The complexity of nitric oxide generation and function in plants

Plants are exposed to environmental stress, in natural and agricultural conditions.Nitric oxide (NO), a small gaseous molecule which plays important roles in plants, has been involved in many physiological processes, and emerged as an important endogenous signaling molecule in the adaptation of plants to biotic and abiotic stress. NO is produced from a variety of enzymatic and non enzymatic sources, which are not yet fully understood. Also, NO and reactive nitrogen species (RNS) can produce posttranslational modifications affecting protein function. Nitrate reductase, a key enzyme in the nitrogen metabolism, is a proposed source of NO in plants which could be affected by posttranslational modifications.Thus, different pathways seem to be involved and can also regulate NO synthesis in the plant cell under physiological or stress conditions. However, how the levels of NO are reached in such time and place to fulfill its functions, are still puzzles to elucidate.

Plants are frequently exposed to environmental stress, both in natural and agricultural conditions.Acclimation to environmental stress results from an integrated series of events, occurring from the anatomical and morphological levels to the cellular, biochemical, and molecular levels (Taiz and Zeiger, 2010).As abiotic stress conditions can lead to growth restriction, acclimation responses become the plant priority.Stress induced-plant growth reduction may be followed by an alteration in the redox state, leading to oxidative and/or nitrosative stress, due to an increase in reactive oxygen species (ROS), or in reactive nitrogen species (RNS) respectively.Also, ROS and RNS can produce protein posttranslational modifications (PTM) affecting their function.Nitrated proteins have been detected in plants of tobacco, soybean, and Arabidopsis (Morot-Gaudry-Talarmain et al., 2002;Jasid et al., 2009;Lozano-Juste et al., 2011).Moreover, nitration and S-nitrosylation have been involved in acclimation to salinity stress.Tanou et al. (2012) showed that ROS/RNS-mediated protein post-translational modifications are a key molecular strategy for signaling transduction and salinity acclimation.
Nitric oxide (NO) is a small gaseous molecule which plays important roles in plants.It has been involved in many physiological processes and emerged as an important endogenous signaling molecule in the adaptation of plants to biotic and abiotic stress.A role for NO, and in some cases S-nitrosothiols (SNOs), has been suggested in a variety of stress responses, including drought, salt, heat, cold and heavy metal stress (Yu et al., 2014).
Most of the published studies demonstrated accumulation of NO under stress conditions (Saxena and Shekhawat, 2013).However, it cannot be considered a general stress response.During plant responses to cadmium stress, NO was increased or decreased, acting as inducer or inhibitor of stress tolerance (Arasimowicz-Jelonek et al., 2011).Also, iron deficiency triggered NO signaling in Arabidopsis thaliana (Chen et al., 2010) but repressed basal NO synthesis in Zea mays (Kumar et al., 2010).
It is possible that NO may confer abiotic stress tolerance in part by functioning as an antioxidant, as has been reported by several authors (Laspina et al., 2005;Hasanuzzaman et al., 2011;Verma et al., 2013).The relevance of NO in stress-induced redox signaling was investigated by treatment of plants with NO donors before or during exposure to abiotic stress conditions.NO treatments either reversed the stress-induced decline, or even further amplified up-regulation of the antioxidant defense system, concomitantly with a reduction in H 2 O 2 accumulation and lipid peroxidation (Hasanuzzaman et al., 2010).
