RESUMEN
Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.
Asunto(s)
Membrana Celular , Microdominios de Membrana , Plantas , Terminología como Asunto , Microdominios de Membrana/metabolismo , Membrana Celular/metabolismoRESUMEN
The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Lipidómica , Proteómica , Arabidopsis/metabolismo , Arabidopsis/genética , Proteómica/métodos , Membrana Celular/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteoma/metabolismo , Esfingolípidos/metabolismo , Fosfolípidos/metabolismoRESUMEN
Plant-microbe interactions (PMIs) are regulated through a wide range of mechanisms in which sterols from plants and microbes are involved in numerous ways, including recognition, transduction, communication, and/or exchanges between partners. Phytosterol equilibrium is regulated by PMIs through expression of genes involved in phytosterol biosynthesis, together with their accumulation. As such, PMI outcomes also include plasma membrane (PM) functionalization events, in which phytosterols have a central role, and activation of sterol-interacting proteins involved in cell signaling. In spite (or perhaps because) of such multifaceted abilities, an overall mechanism of sterol contribution is difficult to determine. However, promising approaches exploring sterol diversity, their contribution to PMI outcomes, and their localization would help us to decipher their crucial role in PMIs.
Asunto(s)
Interacciones Microbiota-Huesped , Plantas , Esteroles , Interacciones Microbiota-Huesped/fisiología , Fitosteroles/metabolismo , Plantas/metabolismo , Plantas/microbiología , Transducción de Señal , Esteroles/metabolismoRESUMEN
Although the functions and structural roles of sterols have been the subject of numerous studies, the reasons for the diversity of sterols in the different eukaryotic kingdoms remain unclear. It is thought that the specificity of sterols is linked to unidentified supplementary functions that could enable organisms to be better adapted to their environment. Ergosterol is accumulated by late branching fungi that encounter oxidative perturbations in their interfacial habitats. Here, we investigated the antioxidant properties of ergosterol using in vivo, in vitro, and in silico approaches. The results showed that ergosterol is involved in yeast resistance to tert-butyl hydroperoxide and protects lipids against oxidation in liposomes. A computational study based on quantum chemistry revealed that this protection could be related to its antioxidant properties operating through an electron transfer followed by a proton transfer mechanism. This study demonstrates the antioxidant role of ergosterol and proposes knowledge elements to explain the specific accumulation of this sterol in late branching fungi. Ergosterol, as a natural antioxidant molecule, could also play a role in the incompletely understood beneficial effects of some mushrooms on health.
RESUMEN
The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.
Asunto(s)
Membrana Celular/metabolismo , Plantas/metabolismo , Esfingolípidos/metabolismo , Biofisica , Polisacáridos/metabolismo , Especificidad de la Especie , Esfingolípidos/químicaRESUMEN
Sphingolipids are fundamental lipids involved in various cellular, developmental and stress-response processes. As such, they orchestrate not only vital molecular mechanisms of living cells but also act in diseases, thus qualifying as potential pharmaceutical targets. Sphingolipids are universal to eukaryotes and are also present in some prokaryotes. Some sphingolipid structures are conserved between animals, plants and fungi, whereas others are found only in plants and fungi. In plants, the structural diversity of sphingolipids, as well as their downstream effectors and molecular and cellular mechanisms of action, are of tremendous interest to both basic and applied researchers, as about half of all small molecules in clinical use originate from plants. Here, we review recent advances towards a better understanding of the biosynthesis of sphingolipids, the diversity in their structures as well as their functional roles in membrane architecture, cellular processes such as membrane trafficking and cell polarity, and cell responses to environmental or developmental signals.
Asunto(s)
Membrana Celular/ultraestructura , Plantas/metabolismo , Esfingolípidos/biosíntesis , Secuencia de Carbohidratos , Comunicación Celular , Membrana Celular/química , Polaridad Celular , Plantas/ultraestructura , Esfingolípidos/química , Estrés FisiológicoRESUMEN
Lipids and proteins modulate both the global order of plasma membrane (PM) and its organization in distinct domains. This raises the question of the influence on PM-ordered domain formation of PM composition, which is finely controlled during cell differentiation. Labeling of plant cell PM with an environment-sensitive probe demonstrated that the level of PM order is regulated during anisotropic expansion observed during both cell regeneration from protoplasts and cell differentiation along the growing root. Indeed, PM order progressively decreased during the polarized growth of regenerated tobacco cells, without observed correlation between this parameter and the kinetics of either cell wall regeneration or cell morphology. This suggests that the dynamics of PM formation and renewal could control the PM organization, maybe by involving the secretory pathway.
