HYDROGEN-PEROXIDE PLAYS A BIVALENT ROLE IN THE REGENERATION OF PROTOPLASTS

Ascorbate peroxidase (APO, EC 1.11.1.11) activity as H2O2 scavenger se ems to be crucial for expression of regenerating potential in cultured protoplasts isolated from Nicotiana tabacum L, leaf mesophyll; protop lasts died soon after p-chloromercuribenzoate (pCMB), which completely inhibits APO, was added to the culture medium. On the contrary, no ch ange in viability and dividing potential of protoplasts was found when catalase activity was impaired by 3-amino-1,2,4-triazole (AMT), compa red with the control. The regulation of APO seemed to be at the transc ription level, as was shown by Northern blot analysis. Protoplasts sti ll survived but lost the dividing potential when peroxidase (POX, EC 1 .11.1.7) activity was inhibited by cyanide (KCN) or dithiothreitol (DT T), which may further suggest that division is dependent on a modifica tion of cell wall plasticity. Apoplastic H2O2 was necessary for ensuri ng cell division since the addition of catalase to the culture medium prevented it; dividing potential was partially recovered when AMT was added to block the activity of exogenously added catalase. Such result s suggest that H2O2 is used as substrate in the POX-mediated cell wall reconstitution. External POX activity appeared to be sufficiently hig h for preventing H2O2 accumulation in the medium; the result was indir ectly confirmed by the capability of POXs to also remove exogenously a dded H2O2. Diamine and polyamine oxidases (EC 1.4.3.6 and 1.5.3.3, res pectively) apparently did not contribute to the H2O2 generation in the apoplast of tobacco leaves and in isolated protoplasts.