In Situ Localisation of Superoxide Generated in Leaves of Alocasia macrorrhiza (L.) Shott under Various Stresses

Abstract

Leaf discs of Alocasia macrorrhiza were treated with various stress factors, including two photo-oxidants, methyl viologen (MV) or riboflavin (RB); three pollutants, sodium bisulphite (NaHSO3), or the heavy metals lead or cadmium; or an osmotic medium, polyethylene glycol 6000. The in situ localisation sites for O2 − generation were identified using specific dye nitro blue tetrazolium as a probe. The level of superoxide production was determined by scanning the blue-stained formazan area and was defined as the percentage of pixels from the stained portion versus the total number of pixels in the entire leaf disc area. All stress factors induced the generation of O2 − in a time- or concentration-dependent pattern. Although superoxide production also was enhanced by longer time periods in untreated discs (control), the degree to which this occurred was less than that measured in leaves treated with either MV or RB. Generation sites were primarily found in the chloroplasts of stomatal guard cells and in the plasma membrane of the epidermis and mesophyll cells, indicating that they were most responsive to stress conditions. Nevertheless, the site of O2 − generation varied among these stress factors.