Nitric oxide increases the enzymatic activity of three ascorbate peroxidase isoforms in soybean root nodules

Abstract

Ascorbate peroxidase is one of the major enzymes regulating the levels of H2O2 in plants and plays a crucial role in maintaining root nodule redox status. We used fully developed and mature nitrogen fixing root nodules from soybean plants to analyze the effect of exogenously applied nitric oxide, generated from the nitric oxide donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine, on the enzymatic activity of soybean root nodule ascorbate peroxidase. Nitric oxide caused an increase in the total enzymatic activity of ascorbate peroxidase. The nitric oxide-induced changes in ascorbate peroxidase enzymatic activity were coupled to altered nodule H2O2 content. Further analysis of ascorbate peroxidase enzymatic activity identified three ascorbate peroxidase isoforms for which augmented enzymatic activity occurred in response to nitric oxide. Our results demonstrate that nitric oxide regulates soybean root nodule ascorbate peroxidase activity. We propose a role of nitric oxide in regulating ascorbate-dependent redox status in soybean root nodule tissue.

Figure 1

Nitric oxide content in soybean nodules as measured after treatment of nodulated soybean with the NO donor DETA/NO at final concentrations of 5 and 10 µM or DETA (negative control for DETA/NO) at a final concentration of 10 µM. Error bars represent the mean (±SE; n = 3) from data that are representative of three independent experiments.

Figure 2

Nodule APX total enzymatic activity in response to treatment with various concentrations of DETA/NO or 10 µM DETA, as measured by a spectrophotometric APX assay (A). Consequence of exogenously applied NO (as 5 and 10 µM DETA/NO) or DETA (10 µM) on nodule H₂O₂ content (B). Error bars represent the means (±SE; n = 3) of three independent experiments.

Figure 3

In-gel assay for nodule APX activity after treatment with 5 and 10 µM DETA/NO (A) or 10 µM DETA (B). The in-gel assay shows responses of different soybean root nodule APX isoforms to DETA/NO or DETA as indicated.

Figure 4

Effect of various concentrations of DETA/NO or DETA on the enzymatic activities of different nodule APX isoforms. Pixel intensities signifying the level of enzymatic activity of nodule APX isoforms, derived from analysis of the intensity of the bands corresponding to each APX isoform, are indicated. In-gel activities (as pixel intensities) in response to treatment with 5 and 10 µM DETA/NO are indicated for APX 1 (A), APX 2 (B) and APX 3 (C). responses of the APX 1, APX 2 and APX 3 isoforms to 10 µM DETA are indicated in (D–F) respectively. Error bars represent the means (±SE; n = 4) of three independent experiments.