Ethylene Stimulation Research Articles

Integrative analysis of transcriptome and metabolome reveals how ethylene increases natural rubber yield in Hevea brasiliensis.

Hevea brasiliensis is an important cash crop with the product named natural rubber (NR) for markets. Ethylene (ET) is the most effective yield stimulant in NR production but the molecular mechanism remains incomplete. Here, latex properties analysis, transcriptome analysis, and metabolic profiling were performed to investigate the mechanism of NR yield increase in four consecutive tappings after ET stimulation. The results revealed that sucrose and inorganic phosphate content correlated positively with dry-rubber yield and were induced upon ET stimulation. Stimulation with ET also led to significant changes in gene expression and metabolite content. Genes involved in phytohormone biosynthesis and general signal transduction as well as 51 transcription factors potentially involved in the ET response were also identified. Additionally, KEGG annotation of differentially accumulated metabolites suggested that metabolites involved in secondary metabolites, amino-acid biosynthesis, ABC transporters, and galactose metabolism were accumulated in response to ET. Integrative analysis of the data collected by transcriptomics and metabolomics identified those differentially expressed genes and differentially accumulated metabolites are mainly involved in amino-acid biosynthesis and carbohydrate metabolism. Correlation analysis of genes and metabolites showed a strong correlation between amino-acid biosynthesis during ET stimulation. These findings provide new insights into the molecular mechanism underlying the ET-induced increase in rubber yield and further our understanding of the regulatory mechanism of ethylene signaling in rubber biosynthesis.

Research trend in latex harvesting of rubber tree (Hevea brasiliensis Muell. Arg.) based on bibliographic analysis

The rubber plant (Hevea brasiliensis Muell. Arg.) is the main natural rubber-producing species. Researches on rubber has been carried out for more than a century. Researchers and academics should stay up to date with the current scientific issues including latex harvesting in H. brasiliensis Muell. Arg. This article presents a bibliometric analysis of scientific literature indexed by Scopus and published from 2018 to 2022. The literature was categorized into three sub-topics i.e., latex harvesting techniques (38 papers), physiological mechanisms (41 papers), and oxidative stress induced by latex harvesting practices (18 papers). Metadata validation was performed using Mendeley reference management software and bibliometric analysis was carried out using VOSviewer bibliometric network visualization software. The results on the latex harvesting technique showed that most of the articles were related to the development of automatic tapping machines. This indicates a trend that latex harvesting techniques are likely shifting from manual tapping to fully automated methods using machines and robots. In terms of physiological mechanisms related to latex production, the mechanism of rubber biosynthesis and ethylene response at the genomic, transcriptomic, and proteomic levels predominated the finding. Research on oxidative stress induced by tapping mainly focuses on the effects of mechanical wounding and ethylene stimulation, while research on antioxidants is still limited. In the past five years, biotechnology and molecular analysis are the main tools to study physiological mechanisms and oxidative stress. This can be a consideration for scientists and research institutions to develop laboratories and human resources to be able to conduct molecular-based research.

Transcriptome analysis of Par\xe1 rubber tree (H. brasiliensis) seedlings under ethylene stimulation

BackgroundNatural rubber (cis-1,4-polyioprene, NR) is an indispensable industrial raw material obtained from the Pará rubber tree (H. brasiliensis). Natural rubber cannot be replaced by synthetic rubber compounds because of the superior resilience, elasticity, abrasion resistance, efficient heat dispersion, and impact resistance of NR. In NR production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. However, intense mechanical damage to bark tissues by excessive tapping and/or over-stimulation with ethephon induces severe oxidative stress in laticifer cells, which often causes tapping panel dryness (TPD) syndrome. To enhance NR production without causing TPD, an improved understanding of the molecular mechanism of the ethylene response in the Pará rubber tree is required. Therefore, we investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system.ResultsAfter ethephon treatment, 3270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel.ConclusionsDynamic changes in gene expression occur specifically in response to ethephon treatment. Certain genes identified may potentially contribute to latex production or TPD suppression. These data provide valuable information to understand the mechanism of ethylene stimulation, and will contribute to improved management practices and/or molecular breeding to attain higher yields of latex from Pará rubber trees.

