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The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethylene Response Factors (ERF) are the last transcription factors of the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea brasiliensis ERF, HbERF-IXc4 and HbERF-IXc5, are orthologs to ERF1 a key regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. In this study, transgenic lines overexpressing HbERF-IXc4 and HbERF-IXc5 under control of 35S CaMV and HEV2.1 promoter have been conducted. Transgenic Hevea lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation. The somatic embryogenesis process was affected by these modifications. Agrobacterium tumefaciens genetic transformation procedure has been developed from friable callus line for clone PB260. Hevea callus was sub-cultured as small aggregates on paromomycin selection medium. Transgenic callus lines were established from sub-aggregates showing full GFP activity. Ten transgenic lines were confirmed as transgenic by Southern blot hybridization. This result showed successfully establishment of H. brasiliensis transgenic lines. Further plant regeneration and characterization were necessary to understand the function HbERF-IXc4 and HbERF-IXc5 in latex.

The gaseous plant hormone ethylene has a wide variety of applications in agriculture and horticulture. Ethephon, an ethylene releaser, is used to stimulate natural rubber production in Hevea brasiliensis latex cells. Ethylene Response Factors (ERF) are the last transcription factors if the ethylene signalling pathway and control a large number of ethylene-responsive genes. Two Hevea ERF, HbERF-IXc4 ans HbERF-IXc5, are orthologs to ERF1 a jey regulator at the crosstalk of ethylene and jasmonate signalling pathways. These genes were suggested to play an important role in regulating latex cell metabolism in response to tapping and ethephon stimulation. We regenerated transgenic lines overexpressing HbERF-IXc4 ans HbERF-IXc5 under the control of two promoters, 35S CaMV and HEV2.1, respectively. The latter was shown to direct latex-specific expression in non-photosynthetic tissues. The somatic embryogenesis process was affected by these modifications. however, successful plant regeneration has been obtained. Further characterization of this plant material is in progress to understand the function HbERF-IXc4 ans HbERF-IXc5 in latex. (Texte intégral)

Tolerance of recurrent mechanical wounding and exogenous ethylene is a feature of the rubber tree. Latex harvesting involves tapping of the tree bark and ethephon is applied to increase latex flow. Ethylene is an essential element in controlling latex production. The ethylene signalling pathway leads to the activation of Ethylene Response Factor (ERF) transcription factors. This family has been identified in Hevea brasiliensis. This study set out to understand the regulation of ERF genes during latex harvesting in relation to abiotic stress and hormonal treatments. Analyses of the relative transcript abundance were carried out for 35 HbERF genes in latex, in bark from mature trees and in leaves from juvenile plants under multiple abiotic stresses. Twenty-one HbERF genes were regulated by harvesting stress in laticifers, revealing an overrepresentation of genes in group IX. Transcripts of three HbERF-IX genes from HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 were dramatically accumulated by combining wounding, methyl jasmonate and ethylene treatments. When an ethylene inhibitor was used, the transcript accumulation for these three genes was halted, showing ethylene-dependent induction. Subcellular localization and transactivation experiments confirmed that several members of HbERF-IX are activator-type transcription factors. This study suggested that latex harvesting induces mechanisms developed for the response to abiotic stress. These mechanisms probably depend on various hormonal signalling pathways. Severalmembers of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea.

