**3.1 Growth parameter**

Experiments were carried out in the presence of different SNP concentrations ranging from 200 to 750 µM. Root length was measured as a parameter to asses the optimal condition. Figure 1 shows that 250 µM SNP brought about a 45% increase in root length, whereas a diminution was observed under the other concentrations. Depending on its dose, NO can promote or inhibit root growth. According to these result, 250 µM SNP was chosen as the concentration to be used in pretreatment.

Fig. 1. Effect of different SNP concentrations on root growth. \* Significant difference (p< 0.05) according to Tukey's test.

#### **3.2 Lipid peroxidation**

Increment in TBARS is a good reflection of oxidative damage to membrane lipids and other vital molecules such as proteins, DNA and RNA. Figure 2 shows that TBARS levels increased 75% respect to controls under salt treatment which is in agreement with results of other studies (Deng et al. 2010).

To complete this analysis, the effect of SNP pre-treatment was evaluated. Figure 2 indicates that in this case, membrane damage was more moderated, as indicated by a 14% augmentation respect to controls. Treatment with SNP alone did not show any difference respect to controls.

#### **3.3 Glutathione content**

GSH is a leading substrate for enzymatic antioxidant functions and it is also a known radical scavenger. Previous reports from our laboratory demonstrated that oxidative stress induces the formation of oxidant species and therefore affects GSH content in soybean plants

Fig. 2. Effect of salinity on TBARS formation and SNP regulation. \* Significant difference (p<0.05) according to Tukey test.

(Balestrasse et al. 2001 and Noriega et al. 2004). Surprisingly, data in Figure 3 show that GSH concentration in soybean roots treated with NaCl was enhanced 3.5-fold respect to controls. Pre-treatment with SNP brought about a 4-fold augmentation respect to controls. Moreover, SNP alone provoked a 2-fold increase respect to controls.

Fig. 3. Effect of salinity on GSH levels and SNP regulation. \* Significant difference (p<0.05) according to Tukey test.

#### **3.4 H2O2 and O2 .- localization** *in situ*

432 Soybean Physiology and Biochemistry

Fig. 2. Effect of salinity on TBARS formation and SNP regulation. \* Significant difference

(Balestrasse et al. 2001 and Noriega et al. 2004). Surprisingly, data in Figure 3 show that GSH concentration in soybean roots treated with NaCl was enhanced 3.5-fold respect to controls. Pre-treatment with SNP brought about a 4-fold augmentation respect to controls. Moreover,

Fig. 3. Effect of salinity on GSH levels and SNP regulation. \* Significant difference (p<0.05)

(p<0.05) according to Tukey test.

according to Tukey test.

SNP alone provoked a 2-fold increase respect to controls.

Accumulation of H2O2 and O2.- were also evaluated *in situ* by histochemical methods as shown in Figure 4a NaCl produced 32% H2O2 spots area versus total root area, while pretreatment with 250 µM SNP prevented this effect and spot area was similar to controls (Figure 4a). Data in Figure 4b showed that roots treated with NaCl produced 41% O2 .- spots area versus total root area. Pretreatments with 250 µM SNP completely prevented the O2 . production induced by NaCl.

Fig. 4. Histochemical detection of H2O2 (A) and O2.- (B) in soybean roots. Experiments were performed as described in Materials and Methods. Pictures are representative of three different experiments with three replicated measurements for each treatment.

#### **3.5 Effect of NO on antioxidant enzyme activities**

We also investigated whether NO can modulate the activities of classical antioxidant enzymes such as CAT and APX. These are the main H2O2-scavenging enzymes that control ROS-mediated responses under biotic and abiotic stresses (Mittler 2002). CAT and APX activities were significantly affected by NaCl (Table 1). They were increased by 47% and 33% in NaCl-treated plants compared to controls, respectively. Moreover, CAT activity significantly augmented up to 24% with respect to controls of SNP-treated plants, whereas APX only showed a mild increase (19%). Heme oxygenase behavior was similar to that found for CAT (Table 1).


Table 1. Antioxidant enzyme activities in soybean roots subjected to 200mM NaCl and 250 µM SNP pretreatment. Enzymatic activities were assayed as described in Materials and Methods. Different letters within columns indicate significant differences (*P* < 0.05) according to Tukey's multiple range test.

