The Properties And Anti-freezing Mechanism Of Key Regulatory Enzyme G6PDH Involved In PPP Of Populous Suaveolens

Populus suaveolens, one of freezing resistant arbor plants, can survive under a temperature of approximately -43.5°C in winter in the distribution of Great Xing'an Mountain, Northeast of China, and consequentially is a better material to study the mechanism of freezing resistance of woody plants. Glucose-6-phosphate dehydrogenase (G6PDH) is the main regulated enzyme, which catalyzes the first irreversible reaction of the oxidative pentose phosphate pathway (PPP), and its main physiological function is to provide NADPH, the reducing agent in a large number of anabolic metabolisms and detoxifation reactions. But little is known about the actual physiological role of G6PDH in the enhancement of freezing resistance induced by freezing in plants, especially in woody plants.In this paper, the changes in the contents of NADPH, NADH, H₂O₂, MDA, soluble protein and endogenous antioxidants, the activities of protective enzymes and some key enzymes involving in the ascorbate-glutathione cycle, pentose phosphate pathway (PPP) and glycolysis as well as freezing resistance (expressed as LT₅₀), and the correlation mentioned above were investigated in detail using P. suaveolens cuttings to explore the physiological function of G6PDH in the enhancement of freezing resistance induced by freezing acclimation at -20°C. The effects of reduced dithiothreitol (DTT_red), pH, various metabolites, metal irons on G6PDH activity, and the catalytic characteristics and kinetic properties of G6PDH purified from P. suaveolens cuttings were studied in order to elucidate the regulation mechanism of G6PDH. Based on the studies mentioned above, the main research works and results were as follows:1. Based on the obtained possible correlation between the elevation of G6PDH and the degree of increase in freezing resistance induced by freezing acclimation, the changes in the contents of NADPH, NADH, H₂O₂, MDA, soluble protein and endogenous antioxidants, the activities of protective enzymes and some key enzymes involving in the ascorbate-glutathione cycle, pentose phosphate pathway (PPP) and glycolysis as well as freezing resistance (expressed as LT₅₀), and the correlation mentioned above were investigated in detail using P. suaveolens cuttings to explore the physiological function of G6PDH in the enhancement of freezing resistance induced by freezing acclimation at -20°C, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase(CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), phosphofructokinase (PFK) and G6PDH,increased the contents of NADPH, NADH, soluble protein, reduced ascorbate (ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG), and decreased the H₂O₂ and MDA contents and LT50 of cuttings. While 2d of de-acclimation at 25°C resulted in a decrease in SOD, POD, CAT, APX, DHAR, MDAR, GR, PFK and G6PDH activities, and caused an increase in NADPH, NADH, H₂O₂, MDA, soluble protein, ASA, GSH, DHA and GSSG contents as well as LT50. The change in G6PDH activity was found to be closely correlated to the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, NADPH, H₂O₂, MDA, soluble protein, ASA, GSH, DHA and GSSG, and to the degree of freezing resistance of cuttings during freezing acclimation. It is suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation is related to the distinct increase in G6PDH activity, which may be involved in the activation of protective enzymes, the regulation of ascorbate-glutathione cycle, the biosynthesis of important substances and antioxidants associated with the elevation of membrane stability, and the induction of freezing resistance of cuttings. Thus, G6PDH may play an important role in the enhancement of freezing resistance in P. suaveolens cuttings.2. A comparison of the changes in the contents of NADPH and NADH, and the activities of G6PDH and PFK in P. suaveolens cuttings was made. A smaller increase caused by freezing acclimation at -20°C observed in PFK activity and NADH content than in G6PDH activity and NADPH content, respectively, was followed by an obvious decrease after 2 d of de-acclimation at 25 °C, suggesting that freezing acclimation have no effect on PFK activity and NADH content, and the response of pentose phosphate pathway and glycolysis to low tempeture stress was significant difference. It is concluded that freezing acclimation might result in an adaptive changes in the pathway of energetic and respiratory metabolism. The response of P. suaveolens cuttings to low temperature may be the effect on the pentose phosphate pathway and its key regulatory enzyme (G6PDH), which are required for the biosynthesis of some important substances (RNA and protein) associated with the enhancement of freezing resistance by providing more NADPH as a reductant.3. During freezing acclimation, a smaller increase was observed in the activities of SOD,POD and CAT than in those of APX,DHAR,MDAR and GR, especially than in ASA,DHA,GSH and GSSG contents. Two days of de-acclimation at 25°C resulted in a larger declining degree of SOD,POD and CAT as compares with APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG were higher in freezing acclimated cuttings than controls, respectively. It is suggested that a higher capacity of the ascorbate-glutathione cycle and H₂O₂ detoxification in freezing acclimated cuttings may be required for the growth and development of de-acclimated cuttings. The significant differences of the detoxification capacity were existed among H₂O₂-degrading enzymes, H₂O₂ are mainly eliminated through APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG involved in the ascorbate-glutathione cycle under low temperature stress.4. The catalytic characteristics and kinetic properties of G6PDH purified from P. suaveolens cuttings were studied in order to elucidate the physiological function and the regulation mechanism of G6PDH. In addition, theenzyme activity in the absence and presence of DTT_red was determined. The results showed that the G6PDH activity was not inactivated by pre-incubation with DTT_red, indicating that the purified enzyme probably presented in cytosol of P. suaveolens. G6PDH was exhibited a pH range of 7.3-8.9, showed the maximum activity around pH 8.2, inhibited by various metabolites such as NADPH, NADH, GTP, UTP, ATP, AMP, ADP, CoA, acetyl CoA, 3-phosphoglycerate(3-PG), fructose-6-phosphate (F6P), erythrose-4-phosphate (E4P) and ribose-5-phosphate (R5P)(all at 1 mmol/L except NADPH and NADH) to different extents. NADPH was the most effective inhibitor of enzyme activity, with an inhibition of 72.0%. The addition of metal irons such as MgCl₂, CaCl₂and KCl (all 1.0 mmol/L) to the standard reaction mixture had no remarkable influence on the cytosolic G6PDH activity. In addition, G6PDH was characterized by K_m value of 360 μmol/L for G6P and 16 μmol/L for NADP, and its V_max value was estimated to be 68 U·mg^-1 protein, suggesting that the activity of purified cytosolic G6PDH was higher from P. suaveolens than from other resported plants.