**Up-Regulation of Heme Oxygenase by Nitric Oxide and Effect of Carbon Monoxide on Soybean Plants Subjected to Salinity**

Guillermo Noriega, Carla Zilli, Diego Santa Cruz, Ethel Caggiano, Manuel López Lecube, María Tomaro and Karina Balestrasse *University of Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas Argentina* 

#### **1. Introduction**

426 Soybean Physiology and Biochemistry

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Salt stress is one of the most important abiotic stresses that adversely affects soybean growth and causes significant crop loss worldwide. Salinity has always been considered a serious constraint on agricultural productivity (Hay & Porter 2006) and affects plant's physiology. Salt stress is a complex phenomenon that involves morphological and developmental changes. Two major components have been identified in this insult, osmotic stress and ion toxicity (Darwish et al. 2009). Higher plants have multiple protective mechanisms against salt stress including ion homeostasis, osmolyte biosynthesis, ROS scavenging, water transport, and transducers of long-distance response coordination. It is generally accepted that many stresses, including salinity, induce an overproduction of ROS, such as H2O2, O2 •-, and HO**.** , and these species are thought to be responsible for the oxidative damage associated with plant stress (Zilli et al. 2009). To counteract the toxicity of ROS, defense systems that scavenge cellular ROS have been developed in plants to cope with oxidative stress via the non-enzymatic and enzymatic systems (Demiral & Turkan 2005; Mandhania et al. 2006)

Nitric oxide (NO) acts as a signaling molecule and mediates multiple physiological processes in plants (Leitner et al. 2009). In addition, it has been implicated in responses to biotic and abiotic stresses, such as disease resistance, salinity , drought, heat stress, among others (Beligni & Lamattina 1999; Romero-Puerta et al. 2004; Corpas et al. 2009). There are several sources of NO in plants, but mainly it can be enzymatically produced by nitrate reductase and nitric oxide synthase-like enzymes (Wilson et al. 2008 and Corpas et al. 2009). NO is a reactive nitrogen species and, depending on its concentration, it produces either protective or toxic effects. A low dose of NO modulates superoxide anion formation and inhibits lipid peroxidation, resulting in an antioxidant function during stress (Boveris et al. 2000 and Santa Cruz et al. 2010). Moreover, microarray studies have shown that NO induces a large number of genes at transcriptional level, among them those of antioxidant enzymes (Parani et al. 2004). It has also been reported that Nitric oxide gives rise to signaling pathways mediating 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 is rather limited.

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 activity of antioxidative enzymes.

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

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 did not provoke any change.
