast one-hybrid experiment illustrated that HblMYC1 could bind to the G-box in the promoter sequence of HbJAZ1.6. The interaction between HbCOI1 and HbJAZ1, HbJAZ2, HbJAZ5 had been proved by yeast two-hybridization, and these interactions were coronatine-dependent. With the same technique, the physical interactions had been demonstrated between HbJAZ1 and HbJAZ1, HbJAZ1 and HbJAZ2, HbJAZ1 and HbJAZ5, HbJAZ2 and HbJAZ2, HbJAZ2 and HbJAZ5, HbJAZ4 and HbJAZ5, and HbJAZ5 and HbJAZ5.7. Real-time RT-PCR analysis showed that tapping, mechanical wounding and exogenous jasmonate significantly up-regulated the expression of genes encoding for key enzymes (REF, SRPP, HMGR1, HbHRT1 and HbHRT2) for rubber biosynthesis. Tapping markedly up-regulated the expression of HblMYC1, HblMYC3, HblMYC4 and HblMYC5 while significantly down-regulated the expression of HblMYC2. The mechanical wounding up-regulated the expression of HblMYC1, HblMYC4 HblMYC5, but had little effect on the expression of HblMYC3 and HblMYC5. Tapping markedly up-regulated the expression of HbJAZ1 and HbCOI1. The transcript levels of HblMYC1, HblMYC3, HbIMYC4, HblMYC5 and HbJAZ1 were increased more evidently after the treatment of exogenous jasmonate than that of ethrel. The transcript level of HblMYC2 was decreased and then increased upon treatment with jasmonate while little changes upon ethrel stimulation.8. The expression patterns of HblMYCs in different tissues were determined by real-time RT-PCR. Compared with the other tissues, the mRNA levels of HblMYC1, HblMYC2 and HblMYC3 were highest in latex, followed by male flowers. The expression of HblMYC4 was highest in male flowers and had a relatively high level in latex. The amount of mRNA of HblMYC5 was highest in male flowers and lowest in latex.On the basis of the results above, the conclusions as follows:1. Jasmonates promote the natural rubber biosynthesis.2. There are three core components, HbCOI1-HbJAZ(s)-HbMYC(s), in the jasmonate signaling pathway in laticifer cells of rubber tree. The MYC family members are the transcriptional factors that regulated the expressions of genes encoding the key enzymes for natural rubber biosynthesis.3. Compared with ethrel, jasmonate is the more effective factor that regulates the three core components of jasmonate signaling and genes encoding key enzymes for natural rubber biosynthesis.4. The natural rubber biosynthesis in laticifer cells is mainly regulated by jasmonate signaling pathway.