RESUMEN
Ascorbic acid is a ubiquitous water soluble antioxidant that plays a critical role in plant growth and environmental stress tolerance. It acts as a free radical scavenger as well as a source of reducing power for several cellular processes. Because of its pivotal role in regulating plant growth under optimal as well as sub-optimal conditions, it becomes obligatory for plants to maintain a pool of reduced ascorbic acid. Several cellular processes help in maintaining the reduced ascorbic acid pool, by regulating its synthesis and regeneration processes. Current study demonstrates that monodehydroascorbate reductase is an important enzyme responsible for maintaining the reduced ascorbate pool, by optimizing the recycling of oxidized ascorbate. Cloning and functional characterization of this important stress inducible gene is of great significance for its imperative use in plant stress management. Therefore, we have cloned and functionally validated the role of monodehydroascorbate reductase gene (mdar) from a drought tolerant variety of Eleusine coracana. The cloned Ecmdar gene comprises of 1437bp CDS, encoding a 478 amino acid long polypeptide. The active site analysis showed presence of conserved Tyr348 residue, facilitating the catalytic activity in electron transfer mechanism. qPCR expression profiling of Ecmdar under stress indicated that it is an early responsive gene. The analysis of Ecmdar overexpressing Arabidopsis transgenic lines suggests that monodehydroascorbate reductase acts as a key stress regulator by modulating the activity of antioxidant enzymes to strengthen the ROS scavenging ability and maintains ROS homeostasis. Thus, it is evident that Ecmdar is an important gene for cellular homeostasis and its over-expression could be successfully used to strengthen stress tolerance in crop plants.
Asunto(s)
Simulación por Computador , Eleusine/enzimología , Eleusine/genética , Genes de Plantas , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Anotación de Secuencia Molecular , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , Fenotipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Estrés Fisiológico/genéticaRESUMEN
Changes in alpha- and beta-amylase activities in African finger millet (Eleusine coracana (L) Gaertener) were followed during germination. Germination on a small scale was performed at 15 degrees C for 1-10 days and at 20, 25 and 30 degrees C for 1-8 days. alpha- and beta-Amylase activities in malt crude extracts of germinated finger millet were evaluated spectrophotometrically using chromogenic methods. The highest alpha-amylase activity was exhibited in malt flour of finger millet germinated at 15 degrees C for 9 days and at 20 degrees C for 6 days, while the highest beta-amylase activity was displayed in the malt flour germinated for 5 days at 30 degrees C. Thermo-stability of these enzymes in malt extracts was also evaluated. Malt extracts incubated at 40 and 50 degrees C for up to 4 h retained about 84 and 64% of alpha-amylase activities, respectively. There was a substantial decrease in alpha-amylase activity to more than 90% when malt extracts were incubated at 70 and 90 degrees C for 40 and 10 min, respectively. beta-Amylase was completely inactivated when the crude extract was incubated at 70 degrees C for only 10 min. At pH 5.4, alpha-amylase displayed maximum catalytic activity at around 45 degrees C. Optimum temperature for beta-amylase activity at pH 6.0 was between 50 and 55 degrees C. Activity staining for alpha-amylase was also performed and three bands of activity were found in malt extract, each possibly representing an isozyme of alpha-amylase from finger millet.