An improved chemiluminescence method for hydrogen peroxide determination in plant tissues

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs. Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H2O2. The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H2O2 in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plant-extracts become unnecessary. To exemplified the high performance of the method, measurements of H2O2 were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H2O2 levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H2O2 scavenging enzyme in leaves of grapevine.