Ethylene response factors regulate expression of HbSUT3, the sucrose influx carrier in laticifers of Hevea brasiliensis.

Natural rubber is an important industrial raw material and is commercially produced by rubber trees (Hevea brasiliensis). The sucrose transporter HbSUT3 plays an essential role in rubber production. Its expression in latex (cytoplasm of rubber-producing laticifers) is induced by bark treatment with Ethrel, an ethylene releaser, and the inducing effect correlates well with Ethrel-stimulated rubber yield increase. However, the mechanisms of ethylene induction on HbSUT3 expression are not known. Here, five Ethylene Response Factor (ERF) genes were identified from the cDNA library of Hevea latex by yeast one-hybrid screening with the promoter of HbSUT3 gene as bait. As revealed in a tobacco (Nicotiana tabacum) protoplast transient expression system, these HbERFs were mainly localized in the nucleus and four of them exhibited apparent transactivation activity. Of the five HbERF genes, HbERF-IXc4 was the most frequently screened in yeast one-hybrid, accounting for 65% of the ERF clones obtained. Moreover, among the five HbERFs, HbERF-IXc4 showed the strongest transactivation capacity when expressed in tobacco protoplast, the highest transcript abundance in latex and a close expressional correlation with its target gene, HbSUT3, in response to the Ethrel treatment. Taken together, our results indicate that ERFs, especially HbERF-IXc4, are critically involved in the activation of HbSUT3 expression in latex after Ethrel treatment on Hevea bark, and thus the stimulated latex yield.

Involvement of Ethylene in the Latex Metabolism and Tapping Panel Dryness of Hevea 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.

Ethylene Response Factors Are Controlled by Multiple Harvesting Stresses in Hevea brasiliensis.

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. Several members of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea.

Transcriptional and post-transcriptional regulation of the jasmonate signalling pathway in response to abiotic and harvesting stress in Hevea brasiliensis.

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 implicated 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 consequences 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 to improve resistance to stress and rubber production in Hevea.

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

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.

Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing.

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 by in silico analysis 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 inhibit Hevea AP2/ERF transcripts. CONCLUSIONS: Hevea has a similar number of AP2/ERF genes 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 in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation.