The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

Leonor Puerto-Galán,Manuel Guinea,Juan M Pérez-Ruiz,Francisco Javier Cejudo

doi:10.1093/jxb/eru512

Abstract

Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

Highlights

Aerobic metabolism inevitably produces reactive oxygen species (ROS) including singlet oxygen, superoxide anions, or hydrogen peroxide
Previous analyses have shown that the level of overoxidation of plastidial 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) (Kirchsteiger et al, 2009) and Srx (Iglesias-Baena et al, 2010), but the contribution of these activities to maintain the redox status of 2-Cys Prxs is not yet known
It is well established that NTRC and Srx exert a great influence on chloroplast 2-Cys Prx overoxidation; the relationship between these activities in the control of the level of 2-Cys Prxs overoxidation, is as yet unknown

Summary

Introduction

Aerobic metabolism inevitably produces reactive oxygen species (ROS) including singlet oxygen, superoxide anions, or hydrogen peroxide These species are highly oxidizing and may have a toxic effect, though they exert important signalling activity. In order to balance the toxic and signalling activities of hydrogen peroxide, its intracellular concentration needs to be tightly regulated In this regard the activity of 2-Cys peroxiredoxins (2-Cys Prxs) has been proposed to exert a relevant function. Monomeric structures, respectively (Rhee and Woo, 2011) Both typical and atypical 2-Cys Prxs have a common reaction mechanism, which depends on two cysteine residues, termed peroxidatic and resolving. The sulfinic form of 2-Cys Prxs can be converted back into the sulfenic form in an ATP-dependent reaction catalysed by sulfiredoxin (Srx) (Biteau et al, 2003; Woo et al, 2003)

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