RESUMO
MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree.
Assuntos
Genoma de Planta/genética , Estudo de Associação Genômica Ampla/métodos , Hevea/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Quitinases/genética , Quitinases/metabolismo , Biblioteca Gênica , Hevea/metabolismo , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Muramidase/genética , Muramidase/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Análise de Sequência de RNARESUMO
Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.
Assuntos
Regulação da Expressão Gênica de Plantas , Hevea/efeitos dos fármacos , Hevea/metabolismo , Látex/metabolismo , Casca de Planta/efeitos dos fármacos , Casca de Planta/metabolismo , Aquaporinas/genética , Proteínas de Plantas/genética , Reação em Cadeia da PolimeraseRESUMO
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
The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.