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Review

Nitric Oxide Regulates Multiple Signal Pathways in Plants via Protein S-Nitrosylation

by
Wei Lin
1,
Jian-Xiu Shang
2,
Xiao-Ying Li
2,
Xue-Feng Zhou
1 and
Li-Qun Zhao
2,*
1
School of Biology and Food Science, Hebei Normal University for Nationalities, Chengde 067000, China
2
Key Laboratory of Molecular and Cellular Biology of Ministry of Education, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China
*
Author to whom correspondence should be addressed.
Curr. Issues Mol. Biol. 2025, 47(6), 407; https://doi.org/10.3390/cimb47060407
Submission received: 24 April 2025 / Revised: 24 May 2025 / Accepted: 28 May 2025 / Published: 30 May 2025
(This article belongs to the Section Molecular Plant Sciences)
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Abstract

Nitric oxide (NO) can perform its physiological role through protein S-nitrosylation, a redox-based post-translational modification (PTM). This review details the specific molecular mechanisms and current detection technologies of S-nitrosylation. It also comprehensively synthesizes emerging evidence of S-nitrosylation roles in plant biological processes, including growth and development, immune signaling, stress responses and symbiotic nitrogen fixation. Furthermore, the review analyzes research progress on the crosstalk between S-nitrosylation and other protein PTMs. Finally, unresolved issues such as the spatio-temporal resolution of SNO-proteome mapping and standardized protocols for reproducibility are pointed out. In summary, this work proposes a roadmap for future research.

1. Nitric Oxide and S-Nitrosylation

Key roles for nitric oxide (NO) in plants have been demonstrated in seed dormancy, embryogenic cell formation, root development and gravitropic bending, flowering, stomatal closure, the growth regulation of pollen tubes, nutrition (particularly ion homeostasis), immunity, and adaptive responses to various abiotic stresses [1,2]. For instance, NO operates through reactive oxygen species (ROS) and classical second messengers, including Ca2+ and cyclic guanosine monophosphate, to impact physiological processes in plants [3,4]. Further, crosstalk exists between NO and key hormones, including auxin, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene, and cytokinins in plant metabolism [5]. However, the molecular mechanisms whereby NO impacts the plant life cycle are poorly understood.
In recent years, S-nitrosylation has emerged as an important NO-dependent protein post-translational modification (PTM) involved in a large variety of cellular functions [6]. During S-nitrosylation, an NO group is covalently linked to the free sulfhydryl group of specific cysteine (Cys) residues in the targeted protein, forming S-nitrosothiol (SNO) [7]. SNO is dynamically labile in response to the intracellular redox status, making S-nitrosylation a highly sensitive mediator of cell signaling [8]. The factors that influence the specific formation of SNO modifications include the redox state of cells or tissues, the subcellular localization and acid–base environment of Cys residues, the local NO concentration and the expression levels of transnitrosylase or denitrosylase [9]. The efficiency of S-nitrosylation is also influenced by the concentration of intracellular ROS and NO radicals [10]. Cys residues with acid–base motifs, high sulfur-atom exposure space and low pKa are more susceptible to SNO modification [11]. It has been reported that the sequence of EXC (where E represents Glu, X indicates any amino acid residue, and C represents Cys) is a putative consensus sequence of S-nitrosylation protein, which has a higher probability of the modification [12].
Due to the development of dedicated proteomic approaches, especially the fluorous affinity tag (FAT)-switch method combined with mass spectrometry (MS), hundreds of S-nitrosylated proteins have been identified in plants [13]. Moreover, S-nitrosylation is accepted as a cell signaling mechanism with important functional implications in various physiological processes [6,14]. The diagram that illustrates NO-mediated S-nitrosylation signaling pathways in plant physiological processes is shown in Figure 1.

