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. The sera were tested on immunoblots and in various immunoas- concentrate was solubilized in 15 ml of 30 mM potassium says of Chlase activity. phosphate, pH 7.0/0.05% sodium cholate (solubilizing Heated Chlase was immobilized on a nitrocellulose mem- buffer) and applied to a Sephadex G-200 column (2.5 x 90 cm) brane and incubated with anti-Chlase sera. Affinity-purified equilibrated with 30 mM potassium phosphate (pH 7.0). The Chlase antibodies were obtained by eluting the bound anti- enzyme was eluted with the same buffer at a flow rate of 12 body at pH 2.3 (22). ml/h. Five-milliliter fractions were collected, and those con- Electrophoresis and Immunoblot Analysis. Electrophoresis taining the highest enzyme activity were combined and of either nonheated or heated proteins was routinely per- loaded onto a phenyl-Sepharose CL-6B column (1 x 10 cm) formed on 14% polyacrylamide gels in the presence of 0.1% equilibrated with 30 mM potassium phosphate (pH 7.0). The SDS and stained with Coomassie brilliant blue R-250 to protein was eluted from the column with a linear sodium visualize the proteins. Nonheated proteins were defined as cholate gradient (0-1.5%) at a flow rate of 15 ml/h. Fractions proteins that were incubated at room temperature for 15 min containing enzyme activity were pooled, dialyzed against in 20 mM Tris HCl, pH 6.8/0.1% SDS/10% glycerol, prior to dialysis buffer, and lyophilized. Final purification was per- SDS/PAGE. Heated proteins were obtained after incubation formed by preparative SDS/PAGE as follows: the lyo- for 10 min at 100°C in the above sample buffer adjusted to 5% philized powder was resuspended in the solubilizing buffer, 2-mercaptoethanol and 1% SDS. Proteins were transferred to incubated for 15 min at room temperature in nonheating nitrocellulose for 2 V-h using the Tris/glycine/5% (vol/vol) sample buffer (20 mM Tris HCl, pH 6.8/10% glycerol/0.1% methanol system for semi-dry electrophoretic transfer (LKB SDS), and separated by SDS/PAGE. The resolved gel was operation manual). Proteins were detected with affinity- sliced to 0.5-cm segments. Segments were incubated over- purified sera and horseradish peroxidase-conjugated second night at 37°C in 5 ml of 30 mM potassium phosphate, pH antibody. 3,3'-Diaminobenzidene and 4-chloro-1-naphthol 7.0/0.5% sodium cholate, and enzyme activity was assayed were used for color development of peroxidase activity (23). in the extracts. The active fractions were filtered through a Immunoassays. Immunoprecipitation of Chlase activity. 0.45-,tm (pore size) filter, dialyzed, lyophilized, resuspended The appropriate serum (50 ,ul) was added to -0.008 unit of in 0.5 ml of 10 mM potassium phosphate, pH 7.0/0.05% Chlase and diluted to 350 ,ul with 50 mM Tris HCl, pH 8.0/150 sodium cholate/10% glycerol, and dialyzed against the same mM NaCl/0.1% Triton X-100/10% glycerol. The reaction buffer overnight. The purified enzyme was kept at -20°C. mixture was incubated for 2 h at room temperature. The This enzyme preparation will be referred to as the solubilized above buffer (100 ,ul) containing 10% (vol/vol) fixed Staphy- enzyme. lococcus aureus Cowan I cells was then added and the Enzyme with a specific activity of 150-250 units per mg of mixture was shaken at room temperature for 1 h. Immune protein was routinely obtained by this purification procedure. complexes were collected by centrifugation in a microcen- Molecular Mass Determination. Molecular mass of non- trifuge for 1 min. Chlase activity was assayed in the super- heated and heated Chlase was determined by SDS/PAGE natant as described above. with the following standard proteins purchased from Sigma: Activity test-antigen capture assay in microtiterplates. A BSA (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3- polyvinylchloride plate was coated with donkey anti-rabbit phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 antibodies at 10 pg/ml and the remaining sites for protein kDa), bovine pancrease trypsinogen (24 