Nitric Oxide Synthesis In Plants

In mammals, NO is generated primarily by nitric oxide synthase(s) (NOSs). NOSs are a group of evolutionarily conserved cytosolic or membrane-bound isoenzymes that convert L-arginine to L-citrulline and NO [16]. In plants, NOS activity has been detected in several plant species when mammalian NOS inhibitors have been used to inhibit NO production [17]. However, no gene or protein with a sequence similar to mammalian NOS has been found in the sequenced A. thaliana genome. Guo et al. (2003) identified an AtNOSl gene with sequence similarity to a distinct NOS that was characterized in the snail Helix pomatia. The role of AtNOSl in NO synthesis in vivo was examined in a homozygous mutant line, in which NO levels were found to be much lower than in wild type plants [18]. However, very recently, a discussion regarding the identity of the AtNOSl gene was generated [19,20,52] since other laboratory was not able to detect NOS activity in the recombinant protein encoded by plant NOS genes [52]. Authors suggested that the AtNOS1 mutant line display lower levels of NO because AtNOS1 deletion would lead to defects in mitochondrial biogenesis. Therefore, the mechanisms for arginine-dependent NO synthesis in plants is still not clear.

However, plant NO synthesis is not as simple as in animals since also nitrite can be a substrate for NO production. Nitrate reductase (NR) is another enzyme capable of producing NO in plants [21]. This enzyme has a primary activity that converts nitrate to nitrite; however, NR can also convert nitrite to NO, via a single electron reduction. It was shown that NO synthesis is stimulated by nitrite [21-23] and this stimulation is attenuated in an A. thaliana NR double mutant (nia1/nia2) [22]. It was shown that NR plays a significant role in basal NO generation in leaves [23] as well as during several plant physiological processes or environmental stimulus [21,22,24,25]. In addition, NO production from nitrite involves also non-enzymatic sources. It has been demonstrated that a non-enzymatic reduction of nitrite to NO occurs in the apoplast of barley aleurone layers [26]. This NO production requires an acidic pH and was accelerated by reducing agents such as phenolic compounds.

NITRIC OXIDE STORAGE, DELIVERY AND DETOXIFICATION IN PLANTS

NO is extremely labile in physiologic environment where iron and oxygen are present. Lifetime of NO free radical is just 6-10 s [27]. In mammals, S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNICs) were proposed to be the main forms of NO storage and transport [27]. These types of molecules can be both low- or high-molecular-weight

(i.e. bound to proteins) compounds. Usually, the protein complexes are more stable as compared to the low-molecular-weight ones [27]. S-nitrosylation of certain proteins gives rise to NO transport-storage forms such as S-nitrosoalbumin [28,29] and S-nitrosohemoglobin

[30]. In plants, S-nitrosohemoglobin is endogenously produced, indicating a conserved role of hemoglobin S-nitrosothiols in NO metabolism among humans, nematodes and plants

[31]. Moreover, hexacoordinate hemoglobin AHbl from A. thaliana metabolizes both NO and GSNO. The nitrate forming reactions involve an Fe3+ intermediate, which is efficiently reduced by NADPH. It was proposed that this hemoglobin takes part in NO detoxification, principally during stresses that produce over-accumulation of NO such as hypoxia [31]. However, the rate of S-nitrosohemoglobin decomposition is low at physiological conditions, suggesting that hemoglobin S-nitrosylation could be a means for NO storage. There is no evidence of hemoglobin transport in the plant vascular system and it is highly improbable that hemoglobin function as NO carrier between plant tissues and organs, as it was suggested for mammals [32]. Therefore, the attention is focused to low-molecular-weight NO-containing compounds that could be responsible for NO trafficking in plants.

In the model plant species A. thaliana, a GSNO reductase (AtGSNORl) was characterized. It was demonstrated that AtGSNORl controls NO bioactivity, suggesting that GSNO is a molecule present in plants that potentially can act as an NO transporter [33]. It is known that an active interconversion exists between RSNO and iron nitrosyl compounds. RSNO and DNIC are capable of interconverting depending on the concentrations of iron, low-molecular-weight thiols and NO in the cell. The levels of both DNIC and RSNO decrease when thiols content decreases. When NO level decreases while the concentration of thiols and iron remains constant, DNIC and RSNO serve as donors of NO which is transferred to the acceptor cell. In addition, it was reported that iron induces the destabilization of RSNO leading to the formation of DNICs [34-36]. This biological system would need a small amount of a reducing agent. Ascorbate could be a good candidate to maintain Fe in its reduced form Fe2+ [37]. Even NO can reduce Fe3+ to Fe2+ when it is linked to high-molecular-weight compounds.

