RESUMO
WRKY transcription factors play important roles in plant growth and developmental processes and various stress responses, and are also associated with jasmonic acid (JA) signaling in the regulation of secondary metabolite biosynthesis in plants. The regulatory networks mediated by WRKY proteins in the latex production of Hevea brasiliensis (the Pará rubber tree) are poorly understood. In this study, one novel WRKY gene (designated HbWRKY83) was identified from the latex of H. brasiliensis, and its functions were characterized via gene expression analysis in both the latex and HbWRKY83-overexpressing transgenic Arabidopsis. HbWRKY83 gene contains an open reading frame (ORF) of 921 bp encoding a 306-amino-acid protein which is clustered with group IIc WRKY TF. HbWRKY83 is a nuclear-localized protein with transcriptional activity. Real-time quantitative PCR analysis demonstrated that the transcription level of HbWRKY83 was up-regulated by exogenous methyl jasmonate, Ethrel (ethylene releaser) stimulation, and bark tapping (mechanical wounding). Compared with the wild-type plants, overexpression of HbWRKY83 improved the tolerance of transgenic Arabidopsis lines to drought and salt stresses by enhancing the expression levels of ethylene-insensitive3 transcription factors (EIN3s) and several stress-responsive genes, including Cu/Zn superoxide dismutases CSD1 (Cu/Zn-SOD1) and CSD2 (Cu/Zn-SOD2), related to reactive oxygen species scavenging. Additionally, these genes were also significantly up-regulated by bark tapping. In combination, these results suggest that HbWRKY83 might act as a positive regulator of rubber production by activating the expression of JA-, ethylene-, and wound-responsive genes in the laticiferous cells of rubber trees.
Assuntos
Hevea , Látex/biossíntese , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Regulação da Expressão Gênica de Plantas , Hevea/genética , Hevea/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Tapping Panel Dryness (TPD) affects latex production in Hevea brasiliensis. This physiological syndrome involves the agglutination of rubber particles, which leads to partial or complete cessation of latex flow. Latex harvesting consists in tapping soft bark. Ethephon can be applied to stimulate latex flow and its regeneration in laticifers. Several studies have reported transcriptome changes in bark tissues. This study is the first report on deep RNA sequencing of latex to compare the effect of ethephon stimulation and TPD severity. Trees were carefully selected for paired-end sequencing using an Illumina HiSeq 2000. In all, 43 to 60 million reads were sequenced for each treatment in three biological replicates (slight TPD trees without ethephon stimulation, and slight and severe TPD trees with ethephon treatment). Differentially expressed genes were identified and annotated, giving 8,111 and 728 in response to ethephon in slight TPD trees and in ethephon-induced severe TPD trees, respectively. A biological network of responses to ethephon and TPD highlighted the major influence of metabolic processes and the response to stimulus, especially wounding and jasmonate depression in TPD-affected trees induced by ethephon stimulation.
Assuntos
Hevea/genética , Compostos Organofosforados/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Hevea/metabolismo , Látex/biossíntese , Casca de Planta/genética , Casca de Planta/metabolismo , Doenças das Plantas/genética , Análise de Componente Principal , RNA de Plantas/química , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Análise de Sequência de RNARESUMO
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.
Assuntos
Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Hevea/fisiologia , Látex/biossíntese , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Genes Reporter , Hevea/genética , Filogenia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Natural rubber (cis-1, 4-polyisoprene) is being produced from bark laticifer cells of Hevea brasiliensis and the popular high latex yielding Indian rubber clones are easily prone to onset of tapping panel dryness syndrome (TPD) which is considered as a physiological syndrome affecting latex production either partially or completely. This report describes an efficient protocol for development of transgenic rubber plants by over-expression of 3-hydroxy 3-methylglutaryl Co-enzyme A reductase 1 (hmgr1) gene which is considered as rate limiting factor for latex biosynthesis via Agrobacterium-mediated transformation. The pBIB plasmid vector containing hmgr1 gene cloned under the control of a super-promoter was used for genetic transformation using embryogenic callus. Putatively transgenic cell lines were obtained on selection medium and produced plantlets with 44% regeneration efficiency. Transgene integration was confirmed by PCR amplification of 1.8â¯kb hmgr1 and 0.6â¯kb hpt genes from all putatively transformed callus lines as well as transgenic plants. Southern blot analysis showed the stable integration and presence of transgene in the transgenic plants. Over expression of hmgr1 transgene was determined by Northern blot hybridization, semi-quantitative PCR and real-time PCR (qRT-PCR) analysis. Accumulation of hmgr1 mRNA transcripts was more abundant in transgenic plants than control. Increased level of photosynthetic pigments, protein contents and HMGR enzyme activity was also noticed in transgenic plants over control. Interestingly, the latex yield was significantly enhanced in all transgenic plants compared to the control. The qRT-PCR results exhibit that the hmgr1 mRNA transcript levels was 160-fold more abundance in transgenic plants over untransformed control. These results altogether suggest that there is a positive correlation between latex yield and accumulation of mRNA transcripts level as well as HMGR enzyme activity in transgenic rubber plants. It is presumed that there is a possibility for enhanced level of latex biosynthesis in transgenic plants as the level of mRNA transcripts and HMGR enzyme activity is directly correlated with latex yield in rubber tree. Further, the present results clearly suggest that the quantification of HMGR enzyme activity in young seedlings will be highly beneficial for early selection of high latex yielding plants in rubber breeding programs.
