Inhibition of Pear Fruit Ripening by Mannose - NCBI

Plant Physiol. (1987) 85, 56-61 0032-0889/87/85/0056/06/$0 1.00/0

Inhibition of Pear Fruit Ripening by Mannose' Received for publication November 14, 1986 and in revised form April 20, 1987

CHRISTOPHER B. WATKINS2 AND CHAIM FRENKEL* Department ofHorticulture and Forestry, Rutgers University, New Brunswick, New Jersey 08903 primary influence of mannose is to sequester phosphate, then some insight into the relationships between phosphate and energy related processes might be obtained. During the course of work on the role of glycosidases in fruit ripening (28), we applied mannose to pear fruit as a potential inhibitor of glycosidase activity and found that this sugar inhibited softening and ethylene production of ripening pears. We have attempted to further describe this inhibition and test the hypothesis that the inhibition of pear fruit ripening by mannose is due to sequestering of phosphate. The approach taken was to examine the influence that applications of mannose had on the levels of G6P,3 F6P, M6P, ATP, and Pi in the fruit. These experiments, together with those using glucosamine and 2-deoxyglucose, lead to the suggestion that mannose is effective in inhibiting fruit ripening both by reducing Pi levels and through toxicity of M6P.

ABSTRACT

Softening of the flesh and the rise in ethylene evolution and respiration associated with ripening in pear (Pyrus communis L.) fruit was delayed when mannose was vacuum infiltrated into intact fruit. The extent of delay could be modified by altering the concentration or the volume of mannose applied to the fruit. Inhibition of ripening was associated with phosphorylation of mannose to mannose 6-phosphate (M6P), and accumulation of M6P was associated with lowered levels of inorganic phosphate (Pi), glucose 6-phosphate (G6P), and ATP in the fruit tissue. Subsequently, however, as the M6P was metabolized, the levels of Pi, G6P, and ATP increased and ripening processes were concomitantly released from inhibition. Hence, the degree of inhibition by mannose or the release from inhibition was related to the level of M6P in the fruit and its rate of metabolism. The data provide correlative evidence to support a view that one inhibitory effect of mannose is depletion of Pi in the cell as a result of phosphorylation of mannose to M6P. Inhibition of ripening by mannose was not alleviated by co-application of glucose as a competitive substrate for the hexokinase(s), or by Pi, presumably the depleted metabolite. Also, incubation of tissue disks with M6P resulted in inhibition of ethylene production and respiration. The structural analogs of mannose, glucosamine, and 2-deoxyglucose, which have been shown to mimic mannose action in several plant tissues, did not cause inhibition of ripening of pear fruit comparable with that associated with mannose. Both analogs stimulated respiration, and glucosamine caused only a small inhibition of softening and ethylene evolution. Another mannose analog, a-methylmannoside, did inhibit fruit ripening though to a lesser extent than mannose. Its influence was also associated with accumulation of M6P and a decrease of Pi levels. We conclude that the mannose effect may, in part, be due to M6P toxicity, as well as by depletion of Pi.

MATERIALS AND METHODS

Plant Material. Pear fruit, Pyrus communis L. cvs Bartlett and Bosc., harvested at the mature green stage, were obtained from Mountain Top Orchard, Glen Gardner, NJ and Geneva, NY, respectively. The fruit were stored at 1°C in air or in hypobaric chambers at one-tenth to one-twentieth atmosphere pressure using purafil-purged air as the ventilating gas. The air flow provided approximately 1.5 volume changes per hour. Hypobaric-stored fruit were removed as required and equilibrated overnight in air at 1°C prior to use. Application of the Test Solutions. A modified vacuum infiltration technique (8) was used to apply the test compounds to the fruit. The fruit were first surface sterilized with 70% ethanol, and then pierced longitudinally with a 13 gauge, sterile needle through the calyx and at 4 equidistant places around the calyx. Compounds were applied in a mannitol carrier solution, the final molarity of the solutions always being 300 mm. The control fruit were infiltrated with 300 mM mannitol. All solutions were sterilized by microfiltration using 0.45 ,m Millipore filters prior to use and between each infiltration. The vacuum applied was adjusted to ensure uniform uptake of the treatment solutions as determined by weight change in a given experiment: the uptake rate for each experiment is given in the figure legends. Forty to fifty fruit were infiltrated per treatment. Measurement of Softening, Ethylene Evolution and Respiration. The treated fruit were kept at ambient temperatures, and fruit samples consisting of 10 pears were taken for the measurement of ethylene and CO2 evolution, and softening, at the required intervals. The fruit were sealed in 20 L glass jars, and 1 ml gas samples taken in triplicate for ethylene and CO2 determinations after 15 and 75 min. Tests showed that the rate of production of both gases was linear during this time. Ethylene was determined using a Hewlett Packard 5890A gas chromato-

