Immunocytolocalization of 1-aminocyclopropane-1 ... - Springer Link

Planta (1994)192:453-460

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9 Springer-Verlag 1994

Immunocytolocalization of 1-aminocyclopropane-l-carboxylic acid oxidase in tomato and apple fruit Cesar Rombaldi 1, Jean-Marc Leli~vre 2, Alain Latch~ ~, Michel Petitprez ~, Mondher Bouzayen 1, Jean-Claude Pech 1 J ENSAT, 145, avenue de Muret, F-31076 Toulouse Cedex, France 2 INRA, Station de Technologic, F-84140 Montfavet Cedex, France Received: 27 July 1993 / Accepted: 22 October 1993

Abstract. The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus • domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed. Key words: 1-Aminocyclopropane-l-carboxylic acid oxidase (immunolocalization) - Fruit ripening Lycopersicon (fruit) - Malus (fruit)

Abbreviations: ACC = 1-aminocyclopropane-carboxylic acid; FP = fusion protein; IgG = immunoglobulin G; PEPCase = phosphoenolpyruvate carboxylase; SP = synthetic peptide; TBS = Tris-buffered saline Correspondence to: J.C. Pech; FAX: (33) 61 42 30 29

Introduction The last step of the biosynthesis of the plant hormone ethylene is the conversion of 1-aminocyclopropane-carboxylic acid (ACC) to ethylene (Yang and Hoffman 1984; Kende 1993). The enzyme catalyzing this conversion, ACC oxidase, was isolated recently thanks to a reversebiochemistry approach in which the gene was discovered first. An antisense gene constructed from the ripening-related clone, pTOM13, reduced ethylene production and ACC-oxidase activity in transgenic tomato plants (Hamilton et al. 1990). This clone's identity as ACC oxidase was confirmed by functional expression in yeast (Hamilton et al. 1991) and in Xenopus oocytes (Spanu et al. 1991). For activity, ACC oxidase required iron (Bouzayen et al. 1991) and ascorbate as essential cofactots and the active enzyme was recovered in vitro for the first time from melon fruit (Ververidis and John 1991). Subsequently, it was isolated from apple (FernandezMaculet and Yang 1992) and avocado fruits (McGarvey and Christoffersen 1992). More recently, it was purified to homogeneity and antibodies were raised (Dong et al. 1992; Dupille et al. 1992, 1993). The subcellular localisation of the enzyme is still not clearly established. Earlier experiments had suggested that ACC oxidase was associated with membranes because of its inhibition by membrane-interacting compounds, osmotic shock and cell disruption (Imaseki and Watanabe 1978; Apelbaum et al. 1981; Mayne and Kende 1986). Plant protoplasts, even though losing most of the ethylene-forming capacity of the whole cells, retained some authentic ACC-oxidase activity. Small amounts of this activity were recovered in isolated vacuoles or tonoplast vesicles (Guy and Kende 1984; Porter et al. 1986; Mitchell et al. 1988). However, Peck et al. (1992) found that intact cells and protoplasts of transformed yeast expressing ACC oxidase contained equal amounts of the protein whereas vacuoles did not contain any detectable levels. By generating a gradient of specific radioactivity of ACC between cell compartments, Bouzayen et al. (1990) showed that ethylene originated from both apoplasmic and intracellular ACC. Since anti-

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C. Rombaldi et al.: Immunocytolocalization of ACC oxidase

b o d i e s a r e n o w a v a i l a b l e , it has b e c o m e p o s s i b l e to add r e s s t h e s u b c e l l u l a r l o c a l i z a t i o n of t h e e n z y m e u s i n g dir e c t i m m u n o c y t o l o g y m e t h o d s . In this p a p e r , we r e p o r t o n t h e i m m u n o c y t o l o c a l i z a t i o n of A C C o x i d a s e in n o r m a l a n d p T O M 13 a n t i s e n s e t o m a t o e s u s i n g t w o t y p e s o f antibody and a variety of immunostaining methods associated with observations by both light and electron microscopy.

