Small Rubber Particle Protein Research Articles

Genome-Wide Analysis of the SRPP/REF Gene Family in Taraxacum kok-saghyz Provides Insights into Its Expression Patterns in Response to Ethylene and Methyl Jasmonate Treatments.

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.

Transcriptome analysis reveals the molecular mechanisms of rubber biosynthesis and laticifer differentiation during rubber seed germination.

The molecular mechanisms underlying the initiation of natural rubber synthesis and laticifer differentiation have not been fully elucidated. In this study, we conducted a time-series transcriptome analysis of five rubber tree tissues at four stages of seed germination. A total of 161,199 DEGs were identified between the two groups, including most 16,673 DEGs (A3 vs B3 and A3 vs C3) and lest 1,210 DEGs (C2 vs D2). We found that the maturation of the seed is accompanied by the formation of laticifer cells in cotyledon. Meanwhile, the analysis of hormones related genes expression may provide effective clues for us to promote the differentiation of laticifer cells in seeds by hormones in the future. In this study, hormone-related gene enrichment analyses revealed that IAA, GA, and CTK were activated in laticifer containing tissues. Similarly, GO and GEGG analysis showed that hormone pathways, especially the auxin pathway, are enriched. Gene expression clustering was analyzed using the short time-series expression miner (STEM), and the analysis revealed four distinct trends in the gene expression profiles. Moreover, we enriched transcription factor (TF) enrichment in cotyledon and embryonic axis tissues, and the MYB type exhibited the most significant difference. Furthermore, our findings revealed that genes related to rubber synthesis exhibited tissue-specific expression patterns during seed germination. Notably, key genes associated with rubber biosynthesis, specifically small rubber particle protein (SRPP) and cis-prenyltransferase (CPT), exhibited significant changes in expression in cotyledon and embryonic axis tissues, suggesting synchronous rubber synthesis with seed germination. Our staining results reveled that laticifer cells were exits in the cotyledon before seed imbibition stage. In conclusion, these results lay the foundation for exploring the molecular mechanisms underlying laticifer differentiation and rubber synthesis during seed germination, deepening our understanding of the initiation stages of rubber biosynthesis and laticifer differentiation.

Interactions of REF1 and SRPP1 rubber particle proteins from Hevea brasiliensis with synthetic phospholipids: Effect of charge and size of lipid headgroup

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to β-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.

The Amplification and Activity Analysis of the Small Rubber Particle Protein (SRPP) Promoter of Eucommia ulmoides (EuSRPP) Revealed That Its Activity Was Regulated by MeJA, GA3, and Drought Pathways

As an important temperate gum source plant, Eucommia ulmoides is widely distributed in China, but the low yield of Eucommia ulmoides gum considerably affects its application as a natural rubber in practical production. The small rubber particle protein (SRPP) gene is an influential participant in the Eucommia ulmoides gum biosynthesis process, and its expression affects the gum content. In this study, the promoter activity of the Eucommia ulmoides SRPP (EuSRPP) gene was analyzed by molecular biology and bioinformatics. In order to understand the molecular regulation mechanism of the EuSRPP genes at the transcriptional level, we first obtained the promoter sequences of the EuSRPP1, 3, 4, 5, 6, and 7 genes via genome walking and PCR amplification experiments. Then, the T3 generation of the transgenic homozygous line was obtained via a genetic transformation of Arabidopsis thaliana mediated by Agrobacterium. The six EuSRPP promoters were expressed in transgenic plants and were stably expressed in the leaves, pollinated flowers, and mature pods. As the transgenic plant grows and develops, promoter activity in the root is barely expressed. In addition, after the transgenic Arabidopsis was treated with methyl jasmonate (1 mmol/L MeJA), gibberellin (1 mmol/L GA3), and drought (20% PEG6000), the activity expression of the six EuSRPP promoters increased first and then decreased. The difference, however, is that EuSRPP1, 3, and 4 reach their strongest GUS activity at 3 h of plant treatment, while EuSRPP5, 6, and 7 reach their strongest activity at 6 h of treatment. Based on all experimental results, for the first time, it has been shown that the expression loci of the six EuSRPP gene promoters were relatively consistent. Second, the expression activity of the promoters of the six EuSRPP genes was different under MeJA, GA3, and drought treatment, suggesting that the promoter activity of the EuSRPP genes was regulated by endogenous hormones and drought pathways.

Cloning and characterization of the promoters of the key genes CPT, SRPP and REF involved in Periploca sepium rubber biosynthesis

Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

14-3-3 Proteins Participate in Regulation of Natural Rubber Biosynthesis in Hevea brasiliensis

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.

Molecular Genetic Research and Genetic Engineering of Taraxacum kok-saghyz L.E. Rodin.

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.

Functional Characterization of TkSRPP Promoter in Response to Hormones and Wounding Stress in Transgenic Tobacco.

Taraxacum kok-saghyz is a model species for studying natural rubber biosynthesis because its root can produce high-quality rubber. Small rubber particle protein (SRPP), a stress-related gene to multiple stress responses, involves in natural rubber biosynthesis. To investigate the transcriptional regulation of the TkSRPP promoter, the full-length promoter PR0 (2188 bp) and its four deletion derivatives, PR1 (1592 bp), PR2 (1274 bp), PR3 (934 bp), and PR4 (450 bp), were fused to β-glucuronidase (GUS) reporter gene and transformed into tobacco. The GUS tissue staining showed that the five promoters distinctly regulated GUS expression utilizing transient transformation of tobacco. The GUS activity driven by a PR0 promoter was detected in transgenic tobacco leaves, stem and roots, suggesting that the TkSRPP promoter was not tissue-specific. Deletion analyses in transgenic tobacco have demonstrated that the PR3 from -934 bp to -450 bp core region responded strongly to the hormones, methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid (SA), and also to injury induction. The TkSRPP gene was highly expressed under hormones and wound-induced conditions. This study reveals the regulation pattern of the SRPP promoter, and provides valuable information for studying natural rubber biosynthesis under hormones and wounding stress.

Dissecting microRNA−Target Gene Pairs Involved in Rubber Biosynthesis in Eucommia ulmoides

MicroRNAs (miRNAs) play essential roles in regulating various development processes in plants. However, their role in regulating rubber biosynthesis in Eucommia ulmoides is largely unknown. Rubber is mainly distributed in the tissue covering the seed (GZ) rather than the periphery (GB) of the pericarp of E. ulmoides during accumulation in May. To investigate the roles of miRNAs in rubber biosynthesis, we conducted high-throughput small RNA sequencing using GZ and GB collected on 11 May (rapid accumulation) and 11 June (reduced accumulation). In total, 12 and 25 miRNAs were either up- or downregulated in GZ in May (GZ511) compared to GB (GB511) in May, while 27 and 38 miRNAs were either up- or downregulated in GZ in May compared to GZ in June (GZ611), respectively. Functional analyses of differentially expressed (DE−) genes targeted by DE-miRNAs revealed that miRNAs may regulate genes involved in rubber biosynthesis. For instance, when Eu-miR45 expression declined, the expression of its predicted target, small rubber particle protein 1 (EuSRPP1), increased in GZ511 vs. GB511 and GZ511 vs. GZ611, possibly resulting in higher rubber accumulation in GZ511. Additionally, we identified potential lncRNA−miRNA−mRNA networks in rubber biosynthesis. Overall, these results indicate that miRNAs play pivotal roles in regulating rubber biosynthesis via miRNA-target gene pairs and lncRNA−miRNA−mRNA networks in E. ulmoides. Our findings will enhance research on the mechanisms of rubber biosynthesis in plants.

Comparative Transcriptome Analysis Identifies Potentially Relevant Genes in Rubber Clones with a High Latex Yield (Hevea brasiliensis)

Since the 1950s, the rubber tree (Hevea brasiliensis) has significantly contributed to Malaysia's agricultural economy due to its capability to produce high-value natural rubber. Due to the presence of cis-1,4-polyisoprene, natural rubber exhibits unique properties such as flexibility, high elasticity, and efficient heat dispersion. Cis-1,4-polyisoprene is synthesized via two distinct metabolic pathways: the isoprenoid and rubber biosynthesis. RNA sequencing was performed on 12 specimens from the bark, leaf, and latex tissues of two rubber clones, RRIM 3001 (high latex yield) and RRIM 712 (low latex yield). After quality assessment, these specimens generated a mean of 73,816,244 clean reads. The differential expression analysis revealed that the Acetyl-CoA-Acetyltransferase (AACT) and Diphosphomevalonate Decarboxylase (MVD) genes (via the MVA pathway), as well as the Small Rubber Particle Protein (SRPP) and Rubber Elongation Factor (REF) genes (via the rubber biosynthesis pathway), were overexpressed in the bark, leaf, and latex tissues of RRIM 3001 compared to RRIM 712. The transcription factor Ethylene Response Factor (ERF) was also found to be more highly expressed in RRIM 3001 than in RRIM 712. Five rubber clones with random yield characteristics were used in quantitative amplification analysis to validate AACT, MVD, SRPP, REF, and ERF genes. The quantitative analysis shows that the five genes were consistently expressed with the yield characteristics of the five random rubber clones.

