Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L

Abstract. The function of aspartic proteinases (EC3.4.23) present in ¯owers of Cynara species is stillunknown. Cardosin A, as a highly abundant asparticproteinase from Cynara cardunculus L., a relative of theartichoke, is synthesised as a zymogen and subsequentlyundergoes proteolytic processing, yielding the matureand active enzyme. Here we report the study of theexpression and localization of cardosin A, as a ®rstapproach to address the question of its physiologicalrelevance. A polyclonal antibody speci®c for cardosin Awas raised against a synthetic peptide corresponding toan amino acid sequence of the enzyme. This antibodywas used to study the organ-speci®c, tissue-speci®c andsubcellular localization of cardosin A by immunoblot-ting, tissue printing and immunogold electron micros-copy. The results showed that expression of cardosin Ais highly restricted to the pistils, and that the enzymeaccumulates mainly in protein storage vacuoles of thestigmatic papillae. Cardosin A is also present, althoughmuch less abundantly, in the vacuoles of the cells of theepidermis of the style. In view of these results, thepossible physiological roles of cardosin A are discussed,namely an involvement in defense mechanisms or pollen-pistil interaction, as well as in ¯ower senescence.

Key words: Aspartic proteinase ± Cardosin ± Cynara ±Pistil ± Stigmatic papilla ± Storage vacuole

Introduction

Aspartic proteinases (APs; EC 3.4.23; Davies 1990;Takahashi 1995) are widely distributed in the plant

kingdom and have been puri®ed and characterized fromseveral species of monocotyledons (Doi et al. 1980;Belozersky et al. 1989; Sarkkinen et al. 1992) dicotyle-dons (Polanowski et al. 1985; Rodrigo et al. 1989;VerõÂ ssimo et al. 1996) and gymnosperms (Salmia et al.1978; Bourgeois and Malek 1991). In common with mostof the APs from retrovirus, bacteria, yeast, fungi andvertebrates, plant APs are inhibited by pepstatin (anhexapeptide from Streptomyces), have an acid pH opti-mum and preferentially cleave peptide bonds betweenhydrophobic residues (Kervinen et al. 1993; Ramalho-Santos et al. 1996). The amino acid sequences of someplant APs have recently been deduced (Runeberg-Rooset al. 1991; Cordeiro et al. 1994b; Asakura et al. 1995).As compared with APs of mammalian and microbialorigins, cDNA sequences of plant APs contain an extrasegment coding for about 100 amino acids which hasbeen suggested to be involved in the vacuolar targetingof these enzymes (Guruprasad et al. 1994).

The plant APs identi®ed so far are vacuolar orsecreted proteins. The barley AP is present in the vacuole(Runeberg-Roos et al. 1994), the APs from hemp andbuckwheat are associated with intracellular proteinbodies (St. Angelo and Ory 1970; Elpidina et al. 1990)and those from the carnivorous pitcher plants aresecreted into the pitcher ¯uid (Toke s et al. 1974). Littleis known about the biological function of these enzymes.It has been proposed that plant APs are involved instorage and extracellular protein hydrolysis but in mostcases the evidence is still circumstantial. The APs presentin the pitchers of the carnivorous plants are thought totake part in the digestion of trapped insects (Toke s et al.1974). In tomato and tobacco leaves, the APs co-localizewith pathogenesis-related proteins and may regulatetheir biological action (Rodrigo et al. 1989, 1991),whereas in wheat seeds the presence of an AP wasassociated with the hydrolysis of storage proteins(Belozersky et al. 1989). The APs from Arabidopsisand barley have been shown to process 2S storagealbumins and barley lectin precursor in vitro, respec-tively (D'Hondt et al. 1993; Runeberg-Roos et al. 1994).In the case of the barley proteinase, the enzyme

Planta (1997) 203: 204±212

Cardosin A, an abundant aspartic proteinase, accumulates in proteinstorage vacuoles in the stigmatic papillae of Cynara cardunculus L.

