In situ mass spectrometry imaging reveals pesticide residues and key metabolic pathways throughout the entire cowpea growth process.

Cowpea plants, renowned for their high edibility, pose a significant risk of pesticide residue contamination. Elucidating the behavior of pesticide residues and their key metabolic pathways is critical for ensuring cowpea safety and human health. This study investigated the migration of pesticide residues and their key metabolic pathways in pods throughout the growth process of cowpea plants via in situ mass spectrometry. To this end, four pesticides--including systemic (thiram), and nonsystemic (fluopyram, pyriproxyfen, and cyromazine) pesticides--were selected. The results indicate the direct upward and downward transmission of pesticides in cowpea stems and pods. Systemic pesticides gradually migrate to the core of cowpea plants, whereas nonsystemic pesticides remain on the surface of cowpea peels. The migration rate is influenced by the cowpea maturity, logarithmic octanol-water partition coefficient (log Kow) value, and molecular weight of the pesticide. Further, 20 types of key metabolites related to glycolysis, tricarboxylic acid cycle, and flavonoid synthesis were found in cowpea pods after pesticide treatment. These findings afford insights into improving cowpea quality and ensuring the safe use of pesticides.

Brassinosteroids Regulate the Water Deficit and Latex Yield of Rubber Trees.

Brassinolide (BR) is an important plant hormone that regulates the growth and development of plants and the formation of yield. The yield and quality of latex from Hevea brasiliensis are regulated by phytohormones. The understanding of gene network regulation mechanism of latex formation in rubber trees is still very limited. In this research, the rubber tree variety CATAS73397 was selected to analyze the relationship between BR, water deficit resistance, and latex yield. The results showed that BR improves the vitality of rubber trees under water deficit by increasing the rate of photosynthesis, reducing the seepage of osmotic regulatory substances, increasing the synthesis of energy substances, and improving the antioxidant system. Furthermore, BR increased the yield and quality of latex by reducing the plugging index and elevating the lutoid bursting index without decreasing mercaptan, sucrose, and inorganic phosphorus. This was confirmed by an increased expression of genes related to latex flow. RNA-seq analysis further indicated that DEG encoded proteins were enriched in the MAPK signaling pathway, plant hormone signal transduction and sucrose metabolism. Phytohormone content displayed significant differences, in that trans-Zeatin, ethylene, salicylic acid, kinetin, and cytokinin were induced by BR, whereas auxin, abscisic acid, and gibberellin were not. In summary, the current research lays a foundation for comprehending the molecular mechanism of latex formation in rubber trees and explores the potential candidate genes involved in natural rubber biosynthesis to provide useful information for further research in relevant areas.

Comprehensive Analysis of BR Receptor Expression under Hormone Treatment in the Rubber Tree (Hevea brasiliensis Muell. Arg.).

Brassinosteroids (BRs) are important for plant growth and development, with BRI1 and BAK1 kinases playing an important role in BR signal transduction. Latex from rubber trees is crucial for industry, medicine and defense use. Therefore, it is beneficial to characterize and analyze HbBRI1 and HbBAK1 genes to improve the quality of the resources obtained from Hevea brasiliensis (rubber tree). Based on bioinformatics predictions and rubber tree database, five HbBRI1s with four HbBAK1s were identified and named HbBRI1~HbBRL3 and HbBAK1a~HbBAK1d, respectively, which were clustered in two groups. HbBRI1 genes, except for HbBRL3, exclusively contain introns, which is convenient for responding to external factors, whereas HbBAK1b/c/d contain 10 introns and 11 exons, and HbBAK1a contains eight introns. Multiple sequence analysis showed that HbBRI1s include typical domains of the BRI1 kinase, indicating that HbBRI1s belong to BRI1. HbBAK1s that possess LRR and STK_BAK1_like domains illustrate that HbBAK1s belong to the BAK1 kinase. BRI1 and BAK1 play an important role in regulating plant hormone signal transduction. Analysis of the cis-element of all HbBRI1 and HbBAK1 genes identified hormone response, light regulation and abiotic stress elements in the promoters of HbBRI1s and HbBAK1s. The results of tissue expression patterns indicate that HbBRL1/2/3/4 and HbBAK1a/b/c are highly expressed in the flower, especially HbBRL2-1. The expression of HbBRL3 is extremely high in the stem, and the expression of HbBAK1d is extremely high in the root. Expression profiles with different hormones show that HbBRI1 and HbBAK1 genes are extremely induced by different hormone stimulates. These results provide theoretical foundations for further research on the functions of BR receptors, especially in response to hormone signals in the rubber tree.

