Cold-induced accumulation of 1-aminocyclopropane 1-carboxylate oxidase protein in Granny Smith apples

Postharvest Biologyand Technology Postharvest Biology and Technology 5 (1995) 11-17

Cold-induced accumulation of 1-aminocyclopropane 1-carboqlate oxidase protein in Granny Smith apples J-M. Lelikvre *ya, L. Tichit a, L. Fillion a, C. Larrigaudikre b, M. Vendrell bye, J-C. Pech d nINRA-Avignon, STPY B.P 91, 84143 Montfavet Cbdex, Fmnce b UdL-IRTA Post Collita, Alcade Rovim Rowe, I77 25006 Lleiah, Spain c CSIC, Jorge Girona 18-26, 08034 Barcelona, Spain d ENSAT, 145, Avenue de Muret, 31076 Toulouse Cidtx, France

Accepted January 12, 1994

Abstract

Preclimacteric Granny Smith apple (Mulus domestica Borkh.) fruits exhibited a sharp increase in ethylene production after 5 days in the cold (4”C), while in the absence of chilling (17°C) ethylene production started to increase only at 35 days. Antibodies raised against a recombinant tomato 1-aminocyclopropane I-carboxylate oxidase - the enzyme catalyzing the conversion of ACC into CzH4 - were used to demonstrate an accumulation of ACC oxidase protein in fruits stored at 4°C which paralleled the increase in ethylene production. Two-dimensional gel electrophoresis indicated that the protein corresponded to one polypeptide species only which was identical in both chilled and non-chilled fruits. These results therefore indicate that ACC oxidase, in addition to ACC synthase, is induced during chilling of pre-climacteric Granny Smith apples and accumulates before any transfer of the fruit to warmer temperatures. Keywords:

Immunoblot;

Ethylene-forming

enzyme; Malus

1. Introduction Exposure to cold temperatures of some late pear varieties (Bose, Passe Crassane, D’Anjou) is absolutely required for normal ripening of the fruit on the tree (Wang et al., 1971) or after harvest (Hansen and Hartman, 1937; Ulrich and Paulin, 1954a). *Corresponding

author. Fax: (33) 90 31 62 58.

Abbreviations: ACC, l-aminocylopropane-1-carboxyhc acid; NEPHGE, non-equilibrium electrophoresis; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0925-5214/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved. SSDI 0925-5214(94)00004-C

pH gradient

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It is now well established that low temperature stimulates autocatalytic ethylene production, a common feature of climacteric fruits, and that ethylene treatment at warmer temperatures can also induce normal ripening and thus replace lowtemperature exposure (Ulrich and Paulin, 1954b; Looney, 1972). A cold treatment, albeit not absolutely required, is able to hasten the onset of the climacteric rise of ethylene production and thus of ripening in Conference pears and in Granny Smith apples (see Jobling et al., 1991). The cold-induced ethylene production follows the general pathway of ethylene biosynthesis: S-adenosyl methionine (SAM) + l-aminocyclopropane-1-carboxylic acid (ACC) + C2H4 (Yang and Hoffman, 1984) and it is generally agreed that the stimulation of ethylene production is due to increased availability of ACC resulting from a stimulation of ACC synthase activity (Yang and Hoffman, 1984). However, recent data suggest that the ability to convert ACC into ethylene is also stimulated during the cold treatment in preclimacteric Granny Smith apples; the capacity to convert ACC into ethylene is induced more rapidly at low temperatures (0” to 5°C) than at 20°C (Jobling et al., 1991; Larrigaudiere and Vendrell, 1993). In this case, low temperatures stimulate ACC oxidase activity earlier than ACC synthase (Jobling et al., 1991). We demonstrate in this work that an accumulation of ACC oxidase protein actually occurs during storage of preclimacteric Granny Smith apples at low temperatures. 2. Materials and methods Plant material and storage Apple (Malus domestica Borkh. cv Granny Smith) fruits were harvested

at the preclimacteric stage in mid-October 1992 in an orchard of the RhGne Valley close to Avignon. Immediately after harvest, ethylene production was checked for all fruits, and those where it was above 0.5 nl h-’ g-l were discarded. Fruits were stored in ventilated rooms at 17°C or 4°C. The ethylene concentration always remained below 0.1 ppm during the course of the experiment. Ethylene measurement