Assuntos
Hevea , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes , Látex/biossíntese , Proteínas de Plantas , Plantas Geneticamente Modificadas , Hevea/genética , Hevea/metabolismo , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/biossíntese , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Natural rubber has unique physical properties that cannot be replaced by products from other latex-producing plants or petrochemically produced synthetic rubbers. Rubber from Hevea brasiliensis is the main commercial source for this natural rubber that has a cis-polyisoprene configuration. For sustainable production of enough rubber to meet demand elucidation of the molecular mechanisms involved in the production of latex is vital. To this end, we firstly constructed rubber full-length cDNA libraries of RRIM 600 cultivar and sequenced around 20,000 clones by the Sanger method and over 15,000 contigs by Illumina sequencer. With these data, we updated around 5,500 gene structures and newly annotated around 9,500 transcription start sites. Second, to elucidate the rubber biosynthetic pathways and their transcriptional regulation, we carried out tissue- and cultivar-specific RNA-Seq analysis. By using our recently published genome sequence, we confirmed the expression patterns of the rubber biosynthetic genes. Our data suggest that the cytoplasmic mevalonate (MVA) pathway is the main route for isoprenoid biosynthesis in latex production. In addition to the well-studied polymerization factors, we suggest that rubber elongation factor 8 (REF8) is a candidate factor in cis-polyisoprene biosynthesis. We have also identified 39 transcription factors that may be key regulators in latex production. Expression profile analysis using two additional cultivars, RRIM 901 and PB 350, via an RNA-Seq approach revealed possible expression differences between a high latex-yielding cultivar and a disease-resistant cultivar.
Assuntos
Vias Biossintéticas/genética , Hevea/genética , Látex/biossíntese , Borracha/metabolismo , Transcriptoma , Hevea/metabolismo , Proteínas de Plantas/genética , RNA Mensageiro , RNA de Plantas , Análise de Sequência de RNA , Fatores de TranscriçãoRESUMO
MAIN CONCLUSIONS: HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene ß-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.
Assuntos
Diferenciação Celular , Hevea/citologia , Látex/biossíntese , Proteínas de Plantas/metabolismo , Adaptação Fisiológica/genética , Sequência de Bases , Desidratação , Secas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/genética , Casca de Planta/citologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ligação Proteica , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Frações Subcelulares/metabolismo , Técnicas de Hibridização Subtrativa , Nicotiana/genética , Ativação Transcricional/genéticaRESUMO
X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.
Assuntos
Aquaporinas/química , Aquaporinas/genética , Genoma de Planta , Hevea/genética , Látex/biossíntese , Proteínas de Plantas/genética , Aquaporinas/isolamento & purificação , Biologia Computacional , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia Estrutural de Proteína , Frações Subcelulares/metabolismoRESUMO
BACKGROUND: Rubber tree (Hevea brasiliensis) is an important industrial crop cultivated in tropical areas for natural rubber production. Treatment of the bark of rubber trees with ehephon (an ethylene releaser) has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. Deciphering the enigma is of great importance for improvement of rubber tree for high yield. RESULTS: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 h (E8) and 24 h (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. CONCLUSIONS: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.