Visualization and metabolome for the migration and distribution behavior of pesticides residue in after-ripening of banana.

Exploring the behavior of pesticide residues in fruits is important for effectively applying pesticides and minimizing the risk of pesticide exposure to humans. However, most studies do not consider in situ visual analysis of residues and migration patterns in fresh fruit samples. We investigated the migration patterns of thiram, propamocarb, imidacloprid and pyraclostrobin in fresh bananas based on ambient mass spectrometry imaging, metabolome and transcriptome analysis. The systemic pesticides entered via lateral penetration and vertical migration over time, which began to internally migrate to the inner core after 6 h. The non-systemic pesticide thiram did not enter the interior of the bananas, and remained only in the peel. The transportation rate of the pesticides increased with the decrease of water-octanol partition coefficient and the relative molecular mass. Moreover, the pesticide migrated fast with the increase of banana ripeness. The pesticides significantly enhanced pyruvate kinase, NADP-dependent malic enzyme, and malate synthase activities in the banana peels through carbohydrate metabolism. The banana pulp was also protected against the external toxicity of pesticides by the ascorbate-glutathione cycle. These results can provide guidelines for the appropriate application of pesticides and their safety evaluation.

ANGPTL8 accelerates liver fibrosis mediated by HFD-induced inflammatory activity via LILRB2/ERK signaling pathways.

INTRODUCTION: High calorie intake is known to induce nonalcoholic fatty liver disease (NAFLD) by promoting chronic inflammation. However, the mechanisms are poorly understood. OBJECTIVES: This study examined the roles of ANGPTL8 in the regulation of NAFLD-associated liver fibrosis progression induced by high fat diet (HFD)-mediated inflammation. METHODS: The ANGPTL8 concentration was measured in serum samples from liver cancer and liver cirrhosis patients. ANGPTL8 knockout(KO) mice were used to induce disease models (HFD, HFHC and CCL4) followed by pathological staining, western blot and immunohistochemistry. Hydrodynamic injection of an adeno-associated virus 8 (AAV8) was used to establish a model for restoring ANGPTL8 expression specifically in ANGPTL8 KO mice livers. RNA-sequencing, protein array, Co-IP, etc. were used to study ANGPTL8's mechanisms in regulating liver fibrosis progression, and drug screening was used to identify an effective inhibitor of ANGPTL8 expression. RESULTS: ANGPTL8 level is associated with liver fibrogenesis in both cirrhosis and hepatocellular carcinoma patients. Mouse studies demonstrated that ANGPTL8 deficiency suppresses HFD-stimulated inflammatory activity, hepatic steatosis and liver fibrosis. The AAV-mediated restoration of liver ANGPTL8 expression indicated that liver-derived ANGPTL8 accelerates HFD-induced liver fibrosis. Liver-derived ANGPTL8, as a proinflammatory factor, activates HSCs (hepatic stellate cells) by interacting with the LILRB2 receptor to induce ERK signaling and increase the expression of genes that promote liver fibrosis. The FDA-approved anti-diabetic drug metformin, an ANGPTL8 inhibitor, inhibited HFD-induced liver fibrosis in vivo. CONCLUSIONS: Our data support that ANGPTL8 is a proinflammatory factor that accelerates NAFLD-associated liver fibrosis induced by HFD. The serum ANGPTL8 level may be a potential and specific diagnostic marker for liver fibrosis, and targeting ANGPTL8 holds great promise for developing innovative therapies to treat NAFLD-associated liver fibrosis.

Genome-wide identification and response stress expression analysis of the BES1 family in rubber tree (Hevea brasiliensis Muell. Arg.).

Brassinolide (BR) plays an important role in plant growth, development, and the adaptation adversity process. Moreover, BRI1-EMS-SUPPRESSOR 1 (BES1) genes are crucial transcription factors (TFs) in the BR signaling pathway. To realize the function of HbBES1 family is helpful to improve genetic resources for rubber tree breeding. Based on the rubber tree database, we used bioinformatics to characterize physicochemical properties, gene structure, cis-elements, and expression patterns. These results indicated that there were nine BES1 members in rubber tree, which we named HbBES1-1 to HbBES1-9 and divided into two groups (I and II) based on their genetic relationships. HbBES1 genes in the same group shared similar gene structures and motifs. Cis-acting element analysis showed that the promoter sequences of HbBES1 genes contained many regulator elements that were related to hormone and stress, indicating that HbBES1 genes might be involved in the regulation of hormone and stress signal pathways. Our analysis of tissue specificity revealed that all of the nine HbBES1 members expressed highly in branches. Gene expression profiles under different hormone treatments showed that the HbBES1 gene family was induced to varying degrees under different hormones, HbBES1-3 and HbBES1-9 were extremely induced by ethylene (ETH). These results lay the foundation for further exploration of the molecular mechanism of the BES1 gene family, especially HbBES1-3 and HbBES1-9, regulating plant stress tolerance in rubber tree.