Materials and methods Material. Tomato (Lycopersicon esculentum Mill. cv. Prisca) fruits were harvested at various stages of ripening in a commercial field near Toulouse (France). Tomato fruit of the Alisa Craig cultivar transformed with ACC-oxidase antisense gene and their untransformed control plants were kindly provided by Prof. D. Grierson (University of Nottingham, School of Agriculture, Sutton Bonington, UK). Apple (Malus • domestica Borkh. cv. Golden delicious) fruit were harvested in mid-September in a commercial orchard near Toulouse (France) and ripened at 20~ Gel electrophoresis and immunoblotting. Proteins were extracted by the trichloroacetic acid (TCA) method described by Meyer et al. (1988). Proteins were separated by SDS-PAGE according to Laemmli (1970), using a Mini Protean II apparatus (Bio-Rad, Richmond, Cal., USA). The molecular-mass markers used were lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), bovine carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa) and phosphorylase b (97.4 kDa). Electrotransfer of polypeptides to a nitrocellulose membrane (using a Mini TransBlot; Bio-Rad) and immunoblotting were performed as described previously (Dupille et al. 1993). Before immunostaining, polypeptides were stained with Ponceau S (Harlow and Lane 1988). Protein concentrations were determined according to Bradford (1976) using the Bio-Rad reagent and bovine serum albumin (BSA) as standard. Synthesis of the peptide and preparation of a fusion protein in E. coli. In order to predict the location of potential continuous/sequential epitopes in the amino-acid sequence derived from pRC13, a reconstruction of the original pTOM13 c D N A (Holdsworth et al. 1987; Hamilton et al. 1991), stretches of amino-acid residues corresponding to hydrophilic, flexible and exposed regions were calculated according to the three distinct methods described by Parker et al. (1986), Karplus and Schulz (1985) and Emini et al. (1985), respectively. Among the twelve predicted stretches, corresponding to putative turns or loops containing hydrophilic/flexible/exposed amino-acid residues, the synthetic peptide (SP) 266VEKEAEESTQVY-277 was selected as an antigen for production of polyclonal antibodies. The chemical synthesis of the peptide, its coupling to keyhole-limpet haemocyanin (KLH; Calbiochem, San Diego, Cat., USA) and the immunization schedule were performed as described by Gazin et al. (1986). A fusion protein was generated in E. coli as previously described (Dupille et al. 1993) using the pRC13 c D N A clone. Polyclonal antibodies were prepared by injecting into rabbits the fusion polypeptide purified to homogeneity together with the polyacrylamide gel used in the last step of purification (Meyer et al. 1988).

PuriJieation of antibodies. The antibodies were purified in a two-step procedure. First, the immunoglobulins G (IgGs) were isolated by passing the serum (100 lal diluted to 1 ml of TBS1 medium consisting of 25 mM Tris-HC1, pH 7.6, 35 mM NaC1 and 0.02%, w/v, NAN3) through a Sephadex G-25 molecular sieve (Pharmacia, Uppsala, Sweeden) and a diethylaminoethyl (DEAE)-Trisacryl M anionexchange column (IBF, Villeneuve-la-Garenne, France) successively, according to Fourcart et al. (1982). The IgGs were further purified by affinity chromatography following the method described by

Fig. IA-C. Specificity of ACC-oxidase antibodies. Immunodetection was performed after transfer to nitrocellulose of the following protein extracts separated by SDS-PAGE: 3 lag of proteins from pRC13-transformed E. coli (lanes 1, 3, 5), 10 lag of proteins from breaker tomato fruit (lanes 2, 4; 6 8; 10, 11) and 10 lag of proteins from pTOM13 antisense tomato fruit (lane 9). A Immunodetection with SP antibodies used as crude serum (lanes 1, 2) or after affinity purification on SP columns (lanes 3, 4); B Immunodetection with FP antibodies used as crude serum (lanes 5, 6) or after affinity purification on SP (lane 7) or FP columns (lanes 8, 9); C Immunodetection with FP antibodies pre-absorbed with FP antigen (lane 10) or with pre-immune serum (lane 11). Sizes of the marker proteins and the 35-kDa antigen ( ~ ) are shown on the left side

