RESUMEN
Cellulose is the most abundant biopolymer in the world, the main load-bearing element in plant cell walls, and represents a major sink for carbon fixed during photosynthesis. Previous work has shown that photosynthetic activity is partially regulated by carbohydrate sinks. However, the coordination of cellulose biosynthesis with carbohydrate metabolism and photosynthesis is not well understood. Here, we demonstrate that cellulose biosynthesis inhibition (CBI) leads to reductions in transcript levels of genes involved in photosynthesis, the Calvin cycle, and starch degradation in Arabidopsis (Arabidopsis thaliana) seedlings. In parallel, we show that CBI induces changes in carbohydrate distribution and influences Rubisco activase levels. We find that the effects of CBI on gene expression and carbohydrate metabolism can be neutralized by osmotic support in a concentration-dependent manner. However, osmotic support does not suppress CBI-induced metabolic changes in seedlings impaired in mechanoperception (mid1 complementing activity1 [mca1]) and osmoperception (cytokinin receptor1 [cre1]) or reactive oxygen species production (respiratory burst oxidase homolog DF [rbohDF]). These results show that carbohydrate metabolism is responsive to changes in cellulose biosynthesis activity and turgor pressure. The data suggest that MCA1, CRE1, and RBOHDF-derived reactive oxygen species are involved in the regulation of osmosensitive metabolic changes. The evidence presented here supports the notion that cellulose and carbohydrate metabolism may be coordinated via an osmosensitive mechanism.
Asunto(s)
Arabidopsis/metabolismo , Metabolismo de los Hidratos de Carbono , Celulosa/biosíntesis , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Benzamidas/farmacología , Supervivencia Celular , Celulosa/antagonistas & inhibidores , Celulosa/genética , Activación Enzimática , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Presión Osmótica , Fotosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polietilenglicoles/farmacología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.
Asunto(s)
Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Fosfatidilinositol 4,5-Difosfato/análisis , Fosfolipasa C delta/química , Fosfolipasa C delta/metabolismo , Liposomas Unilamelares/química , Liposomas Unilamelares/aislamiento & purificación , Animales , Fluorescencia , Micromanipulación , Microscopía Fluorescente , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The chemoselective phosphorylation of hydroxyl containing amino acid derivatives and polyols by phosphoryl chlorides catalyzed by 2-aryl-4-(dimethylamino)pyridine-N-oxides is described.
Asunto(s)
Polímeros/química , Piridinas/química , Aminoácidos/química , Aminoácidos/metabolismo , Catálisis , Compuestos de Fósforo/química , Fosforilación , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMEN
De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound 'proto-organelles', which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction.