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Plant Signal Behav. 2010 March; 5(3): 209–212.
PMCID: PMC2881262

NO says more than ‘YES’ to salt tolerance

Salt priming and systemic nitric oxide signaling in plants


Nitric oxide (NO) is now recognized as an important signaling molecule and there has been an increasing bulk of studies regarding the various functions of NO in plants exposed to environmental stimulus. There is also emerging evidence, although not extensive, that NO plays systemic signaling roles during the establishment of salt tolerance in many plant species. In this mini-review, we highlight several candidate mechanisms as being functional in this NO systemic signaling action. In addition, we outline data supporting that plants possess prime-like mechanisms that allow them to memorize previous NO exposure events and generate defense responses following salt stress.

Key words: nitric oxide, nitrosative stress, priming, salinity, systemic signaling


In recent years, nitric oxide (NO) has been recognized as an important endogenous signaling molecule that orchestrates multiple defense responses to both abiotic and biotic stress.1 There is an ever-growing list of studies demonstrating that exogenous application of NO, possibly acting in conjunction with other signals, enhances salt tolerance in various plant systems.29 Moreover, it has been proposed that exogenously applied NO can induce tolerance to salinity not only in treated tissues but also in distant non-treated tissues, indicating that NO triggers systemic biological responses during salt stress.2,7,8

Salinity imposes both ionic toxicity and osmotic stress to plants, leading to secondary stresses such as nutritional disorders and oxidative/nitrosative events; therefore tolerance to salinity is a multifaceted trait involving a web of various signaling networks and cross-talk between different sensors and signal transduction pathways.10,11 However, the increasing extent of damage caused to agriculture by salinity underscores the need to develop crops and plants with enhanced tolerance to salt stress.1012 Recently, the phenomenon of priming, which involves prior exposure to a stressful factor making a plant more resistant to a future stress imposition, raised major interest among researchers and opened up new perspectives in plant stress physiology.13,14 Since there is now experimental evidence that pre-exposure to NO efficiently protects salt-grown plants,2,7,8 it is likely that a NO pre-treatment can serve as a model pre-stress system to study priming traits affecting plant salt tolerance. This review outlines up-to-date progress in terms of the regulation of NO-mediated plant responses to salinity with an emphasis on the interplay between NO signal transduction pathways and the priming-derived tolerance to salt stress.

Nitric Oxide is Associated with Induced Plant Tolerance to Salinity

Plant response to salt stress are controlled at the molecular level by multiple extracellular and intracellular signals.1012 Increasing evidence, based largely on experiments in which whole plants or cell cultures were treated with NO donors, revealed that NO occupies a central place in this signaling network to induce tolerance against salinity.29,15,16 In parallel, Arabidopsis mutant (Atnoa1) plants with an impaired in vivo NO synthase (NOS) activity exhibit hypersensitivity to salt treatment.6 Although these examples suggest that NO shapes cellular signaling responses against salinity, however, our understanding of how NO participates in this signaling pathway remains rudimentary. Several experiments using NO donors and NO inhibitors indicated that NO serves as a signal in inducing salt tolerance by increasing the Na+/K+ ratio, which was dependent on the increased plasma membrane (PM) H+-ATPase as well as vacuolar (V) H+-ATPase and H+-PPase activities.3,4,9 Therefore, the NO-mediated regulation of Na+ homeostasis and K+ acquisition via increased expression of plasma membrane Na+/H+ antiporter and H+-ATPase-related genes may represent an important salt tolerance mechanism. On the other hand, Uchida et al.2 demonstrated that sodium nitroprusside (SNP, a NO donor) induces not only expression of transcripts for stress-related genes (e.g., sucrose-phosphate synthase, Δ′-pyrroline-5-carboxylate synthase, small heat shock protein 26) but also enhances ROS-scavenging enzymes activities in salt-stressed rice seedlings. Liu and co-workers5 reported that the increase of nitrate reductase-dependent NO production in red kidney bean roots under salt stress enhanced the activities of antioxidant enzymes by controlling the NADPH levels via glucose-6-phosphate dehydrogenase (G-6-PDH) activation. Currently, the antioxidant-based protective effect of NO in salt stress was confirmed in citrus plants. Tanou et al.8 showed that exogenously introduced NO effectively induced antioxidant enzymes activity, promoted the maintenance of the cellular redox homeostasis and mitigated the oxidative damage produced by Fenton-like reaction-mediated OH* generation under salinity. Despite the obvious complexity, these results support the idea that NO exhibits an antioxidant role during the establishment of salt tolerance.