Harlow and Lane (1988). Briefly, the SP or the fusion protein (FP) were coupled to activated CN Br-Sepbarose 4B (Pharmacia). The IgGs recovered from the DEAE-Trisacryl M column were concentrated to 100 lal, adjusted to 150 mM NaC1, and applied to a 2.5-ml affinity column equilibrated with TBS2 medium (TBS1 containing 150 mM NaCI instead of 35 mM). The fixation of antibodies onto the column was performed over a 16-h period at room temperature, by flow-through recycling, in a closed circuit of 2 ml of TBS2 medium containing the antibodies at a flow rate of 0.1 ml.min ~. After washing the column with TBS2 medium to remove unbound IgGs, specific antibodies were eluted with a medium containing 100 mM glycine and 150 mNaC1 M adjusted to pH 2.8. The elution of purified antibodies was monitored with a 280-nm UV recorder. Fractions containing the antibodies were immediately adjusted to pH 7.6 with 200 mM Tris base, pooled and concentrated to the initial volume (100 lal) using Centriprep 10 and Centricon 10 filters (Amicon, Beverly, Mass., USA), successively. Table 1 summarizes

Fig. 2A-L. Immunocytolocalization of ACC oxidase in tomato and apple fruit. A E Immunofluorescence labeling of tomato cells (Prisca cv.). F Immunofluorescence labeling of apple cells (Golden delicious cv.). G-L Silver-enhanced immunogold labeling of tomato cells (Alisa Craig cv). Semithin sections of breaker tomato fruit were treated with pre-immune serum (A), SP antibodies purified on an SP-affinity column (B, arrow indicates a plasmolyzed cell), FP antibodies purified on an SP-affinity column (C), FP antibodies purified on an FP-affinity column (D) and Calcofluor white (E). D and E show the same group of cells. Semithin sections of climacteric apple fruit were treated with FP antibodies purified on an FP-affinity column (F). Semithin sections of breaker tomato fruit were treated with FP antibodies purified on an FP-affinity column (G), FP antibodies pre-absorbed with FP antigen (H), Toluidine blue (I), FP antibodies purified on an FP-affinity column viewed by epifluorescence (J). I and J represent enlargements of cells delimitated by the square in G. Semithin sections of pTOM13 antisense tomato fruit were treated with FP antibodies purified on an FP-affinity column (L). c, cytoplasm; w, cell wall; v, vacuole. A, B, C, F bars = 110 lam; G, H bars = 120 lam; D, E bars = 60 lam; I, J bars = 40 lam; L b a r - 2 0 0 l a m . A , B , C , G , H , L x 2 5 ; D , E , F x 5 0 ; I , J • 100


455

456 the concentration of proteins in SP and FP antibodies at various degrees of purification and their dilution for use in immunoblot and immunocytology.

