The formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding.

BACKGROUND: Although the wound response of plants has been extensively studied, little is known of the rapid occlusion of wounded cell itself. The laticifer in rubber tree is a specific type of tissue for natural rubber biosynthesis and storage. In natural rubber production, tapping is used to harvest the latex which flows out from the severed laticifer in the bark. Therefore, study of the rapid wound-occlusion of severed laticifer cells is important for understanding the rubber tree being protected from the continuously mechanical wounding. RESULTS: Using cytological and biochemical techniques, we revealed a biochemical mechanism for the rapid occlusion of severed laticifer cells. A protein-network appeared rapidly after tapping and accumulated gradually along with the latex loss at the severed site of laticifer cells. Triple immunofluorescence histochemical localization showed that the primary components of the protein-network were chitinase, ß-1,3-glucanase and hevein together with pro-hevein (ProH) and its carboxyl-terminal part. Molecular sieve chromatography showed that the physical interactions among these proteins occurred under the condition of neutral pH. The interaction of ß-1,3-glucanase respectively with hevein, chitinase and ProH was testified by surface plasmon resonance (SPR). The interaction between actin and ß-1,3-glucanase out of the protein inclusions of lutoids was revealed by pull-down. This interaction was pharmacologically verified by cytochalasin B-caused significant prolongation of the duration of latex flow in the field. CONCLUSIONS: The formation of protein-network by interactions of the proteins with anti-pathogen activity released from lutoids and accumulation of protein-network by binding to the cytoskeleton are crucial for the rapid occlusion of laticifer cells in rubber tree. The protein-network at the wounded site of laticifer cells provides not only a physical barrier but also a biochemical barrier to protect the wounded laticifer cells from pathogen invasion.

Jasmonate signalling in the regulation of rubber biosynthesis in laticifer cells of rubber tree, Hevea brasiliensis.

Rubber trees are the world's major source of natural rubber. Rubber-containing latex is obtained from the laticifer cells of the rubber tree (Hevea brasiliensis) via regular tapping. Rubber biosynthesis is a typical isoprenoid metabolic process in the laticifer cells; however, little is known about the positive feedback regulation caused by the loss of latex that occurs through tapping. In this study, we demonstrate the crucial role of jasmonate signalling in this feedback regulation. The endogenous levels of jasmonate, the expression levels of rubber biosynthesis-related genes, and the efficiency of in vitro rubber biosynthesis were found to be significantly higher in laticifer cells of regularly tapped trees than those of virgin (i.e. untapped) trees. Application of methyl jasmonate had similar effects to latex harvesting in up-regulating the rubber biosynthesis-related genes and enhancing rubber biosynthesis. The specific jasmonate signalling module in laticifer cells was identified as COI1-JAZ3-MYC2. Its activation was associated with enhanced rubber biosynthesis via up-regulation of the expression of a farnesyl pyrophosphate synthase gene and a small rubber particle protein gene. The increase in the corresponding proteins, especially that of farnesyl pyrophosphate synthase, probably contributes to the increased efficiency of rubber biosynthesis. To our knowledge, this is the first study to reveal a jasmonate signalling pathway in the regulation of rubber biosynthesis in laticifer cells. The identification of the specific jasmonate signalling module in the laticifer cells of the rubber tree may provide a basis for genetic improvement of rubber yield potential.

Cribiform and intraductal carcinoma in hereditary prostate cancer: clinical and pathological analysis of 20 cases.

Cribiform and intraductal carcinoma are patterns of aggressive prostate carcinoma. This study investigated the clinical and pathological features of hereditary prostate cancer. Twenty cases of hereditary prostate cancer from 11 family lines treated at the First Affiliated Hospital of Zhejiang University School of Medicine between 2016-2022 were included to summarize the clinical and pathological features by analyzing clinical information including follow up the survival of the patients and pathological features. Of the 20 hereditary prostate cancer cases, 19 were radical prostate specimens and 1 was a biopsy specimen. The mean age at diagnosis of the patients was 67.55 years and the mean PSA was 15.44 ng/ml, of which 10 cases had PSA ≥ 10 ng/ml and 5 cases had PSA ≥ 20 ng/ml. Of the 19 radical prostate specimens, Gleason cribriform pattern (Gleason grade 4) of PCa is observed in 15 cases (78.95%), and intraductal carcinoma, usually a rare form, is seen in 9 cases (47.3%). Two cases demonstrated pelvic lymph node metastasis, and 7 cases (35%) belonged to high-risk or very high-risk PCa. One case (5.26%) showed partial deletion of expression of RB1, and 13 cases (68.42%) showed deletion of expression of PTEN. Follow-up was 4-90 months, 2 cases had biochemical recurrence and 1 case died from prostate cancer. The mean age at diagnosis of this group of patients with hereditary prostate cancer was 67.55 years, the mean preoperative PSA was 15.44 ng/ml, and their histomorphology was characterized by a high percentage of intraductal carcinoma and cribriform pattern of the prostate.

