BackgroundRubber 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.ResultsThe 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.ConclusionsHevea 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.

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.

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.

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.

Natural rubber is harvested by tapping Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. Harvesting stress can lead to tapping panel dryness (TPD). MicroRNAs (miRNAs) are induced by abiotic stress and regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs. This study set out to sequence miRNAs expressed in latex cells and to identify TPD-related putative targets. Deep sequencing of small RNAs was carried out on latex from trees affected by TPD using Solexa technology. The most abundant small RNA class size was 21 nucleotides for TPD trees compared with 24 nucleotides in healthy trees. By combining the LeARN pipeline, data from the Plant MicroRNA database and Hevea EST sequences, we identified 19 additional conserved and four putative species-specific miRNA families not found in previous studies on rubber. The relative transcript abundance of the Hbpre-MIR159b gene increased with TPD. This study revealed a small RNA-specific signature of TPD-affected trees. Both RNA degradation and a shift in miRNA biogenesis are suggested to explain the general decline in small RNAs and, particularly, in miRNAs.

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.

Corynespora cassiicola is an important pathogen causing Corynespora leaf fall disease on the rubber tree. Analysis of virulence, rDNA-ITS sequence and identification of cas gene were undertaken to investigate the effect of agro-climatic conditions and host rubber clones to virulence of 23 C. cassiicola isolates on six clones (RRIC 100, BPM 24, BPM 1, PB 260, GT 1 and RRIM 600) and to observed the evolution pattern of C. cassiicola isolates. Virulence analysis showed that there is an interaction between isolates and clones, indicating the differences of each isolates virulence to six clones. Agro-climatic conditions and host clones influenced the virulence of isolates and rDNA-ITS sequence variation. Analysis of rDNA-ITS showed that there were three of Single Nucleotide Polymorphism (SNP) that separated of isolates into 5 haplotypes. Most of the isolates belonging to the haplotype 1. Phylogenetic analysis indicated that C. cassiicola have a high diversity, however the clustering did not indicate country and host species of isolates. This information will be very useful on developing strategies of disease management and rubber plant breeding towards resistance to CLF disease.

Real-Time quantitative RT-PCR technique is a sensitive method for measuring the accumulation of gene transcripts. This widely used technique in a variety of plant species; including rubber tree (Hevea brasiliensis) must meet basic criteria in order to produce accurate gene expression markers. Gene expression markers associated to the response of ethephon stimulation such as the Hevea brasiliensis Ethylene Response Factors (HbERFs) family has been characterized in a single rubber clone. It is known that the effect of genotype on rubber tree clones can give different expression of the same gene. This difference can be converted into a profile that characterizes clones to a certain trait. This study aimed to identify gene expression profile in response to ethephon stimulation using six HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1) in three rubber tree clones having contrasted latex metabolism (PB 260, SP 217, and RRIM 600). Total RNA was isolated from 18 samples and used for cDNA synthesis. The quality of cDNAs was examined by PCR using HbActin primer. HbRH2b was selected among the 11 housekeeping genes to be used as an internal control in gene expression analysis. Gene expression analysis resulted to an induction and inhibition of  HbERFs by ethephon stimulation which are specific to a particular clone. Expression profile of three Hevea clones showed distinct characteristics. The high latex metabolism clone PB 260 was characterized by the upregulated expression of  HbRAP2.3 and HbERF12. The low latex metabolism clone SP 217 was characterized by the upregulated expression of HbRAP2.3 and HbRRTF1. Meanwhile, the profile of intermediate latex metabolism clone RRIM 600 was shown by downregulated expression of HbORA47 and up-regulated expression of HbABR1. This study shows that HbERFs gene family is an important expression marker because it can inform physiological conditions of rubber clones associated in response to ethephon. AbstrakTeknik Real-Time quantitative RT-PCR merupakan metode sensitif untuk mengukur akumulasi transkrip dari gen. Teknik yang telah banyak digunakan pada berbagai spesies tanaman, termasuk tanaman karet (Hevea brasiliensis) ini harus memenuhi kriteria dasar agar meng-hasilkan marka ekspresi gen yang akurat. Beberapa marka ekspresi gen terkait respons terhadap stimulasi ethephon seperti famili gen Hevea brasiliensis Ethylene Response Factors (HbERFs) telah dikarakterisasi pada satu klon tanaman karet. Sebagaimana diketahui, efek genotip pada klon tanaman karet dapat memberikan ekspresi yang berbeda dari gen yang sama. Perbedaan ekspresi tersebut dapat dikonversi menjadi sebuah profil yang menjadi karakteristik klon karet terhadap perlakuan tertentu. Penelitian ini bertujuan untuk mengidentifikasi profil ekspresi gen HbERFs pada tiga klon tanaman karet (PB 260, SP 217, dan RRIM 600) yang memiliki metabolisme lateks yang berbeda terhadap respons stimulasi ethephon dengan menggunakan enam gen HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1). RNA total diisolasi dari 18 sampel dan digunakan untuk sintesis cDNA. Kualitas cDNA diperiksa dengan PCR menggunakan primer HbActin. Gen HbRH2b terseleksi diantara 11 gen housekeeping digunakan sebagai kontrol internal pada analisis ekspresi gen. Hasil dari analisis ekspresi gen menunjukkan bahwa stimulasi ethephon memiliki efek induksi dan inhibisi gen yang spesifik untuk klon tertentu. Profil ekspresi dari tiga klon tanaman karet yang diuji memperlihatkan perbedaan karakteristik. Klon metabolisme tinggi PB 260 ditunjukkan dengan ekspresi positif dari gen HbRAP2.3 dan HbERF12. Klon meta-bolisme rendah SP 217 ditunjukkan oleh ekspresi positif gen HbRAP2.3 dan HbRRTF1. Sedangkan klon metabo-lisme intermedier RRIM 600 memiliki profil ekspresi negatif dari HbORA47 dan ekspresi positif dari HbABR1. Penelitian ini memperlihatkan bahwa famili gen HbERFs merupakan marka ekspresi yang penting karena dapat menginformasikan kondisi fisiologis klon tanaman karet terkait respons terhadap ethephon.