Tissue processing. For immunocytological observations by both light and electron microscopy, small pieces (2 mm 3) of tomato fruit pericarp or apple fruit cortex were taken from the equatorial region and fixed in freshly prepared 2% (w/v) p-paraformaldehyde and 0.2% (v/v) glutaraldehyde in 75 mM Na-cacodylate buffer (pH 7.2, 741 mosmol) at 4~ for 2 h. Then samples were rinsed twice with 10 ml of 200 mM Na-cacodylate buffer (pH 7.2, 371 mosmol) for 10 min at 4~ Tissues were dehydrated in a graded series of ethanol in water (w/v) at decreasing temperatures (4, -10, -20, - 3 5 and -35~ for 1 h for each ethanol concentration. The tissues were finally embedded in LR White resin and polymerized under UV at -20~ as described by VandenBosch (1991). Some samples were fixed in 3% (w/v) paraformaldehyde and 1% (v/v) glutaraldehyde in 50 mM Na-cacodylate (pH 7.2) for 2 h at 4~ After washing twice for 20 min with 200 mM Na-cacodylate (pH 7.2) the tissues were post-fixed with 1% osmium tetroxide for 1 h at room temperature and rinsed in 200 mM cacodylate (pH 7.2). Dehydration and embedding in LR White resin were performed at room temperature and polymerisation was at 50~ for 24 h according to VandenBosch (1991). Light-microscopical immunocytochemistry. Semi-thin sections were cut, dried onto poly-L lysine-coated glass slides, washed for 30 min with 0.5 M NH4C1, and rinsed twice in the following TBST medium: 20mM Tris-HC1 (pH 7.2), 150mM NaCI, 0.05% (v/v) Tween 20, 0.05% (v/v) Triton X-100, 0.02% (w/v) NaN 3, 0.2% (w/v) BSA. Sections were incubated for 2 h in the blocking medium made of 10% (v/v) normal goat serum (Amersham, Little Chalfond, Buckinghamshire, UK) in TBST and then excess serum was removed. Sections were incubated for 4 h in the presence of the primary antibodies raised either against SP or FP. For immunofluorescence labeling, sections were rinsed four times with modified TBST medium (pH 8.2 instead of 7.2) and covered for 1.5 h with fluorescein-isothiocyanate-linked (FITC) goat anti-rabbit IgG (Amersham) diluted 1: 100 in TBST (pH 8.2). After successive washes with TBST (pH 8.2), the sections were treated as follows: incubation for 5 min in 0.01% Calcofluor white (Sigma) in TBST (pH 8.2); washing twice in distilled water; covering with antifading solution (1 mg-ml 1 1,4-phenylenediamine in 90% (v/v) glycerol in TBST minus Triton, Tween and BSA); mounting and viewing in a Leitz (Wetzlar, Germany) fluorescence microscope with excitation and barrier filters BP 450490 and BP 520-525 nm for FITC and BP 340-390 and LP 460 nm for Calcofluor white. For immunogotd labeling, sections were incubated for 45 min in the presence of a complex between 10-nm colloidal gold and goat anti-rabbbit IgG diluted in 1: 50 TBST. Then sections were treated as follows: washing three times in TBST and three times more with distilled water for 10 min, removal of excess water, covering with gold labeling enhancer (Inten-SE, Amersham) for 10 min, and washing with distilled water for 1 h. Sections were observed with a Leitz photomicroscope with normal bright-field imaging and with epipolarized illumination. Some sections were stained with basic Toluidine blue 2.5% (w/v) in sodium borate for 5 min and viewed under normal bright-field imaging. Transmision-electron-microscopical immunocytochemistry. Silver-togold thin sections mounted onto gold grids were blocked for 1 h on 200-gl drops of TBST containing 10% (v/v) of normal goat serum and then transferred and incubated for 2 h on 200-~d drops of TBST containing the purified FP antibodies in TBST. Grids were rinsed with TBST and incubated for 1 h on 200-gl drops of 10- or 15-nm colloidal-gold-conjugated goat antiserum to rabbit immunoglobulins diluted 1:50 in TBST. After three washings with TBS and three rinsings with distilled water, grids were counter-stained with 2.5% (w/v) uranyl acetate in 50% ethanol. The thin sections were viewed in a Philips (Eindhoven, The Netherlands) H300 electron microscope at 75 kV. The immunolabeling of sections of osmoticated

C. Rombaldi et al.: Immunocytolocalization of ACC oxidase tissues embedded in LR White resin required the removal of osmium with 500 mM Na-metaperiodate for 30 min (Craig and Miller 1984). Then grids were rinsed with distilled water and treated for immunogold labeling as above.

Immunocytochemical controls. Specificity of labeling was assessed by several control tests including incubation of tissue sections: (i) with pre-immune serum diluted 1:100; (ii) with pre-absorbed FP antibodies (purified antibodies were incubated in the presence of FP antigen at 30 laM); (iii) in the absence of either primary or secondary antibodies; (iv) in the presence of antibodies raised against phosphoenolpyruvate carboxylase (PEPCase) diluted 1: 500 (Vidal et al. 1980).

Results Specificity o f antibodies. Three peptides considered as suitable epitopes were synthesized chemically a n d used separately for raising antibodies. The serum raised against the peptide 2 6 6 - V E K E A E E S T Q V Y - 2 7 7 showed the highest activity a n d was retained for s u b s e q u e n t work. A n o t h e r type of a n t i b o d y raised against A C C - o x i dase F P had very high t i t r a t i o n since the crude serum or the c o r r e s p o n d i n g IgGs could be used at a 1:3000 dilution (Table 1). The specificity of crude or purified antisera raised against either the synthetic peptide (SP), or the fusion p r o t e i n (FP) was tested o n i m m u n o b l o t s after transfer to nitrocellulose m e m b r a n e s of p r o t e i n extracts separated o n S D S - P A G E gels as illustrated in Fig. 1A a n d 1B, respectively. P r e - a b s o r b e d F P a n t i b o d i e s a n d p r e - i m m u n e serum were used as c o n t r o l (Fig. 1C). The SP crude serum recognized several polypeptides in p R C 1 3 t r a n s f o r m e d E. coli (lane 1) i n c l u d i n g a 3 5 - k D a b a n d . I n p r o t e i n extracts of b r e a k e r tomatoes, the 35-kDa polypeptide was p r e d o m i n a n t l y recognized b u t several non-specific b a n d s were present m a i n l y in the u p p e r part of the gel (lane 2). The very faint non-specific b a n d s observed o n i m m u n o b l o t s o b t a i n e d with the crude serum were completely absent when a n t i b o d i e s were purified by affinity c h r o m a t o g r a p h y . After purification, the SP antibodies recognized a single b a n d in b o t h t r a n s f o r m e d E. coli (lane 3) a n d b r e a k e r t o m a t o e s (lane 4) extracts. The F P crude serum showed a single b a n d in t r a n s f o r m e d E. coli (lane 5) a n d a m a j o r b a n d of 35 k D a in b r e a k e r t o m a toes, with some very faint non-specific staining in the higher-molecular-weight range (lane 6). After affinity purification o n c o l u m n s c o n t a i n i n g either the SP (lane 7) or the F P (lane 8), the a n t i b o d i e s recognized exclusively the 35 k D a polypeptide in total p r o t e i n extracts of b r e a k e r