Synthetic mRNA-based gene therapy for glioblastoma: TRAIL-mRNA synergistically enhances PTEN-mRNA-based therapy.

Glioblastoma (GBM) is characterized as having high molecular heterogeneity and complexity, which can be well revealed by genomic study. A truly effective treatment for GBM should flexibly address its heterogeneities, complexity, and strong drug resistance. This study was performed to explore the effectiveness of an mRNA-based therapeutic strategy using in vitro synthesized PTEN-mRNA and TRAIL-mRNA in tumor cells derived from PTEN-deletion patients. The PTEN gene alterations were revealed by whole-exome sequencing of three paired clinical GBMs and selected as the therapy target. Patient-derived primary glioblastoma stem cells (GBM2) and a DBTRG-cell-derived xenograft were used to detect mRNA's cytotoxicity in vitro and tumor suppression in vivo. Following the successful in vitro synthesis of PTEN-mRNA and TRAIL-mRNA, the combinational treatment of PTEN-mRNA and TRAIL-mRNA significantly suppressed tumor growth compared with treatment with PBS (96.4%), PTEN-mRNA (89.7%), and TRAIL-mRNA (84.5%). The combinational application of PTEN-mRNA and TRAIL-mRNA showed synergistic inhibition of tumor growth, and the JNK pathway might be the major mechanism involved. This study provided a basis for an mRNA-based therapeutic strategy to be developed into an effective patient-tailored treatment for GBM.

Intracranial delivery of synthetic mRNA to suppress glioblastoma.

Owing to messenger RNA's unique biological advantages, it has received increasing attention to be used as a therapeutic, known as mRNA-based gene therapy. It is critical to have an ideal strategy of mRNA gene therapy for glioma, which grows in a special environment. In the present study, we screened out a safe and efficient transfection reagent for intracranial delivery of synthetic mRNA in mouse brain. First, in order to analyze the effect of different transfection reagents on the intracranial delivery of mRNA, the synthetic luciferase mRNA was wrapped with two different transfection reagents and microinjected into the brain at the fixed point. The expression status of delivered mRNA was monitored by a small animal imaging system. The possible reagent-induced biological toxicity was evaluated by behavioral and blood biochemical measurements. Then, to test the therapeutic effect of our intracranial delivery mRNA model on glioma, synthetic modified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA was used as an example of therapeutic application. This model demonstrated that synthetic mRNA could be successfully delivered into the brain using commercially available transfection reagents, and TransIT-mRNA showed better results than in vivo-jetPEI kit. This model can be applied in precise targeting and personalized gene therapy of glioma.

Identification of cis conformation natural rubber and proteins in Ficus altissima Blume latex.

Ficus altissima Blume, a horticultural plant in tropical and warm subtropical regions, can produce natural rubber with comparable molecular weight to the Hevea brasiliensis rubber. The F. altissima latex has an acidic pH (about 4.89). The rubber particle size distribution is a unimodal profile, and the peak frequency is at a size of 4.5 µm. The natural rubber of F. altissima was determined to be a cis conformation via 13C NMR. The Mp (molecular weight of the peak maxima) of the deproteinized F. altissima rubber was 9.34 × 105 Da. LC-MS was used to identify the proteins of rubber particles and serum. The most abundant protein of the creamy rubber particle layer is an acid phosphatase, while the most abundant proteins of serum were an (R)-mandelonitrilelyase and a polygalacturonase inhibitor. Pharmaceutical proteins (ficins) or enzymes related to the biosynthesis of natural medicines (a cannabidiolic acid synthase and two lupeol synthase) were identified in F. altissima latex. The data of this study may be helpful for research on the functions of latex in latex-borne plants and the biosynthesis mechanism of natural rubber.

Case Report: Primary Pulmonary Angiosarcoma With Brain Metastasis.

