Wei-Min Tian

A method for protein extraction from different subcellular fractions of laticifer latex in Hevea brasiliensis compatible with 2-DE and MS

BackgroundProteomic analysis of laticifer latex in Hevea brasiliensis has been received more significant attentions. However, the sticky and viscous characteristic of rubber latex as cytoplasm of laticifer cells and the complication of laticifer latex membrane systems has made it challenge to isolate high-quality proteins for 2-DE and MS.ResultsBased on the reported Borax/PVPP/Phenol (BPP) protocol, we developed an efficient method for protein preparation from different latex subcellular fractions and constructed high-resolution reference 2-DE maps. The obtained proteins from both total latex and C-serum fraction with this protocol generate more than one thousand protein spots and several hundreds of protein spots from rubber particles as well as lutoid fraction and its membranes on the CBB stained 2-DE gels. The identification of 13 representative proteins on 2-DE gels by MALDI TOF/TOF MS/MS suggested that this method is compatible with MS.ConclusionThe proteins extracted by this method are compatible with 2-DE and MS. This protein preparation protocol is expected to be used in future comparative proteomic analysis for natural rubber latex.

Comparative Proteomics of Primary and Secondary Lutoids Reveals that Chitinase and Glucanase Play a Crucial Combined Role in Rubber Particle Aggregation in Hevea brasiliensis

Lutoids are specific vacuole-based organelles within the latex-producing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation.

Cloning and characterization of HbJAZ1 from the laticifer cells in rubber tree (Hevea brasiliensis Muell. Arg.)

Although jasmonate (JA) signaling has recently been well elucidated in Arabidopsis thaliana, the JASMONATE ZIM domain proteins (JAZs) are suggested to have an important role in regulating specific JA responses in plants, not much is known about how the specific JA responses are regulated in plants. Laticifer is the sole site of natural rubber biosynthesis in rubber tree. Here, the full-length cDNA and promoter region of HbJAZ1 gene were cloned and characterized from the laticifer cells in rubber tree by a combination of molecular techniques and bioinformatic analyses. The cDNA was 1,442-bp long containing an 852-bp open reading frame (ORF) encoding a putative protein of 284 amino acids. The predicted HbJAZ1 protein possessed the conservative ZIM and Jas domains, and was homologous to the JAZ members from Arabidopsis and the JAZ homologs from other plant species. The 976-bp promoter region of HbJAZ1 was isolated using PCR-based DNA Walking. TATA box and other core configurations as well as some special cis-acting elements involving in gibberellin-, jasmonic acid- and wound-responsiveness were found within this region. Transcription pattern analysis revealed that HbJAZ expression was strongly up-regulated by tapping and mechanical wounding, but not by ethrel application. Characterization of HbJAZ1 will attribute to understand JA signaling in laticifers, a specialized tissue for natural rubber biosynthesis in rubber trees.

Mechanical wounding-induced laticifer differentiation in rubber tree: An indicative role of dehydration, hydrogen peroxide, and jasmonates

The secondary laticifer in the secondary phloem of rubber tree are a specific tissue differentiating from vascular cambia. The number of the secondary laticifers is closely related to the rubber productivity of Hevea. Factors involved in the mechanical wounding-induced laticifer differentiation were analyzed by using paraffin section, gas chromatography-mass spectrometry (GC-MS), and Northern-blot techniques. Dehydration of the wounded bark tissues triggered a burst of hydrogen peroxide, abscisic acid, and jasmonates and up-regulated the expression of HbAOSa, which was associated with the secondary laticifer differentiation strictly limited to the wounded area. Application of exogenous hydrogen peroxide, methyl jasmonate, and polyethylene glycol 6000 (PEG6000) could induce the secondary laticifer differentiation, respectively. Moreover, 6-Benzylaminopurine, a synthetic cytokinin, enhanced the methyl jasmonate-induced secondary laticifer differentiation. However, the dehydration-induced secondary laticifer differentiation was inhibited by exogenous abscisic acid. Diphenyleneiodonium chloride (DPI), a specific inhibitor of NADPH oxidase, was effective in inhibiting the accumulation of hydrogen peroxide as well as of jasmonates upon dehydration. It blocked the dehydration-induced but not the methyl jasmonate-induced secondary laticifer differentiation. The results suggested a stress signal pathway mediating the wound-induced secondary laticifer differentiation in rubber tree.

Gene expression analysis and SNP/InDel discovery to investigate yield heterosis of two rubber tree F1 hybrids.

