Superoxide dismutases (SOD, EC 1.15.1.1) represent the first line of plant defense against ROS in the array of enzymes that function to protect the plant cells against oxidative stress. For this reason SOD are classified as a chain-breaking group of enzymes since they scavenge superoxide and they yield another form ofROS; hydrogen peroxide (H2O2). This study shows the effect of exogenous NO (as the NO donor DETA/NO) and salt stress on the enzymatic activity of various SOD isoforms in maize leaves. SOD activity was differentially regulated in the presence of NO and salt stress (NaCl) as shown in Figure 3.1 (A-C) and Table 3.1. When protein
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extracts from maize leaves were subjected to native PAGE and monitored for SOD activity, ten different SOD isoforms were observed in the untreated control plants (Figure 3.1A). Incubation of gels in 5 mM H2O2 (Figure 3.1C) and/or 6 mM KCN (Figure 3.2B) before staining for SOD activity identified three MnSOD isoforms (based on their resistance to both H2O2 and KCN inhibition), and seven Cu/Zn-SOD isoforms (inhibited by both H2O2 and KCN).
Figure 3.1 The effect of exogenous NO and salt stress on SOD enzymatic activity in maize. Assays were done on maize leaves taken from plants that were treated with the various treatments at the V1 stage for a period of 21 d (they were at the V3 stage at time of harvest). In- gel activities of various SOD isoforms in response to the following treatments are shown: 10 μM DETA/NO, 10 μM DETA, 150 mM NaCl, 10 μM DETA/NO + 150 mM NaCl, 10 μM DETA + 150 mM NaCl along with the untreated control. Individual SOD isoforms were identified by incubating gels in 6 mM KCN (B) and 5 mM H2O2 (C) respectively.
Under the influence of NO and/or salt stress, the intensities of some SOD isoforms (MnSOD2, MnSOD3, Cu/ZnSOD1, Cu/ZnSOD2, Cu/ZnSOD3, Cu/ZnSOD4, Cu/ZnSOD5, Cu/ZnSOD6 and Cu/ZnSOD7) were preferentially enhanced, whereas
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the intensity of MnSOD1 decreased when compared to the untreated plants (Figure 3.1A and Table 3.1). According to the densitometry analysis, MnSOD1 showed a ± 20% reduction in enzymatic activity in response to the treatment with the nitric oxide donor (DETA/NO) but no changes in response to DETA (used as a control for DETA/NO treatments) in comparison to the untreated plants (Figure 3.1A and Table 3.1). In fact, the DETA treatment had no effect on all the SOD isoforms detected when compared to the untreated controls. However, the activity of MnSOD1 in response to salt stress was reduced to a much lower level than it was in response to exogenously applied NO as indicated by the ± 37% reduction compared to the untreated plants (Table 3.1).
Table 3.1 Relative enzymatic activity of maize SOD isoforms.
R e la ti ve SOD A ct ivit y (Ar b itr ar y Va lu e s) Maize SOD isoforms Treatments
Untreated DETA/NO DETA NaCl DETA/NO +
NaCl DETA + NaCl MnSOD1 1.58 ± 0.076a 1.28 ± 0.065b 1.57 ± 0.076a 1.00 ± 0.052c 1.85 ± 0.090d 1.00 ± 0.050c MnSOD2 1.01 ± 0.043a 1.16 ± 0.055b 1.00 ± 0.041a 1.34 ± 0.063c 1.32 ± 0.061c 1.35 ± 0.065c MnSOD3 1.00 ± 0.050a 1.10 ± 0.065a 1.00 ± 0.050a 1.28 ± 0.064b 1.06 ± 0.053a 1.28 ± 0.064b Cu/ZnSOD1 1.00 ± 0.050a 1.57 ± 0.077b 1.01 ± 0.050a 1.74 ± 0.086c 2.25 ± 0.112d 1.73 ± 0.089c Cu/ZnSOD2 1.00 ± 0.050a 1.23 ± 0.061b 1.00 ± 0.050a 1.75 ± 0.088c 1.44 ± 0.072d 1.77 ± 0.089c Cu/ZnSOD3 1.00 ± 0.050a 1.37 ± 0.068b 1.00 ± 0.050a 1.14 ± 0.057a 1.72 ± 0.086c 1.14 ± 0.057a Cu/ZnSOD4 NA NA NA 1.00 ± 0.050a 1.19 ± 0.060b 1.00 ± 0.050a Cu/ZnSOD5 NA NA NA 1.00 ± 0.050a 0.93 ± 0.047a 1.00 ± 0.050a Cu/ZnSOD6 NA 1.00 ± 0.050a NA 1.465 ± 0.074b 1.175 ± 0.059a 1.467 ± 0.073b Cu/ZnSOD7 1.00 ± 0.050a 1.35 ± 0.068b 0.99 ± 0.050a 1.87 ± 0.094c 1.59 ± 0.08d 1.87 ± 0.094c Data presented in this table are the means ± standard error of three replicates (n = 3). Means marked with different letters in the same row for the same isoform indicate significant differences between treatments at 95% confidence according to the Tukey-Kramer test. The letters NA in the table indicate that very low or no activity was detected.
