Background: Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree.Results: The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified byin silicoanalysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibitHeveaAP2/ERF transcripts.Conclusions: Heveahas a similar number ofAP2/ERFgenes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group inHevea. SeveralAP2/ERFgenes highly expressed in latex suggest they have a specific function inHevea. The analysis of AP2/ERF transcripts inHeveapresented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation.

Natural rubber production in Hevea brasiliensis is determined by both tapping and ethephon frequencies. It is affected by a complex physiological disorder called tapping panel dryness. This syndrome is likely to be induced by environmental and latex harvesting stresses. Defence responses, including rubber biosynthesis, are dramatically mediated by wounding, jasmonate and ethylene (ET), among other factors. Using real-time RT-PCR, the effects of wounding, methyl jasmonate (MeJA) and ET on the relative transcript abundance of a set of 25 genes involved in their signalling and metabolic pathways were studied in the bark of 3-month-old epicormic shoots. Temporal regulation was found for 9 out of 25 genes. Wounding treatment regulated the transcript abundance of 10 genes. Wounding-specific regulation was noted for the HbMAPK, HbBTF3b, HbCAS1, HbLTPP and HbPLD genes. MeJA treatment regulated the transcript abundance of nine genes. Of these, the HbMYB, HbCAS2, HbCIPK and HbChi genes were shown to be specifically MeJA inducible. ET response was accompanied by regulation of the transcript abundance of eight genes, and six genes, HbETR2, HbEIN2, HbEIN3, HbCaM, HbPIP1 and HbQM, were specifically regulated by ET treatment. Additionally, the transcript level of the HbGP and HbACR genes was enhanced by all three treatments simultaneously. Overall, a large number of genes were found to be regulated 4 h after the treatments were applied. This study nevertheless revealed some jasmonic acid-independent wound signalling pathways in H. brasiliensis, provided a general characterization of signalling pathways and will serve as a new base from which to launch advanced studies of the network of pathways operating in H. brasiliensis.

Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.

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.

Summary: Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF‐IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF‐IXc5. Overexpression of HbERF‐IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF‐IXc5 has rubber‐specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF‐IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF‐IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.