#### **3.6 Heme oxygenase-1 activity and gene expression**

Previous findings from our group demonstrated the protective role that HO-1 plays against oxidative stress in soybean plants (Noriega et al. 2004 and Balestrasse et al. 2005). Figure 5 indicates that salt stress caused HO-1 mRNA induction (13%, respect to controls). This enhancement is positively correlated with enzyme activity (Table 1). Pretreatment with 250 µM SNP brought about an augmentation of gene expression in control plants (21%), as well as salt treated plants (27%) (Figure 5). Once again, this behavior was also found when enzyme activity was determinated (Table 1) .These results indicate on one hand, that NO

Fig. 5. HO-1 mRNA expression was analyzed by semi-quantitative RT-PCR as described in Materials and Methods. The 18S amplification band is shown to confirm equal loading of RNA and RT efficiency. Relative HO-1 transcript expression taking control as 1 U. Data are means of three independent experiments and bars indicate SE. \*Significant differences (P < 0.05 according to Tukey test).

induces HO-1 more efficiently than NaCl, and on the other hand, both compounds have a synergic effect on this induction. To asses whether HO-1 is involved in the protection against NaCl exerted by NO, experiments were carried out in plants treated with ZnPPIX, a well known irreversible HO-1 inhibitor. Plants with inhibited HO-1 activity can not cope with NaCl insult (data not shown). We can assume that protection exerted by SNP may be due to the augmentation of the activity of this antioxidant enzyme.

#### **3.7 Effect of NO and CO 3.7.1 Glutathione content**

434 Soybean Physiology and Biochemistry

Control 0.065 ± 0.001a 120 ± 12a 0.0040 ± 0.0010a NaCl 0.073 ± 0.001b 176 ± 9b 0.0053± 0.0012a SNP 0.079 ± 0.002c 149± 2c 0.0047 ± 0.0010a SNP+ NaCl 0.083 ± 0.004c 138 ± 14c 0.0050 ±0.0010a Table 1. Antioxidant enzyme activities in soybean roots subjected to 200mM NaCl and 250 µM SNP pretreatment. Enzymatic activities were assayed as described in Materials and Methods. Different letters within columns indicate significant differences (*P* < 0.05)

Previous findings from our group demonstrated the protective role that HO-1 plays against oxidative stress in soybean plants (Noriega et al. 2004 and Balestrasse et al. 2005). Figure 5 indicates that salt stress caused HO-1 mRNA induction (13%, respect to controls). This enhancement is positively correlated with enzyme activity (Table 1). Pretreatment with 250 µM SNP brought about an augmentation of gene expression in control plants (21%), as well as salt treated plants (27%) (Figure 5). Once again, this behavior was also found when enzyme activity was determinated (Table 1) .These results indicate on one hand, that NO

Fig. 5. HO-1 mRNA expression was analyzed by semi-quantitative RT-PCR as described in Materials and Methods. The 18S amplification band is shown to confirm equal loading of RNA and RT efficiency. Relative HO-1 transcript expression taking control as 1 U. Data are means of three independent experiments and bars indicate SE. \*Significant differences (P <

CAT (pmol/mg protein)

APX (U/mg protein)

Treatment HO-1

according to Tukey's multiple range test.

0.05 according to Tukey test).

(U/mg protein)

**3.6 Heme oxygenase-1 activity and gene expression** 

As already stated, there is a positive relationship between NO content and GSH levels (Figure 3). This result prompted us to investigate whether HO is involved in the regulation of this tripeptide. To fulfill this purpose, experiments were carried out in plants treated with ZnPPIX and then subjected to NO (HO inductor) or CO (HO reaction product) for 48 h before salt stress. Afterwards, GSH content (Figure 6) as well as HO-1 gene expression (Figure 8) was determinated.

Fig. 6. Effect of NO and CO on GSH content. (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.

In plants pretreated with ZnPPIX for 72 h before Hoagland (H) treatment (ZnPPIX/H), GSH level diminished 20% respect to controls (H). Figure 6 shows that NO (ZnPPIX/SNP) as well as CO (ZnPPIX/H) enhanced GSH levels (40% and 15%, respectively).
