**4. Discussion**

In a previous work, we found that SNP pre-treatment ameliorates Cd-induced oxidative stress and modulates HO-1 gene expression in soybean plants (Noriega et al. 2007). Taking into account the fact that NO is involved in various signaling pathways, in the present study we evaluated whether this molecule could enhance HO activity conferring a major protection against salt stress.

Our data demonstrated that, depending on its concentration, NO can improve the plant antioxidant response against salinity. This model was appropriate to determine the beneficial effect of exogenously added NO. While the lower dose of SNP did not reduce the oxidative damage (data not shown), the application of 500 or 750µM SNP showed a deleterious effect suggesting a pro-oxidant behavior of NO at these concentrations (Figure 1).

positive relationship between GSH levels and GR activity. Enzyme activity (GR) diminished 22% respect to controls when HO was inhibited, but an increase was detected in plants

Fig. 7. Effect of NO or CO on GR activity. (H) Control plants, (ZnPPIX/H) plants pretreated with ZnPPIX and then with H; (ZnPPIX/SNP) plants pretreated with ZnPPIX and then with SNP; (ZnPPIX/CO) plants pretreated with ZnPPIX and then with CO as described in Materias

Figure 8 shows HO-1 gene expression under different conditions. ZnPPIX/SNP treatment brought about a 20% augmentation respect to controls. This increase is positively correlated with GSH content and GR activity. On the other hand, CO did not show any effect. It is interesting to note that the enhancement of GSH content is not related to oxidative stress, since TBARS levels in roots of SNP and CO treated plants do not differ from controls. In

In a previous work, we found that SNP pre-treatment ameliorates Cd-induced oxidative stress and modulates HO-1 gene expression in soybean plants (Noriega et al. 2007). Taking into account the fact that NO is involved in various signaling pathways, in the present study we evaluated whether this molecule could enhance HO activity conferring a major

Our data demonstrated that, depending on its concentration, NO can improve the plant antioxidant response against salinity. This model was appropriate to determine the beneficial effect of exogenously added NO. While the lower dose of SNP did not reduce the oxidative damage (data not shown), the application of 500 or 750µM SNP showed a deleterious effect suggesting a pro-oxidant behavior of NO at these concentrations (Figure 1).

and Methods. \* Significant difference (p<0.05) according to Tukey's test.

contrast, HO inhibition brought about an enhancement (28%) in TBARS levels.

**3.7.3 HO-1 gene expression** 

protection against salt stress.

**4. Discussion** 

treated with NO and CO (33% and 26%, respectively).

Fig. 8. Effect of NO or CO on HO-1 gene expression. (H) Control plants, (ZnPPIX/H) plants pretreated with ZnPPIX and then with H; (ZnPPIX/SNP) plants pretreated with ZnPPIX and then with SNP; (ZnPPIX/CO) plants pretreated with ZnPPIX and then with CO as described in Materias and Methods. \* Significant difference (p<0.05) according to Tukey's test.

The pre-treatments with 250 µM SNP effectively ameliorated NaCl-induced oxidative stress, as indicated by the decrease in H2O2 and O2 .- formation (Figure 4), preventing TBARS formation (Figure 2) and enhancing GSH content (Figure 3). The activities of classical antioxidant enzymes, such as CAT and APX were also augmented by SNP treatment, instead of the drastically diminution observed with salinity alone (Table 1). These data are in agreement with reports showing a protective effect of NO in plants subjected to other stresses (Zhao et al. 2004; Shi et al. 2005 and Noriega et al. 2007). Nevertheless, the molecular mechanism that mediates NO enhancement of antioxidant enzyme activities is not completely understood. Interestingly, we found that HO and CAT activities had a similar behaviour with respect to SNP pre-treatment under salinity (Table 1). A recent study showed that the time-course of induction of those enzymes in soybean nodules subjected to Cd stress is related (Balestrasse et al. 2008). These results suggest a close relationship between the signal transduction pathways involved in the response of HO and CAT after oxidative stress generation and support the antioxidant role of HO. In addition, there was a positive correlation between HO-1 transcript levels and enzyme activity (Figure 5 and Table 1). Previous reports have also demonstrated that the enhancement of HO activity is associated with an increase in HO-1 transcript levels and protein content (Yannarelli et al. 2006 and Balestrasse et al. 2008). Although this mechanism can account for the changes observed in HO activity, the incidence of post-translational modifications or different HO isoforms under stress conditions needs to be addressed. Experiments carried out in plants treated with SNP in the absence of NaCl showed that NO itself can up-regulate HO-1 mRNA expression, but to a lesser extent (Figure 5). This observation indicates that a certain balance between NO and ROS is required to trigger the full response. Interestingly, a recent report found that the ROS–NO ratio is important to elicit ROS-activated stress responses and cell death regulation in plant leaves during ozone exposure (Ahlfors et al. 2009). Moreover, new evidence suggests that plastids and peroxisomes are important regulators of NO levels in plants (Corpas et al. 2009 and Gas et al. 2009).

Biliverdin, one of the products of the HO, is an efficient scavenger of ROS and it can account for the antioxidant properties of this enzyme both in animals and plants (Otterbein et al. 2003 and Noriega et al. 2004). More recently, it has been shown that CO released by HO is an important signal molecule for the tolerance mechanisms against cadmium and salt stress (Han et al. 2008). It would be interesting to determine whether CO could also play a role in the defense against salinity in soybean plants.

Pretreatment with ZnPPIX decreased HO-1 expression (Figure 8) and increased parameters of oxidative stress. When the inhibitor was added before NO or CO treatment, HO-1 expression as well as GSH content (Figure 6) and GR activity were increased (Figure 7). These results let us suppose that a close relationship between HO-1 induction and GSH content could exist. Taking together, these data provide evidence of one of the possible roles that NO, as well as CO could play against oxidative insult.