On the other hand, the combination of NaCl and DETA/NO caused an increase of ± 17% in MnSOD1 activity when compared to the untreated plants, whereas the
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combination of NaCl and DETA had a similar effect as the NaCl treatment alone. MnSOD2 showed an increase in activity of ± 15% in response to DETA/NO and ± 33% in response to NaCl, whereas the combined treatment of DETA/NO and 150 mM NaCl resulted in an increase of ± 32% when compared to the control plants. For the MnSOD3 isoform there were no significant changes in the activity for (DETA/NO and „DETA/NO + NaCl‟) treatments when compared to the untreated plants (Figure
3.1A and Table 3.1). However, there was a ± 27% increase in the intensities of the NaCl treated plants compared to the control plants. The results showed that all Cu/ZnSOD isoforms detected in this study were differentially regulated by both NO and salt stress. Cu/ZnSOD1 showed an increase of ± 56% in response to DETA/NO and a ± 74% increase in response to NaCl treatment compared to the untreated plants. However, the highest increase in SOD activity for Cu/ZnSOD1 was observed (a ± 125% increase) in the combined treatment of DETA/NO and NaCl. For Cu/ZnSOD2 an increase in enzymatic activity was observed for the various treatments. Apart from the slight increase in enzymatic activity observed in response to treatment with DETA/NO, both NaCl and the combination of DETA/NO and NaCl resulted in enhanced activity (by ± 75 and ± 44% respectively) for the Cu/ZnSOD2 isoform. For Cu/ZnSOD3, the enzymatic activity was slightly up-regulated in plants treated with salt stress, whereas plants treated with NO (including the NO-treated plants that were subjected to NaCl treatment) showed significant increase in enzymatic activity when compared to the untreated controls plants.
Furthermore, no or very low SOD activity was detected for three Cu/ZnSODs (Cu/ZnSOD4, Cu/ZnSOD5 and Cu/ZnSOD6) in the untreated as well as DETA/NO and DETA treated plants. However, SOD activity was detected in the salt stressed plants including the salt stressed plants supplemented with DETA/NO and DETA
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respectively. For Cu/ZnSOD4, there was no significant change in the intensity in response to NaCl and DETA, whereas plants treated with the combination of DETA/NO and NaCl showed a slightly higher increase in SOD activity when compared to the salt stressed plants alone. No significant changes in activity were detected for Cu/ZnSOD5 in response to treatment with NaCl, DETA/NO + NaCl and DETA treatments respectively. For Cu/ZnSOD6, no activity was detected in the untreated and DETA treated plants whereas the activity in the salt stressed plants was significantly higher than those in the NO-treated plants. However there was no significant difference in activity in the plants treated with NO and the NO-treated plants supplemented with NaCl. For Cu/ZnSOD7, plants treated with DETA/NO resulted in an increase in SOD activity of ± 34% compared to the untreated and DETA treated plants. No significant change in activity was observed for the untreated and DETA treated plants. A similar trend was observed for the salt-stressed plants and those treated with a combination of DETA and NaCl. However, treatment with NaCl resulted in an increase of ± 86% in SOD activity whereas the combination of DETA/NO and NaCl increase the activity by ± 58% when compared to the untreated plants.
3.4.2 The effect of exogenous NO and salt stress on APX and GPX enzymatic