Primary pulmonary angiosarcoma (PPA) is a rare malignant vascular tumor, of which early diagnosis is challenging due to lack of specific clinical manifestations and a low level of suspicion. Here, we report a case of PPA presented with advanced brain metastasis. A 21-year-old patient with 1 week history of headache and mild cough was hospitalized for a head injury. Head MRI showed multiple intracranial lesions with brain edema. Chest CT displayed bilateral pulmonary infiltrates with mediastinal lymph node enlargement. After 2 months of anti-tuberculosis treatment, the patient was readmitted for persistent headache and cough with occasional hemosputum along with worsening pulmonary and intracranial lesions. Despite seizure prophylaxis and control of intracranial pressure and brain edema, his symptoms progressively aggravated, accompanied by cough with bloody sputum, frequent epileptic seizures, and hypotension. He eventually developed coma and died within 3 months of onset of symptoms. An autopsy confirmed PPA with brain metastasis.

Prognostic and Predictive Value of an Immunoscore Signature in Glioblastoma Multiform.

BACKGROUND: Although increasing evidence shows that immune infiltration plays an essential role in glioblastoma (GBM), current prognostic indicators do not accurately represent the risk of immune cells infiltration in patients. It is therefore critical to identify new prognostic markers for GBM. Here, we investigated the effectiveness of using immunoscore to improve risk stratification and prediction of prognosis in GBM patients receiving chemotherapy. METHODS: Using mRNA microarrays and CIBERSORT, we analyzed 22 types of immune cell fractions in 517 GBM samples and characterized an immunoscore using the least absolute shrinkage and selection operator (LASSO) Cox regression model based on the fraction of immune cell types and patients' overall survival. The prognostic and predictive accuracy of immunoscore was verified in the validation cohort and the entire cohort. RESULTS: Using the LASSO model, an immunoscore was developed to classify patients into High and Low immunoscore groups in the training cohort (P < 0.0001) based on the fraction of eight immune cell types. The immunoscore performance was validated in the validation cohort (P < 0.0001) and the entire cohort (P < 0.0001). Furthermore, a nomogram comprising age, IDH1 status, and immunoscore was generated to predict one- and three-year survival rates in the training cohort. The predictive value of the immunoscore was also confirmed in the validation cohort and the entire cohort (C-index: 0.66, 0.67, and 0.68, respectively). In addition, we concluded that patients in the low-immunoscore group may benefit from adjuvant chemotherapy for GBM. CONCLUSION: Immunoscore, an immune-infiltration-based signature, is a reliable prognostic and predictive tool for GBM.

An Efficient and Footprint-Free Protocol for the Transdifferentiation of Hepatocytes Into Insulin-Producing Cells With IVT mRNAs.

BACKGROUND: Direct transdifferentiation of adult somatic cells into insulin-producing cells (IPCs) is a promising approach for cell-based therapies for type 1 diabetes mellitus. Liver cells are an ideal source for generating IPCs because they have regenerative ability and a developmental process similar to that of the pancreas. Pancreas versus liver fate is regulated by TALE homeoprotein (TGIF2) during development. Here, we wanted to investigate whether TGIF2 could enhance the efficiency of transdifferentiation of hepatocytes into IPCs induced by three pancreatic transcription factors (pTFs), i.e., Pdx1, NeuroD, and Mafa, which are crucial for pancreatic development in the embryo. METHODS: The in vitro transcribed (IVT) mRNAs of TGIF2 and the three pTFs were synthesized in vitro and sequentially supplemented in hepatocytes. On day 6, the expression of transcription factors was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and insulin expression was detected by immunofluorescence. Glucose-stimulated insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). The key genes controlling cell polarity and the Wnt/PCP signaling pathway were assayed by qRT-PCR, and the level of JNK protein phosphorylation, which regulates the Wnt/PCP signaling pathway, was detected by western blotting. RESULTS: IVT mRNAs could be efficiently transfected into hepatocytes. Quantitative real-time polymerase chain reaction results revealed that compared with ectopic expression of the three pTFs alone, ectopic expression of the three pTFs plus TGIF2 could strongly reduce hepatic gene expression and subsequently improve the induction of a set of pancreatic genes. Immunofluorescence analysis showed that TGIF2 expression could double the transdifferentiation yield; 30% of the cells were insulin positive if induced by TGIF2 plus the 3 pTFs, while only 15% of the cells were insulin positive if induced by the three pTFs alone. ELISA analysis confirmed that glucose-stimulated insulin secretion was less efficient after transfection with the three pTFs alone. The differentiated cells derived from the addition of TGIF2 mRNA could form islet-like clusters. By contrast, the cells differentiated with the three pTFs did not form clusters under the same conditions. Tgif2 induced transdifferentiation more efficiently by remodeling the expression of genes in the Wnt/PCP pathway. Overexpression of TGIF2 in hepatocytes could activate the expression of key genes controlling cell polarity and genes in the Wnt/PCP signaling pathway, increasing the level of JNK protein phosphorylation. CONCLUSIONS: Our study established a novel footprint-free protocol for efficient transdifferentiation of hepatocytes into IPCs using IVT mRNAs of TGIF2 and 3 pTFs, which paved the way toward a clinical application.