2. Identification of S-Nitrosylated Proteins in Plants: Methodological Aspects

2.1. The Biotin-Switch Technique

The biotin-switch technique (BST) is an efficient tool for identifying S-nitrosylated proteins [15,16]. The method consists of three main steps (Figure 2). First, S-nitrosylated proteins from extracts or recombinants are treated with S-methyl methanethiosulfonate (MMTS) and 2.5% SDS at 50 °C for 30 min with recurrent vortexing to block unmodified Cys residues. Then, MMTS is removed by protein sedimentation with cold acetone, and the proteins are dissolved in RB buffer (25 mM HEPES, 1 mM EDTA, and 1% SDS, pH 7.7). Second, ascorbic acid (ASC) is added to the RB buffer and the mixture is incubated at room temperature for 1 h with recurrent vortexing in the dark. During this step, S-nitrosylated Cys residues are transformed into free Cys thiols by the reducing role of ASC. Because ASC is a poor reducing agent, long incubation times (up to 3 h) and high ASC concentrations (30 mM or more) can greatly enhance detection sensitivity [17]. Third, the free thiols (i.e., originally S-nitrosylated sites) are labeled with a sulfhydryl-specific biotinylating reagent, such as N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide (biotin-HPDP). Notably, steps 2 and 3 occur simultaneously to ensure the immediate biotinylation of the freshly generated thiols. Since MMTS groups decompose when exposed to light, all steps must be performed in complete darkness.
After the reactions are finished, the method used to identify S-nitrosylated proteins in plant extracts is different from that for recombinant proteins. In vivo, proteins labelled with biotinylated are purified by immunoprecipitation with streptavidin beads 12 h at 4 °C. Then, the beads are washed three times with HEN buffer (1.25 mM HEPES, 500 mM EDTA, and 1 mM neocuproine, pH 7.7). The bound proteins are eluted with 10 mM DTT in SDS-PAGE solubilization buffer then subjected to 10% SDS-PAGE at first. Then, the proteins are transferred to a polyvinylidene difluoride membrane for identification using appropriate antibodies. On the contrary, in vitro biotinylated proteins in SDS-PAGE solubilization buffer are firstly subjected to 12% SDS-PAGE. Then, the labeled proteins are analyzed by immunoblotting with the corresponding antibodies or liquid chromatography-tandem MS (LC-MS/MS). Detailed methods of LC-MS/MS detection are described in Section 2.3.

2.2. Other Methods Used to Detect S-Nitrosopeptides

It is difficult for biotin-related reagents to elute from the capture resin, which complicates MS/MS spectral interpretation. Therefore, only several hundred S-nitrosylated proteins have been detected by the BST in plants, limiting the further study of S-nitrosylation. Thus, it is important to find new reagents to label sulfhydryl groups. In recent years, several other labeling reagents, including isotope-coded affinity tags [18], isobaric iodo-TMT tags [19,20], Cys-specific phosphonate adaptable tags [21], bioorthogonal cleavable-linker tags [22], thiol-reactive resin tags [23], and FATs [12] have been found to label and enrich S-nitrosopeptides. These labels are used instead of thiol-reactive biotin in the third step of the process, while the rest of the procedure is similar to the BST. Given the lack of commercial antibodies that can recognize these labels, tag-switch-enriched proteins cannot be subjected to immunoblotting, which limits their application in proteomic studies.
Among these regents, FATs have covalent C–F bonds that prevent unexpected dissociation during MS/MS and reduce the complexity of MS/MS, thus allowing for the identification of low-abundance targets [12]. FATs have been successfully applied to proteomic studies of PTMs, including protein phosphorylation, tyrosine nitration, 4-hydroxy-2-nonenal modification, and S-nitrosylation [24,25]. Recently, Qin et al. (2023) developed an N-(4,4,5,5,6,6,7,7,8,8,9,9,9-trideca-fluorononyl) iodoacetamide tag-based FAT-switch approach, which showed a significantly improved sensitivity and efficiency of enrichment and a greater detection of S-nitrosylated peptides compared to the classic BST [13]. Thus, the FAT-switch method can be used for broadscale S-nitrosylation proteomic analyses. The comparations of BST and FAT-switch techniques are shown in Table 1.
Furthermore, the protein microarray-based approach can investigate low-mass S-nitrosothiols (SNOs) [26]. The numbers of S-nitrosylated Cys residues can be measured by DAN (NO fluorescence probe; Dimethyl diacetoxyfumarate) assay. In this method, the released NO from the thiol group is detected by fluorescence labeling [27].