Assuntos
Etilenos/farmacologia , Hevea/metabolismo , Látex/biossíntese , Compostos Organofosforados/farmacologia , Transcriptoma , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Hevea/genética , Redes e Vias Metabólicas , Anotação de Sequência Molecular , Casca de Planta/genética , Casca de Planta/metabolismo , RNA de Plantas/genética , Análise de Sequência de RNARESUMO
Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.
Assuntos
Etilenos/metabolismo , Hevea/metabolismo , Látex/biossíntese , Doenças das Plantas , Hevea/genética , Látex/metabolismo , Floema/metabolismo , Fosfatos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Sacarose/metabolismo , TranscriptomaRESUMO
Mechanical wounding or treatment with exogenous jasmonates (JA) induces differentiation of the laticifer in Hevea brasiliensis. JA is a key signal for latex biosynthesis and wounding response in the rubber tree. Identification of JAZ (jasmonate ZIM-domain) family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid-derived hormone controls gene expression and related physiological processes in plants. In this work, the full-length cDNAs of six JAZ genes were cloned from H. brasiliensis (termed HbJAZ). These HbJAZ have different lengths and sequence diversity, but all of them contain Jas and ZIM domains, and two of them contain an ERF-associated amphiphilic repression (EAR) motif in the N-terminal. Real-time RT-PCR analyses revealed that HbJAZ have different expression patterns and tissue specificity. Four HbJAZ were up-regulated, one was down-regulated, while two were less effected by rubber tapping treatment, suggesting that they might play distinct roles in the wounding response. A yeast two-hybrid assay revealed that HbJAZ proteins interact with each other to form homologous or heterogeneous dimer complexes, indicating that the HbJAZ proteins may expand their function through diverse JAZ-JAZ interactions. This work lays a foundation for identification of the JA signalling pathway and molecular mechanisms of latex biosynthesis in rubber trees.
Assuntos
Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/genética , Látex/biossíntese , Oxilipinas/metabolismo , Doenças das Plantas , Proteínas de Plantas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar , Dimerização , Resistência à Doença , Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H. brasiliensis latex, resulting in 4325 expressed sequence tags (ESTs) assembled into 1308 unigenes (692 contigs and 617 singletons). Quantitative analyses of the transcription levels of high redundancy clones in the ESTs revealed genes highly and predominantly expressed in laticifers, such as Rubber Elongation Factor (REF), Small Rubber Particle Protein and putative protease inhibitor proteins. HRT1 and HRT2, cis-prenyltransferases involved in rubber biosynthesis, was also expressed predominantly in laticifers, although these transcript levels were 80-fold lower than that of REF. The 5'-upstream regions of these laticifer-specific genes were cloned and analyzed in silico, revealing seven common motifs consisting of eight bases. Furthermore, transcription factors specifically expressed in laticifers were also identified. The common motifs in the laticifer-specific genes and the laticifer-specific transcription factors are potentially involved in the regulation of gene expression in laticifers.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/genética , Látex/biossíntese , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Borracha , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar , Dimetilaliltranstransferase/metabolismo , Etiquetas de Sequências Expressas , Expressão Gênica , Biblioteca Gênica , Hevea/metabolismo , Dados de Sequência Molecular , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
WRKY proteins constitute a large family of transcription factors. In this study, we identified 81 WRKY genes (named HbWRKY1 to HbWRKY81) in the latest rubber tree genome. Tissue-specific expression profiles showed that 74 HbWRKYs were expressed in at least one of the tissues and the other 7 genes showed very low expression in all tissues tested, which suggested that HbWRKYs took part in many cellular processes. The responses of 20 selected HbWRKYs to jasmonic acid (JA) and ethylene (ET) were analyzed in the latex. 17 HbWRKYs responded to at least one treatment, which included 15 HbWRKYs responding to JA treatment, 15 HbWRKYs to ET, which suggested that these HbWRKYs were regulated by JA and ET. We also observed that HbWRKY3, 14, and 55 bind HbSRPP promoter and activate the transcription in yeast. This study suggests that HbWRKY proteins maybe involved in the transcriptional regulation of nature rubber biosynthesis.
