Changes in the isozyme composition of antioxidant enzymes in response to aminotriazole in leaves ofArabidopsis thaliana

Abstract

The changes in isozyme profiles of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and 81utathione reductase (GR) during severe deactivation of total CAT activity by aminotriazole (AT) treatment were investigated in the leaves of Arabidopsis thaliana (Columbia ecotype) in relation to H202-mediated oxidative stress. In spite of striking deactivation of total CAT activity by 0.1 mM AT, there were no significant differences in H202 levels or total leaf soluble protein contents including a Rubisco in both the control and AT-treated leaves. On the other hand, one specific protein band (molecular mass, 66 kD) was observed on the SDS-gel from leaf soluble proteins whose staining intensity was strikingly enhanced by AT treatment for 6 h. However, this band disappeared at 12 h. In the native-gel assays of CAT, POD, APX and GR isozymes, AT remarkably inhibited the expression of the CAT1 isozyme with no effects on CAT2 and CAT3, and generally had no effect on POD isozyme profiles. However, AT stimulated the intensity of activities of pre-existing APX1 and GR1 isozymes. In particular, it induced a new synthesis of one GR isozyme. Therefore, these results collectively suggest that a striking deactivation of total CAT activity by AT in A. thaliana leaves largely results from the suppression of CAT1 isozyme, and that APXl, GR1, and a newly synthesized GR isozyme could complement the role of CAT1 to metabolize H202 into non-toxic water.

A bentazone-resistant mutant of cyanobacterium, Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stress

“…GR activity was located by immersing the gel in 100 mL of 0.25 M Tris-HCl buffer pH 7.5 containing 0.24 mM MTT, 0.4 mM of NADPH, 10 mg of 2,6-dichlorophenolindophenol and 3.4 mM of GSSG. The gel was incubated in dark for 1 h to develop purple bands (Kang et al 1999). For GST the gel was incubated for 10 min each in 0.1 MK-PO 4 buffer pH 6.5 and 0.1 MK-PO 4 buffer pH 6.5 containing 4.5 mM GSH, 1 mM CDNB and 1 mM NBT, followed by 0.1 M TrisHCl buffer pH 9.6 containing 3 mM phenazine methosulphate.…”

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis

“…) as described previously (Kang et al, 1999). Peroxidase activity was determined with guaiacol as an electron donor by measuring the oxidation of guaiacol to tetraguiacol at 470 nm (extinction coefficient, 25.5 mM 21 cm -1 ) with 0.1 mM H 2 O 2 and 17 mM guaiacol (Chance and Maehly, 1955).…”

Aluminum stress in the roots of naked barley

“…CAT bands were yellow on a dark greenfield and were stable for several hr (Woodbury et al, 1971). For APX isozymes, non-denaturing PAGE was performed as above except the carrier buffer with 2 mM ascorbate (Kang et al, 1999;Park, 1999). The gel was prerun for 30 min to allow ascorbate to enter the gel prior to the application of the samples.…”

“…In Situ Enzyme Activity Staining on the Non-Denaturing PAGE Non-denaturing PAGE was performed with slab gel apparatus using a discontinuous system (Kang et al, 1999). The final acrylamide monomer concentration in the 0.75 mm thick slab gels was 7.5% (w/ v) for the separating gel and 4% (w/v) for the stacking gel.…”

“…All extracts were immediately stored at -70~ until being used. However, extracts for the catalase and ascorbate peroxidase assays were made fresh before electrophoresis (Kang et al, 1999).…”