Eur. J. Biochem. 167, 579-583 (1987) 0 FEBS 1987

Effect of ethylene treatment on the concentration of fructose-2,6-bisphosphate and on the activity of phosphofructokinase 2/fructose-2,6-bisphosphatase in banana Emmanuel MERTENS

Molecular Pathology, Bruxelles ' Laboratoire de Physiologie des Organes Vtgttaux apres Ricolte, Centre National de la Recherche Scientifique, Meudon

(Received April 6, 1987) - EJB 87 0399

Pierre MARCELLIN ', Emile VAN SCHAFTINGEN and Henri-Gery HERS

Laboratoire de Chimie Physiologique, Universite Catholique de Louvain and International Institute of Cellular and

Preclimacteric bananas fruits were treated for 12 h with ethylene to induce the climacteric rise in respiration. One day after the end of the hormonal treatment, the two activities of the bifunctional enzyme, phospho- fructokinase 2/fructose-2,6-bisphosphatase started to increase to reach fourfold their initial value 6 days later. By contrast, the activities of the pyrophosphate-dependent and of the ATP-dependent 6-phosphofructo-1 -kinases remained constant during the whole experimental period, the first one being fourfold greater than the second. The concentrations of fructose 2,6-bisphosphate and of fructose 1,6-bisphosphate increased in parallel during 4 days and then slowly decreased, the second one being always about 100-fold greater than the first. The change in fructose 2,6-bisphosphate concentration can be partly explained by the rise of the bifunctional enzyme, but also by an early increase in the concentration of fructose 6-phosphate, the substrate of all phosphofructokinases, and also by the decrease in the concentration of glycerate 3-phosphate, a potent inhibitor of phosphofructo- kinase 2. The burst in fructose 2,6-bisphosphate and the activity of the pyrophosphate-dependent phosphofructo- kinase, which is in banana the only enzyme known to be sensitive to fructose 2,6-bisphosphate, can explain the well-known increase in fructose 1,6-bisphosphate which occurs during ripening.

In ripening fruits, the presence of ethylene has been associ- ated with a large increase in respiratory metabolism, particu- larly in so-called climacteric fruits in which low concentrations of exogenous ethylene induce autocatalytic ethylene produc- tion and accelerate the ripening process [1, 21. During this ethylene-induced burst in respiration, glycolysis is activated as indicated by the 10 - 20-fold increase in Fru(l,6)Pz concen- tration [3-51. The mechanism which allows the increase of the glycolytic flux is not clear; it was therefore of interest to investigate its relationship to Fru(2,6)Pz, a new regulator of glycolysis discovered 6 years ago [6]. Fru(2,6)P2 is a regulatory molecule which promotes glycolysis and restricts gluconeo- genesis in very different types of eukaryotic cells (reviewed in [7]). In higher plants, Fru(2,6)Pz activates PP1-PFK and inhibits cytoplasmic FBPase 1 [7]. A dramatic increase in its concentration has been observed in various situations in which a resumption of metabolic activity occurs [8- 101.

Climacteric fruits, such as banana, are good models to reevaluate the link between ethylene and glycolysis. The aim of the present work was to determine whether Fru(2,6)Pz

Correspondence ro H. G. Hers, Laboratoire de Chimie Physiologique, UCL 75.39,75 Avenue Hippocrate, B-1200 Bruxelles, Belgium

Abbreviations. Fru(2,6)P2, fructose 2,6-bisphosphate; Fru6P, fructose 6-phosphate; Glc6P, glucose 6-phosphate; PPi, inorganic pyrophosphate; PFK 1, 6-phosphofructo-1-kinase; PFK 2, 6-phos- phofructo-2-kinase; FBPase 1, fructose 1,6-bisphosphatase; FBPase 2, fructose 2,6-bisphosphatase; PPi-PFK, pyrophos- phate : fructose 6-phosphate 1 -phosphotransferase.

