Two delta-1-pyrroline-5-carboxylate dehydrogenase isoforms are expressed in Nicotiana plumbaginifolia cultured cells and differentially modulated during the culture growth cycle

delta'-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They slowed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl- at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K-m-values for the two substrate, P5C and NAD(+), the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed.