A forty year journey: The generation and roles of NO in plants.

In this year there is the 40th anniversary of the first publication of plant nitric oxide (NO) emission by Lowell Klepper. In the decades since then numerous milestone discoveries have revealed that NO is a multifunctional molecule in plant cells regulating both plant development and stress responses. Apropos of the anniversary, these authors aim to review and discuss the developments of past concepts in plant NO research related to NO metabolism, NO signaling, NO's action in plant growth and in stress responses and NO's interactions with other reactive compounds. Despite the long-lasting research efforts and the accumulating experimental evidences numerous questions are still needed to be answered, thus future challenges and research directions have also been drawn up.

The Evolution of HD2 Proteins in Green Plants.

In eukaryotes, protein deacetylation is carried out by two well-conserved histone deacetylase (HDAC) families: RPD3/HDA1 and SIR2. Intriguingly, model plants such as Arabidopsis express an additional plant-specific HDAC family, termed type-2 HDACs (HD2s). Transcriptomic analyses from more than 1300 green plants generated by the 1000 plants (1KP) consortium showed that HD2s appeared early in green plant evolution, the first members being detected in several streptophyte green alga. The HD2 family has expanded via several rounds of successive duplication; members are expressed in all major green plant clades. Interestingly, angiosperm species express new HD2 genes devoid of a zinc-finger domain, one of the main structural features of HD2s. These variants may have been associated with the origin and/or the biology of the ovule/seed.

Nitric oxide production mediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana.

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. In this study, we investigated the production and/or function of NO in Arabidopsis thaliana leaf discs and plants elicited by oligogalacturonides (OGs) and challenged with Botrytis cinerea. We provided evidence that OGs triggered a fast and long lasting NO production which was Ca(2+) dependent and involved nitrate reductase (NR). Accordingly, OGs triggered an increase of both NR activity and transcript accumulation. NO production was also sensitive to the mammalian NO synthase inhibitor L-NAME. Intriguingly, we showed that L-NAME affected NO production by interfering with NR activity, thus questioning the mechanisms of how this compound impairs NO synthesis in plants. We further demonstrated that NO modulates RBOHD-mediated reactive oxygen species (ROS) production and participates in the regulation of OG-responsive genes such as anionic peroxidase (PER4) and a ß-1,3-glucanase. Mutant plants impaired in PER4 and ß-1,3-glucanase, as well as Col-0 plants treated with the NO scavenger cPTIO, were more susceptible to B. cinerea. Taken together, our investigation deciphers part of the mechanisms linking NO production, NO-induced effects and basal resistance to B. cinerea.

Are grapevine stomata involved in the elicitor-induced protection against downy mildew?

Stomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H(2)O(2) production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA and elicitors share common signaling events, it led us to question whether stomatal movements and H(2)O(2) production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine-Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production.

An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells.

The pathogenicity of various Streptomyces scabies isolates involved in potato scab disease was correlated with the production of thaxtomin A. Since calcium is known as an essential second messenger associated with pathogen-induced plant responses and cell death, it was investigated whether thaxtomin A could induce a Ca2+ influx related to cell death and to other putative plant responses using Arabidopsis thaliana suspension cells, which is a convenient model to study plant-microbe interactions. A. thaliana cells were treated with micromolar concentrations of thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements with the apoaequorin Ca2+ reporter protein and by external pH measurement. Involvement of anion and calcium channels in signal transduction leading to programmed cell death was determined by using specific inhibitors. These data suggest that this toxin induces a rapid Ca2+ influx and cell death in A. thaliana cell suspensions. Moreover, these data provide strong evidence that the Ca2+ influx induced by thaxtomin A is necessary to achieve this cell death and is a prerequisite to early thaxtomin A-induced responses: anion current increase, alkalization of the external medium, and the expression of PAL1 coding for a key enzyme of the phenylpropanoid pathway.

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.

In vivo imaging of an elicitor-induced nitric oxide burst in tobacco.

A growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti-microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF-2 DA) has been developed which allows bio-imaging of NO in vivo. Here we use the cell-permeable DAF-2 DA, in conjunction with confocal laser scanning microscopy, for real-time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO-induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.

Nitric oxide and salicylic acid signaling in plant defense.

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H(2)O(2)-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IkappaBalpha and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Nitric oxide modulates the activity of tobacco aconitase.

Recent evidence suggests an important role for nitric oxide (NO) signaling in plant-pathogen interactions. Additional elucidation of the role of NO in plants will require identification of NO targets. Since aconitases are major NO targets in animals, we examined the effect of NO on tobacco (Nicotiana tabacum) aconitase. The tobacco aconitases, like their animal counterparts, were inhibited by NO donors. The cytosolic aconitase in animals, in addition to being a key redox and NO sensor, is converted by NO into an mRNA binding protein (IRP, or iron-regulatory protein) that regulates iron homeostasis. A tobacco cytosolic aconitase gene (NtACO1) whose deduced amino acid sequence shared 61% identity and 76% similarity with the human IRP-1 was cloned. Furthermore, residues involved in mRNA binding by IRP-1 were conserved in NtACO1. These results reveal additional similarities between the NO signaling mechanisms used by plants and animals.

Involvement of plasma membrane proteins in plant defense responses. Analysis of the cryptogein signal transduction in tobacco.

Cryptogein, a 98 amino acid protein secreted by the fungus Phytophthora cryptogea, induces a hypersensitive response and systemic acquired resistance in tobacco plants (Nicotiana tabacum var Xanthi). The mode of action of cryptogein has been studied using tobacco cell suspensions. The recognition of this elicitor by a plasma membrane receptor leads to a cascade of events including protein phosphorylation, calcium influx, potassium and chloride effluxes, plasma membrane depolarization, activation of a NADPH oxidase responsible for active oxygen species (AOS) production and cytosol acidification, activation of the pentose phosphate pathway, and activation of two mitogen-activated protein kinase (MAPK) homologues. The organization of the cryptogein responses reveals that the earliest steps of the signal transduction pathway involve plasma membrane activities. Their activation generates a complex network of second messengers which triggers the specific physiological responses. This study may contribute to our understanding of plant signaling processes because elicitors and a variety of signals including hormones, Nod factors, light, gravity and stresses share some common transduction elements and pathways.

Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose.

Reactive oxygen species are believed to perform multiple roles during plant defense responses to microbial attack, acting in the initial defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP acting as a second messenger in tobacco, NO treatment induced dramatic and transient increases in endogenous cGMP levels. Furthermore, NO-induced activation of PAL was blocked by 6-anilino-5,8-quinolinedione and 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked PAL activation, inhibition by 1H-(1,2,4)-oxadiazole[4, 3-a]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants.

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.

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 Kd 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 crytogein concentrations required for biological activity in vivo.