In vivo and in vitro protective role of proline Lokman Öztürk 1 and Yavuz Demir 2, * 1 Faculty of Science and Art, Department of Chemistry, Atatürk University, Erzurum, Turkey; 2 Faculty of Education, Department of Biology, Atatürk University, Erzurum, Turkey; *Author for correspondence (e-mail: ydemir_409@yahoo.com; phone: +90-442-2314009; fax: +90-442-2360955) Received 17 December 2001; accepted in revised form 25 May 2002 Key words: Catalase, Peroxidase, Polyphenoloxidase, Proline Abstract Accumulation of proline in response to environmental stresses seems to be widespread among plants. To eluci- date the role of proline in plant responses, in vivo and in vitro, we studied the effect of proline on catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.14.18.1). In vivo, these three enzymes were activated by proline, while CAT and POD were activated and PPO was inactivated by NaCl. In vitro, CAT and POD were activated and PPO was inactivated by proline. Proline appeared to protect these three enzyme activities. The significance of these findings with regard to environmental stress-induced proline accu- mulation in vivo is discussed. The ability of proline to activate the enzymes may suggest a limited conforma- tional change. These results are important for characterisation of metabolic responses to environmental stresses and can be used as a stress indicator. Introduction In plants, enzymatic browning may be delayed or eliminated by removing the reactants such as oxygen and phenolic compounds or by using PPO inhibitors. Complete elimination of oxygen from fruits during processing is difficult because oxygen is ubiquitous. In order to delay enzymatic browning, PPO inhibitors (ascorbic acid and 2-mercaptoethanol) were used and a significant decrease in PPO activity was found. It was suggested that ascorbic acid could be used safely for elimination of enzymatic browning (Arslan et al. 1997). Plant cells have an active oxygen scavenging sys- tem consisting of multiple defence enzymes such as CAT and POD (Nandwal et al. 2000). An increase in POD activity is a common response to oxidative and abiotic stresses. Enhanced production of oxygen free radicals is responsible for peroxidation of membrane lipids and the degree of peroxidative damage of cells was controlled by the potency of the antioxidative POD enzyme system (Sreenivasulu et al. 1999). Adverse environmental conditions induce the ac- cumulation of ‘stress metabolites’ in plants. Of these metabolites proline is probably the most widespread. It has been shown that proline accumulates under conditions of water shortage, high salinity, chilling, heat, and heavy metal exposure. It plays a major role in osmoregulation and osmotolerance (Demir 2000). Moreover, it has been shown to protect enzymes from inactivation by salinity, heat, chilling and dilution in vitro (Sharma et al. 1998; Demir and Kocaçaliskan 2001). We studied the effects of proline on PPO, CATand POD activities in spinach leaves since these three en- zymes are known to be highly sensitive to environ- mental stresses in vivo and in vitro conditions. Materials and methods Growth conditions Seeds of spinach (Spinacia oleracea L. cv. Gladiatör) were grown in sand culture and supplied with stan- dard 1% Hoaglands (Hoagland and Arnon 1982) so- lution every 2 d. They were maintained in growth chambers under controlled conditions (12 h light/22 259 Plant Growth Regulation 38: 259–264, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.