Latex Diagnosis at the Whole Trunk Level under Different Tapping Systems in Young-tapping Rubber Trees

Background: Numerous rubbers tapping systems have been developed to increase latex yield. The objectives were (i) to test the efficiency of stimulation tapping systems and (ii) to describe the sucrose balance between supply and demand in the latex-producing bark of the rubber tree.Methods: The experiment was conducted at Thepa Research Station in Songkhla province. Eleven-year-old of RRIM600 clone was investigated. The experiment was designed as One Tree Plot Design (OTPD) with 4 tapping systems (Treatment; T) and 4 replications. Treatments were T1: S/3 2d/3, T2: S/6 d3, T3: S/6 d3 with RRIMFLOW and T4: S/6 d3 with LET. Result: S/6 d3 with RRIMFLOW tapping system in young-tapping rubber tree provided significantly highest averaged latex yield per tapping. The average cumulative latex yield was no significant difference comparing with the traditional tapping system. Rubber girth increment had no significant difference among treatments (P greater than 0.05). An averaged sucrose distribution in the trunk level of none stimulation treatments were high to very high sucrose values; however, it was medium sucrose values in the stimulation treatments. Inorganic phosphorus distribution in the trunk level showed medium to high values. Hence, the finding indicated that the use of ethylene stimulation together with tapping system should be considered for rubber tree and to control the balance of sucrose content in the trunk level of rubber tree.

Physiological and Productivity Impact of Mechanical Wounding and Mortex Stimulation on Rubber Clones RRIM 2025 and PB 350

The effects of mechanical wounding involving mechanical boring and tapping followed by the application of Mortex, an ethylene-based stimulant, on Hevea clones RRIM 2025 and PB 350 were investigated. Mortex at three different concentrations of 0.75%, 2.5% and 5% were applied at the tapping panel and the response of both clones in influencing wound-induced ethylene, latex yield, sugar, thiol and proline contents were determined. Data were analysed against control 1 (untapped and unstimulated), control 2 (tapped and unstimulated) and the application of nanosilver solution (Silversol), an ethylene inhibitor containing Ag+. Latex yield of both clones appeared to be induced and dependent on ethylene stimulation. Mortex at 2.5% was suggested to be optimum for high yield stimulation in both clones. Application of 5% Mortex gave the highest yield for both clones and induced highest wound-induced ethylene release in the shaved bark of RRIM 2025. Meanwhile, 2.5% Mortex gave the highest wound ethylene evolution in shaved bark of PB 350. Wounding by tapping alone produced wound ethylene at low levels in both clones. The very low levels of accumulated wound ethylene found in bored holes at 2 cm above tapping cut was likely due to tapping activity and not influenced by Mortex application. Silversol was found to successfully inhibit ethylene action by reducing wound ethylene release in shaved bark of both clones. Reduced sucrose content in the latex with 5% Mortex was observed. In contrast, unstimulated trees and an application of Silversol appeared to reduce latex yield in both clones with a concomitant increase in sucrose content. Suppression of ethylene action by Ag+ also reduced thiol and inorganic phosphorus content, suggesting that Silversol was effective in overcoming the stress effects related to ethylene regulation.

Identification and Characterization of Glycoproteins and Their Responsive Patterns upon Ethylene Stimulation in the Rubber Latex.