Mannose, and its structural analogs glucosamine and 2-deoxyglucose, can inhibit many metabolic processes in plant tissues, including growth, respiration, ion uptake, and photosynthesis (Ref. 12 for review). According to Herold and Lewis (12), the primary mechanism of inhibition is the ready phosphorylation of these compounds to form phosphate esters, which are not further metabolized. In the cell, Pi is lowered, and metabolism may become limited. An obvious consequence of reduced Pi levels, for example, would be a reduction in the regeneration of ATP from ADP and restriction of the rate of energy requiring reactions. The application of mannose has been proposed as a tool to study phosphate metabolism in plant tissues (12). If the ' New Jersey Agricultural Experiment Station, Publication No. D12240-19-86 supported by State funds and United States Hatch Act.

3 Abbreviations: G6P, glucose 6-phosphate; ACC, l-aminocyclopropane-1-carboxylic acid; F6P, fructose 6-phosphate; M6P, mannose 6-

2 Supported by a New Zealand National Research Advisory Council Fellowship. Present address: Division of Horticulture and Processing, D.S.I.R., Private Bag, Auckland, New Zealand.

phosphate. 56

MANNOSE INHIBITION OF PEAR FRUIT RIPENING graph equipped with a flame ionization detector and a Poropak Q column at 100°C, and CO2 by thermal conductivity using a Shimadzu GC 8A fitted with a Poropak R column at 30°C. The fruit were briefly rinsed with deionized water, dried, and flesh firmness measured on opposite sides of each pear with an Effigi fruit tester fitted with a 7.9 mm diameter probe. Preparation and Extraction of Tissue. The same 10 fruit on which fruit firmness was determined were used to obtain tissue samples for later analysis. Two segments, each approximately one-eighth of the fruit, were excised from opposite sides of each fruit, the core tissue removed, and the remaining tissue weighed and sliced into liquid N2. The fruit samples, weighing approximately 200 g per treatment, were freeze-dried, reweighed, pulverized to powder in a Waring Blendor, and stored at -20°C until required. The tissue powder was extracted by a procedure similar to that of Chapin and Bieleski (5). Samples weighing 500 mg were extracted overnight at -20°C in 25 ml methanol:chloroform:formic acid:water (MCFW, 12:5:1:2 v/v) and centrifuged at 5,000g for 10 min. Five ml of chloroform and 10 ml of water were gently mixed with the supernatant and the sample centrifuged (5,000g, 10 min). The aqueous phase containing sugars, amino acids, organic acids, and phosphate esters, was saved and the chloroform phase containing pigments, lipids, and phospholipids discarded. The residue was re-extracted twice with 20 ml of MWF (20:79:1 v/v). Each time, the samples were centrifuged at 10,000g for 10 min and the two extracts were combined with the first aqueous methanol phase. The combined extract was dried at 40°C on a rotary evaporator and placed under vacuum over KOH pellets to remove the formic acid. The extracts were made up to known volume with water and centrifuged at 10,000g for 10 min to remove any particulate matter. Samples were taken for immediate assay of ATP or frozen at -20°C for later assays of Pi and glycolytic intermediates. All extractions were repeated at least once, and all analyses were carried out in triplicate. Analysis of Pi, ATP, G6P, F6P, and M6P. Pi was determined by the method of Fiske and SubbaRow using the Sigma Diagnostic Kit No. 670. ATP was determined by the luciferin-luciferase method. Firefly lantern extract (Sigma FLE-50) was made up according to the instructions of the manufacturer, and after standing overnight at 4°C, it was centrifuged (10,000g, 10 min) immediately before use. Bioluminescence was measured in a custom-built photometer with electronics constructed as described by Blinks et al. (3) and photomultiplier housing, reaction chamber, and shutter assembly designed and built by W. Ward and H. Verny (unpublished data). Aliquots (0.1 ml) of diluted extract were incubated for 10 min in 0.2 ml of buffer (50 mm Hepes + 50 mm Mg acetate, pH 7.5) and distilled water to a final volume of 0.6 ml; 0.1 ml of this solution was rapidly injected into a photometer containing 0.05 ml of luciferin-luciferase, and the peak height recorded. Internal standardization was used in the ATP determinations, and recovery was greater than 95%. G6P, F6P, and M6P were assayed enzymically by measuring the reduction of NADP by G6P dehydrogenase and coupling the required reactions to this reduction in sequential steps with phosphomannose isomerase and phosphoglucose isomerase. The procedures for G6P and F6P were as described by Latzko and Gibbs (17) except that Tricine buffer was used instead of TrisHCI buffer and the NADPH formed was measured by an AMINCO SPF 125 spectrofluorometer rather than by a spectrophotometer. M6P was measured by the addition of 1 unit of phosphomannose isomerase to the assay mixture (10). Effect of Exogenous M6P. The influence of exogenous M6P was studied with tissue disks (8 mm diameter, 2 mm thick) cut from the cortical tissue of Bosc pear fruit using a cork borer and scalpel. Six discs (about 1.2 g) were incubated at 27°C in 25 ml Erlenmeyer flasks containing 3 ml incubation solutions consist-

57

ing of 0, 0.5, 1, 5, and 10 mm M6P (barium salt) or 10 mm mannose in mannitol (made up to 600 mm final concentration), 10 mm Mes buffer (pH 6.1), and 50 mg/ml chloramphenicol. For each treatment, three flasks were flushed with air and sealed with rubber serum caps between 4 to 5 and 9 to 10 h after the start of incubation. Gas samples 1 ml each, were withdrawn from the headspace of the flasks and ethylene and CO2 determined by gas chromatography. Each experiment was repeated three times. RESULTS Effect of Mannose. Infiltration of Bartlett pear fruit with 100 mM mannose inhibited softening, ethylene evolution, and respiration (Fig. 1). Treatment with other sugars such as glucose and galactose had little effect (data not shown). With mannose application, M6P accumulated and Pi levels declined over the first 2 d (Fig. 2). By d 2, the concentration of M6P was 491 nmol/g fresh weight compared with 4 nmol/g fresh weight in control treatments. However, after 2 d the M6P concentration decreased from this maximum by approximately 100 nmol/g fresh weight per day, and this decrease was associated with an increase in Pi levels in the fruit (Fig. 2). Softening and ethylene evolution were progressively inhibited by increasing mannose concentrations. Mannose, at all concentrations tested, inhibited respiration but a dose response was not evident. The inhibitory influence of increasing mannose concentration was closely associated with both the accumulation of M6P and the decrease in Pi levels (Fig. 2). On d 2 the M6P levels were 4, 248, 491, and 536 nmol/g fresh weight in fruit treated with 0, 30, 100, and 300 mm mannose, respectively. By d 4, only 25 nmol/g fresh weight remained in fruit given 30 mM mannose and there was no evidence ofinhibition of softening and ethylene evolution. However, with 300 mm mannose, 159 nmol M6P/g fresh weight was still present at d 8. The patterns of ATP, G6P, and F6P accumulation in the fruit were also altered by mannose treatment (Fig. 3). During the ripening of the control fruit, ATP concentrations increased as shown in other climacteric fruit (25), whereas those of G6P and F6P decreased. Applied mannose reduced ATP concentrations, this being particularly evident at 2 d. G6P and F6P concentrations were also lowered by mannose application, so that on d 2 the levels of ATP, G6P, and F6P in fruit treated with 100 mm fruit were only 38, 25, and 47%, respectively, of the levels of these metabolites in the control treatment. However, as in the case of changes of the Pi levels, those of ATP, G6P, and F6P also increased after 2 d. Recovery of Pi and ATP levels were incomplete with 300 mM mannose (Fig. 3). The metabolism of M6P and the concomitant increase in the levels of the other compounds measured, seemed closely associated with the release from inhibition of softening, ethylene evolution, and respiration

(Fig. 1).

