Nitric oxide and phytohormone interactions: current status and perspectives

1As sessile organisms, plants must rely on highly sophisticated signaling mechanisms to adjust their growth, shape, and metabolism with the constant changes in their environment. Playing a key role in this process, plant hormones integrate a multitude of internal and external cues into coordinated metabolic and developmental responses, which, in turn, maximize plant fitness under diverse ontogenetic and environmental contexts. To effectively carry out such critical function, distinct plant hormones intensively interact among themselves and also with other endogenous signaling substances.

2Among these hormone-interacting molecules, the gaseous free radical nitric oxide (NO) has recently gained special interest in the research community given its involvement in a number of signaling cascades controlling plant responses ranging from seed germination to plant senescence. Whereas great strides have been made in recent years in understanding the mechanistic relationship between NO and phytohormones in certain physiological responses, the exact nature of the interaction between these substances in many developmental, metabolic, and defense events still remains remarkably elusive. In some cases, for instance, it is known that both NO and plant hormones are able to influence a given response, but it is not clear whether they share a common signaling cascade or just modulate the same plant event via parallel, independent signaling pathways.

3As mentioned by, characterizing the precise function of NO in a particular signaling event is more difficult than it might appear. Firstly, the particular chemical characteristics of NO inexorably imply peculiar mechanisms for “sensing” the presence and levels of this signaling molecule. Instead of a unique or very few receptors, NO likely interacts with a wide range of target proteins via direct modification of protein structure (Figure 1). Through these chemical modifications of target proteins, NO may trigger changes in their activities and cellular functions, ultimately leading to the transduction of the NO message into plant responses.

4Among the biologically relevant NO-dependent post-translational modifications (PTMs), the covalent modification of cysteine residues through a processes known as S-nitrosylation (Figure 1A) has been emerging as a critically important mechanism intermediating NO signal transduction in plants. This specific, reversible and regulated NO-dependent PTM has been implicated as potentially controlling the function of components of plant processes as diverse as cellular architecture, photosynthesis, genetic information processing, protection against oxidative stress, defense responses to biotic and abiotic stresses, hormonal signaling, among others. Currently, some of the best characterized examples of S-nitrosylation in plant systems include the modulation of phytohormone biosynthetic enzymes, receptors, and signal transduction proteins, which will be discussed in more detail later in this review. The specificity of this NO-triggered PTM is essentially based on the fact that only cysteine residues surrounded by particular neighboring amino acids seem to be the target of S-nitrosylation.

5A second physiologically relevant NO-dependent PTM depends on the reaction between NO and reactive oxygen species (ROS), such as superoxide, resulting in the production of NO-derived species, such as peroxynitrite (ONOO-), which, in turn, can covalently modify tyrosine residues through a process known as tyrosine nitration (Figure 1B; ). Initially considered an irreversible process, tyrosine denitration is now believed to occur either enzymatically or non-enzymatically. Reinforcing such reversibility in tyrosine nitration, transient, rather than permanent, changes in the abundance of nitrated proteins have already been reported in the literature. More research is required to better define the biological relevance of this NO-dependent protein modification in plants, which apparently may target proteins involved in many basic cellular processes, such as photosynthesis, respiration and nitrogen metabolism

6In addition to S-nitrosylation and tyrosine nitration, a third important NO-dependent PTM involves the binding of NO to transition metal centers of metalloproteins in a process known as metal nitrosylation (Figure 1C). Currently, one of the best characterized examples of metal nitrosylation is the activation of soluble guanylate cyclase (sGC) in animal systems. In plants, although cyclic guanosine monophosphate (cGMP) has already been reported as an important intermediate in several NO-induced processes, including root development, mitochondrial respiration, nodule functioning, and defense responses, more studies are still required to clarify whether metal nitrosylation also regulates plant sGC.

7Regardless of the specific type of NO-triggered PTM considered, these chemical modifications may represent a central mechanism through which NO impacts signaling networks responsible for controlling plant development and metabolism. In responses regulated by plant hormones, for instance, these PTMs might facilitate the influence of NO on hormonal production and/or action via three distinct but non-exclusive mechanisms. The first mechanism implicates NO-dependent chemical modifications of proteins (e.g., transcription factors, regulatory proteins, and channels) whose functions may not be directly implicated in plant hormone metabolism, distribution, or signaling but, instead, may influence the abundance of other proteins more intimately implicated in such specific roles (Figure 2A). In contrast, a second and more direct way involves the NO-triggered PTM of proteins directly associated with the production, degradation, conjugation, transport, perception, or signaling transduction of plant hormones (Figure 2B). For example, in the first mechanism, NO may chemically modify a transcription factor that stimulates the production of a hypothetical enzyme responsible for hormone degradation, whereas in the second mechanism, NO would directly interact and modify the activity, stability, and/or cellular localization of this degradative enzyme (Figures 2A, B). A third possibility recently described in the literature involves the direct chemical reaction between NO-derivates (e.g., peroxynitrite) and certain hormonal species (e.g., zeatin), rendering products with altered biological activity (Figure 2C). Specific examples of all three of these mechanisms of NO–phytohormone interaction will be provided and discussed later in this review.

8Considering that a massive number of proteins, peptides, and other molecules may undergo changes in their structure and activity via direct NO-dependent chemical modifications and an equivalent amount of genes may have their transcription levels influenced by NO, one pertinent question that arises is how NO signals can confer sufficient specificity to trigger coordinated downstream effects. Although answering this question involves a certain degree of speculation at this point in the research of NO signaling in plants, aspects such as spatial and temporal signaling compartmentation and a precise control of NO biosynthesis and removal might possibly be key to explaining how a molecule as small as NO might be responsible for controlling so many plant responses.

9As is the case with cytosolic Ca2+, a strict temporal and spatial regulation of NO levels inside each plant cell might be essential for delivering sufficiently specific NO signals. The transient generation of “NO hot-spots,” in particular plant cell compartments, could lead to compartmentalized protein modifications, and, consequently, the NO signals may be sensed by a specific group of proteins responsible for a particular set of cellular functions. A possible mechanism for assuring such localized action of NO could be the existence of macromolecular modules including all major NO signaling components (e.g., NO biosynthetic enzymes, NO removal enzymes, and targets of NO-dependent PTMs). Although such macromolecular complexes have not yet been described in plants, recent models for NO-mediated stress signaling in animal systems suggest, for instance, that the control of certain membrane calcium channels via reversible S-nitrosylation is facilitated by the close proximity of these channels to the NO-generating enzyme. Therefore, in this case, instead of a global change in cellular NO levels, the transient production of this signaling molecule at particular regions of the animal cell may control the activity of nearby target proteins via reversible S-nitrosylation. As an ultimate consequence, such compartmentation and fine-tuned dynamics of NO production could minimize a certain spatial promiscuity in terms of concomitant occurrence of NO, NO-derivates, and their target proteins.

10A relevant bottleneck for advances in the evaluation of the possible existence of such NO signaling macromolecular modules in plants is the still incipient characterization not only of the targets of NO-dependent PTMs but also of the biosynthetic and removal machinery responsible for controlling NO levels inside the plant cell compartments. Interestingly, though, compartmentalized production of NO has already been reported in plant cells., for example, reported that after challenging epidermal tobacco cells with the elicitor cryptogein, NO accumulation first appeared in the plastids and subsequently in other cell compartments, such as the nucleus and the cytoplasm.