DNICs IN PLANTS

DNICs have been extensively studied in mammals and measured under various different physiological conditions [38-40]. Detection of DNICs in plants by EPR faces the problem that there are relatively high manganese levels in plant tissues. One of the hyperfine splitting components of EPR signal from manganese complexes mask the 2.03 EPR signal of DNICs [36, Graziano, Simontacchi, Puntarulo and Lamattina, unpublished results]. Therefore, the detection limit for DNICs by EPR in plants is higher than in animals. Formation of DNICs from endogenous-free iron and thiol-containing ligands was first demonstrated in isolated leaves from bean and China rose treated with exogenous gaseous NO [41]. Recently, it was shown that treatment with high concentrations of nitrite, a condition that stimulates NO production, induced the formation of DNICs in isolated China rose leaves [23]. Incubation of the leaves with nitrate, a competitive inhibitor of the NR-dependent NO-synthesis reaction, resulted in decreased signal intensity of DNICs, demonstrating that NR activity is the main source for NO synthesis in those conditions. This landmark work demonstrated that DNICs could be synthesized in plants not only from endogenous thiol-containing ligands and iron pools but also from endogenously-produced NO. The concentration of the complexes accumulated over 1 h was found to be 8 nmol-g-1 leaf FW (fresh weight). Thus, the levels of this nitrosylated iron pool are sufficiently high that its formation may function as a metabolic process for maintaining iron bioavailability under conditions of iron starvation [15,23].

PLANT IRON NUTRITION: AN OVERVIEW

Iron fulfills a vital role in virtually all living organisms from bacteria to animals. Iron ability to undertake one electron oxidation-reduction reactions places this molecule in the center of key biochemical processes like oxygen transport, ATP generation and the rapid reaction with free radicals. Iron can vary its redox potential in response to different environmental conditions and place this transition metal as an important intermediate for electron-transfer reactions and thereby with essential properties for electron transport chains in respiration and photosynthesis. The fact that iron deficiency affects more than 30% of the world's population [42] confirms its relevance and places it as a subject for many research projects worldwide.

Iron is a critical element for many central metabolic pathways during the whole plant life cycle. Low iron availability leads to growth arrest and chlorosis while increased amounts of iron leads to increased formation of reactive oxygen species (ROS), loss of selective membrane permeability and generalized tissue damage. Thus, the control of mechanisms that regulate iron homeostasis is crucial for plant survival [43,44]. Under iron-limiting conditions, plants develop iron deficiency symptoms characterized by leaf chlorosis, impaired chloroplast development and decreased expression of photosynthetic proteins [15,45] that could limit plant growth and survival.

In soils and in aerobic environment, iron is mainly in the ferric [Fe3+] form. This iron is not easily accessible for plants because it is highly insoluble at neutral or basic pH. Thereby, plants have evolved mechanisms to facilitate iron acquisition from soil which include the solubilization or chelation of iron into solution. In general, non-graminaceous plants have developed a mechanism named Strategy I based on the reduction of Fe3+ to the more soluble form Fe2+ and its uptake through specific iron transporters. The proteins involved in iron reduction and transport at the root level were already characterized in several plant species [46]. On the other hand, graminaceous plants use a mechanism termed Strategy II which is based on the chelation of Fe3+ through the release of specific Fe3+ chelators called siderophores [43,46,47].

Once iron is inside the root cell it is reduced or de-chelated. However, under aerated conditions part of the iron is oxidized and precipitates as hydroxide or phosphate salt, forming an extracellular or apoplastic iron pool [48]. This pool comprises up to 95% of total root iron content in hydroponic culture and can be used under conditions of iron deficiency [44]. Free iron has to be shielded from oxygen to prevent precipitation and generation of oxygen radicals. It has been proposed that organic acids (citrate) or non-proteinogenic amino acids (nicotianamine) are the molecules responsible for chelation and relocation of iron to the aerial part of the plant through the transpiration stream; however, this process is rather unclear. Once in the leaves, Fe3+-citrate is probably a substrate of a ferric-chelate reductase already described in mesophyll cells [49]. The reduced Fe2+ form is thought to be internalized by cells. The traffic of iron through phloem is far less documented in spite of the confirmed iron presence in phloem. It was suggested that nicotianamine is the main candidate for iron transport in phloem [50]. Nevertheless, a metal-binding protein was demonstrated to transport iron in Ricinus communis [51].