Miguel Ramalho-Santos1*, Jose Pissarra2*, Paula Verõ ssimo1, Susana Pereira2,Roberto Salema2, Euclides Pires1, Carlos J. Faro1

1Departamento de BioquõÂmica, Faculdade de Cieà ncias e Tecnologia, Universidade de Coimbra, Apartado 3126, P-3000 Coimbra, Portugal2Instituto de Botaà nica and Centro de Citologia Experimental, Universidade do Porto, P-4100 Porto, Portugal

Received: 10 December 1996 /Accepted: 14 March 1997

*Both authors contributed equally to this work

Abbreviations: AP = aspartic proteinase; EM = electron micros-copy

Correspondence to: C.J. Faro; Fax: 351 (39) 26798;E-mail: [email protected]

co-localizes with barley lectin precursor, suggestingtherefore that it is probably involved in the processingof this protein in vivo (Runeberg-Roos et al. 1994).

Recently, we isolated and characterized two APsfrom fresh ¯owers of the asteraceous plant Cynaracardunculus L. that belongs to the same genus as theartichoke (VerõÂ ssimo et al. 1996). The enzymes werenamed cardosin A and cardosin B, and were shown to bethe products of two di�erent though related genes,which have probably arisen by gene duplication. Bothcardosins are formed by two polypeptide chains withapparent molecular masses of 31 and 15 kDa forcardosin A, and 34 and 14 kDa for cardosin B.Together, they account for the majority of the solubleprotein in mature stigmas, and such an abundance ofproteases in the mature stigmas of a plant is in itselfunexpected. Although cardosin B is less abundant thancardosin A it has a higher proteolytic activity. CardosinB has a broader speci®city than cardosin A (VerõÂ ssimoet al. 1995; Ramalho-Santos et al. 1996) and in view ofthis result it was proposed that while cardosin B maytake part in general protein digestion, cardosin A mayhave a function in a more speci®c and regulated process(Faro et al. 1995).

Although the cardosins are well known milk-clottingenzymes (Faro et al. 1992; Macedo et al. 1993), theirbiological functions remain to be elucidated. As a ®rstapproach to address this question we have studied theorgan-speci®c, tissue-speci®c and cytological localizationof cardosin A by immunoblotting, tissue printing andimmunogold electron microscopy (EM). In view of theresults, the possible physiological signi®cance of cardo-sin A is discussed.

Materials and methods

Plant material. Seeds, roots, midribs, leaves, bracts, ¯owers andpollen of Cynara cardunculus L. were collected from plants grownfrom seeds supplied by the Botanical Gardens of the University ofCoimbra. Flowers were collected at three stages of development:closed, open and senescent capitula. Roots, leaves and bracts werecut into fragments approx. 1 cm wide. Seeds were stored at roomtemperature and all the other organs were kept at)80 °C until used.

Protein extraction. Corollas, stamens and pistils were obtained bydissection of ¯owers collected from open capitula. Seeds, fragmentsof roots, leaves, midribs or bracts, corollas, stamens, pollen orpistils at di�erent stages of development were ground in a mortarand pestle under liquid nitrogen. The ground tissues were homog-enized at 20% (w/v) in 100 mM Tris-Bicine, 2% (w/v) SDS, 8 Murea, and the homogenates incubated for 2 h at 37 °C, underagitation. The extracts were centrifuged at 3000 g for 15 min andthe supernatant further centrifuged at 12 000 g for 20 min. Thislast supernatant was collected and stored at )20 °C.

Protein determination. Protein concentration was determined usingthe bicinchoninic acid protein assay reagent kit (Pierce, Rockford,Ill., USA) according to the manufacturer's instructions.

Puri®cation of cardosins. Cardosins A and B were puri®ed fromfresh stigmas of Cynara cardunculus L. by a simple two-stepprocedure involving extraction at low pH, gel ®ltration onSuperdex 200 and ion-exchange chromatography on Mono Q

(Pharmacia, Uppsala, Sweden), performed essentially as previouslydescribed (VerõÂ ssimo et al. 1996).

Sodium dodecyl sulfate polyacrylamide gel elecrophoresis (SDS-PAGE ). Puri®ed cardosins A and B or protein extracts describedabove were loaded onto 15% polyacrylamide gels for SDS-PAGE,and electrophoresis was performed in a Mini Protean II (BioRad,Hercules, Calif., USA) apparatus according to the method ofLaemmli (1970). For protein detection gels were stained withCoomassie Brilliant Blue R-250 (Sigma, St. Louis, Mo., USA).

Antibody production. A cardosin A-speci®c peptide bearing thesequence TSSEELQVDCNT was synthesised at the University ofShe�eld (Dr. A. Moir, Krebs Institute, She�eld, UK), puri®ed byReversed-phase HPLC on a C8 column (HPLC Technology,Maccles®eld, UK) and used for the production of an antibodyusing thyroglobulin as carrier, essentially as described by Sam-brook et al. (1989). The thyroglobulin-peptide complex, containing300 lg of the peptide, was emulsi®ed with Freund's completeadjuvant (Sigma) and injected subcutaneously into a New Zealandrabbit, from which preimmune blood had previously been collect-ed. A second injection was made two weeks later using the sameamount of the thyroglobulin-peptide solution emulsi®ed withFreund's incomplete adjuvant and the rabbit was bled two weeksafter this last injection.

Immunoblotting analysis. For immunoblotting analysis the pro-teins separated by SDS-PAGE were transferred onto nitrocellulosemembranes by electroblotting in 10 mM 3-(cyclohexylamino)-1-propane-sulfonic acid (Caps), 10% methanol, pH 11 at 500 mA for1 h. The membranes were incubated in a blocking solution, 2.5%(w/v) of skimmed milk in 0.1% (v/v) Tween 20 (Merck, Darmstadt,Germany) in phosphate-bu�ered saline (PBS: 10 mM Na2HPO4,1.8 mM KH2PO4, (PBS formula) 137 mM NaCl, 2.7 mM KCl, pH7.4), for 45 min at room temperature and then incubated overnightwith a 1:200 dilution of the anti-cardosin A polyclonal antibody inPBS-Tween. The membrane was washed three times with PBS-Tween for 10 min and incubated with swine anti-rabbit IgGconjugated to horseradish peroxidase (Dako, Glostrup, Denmark)at a 1:1000 dilution for 1 h. After washing the membrane threetimes with PBS-Tween for 10 min the peroxidase activity wasdeveloped with 0.01% (w/v) diaminobenzidine (DAB) in PBS-Tween in the presence of 0.1% (v/v) H2O2.

Tissue printing with cardosin A antiserum. Tissue prints wereobtained by pressing freshly cut sections of Cynara cardunculus L.stigmas on a nitrocellulose membrane for 20±30 s. The membranewas air-dried and the tissue prints were developed essentially asdescribed for the electroblots of the SDS-polyacrylamide gels.Controls were performed in the same way except that preimmuneserum was used instead of the primary antibody. Antigen on thetissue prints was localized with a Nikon (Tokyo, Japan) photo-microscope.

Immunogold transmission EM. Post-embedding EM immunocyto-chemistry was performed essentially as described by Pereira et al.(1992). Small pieces of both stigmas and styles were ®xed in 3%(w/v) paraformaldehyde, 0.5% (v/v) glutaraldehyde, 2% (w/v)sucrose, 0.05% (w/v) CaCl2 in 1.25% (w/v) Pipes bu�er (pH 7.2)for 2 h, at room temperature, dehydrated in a water/ethanol seriesand embedded in LR White resin (London Resin Co., Basingstoke,UK). Immunogold labelling of cardosin A was performed inultrathin sections on uncoated grids which were incubated with thecardosin A antiserum (1:400), followed by the colloidal gold-labelled secondary antibodies against rabbit immunoglobulin G(1:30). Control sections were prepared as experimental sectionsexcept that primary antibody was replaced with rabbit preimmuneserum. Grids were stained by uranyl acetate and lead citrate andviewed in an electron microscope (EM 10 C; Zeiss, Oberkochen,Germany).

M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae 205

Light microscopy. Semi-thin sections of the same blocks preparedas described for immuno-EM were collected on glass slides.Sections were stained with 1% (w/v) Azure II in distilled water:1%(w/v) methylene blue in 1% (w/v) sodium borate (1:1, v/v) andphotographed with a Nikon microscope.

Results

Structure of the stigma and style of C. cardunculus. Thestigma of C. cardunculus ¯owers (Fig. 1) is very long andof the papillate type. It has two longitudinal groovesbecause it consists of two portions not completely fused(Fig. 2A,B). A papillate epidermis covers thin-walledparenchyma cells that surround sclerenchyma andvascular tissues (Fig. 2A,B). As can be seen with theelectron microscope the epidermic papillae are unicellu-lar, smooth and the wall is covered with a cuticle. Thecytoplasm is rich in ribosomes and mitochondria and iscon®ned to a thin layer adjoining the cell wall andbetween the large protein storage vacuoles that essen-tially occupy most of the cell volume (data not shown).

The style, of the closed type, is composed of a singlelayer of epidermis with a thick cuticle surroundingseveral layers of vacuolated cortical parenchyma cells.The inner portion of the style is occupied by twovascular bundles and supporting tissue on either side ofa central transmitting tissue (Fig. 2C). The cortical cellsappear roundish in transverse section with the thin layerof cytoplasm adjacent to the cell wall due to the presenceof a large vacuole. The transmitting tissue located in thecenter of the style consists of roundish cells with verythick loosely ®brillar walls that provide the route forpollen tube growth.

Speci®city of the anti-cardosin A polyclonal antibody. Apolyclonal antibody intended to be speci®c for cardosinA was produced against a synthetic peptide bearing the

sequence TSSEELQVDCNT. This sequence corre-sponds to a region in the enzyme with low homologyto cardosin B and predicted to be exposed by analysis ofits amino acid content and by comparison with thethree-dimensional model of the barley grain AP (Guru-prasad et al. 1994).

Fig. 1A,B. In¯orescence ofCynara cardunculus. A Capitulum with aninde®nite number of ¯orets (¯owers) on a common receptacle. BUpper portion of a pistil showing part of the style (sy) and the longstigma (st) in which pollen grains become enmeshed.Bar = 2 mm; ´5 (B)

Fig. 2A±C. Structure of the stigma and the style ofC. cardunculus. A,B Cross-sections of the stigma, near the apex (A) and at the medianregion (B), showing two stigmatic portions not completely united thatconsist of a glandular papillate epidermis (pe), thin-walled parenchy-ma, vascular tissues (vt). sc, sclerenchyma. C Cross-section of the stylewith a typical cuticle-covered epidermis (ep) surrounding parenchymacells. The inner core is occupied by the transmitting tissue (tt) and twovascular bundles (vb). Bars = 100 lm; ´160 (A); ´140 (B); ´190 (C)

206 M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae

On immunoblots of SDS-polyacrylamide gels thepolyclonal antibody showed strong reactivity withcardosin A, and complete absence of reactivity withother pistil proteins (Fig. 3B, lanes 2±4). This speci®citywas further con®rmed when the antibody reactedstrongly with puri®ed cardosin A and not at all withpuri®ed cardosin B (Fig. 3A, lanes 1 and 2, respective-ly).

Organ-speci®c expression of cardosin A. Immunoblottinganalysis of protein extracts from di�erent parts of theplant revealed that expression of cardosin A is speci®-cally restricted to the pistils. Crude extracts were made in100 mM Tris-Bicine, 2%, (w/v) SDS, 8 M urea, andanalysed by immunoblotting using the anti-cardosin Apolyclonal antibody. Protein extracts from seeds, roots,midribs, leaves, bracts, stamens and pollen did not showany reaction with the anti-cardosin A polyclonal anti-body (data not shown). A very faint reaction wassometimes detected with extracts of corolla proteins(data not shown). All of the three stages of pistildevelopment investigated, from closed, open and senes-cent capitula, reacted strongly with the antibody(Fig. 3B, lanes 2, 3 and 4, respectively), con®rming thehigh expression and accumulation of cardosin A in thispart of the plant that had been anticipated in previouspuri®cation and enzymic studies. Coomassie-stainedSDS-polyacrylamide gels showed that cardosin A isindeed highly abundant in extracts of pistil proteins(Fig. 3B, lane 1).

Tissue printing with cardosin A antiserum. Localizationof cardosin A in the stigmas of C. cardunculus was ®rststudied by Western tissue printing. Since it was devel-oped (Cassab and Varner 1987), tissue print immuno-blotting has become a widely used, fast and reliabletechnique for locating proteins in plant tissues. Imagesobtained revealed a high accumulation of the enzyme inthe broad outer region of the upper portion of the stigma(Fig. 4A). Towards the lower portion of the stigma(Fig. 4B) this accumulation appeared to occur essential-ly at the periphery of the stigma, which seems tocorrelate with the position of the conspicuous proteinstorage vacuoles shown in Fig. 2A and B. The marks onthe nitrocellulose visible on Fig. 4A (indicated byarrows) corresponding to the areas of sclerenchyma aredue to their rigidity and to the pressure applied, and donot represent immunolabelling. No reaction was detect-ed in controls, in which preimmune serum was used(data not shown).

Immunocytochemical localization of cardosin A. Immu-nogold EM of the stigma revealed that cardosin A occursmainly in the epidermic papillae where it is con®ned tolarge protein storage vacuoles (Fig. 5A,B). Gold labelis strikingly abundant in these dense protein masses andis virtually absent from cytoplasm and organelles,although a few gold grains can be observed in the cellwalls. Pre-treatment of grids with sodium periodate to

Fig. 3A,B. Speci®city of the anti-cardosin A polyclonal antibody andorgan-speci®c expression of cardosin A.A Ten micrograms of puri®edcardosin A (lane 1) and puri®ed cardosin B (lane 2) were loaded on15% SDS-polyacrylamide gels. Proteins were transferred ontonitrocellulose membranes by electroblotting and probed with theantibody raised against a synthetic cardosin A-speci®c peptide usingthe DAB/peroxidase method. B Ten to twenty micrograms of crudeprotein extracts from pistils from closed (lane 2), open (lane 1 and 3)and senescent (lane 4 ) capitula were separated on 15% SDS-polyacrylamide gels. In lane 1 proteins were stained with Coomassieblue. In all other lanes proteins were transferred and probed in thesame way as described in panel A

Fig. 4A,B. Localization of cardosin A on tissue prints of C.cardunculus stigmas. Tissue prints were obtained by pressing freshlycut sections of the upper (A) or lower (B) portion of stigmas on anitrocellulose membrane. The membrane was air-dried and cardosinA was detected with the anti-cardosin A polyclonal antibody using theDAB/peroxidase method. Staining occurs essentially at the peripheryof the stigma. Arrows indicate sclerenchyma imprints that do notrepresent immunolabelling. Bars = 200 lm; ´45 (A); ´ 68 (B)

M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae 207

oxidize carbohydrates and thus avoid any possible non-speci®c glycan recognition by the serum yielded the samelabelling pattern (data not shown). Gold particles arealso very scarce in preimmune-serum-treated controlsections (Fig. 5C). In the stigma, cortical parenchymacells contain cardosin A since label is also distributed,although less densely, in this type of cell, in largevacuoles over a loose matrix (data not shown). Cells ofvascular and supporting tissues are unlabelled (Fig. 5D).

Fig. 5A±D. Immunocytochemical localization of cardosin A insections of epidermal papillae of the stigma of C. cardunculus. ALongitudinal section of the apical region of an epidermal papilla ofthe stigma. Labelling is abundant in the large protein storage vacuoles(pv). B Higher magni®cation of epidermal papilla showing that goldlabel is largely con®ned to the large protein masses and absent fromcytoplasm and mitochondria (m). Occasionally, a few gold particlescan be observed on the cell walls (cw). C Control section of epidermalpapilla treated with preimmune serum.D Phloem cells from vasculartissue showing no labelling. Bars = 1 lm; ´13 000 (A); ´ 21 000(B); ´ 12 000 (C); ´ 8500 (D)

208 M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae

In the style, it is possible to see that cardosin A ispresent mainly in the epidermal cells where gold labelappears, although less densely than in stigma papillae, inthe large central vacuole over a very loose matrix(Fig. 6A,B). A few subepidermal cells also containcardosin A since gold label appeared over a very loosematrix con®ned to the vacuole (data not shown). Nogold particles were detected over the thin cytoplasmlayer, organelles and cell wall. The cells in the vascular