Natural rubber is an important industrial material, which is obtained from the only commercially cultivated rubber tree, Hevea brasiliensis. In rubber latex production, ethylene has been extensively used as a stimulant. Recent research showed that post-translational modifications (PTMs) of latex proteins, such as phosphorylation, glycosylation and ubiquitination, are crucial in natural rubber biosynthesis. In this study, comparative proteomics was performed to identify the glycosylated proteins in rubber latex treated with ethylene for different days. Combined with Pro-Q Glycoprotein gel staining and mass spectrometry techniques, we provided the first visual profiling of glycoproteomics of rubber latex and finally identified 144 glycosylated protein species, including 65 differentially accumulated proteins (DAPs) after treating with ethylene for three and/or five days. Gene Ontology (GO) functional annotation showed that these ethylene-responsive glycoproteins are mainly involved in cell parts, membrane components and metabolism. Pathway analysis demonstrated that these glycosylated rubber latex proteins are mainly involved in carbohydrate metabolism, energy metabolism, degradation function and cellular processes in rubber latex metabolism. Protein–protein interaction analysis revealed that these DAPs are mainly centered on acetyl-CoA acetyltransferase and hydroxymethylglutaryl-CoA synthase (HMGS) in the mevalonate pathway for natural rubber biosynthesis. In our glycoproteomics, three protein isoforms of HMGS2 were identified from rubber latex, and only one HMGS2 isoform was sharply increased in rubber latex by ethylene treatment for five days. Furthermore, the HbHMGS2 gene was over-expressed in a model rubber-producing grass Taraxacum Kok-saghyz and rubber content in the roots of transgenic rubber grass was significantly increased over that in the wild type plant, indicating HMGS2 is the key component for natural rubber production.

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.

Can fertilization be a driver of rubber plantation intensification?

Improving the productivity of existing rubber plantations is necessary to cope with the growing demand for natural rubber (NR) while limiting the environmental and social impacts linked to the expansion of rubber cultivation at the expense of natural ecosystems and food crops. The effect of fertilization on NR yield is still unclear and is poorly documented particularly in Thailand, the world leader in NR production. Hence, the main objective of this study was to re-assess the possible effects of fertilization on the yield of intensively managed rubber plantations in Thailand. Our main hypothesis was that the effect of fertilization would be higher with intensive latex harvesting practices (high tapping frequency with ethylene stimulation). To test this hypothesis, we set-up a split-plot experiment with four fertilization doses (T1, no fertilization, T2, T3, T4, respectively low, medium and high doses of NPK fertilizer) and two tapping systems (S/2 d2 with and without ethylene stimulation). Here, we present the results of the first three years on dry rubber yield, latex metabolism assessed with the latex diagnosis method, and latex and rubber properties related to their technological properties. Our results showed a positive effect of fertilization on yield from the first year, but the effect was only statistically significant (p < 0.05) in the third year. The maximum effect of fertilization (+13%) compared to the control treatment (T1) was obtained with the highest dose of fertilizer (T4). Cumulatively over the 3-year period, the increase in yield was +5% with the T2 dose, and +8% with the T3 and T4 dose. Contrary to our main assumption, we observed no interaction between fertilization and ethylene stimulation. Latex diagnosis revealed that the effect of fertilization on yield was linked with a direct effect on latex metabolism mainly through an increase in inorganic phosphorus content (Pi). Finally, we observed no detrimental effect of the fertilizer treatments on latex and rubber quality. We can thus conclude that increasing latex yield through fertilization combined with intensive tapping does not involve any identified risks for the quality of rubber. Taken together, our results demonstrate that fertilization can help increase the yield of rubber plantations where intensive latex harvesting is practised. However, in a context of low NR prices for rubber farmers, the economic return of a +8% increase in yield is questionable. Our trial is continuing to assess the long term effects of the fertilization on yield as well as on the growth and nutritional status of the trees, and the nutrient balance of the plantation.

Comparative Proteomic Analysis of the Hevea brasiliensis Latex under Ethylene and Calcium Stimulation.

Calcium ions usually act as a second messenger in the signal transmission process and a major element required by plants. In Hevea, calcium ion could alleviate the negative effects of long-term ethylene application to a certain extent. However, the molecular mechanisms remain unclear. Two-dimensional electrophoresis was used to determine the pattern of protein changes in latex after treatments with calcium and/or ethylene. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression levels of some proteins and genes. STRING software was used to determine the protein-protein interaction network of the identified proteins. Comparative proteomics identified 145 differentially expressed proteins, which represented 103 unique proteins. The abundance change patterns of some proteins involved in signal transduction, rubber particle aggregation, and natural rubber biosynthesis were altered upon calcium stimulation. Quantitative real-time polymerase chain reaction analysis of 29 proteins showed that gene expression did not always maintain the same trend as protein expression. The increased enzyme activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase suggested that calcium can enhance the antistress ability of plants by increasing the activity of their antioxidant enzyme systems. These results supplement the rubber latex proteome, and provide evidence for investigating the molecular mechanisms by which calcium alleviates the negative effects of ethylene stimulation.