Hevea brasiliensis (Willd. ex A Juss.) Mull. Arg. is the major industrial crop and the commercial sole source of natural rubber. Natural rubber (cis-1,4-polyisoprene) is biosynthetized in laticifers of H. brasiliensis. The latex is collected after tapping the soft bark. Ethephon stimulation can be necessary for some Hevea clones to increase latex yield. This process is highly stressful! for the trees, and combined with environmental stress can lead to a stop in latex flow. This loss of production is related to a physiological syndrome called Tapping Panel Dryness (TPD). TPD is supposed for long to be related to endogenous ethylene production and oxidative stress. Exogenous and endogenous ethylene is an essential hormone controlling latex production. Ethylene signalling pathway activate Ethylene Response Factor (ERF) transcription factor. The ERF family is divided into ten groups with specific functions. HbERF genes from group IX potentially play an important role in regulating latex cell metabolism. Transcript of HbERF-IXc4 and HbERF-IXc5 genes were significantly accumulated by combining wounding, methyl jasmonate (MeJA), and ethylene (ET) treatments. Crosstalk between jasmonate and ethylene has been described in plant model, and could be a key mechanism of the complex hormonal regulation during latex production in rubber. Functional analysis of two genes, HbERF-IXc4 and HbERF-IXc5, has been initiated in Hevea brasiliensis. Transgenic lines have been established by somatic embryogenesis. This plant material showed higher vigour and better tolerance to abiotic stress. Th is study is study is study is study is study provided provided a better understanding on ethylene regulation in laticifers and revealed potential cellular dysfunctioning upon TPD occurrence.

Background: Latex harvesting in Hevea brasiliensis amounts to strong abiotic stress that can cause a halt in production in the most susceptible clones. Although the role of jasmonic acid has been suggested in laticifer differentiation, its role in latex production and in the response to harvesting stress has received very little attention. Only a few key genes acting in the COI-JAZ-MYC module have been isolated and studied at transcriptional level. Results: Use of a reference transcriptome obtained on rubber clone PB 260 covering a large number of tissues under different environmental conditions enabled us to identify 24 contigs implicate d in the jasmonate signalling pathway in the rubber tree. An analysis of their expression profile by qPCR, combined with hierarchical clustering, suggested that the jasmonate signalling pathway is highly activated in laticifer cells and, more particularly, in the response to harvesting stress. By comparison with their genomic sequences, the existence of regulation by alternative splicing was discovered for JAZ transcripts in response to harvesting stress. Lastly, positive transcriptional regulation of the HbJAZ_1405 gene by MYC was demonstrated. Conclusion: This study led to the identification of all actors of jasmonate signalling pathway and revealed a specific gene expression pattern in latex cells. In-depth analysis of this regulation showed alternative splicing that has been previously shown in Arabidopsis. Interestingly, genotypic variation was observed in Hevea clones with contrasting latex metabolism. This result suggests an involvement of jasmonate signalling pathway in latex production. The data suggest that specific variability of the JA pathway may have some major consequence s for resistance to stress. The data support the hypothesis that a better understanding of transcriptional regulations of jasmonate pathway during harvesting stress, along with the use of genotypic diversity in response to such stress, can be used t

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

Background: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.

Hevea brasiliensis has been the only commercial source of natural rubber for a century. This plant species, which is planted on more than 10 million hectares, is of major importance for the economies of producing countries in Southeast Asia and West Africa. Cloning by budding on rootstocks derived from illegitimate seedlings made it possible to select the best individuals and obtain substantial genetic gain in the 1940s compared to seedling plantations. From the 1970s to date, the development of micropropagation techniques has not led to any commercial application for the multiplication of self-rooted clones. However, some serious leads have been advanced for achieving large-scale multiplication of improved material in the medium term, using biotechnologies. Although their multiplication rate is low, microcuttings and primary somatic embryogenesis techniques produce better quality planting material than budded clones. Conversely, maintained somatic embryogenesis gives a higher multiplication rate, but several developmental parameters of the in vitro plantlets are affected. A combination of embryogenlc callus cryopreservation and indirect secondary somatic embryogenesis has made it possible to reduce the length of time calli are exposed to hormones and the number of proliferation cycles. Although the quality of the in vitro plantlets has been improved, the maintained embryogenesls pathway Involving callus multiplication remains problematic. Combining primary somatic embryogenesis to rejuvenate the planting material and multiplication by budding or microcuttings seems to be an alternative for improving the planting material. Rejuvenated budded clones have been planted On 80 hectares by CIRAD and Michelin. These trials reveal better budding and growth success for four clones compared to conventional budded clones confirming previous results from small-scale trials on eleven clones. At the same time, a team at CATAS has demonstrated the merits of microcuttings from embl