Nitrate reductase activity changes during a culture cycle of tobacco cells: the participation of a membrane-bound form enzyme

Abstract

The in vivo and vitro nitrate reductase (NR; EC 1.6.6.1) activities of suspension cell cultures were investigated in two strains of Nicotiana tabacum: a photomixotrophic green wild type (WT) and non-photosynthetic green type RubisCo − (SP25). During a batch culture cycle of 21 days, two peaks of in vivo NR activity (NRA) were observed: an early peak at 7 h and a second peak which reached a maximum at 6–8 days. At the beginning of the culture, in vitro activity was detected early for the WT cells compared to SP25 cells for which the activity was very low or absent. During the second peak, in vitro activity was present in the two strains and correlated very well with in vivo activity. Immunoblot experiment suggested that NR 120 kDa polypeptide did not accumulate in the cytosol at the beginning of the first phase of activity and the NR protein seemed to be labile. In contrast, the NR protein was stable during the second peak. On the other hand, it was observed that the dividing cells at 5–8 days seemed to accumulate nitrate, while the non-dividing cells, at the beginning of the culture, are unable to do that. This feature might be related to a change of cell permeability for nitrate because NR activity causing nitrate reduction is better in the second peak than in the first one. In correlation with this likely permeability change, external nitrate concentration modulated the first peak NRA and not the second one. The differential sensitivity of NRA of the two peaks to nitrate, led us to rediscuss the localization site of NR and to focus our experiments on a membrane-bound form of this enzyme.