Enzymes. 6-Phosphofructo-2-kinase (EC 2.7.1 .l05); fructose-2,6- bisphosphatase (EC 3.1.3.46) ; pyrophosphate : fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90).

could be at least partially responsible for the rise in Fru(l,6)Pz associated with ripening.

MATERIALS AND METHODS

Materials

Banana fruit were obtained from a local market. They were preclimacteric and green in color. Bovine serum albumin and polyvinylpyrrolidone were from Sigma (St Louis, USA). Dithiothreitol was from Janssen Chmica (Belgium), and 3 MM papers from Wathman (Springfield Mills, England). Enzymes and others biochemicals were from Boehringer (Mannheim, FRG). Chemicals were from Merck (Darmstadt, FRG) and were of analytical grade. Potato tuber PPi-PFK [Ill, Fru(2,6)Pz 1121 and [2-32P]Fru(2,6)Pz [13] were prepared as described. Fru(6)P was acid-treated to remove contam- inant Fru(2,6)P2 as in [8].

Ethylene treatment and preparations of extracts

Ripening was initiated at 20°C by introducing an air stream containing 15 ppm ethylene during 12 h at a rate of 10 l/min in a 250-1 container. Thereafter, control and treated fruits were stored separately in a 250-1 chamber aerated with H,O-saturated air at a rate of 10 l/min. C 0 2 production was monitored with five fruits per condition. The respiration rate of a single fruit was followed by putting it into a continuous COz-free air flow system which was connected with a Cosma Rubis 3000 infrared gas analyzer. Pulp samples were daily collected by cutting approximately 10 g of the central part of three fruits, transferred to liquid nitrogen and stored at - 80 "C until homogenization. The frozen slices were homog-

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enized with a Potter-Elvehjem device in 10 vol. 50 mM NaOH (for the measurement of Fru(2,6)Pz) or in 6 vol. 5% (w/v) ice- cold perchloric acid, subsequently neutralized with KOH/ KHC03 (for the measurement of other metabolites). Both mixtures were then centrifuged at 5000 x g and the pellets were discarded. For the assay of enzymes, frozen samples were homogenized with a Potter-Elvehjem device in 6 vol. ice-cold buffer containing 10 mM magnesium acetate, 10 mM sodium diethyldithiocarbamate, 10 mM dithiothreitol, 1 mM EDTA, 1 YO bovine serum albumin, 2% poly(ethy1ene glycol), 0.5% polyvinylpyrrolidone and 50 mM Tris/acetic acid pH 7.8. The homogenates were immediately centrifuged for 15 min at 30000 x g; the resulting supernatants were used for enzyme measurements. Bovine serum albumin, sodium diethyldithio- carbamate, polyvinylpyrrolidone and poly(ethy1ene glycol) were added to the extraction medium to avoid irreversible binding of proteins by tannins during the extraction of unripe bananas [14]. It has been checked that, thanks to these additions, the same amount of glucose-6-phosphate dehydro- genase (1.25 U/g) was extracted from ripe and unripe ba- nanas. Furthermore, it has been verified, for all enzymes under investigation, that their activities were additive when samples of ripe and unripe bananas were homogenized together.

Determinations of metabolites Fru(2,6)Pz was assayed as in [a]. Because of its low concen-

tration in tissues, it has been verified that known amounts of this phosphoric ester added to bananas extracts were completely recovered. Glc6P [15], ATP [16], glycerate-3-P [ 171, Fru( 1 ,6)P2 [ 181 and PPi were measured using an Aminco DW2 spectrophotometer in the dual-wavelength mode. PPi was assayed in extracts made with 3% (w/v) ice-cold perchloric acid containing 10 mM EDTA, centrifuged 5 min at 5000 x g and adjusted to pH 5 with KOH/KHC03; PPi was immediately measured in an assay mixture with the same composition as the one used for the measurement of Fru(2,6)Pz except that Fru(2,6)Pz was 0.5 pM and PPi was omitted. Pi was measured by the method of Fiske and Sub- barow [19] and sucrose by the method of Roe [20].