kDa), soybean tryp- binding were blocked by BSA. Serum (50 ,u), in various sin inhibitor (20.1 kDa), and bovine milk a-lactalbumin (14.2 dilutions, was added to each well and incubated for 8 h at 4°C. kDa). The molecular mass of Chlase in solution was esti- Wells were washed with PBS. Antigen (50 ,l) was then added mated on a Sepharose CL-6B column (1.5 x 90 cm) equili- to each well at 2.5 ,ug/ml and incubated overnight at 4°C. The brated with 30 mM potassium phosphate (pH 7.0) and run at wells were washed with PBS and 200 ,Al of reaction buffer a flow rate of 18 ml/h. Thyroglobulin (667 kDa), apoferritin containing Chl-a was added. Chlase activity was assayed (443 kDa), ,3-amylase (200 kDa), BSA (66 kDa), and carbonic after incubation for 2.5 h at 37°C. Downloaded by guest on September 29, 2021 Plant Biology: Trebitsh et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9443

Inhibition test-antibody capture assay in microtiter Chlase plates. A polyvinylchloride plate was coated with Chlase at activity, % ( 50 100 1 ,ug/ml and the remaining sites for protein binding were kDa 1 21.. I. blocked by BSA. Serum (50 ,ul), in various dilutions, was and incubated for 2 h at 37°C. The wells added to each well 66.0 FIG. 1. SDS/PAGE of purified were washed with PBS and then 200 ,ul of reaction buffer Chlase from flavedo of ethylene-treated containing Chl-a was added. Chlase activity was assayed 45.0 - Citrus fruit. Lanes: 1, heated purified 36.0 - after incubation for 2.5 h at 37°C. Activity in the presence of Chlase (1 .g incubated for 10 min at preimmune serum represented 100% Chlase activity. 29.() - 100°C with 5% 2-mercaptoethanol/1% SDS); 2, nonheated purified Chlase (1 ug 24.0- incubated for 10 min at room temperature RESULTS AND DISCUSSION 20.1 - with 0.1% SDS). The gel was stained with Coomassie brilliant blue R-250. Enzyme Purification of Chlase. The purification of Chlase from 10 g activity (percentage of maximum activ- of acetone powder prepared from 3 kg of ethylene-treated ity) of the nonheated protein was local- orange (Citrus sinensis L. Osbeck, cv. Valencia) fruit peel is 14.2 - ized by incubating slices of the resolved summarized in Table 1. The enzyme was purified 400-fold to gel in Chlase assay buffer. homogeneity, with 9% recovery, by gel filtration, adsorption chromatography, and gel electrophoresis. Similar to Chlase Molecular Mass. The molecular mass of partially purified from other sources (14, 16), Citrus Chlase is highly hydro- Citrus Chlase, as determined by Sepharose CL-6B gel filtra- phobic, as indicated by its strong adherence to ethyl-agarose tion, was estimated to be 376 kDa. This value approximates (data not shown). The hydrophobic properties ofthe enzyme those reported for Phaeodactylum tricornutum (28) and for permitted the use of a hydrophobic column (phenyl- soluble Chlase from tea (24). On the other hand, SDS/PAGE Sepharose CL-6B) as one of the purification steps. The of nonheated purified Chlase indicated that enzyme activity preparative SDS/PAGE, used as the last purification step, was associated with the 25-kDa protein band and the molec- was most efficient and enabled us to assign enzymatic ular mass of the heated enzyme protein was 35 kDa (Fig. 1). activity to a single protein band (Fig. 1). These latter values agree with the estimated molecular mass Purified Citrus Chlase had a specific activity of 174 ,mol of Chlase from other plant sources, which has been found to of Chl-a per min per mg of protein (Table 1), which is much range-from 27 to 39 kDa (12, 14, 16-18, 24, 28). The 35-kDa higher than that reported previously (13, 14, 18, 24, 25). For heated form could also be obtained by heating Chlase in example, Chlase from ethylene-treated Citrus unshiu peel nonheating sample buffer, which contained 0.1% SDS and no had a specific activity of 0.069 ,mol of Chl-a per min per mg 2-mercaptoethanol. When incubated at room temperature in of protein (17) and Chlase from sugar beet leaves had a nonheating buffer containing 2-mercaptoethanol, the 25-kDa specific