11Placing NO as an element of a given signaling cascade necessarily implies that changes in its levels or cellular localization might occur during the course of the signaling event. Therefore, characterizing the specific changes in the NO biosynthetic and degradation mechanisms responsible for delivering adequate concentrations of this molecule at the right time and place seems a logical step in any research interested in discriminating the actual role of NO during the regulation of specific plant responses. However, the relevance of the different origins of NO in plants is still poorly understood; as a consequence, controversy and ambiguity are still frequently found in the current literature.

12Besides the non-enzymatic NO production, which is believed to occur only under very specific conditions, so far, seven potential enzymatic sources of NO have been identified in plants (Figure 3A; ). Among them, nitrate reductase (NR) and NO synthase-like (NOS-like) activities are currently considered as the most likely candidates for the production of NO under physiologically relevant conditions. Since the discovery that plant NR could produce NO both under in vitro and in vivo conditions, a great deal of evidence has indicated this enzyme as one of the major plant biosynthetic sources of NO. Supporting this view, pharmacological and genetic approaches in different plant species, organs, tissues, and experimental conditions have revealed that NR inhibition frequently results in decreased NO production. On the other hand, the existence of NOS-like activity in plants is exclusively supported by biochemical and pharmacological evidence since a canonical NOS gene or a mutant deficient in NOS-like-dependent NO production has not been identified in higher plants yet. Thus far, the organism more closely related to higher plants in which such a gene was described is the photosynthetic microalgae Ostreococcus tauri, which belongs to a basal branch of the flowering plant evolutionary tree.

13In 2003, studies revealed that NO Associated1 (AtNOA1), formerly described as AtNOS1, also significantly influences NO generation in Arabidopsis. However, according with the latest consensus in the literature, AtNOA1 encodes a chloroplast-localized cGTPase probably involved in ribosome assembly and subsequent mRNA translation to proteins in this organelle. Therefore, the reduced NO production observed in noa1 mutants is currently interpreted as an indirect outcome of disturbances in chloroplast metabolism due to the lack of AtNOA1 function. More recently, this mutant was crossed with the NR-deficient nia1-nia2 mutant of Arabidopsis, generating a triple mutant (nia1,2noa1,2), which presented no detectable NO production and a range of physiological and developmental disturbances, thereby reinforcing the physiological importance of these pathways for determining the endogenous NO levels in plants.

14Another important and frequently neglected aspect that may influence NO metabolism and signaling in plants is the presence of efficient mechanisms for removing the NO signal from a particular cell type or compartment as soon as it is no longer required. Besides the inherent chemical instability of NO in the presence of oxygen, this molecule might also be removed from plant tissues by several biochemical mechanisms (Figure 3B;; ). Firstly, NO can be removed by reacting with ROS, such as superoxide anions, generating peroxynitrite. Secondly, NO may interact with plant proteins, such as non-symbiotic hemoglobins (nsHbs), which facilitates its oxidation to nitrate. Finally, NO might also react with thiol proteins and peptides, resulting in the formation of S-nitrosothiols. In plant tissues, one of the most abundant low-molecular-mass S-nitrosothiols is the intracellular antioxidant glutathione, which may react with NO or with the NO-derivate N2O3, generating S-nitrosoglutathione (GSNO;; ). The GSNO formed can spontaneously liberate NO or be metabolized by the enzyme S-nitrosoglutathione reductase (GSNOR), originating oxidized glutathione (GSSG) and NH3. Besides being an intracellular NO reservoir, GSNO may also be transported between cells, possibly playing a critical role as a vehicle of the NO signal throughout the plant body.

15Before exploring the general mechanisms underlying the interactions between NO and phytohormones, it is worth mentioning that a great diversity of methodological approaches, experimental designs, and plant models have been used in NO research, which sometimes makes it difficult to directly compare the literature data. In terms of methodological approaches, for instance, a considerable variety of analytical techniques have been employed to determine NO levels in plant systems, including the Griess and the hemoglobin assays, electron spin resonance, laser-based photoacoustic detection, ozone-based chemiluminescence, and various fluorescent probes (reviewed by and ). As expected, these different methods provide distinct information. For example, it is always a challenge to compare results obtained by gas-phase NO detection techniques (e.g., chemiluminescence or laser photoacoustic) with fluorescent methods for in situ NO detection since these two groups of techniques differ greatly in their specificity, spatial resolution, and capacity to indicate the actual concentration of NO inside the target cells. Furthermore, evaluations of NO levels under the same experimental conditions by two or more independent methods, although recommended, are rarely carried out.

16Besides measuring NO itself, alternatives to access NO and other RNS signaling inside the cells, such as the abundance of NO-triggered chemical modifications on proteins and peptides, have also recently drawn increasing attention of the plant research community, providing, in some cases, spectacularly relevant information. A number of technical options are currently available for such proposes, including the determination of S-nitrosothiol levels in plant extracts by reductive gas-phase chemiluminescence, immunolocalization of particular S-nitrosylated thiols or proteins or even proteomic profiling of proteins chemically modified by NO or NO-derivates, which, among other aspects, may facilitate the identification of the specific targets of NO-dependent PTMs in distinct plant responses.

17Another relevant aspect to be considered in NO–phytohormone interaction studies is that the simple observation of changes in NO levels triggered by exogenous plant hormones does not necessarily imply a straightforward relationship between NO and the hormonal stimulus. Firstly, the exogenous application of a signaling substance might potentially induce global, unspecific changes in plant biochemistry, metabolism, and development. Secondly, modifications in NO levels might sometimes result from excessive levels of exogenous hormones; therefore, whenever possible, the actual concentration of particular phytohormone species inside the plant cells and tissues should be determined following the supplementation with these substances. Finally, some plant hormones may affect the biosynthesis and signaling of others; consequently, the establishment of a direct correlation between the pharmacological effect of a specific plant hormone on a given cellular response is not always an easy task. To overcome such a lack of specificity and potentially artificial effects, the use of transgenic and mutant plants with altered production, degradation or signaling of particular hormonal classes as well as a detailed characterization of several elements involved in phytohormone and NO metabolisms and signaling transduction have proven to be a powerful strategy for accessing the mechanistic relationship between these substances.

18Despite these methodological disparities and the limited literature information currently available, there is virtually no doubt that NO and phytohormones interact at multiple, diversified levels. Depending on the signaling cascade, NO has been demonstrated to act either upstream or downstream of plant hormones. Obviously, placing NO downstream of the hormonal stimuli in a signaling route necessarily means that the NO biosynthetic, degradation, conjugation, or deconjugation machinery may be affected at certain point between the perception of hormonal stimulus and the induction of the plant response. Therefore, the time period between the hormonal message input and the detection of changes in endogenous NO levels represents valuable information. In some cases, lag phases compatible with changes in the transcripts level or protein abundance of NO-synthesizing or removal enzymes have been reported. However, under some particular circumstances, the lag phase observed between the application of plant hormones and the rise in NO endogenous levels has been shown to be of just few minutes, which indicates that the post-translational regulation of proteins involved in NO metabolism rather than their de novo synthesis might sometimes be implicated.

19When acting upstream of phytohormones, NO seems able to modulate elements controlling either the plant hormone levels (e.g., biosynthetic, degradation, and conjugation enzymes), distribution (e.g., transport proteins) or signaling (e.g., receptors and signal transduction proteins). This modulation has been shown to occur either at the transcriptional or post-translational levels; however, some post-transcriptional or even translational regulation of hormone-related proteins by NO, although not yet demonstrated, cannot be ruled out.

20Based on the basic information provided thus far, the current state-of-the-art of the interplay between NO and each one of the major classes of plant hormones [i.e., auxins, cytokinins, gibberellins (GAs), abscisic acid (ABA), and ethylene] will now be discussed. Although discussed here in an isolated manner, it is important to keep in mind that very frequently, if not always, plant hormones intensively interact with each other during the induction and establishment of plant responses. However, future studies will still be required to mechanistically explain exactly how distinct plant hormones concomitantly interact with NO to regulate specific plant events.

21Synergistic effects of auxin and NO have been observed during the regulation of a series of plant responses, including root organogenesis, gravitropic responses, root nodule formation, root responses to iron deficiency, activation of cell division and embryogenic cell formation, NR activity stimulation, among others. In virtually all of these cases, NO was identified to function downstream of auxins, apparently through linear signaling pathways. Increased NO production has frequently been observed after exogenous auxin application or in auxin overproducer mutants, being especially evident in plant tissues or cells undergoing auxin-dependent physiological responses. On the other hand, no or weak stimulation in NO production by auxins has been reported in some particular experimental conditions or cell types, suggesting that the auxin-dependent NO production may occur exclusively under specific temporal and spatial contexts.

22Currently, most of the reports on NO and auxin interaction are focused on plant root responses, with relatively little information available on the crosstalk between these two signaling molecules in shoot or reproductive tissues. During the last decade, detailed information about the interaction between NO and auxin during root growth and development was provided by a series of studies conducted by Lamattina and colleagues, including the interplay between these molecules during adventitious roots formation, lateral root development, and root hair initiation and elongation. In almost all of these studies, the removal of NO by scavengers significantly decreased typical auxin-dependent root responses, such as the activation of mitogen-activated protein kinases (MAPKs) during the adventitious root formation and induction of cell cycle genes during lateral root formation.

23Also focusing on root tissues responses, identified a direct correlation between auxin availability, root NO levels and the expression of iron acquisition genes and other Fe deficiency-associated stress responses, providing further support for the action of NO as a downstream element in the auxin signaling pathway. Similarly, a clear spatial correlation was also observed between the asymmetric auxin distribution and the endogenous NO localization during the gravitropic bending in soybean roots and during indeterminate nodule formation in roots of Medicago species infected by auxin-overproducing rhizobia.

24A possible role for NR as the major biosynthetic source of the auxin-induced NO production during some plant root responses has been suggested., for instance, reported that the NO production during the auxin-induced lateral root development in Arabidopsis requires NR activity since the NR-deficient double mutant nia1,nia2 failed to increase NO generation in response to exogenous auxin, whereas no evidence for an involvement of NOS in this response was observed. NR-dependent NO production was also shown to be crucially important for the adequate vesicle trafficking during root hair formation because exogenous NO application completely restored the abnormal vesicle formation and trafficking as well as root hair growth in the nia1,nia2 Arabidopsis mutant. In a few cases, however, such as during the auxin-regulated NO generation under Fe deficiency and during the gravitropic bending in soybean roots, evidence indicates the involvement of not only NR but also NOS and/or NOA1 in the auxin-induced NO generation.

25Considering that many of these root responses, including root hair formation and lateral root development, respond to both auxins and nitrate supply, NR-dependent NO generation might be a key integrator of exogenous and endogenous cues leading to the control of plant root biology. Although the precise mechanism through which auxin trigger NR-dependent NO generation has still not been fully characterized, literature data indicate a promotive effect of this plant hormone on NR protein, activity and gene transcription.

26Besides these impacts of auxin on NO production, recent studies have demonstrated that NO might also modulate auxin metabolism, transport, and signaling. For example, NO has been demonstrated to enhance root indole-3-acetic acid (IAA) levels in cadmium-treated Medicago truncatula seedlings by reducing its degradation via IAA oxidase activity (Figure 4), thereby positively impacting auxin equilibrium and ameliorating cadmium toxicity. In addition, pharmacological treatments and NO-overproducing mutants indicated that, at high concentrations, NO inhibits acropetal auxin transport in Arabidopsis roots by reducing the abundance of the auxin efflux protein PIN-FORMED 1 (PIN1) via a proteasome-independent post-transcriptional mechanism. This NO-dependent decrease in PIN1 protein levels and consequent disturbance in root auxin transport resulted in severe reductions in root meristem size and activity in primary roots due to a reduction in cell division and a promotion in cell differentiation, compromising the root apical meristem maintenance and primary root growth.

27Finally, a direct influence of NO on auxin perception and signal transduction has also been suggested based on the recent demonstration that the auxin receptor protein TIR1 (TRANSPORT INHIBITOR RESPONSE 1) undergoes S-nitrosylation at two particular cysteine residues (cys-140 and cys-480). This S-nitrosylation of TIR1 seems to promote its interaction with AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) proteins, which are transcriptional repressors of genes associated with auxin responses (Figure 4). Being part of an E3 ubiquitin ligase complex, TIR1 marks AUX/IAA proteins to proteasome degradation, de-repressing the expression of auxin-dependent genes. Therefore, as a result, the increased TIR1–AUX/IAA interaction caused by TIR1 S-nitrosylation may facilitate AUX/IAA degradation via proteasome and subsequently promote auxin-dependent gene expression. A possible impact of S-nitrosylation on the capacity of TIR1 to bind auxin could also be a possible outcome of this NO-dependent PTM, but further investigations are still required on this subject.

28Furthermore, evidence indicates that nsHbs might also influence and modify the auxin signaling and action site by modulating the endogenous NO levels., for example, detected a drastic modification in auxin-regulated root morphology and development in transgenic lines of Arabidopsis overexpressing class 1 nsHb, which could be interpreted as the result of changes in the content and/or bioactivity of NO in these plants.

29During the last few years, accumulating evidence has indicated complex and multilevel interactions between NO and cytokinins. Both synergistic and antagonistic interactions between NO and cytokinins have been described depending on the physiological response, plant species and experimental approach. Evidence implying a possible participation of NO in cytokinin signal transduction was first obtained during the accumulation of the red pigment betalaine in Amaranthus caudatus seedlings, which was shown to positively respond not only to cytokinins but also to NO gas or donors. Since then, a number of studies have reported rapid and dose-dependent increases in NO production triggered by μM concentrations of cytokinins in both plant cell cultures and intact seedlings. In Arabidopsis seedlings, for instance, zeatin triggered increases in NO production within 3 min via a biosynthetic mechanism sensitive to arginine analogs and apparently independent of NR activity. However, other evidence revealed unchanged or even lower NO levels after cytokinin treatments or in mutant or transgenic plants with increased cytokinin production. Moreover, no obvious influence of exogenous application or depletion of NO has been observed on some early signaling events leading to the induction of primary cytokinin responses, such as the activation of cytokinin-responsive Arabidopsis response regulator (ARR)5 promoter in seedlings.

