Ethylene Induces De Novo Synthesis of Chlorophyllase, a Chlorophyll

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9441-9445, October 1993 Plant Biology Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in Citrus fruit peel (ripening/senescence/gibbereilin A3/N6-benzyladenine) TOVA TREBITSH, ELIEZER E. GOLDSCHMIDT, AND JOSEPH Riov The Kennedy-Leigh Centre for Horticultural Research, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel Communicated by Kenneth V. Thimann, July 15, 1993 ABSTRACT Chlorophyllase (Chlase; EC 3.1.1.14) was to induce a severalfold increase in Chlase activity (8-10). It extracted from plastid fractions of ethylene-treated orange is not known, however, whether this increase involves de fruit peel and purified 400-fold to homogeneity by gel iftration, novo synthesis of the enzyme protein or activation of a hydrophobic chromatography, and preparative SDS/PAGE of constitutive enzyme. Gibberellin A3 (GA) and N6-benzylad- nonheated protein. SDS/PAGE of nonheated purified enzyme enine (BA) delay the senescent pigment changes and oppose indicated that Chlase activity is associated with a single protein the ethylene-induced loss of Chl (6, 7, 11), but their effect on band migrating at an apparent molecular mass of 25 kDa Chlase activity has not yet been determined. Thus, little is whereas the heated purified enzyme had a molecular mass of35 known about the hormonal and molecular regulation of the kDa. The N-terminal sequence of the purified protein was Chlase enzyme system. determined. The purified enzyme was used as an immunogen While attempting to study the overall regulation of Chlase, for raising antibodies in rabbits. The antiserum was highly we found it essential to adopt an immunological approach. specific and on Western blots recognized both the heated and Chlase has been partially purified and characterized previ- the nonheated form of Chlase. The antibodies also recognized ously from various plant materials (12-16). Citrus Chlase has the solubilized enzyme, as shown by an immunoprecipitation been purified from ethylene-treated Citrus unshiu by assay and by antigen-antibody capture assays in microtiter Shimokawa (17). The Chlase preparations obtained using plates. Treatment with ethylene, which enhances degreening, Shimokawa's procedure (17) were not pure enough, how- increased Chlase activity 12-fold. Immunoblot analyses of ever, for raising anti-Chlase antibodies. The aim of the crude extracts from ethylene-treated fruit detected a strong present study was, therefore, (i) to purify Chlase to homo- protein, while only a trace level of the geneity, (ii) to obtain specific anti-Chlase antibodies, and (iii) signal of the Chlase to examine whether the enhancement of Chlase activity by enzyme protein could be detected in air. Gibberellin A3 and ethylene and its counteraction by GA and BA involve control N6-benzyladenine partly counteracted the ethylene-induced the of the enzyme increase in Chlase activity as well as the immunodetected of synthesis protein. upsurge ofthe Chlase protein. Ethylene appears to enhance the degreening of citrus fruit through de novo synthesis of the MATERIALS AND METHODS Chlase protein, which in turn is inhibited by the senescence- Plant Material. Mature green orange (Citrus sinensis L. delaying regulators, gibberellin A3 and N6-benzyladenine. The Osbeck, cv. Valencia) fruit was harvested from trees grown Chlase enzyme protein may, therefore, serve as a model system in Rehovot, Israel. Fruit was treated with a stream of for studying the hormonal molecular regulation of fruit rip- humidified air with or without ethylene at 80 Al/liter for 72 h ening and senescence. at 25°C in the dark. When indicated, fruit was dipped in GA or BA in water containing 5% (vol/vol) ethanol and 0.02% Senescence of green plant tissues involves breakdown of the Tween 20 for two 30-s periods. After 24 h at room temper- photosynthetic apparatus and destruction of chlorophyll ature, fruit was treated with ethylene or air as above. (Chl). Loss of Chl occurs during senescence of vegetative Enzyme Extraction. The flavedo (the outer colored layer of tissues as well as during fruit ripening. Despite the central Citrus fruit peel) was removed, homogenized in ice-cold 50 role ofChl in the life processes ofplants, little is known about mM Tris-HCl, pH 8.0/0.4 M sucrose, and centrifuged at its catabolism (1). The chlorophyllase (Chlase) system (Chl 12,000 x g for 10 min. The floating chloroplast pellet was chlorophyllidohydrolase, EC 3.1.1.14) was discovered 80 homogenized in the same buffer without sucrose and the years ago by Willstatter and Stoll (2), who suggested that resultant floating pellet was homogenized in acetone at removal of the phytol could be the first step in Chl catabo- -15°C for the preparation of an acetone powder. Acetone lism. Evidence accumulated