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 .
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 . 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 . 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 . 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 . 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 .
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 . 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 . 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 . 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 . 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.