Manipulation of pear fruit ripening with mannose was also examined by comparing different uptake volumes: 'low' uptake (5.7 ml/100 g fruit weight) with 'high' uptake (9.1 ml/100 g fruit weight). An increase of the uptake volume of the mannitol control resulted in little effect on ethylene evolution, but some inhibition of softening and respiration (data not shown). In comparison, an increase of the uptake volume of mannose resulted in a proportionally greater delay of softening, and of ethylene evolution. The inhibition was reflected in the level of M6P and on d 8, substantial levels of M6P (189 nmol/g fresh weight) still remained in the high uptake fruit (cf 12 nmol M6P/ g fresh weight in the low uptake fruit). Cultivars differed in their response to mannose application. Figure 4 shows the more pronounced inhibition of ripening by 100 mm mannose in Bosc pears than shown earlier with Bartlett pears. It is possible that this difference is related to the ripening stage of the fruit as the Bosc pears were climacteric. However, a

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WATKINS AND FRENKEL

Plant Physiol. Vol. 85, 1987

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Glucose did not, however, alleviate the inhibition of softening ethylene evolution by mannose (Fig. 4). The inhibition of respiration by mannose was initially reduced by glucose though O P'sL_A the stimulation of respiration in mannose-treated fruit after 5 d 0 2 4 6 8 0 2 4 6 8 was enhanced by the co-application of glucose. The accumulation of M6P and the lowering of Pi, ATP, G6P, and F6P in DAYS AFTER TREATMENT mannose-treated fruit was also unaffected by the co-application of glucose (Figs. 5 and 6). Thus, at equimolar concentrations of FIG. 2. Changes of M6P and Pi levels in Bartlett pears as influenced by 10, 100, or 300 mm mannose. Mannitol control (0), 30 mm (U), 100 glucose and mannose, glucose did not inhibit mannose uptake by the cells; to the contrary, it is possible that the reverse mM (0), 300 mm (0) mannose. occurred. In corn scutellum, glucose uptake was inhibited by 80% in the presence of equimolar (49 mM) mannose (13). A much greater ratio of glucose to mannose may be needed to overcome the mannose inhibition of metabolism, e.g. in excised roots a ratio of glucose to mannose of 3.5:1.0 was required to alleviate inhibition of growth and respiration caused by mannose co a 1.0

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When 30 mm KH2PO4 was applied with 60 mm mannose to Bartlett pears, Pi did not alleviate the inhibition of either soft~~~~~~~~~~~~~0. 0 260 2 ening or ethylene evolution by mannose (data not shown). In the presence of exogenous Pi and mannose, the levels of M6P were 17 and 27% greater than those from mannose alone on d 2 and 4, respectively. Thus, application of Pi with mannose stim0c ulated M6P accumulation in the tissue. The ATP concentration was also higher in the presence of exogenous Pi than in its absence, even in the mannose-treated fruit. Phosphorylation of mannose by hexokinases requires ATP, and radiotracer studies have shown that the primary metabolic incorporation of exogenous Pi in plant tissues is likely to be via ATP and UTP production (2). Thus, the higher levels of M6P obtained when Pi DAYS AFTER TREATMENT FIG. 3. Changes of ATP, G6P, F6P levels in Bartlett pears as influ- was applied together with mannose may have been due to enced by 30, 100, or 300 mm mannose. Mannitol control (0), 30 mM enhanced phosphorylation of mannose caused by elevated ATP levels. (), 100 mM (0), 300 mm (01) mannose. Short-Term Influence of Mannose. The inhibitory influence more likely explanation is a difference in the rate of M6P of 100 mM mannose on ethylene and respiration over a 24 h metabolism of the fruit cvs. For example, in Bosc fruit the M6P period was examined using climacteric Bosc pears. Inhibition concentration did not decline to 100 nmol/g fresh weight until was evident already at 4 h, and by 12 h, both ethylene production 7 to 8 d (Fig. 5), whereas in Bartlett fruit this value was usually and respiration in mannose-treated pears were inhibited (by 41 reached by 5 to 6 d. and 21%, respectively) compared with the control fruit. The Effect of Glucose or Exogenous Pi. We attempted to overcome accumulation of M6P in mannose-treated fruit was initially rapid the inhibition of ripening by mannose in two ways. First, by but the rate of increased declined steadily over time (Fig. 7), ~~~~~~~~E

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MANNOSE INHIBITION OF PEAR FRUIT RIPENING z

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