Enzymes PPi-PFK and PFK 1 were assayed by the production of

Fru(l,6)Pz in the presence of 5 mM magnesium acetate, 0.15 mM NADH, 1 mM Fru6P, 3.5 mM Glc6P, 50 pg/ml aldolase, 1 pg/ml triosephosphate isomerase, 10 pg/ml glycerol-3-P dehydrogenase and 50 mM Hepes pH 7.5. For PPi-PFK, PPi was 1 mM and Fru(2,6)Pz was 2 pM; for the assay of PFK 1, ATP was 2 mM. PFK 2 activity was assayed as in [21], except that Fru6P and ATP were 5 mM. Glc6P dehydrogenase activity was monitored as in [22]. Phosphoryl- ase activity was measured in the phosphorolytic direction by the production of Glcl P in the presence of 50 mM potassium acetate, 5 mM magnesium acetate, 2.5 mM dithiothreitol, 10mM Pi, 0.25% soluble starch, 0.15mM NADP, 1 pM Glc(l,6)Pz, 0.4 U phosphoglucomutase, 0.7 U Glc6P dehy- drogenase and 25 mM Hepes pH 7.5. Labelling of FBPase 2 was done as in [21]. Acid phosphatase was assayed by the release of p-nitrophenol in the presence of 5 mM magnesium acetate, 100 mM p-nitrophenylphosphate and 250 mM acetate/acetic acid pH 5.0. All enzyme measurements were made at 30°C. One unit is the amount of enzyme that catalyses the conversion of 1 pmol substrate/min under the standard conditions of assay.

RESULTS COz and sucrose production after ethylene treatment

The evolution of these two parameters shown in Fig. 1 is a characteristic feature of the climacteric surge in respiration [l, 21. Untreated fruits remained at the preclimacteric stage during the experiment, as attested by the low COz production and by the fact that they stayed dark green. Ethylene-treated fruits presented a typical respiratory crisis which began after 1 day, as shown by a high COz production; the colour of the peel began to change at the 2nd day; they were well synchronized, as illustrated by the low SEM of sucrose con- centration during its production (Fig. 1).

Concentrations of Fru(2,6)P2 and of other metabolites after ethylene treatment

The concentration of Fru(2,6)Pz in preclimacteric fruits was exceedingly low, whereas that of its isomer, Fru(1,6)Pz was almost 80-fold higher. The concentration of these two bisphosphate esters increased in a similar manner after ethyl- ene treatment to reach respectively 5-fold and 10-fold their initial value between the third and fifth days and then slowly decreased (Fig. 2). Since the two bisphosphate esters are formed from ATP and Fru6P by their respective PFK, we also measured the concentrations of these two precursors. The concentration of ATP showed only minimal variations during the first 4 days and then slowly decreased, whereas that of Glc6P (which was always in equilibrium with Fru6P) showed an early increase and reached a maximum after three days and then decreased to control levels (Fig. 3 b). The concentra- tion of glycerate-3-P, an inhibitor of PFK 2, decreased con- tinuously during the first four days, then remained at about one third of its original value. Pi is a cofactor of PFK 2; its concentration was slightly decreased during the first half of the experiment and then increased to reach 1.5-fold its initial value after 6 days (Fig. 3a). The concentration of PPi (20 nmol/g tissue) remained constant during ripening (not shown).