Nitric Oxide Priming for Salt-Associated Tolerance

Although manipulation of antioxidant genes seems to be a sound approach to counteract salt-induced oxidative stress, however, the genetically complex mechanisms of salt stress tolerance make the task extremely difficult.13 Recently, it is thought that priming factors are able to reduce the effects of salt stress in plants.17 Besides being well-established in the biotic stress, analogous priming phenomena have also been currently characterized under abiotic stress conditions.1012 In general, “defense priming” describes a process in which immunity-like responses to a stress are accelerated, enhanced or potentiated by prior stimulation. It is now recognized that the oxidative stress caused by salinity is also accompanied by an accumulation of NO and other reactive nitrogen species (RNS), leading to the nitrosative stress.18,19 Questions then arose whether the NO-derived nitrosative stress events are involved in the phenomenon of salt priming. In support to this hypothesis, there is evidence showing that salt-stressed plants altered their physiognomy, physiology and metabolism in response to a prior NO experience. A typical example of NO-associated salt priming action is the enhanced tolerance of both halophytes and glycophytes plants to salinity during germination and early seedling stages after pre-exposure of their seeds to NO donors.15,16 One of the first observations that recognized that NO pre-exposure participates in the protection of whole plants against salt stress comes from the work of Uchida et al.2 who showed that the application of 1 µM SNP as a NO donor for 2 days, markedly promoted the survival of rice seedlings following exposure to 100 mM NaCl. In line, NaCl-imposed phenotypic and physiological disturbances in citrus leaves were partially prevented by root pretreatments with 100 µM SNP for 2 days.7,8 These data provide evidence that a prior exposure to NO, as mimicked by SNP pre-exposure, may act as priming agent capable of rendering plants more tolerant to subsequent exposure to salt stress. In support, an early phase of NO generation followed by a later phase of NO formation could have an important role in acclimation of plants to osmotic stress.20 Analogiously, an early NO burst and a subsequent wave of NO generation enhance the resistance of plants to pathogens,21,22 further indicating that the biphasic timing of NO and plant cell contact is a key feature for enhanced defense against both biotic and abiotic stress.

Mechanisms Underlying the NO-Dependent Prime-Like Action Under Salinity

Although NO elicits ion homeostasis and antioxidant-related defense obviously represents a survival cellular response under salinity it remains elusive how NO signaling can induce the whole plant salt tolerance. Significant progress toward the recognition of additional key regulators of NO-responsive signaling network to stressful conditions has been made during the last years. Transcriptomic and proteomic studies led to the identification of numerous NO target genes and proteins under several (patho) physiological processes.2327 However the identification and characterization of the specific targets of NO in salt-stressed plants is only beginning to emerge. A recent proteomic study by Tanou et al.7 in citrus plants provides a global survey of the proteins whose accumulation levels are controlled by salt stress. Intriguingly, root pre-treatment with SNP prior to impose salt stress reversed a large part of the NaCl-responsive proteins. These SNP/NaCl-responsive proteins, which are mainly involved in photosynthesis (corresponding to enzymes playing a role in the Calvin-Benson cycle), defense mechanism and energy/glycolysis, are potentially important for salt tolerance as these may exert a NO-mediated priming effect by acclimating citrus plants before the actual experience of salt stress. The data discussed above indicate that a NO pre-exposure can specifically modify protein expression signatures, and that a NO specific function is needed for a proper salt tolerance response.

