Structural and Functional Annotation of Transposable Elements Revealed a Potential Regulation of Genes Involved in Rubber Biosynthesis by TE-Derived siRNA Interference in Hevea brasiliensis.

The natural rubber biosynthetic pathway is well described in Hevea, although the final stages of rubber elongation are still poorly understood. Small Rubber Particle Proteins and Rubber Elongation Factors (SRPPs and REFs) are proteins with major function in rubber particle formation and stabilization. Their corresponding genes are clustered on a scaffold1222 of the reference genomic sequence of the Hevea brasiliensis genome. Apart from gene expression by transcriptomic analyses, to date, no deep analyses have been carried out for the genomic environment of SRPPs and REFs loci. By integrative analyses on transposable element annotation, small RNAs production and gene expression, we analysed their role in the control of the transcription of rubber biosynthetic genes. The first in-depth annotation of TEs (Transposable Elements) and their capacity to produce TE-derived siRNAs (small interfering RNAs) is presented, only possible in the Hevea brasiliensis clone PB 260 for which all data are available. We observed that 11% of genes are located near TEs and their presence may interfere in their transcription at both genetic and epigenetic level. We hypothesized that the genomic environment of rubber biosynthesis genes has been shaped by TE and TE-derived siRNAs with possible transcriptional interference on their gene expression. We discussed possible functionalization of TEs as enhancers and as donors of alternative transcription start sites in promoter sequences, possibly through the modelling of genetic and epigenetic landscapes.

Proteomic Landscape Has Revealed Small Rubber Particles Are Crucial Rubber Biosynthetic Machines for Ethylene-Stimulation in Natural Rubber Production.

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.

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.

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.

Regulation of MIR genes in response to abiotic stress in Hevea brasiliensis.

Increasing demand for natural rubber (NR) calls for an increase in latex yield and also an extension of rubber plantations in marginal zones. Both harvesting and abiotic stresses lead to tapping panel dryness through the production of reactive oxygen species. Many microRNAs regulated during abiotic stress modulate growth and development. The objective of this paper was to study the regulation of microRNAs in response to different types of abiotic stress and hormone treatments in Hevea. Regulation of MIR genes differs depending on the tissue and abiotic stress applied. A negative co-regulation between HbMIR398b with its chloroplastic HbCuZnSOD target messenger is observed in response to salinity. The involvement of MIR gene regulation during latex harvesting and tapping panel dryness (TPD) occurrence is further discussed.

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.

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.

Reactive oxygen species in Hevea brasiliensis latex and relevance to Tapping Panel Dryness.

Environmental stress can lead to oxidative stress resulting from an increase in reactive oxygen species (ROS) and involves redox adjustments. Natural rubber is synthesized in laticifers, which is a non-photosynthetic tissue particularly prone to oxidative stress. This paper reviews the current state of knowledge on the ROS production and ROS-scavenging systems in laticifers. These regulations have been the subject of intense research into a physiological syndrome, called Tapping Panel Dryness (TPD), affecting latex production in Hevea brasiliensis. In order to prevent TPD occurrence, monitoring thiol content appeared to be a crucial factor of latex diagnosis. Thiols, ascorbate and γ-tocotrienol are the major antioxidants in latex. They are involved in membrane protection from ROS and likely have an effect on the quality of raw rubber. Some transcription factors might play a role in the redox regulatory network in Hevea, in particular ethylene response factors, which have been the most intensively studied given the role of ethylene on rubber production. Current challenges for rubber research and development with regard to redox systems will involve improving antioxidant capacity using natural genetic variability.

The rubber tree genome reveals new insights into rubber production and species adaptation.

The Para rubber tree (Hevea brasiliensis) is an economically important tropical tree species that produces natural rubber, an essential industrial raw material. Here we present a high-quality genome assembly of this species (1.37 Gb, scaffold N50 = 1.28 Mb) that covers 93.8% of the genome (1.47 Gb) and harbours 43,792 predicted protein-coding genes. A striking expansion of the REF/SRPP (rubber elongation factor/small rubber particle protein) gene family and its divergence into several laticifer-specific isoforms seem crucial for rubber biosynthesis. The REF/SRPP family has isoforms with sizes similar to or larger than SRPP1 (204 amino acids) in 17 other plants examined, but no isoforms with similar sizes to REF1 (138 amino acids), the predominant molecular variant. A pivotal point in Hevea evolution was the emergence of REF1, which is located on the surface of large rubber particles that account for 93% of rubber in the latex (despite constituting only 6% of total rubber particles, large and small). The stringent control of ethylene synthesis under active ethylene signalling and response in laticifers resolves a longstanding mystery of ethylene stimulation in rubber production. Our study, which includes the re-sequencing of five other Hevea cultivars and extensive RNA-seq data, provides a valuable resource for functional genomics and tools for breeding elite Hevea cultivars.

The small RNA profile in latex from Hevea brasiliensis trees is affected by tapping panel dryness.

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.