RESUMO
Natural rubber (NR) is synthesized by the rubber transferase (RTase) on rubber particles (RPs) in latex. Due to the heterogeneity of the RPs in latex, it is difficult to precisely characterize the RTase activity. In this study, we separated the RPs of Hevea brasiliensis with different particle size distributions, via stepwise centrifugations. Analyses of protein compositions and size distributions of NR in the RPs suggest that RPs in Hevea latex can be categorized into two distinct subclasses, the larger RPs (termed 1kRP, 2kRP, and 8kRP) and the smaller RPs (termed 20kRP and 50kRP). Precise enzymatic assays using the RPs revealed that 50kRP showed the highest RTase activity, whereas the larger RPs, which had been regarded to have quite low activity, also exhibited a comparable activity to the smaller RPs. Immunological detections of cis-prenyltransferases in the RPs showed that the abundance of these enzymes correlates with the extent of RTase activity.
Assuntos
Hevea/metabolismo , Tamanho da Partícula , Borracha/química , Western Blotting , Centrifugação , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica de VarreduraRESUMO
Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.
Assuntos
Vias Biossintéticas , Hevea/genética , Hevea/metabolismo , Borracha/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteoma/análiseRESUMO
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
Cosmopolites sordidus is an important pest on banana plantations worldwide. The chemistry of the aggregation pheromone of this insect has been recently resolved and here we present the first evidence from field trails that sordidin, a compound from the male releases aggregation pheromone, attracts significant number of weevils only if host plant odors are also present. Sordidin attracts few insects when it is presented without the host plant tissue. However, the attractiveness of host plant tissue increases more than tenfold when it is presented simultaneously with sordidin in field traps. We confirm experimentally that sordidin may be used as part of a system for mass trapping and monitoring this insect.