activity of 4.08 ,mol of Chl-a per min per mg of band was retained but an additional 26.5-kDa band appeared protein (12). (data not presented). It seems that the active unit of Chlase Characteristics of Purified Chlase. The pH optimum for the is tightly packed and only heating can unfold the protein into activity of purified Chlase was about pH 7.5, similar to that a rod-like molecule that migrates according to its presumed of crude Citrus Chlase (26). The high activity of Chlase molecular mass of 35 kDa (29). (70-80%) was evident from pH 5.0 to 9.0, as in Chlorella The occurrence of various multiple forms of Chlase, in- protothecoides (15). The purified Chlase, like the crude cluding the high molecular forms obtained by gel flltration, Citrus Chlase (26), was active in temperatures from 30°C to might all be explained by reversible associations, aggrega- 60°C with an optimum at 45°C. The isoelectric point (pI) was tion, or other alterations during extraction and purification estimated to be 4.3 using ampholyte (pH range 3-10), similar (12). It may still be questioned whether the 25- to 35-kDa to Chlase from Chlorella protothecoides (15) and rye (27) (pI polypeptide constitutes the in vivo enzyme or whether it 4.5). The Km value of the purified Chlase was estimated to be represents an active subunit of a larger protein (28). Never- 1.76 ,uM. The fact that the purified Chlase is active over a theless, based on the data presented, it seems that the broad range of pH values and temperatures as well as its smallest active unit of Citrus Chlase has a molecular mass of highly hydrophobic nature may indicate a tightly packed 35 kDa. molecular structure that protects the active site, at least Antibody Specificity. The affinity-purified antiserum was under the conditions of our aqueous in vitro assay system. highly specific and identified the nonheated and heated forms of Chlase on Western blots. Nonheated Chlase was immuno- Table 1. Purification of ethylene-enhanced Chlase from detected at 25 kDa but faint traces were also detected at 35 Citrus peel kDa (heated form) and at 41.5 kDa (Fig. 2). The immunode- Specific Overall Total activity, fold Chlase Protein, activity, units/ purifi- Recov- activity, % Purification step mg units mg cation ery, % kDa 1 2 0 50 100 Chromoplast acetone FIG. 2. Immunoblot of purified Chlase powder 170.00 72.2 0.425 0.0 100.0 66.0 from flavedo of ethylene-treated Citrus Sodium cholate (1%) 114.00 70.7 0.620 1.5 97.9 45.0 - fruit. Lanes: 1, heated purified Chlase (1 (NH4)2SO4 (80%) 51.87 47.9 0.923 2.2 66.3 36.0 ,g incubated for 10 min at 100°C with 5% .. Sephadex G-200 19.89 40.9 2.058 4.9 56.6 ... .., 2-mercaptoethanol/1% SDS); 2, non- 29.0 heated purified Chlase (1 ,ug incubated Phenyl-Sepharose :...... CL-6B 1.80 12.7 7.056 16.6 17.6 24.0 - for 10 min at room temperature with 0.1% Preparative SDS). Proteins were separated by SDS/ 0.04 6.7 173.884 409.8 9.3 20.1 - PAGE, electroblotted to nitrocellulose, SDS/PAGE and visualized as described. Enzyme ac- One unit of enzyme activity is defined as the amount of enzyme tivity (percentage of maximum activity) hydrolyzing 1 pmol ofChl-a per min. Extraction with sodium cholate ofthe nonheated protein was localized by was performed after extraction of proteins with 4% ammonium 14.2 incubating slices of the resolved gel in sulfate. Chlase assay buffer. Downloaded by guest on September 29, 2021 9444 Plant Biology: Trebitsh et al. Proc. Natl. Acad Sci. USA 90 (1993) 0.40 Table 2. Chlase activity in ethylene-treated (72 h) Citrus fruit peel

