RESUMO
⢠Floral scent is a complex trait of biological and applied significance. To evaluate whether scent production originating from diverse metabolic pathways (e.g. phenylpropanoids and isoprenoids) can be affected by transcriptional regulators, Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) transcription factor was introduced into Rosa hybrida. ⢠Color and scent profiles of PAP1-transgenic and control (ß-glucuronidase-expressing) rose flowers and the expression of key genes involved in the production of secondary metabolites were analyzed. To evaluate the significance of the scent modification, olfactory trials were conducted with both humans and honeybees. ⢠In addition to increased levels of phenylpropanoid-derived color and scent compounds when compared with control flowers, PAP1-transgenic rose lines also emitted up to 6.5 times higher levels of terpenoid scent compounds. Olfactory assay revealed that bees and humans could discriminate between the floral scents of PAP1-transgenic and control flowers. ⢠The increase in volatile production in PAP1 transgenes was not caused solely by transcriptional activation of their respective biosynthetic genes, but probably also resulted from enhanced metabolic flux in both the phenylpropanoid and isoprenoid pathways. The mechanism(s) governing the interactions in these metabolic pathways that are responsible for the production of specialized metabolites remains to be elucidated.
Assuntos
Flores/metabolismo , Odorantes , Proteínas de Plantas/metabolismo , Propanóis/metabolismo , Rosa/metabolismo , Terpenos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antocianinas/metabolismo , Abelhas/fisiologia , Vias Biossintéticas/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Humanos , Proteínas Associadas a Pancreatite , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Rosa/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Compostos Orgânicos Voláteis/análiseRESUMO
The shikimate pathway of plants mediates the conversion of primary carbon metabolites via chorismate into the three aromatic amino acids and to numerous secondary metabolites derived from them. However, the regulation of the shikimate pathway is still far from being understood. We hypothesized that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme regulating flux through the shikimate pathway. To test this hypothesis, we expressed a mutant bacterial AroG gene encoding a feedback-insensitive DAHPS in transgenic Arabidopsis plants. The plants were subjected to detailed analysis of primary metabolism, using GC-MS, as well as secondary metabolism, using LC-MS. Our results exposed a major effect of bacterial AroG expression on the levels of shikimate intermediate metabolites, phenylalanine, tryptophan and broad classes of secondary metabolite, such as phenylpropanoids, glucosinolates, auxin and other hormone conjugates. We propose that DAHPS is a key regulatory enzyme of the shikimate pathway. Moreover, our results shed light on additional potential metabolic bottlenecks bridging plant primary and secondary metabolism.
Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Escherichia coli/enzimologia , Retroalimentação Fisiológica , Redes e Vias Metabólicas , Ácido Chiquímico/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Flores/efeitos dos fármacos , Flores/genética , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lignina/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Caules de Planta/efeitos dos fármacos , Caules de Planta/genética , Plantas Geneticamente Modificadas , Análise de Componente Principal , Triptofano/análogos & derivados , Triptofano/farmacologiaRESUMO
Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.
