Search Results (12)

Allergenic proteins in natural rubber latex (NRL) pose significant health risks, particularly in rubber gloves. This study evaluated RNA interference (RNAi) technology for silencing HbREF (rubber elongation factor) and HbSRPP (small rubber particle protein) genes in Hevea brasiliensis to reduce latex allergen content. Double-stranded RNA (dsRNA) targeting these genes demonstrated high stability at 25–37 °C for 6 h and under UV/outdoor conditions for 72 h, but degraded rapidly above 50 °C. Among the three delivery methods tested, direct injection achieved the highest efficiency (>90% gene silencing within 12 h), followed by root drenching (54–84%) and foliar spray (46–70%). HbREF silencing achieved 98–99% expression reduction within 3 h, while HbSRPP showed dose-dependent responses (70–90% silencing) without off-target effects. Gene silencing affected downstream rubber synthesis genes HbCPT (cis-prenyltransferase) and HbRME (rubber membrane elongation protein) (37–58% reduction) while upstream genes remained unaffected. HbREF silencing reduced Hev b1 allergen by 64.04% and Hev b3 by 12.51%, whereas HbSRPP silencing decreased Hev b3 by 71.54% and Hev b1 by 13.48%. Both treatments caused only a 11–13% reduction in dry rubber content. This RNAi approach effectively reduces major latex allergens while maintaining rubber production, demonstrating commercial potential for developing hypoallergenic rubber products through precision agriculture biotechnology. Full article

Natural rubber production currently relies heavily on a single species, Hevea brasiliensis, underscoring the urgent need to identify alternative sources to alleviate the strain on natural rubber production. The rubber elongation factor (REF) and small rubber particle protein (SRPP), both members of the REF/SRPP gene family, are crucial for natural rubber biosynthesis. However, research on the REF gene has predominantly focused on H. brasiliensis and Taraxacum kok-saghyz. We conducted a comprehensive genome-wide identification and characterization of the REF gene, identifying 87 REF protein sequences across 17 plants species. We observed a significant increase in the copy numbers and expression of REF genes in rubber-producing plants. Notably, in H. brasiliensis, T. kok-saghyz, Eucommia ulmoides, Lactuca sativa, and other rubber-yielding species, the number of REF genes has markedly increased. Furthermore, some REF genes in H. brasiliensis form a distinct clade in phylogenetic analyses and exhibit differences in conserved motif arrangements and tertiary protein structures compared to other REF genes. These findings suggest that REF genes in rubber-producing plants may have undergone independent evolution, leading to changes in copy number and structure. These alterations could contribute to the production of natural rubber in these species. The results of this study provide a scientific basis for further research into the mechanisms of rubber production in plants and for identifying potential rubber-producing species. Full article

Natural rubber is a crucial industrial material, and it is primarily harvested from the latex of the rubber tree Hevea brasiliensis by tapping the tree trunk. During the regular tapping process, mechanical damage seriously affects latex reproduction and rubber yield, but the molecular mechanisms on tapping stimulation remain unclear. In this study, we firstly determined the changed physiological markers on latex regeneration, overall latex yield, and latex flow time during the tapping process. Then, we combined proteomics and transcriptomics analyses of latex during tapping and identified 3940 differentially expressed genes (DEGs) and 193 differentially expressed proteins (DEPs). Among them, 773 DEGs and 120 DEPs displayed a persistent upregulation trend upon tapping. It is interesting that, in the detected transcription factors, basic helix-loop-helix (bHLH) family members occupied the highest proportion among all DEGs, and this trend was similarly observed in DEPs. Notably, 48 genes and 34 proteins related to natural rubber biosynthesis were identified, and most members of small rubber particle protein (SRPP) and rubber elongation factor (REF) showed a positive response to tapping stimulation. Among them, SRPP6 and REF5 showed significant and sustained upregulation at the gene and protein levels following tapping, indicating their pivotal roles for post-tapping rubber biosynthesis. Our results deepen the comprehension of the regulation mechanism underlying tapping and provide candidate genes and proteins for improving latex production in the Hevea rubber tree in future. Full article