r- Chlase activity, ,umol per 'IO 0.30 Q1D Chl, Mg/cm2 h per g (FW) 0 Treatment Air Ethylene Air Ethylene ;^~ 0.20 Harvest day 28.5 ± 0.95 0.23 ± 0.051 ._ 0.10 Control 29.6 ± 0.34 9.6 ± 0.66 0.27 ± 0.017 3.09 ± 0.09 ± ± ± 1.83 ± 0.17 (A GA3 26.6 1.67 19.7 2.19 0.30 0.078 Ct ± ± ± ± r--,-l BA 30.0 1.86 16.6 1.41 0.68 0.061 2.97 0.70 u 0.00 F-I Acetone powders prepared from chloroplast fragments were ex- -i tracted by 0.24% Triton X-100 and assayed for Chlase activity as -0.10 described. FW, fresh weight. 1:20 1:10 1:5 Serum dilution Chlase was immunodetected also in crude acetone pow- ders from green leaves of citrus, Bougainvillea glabra, and FIG. 3. Activity of Chlase immobilized by Chlase polyclonal Hibiscus (Hibiscus rosa-sinensis) and from fruits of tomato antibodies. A polyvinylchloride plate was coated with donkey anti- (Licopersicon esculentum Mill.) and cucumber (Cucumis rabbit antibodies at 10 pg/ml and the remaining sites for protein binding were blocked by BSA. Serum (50 p4), in various dilutions, sativus L.). Significant Chlase activity was detected in all was added to each well and incubated for 8 h at 4°C. Wells were these species. washed with PBS. Antigen was added to each well (50 ILI) at 2.5 N-Terminal Sequence of Chlase. N-terminal sequence of pg/ml and incubated overnight at 4°C. The wells were washed with Chlase was determined to be NH2-Ala-Thr-Leu-Pro-Val-Phe- PBS and 200 Au ofreaction buffer containing Chl-a was added. Chlase Thr-Arg-Gly-Ile-Tyr-. More than half of the determined activity was assayed after incubation for 2.5 h at 37°C. Bars: open, amino acids are hydrophobic. The N-terminal sequence was preimmune serum; shaded, immune serum. not significantly homologous to sequences appearing in the GenBank/EMBL, Swiss Prot, and Protein Identification tected 25-kDa protein corresponded to enzymatic activity Resource data banks as searched by Genetics Computer detected in the resolved gel (Fig. 2). When heated, the Group software (up to the November 1992 release) (31). immunodetected mass of the Chlase protein was 35 kDa and Ethylene-Induced de Novo Synthesis of Chlase. Degreening traces of two additional proteins with higher molecular of Citrus fruits by ethylene has been associated with an masses of 53 kDa and 60 kDa were observed. Further upsurge of Chlase activity (9, 10). Ethylene enhanced Chlase characterization of the various molecular masses detected is activity 5-fold within 24 h (data not shown) and 12-fold within required. A comparable pattern of several polypeptides was 72 h (Table 2). Chlase was immunodetected in both non- seen in Phaeodactylum (28) and canola (30). heated and heated protein extracts from ethylene-treated Various immunoassays of Chlase activity showed that the fruit. Chlase protein was already detected after 24 h of antibodies recognized the solubilized enzyme. Seventy per- ethylene treatment and increased further up to 7 days (Fig. 5). cent of Chlase activity was immunoprecipitated from the Chlase protein could not be detected in air-treated fruit. solute when serum diluted 1:50 was used. The antibody Recently, it has been reported that all polypeptides whose in capture assay also detected an increase in Chlase activity vivo synthesis increased during ripening of orange flavedo when serum was diluted 1:20 (Fig. 3). In addition, antibody- also increased after ethylene treatment, including an uniden- induced inhibition of Chlase activity using antigen capture tified protein of molecular mass within the range of 25-30 assay revealed that enzyme activity was significantly inhib- kDa, which may correspond to Chlase (32). Our data indicate ited compared to the activity in preimmune serum (Fig. 4). that Citrus Chlase, similar to ceilulase in avocado (33), The cumulative evidence shows that these antibodies were polygalacturonase in tomato (34), and many proteins of specifically directed against the Chlase. unknown function (32), is indeed synthesized de novo after ethylene treatment, and therefore, probably takes part in the 110 ripening process. Inhibition of Ethylene-Induced Chlase by GA and BA. Both 100 GA and BA opposed the ethylene-induced loss of Chl (Table $ 90 1 2 3 4 5 6 7 8 kDa UE 80 106 iL) 80 ucis q 70 - 49.5 60 ...... 