Ethylene production of individual fruits was measured after having been kept for 1 h in a sealed 750 ml flask; 0.5 ml of gas was withdrawn and injected into a gas chromatograph. Gas components were separated on an 80-100 mesh alumina column (1.5 m long and l/8 inch in diameter) and detected by a flame ionisation detector. Temperatures of oven, detector and injector were 115°C 160°C and 16O”C, respectively, and the flow of the carrier gas was 30 ml min-‘. Sensitivity of detection was 0.01-0.03 ppm of ethylene. Protein extraction, electrophoresis and immunoblotting

Total proteins were extracted as described by Meyer et al. (1988): pieces of apple cortex were immediately frozen in liquid N2 after peeling, ground to a powder and mixed with the phenol emulsion. After centrifugation the proteins in the phenol phase were precipitated with 4 volumes of methanol supplemented with 0.1 M

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NH40Ac. After washing, the pellet was resuspended in Laemmli buffer (Laemmli, 1970) for SDS-PAGE, or O’Farrell buffer (O’Farrell et al., 1977) for NEPHGE. SDSPAGE was run in a BIO-RAD mini Protean II apparatus according to the recommendations of the manufacturer. The NEPHGE was run according to Meyer et al. (1988) in capillary tubes at 800 V. h (100 V/200 V/300 V, 30 min/45 min/l20 min). Blotting was carried out as described by Towbin et al., 1979. In these conditions, transfer of acidic proteins below 70 kDa on the blot was quantitative. All proteins were visualised on the blot after staining with Ponceau Red S (Sambrook et al., 1989). Western immunoblotting was performed as previously described (Dupille et al., 1993). The serum used was obtained after immunization of rabbits with a recombinant tomato ACC oxidase (Dupille et al., 1993) encoded by TOM 13 cDNA clone (Hamilton et al., 1991). Nitrocellulose membranes were scanned with an LKB Ultroscan XL apparatus to quantify the colour signals. 3. Results and discussion Apple fruits harvested in mid-October 1992 and kept at 17°C started their climacteric about 35 days later. Their rate of ethylene production remained low and very stable during the first 30 days (Fig. 1A). In fruits stored at 4”C, ethylene production dropped slightly during the first 5 days and then increased continuously to reach a rate (measured at 4°C) of 5.1 nl g-’ h-’ after 24 days (Fig. 1A). After transfer to 17°C the pattern and rate of ethylene production were dependent upon the duration of exposure at low temperature (Fig. 1B). Fruits previously stored for 11 days at 4°C and having an initial ethylene production rate of 2.7 nl g-’ h-l exhibited first a doubling of ethylene production immediately after transfer at 17°C followed by a second increase to reach a maximum of 33.4 nl g-’ h-’ after 144 h at 17°C. Fruits held for 24 days at 4°C with an initial production rate of 5.1 nl gg’ h-’ underwent a sharp burst in ethylene emission during the first 6 h after transfer to 17°C followed by a small decline until 24 h. Afterwards ethylene production increased again at a slower rate to reach a maximum of 73 nl g-t h-’ after 120 h at 17°C. Similar results were observed previously by two groups (Jobling et al., 1991; Larrigaudiere and Vendrell, 1993). However, both groups stressed the events that occur upon rewarming and they did not give direct information about the level of the ACC oxidase protein in fruits during the cold treatment. Antibodies raised against ACC oxidase have been recently obtained (Dupille et al., 1993) and shown to recognize the purified enzyme of climacteric Golden Delicious apple fruit. We could follow the accumulation of ACC oxidase protein in Granny Smith fruits stored at either 4°C or 17°C. The specificity of the antibodies was examined using protein extracts of fruits stored for 24 days at 4°C. T.vo bands were visible at 62 and 38 kDa on immunoblots made with the crude serum at a 1: 3000 dilution (Fig. 2, lanes 7 and 8). However, the 62 kDa band was still recognized when the serum was pre-absorbed with the recombinant ACC oxidase antigen, while the signal corresponding to the 38 kDa protein was greatly attenuated (data not shown). It can thus be concluded that oniy the 38 kDa protein was related to ACC oxidase.

