**2. Materials and methods**

#### **2.1 Plant material and treatments**

Surface sterilized soybean seeds (*Glycine max.* L.) (A6445RG) were germinated for 10 days in plastic pots containing vermiculite in controlled environmental chambers, with a photoperiod of 16 h, photon flux density of 175 µmol m-2 s-1, and a day/night regime of 25/20ºC. Afterwards, they were pretreated hydroponically with different sodium nitroprusiate concentrations (250-750 µM) for 72 h and then with NaCl (200 mM) for 48 h.

Carbon monoxide was generated from H2SO4 and formic acid (HCOOH). Stock solution was prepared by bubbling CO in a Hoagland solution for 40 min and was immediately diluted (50%) to perform analysis.

Plants were then harvested. When the effect of Zn-protoporphyrin IX (ZnPPIX) was investigated, roots were pretreated with 22 µM ZnPPIX during 4 h before addition of NaCl.

responses of specific genes to ultraviolet-B (UV-B) radiation, such as chalcone synthase and phenylalanine ammonia lyase (Mackerness et al. 2001). However, information about the role that NO plays in regulation of antioxidant enzymes to counteract salt-induced oxidative stress

Nitric oxide is believed to act as a signal molecule mediating responses to both biotic and abiotic stresses in plants (reviewed in Xuan et al. 2010 and Nürnberger & Scheel 2001) and its presence has been shown to induce seed germination (Liu et al. 2010), to affect growth and development of plant tissue (Beligni & Lamatina 2001, to increase iron homeostasis (Martin et al. 2009), to regulate plant maturation and senescence (Yaacov et al. 1998 and Jasid et al. 2009) to mediate abscisic acid-induced stomatal closing (Garcia-Mata & Lamattina, 2007). Recently, a few studies suggested that NO can play a role in protecting plants from oxidative stresses (Shantel et al. 2008) and NO-donor treatment protected plants from damage by increasing the

Heme oxygenase catalyzes the oxidative degradation of heme and has well-known antioxidant properties in mammals by mean of its products biliverdin IXα and carbon monoxide (CO) (Kikuchi et al. 2005). One of the three known mammalian isoforms, heme oxygenase-1 (HO-1), is induced in animal tissues by many factors including its own substrate heme, heavy metals, UV-A radiation among others (Tomaro & Batlle 2002). While earlier studies pointed to plant HO as a source of phytochrome chromophore (Terry et al. 2002), more recent works showed that HO synthesis increases in soybean plants subjected to oxidative stress conferring resistance to a subsequent insult (Noriega et al. 2004; Balestrasse et al. 2005). Moreover, we have recently demonstrated that ROS are involved in HO-1 upregulation in soybean leaves subjected to UV-B radiation (Yannarelli et al. 2006 and Santa Cruz et al. 2010). We hypothesized that NO may also participate in this process, as it

The aim of the present study was to investigate whether NO or CO could protect soybean against salt-induced oxidative stress through the modulation of HO activity. Soybean plants were subjected to salt stress after pre-treatments with different concentrations of sodium nitroprussiate (SNP), a well-characterized NO-donor or CO. Overall, our results indicate that in soybean plants NO is involved in the signaling pathway leading to HO-1 upregulation under salinity, and that a balance between NO and ROS is important to trigger the antioxidant response against oxidative stress. On the other hand pretreatment with CO

Surface sterilized soybean seeds (*Glycine max.* L.) (A6445RG) were germinated for 10 days in plastic pots containing vermiculite in controlled environmental chambers, with a photoperiod of 16 h, photon flux density of 175 µmol m-2 s-1, and a day/night regime of 25/20ºC. Afterwards, they were pretreated hydroponically with different sodium nitroprusiate

Carbon monoxide was generated from H2SO4 and formic acid (HCOOH). Stock solution was prepared by bubbling CO in a Hoagland solution for 40 min and was immediately diluted

Plants were then harvested. When the effect of Zn-protoporphyrin IX (ZnPPIX) was investigated, roots were pretreated with 22 µM ZnPPIX during 4 h before addition of NaCl.

concentrations (250-750 µM) for 72 h and then with NaCl (200 mM) for 48 h.

regulates the oxidative status and mediates other UV-B responses.

is rather limited.

activity of antioxidative enzymes.

did not provoke any change.

**2. Materials and methods** 

(50%) to perform analysis.

**2.1 Plant material and treatments** 

Controls were incubated in buffer. For fresh weight determination, plants were filtered, washed three times with distilled water, kept on filter paper for a few minutes to remove of excess liquid and weighed. Three different experiments were performed, with three replicated measurements for each parameter assayed