in recent years points indeed to powders were lyophilized and stored at -20°C. the critical role ofChlase in the initial steps ofChl catabolism. For determination of Chlase in crude extracts, 30 mg of Accumulation of chlorophyllide, which is the immediate acetone powder was stirred with 5 ml of 5 mM potassium product of the Chlase reaction, has been demonstrated in phosphate, pH 7.0/50 mM KCl/0.24% Triton X-100 for 60 senescing Citrus fruit (3); in leaves of Citrus, Melia, and min at 30°C. The extract was filtered through glass wool and Pisum (4); and in a nonyellowing Festuca mutant (5). Recent centrifuged at 12,000 x g for 10 min. The supernatant was in vivo and in vitro studies of Chl breakdown in various used for Chlase assay and immunodetection. species revealed the presence of additional catabolic prod- Enzyme Assay and Protein Determination. Aliqots of en- ucts that could be derived only from chlorophyllide (3, 5). zyme were incubated at 37°C in 100 mM sodium phosphate, The effect of exogenous growth regulators on the loss of pH 7.0/0.24% Triton X-100/0.2 ,umol of Chl-a dissolved in Chl in senescing Citrus fruit peel is well documented (6-9). 100% acetone. The reaction was stopped by transferring 0.5 Ethylene, which accelerates the loss of Chl, has been shown ml of the reaction mixture to centrifuge tubes containing acetone/hexane/10 mM KOH, 4:6:1 (vol/vol). The mixture The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: BA, N6-benzyladenine; Chl, chlorophyll; Chlase, in accordance with 18 U.S.C. §1734 solely to indicate this fact. chlorophyllase; GA, gibberellin A3; BSA, bovine serum albumin. 9441 Downloaded by guest on September 29, 2021 9442 Plant Biology: Trebitsh et al. Proc. Natl. Acad Sci. USA 90 (1993) was shaken and centrifuged at 8000 x g for 5 min to separate anhydrase (29 kDa) were used as the standard proteins the phases. Chlorophyllide a was determined in the acetone (Sigma) for determining the molecular mass. The void volume phase spectrophotometrically, using an extinction coefficient of the column was determined with blue dextran. of 74.9 mM-1cm-1 at 667 nm (18). N-Terminal Sequencing. The purified Chlase was trans- One unit of enzyme activity was defined as the amount of ferred into a Problott membrane (Applied Biosystems). enzyme hydrolyzing 1 umol of Chl-a per min at 37°C. N-terminal sequencing was conducted with an Applied Bio- Protein concentration of enzyme extracts containing Tri- systems model 475A system, which includes a gas-phase ton X-100 was determined by the Biuret method (19); other- protein sequencer (model 470A) and a synchronized phenyl- wise, it was determined with the Bradford dye-binding assay thiohydantoin analyzer (model 120A) driven by a control and (Bio-Rad), using bovine serum albumin (BSA) as a standard. data analysis module (model 900A). Determination of Chl Content. Chl content was measured Preparation of Antigen and Antibody Production. Purified according to Moran and Porath (20). Five flavedo disks (11 enzyme (20 ,ug) was incubated for 10 min at 100°C in sample mm in diameter) were incubated overnight in 5 ml of N,N- buffer/5% (vol/vol) 2-mercaptoethanol/1% SDS and then dimethylformamide at 4°C and the Chl content was deter- separated by SDS/PAGE. After electrophoresis, the gel was mined spectrophotometrically. washed with deionized water, stained for 10 min with 0.05% Enzyme Purification. Soluble proteins were extracted by Coomassie brilliant blue R-250, and destained with several homogenizing and stirring 10 g ofacetone powder with 300 ml changes ofwater. The appropriate protein band was cut from of 10 mM potassium phosphate, pH 7.0/4% (vol/vol) the gel and stored at -20°C. (NH4)2SO4 for 60 min at 30°C. After centrifugation at 20,000 Prior to injection, the gel was fragmented by repeatedly x g for 20 min, no Chlase activity was detected in the passing it through a syringe in the presence of phosphate- supernatant. The precipitate was resuspended in 300 ml of 10 buffered saline (PBS) (21). Antibodies to Chlase were gen- mM potassium phosphate, pH 7.0/1% sodium cholate/10% erated in female rabbits by subcutaneous injection of20 jig of (vol/vol) glycerol and stirred for 60 min at 30°C. After protein, followed by several booster injections every 4-6 pelleting insoluble material at 20,000 x g for 20 min, Chlase weeks. Finally, to enhance sera activity against the active was precipitated out from the supernatant by adding solid enzyme, 20 ,ug of solubilized enzyme was again injected (NH4)2SO4 to a final concentration of 80% saturation, dia- followed by additional booster injections. After the second lyzed overnight against 1 mM potassium phosphate (pH 7.0) and each ofthe following injections, rabbits were bled and the (dialysis buffer), and concentrated by lyophilization.

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