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Fig. 1. Ethylene production in Granny Smith apple fruits (A) during storage, and (B) following transfer at 17°C after 11 (-) and 24 days (0) of storage at 4°C. Ethylene measurements were performed at 4°C (0) or 17°C (0) in (A) and at 17’C except for time 0 (4°C) in (B). Each value is the average of measurements performed individually on 3 fruits.

ACC oxidase protein was not detectable at harvest (Fig. 2, lane 1) or after 18 days at 17°C (Fig. 2, lane 2). It was also absent in some fruits kept for 38 days at 17°C (Fig. 2, lane 3) which produced negligible amounts of ethylene (below 0.01 nl h-i g-*) but was abundant in fruits evolving large amounts (36 nl h-’ g-l; Fig. 2, lane 4) of the hormone. Cold storage hastened the appearance of ACC oxidase. After 11 days at 4”C, a slight signal was already visible on immunoblots (Fig. 2, lane 6) and all fruits held for 24 days at 4°C exhibited an amount of ACC oxidase protein (Fig. 2, lanes 7 and 8) which was similar or even higher than in fruits stored for 38 days at 17°C (compare lanes 7 and 8 to lane 4). To estimate the relative amounts of ACC oxidase antigen in each fruit, the areas of the signals obtained after scanning of Western blots were compared. The ratio between the ethylene production of two

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Fig. 2. Amounts of ACC oxidase protein in preclimacteric Granny Smith fruits stored at 17°C or 4°C. Proteins (8 pg per lane) for each fruit were separated on SDS-PAGE, transferred and immunoblotted with anti-ACC oxidase crude serum and anti-rabbit IgG goat immunoglobulins conjugated to alkaline phosphatase. Lanes 1-4: fruits stored at 17°C for 0 (l), 18 (2), and 38 (3 and 4) days after harvest. Lanes 5-8: fruits stored at 4% for 5 (5), 11 (6), and 24 (7 and 8) days. The long arrow indicates the signal related to the 38 kDa ACC oxidase, while the short one corresponds to a non-specific signal of 62 kDa.

fruits was found to be in the same order of magnitude as the ratio between their amounts of antigen. For instance, the fruit corresponding to lane 6 (Fig. 2) produced 5.5 times less ethylene and contained 4.2 to 6.8 times less ACC oxidase than the fruit corresponding to lane 8 (Fig. 2) SDS-PAGE did not reveal any difference in the molecular weight of ACC oxidase protein induced by cold or during ripening at 17°C (Fig. 2). Separation of proteins by 2-D electrophoresis indicated that one polypeptide species only corresponding to the 38 kDa protein was present in protein extracts from apples stored either at 4°C or 17°C or in a mixture of both (data not shown). These data therefore demonstrate that the amount of ACC oxidase protein increased significantly in early-mature Granny Smith apples stored at 4°C before any transfer to a warmer temperature, when compared with fruits held at 17°C. This is in contrast with the extremely slow evolution of ripening of these fruits held at 4°C: changes in firmness, in acidity, and in colour of the peel are barely detectable at this temperature (C. Larrigaudiere, unpublished results) during several months. The rate of ethylene production measured at 4°C or after transfer at 17°C seemed correlated with the amount of the enzyme accumulated in fruits stored at 4°C even though it is difficult to conclude that ACC conversion into ethylene is the rate limiting step from the data shown here. It is likely that de mvo synthesis of the protein is also involved in the cold as during the normal ripening process of apples. An increase in mRNA concentrations for ACC oxidase occurs during ripening of Golden Delicious apples (Dong et al., 1992; Dilley et al., 1993). Furthermore, Wilson et al. (1990) have shown that a mRNA encoding a 38 kDa polypeptide, possibly related to ACC oxidase,