Assuntos
Genes de Plantas , Hevea/genética , Família Multigênica , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Látex/biossíntese , Dados de Sequência Molecular , Especificidade de ÓrgãosRESUMO
MYC2 transcription factor is a key component of the core module COI1-JAZ-MYC2 of jasmonate signaling in Arabidopsis, but the MYC transcription factor (s) associated with jasmonate signaling in jasmonate-responsive laticifer cells remains to be identified. Two full-length cDNAs, designated HblMYC1 and HblMYC2, were isolated from laticifer cells in Hevea brasiliensis by the method of RACE. HblMYC1 contained 1431bp ORF encoding a putative protein of 476 amino acids while HblMYC2 contained 1428bp ORF encoding a putative protein of 475 amino acids. Bioinformatic analysis showed that the putative proteins, HblMYC1 and HblMYC2, possessed a bHLH domain and were most related to the MYC2 among the selected 27 MYC members with identified functions in Arabidopsis. In addition to the presence of cis-regulatory elements involving jasmonate responsiveness in the promoter regions of HblMYC1 and HblMYC2, the abscisic acid-, salicylic acid- and gibberellin-responsive elements were found in the promoter region of HblMYC1. Transcripts of HblMYC1 and HblMYC2 were most abundant in latex, relatively low in male flowers and nearly undetected in bark tissues and roots by real-time RT-PCR analysis. Regular tapping, mechanical wounding, and ethrel remarkably up-regulated HblMYC1 expression, but had little effect on the expression of HblMYC2 in laticifer cells. Successive tapping, however, significantly down-regulated the expression of HblMYC2 while up-regulating the expression of HblMYC1. The HblMYC2 expression took a mutual ebb and flow relationship with the HblMYC1 expression upon treatment with methyl jasmonate. Characterization of HblMYC1 and HblMYC2 will contribute to the understanding of jasmonate signaling in laticifiers, a kind of specialized tissue for natural rubber biosynthesis in Hevea brasiliensis.
Assuntos
Hevea/fisiologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Acetatos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Ciclopentanos/farmacologia , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice/genética , Hevea/efeitos dos fármacos , Hevea/genética , Látex/biossíntese , Dados de Sequência Molecular , Compostos Organofosforados/farmacologia , Oxilipinas/farmacologia , Filogenia , Casca de Planta/genética , Folhas de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Raízes de Plantas/genética , Brotos de Planta/genética , Regiões Promotoras Genéticas/genética , RNA de Plantas/genética , Alinhamento de Sequência , Transdução de Sinais , Fatores de Transcrição/fisiologiaRESUMO
The aim of this study was to evaluate the surgical use of the natural latex biomembrane in diaphragmatic injuries produced experimentally in rabbits. Fifteen healthy adult male and female New Zealand rabbits were employed. The rabbits were assigned to the experimental groups I, II, III, IV and V and analyzed on the 15th, 30th, 45th, 60th and 90th days post surgery, respectively. The surgical procedure consisted in the access to the diaphragm at the eighth right intercostal space, removal of a circle portion of approximately 1.5 cm in diameter following surgical repair with a latex membrane. Macroscopically, it was observed an excellent healing process during the experimental period. The clinical observations, complemented by the histological analysis, indicate that the latex membrane is useful for repair of traumatic inuries of the diaphragm of rabbits.(AU)
Assuntos
Animais , Coelhos , Procedimentos Cirúrgicos Operatórios/veterinária , Diafragma/lesões , Hevea/fisiologia , Látex/biossínteseRESUMO
Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hevea/enzimologia , Compostos Organofosforados/farmacologia , Proteínas Quinases/genética , DNA Complementar/isolamento & purificação , Perfilação da Expressão Gênica , Genes de Plantas , Hevea/genética , Látex/biossíntese , Fases de Leitura Aberta/genética , Reguladores de Crescimento de Plantas , Estruturas Vegetais/química , RNA Mensageiro/análise , BorrachaRESUMO
Efficient sucrose loading in rubber-producing cells (laticifer cells) is essential for retaining rubber productivity in Hevea brasiliensis, but the molecular mechanisms underlying the regulation of this process remain unknown. Here, we functionally characterized a putative Hevea SUT member, HbSUT3, mainly in samples from regularly exploited trees. When expressed in yeast, HbSUT3 encodes a functional sucrose transporter that exhibits high sucrose affinity with a K(m) value of 1.24 mm at pH 4.0, and possesses features typical of sucrose/H(+) symporters. In planta, when compared to the expression of other Hevea SUT genes, HbSUT3 was found to be the predominant member expressed in the rubber-containing cytoplasm (latex) of laticifers. The comparison of HbSUT3 expression among twelve Hevea tissues demonstrates a relatively tissue-specific pattern, i.e. expression primarily in the latex and in female flowers. HbSUT3 expression is induced by the latex stimulator Ethrel (an ethylene generator), and relates to its yield-stimulating effect. Tapping (the act of rubber harvesting) markedly increased the expression of HbSUT3, whereas wounding alone had little effect. Moreover, the expression of HbSUT3 was found to be positively correlated with latex yield. Taken together, our results provide evidence favouring the involvement of HbSUT3 in sucrose loading into laticifers and in rubber productivity.
