Báo cáo lâm nghiệp: "Extraction and study of enzymes linked to malate metabolism in tree leaves"

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Extraction and study of enzymes linked to malate metabolism in tree leaves D. Gerant, A. Citerne, C. Fabert P. Dizengremel BP 239, 54506 Laboratoire de Physiologie Université de Nancy I, V6g6tale et Foresti6re, Vandoauvre, France Introduction Materials and Methods Oak leaves (Quercus pedonculata) were col- It is well established that malate plays a lected in a local forest and spruce needles central role in plant cellular metabolism (Picea abies) in Donon (Vosges mountains). (Lance and Rustin, 1984). Malate is the Twigs were cut and kept at 4°C until use. Based organic acid which can be accumulated to on previous studies, the extraction buffer was the highest level in plant cells and its inter- sufficiently protective against deleterious com- pounds, such as phenolic compounds, tannins nal concentration can display normal daily and terpenoids (Gerant et al., 1988). fluctuations and can also present perma- Fresh tissues were homogenized in a potter nent or long-term changes with environ- grinder at 4°C in the presence of the extraction mental conditions (Lance and Rustin, buffer and centrifuged at 50 000 x g for 30 min. 1984). Enzymes implicated in synthesis The supernatant was collected and desalted on a Sephadex G-25 column (Pharmacia PD10). and catabolism of this substrate are widely Enzymatic activities were measured ac- distributed in the cellular compartments. If cording to Hatch (1978) for fumarase these enzymes have become well known (EC 4.2.1.2), Davis and Patil (1975) for NAD- in herbaceous plants (Macrae, 1971; malic enzyme (EC 1.1.1.39), Queiroz (1968) for Davis and Patil, 1975; Wiskich and Dry, NADP-malic enzyme (EC 1.1.1.40). 1985; Artus and Edwards, 1985), very few studies have been made on woody plants (Pitel and Cheliak, 1985, 1986; Weimar Results and Rothe, 1987). In this study, the extrac- tion of enzymes linked to malate metabo- lism, particularly those located in mito- Two of the studied enzymes are consid- chondria, was investigated in coniferous ered as mitochondrial markers: fumarase and deciduous leaves. Particular attention implicated in the tricarboxylic acid cycle was devoted to the variations in enzyme and NAD-malic enzyme which generates capacities during the growing season. pyruvate internally. By contrast, NADP- Finally, the first steps of purification of malic enzyme catalyzing the same reac- these enzymes are presented and dis- tion as NAD-malic enzyme is located in cussed. the cytosol.
activities. NAD-malic enzyme has an The extraction of these enzymes from absolute requirement for Mn whereas , 2+ oak leaves and spruce needles was per- formed during the growing season: May NADP-malic enzyme can work without + 2 Mg (data not shown). These enzymes 1988!July 1988 for oak (Fig. 1A) and June 1987-March 1988 for spruce (Fig. 1 B). are eluted in different fractions from an Samples were made on the leaves of the ion-exchange chromatography column. first flush for oak leaves (May 1988) and When NAD-malic enzyme is strongly in- on the needles of 1987 for spruce hibited by NaCl, NADP-malic enzyme is needles. not. From these results, it is evident that the elution buffer used in ion-exchange The enzymatic capacities increased chromatography was not appropriate for a markedly during the growing season good separation of the two malic enzymes (March) of spruce and during the new and to detect the presence of isoforms. flush (June) in oak, except NAD-malic The purification study must continue to enzyme. Thereafter, they remained cons- evaluate the elution buffer and the purifi- tant or slightly decreased during the rest of cation of samples from different periods of the time. The highest enzyme capacities tree growth. Seasonal variations of the observed for NAD-malic enzyme and were enzymatic capacities have been shown for fumarase for spruce and oak leaves, res- both spruce needles and oak leaves. For pectively. each species, the specific behavior of the Actually, no evidence has been pre- enzymes could reveal differences in ener- sented on the fact that these variations gy production in deciduous (oak) and must be related to an increased activity or evergreen (spruce) trees. Moreover, it to the appearance of different isoforms of would be of interest to correlate the phy- these enzymes. From these results, it siological seasonal changes known to became obvious that the purification of occur in gaseous exchanges (Gerant et these enzymes was necessary. They were al., 1988) with the behavior of the investigated by using an ion-exchange enzymes. chromatography column. The extracts were applied to a DEAE-Trisacryl column (25 x 1.6 cm) at a flow rate of 1 ml/min. Fifty fractions of 2 ml were collected and References tested for enzyme activities. For oak leaves (Fig. 2A) and spruce Artus N.N. & Edwards G.E. (1985) NAD malic needles (Fig. 2B), NADP-malic enzyme enzyme from plants. FEBS Lett. 182, 225-233 was the first to be eluted (fractions 22-24), Davis D.D. & Patil K.D. (1975) The control of then fumarase activity was recovered in NAD-specific malic enzyme from cauliflower fractions 24-27. By contrast, NAD-malic bud mitochondria by metabolites. Planta 6, 197- 211 enzyme was weakly (spruce) or not de- tectable (oak); NaCl inhibited this activity. Gerant D., Citerne A., Namysl C., Dizengremel P. & Pierre M. (1988) Studies of some respirato- ry enzymes in foliar organs and root systems of spruce and oak trees. Relation with forest de- cline. In: Air Pollution Research Report Vol. 16, Discussion (Bervees J., Mathy P. & Evers P., eds.), E. Guyot SA, Brussels, pp. 109-118 8 Hatch M.D. (1978) A simple spectrophotometric Spruce needles and oak leaves possess assay for fumarate hydratase in crude tissue potential NAD- and NADP-malic extracts. Anal. Biochem. 85, 271-275 enzyme
Lance C. & Rustin P. (1984) The central role of Queiroz O. (1968) Sur le m6tabolisme acide malate in plant metabolism. Physiol. Veg. 22, des crassulac-6es. Ill. Variations d’activité 625-641 enzymatique sous I’action du photop6riodisme et du thermopèriodisme. Physiol. Veg. 6, 117- Macrae A.R. (1971) Malic enzyme activity in 136 mitochondria. Phytochemistry 10, 2343- plant 2347 Weimar M. & Rothe G. (1987) Preparation of extracts from mature spruce needles for enzy- Pitel J.A. & Cheliak W.M. (1985) Methods to matic analysis. Physiol. Plant. 69, 692-698 extract NAD-malate dehydrogenase from white spruce needles. Physiol. Plant. 65, 129-134 Wiskich J.T. & Dry I.B. (1985) The tricarboxylic Pitel J.A. & Cheliak W.M. (1986) Effectiveness acid cycle in plant mitochondria. In: Encyclo- of protective agents for increasing activity of pedia of Plant Physiology, Higher Plant Physi- five enzymes from vegetative tissues of white ology. (Douce R. & Day D.A., eds.), Springer- spruce. Can. J. Bot. 64, 39-44 Verlag, Berlin, pp. 281-313 3