Abstract
SummaryAscorbic acid-glutathione (AsA-GSH) cycle represents important antioxidant defense system in planta. Here we utilized Oncidium cytosolic ascorbate peroxidase (OgCytAPX) as a model to demonstrate that CytAPX of several plants possess dual catalytic activity of both AsA and GSH, compared with the monocatalytic activity of Arabidopsis APX (AtCytAPX). Structural modeling and site-directed mutagenesis identified that three amino acid residues, Pro63, Asp75, and Tyr97, are required for oxidization of GSH in dual substrate catalytic type. Enzyme kinetic study suggested that AsA and GSH active sites are distinctly located in cytosolic APX structure. Isothermal titration calorimetric and UV-visible analysis confirmed that cytosolic APX is a heme-containing protein, which catalyzes glutathione in addition to ascorbate. Biochemical and physiological evidences of transgenic Arabidopsis overexpressing OgCytAPX1 exhibits efficient reactive oxygen species-scavenging activity, salt and heat tolerances, and early flowering, compared with Arabidopsis overexpressing AtCytAPX. Thus results on dual activity CytAPX impose significant advantage on evolutionary adaptive mechanism in planta.
Highlights
Plants generate reactive oxygen species (ROS) continuously as by-products of various metabolic pathways and stresses in different cell compartments
Both AsA and GSH are ubiquitous in eukaryotic organisms, but only AsA is specific and highly abundant in plants, where it is essential for growth and development (Foyer and Noctor, 2016)
We found that Oncidium cytosolic APX1 (OgCytAPX1) and those in some plant species can catalyze AsA and GSH as electron donors to scavenge H2O2
Summary
Plants generate reactive oxygen species (ROS) continuously as by-products of various metabolic pathways and stresses in different cell compartments. The pathway contains ascorbate peroxidase (APX) together with dehydroascorbate reductase (DHAR) and glutathione reductase (GR), in addition to antioxidant metabolites AsA, GSH, and NADPH. AsA is the most important reducing substrate for H2O2 detoxification, with the oxidized product being dehydroascorbate (DHA). The fundamental function of GSH is in thiol-disulfide interactions, in which reduced GSH is interchangeable with the oxidized form, GSSG (Frendo et al, 2013). Both AsA and GSH are ubiquitous in eukaryotic organisms, but only AsA is specific and highly abundant in plants, where it is essential for growth and development (Foyer and Noctor, 2016)
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