Calcium-Dependent Protein Kinase Family Genes Involved in Ethylene-Induced Natural Rubber Production in Different Hevea brasiliensis Cultivars.

Natural rubber latex production can be improved by ethylene stimulation in the rubber tree (Hevea brasiliensis). However, the expression levels of most functional proteins for natural rubber biosynthesis are not induced after ethylene application, indicating that post-translational modifications, especially protein phosphorylation, may play important roles in ethylene signaling in Hevea. Here, we performed a comprehensive investigation on evolution, ethylene-induced expression and protein–protein interaction of calcium-dependent protein kinases (CPKs), an important serine/threonine protein kinase family, in Hevea. Nine duplication events were determined in the 30 identified HbCPK genes. Expression profiling of HbCPKs in three rubber tree cultivars with low, medium and high ethylene sensitivity showed that HbCPK6, 17, 20, 22, 24, 28 and 30 are induced by ethylene in at least one cultivar. Evolution rate analysis suggested accelerated evolution rates in two paralogue pairs, HbCPK9/18 and HbCPK19/20. Analysis of proteomic data for rubber latex after ethylene treatment showed that seven HbCPK proteins could be detected, including six ethylene-induced ones. Protein–protein interaction analysis of the 493 different abundant proteins revealed that protein kinases, especially calcium-dependent protein kinases, possess most key nodes of the interaction network, indicating that protein kinase and protein phosphorylation play important roles in ethylene signaling in latex of Hevea. In summary, our data revealed the expression patterns of HbCPK family members and functional divergence of two HbCPK paralogue pairs, as well as the potential important roles of HbCPKs in ethylene-induced rubber production improvement in Hevea.

Low frequency tapping systems applied to young-tapped trees of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. in Southern Thailand

A declining rubber price and labor shortages in the context of climate variability are problems for rubber smallholders. A low frequency tapping system that may be a solution to these problems was tested in southern Thailand using eight-year-old trees of the RRIM600 clone at the Thepa Research Station, Songkhla province. The experimental design was a randomized complete block with five treatments defined in Table 1 and abbreviated to: T1: S/3 d1 2d/3; T2: S/2 d2; T3: S/2 d3 ET 2.5% Pa1 (1) 8/y (m); T4: S/3 d2 ET 2.5% Pa1 (1) 4/y (m); and T5: S/3 d3 ET 2.5% Pa1 (1) 12/y (m). There were three replications (elementary plot) with 10 trees per treatment in each elementary plot. The results showed that low frequency tapping systems (d3) with stimulation resulted in an equivalent yield in cumulative latex production compared with the other tapping systems and also had higher latex production per tapping. Bark consumption was less in the low frequency tapping systems leading to the possibility of lengthening the economic lifespan of the tapping panels of the tree. A low frequency tapping treatment with stimulation eight times per year induced decreases in the total solids, sucrose and reduced thiol contents; however, the inorganic phosphorus content increased, as is usually seen with the use of ethylene stimulation. The response of rubber trees to a low frequency tapping system should now be tested in the long term.

Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF138,175,258 and SRPP117,204,243, were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF175 or REF258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

Stimulation affecting latex physiology and yield under low frequency tapping of rubber (Hevea brasiliensis) clone RRIM 600 in southern Thailand

Tapping and ethylene stimulation induce s the dynamic change of latex cell metabolism. Those changes under the implement of low frequency tapping systems with ethylene stimulation were investigated during the both periods ( low yield and the high yield) of the year of production. The experiment was established at Thepa Research Station, Songk hla province by using 9 - year - old rubber trees (clone RRIM 600). An experiment was arranged as One Tree Plot design, there were five treatments following T1: S/3 d1 2d/3, T 2: S/2 d2, T3: S/2 d3 ET 2.5% Pa1(1) 8/y (m), T4: S/3 d2 ET 2.5% Pa1(1) 4/y (m) and T 5: S/3 d3 ET 2.5% Pa1(1) 12/y (m). There were three replicates in each treatment. It was found that ethylene stimulation affected the responses of initial flow rate (IFR), plugging index (PI), average latex yield (AY) and sucrose content (Suc). There was a significant difference among the treatments in the both periods. After ethylene stimulation, IFR of the T3 and T5 were superior in the low yield period and showed inferior in the high yield period compared with the T1. PI rapidly decreased in the ethylene application treatments in the both periods. AY of the T3 was the highest in the low yield period and AY of the ethylene stimulation treatments was higher than non - stimulated treatments in the high yield period. Suc of the T3 only increased on the first ta pping day after stimulation in the both periods. Expression to the ethylene stimulation in the low yield period was higher than the high yield period. It was remarkable that the stimulation was effecti vely expressed during a full canopy stage. With the pos itive impact of stimulation on latex physiological parameters, latex yield under low frequency tapping could be compe nsated by ethylene application. (Resume d'auteur)

Identification and functional characterization of HbOsmotin from Hevea brasiliensis

Latex in the laticiferous cell network of Hevea brasiliensis tree is composed of cytoplasm that synthesizes natural rubber. Ethylene stimulation of the tree bark enhances latex production partly by prolonging the duration of latex flow during the tapping process. Here, we identified an osmotin-like cDNA sequence (HbOsmotin) from H. brasiliensis that belongs to the pathogenesis-related 5 (PR-5) gene family. The HbOsmotin protein is present in the lutoids of latex in H. brasiliensis, whereas in onion epidermal cells, this protein is predominantly distributed around the cell wall, suggesting that it may be secreted from the cytoplasm. We investigated the effects of exogenous ethylene on HbOsmotin transcription and protein accumulation in rubber latex, and further determined the protein function after osmotic stress in Arabidopsis. In regularly tapped trees, HbOsmotin expression was drastically inhibited in rubber latex after tapping, although the expression was subsequently recovered by ethylene stimulation. However, in virgin plants that had never been tapped, exogenous ethylene application slightly decreased HbOsmotin expression. HbOsmotin overexpression in Arabidopsis showed that HbOsmotin reduced the osmotic stress tolerance of the plant, which likely occurred by raising the water potential. These data indicated that HbOsmotin may contribute to osmotic regulation in laticiferous cells.

INFLUENCES OF ETHYLENE STIMULATION OF RUBBER TREES (Hevea brasilliensis) ON THE EXTRACTIVES AND FUNGAL RESISTANCE OF LUMBER

ABSTRACT Ethylene stimulation increases the rubber latex yield of live rubberwood (Hevea brasiliensis). Lumber samples from ethylene treated rubberwood (TRW) and from untreated rubberwood (URW) were compared mainly for their resistance to fungi, differences in the chemical composition between TRW and URW, and the antifungal activities of their aqueous extracts. The TRW had significantly higher lignin and extractives contents than the URW, but the TRW had comparatively poor resistance to fungal rotting. The white rot fungus Ganoderma lucidum and the brown rot fungus Gloeophyllum striantum caused in vitro significantly higher mass loss in TRW than in URW. This might be related to the phenolic compounds 2,4-ditert-butylphenol and 4-hydroxy-3,5- dimethoxy-benzaldehyde. The aqueous wood extracts strongly inhibited growth of G. lucidum, with lesser effects on the other fungi tested. Caffeine was detected in the TRW, but not the URW. However, the caffeine degraded so quickly that it had no effect on the 6 and 12 weeks fungal resistances of wood samples.