Transcriptomics and proteomics profiles of Taraxacum kok-saghyz roots revealed different gene and protein members play different roles for natural rubber biosynthesis

The Russian dandelion (Taraxacum kok-saghyz L. Rodin, TKS) is a potential crop for natural rubber production. Our proteomics results have revealed that both methylerythritol (MEP) and mevalonate (MVA) pathways are important for natural rubber biosynthesis (NRB) in TKS roots. In this study, morphological characteristics and rubber content in TKS roots during five development stages were determined and more laticifer cells, rubber particles, and natural rubber were observed in older TKS roots. Combination of transcriptomics and proteomics was further performed to gain an overall realizing of the principle developmental mechanism and metabolic regulation processes in TKS roots and determine the key genes and proteins for NRB. From the detected TKS roots, 34,710 genes and 3695 proteins were positively identified. Among them, 22194 genes and 2145 proteins were differentially expressed during root development, and their correlation coefficients were calculated. GO and KEGG analysis showed that most up-regulated genes and proteins were involved in carbon metabolic process. A total of 102 NRB-related gene and protein members, including 10 cis-isoprene transferases (CPT), 8 small rubber particle proteins (SRPP), 2 rubber elongation factors (REF), as well as one HRT1-REF bridging protein (HRBP), were identified. Our omics data revealed that different family members of genes and proteins play different roles for NRB in TKS roots. Among them, CPT7, SRPP5, SRPP6 and SRPP9 might play more important roles for NRB in TKS roots.

The relationship between EuSRPP1 gene expression and rubber biosynthesis in Eucommia ulmoides Oliver (Du-zhong)

Because of the unique properties, trans-polyisoprene rubber (TPI) has gradually realized that it has great development potential. Eucommia ulmoides Oliver (E. ulmoides Oliver), also named Du-zhong, is one of the few woody plants producing TPI. The significant expansion of small rubber particle protein and rubber elongation factor (SRPP/REF) genes in E. ulmoides Oliver may be associated with the producing of TPI. To understand the relationship between EuSRPP1 gene expression and rubber biosynthesis in E. ulmoides Oliver, in this study, an overexpression transgenic (OT) vector of EuSRPP1 driven by the act promoter and an RNA interference (RNAi) expression vector of EuSRPP1 were constructed. Then, Agrobacterium-mediated genetic transformation of E. ulmoides Oliver was conducted to obtain the OT and RNAi seedlings. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) showed that the EuSRPP1 expression levels in OT seedlings significantly increased, and were 2.53 × and 3.68 × higher in OT-1 and OT-2 seedlings, respectively, than the wild-type (WT) seedlings, Meanwhile, the expression levels of E. ulmoides Isopentenyl Diphosphate Isomerase (EuIPPS) and E. ulmoides Farnesyl Diphosphate Synthase (EuFPS) genes in OT plants, which are related to rubber biosynthesis synthesis, were also up-regulated. To the contrary, EuSRPP1 gene expression levels in the RNAi-1 and RNAi-2 seedlings significantly decreased, which were 3.92% and 42.38% of the WT seedlings, respectively. EuIPPS and EuFPS genes expression levels in the RNAi seedlings were also down-regulated. The TPI content analysis revealed that the TPI content in the overexpression EuSRPP1 callus increased to 1.85 × that of the WT callus, whereas that in the RNAi callus decreased to only 52% of the WT callus. Molecular weights of rubber were significantly different in OT callus (2.31 × 106 Da) and RNAi callus (0.36 × 106 Da). Observation of the rubber particles revealed that the rubber particle electron density was higher in OT seedlings, and the immunogold localization results showed that EuSRPP1 was localized to rubber particles. These results indicate that the EuSRPP1 expression level influences the chain elongation, particle development, and content of rubber in E. ulmoides Oliver.

Optimization of a protein extraction method from natural rubber sheets made of Hevea brasiliensis latex

A method to extract proteins from natural rubber (NR) samples has been developed to characterize the protein composition of NR by SDS-PAGE electrophoresis. While the incubation of NR pieces in aqueous buffers did not provide satisfactory results (no protein bands detected by SDS-PAGE), the solubilization of NR pieces in an organic solvent, prior to centrifugation and protein extraction from the obtained pellet, was found efficient to highlight protein bands by SDS-PAGE. Pure cyclohexane was used to solubilize NR pieces and resulted in a low share of nitrogen in the pellet (29%). The addition of 10% (v/v) ethanol to cyclohexane allowed to significantly increase this share to 73% and to detect 13 protein bands by SDS-PAGE. The molecular weights of the four most intense bands suggest the presence in NR of the following proteins: rubber elongation factor, small rubber particle protein, hevamine, and β-1-3 glucanase.

