David Wendehenne

Nitric oxide: comparative synthesis and signaling in animal and plant cells

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.

Early Signaling Events Induced by Elicitors of Plant Defenses

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.

Analysis of Nitric Oxide Signaling Functions in Tobacco Cells Challenged by the Elicitor Cryptogein

Nitric oxide (NO) has recently emerged as an important cellular mediator in plant defense responses. However, elucidation of the biochemical mechanisms by which NO participates in this signaling pathway is still in its infancy. We previously demonstrated that cryptogein, an elicitor of tobacco defense responses, triggers a NO burst within minutes in epidermal sections from tobacco leaves (Nicotiana tabacum cv Xanthi). Here, we investigate the signaling events that mediate NO production, and analyze NO signaling activities in the cryptogein transduction pathway. Using flow cytometry and spectrofluorometry, we observed that cryptogein-induced NO production in tobacco cell suspensions is sensitive to nitric oxide synthase inhibitors and may be catalyzed by variant P, a recently identified pathogen-inducible plant nitric oxide synthase. NO synthesis is tightly regulated by a signaling cascade involving Ca2+ influx and phosphorylation events. Using tobacco cells constitutively expressing the Ca2+ reporter apoaequorin in the cytosol, we have shown that NO participates in the cryptogein-mediated elevation of cytosolic free Ca2+ through the mobilization of Ca2+ from intracellular stores. The NO donor diethylamine NONOate promoted an increase in cytosolic free Ca2+ concentration, which was sensitive to intracellular Ca2+ channel inhibitors. Moreover, NO appears to be involved in the pathway(s) leading to the accumulation of transcripts encoding the heat shock protein TLHS-1, the ethylene-forming enzyme cEFE-26, and cell death. In contrast, NO does not act upstream of the elicitor-induced activation of mitogen-activated protein kinase, the opening of anion channels, nor expression of GST, LOX-1, PAL, and PR-3 genes. Collectively, our data indicate that NO is intimately involved in the signal transduction processes leading to cryptogein-induced defense responses.

Nitric Oxide Contributes to Cadmium Toxicity in Arabidopsis by Promoting Cadmium Accumulation in Roots and by Up-Regulating Genes Related to Iron Uptake

Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd2+), a nonessential and toxic metal. We demonstrate that Cd2+ induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd2+. By analyzing the incidence of NO scavenging or inhibition of its synthesis during Cd2+ treatment, we demonstrated that NO contributes to Cd2+-triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up- and down-regulated during Cd2+ treatment. Forty-three genes were identified encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd2+-treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NOs ability to promote Cd2+ accumulation in roots. This analysis also highlights that NO is responsible for Cd2+-induced inhibition of root Ca2+ accumulation. Taken together, our results suggest that NO contributes to Cd2+ toxicity by favoring Cd2+ versus Ca2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

Involvement of Free Calcium in Action of Cryptogein, a Proteinaceous Elicitor of Hypersensitive Reaction in Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245–1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.

Nitric oxide in plants: the biosynthesis and cell signalling properties of a fascinating molecule

Nitric oxide (NO) is both a gaseous free radical and a versatile cell-signalling effector that plays important roles in diverse (patho)physiological processes. In ani mals, NO production is catalyzed predominantly by nitric oxide synthases (NOS), which are heme-contain ing proteins related to the cytochrome P450 family. These enzymes catalyze the conversion of L-arginine to L-citrulline and NO using NADPH and molecular oxy gen as cosubstrates, and employ FAD, FMN, tetrahy drobiopterin (BH4), and calmodulin (CaM) as cofactors (Bogdan 2001). The biological effects of NO are medi ated by posttranslational modification of cysteine resi dues and transition metal centers, a key process referred as nitrosylation (Stamler et al. 2001). Because of its high biological reactivity, NO production by NOS is tightly regulated to control the specificity of its signalling as well as to limit its toxicity (Kone et al. 2003). In recent years, NO has also become an increasingly popular target of investigation in plants. NO has been implicated in disease resistance, stomatal closure, re sponses to abiotic stress, iron homeostasis, and in vari ous developmental processes (Neill et al. 2002a, b; Wendehenne et al. 2004). A major advance in our understanding of NO functions in plants has been the identification of enzymes that catalyze NO synthesis. Nitrate reductase (NR) was the first enzymatic source of NO to be identified (Yamasaki and Sakihama 2000). In addition to its role in nitrate reduction, NR catalyzes the reduction of nitrite to NO using NAD(P)H as co-factor. As recently discussed by Meyer et al. (2004), it remains

Integrated Signaling Network Involving Calcium, Nitric Oxide, and Active Oxygen Species but Not Mitogen-Activated Protein Kinases in BcPG1-Elicited Grapevine Defenses

We have already reported the identification of the endopolygalacturonase 1 (BcPG1) from Botrytis cinerea as a potent elicitor of defense responses in grapevine, independently of its enzymatic activity. The aim of the present study is the analysis of the signaling pathways triggered by BcPG1 in grapevine cells. Our data indicate that BcPG1 induces a Ca2+ entry from the apoplasm, which triggers a phosphorylation-dependent nitric oxide (NO) production via an enzyme probably related to a NO synthase. Then NO is involved in (i) cytosolic calcium homeostasis, by activating Ca2+ release from internal stores and regulating Ca2+ fluxes across the plasma membrane, (ii) plasma membrane potential variation, (iii) the activation of active oxygen species (AOS) production, and (iv) defense gene expression, including phenylalanine ammonia lyase and stilbene synthase, which encode enzymes responsible for phytoalexin biosynthesis. Interestingly enough, mitogen-activated protein kinase (MAPK) activation is independent of this regulation pathway that closely connects Ca2+, NO, and AOS.

Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases

Much attention has been paid to nitric oxide (NO) research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.

Evidence for specific, high-affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane

Binding of cryptogein, a proteinaceous elicitor, was studied on tobacco plasma membrane. The binding of the [125I]cryptogein was saturable, reversible and specific with an apparent K d of 2 nM. A single class of cryptogein binding sites was found with a sharp optimum pH for binding at about pH 7.0. The high‐affinity correlates with cryptogein concentrations required for biological activity in vivo.

S-nitrosylation: an emerging post-translational protein modification in plants.

Increasing evidences support the assumption that nitric oxide (NO) acts as a physiological mediator in plants. Understanding its pleiotropic effects requires a deep analysis of the molecular mechanisms underlying its mode of action. In the recent years, efforts have been made in the identification of plant proteins modified by NO at the post-translational level, notably by S-nitrosylation. This reversible process involves the formation of a covalent bond between NO and reactive cysteine residues. This research has now born fruits and numerous proteins regulated by S-nitrosylation have been identified and characterized. This review describes the basic principle of S-nitrosylation as well as the Biotin Switch Technique and its recent adaptations allowing the identification of S-nitrosylated proteins in physiological contexts. The impact of S-nitrosylation on the structure/function of selected proteins is further discussed.