Barley root tip peroxidases convert DAF-FM and DAR-4M to an NO-independent fluorescent product using H₂O₂ derived...

The aim of our study was to investigate the possible involvement of barley root tip peroxidases and polyamine oxidases in the conversion of DAF-FM or DAR-4M into an NO-independent fluorescent product after the exogenous application of polyamines. Application of spermidine or spermine into the incubation medium increased H₂O₂ production by root tip segments in a dose-dependent manner. This spermidine- or spermine-induced increase in H₂O₂ production was accompanied by intensified fluorescence of both DAF-FM and DAR-4M in a polyamine dose-dependent manner, similarly to exogenously added H₂O₂. On the contrary, exogenous putrescine neither evoked H₂O₂ production nor increased DAF-FM or DAR-4M fluorescence. Application of guazatine, a polyamine oxidase inhibitor, into the incubation medium inhibited both H₂O₂ production and DAF-FM or DAR-4M fluorescence. Spermidine- or spermine-induced DAF-FM or DAR-4M fluorescence decreased with an increasing amount of catalase or guaiacol, a competitive substrate for peroxidase, in the incubation medium. Exogenous application of indole-3-acetic acid, a well-known activator of NO generation in roots, but not of H₂O₂, spermidine or spermine, induces NO accumulation in the root tips. Exogenous application of spermidine or spermine to plant tissues with high polyamine oxidase and peroxidase activity, as are the barley root tips, generates an NO-independent fluorescence signal from either DAF-FM or DAR-4M, giving a false positive signal for NO emission.