Isoliquiritigenin inhibits TGF-ß1-induced fibrogenesis through activating autophagy via PI3K/AKT/mTOR pathway in MRC-5 cells.

Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-ß1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-ß1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-ß1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-ß1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-ß1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.

ANGPTL8 enhances insulin sensitivity by directly activating insulin-mediated AKT phosphorylation.

ANGPTL8 is a 22-KDa protein in the angiopoietin-like family. It is a liver-derived hormone that dynamically regulates glucose metabolism after refeeding. The mechanism of its regulation of glucose metabolism is unclear. We analyzed the effect of ANGPTL8 overexpression on glucose tolerance in the mouse liver by tail vein hydrodynamic transfection. The mechanism of ANGPTL8 improving insulin sensitivity was analyzed by the overexpression or knockdown of ANGPTL8 in mouse primary hepatocytes through in vitro synthetic mRNA and siRNA technology. The key site of ANGPTL8 protein regulating this signal pathway was screened by DNA point mutation and fragment truncation. The results showed that ANGPTL8 may directly regulate AKT protein phosphorylation in the insulin-mediated PI3K/AKT signaling pathway to improve insulin sensitivity. Ser94 and Thr98 are the key sites of ANGPTL8 protein in activating AKT protein phosphorylation. Present results indicate that ANGPTL8 may be a potential new agent to reduce postprandial blood glucose.

Therapeutic Prospects of mRNA-Based Gene Therapy for Glioblastoma.

The treatment of glioblastoma has been a big challenge for decades in the oncological field mainly owing to its unique biological characteristics, such as high heterogeneity, diffusing invasiveness, and capacity to resist conventional therapies. The mRNA-based therapeutic modality holds many superior features, including easy manipulation, rapid and transient expression, and adaptive convertibility without mutagenesis, which are suitable for dealing with glioblastoma's complexity and variability. Synthetic anticancer mRNAs carried by various vehicles act as the ultimate attackers of the tumor across biological barriers. In this modality, specifically targeted glioblastoma treatment can be guaranteed by adding targeting molecules at certain levels. The choice of mRNA-bearing vehicle and administration method is a fully patient-tailored selection. This review covers the advantages and possible limitations of mRNA-based gene therapy, the in vitro synthesis of mRNA, the feasible methods for synthetic mRNA delivery and clinical therapeutic prospects of mRNA-based gene therapy for glioblastoma.

Identification of a Specific Gene Module for Predicting Prognosis in Glioblastoma Patients.

Introduction: Glioblastoma (GBM) is the most common and malignant variant of intrinsic glial brain tumors. The poor prognosis of GBM has not significantly improved despite the development of innovative diagnostic methods and new therapies. Therefore, further understanding the molecular mechanism that underlies the aggressive behavior of GBM and the identification of appropriate prognostic markers and therapeutic targets is necessary to allow early diagnosis, to develop appropriate therapies and to improve prognoses. Methods: We used a weighted gene co-expression network analysis (WGCNA) to construct a gene co-expression network with 524 glioblastoma samples from The Cancer Genome Atlas (TCGA). A risk score was then constructed based on four module genes and the patients' overall survival (OS) rate. The prognostic and predictive accuracy of the risk score were verified in the GSE16011 cohort and the REMBRANDT cohort. Results: We identified a gene module (the green module) related to prognosis. Then, multivariate Cox analysis was performed on 4 hub genes to construct a Cox proportional hazards regression model from 524 glioblastoma patients. A risk score for predicting survival time was calculated with the following formula based on the top four genes in the green module: risk score = (0.00889 × EXPCLEC5A) + (0.0681 × EXPFMOD) + (0.1724 × EXPFKBP9) + (0.1557 × EXPLGALS8). The 5-year survival rate of the high-risk group (survival rate: 2.7%, 95% CI: 1.2-6.3%) was significantly lower than that of the low-risk group (survival rate: 8.8%, 95% CI: 5.5-14.1%). Conclusions: This study demonstrated the potential application of a WGCNA-based gene prognostic model for predicting the survival outcome of glioblastoma patients.

