Abstract
Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.
Highlights
Plants being sessile in nature are inevitably exposed to various abiotic and biotic stresses (Apel and Hirt, 2004; Mittler et al, 2004)
We found that the second Size exclusion chromatography (SEC) fraction (F2) showed a band of proteins between 158 and 440 kDa, whereas the F3 fraction corresponding to the dimeric form of AtAPX1 appeared below 158 kDa, representing the major peak in SEC analysis
As AtAPX1 appears in multimeric forms ranging from low molecular weight (LMW) to HMW form, with dual function, acting both as a peroxidase and a molecular chaperone, we investigated the connection between its structural conformations and functions
Summary
Plants being sessile in nature are inevitably exposed to various abiotic and biotic stresses (Apel and Hirt, 2004; Mittler et al, 2004). RONS play an important signaling role in plants and act as key regulators of various metabolic and physiological processes (Mittler et al, 2011; Baxter et al, 2014; Del Rio, 2015). Reactive oxygen species, such as superoxide radical, as well as reactive nitrogen species, such as nitric oxide, interact with each other to yield another type of RONS, peroxynitrite, indicating the cross talk between RONS (Halliwell, 2006; Radi, 2013; Khan et al, 2014). These modifications either reversibly or irreversibly alter the stability, structure, and function of proteins (Begara-Morales et al, 2013, 2014; Radi, 2013; Yang et al, 2015)
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