Laccase activity could contribute to cell-wall reconstitution in regenerating protoplasts

Laccase (EC 1.10.3.1) activity was measured in regenerating and non-regenerating protoplasts isolated from tobacco (Nicotiana tabacum, L.) leaves. Laccase activity diminished soon after isolation: thereafter,it steadily increased in regenerating protoplasts during a 6-day culture period, whereas it was undetectable in non-regenerating protoplasts. A different pattern of isoforms was expressed in protoplasts by comparison with the donor tissue. Polyphenol oxidizing activity was detected also in the spent medium but it was not possible to definitely determine if it was due to laccase or to peroxidase (POX, EC 1.11.1.7) activity. New isoPOXs were quickly expressed soon after protoplast isolation as well, but POX activity remained negligible during the first day in culture. Leaf wounding induced an immediate stimulation of laccase activity whereas POX activity increased very slowly and peaked only after 4 days. Therefore, laccase could be the only effective polymerizing enzyme during the first day of protoplast culture and could contribute in the first steps of healing in wounded leaves, substituting for POX activity in cell wall reconstitution when hydrogen peroxide is not yet available.