2.3. LC-MS/MS

Biotinylated proteins are analyzed by LC-MS/MS to identify S-nitrosylated residues [28]. LC-MS/MS involves the analysis of ionized molecules to determine their structure, molecular weight, and abundance. Schematic illustration of the procedure of site-specific S-nitrosylation analysis by LC-MS/MS is shown in Figure 3. In biology, two ionization techniques are mainly used: matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). Both MALDI and ESI allow proteins or peptides to be ionized with high sensitivity [29,30]. Importantly, MALDI is less affected by salt impurities, and it is commonly chosen for analyzing peptides obtained from electrophoresis gels through peptide mass fingerprinting.
Based on the mass-to-charge change of a specific PTM, LC-MS/MS can be effectively used to detect and identify the modification. Importantly, MS/MS experiments are required to confirm that the mass shift detected in the original ion is also found in the fragment ions carrying the labelled amino acid residue. During the application of BST, S-nitrosylated Cys residues can be detected by ionization after biotinylation. The labelled peptides are separated, enriched, and identified by MALDI to find an addition of 428 Da [31].
To identify proteins, MS/MS spectra made by LC-ESI-MS are updated in the BioTools 2.0 platform to search the NCBInr database using a licensed version of the Mascot v.2.2.04 search engine (www.matrixscience.com, accessed on 20 March 2025; Matrix Science, Israel, India, South Korea, et al.). For reduced samples, carbamidomethyl Cys is defined as a fixed modification from treatment with iodoacetamide, but oxidized methionine is regarded as a variable modification. Additional settings include a peptide mass tolerance of 0.5 Da for the parental ion mass and fragment ion masses and one missed cleavage site. For non-reduced samples, biotin-HPDP Cys modification is defined as a variable modification.

3. Functions of S-Nitrosylation in Plants

NO performs its physiological role primarily through protein S-nitrosylation, a redox-based PTM that is largely determined by the local concentration of NO and the structure of the modified protein [32,33,34]. S-nitrosylation is involved in various signaling pathways, including those affecting plant growth, development, immune responses, stress responses and symbiotic nitrogen fixation [6].

3.1. Growth and Development

NO-mediated S-nitrosylation participates in multiple phases of plant growth and development through regulating hormone signaling [35,36,37]. The functions of S-nitrosylated proteins related to growth and development are summarized in Table 2.

3.1.1. ABA Signaling

ABA is involved in seed germination and early seedling growth [38]. The application of exogenous NO breaks seed dormancy and alleviates the inhibitory effects of ABA on seed germination and early seedling growth [39,40]. For example, NO induces the S-nitrosylation of SnRK2.6 at Cys-137 and decreases the kinase activity of SnRK2.6, thereby inhibiting ABA signaling and promoting seed germination [41]. ABA insensitive5 (ABI5) is the core transcription factor that represses seed germination in ABA signaling. The S-nitrosylation of ABI5 promotes seed germination and seedling growth through triggering ABI5 degradation [42,43]. MYB30 triggers an NO-induced decrease in ABA content during germination [44]. The S-nitrosylation of MYB30 controls seed dormancy and germination by enhancing its transcriptional activity in Arabidopsis thaliana (Arabidopsis) [45]. These findings show that S-nitrosylation is important for seed dormancy and germination.

3.1.2. Auxin Signaling

NO-mediated S-nitrosylation regulates plant growth and development through auxin signaling [16,46,47]. For example, the S-nitrosylation of ROP2 reduces the rate of auxin transport by changing the intracellular localization of ROP2, thereby inhibiting root growth in Arabidopsis [48]. In plants, SKP1/CULLIN1/F-Box protein (SCF)-type E3 ubiquitin ligases are essential for the perception of auxin. The S-nitrosylation of TIR1 and SKP1 enhances protein–protein interactions to control the expression of auxin-responsive genes [49,50]. The S-nitrosylation of IAA17 at Cys-70 inhibits its interaction with TIR1, thereby negatively regulating auxin signaling [51]. These studies provide unique molecular insights into the redox-based auxin signaling in plant growth and development.

