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Polymeric nanoparticles have emerged as carrier systems for molecules that release nitric oxide (NO), a free radical involved in plant stress responses. However, to date, nanoencapsulated NO donors have not been applied to plants under realistic field conditions. Here, we verified the effects of free and nanoencapsulated NO donor, S-nitroso-mercaptosuccinic acid (S-nitroso-MSA), on growth, physiological and biochemical parameters of neotropical tree seedlings kept under full sunlight in the nursery for acclimation. S-nitroso-MSA incorporation into chitosan nanoparticles partially protected the NO donor from thermal and photochemical degradation. The application of nanoencapsulated S-nitroso-MSA in the substrate favoured the growth of seedlings of Heliocarpus popayanensis , a shade-intolerant tree. In contrast, free S-nitroso-MSA or nanoparticles containing non-nitrosated mercaptosuccinic acid reduced photosynthesis and seedling growth. Seedlings of Cariniana estrellensis , a shade-tolerant tree, did not have their photosynthesis and growth affected by any formulations, despite the increase of foliar S-nitrosothiol levels mainly induced by S-nitroso-MSA-loaded nanoparticles. These results suggest that depending on the tree species, nanoencapsulated NO donors can be used to improve seedling acclimation in the nursery.

Nitric oxide (NO), a gaseous, redox-active small molecule, is gradually becoming established as a central regulator of growth, development, immunity and environmental interactions in plants. A major route for the transfer of NO bioactivity is S-nitrosylation, the covalent attachment of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). This chemical transformation is rapidly emerging as a prototypic, redox-based post-translational modification integral to the life of plants. Here we review the myriad roles of NO and SNOs in plant biology and, where known, the molecular mechanisms underpining their activity.

Nitric oxide (NO{bullet}) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO{bullet} and S-nitrosated thiol proteins. MitoSNO1-induced NO{bullet} production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO{bullet} generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO{bullet} donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

Nitric oxide (NO) is emerging as a key regulator of diverse plant cellular processes. A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). Total cellular levels of protein S-nitrosylation are controlled predominantly by S-nitrosoglutathione reductase 1 (GSNOR1) which turns over the natural NO donor, S-nitrosoglutathione (GSNO). In the absence of GSNOR1 function, GSNO accumulates, leading to dysregulation of total cellular S-nitrosylation. Here we show that endogenous NO accumulation in Arabidopsis, resulting from loss-of-function mutations in NO Overexpression 1 (NOX1), led to disabled Resistance (R) gene-mediated protection, basal resistance and defence against nonadapted pathogens. In nox1 plants both salicylic acid (SA) synthesis and signalling were suppressed, reducing SA-dependent defence gene expression. Significantly, expression of a GSNOR1 transgene complemented the SNO-dependent phenotypes of paraquat resistant 2-1 (par2-1) plants but not the NO-related characters of the nox1-1 line. Furthermore, atgsnor1-3 nox1-1 double mutants supported greater bacterial titres than either of the corresponding single mutants. Our findings imply that GSNO and NO, two pivotal redox signalling molecules, exhibit additive functions and, by extension, may have distinct or overlapping molecular targets during both immunity and development.

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excessl (noel), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H₂O₂) in the leaves, which consequently promoted NO production via the activation of nitrate reduclase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H₂O₂-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reducíase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H₂O₂-induced leaf cell death in rice.

Nitric oxide (NO) and ethylene are signalling molecules that are synthesized in response to oxygen depletion. Non-symbiotic plant haemoglobins (Hbs) have been demonstrated to act in roots under oxygen depletion to scavenge NO. Using Arabidopsis thaliana plants, the online emission of NO or ethylene was directly quantified under normoxia, hypoxia (0.1–1.0% O2), or full anoxia. The production of both gases was increased with reduced expression of either of the Hb genes GLB1 or GLB2, whereas NO emission decreased in plants overexpressing these genes. NO emission in plants with reduced Hb gene expression represented a major loss of nitrogen equivalent to 0.2mM nitrate per 24h under hypoxic conditions. Hb gene expression was greatly enhanced in flooded roots, suggesting induction by reduced oxygen diffusion. The function could be to limit loss of nitrogen under NO emission. NO reacts with thiols to form S-nitrosylated compounds, and it is demonstrated that hypoxia substantially increased the content of S-nitrosylated compounds. A parallel up-regulation of Hb gene expression in the normoxic shoots of the flooded plants may reflect signal transmission from root to shoot via ethylene and a role for Hb in the shoots. Hb gene expression was correlated with ethylene-induced upward leaf movement (hyponastic growth) but not with hypocotyl growth, which was Hb independent. Taken together the data suggest that Hb can influence flood-induced hyponasty via ethylene-dependent and, possibly, ethylene-independent pathways.

S-nitrosothiols are labile thiol-NO adducts formed in vivo primarily by metalloproteins such as NO synthase, ceruloplasmin, and hemoglobin. Abnormal S-nitrosothiol synthesis and catabolism contribute to many diseases, ranging from asthma to septic shock. Current methods for quantifying S-nitrosothiols in vivo are suboptimal. Samples need to be removed from the body for analysis, and the S-nitrosothiols can be broken down during ex vivo processing. Here, we have developed a noninvasive device to measure mammalian tissue S-nitrosothiols in situ non-invasively using ultraviolet (UV) light, which causes NO release in proportion to the S-nitrosothiol concentration. We validated the assay in vitro; then, we applied it to measure S-nitrosothiols in vivo in rats and in humans. The method was sensitive to 0.5 µM, specific (did not detect other nitrogen oxides), and was reproducible in rats and in humans. This noninvasive approach to S-nitrosothiol measurements may be applicable for use in human diseases.

To a large extent, the dense extracellular matrix (ECM), which tightly connects tumor cells to arm the tumor into an intractable fortress, significantly decreases the nanoparticles delivery efficacy and overall performance in cancer treatments. Therefore, it is necessary to transform the dense stroma of solid tumors to loose state, which could realize deep penetration of nanomedicine and enhance cancer treatment effects. Here, we fabricated a protein-free collagen nanosweeper, triphenylphosphonium bromide (TPP) coated and S-nitrosothiols loaded mini-sized Au@silica nanorod (Au@SiO -SNO/PEG/TPP, GSNP-TPP), to clear the transport barriers of nanoparticles as well as elevate enhanced permeability and retention (EPR) effect, thus alleviating the diffusion resistance and realizing further penetration of nanoparticles. By modifying the Au@silica with thermo-sensitive S-nitrosothiols, the carrier could release the nitric oxide (NO) due to the surface overheat as well as perform photothermal therapy (PTT) under near-infrared (NIR) laser irradiation. The level of collagen depletion was observed via western blotting and immunofluorescent staining. In addition, the dual-imaging and antitumor efficiency of GSNP-TPPs were evaluated with the HeLa tumor-bearing mouse model. On one hand, the released NO could deplete collagen by activating matrix metalloproteinases (MMPs) to break collagen fibers, thus loosening the dense ECM to enhance the cellular internalization. On the other hand, with the mitochondrial-targeted effect of TPP, the diffusible NO in tumor might rapidly interact with superoxide anion (O Ÿ ) to produce highly toxic and powerful reactive nitrogen species (RNS) -- peroxynitrite (ONOO ), which resulted in mitochondrial damage to induce cell apoptosis. With the unique properties of mini-sized gold nanorods, the formulated nanoparticles exhibited good computed tomography (CT) and multi-spectral optoacoustic tomography (MSOT) imaging effects in precisely locating and monitoring tumor. Moreover, the antitumor efficacy of GSNP-TPPs + laser group was further confirmed by ex-vivo histological analysis of tumor tissue. This work points out a strategy to overcome the obstacle standing in nanoparticles penetration, and opens the door of further exploitation of NO-related theranostic systems.

Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding. As protein synthesis takes place, newly synthesized polypeptide chain passes through the ribosomal exit tunnel, which can accommodate up to 70 residues in the case of a helical peptide. Here the authors show that oxidation of cysteine residues in the nascent chain can occur within the ribosome exit tunnel, where sufficient space exists for the formation of disulfide bonds.