Quantitative determination of superoxide in plant leaves using a modified NBT staining method.

INTRODUCTION: In plants, the ROS (reactive oxygen species) level is tightly regulated because their accumulation produces irreversible damage leading to cell death. However, ROS accumulation plays a key role in plant signaling under biotic or abiotic stress. Although various methods were reported to evaluate ROS accumulation, they are restricted to model plants or provide only qualitative information. OBJECTIVE: Develop a simple method to quantify superoxide radicals produced in plant tissues, based on the selective extraction of the formazan produced after nitroblue tetrazolium (NBT) reduction in histochemical staining. METHODOLOGY: Plant leaves were stained with a standard NBT method and the formazan precipitated in tissues was selectively extracted using chloroform. The organic phase was dried and formazan residue dissolved in dimethylsulfoxide-potassium hydroxide and quantified by spectrophotometry. The method was tested in strawberry plant leaves under different stressing conditions. RESULTS: Formazan extracted from leaves subjected to stress conditions showed similar absorption spectra to those obtained from standard solutions using pure formazan. Calibration curves showed a linear relationship between absorbance and formazan amounts, within the range 0.5-8 µg. Outcomes suggested that formazan was retained in the solid residue of leaf tissues. This protocol allowed us to quantify superoxide radicals produced under different stress conditions. CONCLUSIONS: Chloroform allowed a selective formazan extraction and removal of potential endogenous, exogenous or procedural artefacts that may interfere with the quantitative determination. This protocol can be used to quantify the superoxide produced in plant tissues using any traditional qualitative NBT histochemical staining method.

Effect of solvent on tetrazolium reaction.

The rate of color development of a tetrazolium formazan is shown to be inversely proportional to the dielectric constant of the solvent medium and directly proportional to the hydrogen-bonding capability of solvent mixtures having the same dielectric constant. The geometric isomers of the formazans have different absorbance maxima, and the wavelength of maximum absorbance of a mixture of formazans in different solvents depends upon which isomer predominates in that solvent. The trans-syn-isomer (blue form) of blue tetrazolium has a maximum absorbance at 625 nm in dimethylformamide while the trans-anti-isomer (red form) absorbs at 517 nm in methanol. The absorbance maxima of the corresponding isomers of the formazans of triphenyltetrazolium occur at 535 and 485 nm, respectively. Water and/or methanol (to a lesser extent) are important in the stabilization of the trans-anti-isomer, since the small size of these two substances allows them to form strong intermolecular hydrogen bonds with one or both nitrogen atoms of the azo linkage, thereby preventing the formation of the intramolecular hydrogen bonding exhibited by the trans-syn-isomer. The formazan produced by the reaction of corticosteroids with tetrazolium in strongly basic media can lose a reduction unit and be reoxidized to the tetrazolium. This reaction is solvent dependent and occurs at a much faster rate in chloroform than in alcohol USP.