Hydrogen peroxide plays a bivalent role in the regeneration of protoplasts

Ascorbate peroxidase (APO, EC 1.11.1.11) activity as H2O2 scavenger seems to be crucial for expression of regenerating potential in cultured protoplasts isolated from Nicotiana tabacum L, leaf mesophyll; protoplasts died soon after p-chloromercuribenzoate (pCMB), which completely inhibits APO, was added to the culture medium. On the contrary, no change in viability and dividing potential of protoplasts was found when catalase activity was impaired by 3-amino-1,2,4-triazole (AMT), compared with the control. The regulation of APO seemed to be at the transcription level, as was shown by Northern blot analysis. Protoplasts still survived but lost the dividing potential when peroxidase (POX, EC 1.11.1.7) activity was inhibited by cyanide (KCN) or dithiothreitol (DTT), which may further suggest that division is dependent on a modification of cell wall plasticity. Apoplastic H2O2 was necessary for ensuring 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 results suggest that H2O2 is used as substrate in the POX-mediated cell wall reconstitution. External POX activity appeared to be sufficiently high for preventing H2O2 accumulation in the medium; the result was indirectly confirmed by the capability of POXs to also remove exogenously added H2O2. Diamine and polyamine oxidases (EC 1.4.3.6 and 1.5.3.3, respectively) apparently did not contribute to the H2O2 generation in the apoplast of tobacco leaves and in isolated protoplasts.