Role of caprin-1 in carcinogenesis.

RNA-binding proteins serve an essential role in post-transcriptional gene regulation. Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is an RNA-binding protein that participates in the regulation of cell cycle control-associated genes. Caprin-1 acts alone or in combination with other RNA-binding proteins, such as RasGAP SH3-domain-binding protein 1 and fragile X mental retardation protein. In the tumorigenesis process, caprin-1 primarily functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 holds the potential to be used as a biomarker and a target for the development of novel therapeutics. The present review describes this newly identified putative oncogenic protein and its possible impact on tumorigenesis.

Involvement of HbMC1-mediated cell death in tapping panel dryness of rubber tree (Hevea brasiliensis).

Tapping panel dryness (TPD) causes a significant reduction in the latex yield of rubber tree (Hevea brasiliensis Muell. Arg.). It is reported that TPD is a typical programmed cell death (PCD) process. Although PCD plays a vital role in TPD occurrence, there is a lack of detailed and systematic study. Metacaspases are key regulators of diverse PCD in plants. Based on our previous result that HbMC1 was associated with TPD, we further elucidate the roles of HbMC1 on rubber tree TPD in this study. HbMC1 was up-regulated by TPD-inducing factors including wounding, ethephon and H2O2. Moreover, the expression level of HbMC1 was increased along with TPD severity in rubber tree, suggesting a positive correlation between HbMC1 expression and TPD severity. To investigate its biological function, HbMC1 was overexpressed in yeast (Saccharomyces cerevisiae) and tobacco (Nicotiana benthamiana). Transgenic yeast and tobacco overexpressing HbMC1 showed growth retardation compared with controls under H2O2-induced oxidative stress. In addition, overexpression of HbMC1 in yeast and tobacco reduced cell survival after high-concentration H2O2 treatment and resulted in enhanced H2O2-induced leaf cell death, respectively. A total of 11 proteins, rbcL, TM9SF2-like, COX3, ATP9, DRP, HbREF/Hevb1, MSSP2-like, SRC2, GATL8, CIPK14-like and STK, were identified and confirmed to interact with HbMC1 by yeast two-hybrid screening and co-transformation in yeast. The 11 proteins mentioned above are associated with many biological processes, including rubber biosynthesis, stress response, autophagy, carbohydrate metabolism, signal transduction, etc. Taken together, our results suggest that HbMC1-mediated PCD plays an important role in rubber tree TPD, and the identified HbMC1-interacting proteins provide valuable information for further understanding the molecular mechanism of HbMC1-mediated TPD in rubber tree.

Transcriptomic analyses reveal molecular mechanisms underlying growth heterosis and weakness of rubber tree seedlings.

BACKGROUND: Breeding rubber tree seedling with growth heterosis is vital for natural rubber production. It is the prerequisites for effectively utilizing growth heterosis to elucidate its molecular mechanisms, but the molecular mechanisms remain poorly understood in rubber tree. To elucidate seedling growth heterosis, we conducted comparative transcriptomic analyses between the two hybrids and their parents. RESULTS: By identifying and comparing differently expressed genes (DEGs), we found that the hybrids (BT 3410 and WC 11) show significantly differential expression profiles from their parents (PR 107 and RRIM 600). In BT 3410-parent triad, 1092 (49.95%) and 1094 (50.05%) DEGs indicated clear underdominance or overdominance, respectively. Whereas in WC 11-parent triad, most DEGs (78.2%, 721) showed low- or high-parent dominance; 160 (17.35%) exhibited expression patterns that are not statistically distinguishable from additivity, and 8 (0.87%) and 33 (3.58%) DEGs exhibited underdominance and overdominance, respectively. Furthermore, some biological processes are differentially regulated between two hybrids. Interestingly, the pathway in response to stimulus is significantly downregulated and metabolic pathways are more highly regulated in BT 3410. CONCLUSIONS: Taken together, the genotypes, transcriptomes and biological pathways (especially, carbohydrate metabolism) are highly divergent between two hybrids, which may be associated with growth heterosis and weakness. Analyzing gene action models in hybrid-parent triads, we propose that overdominance may play important roles on growth heterosis, whereas dominance on hybrid weakness in rubber tree seedlings. These findings bring new insights into our understanding of growth heterosis of rubber tree seedling.

Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).