50 - 27.5 0 2 4 6 8 10 12 -18.5 Serum, ,Al per well FIG. 4. Inhibition ofChlase enzyme activity by Chlase polyclonal FIG. 5. Immunoblot of ethylene-induced de novo synthesis of antibodies. A polyvinylchloride plate was coated with Chlase at 1 Chlase during a 7-day treatment. Total proteins were solubilized from ,.g/ml and the remaining sites for protein binding were blocked by acetone powder with 0.24% Triton X-100 and heated prior to BSA. Serum (50 p1) in various dilutions was added to each well and electrophoresis. Lanes: 1-4, air-treated; 1, harvest day; 2, 2 days; 3, incubated for 2 h at 37°C. The wells were washed with PBS and then 4 days; 4, 7 days; lanes 5-8, ethylene-treated; 5, 1 day; 6, 2 days; 7, 200 p1 ofreaction buffer containing Chl-a was added. Chlase activity 4 days; 8, 7 days. Positions of low-range prestained SDS/PAGE was assayed after incubation for 2.5 h at 37°C. Activity in the standards (Bio-Rad) are indicated. The SDS/PAGE gel was blotted presence of preimmune serum represented 100% Chlase activity. onto nitrocellulose and visualized. Downloaded by guest on September 29, 2021 Plant Biology: Trebitsh et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9445

1 2 3 4 5 6 7 kDa We thank Dr. Orith Leitner and Mr. Mordechai Gabbay (Antibody __§,...... Laboratory, Biological Services, Weizmann Institute, Rehovot) for 1,,. -106 their assistance in raising antibodies. N-terminal sequencing was ,.j, .. - 80 | il! r,, carried out by Dr. Ariel Gaathon (Bletterman Laboratory for Mac- 11|, .''., romolecular Research in the Faculty of Medicine, The Hebrew '''''. - 49.5 t;.. University of Jerusalem). The expert technical assistance of Ms. "': .' _ 32.5 Naomi Agur, Mr. David Galili, and Ms. Zehava Mor is gratefully tsi acknowledged. We thank Dr. Doron Holland (Volcani Center, Ag- ,iRi:,! ... ., - 27.5 ricultural Research Organization, Bet-Dagan) for valuable discus- | ,^..'..:' .. sions during preparation of the manuscript. M...... I i. ,b '. .. : .18.5 ..w., 1. Hendry, G. A. F., Houghton, J. D. & Brown, S. B. (1987) New Phytol. 107, 255-302. FIG. 6. Immunoblot of Citrus peel proteins. Total proteins were 2. Willstatter, R. & Stoll, A. (1913) Untersuchungen uber Chlorophyll solubilized from acetone powder by 0.24% Triton X-100 and heated (Springer, Berlin). prior to electrophoresis. Lanes: 2-4, air-treated (72 h); 1, harvest 3. Shimokawa, K., Hashizume, A. & Shioi, Y. (1990) Plant Physiol. day; 2, air; 3, GA at 25 ppm; 4, BA at 25 ppm; lanes 5-7, Suppl. 93, S150 (abstr.). ethylene-treated (72 h, 80 ppm); 5, ethylene; 6, GA at 25 ppm; 7, BA 4. Rise, M. & Goldschmidt, E. E. (1990) Plant Sci. 71, 147-151. at 25 ppm. Positions of low-range prestained SDS/PAGE standards 5. Matile, P., Duggelin, T., Schellenberg, M., Rentsch, D., Bortlik, K., (Bio-Rad) are indicated. The SDS/PAGE gel was blotted onto Peisker, C. & Thomas, H. (1989) Plant Physiol. Biochem. 27, nitrocellulose and visualized. 595-604. 6. Garcia-Luis, A., Fornes, F. & Guardiola, J. L. (1986) Physiol. Plant. 68, 271-274. 2) as reported (6, 7, 11, 35). Similarly, GA and BA counter- 7. Goldschmidt, E. E., Aharoni, Y., Eilati, S. K., Riov, J. & Monse- acted the ethylene-induced increase on Chlase activity (Table lise, S. P. (1977) Plant Physiol. 59, 193-195. 2). Immunoblots of crude acetone extracts clearly showed 8. Purvis, A. C. & Barmore, C. R. (1981) Plant Physiol. 68, 854-856. that whereas ethylene markedly increased Chlase protein, 9. Shimokawa, K., Shimade, S. & Yaeo, K. (1978) Sci. Hortic. to a extent A (Amsterdam) 8, 129-135. GA, and lesser BA, reduced this effect (Fig. 6). 10. Amir-Shapira, D., Goldschmidt, E. E. & Altman, A. (1987) Proc. similar pattern was observed in air-treated fruit; low levels of Natl. Acad. Sci. USA 84, 1901-1905. Chlase protein were detected in control and BA-treated fruit 11. Eilati, S. K. & Goldschmidt, E. E. (1969) Experientia 25, 209-210. but Chlase protein could not be detected in GA-treated fruit. 12. Bacon, M. F. & Holden, M. (1970) Phytochemistry 9, 115-125. It should be noted that BA acted somewhat differently than 13. Fernandez-Lopez, J. A., Almela, L., Almansa, M. S. & Lopez- Roca, J. M. (1992) Phytochemistry 31, 447-449. GA. Although BA diminished the ethylene-induced loss of 14. Moll, W. A. W. & Stegwee, D. (1978) Planta 140, 75-80. Chl, it did not reduce Chlase activity. This observation is 15. Tamai, H., Shioi, Y. & Sasa, T. (1979) Plant Cell Physiol. 