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.

An integrative transcriptomic and genomic analysis reveals novel insights into the hub genes and regulatory networks associated with rubber synthesis in H. brasiliensis

Rubber tree (Hevea brasiliensis) is an important tropical plant that produces natural rubber; however, the hub genes and regulatory networks associated with rubber synthesis remain largely unclear. In this work, 129 publically available RNA-seq datasets were collected to perform gene co-expression network analysis in rubber tree. In total, 25 co-expressed gene modules (M1-M25) were identified, and the molecular functions and hub genes of each module were systematically analyzed. Associations between modules and tissues/stresses revealed that modules were significantly associated with stresses in the tissues where the modules were specifically highly expressed. Notably, module M7 was highly expressed in latex and dramatically responsive to ethylene, jasmonate, and tapping panel dryness treatments. Besides, the genes from M7 were significantly enriched in isoprenoid metabolism, indicating a great contribution of this module to rubber biosynthesis. The co-expression network of rubber biosynthesis genes from M7 revealed that small rubber particle protein 1 (SRPP1), cis-prenyltransferase 2 (CPT2), and rubber elongation factor 1 (REF1) were the most connected hub genes participating in rubber biosynthesis. Two candidate cis-regulatory motifs and three transcription factors that might regulate rubber biosynthesis were further identified. Genomic evolutionary analysis suggested that the expansion of two independently evolved REF/SRPP clusters, II (containing SRPP1) and V (containing REF1), was critical for rubber synthesis. Together, this integrative transcriptomic and genomic analysis provides novel insights into the hub genes and regulatory networks underlying rubber synthesis in H. brasiliensis.

A label-free shotgun proteomics analysis of macadamia nut.

In this study, we present a systematic proteomic overview of macadamia nut using a label-free shotgun proteomic approach. We identified 947 proteins in 723 clusters and gene ontology analysis revealed proteins across 46 functional categories including carbohydrate metabolism (10%), protein metabolic processes (5%), amino acid metabolism (4%), transport (4%), stress response (3%), lipid metabolism (3%), protein folding (3%) and defense response (1.4%). The defense response proteins accounted for 24% of the total peptide abundance. The vicilin-like macadamia antimicrobial peptides 2-3 (MiAMP2) was the most abundant protein, followed by glyceraldehyde-3-phosphate dehydrogenase 3, 11S legumin-like protein, 2-phospho-D-glycerate hydrolase and heat shock 70kDa protein among others. The cascading of amino acid and carbohydrate metabolic pathways in macadamia nut were constructed against reference maps from KEGG and proposed for the first time. Results were also indicative of useful protein candidates with possible allergenic potential and cross-reactivity in macadamia nut. The in-silico analysis revealed homology and linear epitope similarities to known allergens such as conglutin β allergen from lupin, Jug r2 vicilin allergens from walnut, Ara h3 11S globulin from peanut, small rubber particle protein Hev b3, hevein, enolase 2, HSP 70kDa Cla h4, Der f28 allergen, and methylglyoxalases. Label-free shotgun proteomics reveal valuable insights into the genetic and biological makeup of macadamia nut proteome and provide guidance on protein candidates with allergenic potential for further immunological investigation. Data are available via ProteomeXchange with identifier PXD015364.

Histone deacetylase interacts with a WRKY transcription factor to regulate the expression of the small rubber particle protein gene from Hevea brasiliensis

Small rubber particle protein (SRPP) is an abundant rubber particle-associated protein that serves as a co-factor in the biosynthesis of natural rubber in Hevea brasiliensis. However, knowledge of SRPP gene (HbSRPP) expression in transcriptional regulation remains limited. In this work, the yeast one-hybrid experiment was performed to obtain the transcription factors that regulated HbSRPP and further study the natural rubber biosynthesis at the transcriptional level. A group I-type WRKY transcription factor (designated as HbWRKY14) was obtained, and expression analysis results showed that HbWRKY14 was highly expressed in latex. Moreover, methyl jasmonate and ethrel upregulated HbWRKY14 expression. HbWRKY14 can bind to the promoter of HbSRPP and showed capability to suppress the HbSRPP promoter. Thus, HbWRKY14 functions as a negative regulator of HbSRPP expression. Furthermore, H. brasiliensis histone deacetylase (named HbHDA3) interacted with HbWRKY14 and such interaction might relieve the HbWRKY14-mediated HbSRPP transcription inhibition.

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.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz - role of cis-prenyltransferase-like 1 protein.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T.koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T.koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T.koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T.koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass Taraxacum Kok-saghyz.

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.