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Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 microM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that "donors are compounds that spontaneously break down to release NO" and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.

Summary• Participation of nitric oxide (NO) in cross-talk between ivy pelargonium ( Pelargonium peltatum ) leaves and Botrytis cinerea was investigated using electrochemical and biochemical approaches.• In response to the necrotroph, leaves initiated a near-immediate NO burst, but the specificity of its generation was dependent on the genetic makeup of the host plant.• In the resistant cultivar, a strong NO burst was followed by a wave of secondary NO generation, shown by bio-imaging with DAF-2DA. The epicentre of NO synthesis was located in targeted cells, which exhibited a TUNEL-positive reaction. Soon after the challenge, an elevated concentration of hydrogen peroxide (H 2 O 2 ) was correlated with a reversible inhibition of catalase (CAT), ascorbate peroxidase (APX), and suppression of ethylene synthesis. The induced NO generation initially expanded and then gradually disappeared on successive days, provoking noncelldeath-associated resistance with an enhanced pool of antioxidants, which finally favoured the maintenance of homeostasis of surrounding cells.• By contrast, in the susceptible pelargonium, a weak NO burst was recorded and further NO generation increased only as the disease progressed, which was accompanied by very intensive H 2 O 2 and ethylene synthesis. The pathogen colonizing susceptible cells also acquired the ability to produce considerable amounts of NO and enhanced nitrosative and oxidative stress in host tissues.

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