Detecting hydrogen peroxide in leaves in vivo - a comparison of methods

Four hydrogen peroxide detecting probes, 3,3'-diaminobenzidine (DAB), Amplex Red (AR), Amplex Ultra Red (AUR) and a europium-tetracycline complex (Eu3Tc) were infiltrated into tobacco leaves and tested for sensitivity to light, toxicity, subcellular localization and capacity to detect H2O2 in vivo. In the absence of leaves, in water solutions, AUR was very much sensitive to strong light, AR showed slight light sensitivity, while DAB and Eu3Tc were insensitive to irradiation. When infiltrated into the leaves, the probes decreased the photochemical yield (Phi(PSII)) in the following order of effect AR > DAB > AUR > Eu3Tc. With the exception of Eu3Tc, all probes stimulated the build-up of non-photochemical quenching either temporally (DAB, AUR) or permanently (AR), showing that their presence may already limit the photosynthetic capacity of leaves, even in the absence of additional stress. This should be taken into account when using these probes in plant stress experiments. Confocal laser scanning microscopy studies with the three fluorescent H2O2 probes showed that the localizations of Eu3Tc and AUR were mainly intercellular. AR partly penetrated into leaf chloroplasts but probably not into the thylakoid membranes. Photosynthesis-related stress applications of AR seem to be limited by the low availability of internal leaf peroxidases. Applications of AR for kinetic H2O2 measurements would require a co-infiltration of external peroxidase, imposing another artificial modifying factor and thus taking experiments further from ideal, in vivo conditions. Our results suggest that the studied H2O2 probes should be used in leaf studies with caution, carefully balancing benefits and artifacts.