A semi-quantitative histochemical method for assessment of biochemical responses to osmotic stress in Coffea arabica leaf disks.

ABSTRACT

A simple method set for assessing biochemical changes associated with osmotic stress responses was developed using coffee (Coffea arabica L.) leaf disks. Stress was induced by polyethylene glycol (PEG) exposure. Quantitative evaluation of tissue physiological stress parameters was carried out using analytical methods to validate the conversion of classic qualitative histochemical tests for localizing lipid peroxidation, hydrogen peroxide, and total xanthine alkaloids into semi-quantitative assays. Relative electrolyte leakage (EL%) and chlorophyll content (SPAD index) were also recorded. EL% levels of treated disks were higher than those of control ones, whereas SPAD indexes were comparable. Histochemical localization indicated that levels of lipid peroxidation, H2O2, and total xanthines were also higher under osmotic stress than in control conditions. Semi-quantitative data obtained by image processing of histochemical staining consistently matched quantitative evaluations. Chromatographic analyses revealed that theophylline and caffeine concentrations increased in the presence of PEG, whereas theobromine remained constant in relation to the control. The methods herein described can be useful to rapidly acquire initial data regarding biochemical osmotic stress responses in coffee tissues based on simple staining and imaging steps. Moreover, it is likely that the same method may be applicable to other types of stresses and plant species upon minor adjustments.