3.1.3. Other Hormone Signaling Pathways

S-nitrosylation also participates in other hormone signaling pathways. For instance, the S-nitrosylation of RGA represses gibberellin signaling to balance growth and stress responses [52]. The S-nitrosylation of AHP1 at Cys-115 negatively regulates cytokinin signaling by inhibiting its phosphorelay activity [16]. Meanwhile, NO inhibits ABA signaling in guard cells via the S-nitrosylation of OST1 [53]. Therefore, a close relationship exists between NO and phytohormones during plant growth and development. Currently, there are more studies on auxin- and ABA-related proteins than other hormone-associated proteins. Future studies should focus on the S-nitrosylation of ethylene-, JA-, and cytokinin-associated proteins.
Table 2. Plant S-nitrosylated proteins in the regulation of growth and development.
Table 2. Plant S-nitrosylated proteins in the regulation of growth and development.
SpeciesProteinThe Detecting
Techniques		FunctionsResultReference
Arabidopsis thalianaSnRK2.6BSTInhibits enzyme activityPromotes seed germination[41]
Arabidopsis thalianaABI5BST;
LC-MS/MS		Promotes ABI5 degradationPromotes seed germination[42]
Arabidopsis thalianaMYB30BST; LC-MS/MSEnhances MYB30 transcriptional activityPromotes seed germination[45]
Arabidopsis thalianaROP2BSTReduces the auxin transport rateInhibits root growth[48]
Arabidopsis thalianaTIR1BSTEnhances TIR1-Aux/IAA interactionActivates auxin signaling pathway[47]
Arabidopsis thalianaSKP1BST; LC-MS/MSEnhances SKP1 binding to CUL1-TIR1Enhances auxin signal transduction[49]
Arabidopsis thalianaIAA17BST; LC-MS/MS; DAN AssayInhibits its interaction with TIR1Negatively regulates auxin signaling[51]
Arabidopsis thalianaRGABST; LC-MS/MS; DAN AssayInhibits its interaction with the F-box proteinCoordinates growth and stress responses[52]
Arabidopsis thalianaAHP1BST; LC-MS/MS; DAN AssayInhibits AHP1 phosphorylationInhibits cytokinin reactions[15]
Arabidopsis thalianaOST1BSTInhibits OST1 activityNegatively regulates ABA signaling[53]

3.2. Immune Response

NO participates in plant immune responses, including immune-related defensive gene expression, SA-mediated immune responses, and programmed cell death (PCD) [54,55]. Crosstalk between S-nitrosylation and multiple nodes of immune signaling has been reported [14]. The regulatory methods of immune-related S-nitrosylated proteins are shown in Table 3.

3.2.1. Immune-Related Gene Expression

S-nitrosylation can regulate the expression of immunity-related genes. For example, NO mediates the S-nitrosylation of two transcription factors, NPR1 and TGA1, to increase the expression of immunity-related genes [56]. The transcription factor SRG1 is a central site of NO bioactivity in plant immunity. The S-nitrosylation of SRG1 at Cys-87 presumably increases the transcription of immune repressors, contributing to dampening of the immune response [57]. In addition, the S-nitrosylation of SRG3 plays a positive role in plant immune response [58]. These studies illustrate that S-nitrosylation regulates immune signaling by controlling the expression of immunity-related transcription factors.

3.2.2. Regulating Protein Activity

The phytohormone SA plays a critical role in plant immune signaling, helping to trigger the hypersensitivity response and activate systemic acquired resistance [59,60]. NO-induced S-nitrosylation supports the role of SA signaling network in immune responses. The S-nitrosylation of SA-binding protein 3 (AtSABP3) at Cys-280 suppresses its binding activity with the immune activator to resistant against PstDC3000 (avrB) [61]. The S-nitrosylation of small ubiquitin-like modifier (SUMO)-conjugating enzyme 1 (SCE1) at Cys-139 negatively regulates plant immunity by inhibiting its sumoylation [62]. NPR1, which locates in the cytoplasm as an oligomer, is a master regulator of SA-mediated defensive genes. The S-nitrosylation of NPR1 at Cys-156 facilitates its oligomerization to maintain protein homeostasis during plant immune response [46]. These data suggest that NO mediates the S-nitrosylation of SA-related proteins during defensive responses.

