QUANTITATIVE DETERMINATION OF SUPEROXIDE IN PLANT LEAVES USING A MODIFIED NBT STAINING METHOD

GRELLET BOURNONVILLE; DIAZ RICCI, JUAN CARLOS

PHYTOCHEMICAL ANALYSIS

JOHN WILEY & SONS LTD

Lugar: Wolverhampton, UK; Año: 2010

0958-0344

Introduction- In plants, ROS (reactive oxygen species) levels are tightly regulated because their accumulation can produce structural and functional irreversible damages, leading to cell death. Despite, in many cases, that accumulation plays a key role in plants as signal molecules, after biotic or abiotic stress conditions. Various methods to study ROS accumulation were reported, however they are restricted to model plants or only provide qualitative results. Objective- Develop a single method to quantify superoxide radicals accumulation in plant tissues, based on selective extraction and dissolution of formazan obtained from widely known nitroblue tetrazolium hystochemical stain. Methodology- Leaf samples of strawberry plants were stained with classic nitroblue tetrazolium method, and formazan deposits were selectively extracted using chloroform. Organic phase was completely dried and formazan residues was dissolved in dimethylsulfoxide-potassium hydroxide and quantified by spectrofotometry. The method was assayed in plant leaves under different stress conditions, and generating superoxides or formazan, exogenously. Results- Formazan extracted from leaves subject to stress conditions, shown similar absorption spectra to those obtained from formazan standard solutions. Calibration curve shown a linear relationship between absorbance and formazan amounts, within the range 0,5 – 8 micrograms. However, certain formazan retention in leaf tissues, was observed. These results allowed us quantify superoxide radicals produced under different stress conditions.