In plants, NO is produced from a variety of enzymatic and non enzymatic sources, which are not yet fully understood, and are still under study (Table 1).Higher plants seem to have lost nitric oxide synthases (NOSs) in the course of evolution (Fröhlich and Durner, 2011).NOSs are present in almost all known organisms except plants, where neither the gene nor any protein with high sequence similarity to known NOS have been found (Lamattina et al., 2003).However, a NOS-like activity L-Arginine (L-Arg) dependent, has been highly reported in plants, together with a nitrate reductase (NR) dependent pathway.Rasul et al. (2012) have suggested that L-Arg and NR pathways are co-involved in NO production and do not work independently.Part of the NO produced by L-Arg dependent pathway could be oxidized to nitrite, providing substrate for NR-dependent NO synthesis.Soybean cotyledons, growing in the presence of ammonia (without nitrate), were able to produce similar amounts of NO showing that different sources could operate for NO accumulation in soybean cotyledons (e.g.nitrite-and L-Arg-dependent sources).It is likely that under different physiological or stress conditions, one pathway could result more operative depending on the substrate availability to maintain or increase NO generation supporting the required levels (Galatro et al., 2014).However, NO generation was reduced in ammonium-fed tobacco plants where nitrogen assimilation bypassed the NR step, and compromised immune responses (Gupta et al., 2013).
NO is a free radical which diffuses readily through biological membranes and has a biological half-life ranging from 5 to 15 s (Gupta et al., 2011).This short half-life reflects the highly reactive nature of the molecule: it reacts with metal complexes and other radicals, and with biomolecules such as nucleic acids, proteins and lipids (Gupta et al., 2011).Prolonged exposure to stress may result in enhanced production of NO and its derivatives, resulting in nitrosative stress.The nitrosylation of lipids, proteins and nucleic acids leads to severe metabolic impairment and degradation of cellular metabolites leading to programmed cell death (Krasylenko et al., 2010;Misra et al., 2011).S-nitrosylation of proteins, also known as S-nitrosation, constitutes the most studied and described NO-dependent posttranslational modification in plants.It refers to the reversible covalent binding of an NO moiety to the thiol group of a cysteinyl residue (Cys) of a target protein, to produce an S-nitrosothiol (SNO) (Astier et al., 2012).S-nitrosylation may be integral to NO function during a variety of cellular processes (Simontacchi et al., 2013).Depending on the target protein concerned, this PTM will lead to a modification of its enzymatic activity or its protein function.
Polyamines (PAs), nitrogenous aliphatic compounds, also appear to be involved in the regulation of NR activity.PAs form H 2 O 2 during their catabolism, and are also NO producers by still unknown mechanisms (Yamasaki and Cohen, 2006).NO mediates spermine-induced reduction in root elongation in wheat plants (Groppa et al., 2008).The PAs putrescine, spermidine and spermine induced a biphasic response in NR activity, inhibiting the enzyme activity at short incubation times (3h) and stimulating it at longer exposition times (21h) (Rosales et al., 2012).NO is involved in this response, which could be reverted employing the NO scavenger cPTIO.
In addition to the multiple metabolic pathways that lead to NO formation, they can occur in different cell compartments (Table 1).Chloroplasts are proposed sites of NO generation under physiological and stress conditions (Foissner et al. 2000;Arnaud et al., 2006;Jasid et al., 2006;Galatro et al., 2013;Tewari et al., 2013).Gas et al. (2009) proposed that chloroplasts are key players for the control of NO levels in the plant cell.It was shown that chloroplast function positively affects NO levels not only in this organelle, but also in the whole tissue (Galatro et al., 2013).NO detection go along with maximum chlorophyll content, and quantum yield of photosystem II (ΦPSII) in soybean cotyledons highlighting a role for chloroplast functionality in NO generation as it was previously proposed (Galatro et al., 2013).
Thus, different pathways are involved, work together, and also modulate NO production in the plant cell under physiological or stress conditions.However, how the levels of NO are reached in such time and place to fulfill their functions, and how NO can regulate its own synthesis, are still puzzles to elucidate.
NO metabolism in plants is still a challenge.It is needful to identify the ways and sources of NO formation as well as the entry points of NO at the signaling metabolic network during normal or stress physiology.Other intriguing point is how PAs contribute to NO generation, and the physiological significance of other proposed sources (as hydroxilamine or non-enzimatic NO generation).
The elucidation of NO multiple pathways in plants will help to understand plant strategies to withstand stress and, in this way, to contribute to develop plants species with higher tolerance to stress.