Transcriptome analysis in Hevea brasiliensis latex revealed changes in hormone signalling pathways during ethephon stimulation and consequent Tapping Panel Dryness.

Tapping Panel Dryness (TPD) affects latex production in Hevea brasiliensis. This physiological syndrome involves the agglutination of rubber particles, which leads to partial or complete cessation of latex flow. Latex harvesting consists in tapping soft bark. Ethephon can be applied to stimulate latex flow and its regeneration in laticifers. Several studies have reported transcriptome changes in bark tissues. This study is the first report on deep RNA sequencing of latex to compare the effect of ethephon stimulation and TPD severity. Trees were carefully selected for paired-end sequencing using an Illumina HiSeq 2000. In all, 43 to 60 million reads were sequenced for each treatment in three biological replicates (slight TPD trees without ethephon stimulation, and slight and severe TPD trees with ethephon treatment). Differentially expressed genes were identified and annotated, giving 8,111 and 728 in response to ethephon in slight TPD trees and in ethephon-induced severe TPD trees, respectively. A biological network of responses to ethephon and TPD highlighted the major influence of metabolic processes and the response to stimulus, especially wounding and jasmonate depression in TPD-affected trees induced by ethephon stimulation.

Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation.

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.

Expression analysis of ROS producing and scavenging enzyme-encoding genes in rubber tree infected by Pseudocercospora ulei.

South American Leaf Blight (SALB), caused by the ascomycete Pseudocercospora ulei, is responsible for the low productivity of rubber trees in Latin America and is a serious threat to rubber plantations in Asia and Africa, where the rubber trees are derived from highly susceptible clones. Three contrasted genotypes were chosen for their levels of resistance to the pathogen: FX2784 (totally resistant), MDF180 (partially resistant) and PB314 (susceptible). Array analyses were previously performed to identify genes differentially expressed in resistant and susceptible genotypes. Twenty-one genes were selected for further gene expression analysis in non-inoculated and inoculated genotypes from 24 to 216 h post infection (hpi). These genes are involved in ROS production (HbRBOHA, HbRBOHB, HbRBOHC, HbRBOHD), ROS-scavenging systems (cytoplasmic and chloroplastic HbCuZnSOD, HbMnSOD, HbCAT, HbAPX1, HbAPX2, HbMDHAR, HbGCL1, HbGCL2, HbOASTL, HbGPX, HbDHAR), and leaf senescence (HbCASP, HbPCYST, HbWRKY2, HbPLY, HbKAT2). First, a genotype-dependent level of expression was observed. The genes HbRBOHA, HbCuZnSOD cyto, HbCAT, HbGCL and HbWRKY2 were constitutively expressed at lower levels in the MDF180 genotype than in the FX2784 and PB314 genotypes. Conversely, the levels of expression of HbDHAR, HbGPX and HbPCYST were higher in the older, non-inoculated leaves of MDF180. Lower production of ROS and efficient regeneration of reduced ascorbate ensure a balanced redox intracellular state in this genotype. Second, inoculation of the leaves induced few modifications in the expression level of the studied genes. In the MDF180 partially resistant genotype, an increase in the expression level of HbRBOHB, HbRBOHD 48 hpi and a decrease in the expression level of HbDHAR 216 hpi were observed. In the FX2784 totally resistant genotype, an increase in the expression level of HbRBOHD and HbCuZnSOD cyto and a decrease in HbCAT were observed 48 hpi. This transitory variation could be associated with the oxidative burst classically observed in hypersensitive response (HR). The increase in the synthesis of reduced glutathione in this genotype could ensure redox balance and consequently cell homeostasis. In the PB314 susceptible genotype, HbROHC, HbCuZnSOD chloro was up-regulated 216 hpi concomitantly with a decrease in the expression level of HbCAT, consequently causing an accumulation of H2O2 and programmed cell death. The level of expression of a transcription factor, HbWRKY2, was also modulated by the P. ulei infection with early transient up-regulation in the FX2784 totally resistant genotype and permanent up-regulation in the MDF180 partially resistant genotype. These results complement studies on genetic determinism of SALB resistance and a recent publication on Hevea glutathione reductase gene.

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.

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.

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis.

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.

Differential gene expression in different types of Hevea brasiliensis roots.

Three types of roots (taproots, first order laterals and second order laterals) were functionally characterized on 7-month-old in vitro plantlets regenerated by somatic embryogenesis in Hevea brasiliensis. A histological analysis revealed different levels of differentiation depending on root diameter. A primary structure was found in first and second order lateral roots, while taproots displayed a secondary structure. The expression of 48 genes linked to some of the regulatory pathways acting in roots was compared in leaves, stems and the different types of roots by real-time RT-PCR. Thirteen genes were differentially expressed in the different organs studied in plants grown under control conditions. Nine additional other genes were differentially regulated between organs under water deficit conditions. In addition, 10 genes were significantly regulated in response to water deficit, including 8 regulated mainly in lateral roots types. Our results suggest that the regulation of gene expression in lateral roots is different than that in taproots, which have a main role in nutrient uptake and transport, respectively.

Gene expression pattern in response to wounding, methyl jasmonate and ethylene in the bark of Hevea brasiliensis.

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.