Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultu red Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were f ound to be endowed with L-galactono-gamma-lactone dehydrogenase (GLDH) (EC 1.3.2.3) an enzyme that converts L-galactono-gamma-lactone into ASC. Cellul ar fractionation of TBY-2 protoplasts indicated that this enzyme is exclusi vely localised in mitochondria and associated to the membrane fractions. Du ring the growth cycle of TBY-2 cell culture, GLDH transiently increased, re aching the maximum value on the third day of culture, at the beginning of t he exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activiti es of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical red uctase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5 .1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. Ho wever, the scale of the increases varied being about 4-fold for APX and AFR R, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC a nd GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorb ic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH meta bolisms during the growth cycle of TBY-2 cell suspension cultures is also d iscussed. (C) 2000 Editions scientifiques et medicales Elsevier SAS.