Fig. 3A-F. Transmission electron micrographs showing localization of ACC oxidase in tomato fruit. Tissues were fixed using 0.2% glutaraldehyde without post-fixation with osmium (A-C) or 2% glutaraldehyde plus post-fixation with osmium (D-F) as described in Material and methods. Ultrathin sections were treated with FP antibodies purifed on an FP-affinity column (A, B, D), with FP antibodies pre-absorbed with FP antigen (C, E) or with PEPCase antibodies (F). c, cytoplasm; w, cell wall, v, vacuole; is, intracellular space; p, plasma membrane; g, Golgi apparatus; er, endoplasmic reticulum; m, mitochondrion; re, vesicle. A, B, D, E x 20 000, bars =0.751am;C,F x 30000, bars = 0 . 5 0 g m


457

458


Table 1. Characteristics of the different types of polyclonal antibody used for the cytolocalization of ACC oxidase in fruits. Antibodies were purified after isolation of IgG by DEAE chromatography on antigen affinity columns as described in Material and methods Method

Type of antibody

Concentration of proteins and dilution of antibodies used in immunodetection Synthetic peptide (SP)

Crude serum DEAE chromatography Total IgG Antigen aff• column containing: synthetic peptide SP Purified antibodies fusion protein FP Purified antibodies a

Fusion protein (FP)

Proteins (lag ~tl- ~)

Dilution

Proteins (~tg.~tl ~)

Dilution

35 9

1: 100 1: 100

50 23

1:3000 1:3000

40.10 -3 a -

1:30 -

18' 10-3" 32" 10 3a

1:20 1:30

These values represent a fraction of specific IgGs corresponding to the retention capacity of the affinity column

normal tomatoes. In breaker pTOM13 antisense tomatoes, a very weak band was observed at 35 kDa (lane 9). Control immunoblots performed using the FP antibodies treated with an excess of the corresponding antigen (lane 10) and the pre-immune serum (lane 11) did not exhibit any labeling.

lmmunocytolocalization of ACC oxidase. We first examined the subcellular localization of ACC oxidase under light microscopy by immunofluorescence and immunogold labeling (Fig. 2). Breaker tomato cells treated with a pre-immune serum exhibited only a pale-yellow autofluorescence (Fig. 2A), while those treated with SP (Fig. 2B) or FP antibodies (Fig. 2C), purified on an SP affinity column, were brightly and uniformly labeled in yellow-green at the periphery of all cells. The plasma membrane and cytoplasm showed weak fluorescence as clearly illustrated in plasmolyzed cells (arrow at the upper-right corner of Fig. 2B). Immunofluorescence labeling (Fig. 2D) and Calcofluor-white staining, performed on the same semi-thin section (Fig. 2E), were exactly superimposable, indicating that ACC-oxidase protein was mainly localized at the cell wall. Climacteric apple cells displayed the same pattern (Fig. 2F) with occasional labeling in the cytosol (arrows). Labeling with the silver-enhanced immunogold technique of tomato cells incubated in the presence of FP antibodies purified on an FP affinity column showed that silver grains were concentrated at the periphery of the cell (Fig. 2G). Cells delimitated by the square in Fig. 2G were examined at a higher enlargement after staining with basic Toluidine blue (Fig. 2I) and silver-enhanced immunogold viewed by epifluorescence microscopy (Fig. 2J). By comparing Fig. 2I and 2J, it appears that ACC oxidase was located primarily at the cell wall, the thin cytosolic compartment visible with Toluidine staining being almost devoid of immunogold labeling. Semithin sections incubated in the presence of pre-absorbed FP antibodies (Fig. 2H) or in the absence of primary or secondary antibodies (not shown) were essentially free of labeling in all regions of the cells. Most interestingly, cells of pTOM13 antisense tomatoes in which the expression of the ACC-oxidase gene was greatly reduced, exhibited very weak labeling when incubated in the presence of