20, supported by the abundance of Chlase protein in BA-treated 1141-1145. fruit. Differences in the response of senescent pigment 16. Terpstra, W. (1978) Physiol. Plant. 44, 329-334. changes to GA and BA were also described by Garcia-Luis 17. Shimokawa, K. (1982) Phytochemistry 21, 543-545. 18. McFeeters, R. F., Chichester, C. 0. & Whitaker, J. R. (1971) Plant et al. (6). The mechanisms underlying the opposing effect of Physiol. 47, 609-618. ethylene and the senescence-delaying regulators, GA and 19. Bergmeyer, H. U. (1974) Methods ofEnzymatic Analysis (Academ- BA, are presently unknown, but progress in the elucidation ic, New York), 2nd Ed., Vol. 1, pp. 174-176. of hormonal antagonisms has been reported in other plant 20. Moran, R. & Porath, D. (1980) Plant Physiol. 65, 478-479. systems (36, 37). 21. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY). 22. Smith, D. E. & Fisher, P. A. (1984) J. Cell Biol. 99, 20-28. CONCLUSIONS 23. Young, P. R. (1989) J. Immunol. Methods 121, 295-296. 24. Kuroki, M., Shioi, Y. & Sasa, T. (1981) Plant Cell Physiol. 22, Pigment changes are an integral part of the senescence 717-725. syndrome in all chlorophyllous plant tissues. The purification 25. Shioi, Y. & Sasa, T. (1986) Methods Enzymol. 123, 421-427. 26. Amir-Shapira, D., Goldschmidt, E. E. & Altman, A. (1986) Plant of Chlase to homogeneity and its immunodetection in plant Sci. 43, 201-206. tissues are important steps toward elucidation of the physi- 27. Tanaka, K., Kakuno, T., Yamashita, J. & Horio, T. (1982) J. ological significance of Chlase in Chl catabolism in plants. Biochem. (Tokyo) 92, 1763-1773. The N-terminal sequence of the Chlase protein will enable 28. Lambers, J. W. J., Velthuis, H. W. & Terpstra, W. (1985) Biochim. further study of the molecular control of Chlase biogenesis. Biophys. Acta 831, 213-224. 29. Reynolds, J. A. & Tanford, C. J. (1970) J. Biol. Chem. 245, 5161- The involvement of molecular genetic mechanisms in eth- 5165. ylene-induced ripening phenomena has repeatedly been dem- 30. Johnson-Flanagan, A. M. & McLachlan, G. (1990) Physiol. Plant. onstrated in recent years, although the pigment changes have 80, 460-466. not been closely examined so far (38, 39). The ethylene- 31. Devereux, G., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids induced increase in Chlase activity (Fig. 5) has been shown Res. 12, 387-395. 32. Alonso, J. M., Garcia-Martinez, J. L. & Chamarro, J. (1992) Phys- in the present study to involve de novo synthesis of the iol. Plant. 85, 147-156. Chlase protein, but further studies are required to determine 33. Christoffersen, R. E., Warm, E. & Laties, G. C. (1982) Planta 155, the ethylene-dependent step in the molecular control of 52-57. Chlase biosynthesis. 34. DeliaPenna, D., Alexander, D. C. & Bennett, A. B. (1986) Proc. In a recent review, Theologis et al. (39) distinguished Natl. Acad. Sci. USA 83, 6420-6424. 35. Dostal, H. C. & Leopold, A. C. (1967) Science 158, 1579-1580. between ripening processes that are transcriptionally ethyl- 36. Jacobsen, J. V. & Gubler, F. (1992) in Progress in Plant Growth ene-dependent and others that are not. Observations on Regulation, eds. Karssen, C. M., Van Loon, L. C. & Vreugdnhil, antisense tomato mutants suggest that Chl breakdown may D. (Kluwer, Dordrecht, The Netherlands), pp. 116-127. not be entirely ethylene-dependent (39). It should be noted 37. Tucker, M. L., Sexton, R., del Campillo, E. & Lewis, L. N. (1988) that Chlase and are detectable Plant Physiol. 88, 1257-1262. protein activity throughout 38. Gray, J., Picton, S., Shabbeer, J., Schuch, W. & Grierson, D. (1992) leaf and fruit ontogenesis, suggesting a different type of Plant Mol. Biol. 19, 69-87. control from that of other ripening-related enzymes that are 39. Theologis, A., Zarembinski, T. I., Oeller, P. W., Liang, X. & Abel, completely absent prior to the onset of ripening. S. (1992) Plant Physiol. 100, 549-551. Downloaded by guest on September 29, 2021