* Author for correspondence. Email: linsz2002@163.com Forestry Studies in China, 6(4): 1–7 Characterization and Role of Glucose-6-phosphate Dehydrogenase of Populus suaveolens in Induction of Freezing Resistance Lin Shanzhi * Guo Huan Liu Wenfeng Lin Yuanzhen Zhang Qian Hu Dongmei Zhu Baoqing Zhang Zhiyi Key Laboratory for Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing 100083, P. R. China ABSTRACT Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) was purified from the leaves of 8-week-old Populus suaveolens cuttings. The enzyme activity in the absence and presence of reduced dithiothreitol (DTT red ) was determined. The results show that the G6PDH activity is not inactivated by pre-incubation with DTT red , indicating that the purified enzyme probably pre- sented in cytosol of P. suaveolens. The catalytic characteristics and kinetic parameters of cytosolic G6PDH purified from P. suaveolens cuttings were also studied. The results show that G6PDH is characterized by K m value of 360 μmol·L –1 for G6P and 16 μmol·L –1 for NADP, a pH range of 7.3–8.9, and the maximum activity around pH 8.2. The enzyme activity is inhibited by various metabolites such as NADPH, NADH, GTP, UTP, ATP, AMP, ADP, CoA, acetyl CoA, fructose-6-phosphate (F6P), eryth- rose-4-phosphate (E4P), ribose-5-phosphate (R5P) and 3-phosphoglycerate (3-PG) (all at 1 mmol·L –1 except for NADPH and NADH) to different extents. NADPH is the most effective inhibitor of enzyme activity, with an inhibition of 72.0%. The addition of metal ions such as MgCl 2 , CaCl 2 and KCl (all 1.0 mmol·L –1 ) to the standard reaction mixture has no remarkable influence on the cytosolic G6PDH activity. However, CdCl 2 (1.0 mmol·L –1 ) causes high inhibitory effect on the enzyme activity. To explore the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation, the changes in the cytosolic G6PDH activity and freezing resistance (expressed as LT 50 ) of P. suaveolens cuttings during freezing acclimation at –20 °C were investigated. The results reveal that freezing acclimation decreases LT 50 of cuttings, and increases the activity of cytosolic G6PDH compared with control ones, while 2 d of de-acclimation at 25 °C result in a decrease in cytosolic G6PDH activity, and caused an increase in LT 50 . Furthermore, the change in cytosolic G6PDH activity is found to be closely correlated to the degree of freezing resistance of cuttings during freez- ing acclimation. It is suggested that cytosolic G6PDH may be involved in the induction of freezing resistance of cuttings. KEY WORDS Populus suaveolens, freezing acclimation, freezing resistance, LT 50 , G6PDH [Supported by the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University] 1 Introduction Glucose-6-phosphate dehydrogenase (G6PDH) is the main regulated enzyme, which catalyzes the first irreversible reaction of the oxidative pentose phos- phate pathway (OPPP), and its main physiological function is to provide NADPH for reductive biosyn- theses (Copeland and Turner 1987, Graeve et al. 1994, von Schaewen et al. 1995, Dennis et al. 1997). In re- cent years, the studies of G6PDH have been focused on kinetic properties (Wendt et al. 2000, Semenikhina et al. 1999, 2001, Esposito et al. 2001, Hauschild and von Schaewen 2003), isoenzyme (Hong and Copeland 1991, Huppe and Turpin 1996, Esposito et al. 2001, Hauschild and von Schaewen 2003), subcellular dis- tribution (Debnam and Emes 1999), the correlation between enzyme activity and nitrate or ammonia as- similation (Bowsher et al. 1992, Redinbaugh and Campbell 1998) and analysis of cDNA sequence (Graeve et al. 1994, von Schaewen et al. 1995, Schnarrenberger et al. 1995, Wenderoth et al. 1997, Wendt et al. 1999, 2000, Knight et al. 2001). But the above-mentioned studies only dealt with herbaceous plants. It was first found that the G6PDH activity in poplar twigs exhibited a marked increase in fall and winter followed by a gradual decrease in spring (Sagisaka 1972). Sagisaka (1985) reported that G6PDH inactiva- tion resulted in the injuries of poplar twigs during storage in a frozen state. In addition, an increase in G6PDH activity induced by low temperature was ob- served in alfalfa (Krasnuk et al. 1976), ryegrass (Bre- demeijer and Esselink 1995), soybean (Van Heerden et al. 2003), banana (Lin et al. 2001), and Populus to- mentosa (Lin and Zhang 2001). But up to now the actual role of G6PDH in the enhancement of freezing resistance induced by freezing acclimation in plants has not been clarified yet. Moreover, little is known about the correlation between the change in G6PDH activity and the degree of increase in freezing resis-