Asunto(s)
Membrana Celular/metabolismo , Pared Celular/metabolismo , Nicotiana/metabolismo , Células Vegetales/metabolismo , Diferenciación Celular/fisiología , Cinética , Protoplastos/metabolismoRESUMEN
The plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Fosfolípidos/química , Fitosteroles/química , Esfingolípidos/química , Interacciones Microbiota-Huesped/fisiología , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Fitosteroles/metabolismo , Células Vegetales/química , Células Vegetales/ultraestructura , Plasmodesmos/química , Plasmodesmos/metabolismo , Esfingolípidos/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
Since the publication of the fluid mosaic as a relevant model for biological membranes, accumulating evidence has revealed the outstanding complexity of the composition and organization of the plant plasma membrane (PM). Powerful new methodologies have uncovered the remarkable multiscale and multicomponent heterogeneity of PM subcompartmentalization, and this is emerging as a general trait with different features and properties. It is now evident that the dynamics of such a complex organization are intrinsically related to signaling pathways that regulate key physiological processes. Listing and linking recent progress in precisely qualifying these heterogeneities will help to draw an integrated picture of the plant PM. Understanding the key principles governing such a complex dynamic organization will contribute to deciphering the crucial role of the PM in cell physiology.
Asunto(s)
Membrana Celular/fisiología , Fenómenos Fisiológicos de las Plantas , Transducción de SeñalRESUMEN
The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.
Asunto(s)
Metabolismo de los Lípidos , Microdominios de Membrana/metabolismo , Nicotiana/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Pared Celular/ultraestructura , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Proteínas de Plantas/metabolismo , Protoplastos , Nicotiana/ultraestructuraRESUMEN
Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.
Asunto(s)
Colesterol/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Lisofosfolípidos/metabolismo , Microdominios de Membrana/metabolismo , Receptores Depuradores de Clase B/metabolismo , Esfingomielinas/metabolismo , Células CACO-2 , Humanos , Gotas Lipídicas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.
Asunto(s)
Membrana Celular/química , Nicotiana/química , Nicotiana/fisiología , Proteínas de Plantas/análisis , Fenómenos Biofísicos , MicroscopíaRESUMEN
Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Proteínas Fúngicas/farmacología , NADPH Oxidasas/metabolismo , Nicotiana/enzimología , Estallido Respiratorio , Línea Celular , Análisis por Conglomerados , Activación Enzimática/efectos de los fármacos , Mutación con Ganancia de Función/genética , Silenciador del Gen , MicroARNs/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Ácidos Fosfatidicos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Inhibidores de Proteínas Quinasas/farmacología , Estallido Respiratorio/efectos de los fármacos , Nicotiana/efectos de los fármacos , Nicotiana/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.
Asunto(s)
Membrana Celular/fisiología , Resistencia a la Enfermedad/fisiología , Fluidez de la Membrana/fisiología , Arabidopsis/fisiología , Membrana Celular/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Enfermedades de las Plantas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Espectrometría de Fluorescencia , Nicotiana/fisiologíaRESUMEN
The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.
Asunto(s)
Membrana Celular/química , Lípidos de la Membrana/química , Nicotiana/química , Esfingolípidos/química , Técnicas de Cultivo de Célula/métodos , Membrana Celular/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Glicoesfingolípidos/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microscopía Confocal , Modelos Moleculares , Fitosteroles/química , Fitosteroles/metabolismo , Hojas de la Planta/química , Esfingolípidos/metabolismo , Nicotiana/citología , Nicotiana/metabolismoRESUMEN
The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than ß-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains.
Asunto(s)
Membrana Celular/química , Lípidos/análisis , Lípidos de la Membrana/análisis , Plantas/química , 1,2-Dipalmitoilfosfatidilcolina/análisis , Línea Celular , Colesterol/análogos & derivados , Colesterol/análisis , Imagenología Tridimensional , Lípidos/química , Lípidos de la Membrana/química , Microscopía Confocal , Modelos Moleculares , Fosfatidilcolinas/análisis , Fitosteroles/análisis , Espectrometría de Fluorescencia , Esfingolípidos/análisisRESUMEN
The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.
Asunto(s)
Proteínas Fúngicas/farmacología , Nicotiana/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Esfingolípidos/metabolismo , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Nicotiana/citología , Nicotiana/efectos de los fármacosRESUMEN
Nitric oxide (NO) has many functions in plants. Here, we investigated its interplays with reactive oxygen species (ROS) in the defence responses triggered by the elicitin cryptogein. The production of NO induced by cryptogein in tobacco cells was partly regulated through a ROS-dependent pathway involving the NADPH oxidase NtRBOHD. In turn, NO down-regulated the level of H2O2. Both NO and ROS synthesis appeared to be under the control of type-2 histone deacetylases acting as negative regulators of cell death. Occurrence of an interplay between NO and ROS was further supported by the finding that cryptogein triggered a production of peroxynitrite (ONOO(-)). Next, we showed that ROS, but not NO, negatively regulate the intensity of activity of the cryptogein-induced protein kinase NtOSAK. Furthermore, using a DNA microarray approach, we identified 15 genes early induced by cryptogein via NO. A part of these genes was also modulated by ROS and encoded proteins showing sequence identity to ubiquitin ligases. Their expression appeared to be negatively regulated by ONOO(-), suggesting that ONOO(-) mitigates the effects of NO and ROS. Finally, we provided evidence that NO required NtRBOHD activity for inducing cell death, thus confirming previous assumption that ROS channel NO through cell death pathways.