As an important industrial material, natural rubber is mainly harvested from the rubber tree. Rubber tree breeding is inefficient, expensive and time-consuming, whereas marker-assisted selection is a feasible method for early selection of high-yield hybrids. We thus sequenced and analyzed the transcriptomes of two parent rubber trees (RRIM 600 and PR 107) and their most productive hybrids (RY 7-33-97 and RY 7-20-59) to understand their gene expression patterns and genetic variations including single nucleotide polymorphisms (SNPs) and small insertions/deletions (InDels). We discovered >31,000 genetic variations in 112,702 assembled unigenes. Our results showed that the higher yield in F1 hybrids was positively associated with their higher genome heterozygosity, which was further confirmed by genotyping 10 SNPs in 20 other varieties. We also showed that RY 7-33-97 and RY 7-20-59 were genetically closer to RRIM 600 and PR 107, respectively, in agreement with both their phenotypic similarities and gene expression profiles. After identifying ethylene- and jasmonic acid–responsive genes at the transcription level, we compared and analyzed the genetic variations underlying rubber biosynthesis and the jasmonic acid and ethylene pathways in detail. Our results suggest that genome-wide genetic variations play a substantive role in maintaining rubber tree heterosis.

Molecular and biochemical characterization of a cyanogenic β-glucosidase in the inner bark tissues of rubber tree (Hevea brasiliensis Muell. Arg.)

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of β-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both β-glucosidase and linamarase and was thus characterized as a cyanogenic β-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic β-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.

Characteristics of protein-storing cells associated with a 67 kDa protein in Hevea brasiliensis

Abstract The protein-storing cells (PSCs) in Hevea brasiliensis were studied by using light- and electron-microscopy and SDS-PAGE. The cells were found in stem and root where secondary phloem was developed. They are a special kind of phloem parenchyma cell which accumulate in their central vacuoles large amounts of protein, fibril-like under an electron microscope, and have few plastids with very small starch grains. Their distribution is strictly restricted to the secondary phloem axial system where they exactly sequestered in functional phloem or slightly over it. A 67 kDa protein was always found in the tissues where the PSCs were observed. During the first seasonal growth flush, the 67 kDa protein in the terminal branchlet exhibits marked quantitative fluctuation which is consistent with the change of the vacuole protein inclusion of the PSCs in the branchlet. These facts suggested that the 67 kDa protein might be the major part of the vacuole protein of the PSCs. Considering the differences between the PSCs in Hevea and the PSCs in the other trees studied, we define two types of PSCs: Hevea-type, which are the cells specialized for protein storage and Populus-type, which are ordinary parenchyma cells accumulating protein and starch.

Transcriptome Analysis of the Signalling Networks in Coronatine-Induced Secondary Laticifer Differentiation from Vascular Cambia in Rubber Trees

The secondary laticifer in rubber tree (Hevea brasiliensis Muell. Arg.) is a specific tissue within the secondary phloem. This tissue differentiates from the vascular cambia, and its function is natural rubber biosynthesis and storage. Given that jasmonates play a pivotal role in secondary laticifer differentiation, we established an experimental system with jasmonate (JA) mimic coronatine (COR) for studying the secondary laticifer differentiation: in this system, differentiation occurs within five days of the treatment of epicormic shoots with COR. In the present study, the experimental system was used to perform transcriptome sequencing and gene expression analysis. A total of 67,873 unigenes were assembled, and 50,548 unigenes were mapped at least in one public database. Of these being annotated unigenes, 15,780 unigenes were differentially expressed early after COR treatment, and 19,824 unigenes were differentially expressed late after COR treatment. At the early stage, 8,646 unigenes were up-regulated, while 7,134 unigenes were down-regulated. At the late stage, the numbers of up- and down-regulated unigenes were 7,711 and 12,113, respectively. The annotation data and gene expression analysis of the differentially expressed unigenes suggest that JA-mediated signalling, Ca2+ signal transduction and the CLAVATA-MAPK-WOX signalling pathway may be involved in regulating secondary laticifer differentiation in rubber trees.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis of the Gene Expression in Laticifers on the Basis of Latex Flow in Rubber Tree (Hevea brasiliensis Muell. Arg.)

Latex exploitation-caused latex flow is effective in enhancing latex regeneration in laticifer cells of rubber tree. It should be suitable for screening appropriate reference gene for analysis of the expression of latex regeneration-related genes by quantitative real-time PCR (qRT-PCR). In the present study, the expression stability of 23 candidate reference genes was evaluated on the basis of latex flow by using geNorm and NormFinder algorithms. Ubiquitin-protein ligase 2a (UBC2a) and ubiquitin-protein ligase 2b (UBC2b) were the two most stable genes among the selected candidate references in rubber tree clones with differential duration of latex flow. The two genes were also high-ranked in previous reference gene screening across different tissues and experimental conditions. By contrast, the transcripts of latex regeneration-related genes fluctuated significantly during latex flow. The results suggest that screening reference gene during latex flow should be an efficient and effective clue for selection of reference genes in qRT-PCR.

A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles

The extraction of high‐purity proteins from the washing solution (WS) of rubber particles (also termed latex‐producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high‐purity WS proteins. We improved our current borax and phenol‐based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol‐based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high‐quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP‐extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.