A body of evidence suggests that the majority of NO-affected proteins seem to be regulated by S-nitrosylation, which occurs by oxygen-dependent chemical reactions or by the transfer of NO from a nitrosothiol to a sulfhydryl group of a cysteine residue (Cys).28 Cysteine residues may be also oxidized upon oxidative stress29 and this oxidative carbonylation of proteins could contribute to cellular signaling.30 In addition, protein tyrosine nitration, the addition of a nitro group to one of the two equivalent ortho carbons of the aromatic ring of tyrosine residues, has emerged as a surrogate marker reflecting the nitrosative stress conditions associated with NO-based signaling.28,29,31 The study of Tanou et al.7 also revealed that NO pre-exposure before salt stress in citrus alleviated salinity-induced protein carbonylation and provoked protein S-nitrosylation, thereby providing strong evidence that NO achieves its priming function through chemical crocs-talk among different Cys post-translational modifications; these redox-based modifications have been implied in controlling the activity of several proteins (e.g., rubisco activase, glyceraldehyde-3-phosphate dehydrogenase, ATP synthase, aldehyde dehydrogenase and transketolase) under salinity. On this basis, it is possible that S-nitrosylation may lock the structure of specific proteins in a state under which they are less sensitive to irreversible carbonylation provoked by salt-induced ROS overproduction.7 Meanwhile, in salt-stressed olive plants a significant increase in the number of proteins that undergo tyrosine nitration has been detected by immunoblotting and confocal laser scanning microscopy analysis using a specific antibody against 3-nitrotyrosine.18,19 These results support the idea that, along with a general proteome reprogramming, protein posttranslational modifications, together or separately, could contribute to NO cell signaling during salt stress.

Evidence for NO Systemic Signal Signature Induced Tolerance against Salinity

Nowadays, the signaling function of NO and the coordinating response are a central theme in plant physiology. An interesting point in the previously mentioned works of Uchida et al.2 and Tanou et al.7,8 is that root applied pre-treatments with a NO donor induced salt-specific responses in the leaves of the pre-treated plants. These two observations may be linked, in that NO elicits systemic responses able to face salt stress conditions. Supportively, Piterkova et al.22 provided evidence for the transmission of NO systemic responses throughout the tomato plants exposed to biotrophic pathogen infection, signifying that the systemic NO action is a key metabolic process required to achieve efficient resistance against different types of stress. In addition, it has previously been shown that the priming mechanism involved in the systemically induced tolerance phenomena against salinity. For example, root colonization by the endophytic fungus Piriformospora indica confers enhanced tolerance to salt stress in distal leaves of barley.17 Thus, a question arises: how NO signaling function can be systemically transmitted under salt stress?

An important factor of NO signaling regulation under salinity is its spatial production which is tightly linked with the aspect of NO mobility and communication between different cellular compartments. Using the specific NO-sensing fluorochrome 4,5-diaminofluorescein diacetate (DAF-2 DA), NO was detected in vascular bundles of olive and citrus leaves exposed to high salinity and was suggested to have specific functions in the salt stress signaling responses.8,18,19 Another important aspect of NO signaling during salt stress is the generation of S-nitrosoglutathione (GSNO), a transporting glutathione-bound NO molecule. Cross-reactivity with an anti-GSNO antibody and immunofluorescence microscopy analysis revealed that GSNO was mainly localized in spongy mesophyll and vascular tissues of leaves exposed to salt stress.19 Together with previous finding that GSNO produced during pathogen attack is loaded into the phloem, systemically dispersed, and unloaded to initiate systemic acquired resistance (SAR) activation,32 the collective data highlight that the redistribution of NO reaction products from vascular tissues could serve as a mobile signal in stress-associated defense processes. In addition, Valderrama et al.19 found evidence for tyrosine nitration of phloem proteins upon salt exposure. One mode of NO action in the phloem could be, therefore, the tyrosine nitration of proteins, which meets a variety of important criteria that underscore NO signaling function under stressful conditions.18,28,29,31 Furthermore, several molecules, such as salicylic acid, lipid-derived molecules, cytosolic Ca2+, peptides or RNA species have been suggested to be putative short- or long-distance phloem signals mediating the onset of an array of NO-dependent defense mechanisms.32 However, it is likely also that NO does not act alone, but interacts with other salt-depended signaling molecules in order to establish systemic defense responses. For example, phytohormones (e.g., auxin, abscisic acid, ethylene, jasmonic acid) may be transported from salt-treated roots to leaves and therein to induce NO synthesis or alternatively these molecules could trigger NO transport throughout the plant. Other important systemic signaling functions are achieved through NO effect on major protein kinases, such as mitogen-activated protein kinases (MAPKs), which play major role in plants responses to salt stress.33 It was shown that NO can exert its modulating function on MAPKs activity through the whole plant, as exposure of A. thaliana roots to SNP-driven nitrosative stress can induce a rapid activation of protein kinases with MAPK-like properties in shoots.34 Another possibility is that NO systemic signaling function may be mediated by arginine-dependent NO production. In fact, de novo arginine biosynthesis in leaves has been described as a response of plants to salinity35 whereas an enzymatic L-arginine-dependent production of NO (NOS activity) has been demonstrated in leaf extracts from salt stressed plants.19 However, the possibility for a nitrite-dependent inducible NO synthesis or transport in phloem tissue can not be excluded. Another scenario might be that NO exhibits its signaling action through the ‘H2O2 and NO signaling cross-talk’, as classically described in the stomata.36 In this regard, Tanou et al.7 showed that tolerance to salinity could arise in planta through interaction of NO with H2O2.