30Examples of synergistic interaction between cytokinins and NO include the control of leaf senescence, programmed cell death (PCD; ), photosynthesis adaptability to drought stress, cell division, and differentiation, among others. Studies of the integrated influence of NO and cytokinins on plant senescence program have demonstrated that natural, dark- or dehydration-induced leaf senescence can be minimized by exogenous NO application. In addition, mutant or transgenic plants exhibiting decreased NO levels usually display precocious senescence in detached leaves and intact plants, which can sometimes be alleviated by exogenous cytokinin supplementation. Although still limited in terms of current commercial application, this antisenescence trait of NO and cytokinins has been proven to extend post-harvest life of agronomically relevant fruits and vegetables.

31Further indicating a protective and antisenescence role of NO and cytokinins, reported increased NO levels during the cytokinin-induced photosynthetic adaptability to drought stress and described a good correlation between NO production and NR activity during this adaptive plant response to water limitation. In contrast, however, NOS-like-dependent increases in NO generation have been suggested to act as an intermediate during the acceleration of cell apoptosis induced by high cytokinin dosages since cell death was alleviated when cytokinins were supplied along with NOS inhibitors or NO scavengers to Arabidopsis cell cultures.

32The interaction between cytokinins and NO during the regulation of plant cell division has also been recently studied in more detail. Among other evidence, NO deficiency caused either by loss of the gene NOA1 or due to NO scavenger treatments was demonstrated to result in severe inhibition of cytokinin-induced transcriptional activation of the cell cycle gene CYCD3;1 (CYCLIN-D3;1) and the subsequent callus initiation from somatic plant tissues, implying that NO may act downstream of cytokinins in the control of plant cell mitotic cycles. In this study, roots of Atnoa1 mutant were described as severely impaired in cytokinin-induced NO production and less sensitive to cytokinins than wild-type (WT) ones.

33Contrary to the above described synergistic relationships between NO and cytokinins, literature data have also suggested an opposite interaction between these signaling molecules in some plant responses. Studies conducted on epidermal strips of Vicia faba indicated that exogenous cytokinins efficiently reduced NO generation in guard cells exposed to the NO donor sodium nitroprusside (SNP) as well as promoted stomata reopening under dark condition due to the abolishment of the dark-induced increases in endogenous NO, which was interpreted as evidence of a potential scavenging action of cytokinins on the NO produced under these situations.

34Consistent with these results, also observed a negative correlation between endogenous cytokinin and NO levels in transgenic tobacco plants with either increased or decreased cytokinin levels. More recently, reported that cytokinins might intimately participate in NO catabolism since some cytokinin species, such as zeatin, can chemically react with peroxynitrite, leading to the production of cytokinin derivates with virtually no biological activity (Figure 5). Moreover, these authors verified that exogenous zeatin alleviates the severity of the phenotypes attributed to excessive NO levels in the Arabidopsis NO-overproducer nox1 (nitric oxide overexpression 1) mutant, and this same ameliorative effect was observed when nox1 plants were crossed with a cytokinin-overproducing mutant. Based on these biological and chemical data, postulated that these two signaling molecules (NO and cytokinins) might interact by modulating each other’s homeostatic levels and bioactivity (Figure 5). Such peculiar mechanism of interaction between cytokinins and NO, in which one of the substances directly interferes with the levels of another simply by a chemical combination of two molecules (Figure 2C), is quite different from the interaction at biosynthetic or signaling levels usually observed for other NO–phytohormone crosstalks (Figures 2A, B) and certainly deserves further attention.

35Additionally, strong evidence indicating a direct impact of NO on the cytokinin signaling pathway has recently been uncovered. Besides corroborating previous observations that Arabidopsis mutant lines with excessive NO levels display more limited responsiveness to cytokinins, revealed that the phosphorelay mechanism central to the signaling transduction of this hormonal class can be severely impaired by the S-nitrosylation of a particular cysteine residue (cys 115) of the HISTIDINE PHOSPHOTRANSFER PROTEIN 1 (AHP1), hindering the transfer of phosphoryl groups from cytokinin receptors to AHP1 and subsequently to response regulators (ARRs; Figure 5). Confirming the importance of this NO-dependent post-translational protein modification for the cytokinin signal transduction, these authors have demonstrated that non-nitrosylatable mutation of AHP1 consistently relieved the inhibitory effect of NO on cytokinin responses whereas a nitrosomimetic mutation of this protein severely compromised cytokinin responses.

36An additional, less direct way through which cytokinins might modulate NO levels in plant systems seems to rely on the regulatory effect of these hormones on the expression of nsHbs. Cytokinin-triggered changes in the expression of certain nsHbs have been described for several plant models. Moreover, transgenic and mutant plants with altered levels of particular nsHb classes have frequently displayed alterations in plant responses typically controlled by cytokinins. For instance, marked changes in shoot organogenesis and altered expression of genes associated with cytokinin perception and signaling have been observed in Arabidopsis lines silencing or overexpressing class 1 or class 2 nsHbs. In the transgenic lines overexpressing nsHbs, cytokinin feedback repressors (Type-A ARRs) were repressed, whereas cytokinin activators (Type-B ARRs) and receptors were stimulated (Figure 3), culminating in a higher sensitivity of the tissues to the cytokinin-induced shoot organogenesis. Unfortunately, NO content was not measured in these transgenic lines; therefore, a direct correlation between the higher responsiveness to cytokinins observed in nsHb overexpressing lines and their possibly lower NO levels could not be established.

37Both important “stress-related” molecules, NO and ABA intensively crosstalk during certain signaling cascades triggered by environmental challenges, such as water limitation and UV-B radiation, which ultimately leads to the induction of plant adaptive responses, such as stomatal closure and antioxidant defenses. During the induction of these plant stress responses, NO mainly acts as a downstream element in the ABA signaling pathway since the impairment in NO production or its removal from tissues usually decreases or even eliminates ABA responses while the inhibition of ABA production typically does not affect the induction of these responses by exogenous NO application. On the other hand, during the regulation of certain developmental events not directly linked to plant stress responses, such as seed dormancy breaking, NO seems to counteract ABA effects, suggesting a certain level of specificity in the NO–ABA interaction mechanisms, which may depend on the physiological events under analysis (e.g., stomatal closure versus seed dormancy release) or even the type of plant cell, tissue, or organ considered (e.g., guard cell versus seed tissues).

38In some cases, such as during the induction of stomatal closure and during the up-regulation of the gene transcription and activities of antioxidant enzymes, ABA-induced NO generation seems to depend on H2O2 synthesis, suggesting this ROS as a mediator in NO-dependent ABA responses (Figure 6). In addition, the calcium/calmodulin system and MAPKs have also being identified as downstream elements of NO signaling during the regulation of plant antioxidant defenses induced either by ABA or H2O2. Moreover, cGMP has also been demonstrated to participate in NO-dependent ABA signaling, apparently acting downstream of NO and upstream of cytosolic Ca2+ (Figure 6; ). Similarly, type 2C protein phosphatases (PP2Cs), which acts as negative regulators of ABA signaling, have also been suggested to play a role as putative crosstalk elements between ABA receptors and NO-mediated ABA signal transduction, possibly acting downstream of NO in the complex networks controlling ABA-triggered stomatal closure.

39Since the discovery that NO scavengers could reduce ABA-induced stomata closure in turgid leaves of different plant species, intensive research has been dedicated to characterize the mechanisms underlying the interplay between these two molecules in guard cell signaling networks (reviewed in;; ), leading to the identification of several NO targets during the ABA-induced guard cell responses. Among these targets, plasma membrane calcium-dependent anion channels and inward-rectifying K+ channels have been demonstrated to be activated and deactivated, respectively, by NO as a consequence of increases in guard cell cytoplasmatic Ca2+ levels (Figure 6) due to NO-triggered release of this anion from intercellular stores.

40Evidence for the involvement of protein phosphorylation upstream of intracellular calcium release has also been obtained, implicating protein kinases as additional targets of NO action within ABA-regulated guard cell signaling. Moreover, NO has also been reported to directly modulate calcium-independent outward-rectifying K+ channels possibly by post-translationally modifying these channels or closely associated regulatory proteins. As a final consequence, this NO-dependent modulation of both Ca2+-dependent and Ca2+-independent ion channels at the plasma membrane of guard cells facilitates osmotic solute loss, thereby reducing guard cell turgor and promoting stomatal closure.

41It is worth mentioning that NO has been suggested to play a role as a second messenger shared by multiple hormonal signaling cascades involved in the intricate guard cell network responsible for coordinating stomatal movement in higher plants, mediating not only the ABA signal but also ethylene, salicylic acid (SA; ), methyl jasmonate, auxin, and cytokinins. Curiously, though, NO apparently is not an absolute requirement during the ABA signaling cascades leading to stomatal closure or the inhibition of light-induced stomatal opening; therefore, the existence of both NO-dependent and NO-independent pathways in ABA-induced guard cell responses is currently being suggested. Of course, more studies are clearly needed to better characterize a possible integrative, but apparently non-essential, role of NO during the regulation of stomatal movements by distinct environmental and hormonal stimuli.

42At least in bromeliads, NO and ABA also seem to intensively interact to control Crassulacean acid metabolism (CAM) expression, which, in turn, facilitates the survival of these plants under water- and nutrient-limited environments. As during the regulation of stomatal movements, NO apparently acts downstream of ABA and upstream of cytosolic calcium in the ABA-dependent signaling cascade leading to the up-regulation of the CAM machinery (Figure 6), and does not participate in the ABA-independent pathway also responsible for the regulation of this plant stress response. The regulation of CAM expression in bromeliads as well as the control of stomata movements in Arabidopsis seem to have NR activity as the main source of the ABA-induced NO production.

43While a number of pharmacological and genetic studies have reported higher endogenous NO levels following increases in plant tissue ABA concentration (i.e., NO action downstream of ABA; ), NO-triggered changes in ABA biosynthesis and catabolism (i.e., NO action upstream of ABA) have rarely been described. In one of the few examples, reported that during the seed dormancy breaking in Arabidopsis, a rapid accumulation of NO in the endosperm layer preceded a decrease in ABA concentration, which was associated with a pronounced rise in the transcript and protein levels of the ABA 8′-hydroxylase CYP707A2, a key enzyme in ABA catabolism. Moreover, exogenous NO and the NO scavenger carboxy-PTIO (cPTIO), respectively, induced and impaired CYP707A2 transcript accumulation during the imbibition period, further suggesting that the promotive effect of NO on seed dormancy break might indeed be associated with a stimulation of ABA catabolism.

44In addition to modulating ABA catabolism, NO has also been described to affect the sensitivity of plant cells to ABA. reported that the NO donor SNP enhanced germination of dormant Arabidopsis seeds by decreasing the seed sensitivity to exogenous ABA. More recently, genetic evidence supporting this inhibitory effect of NO on ABA sensitivity was obtained by,, who observed that the depletion of endogenous NO levels resulting from the generation of the nia1,2noa1-2 Arabidopsis triple mutant clearly led to ABA hypersensitivity. Among other features, this triple mutant displayed enhanced seed dormancy, decreased seed germination, and reduced seedling establishment in the presence of exogenous ABA, reinforcing the hypothesis that NO production during seed germination and initial seedling development counteracts the ABA inhibitory effects on these events. Interestingly, this ABA hypersensitivity continued through the post-germinative vegetative development of this triple mutant, as evidenced by the presence of increased expression of ABA-responsive genes, extreme drought resistance phenotype as well as higher responsiveness to ABA during stomatal closure. Curiously, dehydration- and ABA-dependent stomatal closure normally occurred in the presence of undetectable NO production in guard cells, corroborating the existence of a NO-independent pathway in this guard cell response. Whether NO exerts its effects directly on ABA receptors or on some downstream element of ABA signaling cascade is obviously an important question that remains to be answered.

45Nitric oxide has also been reported to influence several plant developmental events in which GAs play crucial roles, such as seed germination, hypocotyl elongation, acquisition of photomorphogenic traits, primary root growth, reorientation, and growth of pollen tubes, among others; however, thus far, the actual interaction between NO and GAs has been described for only a limited number of these physiological events. In fact, most of our current knowledge of the mechanisms underlying the interplay between GAs and NO is restricted to the regulation of seed germination and the inhibition of hypocotyl elongation during seedling de-etiolation. During the control of these responses, NO has been described to act upstream of GA, regulating both GA biosynthesis and perception/transduction.

46A certain level of antagonism between NO and GAs has been observed for most of the physiological processes in which both of these signaling compounds participate. A mounting body of evidence has indicated that DELLA proteins apparently represent a key crosstalk component between GA and NO signaling interactions (Figure 7; ). DELLA proteins are a relatively small family of transcriptional regulators notably important for the integration of diverse hormonal signals, such as GAs, ethylene, jasmonate (JA), and ABA. During GA signaling transduction, for instance, the hormonal molecules interact with GA INSENSITIVE DWARF1 (GID1) receptors, which, in turn, binds a DELLA protein and subsequently directs the GA–GID1–DELLA complex to the E3 ubiquitin ligase SLEEPY1 (SLY1), thereby promoting DELLA degradation at the proteasome (Figure 7). Given that DELLAs mainly act by repressing the transcription of GA-regulated genes, the perception and transduction of the GA signal leads, as a final result, to a decrease in DELLA concentration into the cell and a consequent induction of GA-responsive genes.

47Interestingly, recent studies have indicated that NO triggers the opposite effect on cellular DELLA concentration, promoting the accumulation of this protein and a consequent negative impact on GA signal transduction (Figure 7). Essentially, this NO-driven DELLA accumulation can be interpreted as a reduction in tissue sensitivity to GA since a larger number of GA–GID1–DELLA complexes will need to be formed in order to mark an adequate quantity of DELLA proteins for proteasome degradation, thereby leading to a satisfactory level of transcriptional de-repression of GA-regulated genes. This differential effect of NO and GAs on DELLA regulation might account, at least in part, for the antagonism observed between these two signaling compounds during the regulation of physiological processes, such as hypocotyl elongation and primary root growth in Arabidopsis.