Properties of PFK 2 and FBFase 2

PFK 2 activity increased during the climacteric stage to reach maximal activity in ripe fruits, fourfold higher than in unripe fruits (Fig. 4). Since the activities were additive when samples of ripe and unripe fruits were homogenized together (see methods), the increase in activity could not be attributed to the disappearance of an inhibitor nor to the appearance of an activator. Because of its very low activity in banana the enzyme could not be extensively purified, but it could be recovered from both unripe and ripe fruits with a yield of 84- 88% by precipitation in the presence of 18% poly(ethy1ene glycol); this fraction was used for the investigation of some kinetic properties of the enzyme. It was found that the Michaelis constant of climacteric and preclimacteric PFK 2 for its two substrates was approximatively the same (respec- tively 50 pM and 60 pM for Fru6P, and 180 pM and 150 pM for ATP when measured at 2 mM Pi and 2 mM of the other substrate). The activity of FBPase 2 could not be quantita- tively estimated in crude extracts because of the presence of a large excess of non-specific phosphatases. The low-K, enzyme could be identified by its ability to form a radioactive phosphoprotein in the presence of [2-32P]Fru(2,6)P2, which was retained on filter paper [21]. Using this method, we ob- served an enrichement in FBPase2 in the extracts of

581

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0 W .

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150

0 1 3 5 7

TIME AFTER ETHYLENE TREATMENT (days)

Fig. 1. Formation of C 0 2 and sucrose in banana after ethylene treat- ment. Values shown are means & SEM of three differents bananas for sucrose and five differents bananas for C 0 2

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FRU( 2,6)P2

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FRU (t6)Pz p 5rezZPz 1 3 5 7

TIME AFTER ETHYLENE TREATMENT (days)

Fig. 2. Concentration of Fru(2,6)P2 and Fru(l,6)P2 in banana after ethylene treatment. Values shown are means SEM of three differents bananas

climacteric fruits, which was parallel to that of PFK 2 (not shown) .

Other enzymes Both ripe and unripe bananas contained approximatively

0.45 unit PPi-PFK/g tissue, when the enzyme was measured in the forward direction in the presence of 2 pM Fru(2,6)P2. No activity could be detected in the absence of Fru(2,6)Pz,

J I I I

I I I L 1 3 5 7

TIME AFTER ETHYLENE TREATMENT (days)

Fig. 3. Concentration of glycerate-3-P,Pi, Glc6F' and ATP in banana after ethylene treatment. Values shown are means SEM of three differents bananas

150

- m . - 3 100 t c 2 I- 0 a N Y

50

1 3 5 7

TIME AFTER ETHYLENE TREATMENT (days)

Fig. 4. Effect of ethylene treatment on PFK 2 activity. Values shown are means f SEM of three experiments

indicating a complete dependence towards this activator. The K, for PPi was 4 pM with 1 mM Fru6Pand 1 pM Fru(2,6)P2. As reported for the potato enzyme [ll], the K, for Fru(2,6)P2 was 6 nM in the forward direction, in the presence of 1 mM PPi and 2 mM Fru6P; in the reverse direction, the K , was 1.1 pM with 25 pM Fru(1,6)Pz and 5 mM Pi. PFK 1 activity was fourfold lower than PPi-PFK. It remained stable during

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- ATP-PFK - I I

the climacteric period (Fig. 4), and was not affected by the presence of Fru(2,6)P2 (not shown). We could not detect a magnesium-dependent specific fructose 1,6-bisphosphatase in extracts of banana fruit. Phosphorylase activity was high (1 - 1.5 pmol x min- ' x g- ') when measured in the phosphoro- lytic direction; its activity slightly increased during ripening (Fig. 5). In confirmation of previous work [24], we also ob- served an increase in the activity of an unspecific acid phosphatase (optimum pH = 6) during ripening (Fig. 5).

DISCUSSION

PFK 1 versus PPi-PFK and the role of Fru(2,6)P2 The 10-fold increase in the concentration of Fru(1,6)Pz

that we observed during the climacteric peak is in confirma- tion of previous work [l -31. Our observation that there is a simultaneous 5 - 6-fold increase in the concentration of Fru(2,6)P2 put further light on the mechanism by which this change could occur.

