Influence of sodium chloride on the dehydroascorbate reductase activity in Arabidopsis thaliana catalase 2 knokout mutant

Aim. To better understand the mechanisms of abiotic stress resistance in plants, it is important to clarify the role of individual antioxidant enzymes from the same multiproteinic family in the response to stress. It is known that the loss of some isoforms of antioxidant enzymes can be compensated by activation of other enzymes. However, the functional interaction of the ascorbate-glutathione cycle enzymes with catalase under salt stress still remains unexplored. Respectively, we determined the activity of DHAR in knock-out mutants of Arabidopsis thaliana under salt stress. Methods. The DHAR activity was determined in the knock-out line cat2 and in wild-type (WT) Arabidopsis plants after various regimes of treatment with sodium chloride. Results. After treatment with 200 mM sodium chloride in the dark, activation of DHAR was found after 8 hours in WT plants and after 4 hours in the knock-out line cat2. However stress treatment under illumination resulted in significant increase in DHAR activity after 8 hours in both studied lines. In this case, DHAR activity in cat2 was lower than in WT, whereas in non-treated plants or upon stress treatment in the dark no difference between the tested lines was detected. Conclusions. The obtained data indicate that under salt stress conditions, changes in the DHAR activity are included into functional rearrangements of the antioxidant system in cat2 line, which compensate the loss of activity of CAT2 isoenzyme.Keywords: dehydroascorbate reductase, antioxidants, reactive oxygen species (ROS), salt stress, Arabidopsis thaliana

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.