48In addition, studies performed on nia1,2noa1-2 seedlings revealed that this NO-deficient mutant presents defective DELLA accumulation associated with an up-regulation of the E3 ubiquitin ligase SLY1 (Figure 7), resulting in increased GA sensitivity and deficient de-etiolation under red light. Further emphasizing the potential role for DELLAs in the GA–NO antagonistic interactions, exogenous NO was also demonstrated to induce the accumulation of GA-regulated DELLA proteins, very likely by negatively regulating the GID1–SLY1 system of DELLA tagging for degradation (Figure 7). However, as pointed out by, the regulation of DELLA turnover and activity may represent the main but not the only target for NO action in regulating plant growth and other GA-mediated developmental responses since DELLA-independent mechanisms might also be implicated.

49Besides the negative action of NO on GA signaling network, a mutual antagonism controlling the endogenous levels of these two signaling molecules has also recently been proposed (Figure 7). Supporting this suggestion, etiolated seedlings of the GA-deficient Arabidopsis mutant ga1-3 have been shown to exhibit NO levels significantly higher than those observed in the WT genotype. Moreover, both ga1-3 mutant and WT seedlings showed reduced NO levels after GA3 supplementation, thereby suggesting that GAs negatively modulates NO production. On the other hand, WT Arabidopsis seedlings treated with SNP presented a significant reduction in endogenous GA levels. Based on a detailed analysis of the expression of Arabidopsis genes involved in GA biosynthesis (GA20oxidase and GA3oxidase) and catabolism (GA2oxidase), GA20ox3 was identified as the only gene significantly up-regulated in the NO-deficient nia1,2noa1-2 mutant and down-regulated in NO-treated WT seedlings.

50Under certain circumstances, however, NO seems to play a stimulatory rather than inhibitory role in the GA biosynthetic machinery. Exemplifying such a synergist relationship, reported that NO generation was required for the transcription of two GA3oxidase genes (GA3ox1 and GA3ox2) during the Arabidopsis seed dormancy breaking. Another indication of the positive interaction between GA and NO has recently been reported in wheat roots, for which the SNP-induced apical growth was associated with increased GA3 levels.

51Apart from the above-mentioned evidence of NO acting upstream of GA, a certain level of uncertainty remains as to whether NO and GA actually share a common signaling route or just act through parallel, independent cascades during the regulation of some plant responses. During seed dormancy breaking, for instance, although there is virtually no doubt that both of these signal molecules promote germination in a number of species, whether and how NO and GA interact during this process still needs further characterization.

52In fact, whereas a mounting body of evidence indicates that NO selectively interferes in some specific GA-induced events associated with the seed germination process, such as the longevity of cereal aleurone cells, transcription of Myb transcription factor (GAMYB), and amylase synthesis, for some other responses associated with the germination process, no indications of additive or antagonistic responses have been found when both GA and NO were exogenously applied. In addition, a rapid burst in NO production has been detected during early seed germination, which has been speculated to be temporally dissociated from the action of GAs at later stages of seed germination.

53Regardless of whether or not NO and GA share a common signaling cascade during seed dormancy breaking, the stimulation of seed germination by either of these substances can be blocked by sufficiently high concentrations of ABA. Considering that NO may stimulate germination not only by breaking seed dormancy but also by alleviating the influence of environmental factors inhibitory to the germination process, a NO-hormonal network much more complex than the interaction between NO, GA, and ABA might possibly be involved in the regulation of this critically important step in the plant life cycle.

54A significant number of the currently available reports on the interaction between NO and ethylene suggest an antagonistic relationship between these two gaseous molecules. The first and presently most explored plant phenomenon in which NO was demonstrated to counteract ethylene production and action is the control of fruit ripening and the regulation of leaf and flower senescence. For these responses, ethylene has long been identified as a key promotive signal, and a large number of reports indicate that the production and perception mechanisms of this plant hormone are under strict regulation, depending not only on the plant developmental program but also on a number of environmental factors. Additional studies revealed that exogenous application of NO, either by direct fumigation or by means of NO-releasing chemicals, delays senescence of both vegetative and reproductive organs by negatively regulating a number of elements involved in ethylene production. Corroborating this pharmacological evidence, measurements of ethylene and NO emission during either fruit ripening or plant senescence revealed an opposite trend between these gases, in which ethylene production increases, whereas NO levels decrease during the induction and establishment of these processes.

55Recent studies have revealed that the inhibition of fruit ethylene production by NO may be attributed to a reduction in the transcript level and/or activity of key ethylene biosynthetic enzymes. In vegetative and reproductive plant tissues, ethylene production depends on the conversion of the S-adenosyl methionine (SAM), derived from “Yang cycle,” into the immediate ethylene precursor 1-aminocyclopropane 1-carboxylic acid (ACC) through ACC synthase (ACS) activity (Figure 8). The ACC formed may be subsequently converted to ethylene due to the activity of a second enzyme, the ACC oxidase (ACO; ). Since the abundance of ACC, ACS, and ACO in plant tissues represents a critical aspect for determining ethylene production rates, an inhibitory effect of NO on any of these elements can be expected to be an efficient mechanism for down-regulating ethylene synthesis.

56As revealed by a series of studies on climacteric fruits, exogenous NO indeed has the capacity to modulate both the transcription and the activity of both ACS and ACO (Figure 8), consequently impacting not only the levels of ethylene production but also the accumulation of ACC. In tomato fruits, for instance, although the expression of all ACS homologs remained virtually unchanged following NO fumigation, the transcript abundance of ACO genes, such as LeACO1, LeACOH2, and LeACO4, and the levels of ethylene emission were reduced and/or delayed when NO was applied before the start of the ripening process. In banana fruits, on the other hand, NO negatively impacted the expression of both ACS and ACO homologs, leading to a reduction in ACO activity and ethylene emission as well as an accumulation of ACC.

57Apart from controlling the transcript levels of ACS and ACO, NO may also regulate ACS activity via S-nitrosylation and influence ACO activity by a mechanism involving the direct binding of NO to the enzyme, resulting in the ACO–NO binary complex, which is then chelated by ACC to produce the ternary stable complex ACO–NO–ACC (Figure 8). Currently, the impacts of S-nitrosylation on ACS activity remain uncharacterized, and the occurrence of the ACO–NO–ACC ternary complex is exclusively described during in vitro studies conducted on recombinant ACO; therefore, the actual in vivo implications of such regulatory mechanisms still need further elucidation. Nevertheless, the hypothetical formation of an ACO–NO–ACC complex has already been inferred as possibly responsible for the reduction of ACO activity in climacteric peach (Prunus persica) fruits subjected to NO fumigation, which resulted in a concomitant decrease in ethylene emission and accumulation of ACC. In this specific case, the NO-induced reduction of ACO activity was accompanied by an increment in the accumulation of the non-volatile ACC metabolite 1-malonyl aminocyclopropane-1-carboxylic acid (MACC; Figure 8), which was interpreted as a secondary effect of NO during the ripening of these fruits.