3.2.3. Regulating Programmed Cell Death

In Arabidopsis, at the onset of the hypersensitivity response, the S-nitrosylation of the bacterial effector HopAI1 inhibits its phosphothreonine lyase activity to activate programmed cell death (PCD) [63]. The S-nitrosylation of the NADPH oxidase RBOHO regulates PCD in plant immunity by abolishing its ability to synthesize reactive oxygen intermediates [64]. The roles of S-nitrosylation in cellular reprogramming are emerging research direction [14].
Table 3. Plant S-nitrosylated proteins in response to immune stress.
Table 3. Plant S-nitrosylated proteins in response to immune stress.
SpeciesProteinThe Detecting
Techniques		FunctionsResultsReference
Arabidopsis thalianaNPR1/
TGA1		BST;
LC-MS/MS		Enhances the DNA binding activityRegulates plant defense response[56]
Arabidopsis thalianaSRG1BST;
LC-MS/MS		Decreases DNA binding and transcriptional inhibition activityAttenuates the plant immune response[57]
Arabidopsis thalianaSRG3BSTAbolishes the activity for SRG3Positively regulates plant immunity[58]
Arabidopsis thalianaSABP3BST;
LC-MS/MS		Suppresses its binding activityModulates the plant defense response[61]
Arabidopsis thalianaSCE1BSTIncreases SUMO1/2 conjugate levelsImpairs the immune response[62]
Arabidopsis thalianaHopAl1BST;
DAN Assay		Inhibits phosphothreonine lyase activityActivates cell death[63]
Arabidopsis thalianaRBOHOBST;
LC-MS/MS		Decreases enzyme activityDisrupts cell death[64]

3.3. Abiotic Stress

Plants are very sensitive to environmental changes during their life. As a result, crop yields can be dramatically affected by environmental factors [65]. Therefore, it is crucial to explore the mechanism of S-nitrosylation in abiotic stresses. The regulatory methods of S-nitrosylated proteins related to abiotic stress are summarized in Table 4.

3.3.1. Improving Salt Tolerance of Plants

NO-mediated S-nitrosylation is an important signaling mechanism that positively regulates plant salt tolerance [1]. For instance, in Solanum lycopersicum, 1054 putative S-nitrosylated proteins involved in salt tolerance-related reactions have been identified by MS/MS [66]. Elsewhere, the S-nitrosylation of GAPDH increases protein activity to respond to salt stress in Nicotiana tabacum [67]. Further, the S-nitrosylation of ACOh4 promotes the synthesis of ethylene to enhance plant salt resistance [68], while the S-nitrosylation of PRMT5 enhances its methyltransferase activity during salt stress [34]. Recently, we reported that the S-nitrosylation of RAB7 regulated ion homeostasis to promote salt tolerance in Arabidopsis [69]. The S-nitrosylation of SIP5CR at Cys-5 increases its NAD(P)H affinity and enzymatic activity to adapt to salt stress [70]. In conclusion, S-nitrosylation increases plant salt tolerance by regulating different physiological functions.

3.3.2. Improving Temperature Tolerance of Plants

Extreme temperature is a major abiotic stressor that limits plant growth [71]. Plants have developed various mechanisms to increase the tolerance of extreme temperature. For example, in Ganoderma lucidum, the NO-mediated S-nitrosylation of aconitase decreases the production of ROS through regulating ganoderic acid biosynthesis under heat stress [72]. In Arabidopsis, some S-nitrosylated proteins have been identified in response to cold stress [33]. Brassinosteroids induce protein S-nitrosylation to alleviate low-temperature stress in Mini Chinese Cabbage (Brassica campestris L.) [73]. In Brassica juncea, some pathogenesis-related and photosynthetic proteins have been identified to be S-nitrosylated under cold stress [74]. S-nitrosylation might increase plant tolerance to extreme temperature by reducing the level of oxidative damage.