purified FP antibodies (Fig. 2L), while untransformed tomatoes of the same variety were strongly labeled (Fig. 2G and J). Crude FP antibodies gave the same pattern of labeling as purified antibodies (not shown). Ultrathin sections were labeled using the immunogold technique and viewed by electron microscopy (Fig. 3). Figure 3(A, B) shows that gold labeling resulting from incubation with purified FP antibodies was mostly, if not all, located in all regions of the cell wall. Antigen to ACC oxidase was almost absent from the cytoplasm, vacuole, and intercellular space (Fig. 3A, B). Occasionally, a few randomly distributed gold particles were located over the cytoplasm. Control cells incubated in the presence of preabsorbed FP antibodies exhibited no labeling (Fig. 3C). Because the presence of osmium and high concentrations of glutaraldehyde have been shown, in some cases, to alter the antigenicity of the target protein, the tissues were fixed with low concentrations of glutaraldehyde (0.2%) and without post-fixation with osmium. In these conditions, the endomembrane system was not well defined though the structure of the cytosol could be easily distinguished with its main organelles and its large number of vesicles characteristic of ripening fruits. Fixation with 1% glutaraldehyde and post-fixation with osmium allowed better preservation of the ultrastructure (Fig. 3D, E, F) without strongly affecting antigenicity. Gold particles were again observed almost exclusively in the cell wall (Fig. 3D). No labeling was present when preabsorbed FP antibodies were used (Fig. 3E) or when primary or secondary antibodies were omitted (data not shown). Antibodies raised against PEPCase, a cytosolic marker enzyme, gave intense labeling in the cytosol (Fig. 3F), showing that the methods used for the preparation of tissues preserved the subcellular compartmentation of this antigen in the cell.

Discussion

The immunocytolabeling studies presented here demonstrate that the antigens representing ACC oxidase are located in the cell wall of tomato and apple fruits. Two kinds of antigen were used to raise specific antibodies against ACC oxidase: (i) a synthetic peptide of 12

C. Rombaldi et al.: Immunocytolocalization of ACC oxidase residues covalently linked to a carrier protein and (ii) a fusion protein, denaturated by SDS-PAGE, corresponding to the complete amino-acid sequence encoded by pRC13 c D N A clone (Dupille et al. 1993). The FP conferred genuine ACC-oxidase activity to transformed E. coli cells (data not shown) and was equally recognized as a single band of 35 kDa by the antigen-affinity-purified antibodies raised against FP and SP. The high affinity and specificity of the antibodies towards ACC oxidase is attested by their ability to recognize ACC oxidase of different origins. Both FP and SP antibodies give a positive cross-reaction with: (i) the active FP from which they originate, (ii) the in-vitro translation products of pMEL1, a c D N A clone encoding ACC oxidase in melon (Balagu~ et al. 1993), (iii) a 35-kDa polypeptide in tomato protein extracts, and (iv) the native apple fruit ACC oxidase purifed to homogeneity (Dupille et al. 1993). In addition, breaker tomatoes from transgenic plants with an antisense pTOM13 gene show only very faint immunostaining, demonstrating that our antibodies do not recognize proteins structurally unrelated to ACC oxidase. Furthermore, the method used for tissue processing and immunostaining are reliable since PEPCase antibodies, used as a control, were able to recognize their antigen specifically in the cytosol, indicating that cell compartmentation was preserved by the fixation techniques employed. Finally, changing conditions for tissue fixation and resin inclusion did not change the labeling pattern of either PEPCase or ACC oxidase. All the data presented in this work suggest that the antigen corresponding to ACC oxidase is located primarily in the cell wall of ripening fruits. This result is consistent with a previous demonstration that ethylene could be synthesized from apoplasc ACC in plant cells grown in vitro (Bouzayen et al. 1990), and could be an explanation for the low capacity of ethylene production exhibited by isolated protoplasts (Porter et al. 1986). Predictions made from the analysis of c D N A clones encoding ACC oxidase do not support the targeting of the protein at the cell wall. None of the deduced amino-acid sequences of ACC oxidases isolated from various plant tissues contain an N-terminal consensus signal for secretion through the endoplasmic-reticulum pathway (Balagu6 et al. 1993). In tomato, three genes, which are probably differentially regulated, have been found to encode ACC oxidase, but none of the deduced sequences contains a transmembrane-spanning domain usually necessary for protein secretion (Bouzayen et al. 1993). Protein-sequence analysis revealed at least one potential glycosylation site conserved in melon, tomato, apple, carnation and located at amino acids 99-101 (Balagu6 et al. 1993). However, several experiments have shown that the protein has no Nlinked glycosidic residue (Peck et al. 1992; Dupille et al. 1993). The present findings raise the question of the targeting mechanism of ACC oxidase to the cell wall. It may be that a non-conventional signal sequence is involved in the secretion of the protein as shown in yeast for the export of cytosolic proteins into the vacuole (Chiang and Schekman 1991). Another example is the ]3-subunit of interleukin-1, an animal cytokin, which, though lacking a conventional transit peptide and glycosylation,