Fig. 6A±D. Localization of cardosin A in sections of stylar epidermalcells. A gold label is distributed over a loose vacuolar matrix in anepidermal cell. cw, cell wall. B Basal region of epidermal cell andportion of an adjoining subepidermal cell. Labelling is restricted to thevacuole (v), being absent from the cell wall, cytoplasm (cy) andorganelles. C Xylem parenchyma adjoining a xylem conductingelement (xy) without signi®cant labelling. D Transmitting tissue cellswith a very large extracellular matrix (tt) and parenchyma cells (p)showing no labelling. Bars = 1 lm; ´ 15 000 (A); ´ 20 800(B); ´ 24 300 (C); ´ 13 200 (D)

M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae 209

bundle and transmitting tissues are unlabelled(Fig. 6C,D).

Discussion

In this paper it is shown by biochemical means and byimmunocytochemistry that the abundant AP from C.cardunculus, cardosin A, accumulates in protein storagevacuoles of the stigmatic papillae and in vacuoles ofepidermal cells of the style. Expression of cardosin Awas found to be highly restricted to the pistils. Althoughthe strongest reaction on immunoblots was observed inthe mature, open capitula stage, comparable levels ofexpression of cardosin A were detected in the closed andsenescent capitula stages. If the enzyme is in fact presentand plays a role in other parts of the plant, it could notbe detected by our immunoassay.

Immunogold EM has proved cardosin A to be avacuolar protein. Previous studies indicated thatcardosin A is a glycoprotein, both of its subunitsbeing glycosylated (Faro et al. 1995). It seems there-fore that the enzyme enters the secretory pathway,undergoes glycosylation in the ER and Golgi appara-tus and is probably deposited in a precipitated form,due to its high quantity. Nevertheless, a possible tra�cbetween these protein storage vacuoles and the cellwall and extracellular space cannot, in our view, beexcluded.

Both cardosin A and cardosin B have been shown todi�er signi®cantly from cyprosins (VerõÂ ssimo et al.1996), other APs also isolated from the ¯owers ofC. cardunculus, which were initially named cynarases(Heimgartner et al. 1990). While cardosins A and B arethe product of di�erent genes, cyprosins are thought tobe di�erent forms of the same enzyme. Cyprosins weresaid to accumulate in the epidermal cell layer of thestyles (Cordeiro et al. 1994a), whereas cardosin A occursmostly in the stigmatic papillae. Unfortunately, no datais available on the subcellular localization of cyprosins.In any case it seems reasonable to admit the existence ofheterogeneity within the aspartic proteinases fromC. cardunculus. Whether this heterogeneity has a phys-iological signi®cance remains to be elucidated.

As in other organisms, plant proteases are involved ina diverse array of processes (Vierstra 1993; Callis 1995).Nevertheless, and despite all the present lines ofresearch, the restricted expression and high accumula-tion of a protease in stigmas has not, to the best of ourknowledge, been reported until now. Plant APs havebeen identi®ed in other parts of plants, usually seeds orleaves, but not in ¯owers (Kervinen et al. 1995), and notin abundant quantities (Boller 1986). They have beensuggested to be involved in protein turnover (Belozerskiet al. 1989; Rodrigo et al. 1989, 1991) or speci®cproteolytic processing of other proteins (D'Hondt et al.1993; Runeberg-Roos et al. 1994), but their physiologyremains still largely unknown. The results presentedhere, demonstrating the restricted location of cardosinA, along with previous results concerning its highexpression and narrow speci®city (VerõÂ ssimo et al.

1995, 1996), make this enzyme a good model for thestudy of plant AP function.

In view of the results discussed above, we discussthree main possibilities for the function of cardosin A, asworking hypotheses. An involvement in ¯ower senes-cence seems to us to be at least a secondary function ofthe enzyme. The activity of the enzyme could contributeto the release of seeds and eventually to the annualsenescence of the above-ground shoot of the plant, aperennial Asteraceae. Participation of proteases in celldeath programs is a well known fact both in animals(Kumar 1995) and in plants (Dalling and Nettleton1986). However, it has been proposed that vacuolarproteases do not participate in protein breakdownduring senescence, except perhaps in a terminal stage,when the vacuolar membrane is broken (Boller 1986). Insupport of this hypothesis we have found that cardosinA puri®ed from senescent ¯owers is still active, and thatthis activity remains present in dried ¯owers for severalyears.