58Besides stimulating the irreversible conversion of ACC into MACC, NO may also negatively impact the turnover of SAM, which is the main precursor molecule for ACC synthesis. Supporting this assumption, proteomic analysis of Arabidopsis plants revealed that the methylmethionine cycle enzymes adenosyl homocysteinase (SAHase), methionine synthase (MET synthase) and methionine adenosyltransferase (MAT, also known as SAM synthase), whose activities are responsible for the production of homocysteinase (HCY), methionine (Met), and SAM, respectively, may undergo S-nitrosylation (Figure 8). In addition, similar analyses conducted on GSNO-treated protein extracts of Kalanchoe pinnata and Brassica juncea also identified cobalamin-independent MET synthases as a common target of S-nitrosylation. Whereas the influence of S-nitrosylation on the activities of SAHase and MET synthase has yet to be determined, a detailed study conducted by revealed that the activity of MAT1, one of the three Arabidopsis MAT isoforms, is indeed suppressed via S-nitrosylation at cys-114, having as a logical consequence the depletion of the SAM pool and a reduction in ethylene production. Curiously, the study conducted by was the first detailed characterization of S-nitrosylation in plant systems, opening up a new window of opportunities for accessing the actual relevance of this NO-dependent post-translational regulatory mechanism in plant signaling.

59In contrast to the above-mentioned evidence of an antagonistic relationship between NO and ethylene during the maturation, senescence, and abscission of plant organs, a number of reports have also indicated that NO donors, such as SNP, might sometimes stimulate, rather than negate, ethylene production in certain plant materials, such as non-senescent leaf tissues of Arabidopsis, tobacco, and maize and apple embryos. In tobacco leaves, for instance, SNP infiltration has been show to stimulate ACS expression, whereas in Arabidopsis roots the application of GSNO positively impacted the transcript levels of not only ACS but also other key ethylene biosynthetic enzymes, such as SAM synthetases, ACOs, and 5-methylthioribose kinase (MTK; ). Further emphasizing a stimulatory influence of NO on ethylene biosynthesis, ethylene production is usually elevated when the NO accumulation is promoted via suppression of nsHbs gene expression. Similarly, the increased NO production observed in transgenic tobacco lines expressing mammalian NOS were accompanied by a higher expression of ACO and some other ethylene-related genes. Moreover, a concomitant increase in both ethylene and NO emission has been consistently observed both in tobacco leaves undergoing bacterially triggered hypersensitive response and in Arabidopsis and cucumber (Cucumis sativus) roots subjected to Fe deficiency.

60Besides these indications of a positive influence of NO on ethylene production, some data also seem to support a stimulatory role of ethylene on NO production under certain circumstances. Earlier in the research of NO–ethylene interaction in plants, reported that exogenous ACC induced significant increases in both ethylene and NO emission in pea (Pisum sativum) leaves. More recently, have also detected increased NO levels in the root subapical region of Arabidopsis and cucumber plants exposed to ACC. In addition, these authors reported that inhibitors of ethylene biosynthesis and action completely abolished the increases in NO levels in roots of plants subjected to Fe deficiency. In contrast, ethylene supplementation or depletion, respectively, repressed and promoted NO production during the abscission of mature olive fruits, which apparently indicates that under certain circumstances ethylene may negatively, rather than positively, impact the endogenous NO levels. In agreement with this, ethylene has sometimes been shown to induce class 1 nsHbs, which in turn may lead to reductions in tissue concentration of NO.

61Surprisingly, the possible influence of NO on ethylene signal transduction elements has remained virtually unexplored, both during antagonistic (e.g., fruit ripening and leaf senescence) and synergistic (e.g., plant defense to biotic stresses and Fe deficiency) interactions between these signaling substances. Therefore, it is currently unknown whether NO might regulate the transcripts levels or activities of receptors, signal transduction proteins and/or transcription factors involved in ethylene signaling, which would very likely impact the sensitivity of the plant tissues to this plant hormone. In one of the few studies on this line, demonstrated that the dark-induced early senescence phenotype of the Arabidopsis NO-deficient mutant noa1 was suppressed by mutation in ETHYLENE INSENSITIVE 2 (EIN2) and indicated that this protein might act downstream of NO signaling, possibly playing a key role as a crosstalk point between ethylene and NO signaling cascades.

62Besides interacting with the five “classical” phytohormone classes, NO has also been reported to crosstalk with other plant hormones, including JAs, SA, polyamines, and brassinosteroids. Some of these interactions, such as the interplay between NO, SA, and JA in plant defense responses, have been investigated in great detail, uncovering impressively complex NO–phytohormone interaction networks. A detailed discussion about these interactions is beyond the scope of the present work; instead, just some brief, general comments, and examples of these NO–phytohormone crosstalks will be provided below.

63As recently reviewed by and, during the induction of plant defense responses against biotic challenges, NO positively impacts the production of both SA and JA and, at the same time, NO modulates SA signaling by controlling the oligomerization status of the translational activator NON-EXPRESSER OF PATHOGENESIS-RELATED GENE1 (NPR1) via S-nitrosylation at cys156 (; Figure 9). S-nitrosylation of NPR1 facilitates its oligomerization and permanence in the cytosol, where it may interact with SA receptors (NPR3/4). Following such interaction with NPR3/4, the S-nitrosylated cys156 of NPR1 is reduced, promoting NPR1 monomer formation and its consequent migration to the nucleus, where this protein may interact with several TGA-class transcription factors that subsequently activate promoters of SA-responsive genes (; Figure 9). In contrast, the presence of the S-nitrosylated, oligomeric form of NPR1 in the cytosol facilitates the repression of JA-triggered responses. Consequently, this NO-dependent PTM of NPR1 seems to play a key integrative role during the hormonal signaling cascades leading to coordinated plant immunity responses. In parallel, S-nitrosylation of SA-BINDING PROTEIN 3 (SABP3) at cys280, which takes place during late stages of bacterial infection, represses its capacity to bind SA, and antagonizes the expression of plant immunity responses, thereby representing a negative feedback loop apparently essential for the correct regulation of SA-modulated plant defense against biotic challenges (Figure 9).

64Accumulating evidence indicates that NO might also mediate both developmental and stress responses induced by polyamines. Briefly, very rapid NO production has been observed in plant tissues exposed to mM concentration of polyamines, which has sometimes been interpreted as an indication of a potential NO biosynthetic pathway involving the catabolism of these plant hormones. Given the absence of a lag phase between the application of polyamines and the rise in NO endogenous levels, it is currently assumed that these hormones might be directly converted to NO by the action of one or more enzymes, whose identities are yet to be determined. So far, it is only known that the polyamine-induced NO production can be quenched by mammalian NOS inhibitors and is not affected in Arabidopsis NR-deficient mutants. Whether polyamines act as substrates, cofactors, or signals for promoting NO synthesis also needs to be better determined; therefore, monitoring the formation of 15NO from isotopic-labeled polyamines in plant tissues or extracts seems an important experiment in future studies. A possible influence of NO on polyamine metabolism has been demonstrated in some studies but not in others; consequently, this topic still deserves further investigation.

65Considering that polyamines and ethylene share SAM as a common precursor, all the basic NO-dependent mechanisms controlling the SAM pools discussed earlier in this review (Figure 8) might also indirectly affect polyamine synthesis in plants. In addition, we also need to keep in mind that L-arginine is substrate for the production of polyamines, via arginase and arginine decarboxylase activities, as well as NO, via NOS-like activities; therefore, the availability of this particular amino acid might also influence NO/polyamine connections in plants and other organisms. In mammals, for instance, the occurrence of an arginine switch, in which NOS and arginase compete for arginine, seems to be supported by a great deal of experimental evidence. In parallel, literature data in the animal field also indicates that polyamines such as spermidine and spermine influence NO production via NOS activity during diverse physiological responses, which may represent an important source of information to guide current and future research on the NO and polyamine interactions in plants. The polyamine precursor agmatine, for example, has been demonstrated to act either as an alternative substrate or a competitive inhibitor of mammalian NOS, depending on the isoform or physiological process taken under consideration, thereby indicating a possible role for this compound as an endogenous regulator of NO generation in mammals. In plants, the ameliorative effects of both polyamines and NO under stressful conditions might represent an important driving force to stimulate further studies on the interaction between these critically important signaling compounds.