Taraxacum kok-saghyz (TKS) is a model plant and a potential rubber-producing crop for the study of natural rubber (NR) biosynthesis. The precise analysis of the NR biosynthesis mechanism is an important theoretical basis for improving rubber yield. The small rubber particle protein (SRPP) and rubber elongation factor (REF) are located in the membrane of rubber particles and play crucial roles in rubber biosynthesis. However, the specific functions of the SRPP/REF gene family in the rubber biosynthesis mechanism have not been fully resolved. In this study, we performed a genome-wide identification of the 10 TkSRPP and 2 TkREF genes’ family members of Russian dandelion and a comprehensive investigation on the evolution of the ethylene/methyl jasmonate-induced expression of the SRPP/REF gene family in TKS. Based on phylogenetic analysis, 12 TkSRPP/REFs proteins were divided into five subclades. Our study revealed one functional domain and 10 motifs in these proteins. The SRPP/REF protein sequences all contain typical REF structural domains and belong to the same superfamily. Members of this family are most closely related to the orthologous species T. mongolicum and share the same distribution pattern of SRPP/REF genes in T. mongolicum and L. sativa, both of which belong to the family Asteraceae. Collinearity analysis showed that segmental duplication events played a key role in the expansion of the TkSRPP/REFs gene family. The expression levels of most TkSRPP/REF members were significantly increased in different tissues of T. kok-saghyz after induction with ethylene and methyl jasmonate. These results will provide a theoretical basis for the selection of candidate genes for the molecular breeding of T. kok-saghyz and the precise resolution of the mechanism of natural rubber production. Full article

The rubber elongation factor (REF) is the most abundant protein in the latex of Hevea brasiliensis, which is closely related to natural rubber biosynthesis. In order to gain a deeper understanding of the transcriptional regulation mechanism of HbREF1, a 1758 bp genomic DNA fragment of the HbREF1 promoter was isolated. Promoter sequence analysis revealed several transcription factor binding sites in the HbREF1 promoter, such as bZIP, bHLH, EIL, AP2/ERF, MYB, and Trihelix. To assess the promoter activity, a series of HbREF1 promoter deletion derivatives were created and fused with firefly luciferase (LUC). The LUC image demonstrated that all of the HbREF1 promoters exhibited transcriptional activity. Furthermore, the assay revealed the presence of multiple regulatory elements within the promoter region that negatively regulate the transcriptional activity. Subsequent analysis of the transcriptional activity following treatment with phytohormones identified an ABA-responsive element located between −583 bp and −200 bp, an ET-responsive element between −718 bp and −583 bp, a JA-responsive element between −1758 bp and −1300 bp, and a SA-responsive element between −1300 bp and −718 bp. These results were largely consistent with the predictions of cis-acting elements. This study has established significant groundwork for future investigations into the regulatory mechanism of HbREF1. Full article

Plant 14-3-3 proteins mediate a wide range of functionally diverse proteins through protein–protein interactions that are typically phosphorylation-dependent. However, the interactions between 14-3-3 proteins and the major regulators of nature rubber (NR) biosynthesis in H. brasiliensis have not been fully elucidated. In this study, we obtained 81 essential client proteins that interacted with H. brasiliensis 14-3-3 proteins (HbGF14s) through yeast two-hybrid (Y2H) screening. These interaction partners were involved in plant signal transduction, metabolism, development, and NR biosynthesis including small rubber particle protein (SRPP), rubber elongation factor (REF), and MYC2, etc. Furthermore, the interaction of HbGF14c and HbSRPP of H. brasiliensis was confirmed in plants through bimolecular fluorescence complementation (BiFC) assays and in vitro with Pull-down assays. Specifically, the RVSSYLP motif was found to mediate the interaction between HbSRPP and HbGF14c. The findings of this study provide a theoretical basis for the elucidation of the molecular regulation mechanism of the 14-3-3 proteins involved in NR biosynthesis, which could be used to enhance the production of rubber trees through genetic improvement. Full article