459 is secreted without going through the endoplasmic reticulum pathway (Rubartelli et al. 1990). In yeast transformed with a c D N A homolog of pRC13, Peck et al. (1992) found that 84% of ACC-oxidase activity was recovered in protoplasts but was absent from the vacuole. Since a consistent amount of ACC oxidase was associated with particulate fractions the authors hypothesized that a protein-protein interaction occurs involving a putative leucine zipper motif. In spite of the absence of a typical transit peptide, the conserved region between amino acids 120 and 145 has the potential to form an amphipatic 0~-helix which would be long enough to span the membrane lipid layer (Kende 1993), and contains multiple leucine residues which may be involved in a leucine zipper formation. Furthermore, a short region of 21 amino acids near the C-terminus, predicted by computer-assisted structural analysis to be an alpha helix, indicates the possibility of interactions of ACC oxidase with membranes (Balagu6 et al. 1993). However, the presence of the protein in the cell wall demonstrated in this study, i.e. not closely associated to the plasma membrane, does not support this possibility. Only very little and infrequent staining was observed in the cytosol while previous data had shown that ethylene could be generated from both apoplasmic and intracellular ACC (Bouzayen et al. 1990). Also, plant protoplasts, even though loosing most of the original activity of the whole cell remain capable of generating ethylene from ACC (Porter et al. 1986; Bouzayen et al. 1990). These data strongly suggest that at least part of the enzyme is present in the internal compartments of the cell. The faint and inconstant immunolabeling of this putative intracellular (cytosolic) ACC oxidase may be due to a reduced accessibility to or to a weak recognition by the antibodies. However, the PEPCase antigen, used in this study as a cytosolic marker could always be detected on the same tissue section. Furthermore, the leakage of a cytosolic ACC-oxidase antigen during tissue processing seems unlikely since the PEPCase antigen remained located exclusively in the cytosol. In conclusion, in ripening tomato and apple fruit, the antigen corresponding to ACC oxidase appears to be located primarily in the cell wall and therefore is not membrane-bound. Such a massive presence of the ACC oxidase in the apoplastic space can be interpreted in two different ways. Either the protein indeed acts as ACC oxidase in the cell wall space, or the protein has, in this compartment, a different hitherto unknown function. These latter hypotheses, as well as the mechanisms underlying the targeting to the cell wall, remain to be elucidated. This work represents some of the research submitted by C. Rombaldi in partial fulfillment of the requirements for the Doctorate degree. It was partly carried out at the Laboratoire de Biologie Mol6culaire des Relations Plantes-Microorganismes (CNRS/INRA, Toulouse-Auzeville, France) with generous assistance and advice from Dr. G. Truchet and F. de Billy. The authors are grateful to Professor D. Grierson and Dr. A. Hamilton (University of Nottingham, UK) for providing us with pTOM13 antisense tomatoes and the latter for critical reviewing of the manuscript. The PEPCase antibodies were kindly provided by Dr. J.Vidal (CNRS, Orsay,

460 France). This work was supported by the Minist6re de l'Education Nationale (JE 179), the EEC (ECLAIR Grant AGRE 015) and by CAPES-COFECUB (doctoral fellowship to C. Rombaldi).

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