3.3.3. Improving Oxidative Tolerance of Plants

Some redox homeostasis-related proteins can be S-nitrosylated in plants. In Arabidopsis, the S-nitrosylation of hemoglobin AHb1 reduces NO emission under hypoxic stress [75]. The S-nitrosylation of GSNOR1 induces selective autophagy, which enhances plant tolerance to low-oxygen stress [76]. The S-nitrosylation of peroxidase APX1 enhances protein activity to adapt to oxidative stress [77]. These results demonstrate that S-nitrosylation has emerged as a connection between ROS and nitrogen species signaling. However, the crosstalk between NO and ROS requires further study.
Table 4. Plant S-nitrosylated proteins in response to abiotic stress.
Table 4. Plant S-nitrosylated proteins in response to abiotic stress.
SpeciesProteinThe Detecting
Techniques		FunctionsResultsReference
Nicotiana tabacumGAPDHBSTEnhances protein kinases activityResponses to salt stress[67]
Arabidopsis thalianaACOh4BST; LC-MS/MSPromotes the synthesis of ethyleneEnhances the salt resistance [68]
Arabidopsis thalianaRAB7BST; LC-MS/MSEnhances protein activityMaintains the ionic balance under salt stress[69]
Arabidopsis thalianaPRMT5BST; LC-MS/MS; DAN AssayEnhances methyltransferase activityEnhances stress tolerance[34]
Solanum lycopersicum L.SIP5CRBSTBoosts both SlP5CR activity and proline synthesisEnhances stress tolerance[70]
Ganoderma lucidumAconBST; LC-MS/MSRegulates ganoderic acid biosynthesisEnhances heat stress[72]
Arabidopsis thalianaAHb1BSTReduces NO emissionEnhances hypoxic stress[75]
Arabidopsis thalianaAPX1BST; LC-MS/MS; DAN AssayEnhances APX1 activityEnhances the antioxidative capacity[77]
Arabidopsis thalianaGSNORBST; LC-MS/MS; DAN Assay;Induces the autophagic degradation of GSNOREnhances hypoxic stress[76]
Helianthus annuusGAPDHBST; LC-MS/MSRegulates protein activityResponses to salt stress[78]
Helianthus annuusMDHARBST; LC-MS/MSRegulates protein activityResponses to salt stress[78]
NO plays a central role in abiotic stress [79]; it undergoes extensive crosstalk with other signaling molecules (e.g., ROS, hormonal signals, and epigenetic regulation) [80]. Current research on NO in abiotic stress mainly focuses on aspects such as antioxidant defense, signaling pathways, stomatal movement and osmotic regulatory substances [81]. However, the study of the spatial distribution of NO is still scarce. In the future, it is necessary to integrate molecular biology, nanotechnology and artificial intelligence to investigate precise NO signaling networks, providing new strategies for sustainable agriculture.

3.4. Role of S-Nitrosylation in Legumes

It has been reported that S-nitrosylated proteins exists in legumes [82]. Glutathione peroxidases (Gpxs) are highly expressed in nodules of Lotus japonicus. The S-nitrosylation of LjGpx1 and LjGpx3 inhibit enzyme activity to realize antioxidative functions [83]. In Medicago truncatula, the activity of plastid glutamine synthetase was inhibited by S-nitrosylation [84]. These results imply that S-nitrosylation plays a role in symbiotic nitrogen fixation. However, the mechanism of S-nitrosylation in symbiosis still needs more research.

4. Denitrosylation and Transnitrosylation

It has been reported that certain proteins have denitrosylase activities. Denitrosylation refers to the process by which a nitrosyl group (-NO) is removed from a Cys residue of an S-nitrosylated protein. Emerging evidence also indicates that a small group of S-nitrosylated proteins can transfer their NO moiety to another protein and nitrosylate the latter in a process termed transnitrosylation [85]. Several enzymes, including hemoglobin, superoxide dismutase 1, S-nitrosoglutathione reductase, and protein-disulfide isomerase, have been shown to possess either transnitrosylase or denitrosylase activities [86]. The discovery of denitrosylases and transnitrosylases have revealed an enzyme-based mechanism vital for the specificity of NO signaling.
Thiol-disulfide oxidoreductase thioredoxin h5 (TRXh5) can denitrosylate NPR1, which is thought to facilitate monomerization by preventing the formation of intermolecular disulfide bonds. Additionally, evidence suggests that TRXh5 is a potent protein-SNO denitrosylase with a wide set of endogenous targets [87]. ROG1 transnitrosylates GSNOR1 to promote its degradation, thereby regulating NO signaling and eventually various physiological responses [88]. Unlike other types of protein modification enzymes, identified denitrosylases and transnitrosylases remain largely uncharacterized due to a lack of consensus around conserved functional domains. Therefore, it is important to identify and characterize the adjustment mechanisms of denitrosylation and transnitrosylation.