As was highlighted above, much is known about iron uptake mechanisms at the root level, but there is no clear evidence about how plants transport iron between shoot and roots and also between cells and inside the cells. Indeed, iron mobilization inside leaf mesophyll cells is a limiting step for iron acquisition [49]. Moreover, little is known about the molecules and cellular regulatory processes that sense and/or perceive iron status in plants and coordinate the molecular changes that finally execute the adjustment of adaptive responses leading to iron homeostasis. Very poor and fragmented information is also available on the molecules and regulatory mechanisms involved in intracellular iron transport and even in the compartmentalization and delivery of iron within the cells and organelles.

NITRIC OXIDE AND DNICs PARTICIPATION IN PLANT IRON METABOLISM

Recent works have demonstrated that NO is emerging as a new player in plant iron metabolism. It was shown that, in maize plants growing under low-iron conditions, application of the NO donors sodium nitroprusside (SNP) or S-nitroso-acetylpenicillamine (SNAP) or even gaseous NO completely prevent the development of iron-deficiency symptoms. Moreover, NO was able to revert the iron deficiency phenotype of the ys1 and ys3 maize mutants, impaired in iron acquisition. Interestingly, total iron content in those plants was not increased by NO treatment, suggesting that probably an NO-mediated improved efficiency in iron mobilization within the plant occurs [15]. Thereafter, it was found that iron-deficiency symptoms were also prevented by NO in tomato [Graziano and Lamattina, unpublished results], a plant belonging to the Strategy I responses for iron acquisition. Interestingly, as was previously described in young human patients with anemia [53], plants respond with an induction of NO production when growing under iron-deficient conditions. The source for this NO synthesis might be NR, since NO accumulation was blocked by the NR inhibitor tungstate and not by the NOS-inhibitor l-NAME [Graziano and Lamattina, unpublished results].

In order to explain NO participation in plant iron availability, we have previously proposed that a complex between iron and NO is synthesized in the form of low-molecular-weight DNIC [54]. It was also proposed that there is an interconversion between low-molecular-weight S-nitrosothiols (RSNO) and DNIC, which equilibrium depends on the NO and iron state of the plant. During low-iron supply, it has been reported that glutathione (GSH) levels are increased in roots [55] as well as NO production [Graziano and Lamattina, unpublished results]; therefore, RSNO synthesis may be stimulated under iron-deficient conditions in plant roots. Since iron destabilizes RSNO and induces the formation of DNICs, iron uptake from soil to the root cell would favor DNIC synthesis. These low-molecular-weight complexes may be transported to the aerial parts of the plant and also between and inside cells. According to the equilibrium between synthesis and degradation of DNICs, these complexes would decompose inside leaf mesophyll cells releasing iron (Fig. 1). This would be a mechanism for transporting not only iron but also NO between plant organs and cells and even

Fig. 1. Simplified model that represents the putative NO and DNIC involvement in plant iron sensing and transport. Iron deficiency stimulates GSH (RS-) and NO production in root cells, inducing RSNO synthesis. Iron uptake from soil destabilizes RSNO and favors DNIC formation. DNIC is transported to the aerial tissues through the plant vascular system and is decomposed inside leaf mesophyll cells releasing iron. RSNO could be newly synthesized in leaf cells and transported to roots.

Fig. 1. Simplified model that represents the putative NO and DNIC involvement in plant iron sensing and transport. Iron deficiency stimulates GSH (RS-) and NO production in root cells, inducing RSNO synthesis. Iron uptake from soil destabilizes RSNO and favors DNIC formation. DNIC is transported to the aerial tissues through the plant vascular system and is decomposed inside leaf mesophyll cells releasing iron. RSNO could be newly synthesized in leaf cells and transported to roots.

within organelles. When NO production is stimulated or NO is exogenously applied, it was shown that DNIC formation is induced [23]. Therefore, the addition of NO donors to plants growing under low-iron supply could stimulate DNIC formation and, consequently, iron mobilization.

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