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.

Genomic insight into domestication of rubber tree.

Understanding the genetic basis of rubber tree (Hevea brasiliensis) domestication is crucial for further improving natural rubber production to meet its increasing demand worldwide. Here we provide a high-quality H. brasiliensis genome assembly (1.58 Gb, contig N50 of 11.21 megabases), present a map of genome variations by resequencing 335 accessions and reveal domestication-related molecular signals and a major domestication trait, the higher number of laticifer rings. We further show that HbPSK5, encoding the small-peptide hormone phytosulfokine (PSK), is a key domestication gene and closely correlated with the major domestication trait. The transcriptional activation of HbPSK5 by myelocytomatosis (MYC) members links PSK signaling to jasmonates in regulating the laticifer differentiation in rubber tree. Heterologous overexpression of HbPSK5 in Russian dandelion (Taraxacum kok-saghyz) can increase rubber content by promoting laticifer formation. Our results provide an insight into target genes for improving rubber tree and accelerating the domestication of other rubber-producing plants.

Detection of cucumber mosaic virus isolates from banana by one-step reverse transcription loop-mediated isothermal amplification.

Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials.

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

BACKGROUND: Proteomic 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. RESULTS: Based 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. CONCLUSION: The 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.

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree.

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense.

Comparative transcriptome analysis reveals phytohormone signalings, heat shock module and ROS scavenger mediate the cold-tolerance of rubber tree.

Two contrasting cold response rubber tree clones, the cold-resistant '93-114' and cold-sensitive 'Reken501', were subject to a global transcriptome response assessing via high-throughput RNA-seq technique and comprehensive bioinformatics analysis using the referenced rubber tree genome with the purpose of exploring the potential molecular cues underlying the tolerance of rubber trees to cold stress. As a result, a total of 1919 genes had significantly higher expression, while 2929 genes had significantly lower expression in '93-114' than in 'Reken501' without cold stress. Upon cold stress, the numbers of genes with significantly higher expression decreased to 1501 at 1 h treatment and to 1285 at 24 h treatment in '93-114' than that of 'Reken501', conversely, the numbers of genes with significantly lower expression increased to 7567 at 1 h treatment and to 5482 at 24 h treatment. Functional annotation of the differentially expressed genes between '93-114' and 'Reken501' suggests that down-regulation of auxin and ethylene signaling and activation of heat shock module and ROS scavengers is a primary strategy for H. brasiliensis to cope with cold stress. Our identified vital differentially expressed genes may be beneficial for elucidation of the molecular mechanisms underlying cold tolerance and for genetic improvement of H. brasiliensis clones.

Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity.

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.

Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensis Muell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum.

Ethrel is the most effective stimuli in prolonging the latex flow that consequently increases yield per tapping. This effect is largely ascribed to the enhanced lutoid stability, which is associated with the decreased release of initiators of rubber particle (RP) aggregation from lutoid bursting. However, the increase in both the bursting index of lutoids and the duration of latex flow after applying ethrel or ethylene gas in high concentrations suggests that a new mechanism needs to be introduced. In this study, a latex allergen Hev b 7-like protein in C-serum was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). In vitro analysis showed that the protein acted as a universal antagonist of RP aggregating factors from lutoids and C-serum. Ethrel treatment obviously weakened the effect of C-serum on RP aggregation, which was closely associated with the increase in the level of the Hev b 7-like protein and the decrease in the level of the 37 kDa protein, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting analysis and antibody neutralization. Thus, the increase of the Hev b 7-like protein level or the ratio of the Hev b 7-like protein to the 37 kDa protein in C-serum should be primarily ascribed to the ethrel-stimulated prolongation of latex flow duration.

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.

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.

Rapid and efficient isolation of high-quality small RNAs from recalcitrant plant species rich in polyphenols and polysaccharides.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.

Molecular and biochemical characterization of a cyanogenic ß-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 ß-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both ß-glucosidase and linamarase and was thus characterized as a cyanogenic ß-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 ß-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.