Alternatively, it is possible that the cell-to-cell and/or tissue-to-tissue NO signaling can be mediated by the various RNS which were generated during the strong interaction of NO with ROS, particularly evidenced under salt-derived oxidative/nitrosative stress situations.19 Hence, it is possible that in all these situations, NO does not act in isolation, but in concert with other signals, such as ROS and hormones, to control the development salt tolerance mechanisms. Clearly, the possible existence of a systemic NO signaling action, the mechanism by which this occur, and its potential role in the regulation of salt tolerance responses are all worthy of further investigation.

Critically, it is significant to note that, in parallel to salt-specific NO systemic signaling, alternative modes of NO action such as constitutive signaling mechanism, i.e., continuously active at physiological conditions, may also occur.8 Finally, it is worth mentioning that NO is typically applied to NaCl-treated experimental plants through various NO donors which may not mimic physiological situations. Therefore, it is possible that the signal transduction pathway activated by exogenously applied NO donors-produced NO may differ from the pathway induced by salt stress-driven endogenously NO generation, as previously suggested for the extracellular and intracellular ROS signaling in salt-stressed plant cells.37

Conclusions and Perspectives

The recent evidence discussed here documents that NO acts as transmissible signal mediating long-distance plant salt tolerance responses. Nitric oxide bound to proteins or indirectly, that is, following interaction of NO with signaling molecules such as ROS or phytohormones seems to be a good candidate for such a signaling function. However, our limited knowledge of NO-associated specific metabolism together with the ongoing debate relative to the source of NO in plants is still a major gap in our understanding of NO specific function under physiological and salinity conditions. Therefore, continuous application of newly available sophisticated methodology, such as genome-wide expression analysis, proteomics, metabolomics, forward and reverse genetics will identify the NO-responsive gene, protein and metabolite networks and will contribute to the generation of new hypotheses. Particularly, how NO can exert important redox-based protein posttranslational modifications is a crucial piece of the salt tolerance puzzle. A major challenge in our attempts to understand how NO regulates signal transduction under salinity is also to determine the precise site of NO generation, metabolism and action. Finally, an understanding of the cross-talk between signaling pathways linked to reactive oxygen and nitrogen species is also a major issue for elucidating the mechanisms underlying nitric oxide-dependent plant salt tolerance.


The authors thank Dr. Dominique Job (CNRS/Bayer CropScience Joint Laboratory, Lyon, France) for the thoughtful remarks and the overall contribution. G.T. is grateful to the State Scholarship’s Foundation of Greece for a fellowship. We apologize to the colleagues whose work could not be cited here because of space restrictions.



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