Assuntos
Hevea/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Borracha/metabolismo , Sacarose/metabolismo , Transporte Biológico , Clonagem Molecular , Expressão Gênica/efeitos dos fármacos , Genes de Plantas , Hevea/genética , Látex/biossíntese , Proteínas de Membrana Transportadoras/genética , Compostos Organofosforados/farmacologia , RNA de Plantas , Saccharomyces cerevisiae/genéticaRESUMO
Rubber tree (Hevea brasiliensis) is an important industrial crop for natural rubber production. Ethylene, as a stimulant of latex production in H. brasiliensis, has been widely used in commercial latex production. However, the mechanism of ethylene action are not completely elucidated, especially in molecular aspect. Here, we focus on the molecular biological progression of ethylene stimulation of latex production. Our data and all previous information showed ethylene had little direct effect on accelerating rubber biosynthesis. The prolonged latex flow and acceleration of sucrose metabolism by ethylene may be the main reasons for the stimulation of latex yield by ethylene.
Assuntos
Etilenos/metabolismo , Hevea/metabolismo , Látex/biossíntese , Compostos Organofosforados/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Borracha/metabolismo , Sacarose/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/efeitos dos fármacos , Hevea/genéticaRESUMO
Natural rubber is synthesized in specialized articulated cells (laticifers) located in the inner liber of Hevea brasiliensis. Upon bark tapping, the laticifer cytoplasm (latex) is expelled due to liber tissue turgor pressure. In mature virgin (untapped) trees, short-term kinetic studies confirmed that ethylene, the rubber yield stimulant used worldwide, increased latex yield, with a concomitant decrease in latex total solid content, probably through water influx in the laticifers. As the mature laticifers are devoid of plasmodesmata, the rapid water exchanges with surrounding liber cells probably occur via the aquaporin pathway. Two full-length aquaporin cDNAs (HbPIP2;1 and HbTIP1;1, for plasma membrane intrinsic protein and tonoplast intrinsic protein, respectively) were cloned and characterized. The higher efficiency of HbPIP2;1 than HbTIP1;1 in increasing plasmalemma water conductance was verified in Xenopus laevis oocytes. HbPIP2;1 was insensitive to HgCl(2). In situ hybridization demonstrated that HbPIP2;1 was expressed in all liber tissues in the young stem, including the laticifers. HbPIP2;1 was up-regulated in both liber tissues and laticifers, whereas HbTIP1;1 was down-regulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Ethylene-induced HbPIP2;1 up-regulation was confirmed by western-blot analysis. The promoter sequences of both genes were cloned and found to harbor, among many others, ethylene-responsive and other chemical-responsive (auxin, copper, and sulfur) elements known to increase latex yield. Increase in latex yield in response to ethylene was emphasized to be linked with water circulation between the laticifers and their surrounding tissues as well as with the probable maintenance of liber tissue turgor, which together favor prolongation of latex flow.
Assuntos
Aquaporinas/metabolismo , Etilenos/farmacologia , Hevea/citologia , Hevea/metabolismo , Látex/biossíntese , Proteínas de Plantas/metabolismo , Água/fisiologia , Sequência de Aminoácidos , Animais , Aquaporinas/química , Aquaporinas/genética , Sequência de Bases , Western Blotting , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hevea/efeitos dos fármacos , Hevea/genética , Cinética , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Casca de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , XenopusRESUMO
BACKGROUND AND AIMS: The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. METHODS: Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. KEY RESULTS: Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. CONCLUSION: The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.
Assuntos
Etilenos/farmacologia , Hevea/citologia , Hevea/metabolismo , Látex/biossíntese , Sacarose/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Transporte Biológico/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Hevea/enzimologia , Hevea/genética , Hibridização In Situ , Látex/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Filogenia , Casca de Planta/efeitos dos fármacos , Casca de Planta/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Árvores/efeitos dos fármacos , Árvores/genéticaRESUMO
In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.