Molecular mechanism underlying ethylene stimulation of latex production in rubber tree (Hevea brasiliensis)

Ethephon (an ethylene releaser) is commonly used to enhance latex productivity of rubber tree ( Hevea brasiliensis ), but the underlying mechanism has been elusive. The review presents the recent advances in understanding the molecular mechanism of increasing latex production in rubber tree by ethylene application. Rubber tree (Hevea brasiliensis) is currently the only commercial sources of natural rubber which is an important industrial material. Application of ethephon (an ethylene generator) has been a measure extensively adopted in worldwide rubber plantations to increase the productivity of rubber tree since the 1960s. In the recent years, adoption of genomic, transcriptomic, and proteomic approaches has brought great progress in understanding the mechanism of ethylene stimulation of latex yield. In this review, an update on the molecular mechanism of ethylene stimulation of latex production in H. brasiliensis is presented. Ethylene stimulation can be roughly ascribed to the acceleration of latex regeneration and the prolongation of the flow of latex. The relevant biochemical pathways and processes are comprehensively reviewed. The possibility that the limiting factors of the biosynthesis of rubber identified in bark glycolysis and photosynthetic carbon fixation can be utilized in molecular diagnostic and genetic engineering for rubber high-yielding variety improvement is discussed. Unsolved problems of the ethylene stimulation and a major challenge to understand the regulatory mechanism of the ethylene stimulation are also indicated.

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.

Molecular mechanism of ethylene stimulation of latex yield in rubber tree (Hevea brasiliensis) revealed by de novo sequencing and transcriptome analysis.

BackgroundRubber tree (Hevea brasiliensis) is an important industrial crop cultivated in tropical areas for natural rubber production. Treatment of the bark of rubber trees with ehephon (an ethylene releaser) has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. Deciphering the enigma is of great importance for improvement of rubber tree for high yield.ResultsDe novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 h (E8) and 24 h (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified.ConclusionsThe rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2587-4) contains supplementary material, which is available to authorized users.

Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray.

Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ –2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis.

De novo Transcriptome Assembly of Floral Buds of Pineapple and Identification of Differentially Expressed Genes in Response to Ethephon Induction.

A remarkable characteristic of pineapple is its ability to undergo floral induction in response to external ethylene stimulation. However, little information is available regarding the molecular mechanism underlying this process. In this study, the differentially expressed genes (DEGs) in plants exposed to 1.80 mL·L−1 (T1) or 2.40 mL·L−1 ethephon (T2) compared with Ct plants (control, cleaning water) were identified using RNA-seq and gene expression profiling. Illumina sequencing generated 65,825,224 high-quality reads that were assembled into 129,594 unigenes with an average sequence length of 1173 bp. Of these unigenes, 24,775 were assigned to specific KEGG pathways, of which metabolic pathways and biosynthesis of secondary metabolites were the most highly represented. Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority were involved in metabolic and cellular processes, cell and cell part, catalytic activity and binding. Gene expression profiling analysis revealed 3788, 3062, and 758 DEGs in the comparisons of T1 with Ct, T2 with Ct, and T2 with T1, respectively. GO analysis indicated that these DEGs were predominantly annotated to metabolic and cellular processes, cell and cell part, catalytic activity, and binding. KEGG pathway analysis revealed the enrichment of several important pathways among the DEGs, including metabolic pathways, biosynthesis of secondary metabolites and plant hormone signal transduction. Thirteen DEGs were identified as candidate genes associated with the process of floral induction by ethephon, including three ERF-like genes, one ETR-like gene, one LTI-like gene, one FT-like gene, one VRN1-like gene, three FRI-like genes, one AP1-like gene, one CAL-like gene, and one AG-like gene. qPCR analysis indicated that the changes in the expression of these 13 candidate genes were consistent with the alterations in the corresponding RPKM values, confirming the accuracy and credibility of the RNA-seq and gene expression profiling results. Ethephon-mediated induction likely mimics the process of vernalization in the floral transition in pineapple by increasing LTI, FT, and VRN1 expression and promoting the up-regulation of floral meristem identity genes involved in flower development. The candidate genes screened can be used in investigations of the molecular mechanisms of the flowering pathway and of various other biological mechanisms in pineapple.