Another possibility is an involvement in defenseagainst pathogens. Although the best-known plantdefensive enzymes are chitinases and b-1,3-glucanases(Bell 1981), thought to be involved in digestion of cellwalls of invading fungi, vacuolar proteases have beenproposed to have defensive roles (Boller 1986). In thisperspective, the bursting of the vacuoles in a hypersen-sitive reaction to invasion could lead to an arrest of thegrowth of the pathogen and eventually to its destructionthrough the action of proteases. However, the questionof the possible target of these enzymes is not clear. Onerelated ®eld of research is the study of plant proteaseinhibitors that contribute to resistance against insects orpathogens (Ryan 1990). It is believed that due to theaction of these inhibitors in the digestive tract of insectsa feedback mechanism leads to a pernicious hyperpro-duction of digestive proteinases (Ryan 1990). The samepernicious e�ects could possibly be exerted directly bycardosin A if active in the digestive tract of herbivorousinsects, thus protecting the valuable and vulnerablestigma from attack. The massive amounts in whichcardosin A accumulates in the periphery of the stigmaseem to favour this hypothesis.

Finally, an involvement of cardosin A in the pollen-pistil interaction must be considered. The stigmaticpapillae are where pollen ®rst contacts and interactswith the female sporophytic tissues in its way to theovary, being captured and adhering to the stigmasurface, hydrating and germinating (Heslop-Harrison1975). All these processes certainly involve molecularevents of recognition, signalling, and response (Elle-man and Dickinson 1994). The fact that cardosin A islocated in the vacuole of the papillae and not at thestigma surface does not impede its interaction withpollen. It has been shown that capture of pollenstimulates secretion by the papillae (Elleman andDickinson 1990) and that secreted glycoproteins maylater return to an inactive pool in the vacuole (Robertset al. 1984; Sarker et al. 1988). It has also recentlybeen found that phytohemagglutinin-E, an extensivelystudied vacuolar seed protein, is found both in

210 M. Ramalho-Santos et al.: Aspartic proteinase in vacuoles of stigmatic papillae

vacuoles and cell walls of bean roots (Kjemtrup et al.1995). In C. cardunculus, it is possible that cardosin Aparticipates in extracellular events of protein break-down eventually important for the proper adhesionand/or germination of pollen. The initial stages of thepollen-pistil interaction in members of the Compositae(Asteraceae) have been studied (Elleman et al. 1992).Both stigma and pollen respond to pollination withthe production of an abundant extracellular electron-opaque matrix into which the pollen tubes initiallygrow. However, the molecular nature of this matrixand the mechanisms underlying the interaction ofpollen- and stigma-derived factors is not yet clear. Itwill be interesting to investigate if a stigmatic secretionalso occurs upon pollination in C. cardunculus andwhether or not cardosin A is secreted during thisprocess. In any case, a possible role of cardosin A inthe pollen-pistil interaction in C. cardunculus wouldrepresent a new molecular mechanism, since aninvolvement of stigmatic proteases in the pollen-pistilinteraction has not, to the best of our knowledge, beenreported (Knox 1984; Clarke and Newbigin 1993).

In summary, cardosin A may eventually have aprimary function in the interaction with pathogens orpollen or even both, since defense and pollen-recognitionmechanisms are currently thought to be related (Dic-kinson 1993), and a secondary function in the annualsenescence of the ¯ower.

The authors thank Arthur Moir (Krebs Institute, University ofShe�eld, UK) for the peptide synthesis and assistance in theproduction of the anti-cardosin A polyclonal antibody. The skillfulphotographic assistance of Mrs Andrea Costa is gratefullyacknowledged. The research was supported by Junta Nacional deInvestigacË aÄ o Cientõ ®ca e Tecnolo gica (JNICT), Portugal. MiguelRamalho-Santos and Paula Verõ ssimo are recipients of fellowshipsfrom the PRAXIS XXI program (JNICT).

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