5. The Crosstalk Between S-Nitrosylation and Other Protein PTMs

S-nitrosylation can regulate other protein PTMs, including phosphorylation, acetylation, ubiquitylation, and methylation, as part of its involvement in plant physiological processes. In Arabidopsis, the S-nitrosylation of PRMT5 enhances its methyltransferase activity in abiotic stress [34]. It has been reported that S-nitrosylation represses the phosphorylation of cytokinin signaling pathway but increases histone acetylation through the inhibition of histone deacetylases during stress responses [16,89].
Previous studies showed a crosstalk between S-nitrosylation and other redox-related modifications. It was reported that 639 proteins could be both persulfidated and S-nitrosylated (e.g., NADP-isocitrate dehydrogenase) [90]. María et al. showed that the content of H2S and NO increased significantly in sweet peppers during the mature period [91]. Therefore, there is an inevitable connection between H2S and NO in plant stress response. In addition, NO enhances the drought tolerance of Antiaris toxicaria seeds via protein S-nitrosylation and carbonylation [92]. The study has revealed that S-nitrosylation controls ROS and reactive nitrogen species (RNS) homeostasis in plants [93]. However, the mechanism of S-nitrosylation in the regulation of other protein PTMs is not clear.

6. Conclusions and Perspectives

In recent years, significant progress has been made in understanding the biochemical and biological functionality of protein S-nitrosylation. S-nitrosylation affects many physiological processes by controlling protein functions such as protein activity, binding activity, and transcriptional activity [8] (Figure 4). Therefore, S-nitrosylation plays important roles in plant biology and agricultural science.
However, the current research on S-nitrosylation has certain limitations and challenges. Firstly, the function of S-nitrosylated proteins has been widely studied in Arabidopsis, but it is rarely studied in crops [6]. A report related to S-nitrosylation in wheat was based on proteomic analysis and did not address the detailed role of S-nitrosylation in abiotic stress [94]. Since the aim of much research is to improve crop stress resistance, the application of S-nitrosylation to crops is crucial. Secondly, the research on S-nitrosylation involved in plant physiological processes is not sufficient. There are more studies on S-nitrosylated proteins participating in the seed germination stage than the seedling stage. Similarly, there are more studies on S-nitrosylated proteins related to the salt stress response than the drought stress response [95]. Therefore, further research is needed to explore extensive studies of S-nitrosylation related to plant growth and development. Thirdly, many proteins can be S-nitrosylated, but only some of the modification sites play roles during plant metabolism. Deeper research is needed to investigate the functional mechanism of S-nitrosylation.
Furthermore, we identified several research gaps around S-nitrosylation. Firstly, there is a lack of a reliable method to predict S-nitrosylation sites. The specific microenvironments around the S-nitrosylated cysteine residues (e.g., acidic residues, hydrogen bond networks) require further investigation. Secondly, it is valuable to integrate analyses of S-nitrosylation, transcriptomic and metabolomic data during stress response. Thirdly, there is a lack of a cross-species comparative study on S-nitrosylation. Such a study would uncover the conservation and diversity of S-nitrosylation during evolution.
The BST has been largely used because it avoids the instability of protein SNO groups. However, several limitations of this method have been reported in recent years. First, the BST is time-consuming and complicated, making it particularly inapposite for monitoring dynamic changes of S-nitrosylated proteins in vivo [96]. Second, the biotin-switch method is indirect and highly dependent on the complete alkylation of all free thiols. Third, S-nitrosylation is a low-abundance modification, so even low levels of uncapped thiols can lead to a high false-positive rate. Another source of false positives is sunlight-driven disulfide reduction, which can be eliminated by performing all procedures in complete darkness [97]. This unfortunate restraint makes sample preparation more tedious. Therefore, the development of a highly sensitive and user-friendly method should urgently be explored in the near future.
In conclusion, S-nitrosylation is a promising area in plants. Associated research will provide a theoretical basis for agricultural production and make an important contribution to plant biology.

Author Contributions

Conceptualization, W.L. and J.-X.S.; data curation, X.-Y.L. and X.-F.Z.; writing—original draft preparation, W.L. and L.-Q.Z.; writing—review and editing, W.L.; visualization, W.L. and J.-X.S.; funding acquisition, W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Hebei Normal University for Nationalities, grant number DR2024017 to W.L.