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accumulated in Conference pears stored at low temperature. Also, we have recently observed that the ACC oxidase protein accumulates in Passe-Crassane pear fruits during storage at 4°C (unpublished results). The cold-induced synthesis of ACC oxidase seems therefore to be a widespread phenomenon in winter (late) pome fruits. How the cold signal is involved in the expression of ACC oxidase gene remains to be elucidated. Acknowledgements The authors are grateful to Dr. G. Albagnac (INRA Avignon) for critically revising the manuscript, Dr. A. Latch6 for helpful discussions and C. Rombaldi for the gift of purified recombinant tomato ACC oxidase protein. This project was funded in part by a grant from Region Provence-Alpes-C&e d’Azur to JML References Dilley, D.R., Kuai, J., Poneleit, L., Zhu, Y., Pekker, Y., Wilson, I.D., Burmeister, D.M., Gran, C. and Bowers, A., 1993. Purification and characterization of ACC oxidase and its expression during ripening in apple fruit. In: J.C. Pech, A. Latch6 and C. Balagut (Editors), Cellular and Molecular Aspects of the Plant Hormone Ethylene. Kluwer, Dordrecht, pp. 46-52. Dong, J.G., Femandez-Maculet, J.C. and Yang, SF., 1992. Purification and characterization of l-aminocyclopropane-1-carboxylate oxidase from apple fruit. Proc. Natl. Acad. Sci. USA, 89: 9789-9793. Dupille, E., Rombaldi, C., Leli&vre, J.M., Cleyet-Marel, J.C., Pech, J.C. and Latch& A., 1993. Purification, properties and partial amino-acid sequence of I-amino-cyclopropane-lcarboxylic acid oxidase from apple fruits. Planta, 190: 65-70. Hamilton, A.J., Bouzayen, M. and Grierson, D., 1991. Identification of a tomato gene for ethyleneforming enzyme by expression in yeast. Proc. Natl. Acad. Sci. USA, 88: 7434-7437. Hansen, E. and Hartman, H., 1937. Effect of ethylene and certain metabolic gases upon respiration and ripening of pears before and after cold storage. Plant Physiol., 12: 441-454. Jobling, J., McGlasson, W.B. and Dilley, D.R., 1991. Induction of ethylene synthesizing competency in Granny Smith apples by exposure to low temperature in air. Postharvest Biol. Technol., 1: 111-118. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Larrigaudibre, C. and Vendrell, M., 1993. Short-term activation of the conversion of l-aminocyclopropane-1-carboxylic acid to ethylene in rewarmed Granny Smith apples. Plant Physiol. Biochem., 31: 585-592. Looney, N.E., 1972. Interaction of harvest maturity, cold storage and two growth regulators on ripening of Bartlett pears. J. Am. Sot. Hortic. Sci., 97: 81-83. Meyer, Y., Grosset, J., Char-tier, Y. and Cleyet-Marel, J.C., 1988. Preparation by 2-dimensional electrophoresis of protein for antibody production: antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts. Electrophoresis, 9: 704-712. O’Farrell, P.Z., Goodman, N.M. and G’Farrell, PH., 1977. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell, 12: 1133-1142. Sambrook, J., Fritsch, E.F. and Maniatis T, 1989. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Towbin, H., Staehelin, T and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Ulrich, R. and Paulin, A., 1954a. Sur la complexitd des conditions thermiques de la maturation des

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poires Passe-Crassane. CR. Acad. Agric. Fr., 40: 280-282. Ulrich, R. and Paulin, A., 1954b. Sur la maturation des poires Passe-Crassane. CR. Acad. Agric. Fr., 40: 603-604. Wang, C.Y. and Adams, D.O., 1982. Chilling-induced ethylene production in cucumbers (Cucumis sativus L.). Plant Physiol., 69: 424-427. Wang, C.Y., Mellenthin, W.M. and Hansen, E., 1971. Effect of temperature on development of premature ripening in Bartlett pears. J. Am. Sot. Hortic. Sci., 96: 122-125. Wilson, I.D., ‘Ibcker, G.A., Knee, M. and Grierson, D., 1990. Changes in mRNA during low temperature storage and ripening of pears. Phytochemistry, 29: 2407-2409. Yang, SE and Hoffman, N.E., 1984. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol., 35: 155-189