Natural rubber (NR) remains an indispensable raw material with unique properties that is used in the manufacture of a large number of products and the global demand for it is growing every year. The only industrially important source of NR is the tropical tree Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg., thus alternative sources of rubber are required. For the temperate zone, the most suitable source of high quality rubber is the Russian (Kazakh) dandelion Taraxacum kok-saghyz L.E. Rodin (TKS). An obstacle to the widespread industrial cultivation of TKS is its high heterozygosity, poor growth energy, and low competitiveness in the field, as well as inbreeding depression. Rapid cultivation of TKS requires the use of modern technologies of marker-assisted and genomic selection, as well as approaches of genetic engineering and genome editing. This review is devoted to describing the progress in the field of molecular genetics, genomics, and genetic engineering of TKS. Sequencing and annotation of the entire TKS genome made it possible to identify a large number of SNPs, which were subsequently used in genotyping. To date, a total of 90 functional genes have been identified that control the rubber synthesis pathway in TKS. The most important of these proteins are part of the rubber transferase complex and are encoded by eight genes for cis-prenyltransferases (TkCPT), two genes for cis-prenyltransferase-like proteins (TkCPTL), one gene for rubber elongation factor (TkREF), and nine genes for small rubber particle proteins (TkSRPP). In TKS, genes for enzymes of inulin metabolism have also been identified and genome-wide studies of other gene families are also underway. Comparative transcriptomic and proteomic studies of TKS lines with different accumulations of NR are also being carried out, which help to identify genes and proteins involved in the synthesis, regulation, and accumulation of this natural polymer. A number of authors already use the knowledge gained in the genetic engineering of TKS and the main goal of these works is the rapid transformation of the TKS into an economically viable rubber crop. There are no great successes in this area so far, therefore work on genetic transformation and genome editing of TKS should be continued, considering the recent results of genome-wide studies. Full article

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. Full article

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. Full article

The rubber grass Taraxacum kok-saghyz (TKS) contains large amounts of natural rubber (cis-1,4-polyisoprene) in its enlarged roots and it is an alternative crop source of natural rubber. Natural rubber biosynthesis (NRB) and storage in the mature roots of TKS is a cascade process involving many genes, proteins and their cofactors. The TKS genome has just been annotated and many NRB-related genes have been determined. However, there is limited knowledge about the protein regulation mechanism for NRB in TKS roots. We identified 371 protein species from the mature roots of TKS by combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Meanwhile, a large-scale shotgun analysis of proteins in TKS roots at the enlargement stage was performed, and 3545 individual proteins were determined. Subsequently, all identified proteins from 2-DE gel and shotgun MS in TKS roots were subject to gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and most proteins were involved in carbon metabolic process with catalytic activity in membrane-bounded organelles, followed by proteins with binding ability, transportation and phenylpropanoid biosynthesis activities. Fifty-eight NRB-related proteins, including eight small rubber particle protein (SRPP) and two rubber elongation factor(REF) members, were identified from the TKS roots, and these proteins were involved in both mevalonate acid (MVA) and methylerythritol phosphate (MEP) pathways. To our best knowledge, it is the first high-resolution draft proteome map of the mature TKS roots. Our proteomics of TKS roots revealed both MVA and MEP pathways are important for NRB, and SRPP might be more important than REF for NRB in TKS roots. These findings would not only deepen our understanding of the TKS root proteome, but also provide new evidence on the roles of these NRB-related proteins in the mature TKS roots. Full article

Natural rubber is a kind of indispensable biopolymers with great use and strategic importance in human society. However, its production relies almost exclusively on rubber-producing plants Hevea brasiliensis, which have high requirements for growth conditions, and the mechanism of natural rubber biosynthesis remains largely unknown. In the past two decades, details of the rubber chain polymerization and proteins involved in natural rubber biosynthesis have been investigated intensively. Meanwhile, omics and other advanced biotechnologies bring new insight into rubber production and development of new rubber-producing plants. This review summarizes the achievements of the past two decades in understanding the biosynthesis of natural rubber, especially the massive information obtained from the omics analyses. Possibilities of natural rubber biosynthesis in vitro or in genetically engineered microorganisms are also discussed. Full article

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 REF_138,175,258 and SRPP_117,204,243, 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. Full article