Conflicts of Interest

There are no conflicts of interest among the authors.

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Cimb 47 00407 g001
Figure 1. A proposed model of NO-mediated S-nitrosylation signaling. NO induces S-nitrosylation of A protein and the S-nitrosylated A (A-SNO) may subsequently transnitrosylate B at cysteine residues. A-SNO may also participate in regulation of other protein PTM. The NO-mediated S-nitrosylation regulates various physiological through different pathways.
Figure 1. A proposed model of NO-mediated S-nitrosylation signaling. NO induces S-nitrosylation of A protein and the S-nitrosylated A (A-SNO) may subsequently transnitrosylate B at cysteine residues. A-SNO may also participate in regulation of other protein PTM. The NO-mediated S-nitrosylation regulates various physiological through different pathways.
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Figure 2. The schematic diagram of biotin-switch technique (BST) for detection of S-nitrosylated proteins. (a) Reaction steps for labeling S-nitrosylated proteins. 1, Unmodified Cys residues are blocked by MMTS. 2, The ASC specifically reduces S-nitrosylated Cys residues. 3, The newly liberated thiols are substituted by biotin-HPDP. (b) The structural formula of MMTS. (c) The structural formula of ASC. (d) The structural formula of biotin-HPDP.
Figure 2. The schematic diagram of biotin-switch technique (BST) for detection of S-nitrosylated proteins. (a) Reaction steps for labeling S-nitrosylated proteins. 1, Unmodified Cys residues are blocked by MMTS. 2, The ASC specifically reduces S-nitrosylated Cys residues. 3, The newly liberated thiols are substituted by biotin-HPDP. (b) The structural formula of MMTS. (c) The structural formula of ASC. (d) The structural formula of biotin-HPDP.
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Figure 3. Schematic illustration of the procedure of site-specific S-nitrosylation analysis by LC-MS/MS.
Figure 3. Schematic illustration of the procedure of site-specific S-nitrosylation analysis by LC-MS/MS.
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Figure 4. The functions of S-nitrosylated proteins in various physiological processes. NO-mediated protein S-nitrosylation affects plant physiological processes by altering protein structure and function.
Figure 4. The functions of S-nitrosylated proteins in various physiological processes. NO-mediated protein S-nitrosylation affects plant physiological processes by altering protein structure and function.
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Table 1. The comparative table of BST and FAT-switch techniques.
Table 1. The comparative table of BST and FAT-switch techniques.
BST TechniquesFAT-Switch Techniques
Sensitivity and efficiencyLowHigh
SpecificityHighHigh
CostMiddleMiddle
Limitation of methodTime-consuming and tedious; Unsuitable for monitoring dynamic changes in S-nitrosylationLack of commercial antibodies; Unsuitable for monitoring dynamic changes in S-nitrosylation
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Lin, W.; Shang, J.-X.; Li, X.-Y.; Zhou, X.-F.; Zhao, L.-Q. Nitric Oxide Regulates Multiple Signal Pathways in Plants via Protein S-Nitrosylation. Curr. Issues Mol. Biol. 2025, 47, 407. https://doi.org/10.3390/cimb47060407

AMA Style

Lin W, Shang J-X, Li X-Y, Zhou X-F, Zhao L-Q. Nitric Oxide Regulates Multiple Signal Pathways in Plants via Protein S-Nitrosylation. Current Issues in Molecular Biology. 2025; 47(6):407. https://doi.org/10.3390/cimb47060407

Chicago/Turabian Style

Lin, Wei, Jian-Xiu Shang, Xiao-Ying Li, Xue-Feng Zhou, and Li-Qun Zhao. 2025. "Nitric Oxide Regulates Multiple Signal Pathways in Plants via Protein S-Nitrosylation" Current Issues in Molecular Biology 47, no. 6: 407. https://doi.org/10.3390/cimb47060407

APA Style

Lin, W., Shang, J.-X., Li, X.-Y., Zhou, X.-F., & Zhao, L.-Q. (2025). Nitric Oxide Regulates Multiple Signal Pathways in Plants via Protein S-Nitrosylation. Current Issues in Molecular Biology, 47(6), 407. https://doi.org/10.3390/cimb47060407

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