66Generation of information about whether and how NO and brassinosteroids interact has only recently begun. In one of these studies, demonstrated that nM concentrations of brassinosteroids promoted rapid increases in the NO levels of leaf mesophyll cells, which together with some other evidence allowed the authors to place NO as a possible intermediate in the brassinosteroid-induced ABA biosynthesis in maize leaves. More recently, reported that NR and NOS-like activities are probably involved in the brassinosteroid-induced NO production in Arabidopsis and that NO very likely mediates brassinosteroid-triggered modifications in plant root architecture.

67Despite the methodological difficulties and conceptual complexity intrinsically involved in the elucidation of the exact mechanisms responsible for interconnecting plant hormones and NO signaling during the coordination of plant metabolism and development, some cutting edge insights into the NO–phytohormone crosstalks have recently been achieved.

68Many downstream and upstream components of the NO signaling cascades have been identified, and NO-dependent PTMs, notably S-nitrosylation, have emerged as critical mechanisms controlling key elements involved in plant hormone production and signaling. As highlighted in the course of this review, by chemically modifying these hormone-related proteins, NO may modify plant hormone metabolism and signaling at multiple, diversified levels. The identification and functional analysis of the protein targets of NO-dependent PTMs and whose action determines the delicate hormonal homeostasis in plants has been, and will probably continue to be, an approach of upmost relevance in NO–phytohormone studies.

69Currently, the physiological relevance of NO-dependent chemical modifications of phytohormone-related proteins has been poorly investigated in planta; therefore, this remains a rich area for future investigation. Clarifying how these NO-triggered PTMs, particularly S-nitrosylation and tyrosine nitration, actually control protein activity, subcellular localization as well as protein–protein, protein–DNA, protein–cofactors, or even protein–hormone binding capacity will inexorably involve the use of a wide range of experimental strategies and methodological approaches, some of which are currently available (e.g., overexpression of modified proteins in mutant genetic backgrounds) and others yet to be developed. Since some proteins are targets of multiple NO-dependent PTMs, sometimes even involving different types of these chemical modifications (e.g., both S-nitrosylation and nitration;; ), it would be enlightening to determine the impacts of concomitant NO-triggered modifications on the same protein.

70Moreover, characterizing how these target proteins are chemically modified by NO and NO-derivates at the right time and place seems to be another promising area of progress in NO–phytohormone interactions. Addressing this question inevitably implies dealing with several critical aspects of the NO physiology that still require further elucidation. Firstly, the basic mechanisms responsible for NO production, removal, and transport in plants continues to represent a critical impediment for advances in the clarification of how NO levels are temporally and spatially controlled by plant hormones and other stimuli. A fine-tuned equilibrium between NO production and removal (e.g., biosynthesis versus degradation, conjugation versus deconjugation) might possibly exist to determine both the localization and the concentration of NO and NO-derivates within the plant cells. Secondly, given the impressive diversity of target proteins, which are ubiquitously distributed within the plant cells, the existence of a certain subcellular compartmentation in NO production and action is an assumption that urgently needs to be investigated in greater detail. Moreover, a concentration-dependent action mode for NO has also been proposed, in which distinct responses may be triggered depending on the abundance of this free radical. Obviously, the development of more sensitive and specific means to determine the subcellular localization and concentration of NO and NO-derivates is critical for further advances in this area. Thirdly, considering that both S-nitrosylation and tyrosine nitration are apparently reversible events, more conclusive studies on the denitrosylation and denitration systems as well as the general turnover of S-nitrosylated and nitrated proteins in plant cells also seems a logical requirement for a deeper understanding of the dynamics of these regulatory processes. Similar to the action of protein phosphatases during the regulation of protein phosphorylation, denitrosylases and denitrases may possibly play an important role in defining the kinetics of the NO impacts on plant signaling cascades.

71Another aspect that also deserves further attention is the potential existence of feed-forward cycles, in which NO modulates the production and/or signaling of specific plant hormones and these same hormonal species influence the machinery responsible for controlling NO endogenous levels. As described in the course of this review, accumulating pharmacological and genetic evidence demonstrates that representatives of virtually all classes of plant hormones may impact, at least at a certain degree, the endogenous concentration and/or distribution of NO and, also very frequently, literature data seems to indicate that changes in NO levels might trigger alterations on the metabolism and/or signaling of many, if not all, hormonal classes. It is not clear, however, whether these processes occur at the same place and time, which is critical for generating authentic feed-forward cycles involving these signaling substances. Naturally, a more complete characterization of the actual impacts of specific plant hormones on the NO biosynthetic and removal machinery (e.g., NR, NOA1, GSNOR, nsHbs) seems a key step in such research topic.

72Additionally, as also discussed earlier in this review, NO might affect the signaling transduction of certain hormones, such as auxins and GAs, by modulating signaling elements (e.g., receptor, signaling transduction molecules) that impact the general dynamics of ubiquitination and proteasome-dependent degradation of repressor proteins (e.g., AUX/IAA and DELLA proteins). Interestingly, in animal systems, NO has consistently been shown to influence protein stability via regulation of ubiquitination and proteasome-dependent proteolysis and, at least in humans, ubiquitin ligases themselves are targets of S-nitrosylation. Considering that several plant hormone signaling transduction mechanisms are based on the ubiquitination and subsequent proteasome-dependent degradation of repressor proteins, investigating whether NO might also directly affect this protein degradation labeling system in plants seems a promising venue for uncovering additional mechanisms possibly involved in NO signaling in plant systems.

73Another intriguing question that remains to be answered is how plants can distinguish endogenously produced NO signals from the NO naturally present in the environment (e.g., atmosphere, rhizosphere). Whereas the gaseous and highly diffusible nature of NO may promote certain movement of this molecule inside the plant tissue and at the plant–environment interface, the high reactivity and inherent instability of NO may possibly limit the diffusion of this free radical through biological tissues. In several aspects, this seems a relevant and challenging question to be answered in the future.

74Finally, we must remain open-minded to conceive increasingly complex NO–phytohormone interconnection nodes since new targets of NO-dependent PTMs and other upstream and downstream elements of NO signaling cascades will likely be identified in the future. At the same time, more complete pictures mechanistically explaining how multiple plant hormones may simultaneously interact with NO to control specific plant responses might also emerge, very likely leading to exciting new models of NO–phytohormone interaction networks. Moreover, this whole scenario will be further complicated when the intensive research conducted today in a restricted number of plant models (e.g., Arabidopsis, tomato, rice) is extended to a broader range of plant species and environmental contexts. Altogether, this knowledge will improve our ability to define the actual roles of NO during the regulation of the distinct plant responses controlled by this multipurpose signaling molecule and may also lead to new opportunities to manipulate NO–phytohormone interactions and, thus, regulate plant growth, development, and metabolism.

75The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.