NO signaling functions in the biotic and abiotic stress responses

Over the past two decades, it has been recognized that nitric oxide (NO) plays an important role in diverse mammalian physiological processes. NO regulates physiological processes by modulating the activity of proteins principally by nitrosylation, a process referring to the binding of NO to a transition metal centre or cysteine residues [1]. An important class of proteins that constitutes key targets of NO is that of the Ca²⁺ channels including plasma membranes as well intracellular Ca²⁺ channels. NO modulates these channels directly by nitrosylation, but also indirectly via the second messenger cyclic GMP (cGMP) and/or cyclic ADP ribose (cADPR). Therefore, NO emerges as a key messenger governing the overall control of Ca²⁺ homeostasis [2].

In the late 1990s, NO also became an increasingly popular target for investigation in plants. As in mammals, NO fulfils a broad spectrum of signaling functions in (patho)physiological processes in plants [3]. Here, we summarise studies published in recent years that provide novel insights into the signaling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). It focuses particularly on the cross-talk operating between NO and Ca²⁺.

Over the past few years, we have studied the functions of NO in plant cells challenged with elicitors of defense responses. One elicitor has been used primarily: cryptogein, a 10 kDa elicitor produced by the oomycete Phytophthora cryptogea [4].

Using the NO sensitive fluorophore 4,5-diaminofluorescein diacetate (DAF-2DA), we reported the real-time imaging of NO production in epidermal tobacco cells treated with cryptogein [5]. After elicitation with the elicitor, the earliest burst of NO was in the chloroplasts, where NO production occurred within 3 minutes. The level of fluorescence increased with time, and after 6 minutes NO was also found along the plasma membrane, in the nucleus and most probably in peroxisomes. To investigate the signaling events that mediate NO production, and to analyse NO signaling activities in the cryptogein transduction pathway, a spectrofluorometric assay using DAF-2DA was developed to follow NO production in tobacco cultured cells. As observed in tobacco epidermal tissue, cryptogein induced a fast and transient NO production in tobacco cell suspensions [6]. This production was completely suppressed in the presence of the NO scavenger cPTIO, and was reduced by 55 to 85% by mammalian nitric oxide synthase inhibitors. By contrast, inhibitors of nitrate reductase inhibitors, a plant NO source, had no effect on cryptogein-induced NO production.

Calcium signals are thought to play an important role in the tobacco cells response to cryptogein [7]. To investigate whether NO was active in this process, the recombinant aequorin technology was used. Aequorin is a photoprotein from Aequora victoria which undergoes a conformational change and emits luminescence when occupied by Ca²⁺. Using transgenic Nicotiana plumbaginifolia cell suspensions that constitutively express aequorin in the cytosol, it was shown that cryptogein triggers a biphasic increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) resulting from an influx of extracellular Ca²⁺ and Ca²⁺ release from internal stores [7]. When cryptogein-triggered NO production was suppressed by cPTIO or inhibitors of mammalian NOS, the intensity of the first [Ca²⁺]_cyt increase was reduced by almost 50% whereas the second [Ca²⁺]_cyt peak was unaffected. We further provided evidence that NO appears to contribute to the elicitor-induced [Ca²⁺]_cyt elevation by promoting the release of Ca²⁺ from intracellular Ca²⁺ stores into the cytosol [6].

Recently, the role of NO in controlling Ca²⁺ homeostasis was investigated more thoroughly. Its has been shown that NO, released by the sulphur-free NO donor DEA-NONOate, elicits within minutes a transient influx of extracellular Ca²⁺ and a synchronized increase of [Ca²⁺]_cyt in aequorin-transformed tobacco cells [6]. As predicted from a pharmacological study, the channels responsible for NO-induced [Ca²⁺]_cyt elevation include voltage-dependent Ca²⁺ channels of the plasma membrane and intracellular Ca²⁺ channels sensitive to RYR and IP₃R inhibitors. This observation paralleled the situation encountered in animal cells in which almost all the molecules involved in the control of Ca²⁺ homeostasis seem to be modulated by NO [2]. Recent evidence from our laboratory suggest that NO mediates [Ca²⁺]_cyt through multiple mechanisms including phosphorylation-dependent processes, cADPR and plasma membrane depolarisation.

Our data, along with those from other studies, highlight the crucial role of NO in protecting plants against pathogens by promoting Ca²⁺ mobilization but also defense- and stress-related gene expression and HR [6]. Besides pathogen attack, abiotic stressors, such as drought, salinity and extreme temperature are serious threats to agriculture. In the recent years, a significant amount of work has gone into investigating NO synthesis and functions in plants exposed to abiotic stressors. For example, it was shown both in tobacco leaf peels and tobacco suspension cells that high temperature, osmotic stress, or salinity, generate a rapid and significant surge in NO levels [8]. In contrast, light stress and mechanical injury had no apparent effect on NO production in tobacco and/or tomato. Thus, although NO synthesis can be triggered by several, disparate abiotic stressors, it cannot be considered a universal plant stress response.

Plants express adaptive response to allow them to confer tolerance to environmental stresses and ensure survival. NO function is signal transduction pathways during this response. Although the precise signaling functions of NO are poorly understood, its capacity to modulate Ca²⁺ homeostasis provides an extraordinary and remarkably effective way of conveying information. Little is known about the signaling consequence of the NO/Ca²⁺ crosstalk but it is likely that modulation of the expression of stress-related gene may occur.

Affiliations

1. Plante-Microbe-Environnement, UMR INRA 1088/CNRS 5184/Universitŭ de Bourgogne, 17 rue Sully, BP 86510, Dijon, 21065 Cedex, France
   • David Wendehenne
   • , Olivier Lamotte
   • , Elodie Vandelle
   • , Cécile Courtois
   • , Laurent Barnavon
   • , Marc Bentéjac
   •  & Alain Pugin
2. Plant Sciences Group, School of Biological Sciences, University of Auckland, Private Bag, Auckland, 92019, New Zealand
   • Kevin Gould
3. Institute of Biochemical Plant Pathology, GSF-National Research Center for Environment and Health, Neuherberg, D-85764, Germany
   • Jörg Durner
4. UMR CNRS 6161, Laboratoire de Transport des Assimilats, UFR Sciences-Bâtiment Botanique, 40 avenue de Recteur Pineau, Poitiers, 86022 Cedex, France
   • David Lecourieux

Corresponding author

Correspondence to David Wendehenne.

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