Fru(l,6)P2 can be formed in plants by both PFK 1 and PPi-PFK, and the respective role of these two enzymes in glycolysis has not been defined yet. As shown in Fig. 5, when measured under optimal conditions, the activity of PPi-PFK was about fourfold that of PFK 1 and the concentration of the two enzymes remained unchanged during ripening. The activity of both enzymes would be favoured by the rise in the concentration of their common substrate, Fru6P. More important is the fact that PPi-PFK is extremely sensitive to the stimulation by Fru(2,6)P2, whereas PFK 1 is completely insensitive to this important glycolytic stimulator. In addition, PPi concentration (20 nmol/g) was at least fivefold above the K,,, (4 pM) of PP,-PFK, suggesting that PPi-PFK could operate towards glycolytic direction. It is clear, therefore, that if the observed rise in Fru(2,6)P2 concentration has a role to play in the increased glycolytic rate of climacteric, it must be by a stimulation of PPi-PFK, which in banana is the only enzyme known to be sensitive to this effector. We were, in- deed, unable to detect any activity of fructose-l,6-bi- sphosphatase, which in many plants is the other enzyme the activity of which is affected by Fru(2,6)P2. A word of caution should be added, however, since the concentration of

Fru(2,6)P2 in green bananas (50 pmol/g) could be already saturating for PPi-PFK. Indeed, the K, for the activator is close to 5 nM under optimal conditions, but could be largely increased by the concentrations of Pi [ l l ] and of glycerate-3- P [23], normally present in the cell.

We cannot, therefore, discard the possibility that Fru(2,6)P2 could play another, still unsuspected role in fruit ripening. It is, indeed, remarkable that a large increase in the concentration of Fru(2,6)P2 has been observed in several other conditions characterized by the resumption of metabolic activity after some kind of dormancy such as the germination of fungal spores [9] and of seeds [lo] and after slicing Jerusalem artichoke tubers [8].

The control of Fru(2,6)P2 concentration

The concentration of Fru(2,6)P2 in cells depends on the relative activities of PFK 2 and of FBPase 2. Whereas the first of these two enzymes could be easily characterized in an extract of banana, the second, because of a large excess of unspecific phosphatase, could only be detected, in a semi- quantitative way, by the formation of a [32P]phosphoprotein upon incubation in the presence of [2-32P]Fru(2,6)Pz. The parallel variation of the two activities during ripening is in agreement with the hypothesis that in banana, as in spinach leaves [21], the two enzymes are part of a single bifunctional protein. Each enzymic activity can be controlled by the amount of enzyme as well as by the concentration of sub- strates and effectors. There was actually a remarkable fourfold increase in the activity of PFK 2/FBPase 2 that started 24 h after the end of the ethylene treatment and ex- tended over all the experimental period. Since the kinetic properties of the enzyme were not modified, it appears most likely that one effect of ethylene was simply to stimulate the synthesis of the bifunctional protein. This effect is similar to that already reported [23] for an unspecific acid phosphatase, whereas other enzymatic activities that were measured, in- cluding that of PFK 1, remained stable throughout the pro- cess of ripening; it is therefore likely that the twofold increase in the activity of PFK 1 , reported by others [4] is attributable to the difficulty in extracting proteins from unripe bananas (see Methods). It is also worth mentioning that an approx- imately 30% increase in PFK 2 activity was observed in carrot 21 h after ethylene treatment [25]. Since the protein is bi- functional, an increase of its concentration after ethylene treatment is an indication that it could play a role in the process of ripening, without it being possible to predict which one of its two antagonistic activities will prevail over the other. It is therefore not surprising that the increase in Fru(2,6)P2 concentration and that in PFK 2 activity did not completely coincide. The two factors that may explain the early rise in concentration are the increased concentration of Fru6P, a substrate of PFK 2, and the decreased concentration of glycerate-3-P, a potent inhibitor of the same enzyme. The latter decrease seems to be an early effect of the ethylene treatment.

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