STYLAR PEROXIDASE AND INCOMPATIBILITY REACTIONS IN PETUNIA HYBRIDA

Transcription

1 J. Cell Sd. 82, 1-10 (1986) Printed in Great Britain The Company of Biologists Limited 1986 STYLAR PEROXIDASE AND INCOMPATIBILITY REACTIONS IN PETUNIA HYBRIDA LUISA CARRARO, GIULIANA LOMBARDO AND F. M. GEROLA Dipartimento di Biologia, Sezione di Botanica Sistematica Universitd degli Studi dimilano, Via Celoria 26, 201'33 Milano, Italy SUMMARY Self-, cross- and non-pollinated styles of Petunia hybrida were treated with 3-3'-diaminobenzidine for the ultrastructural localization of peroxidase activity. Wall peroxidases were absent in cross-pollinated styles, but they were detectable as an osmiophilic deposit on the cell walls of the outer portion of the transmitting tissue in self- and non-pollinated styles. The cell layer showing peroxidase activity was thicker in the self-pollinated styles than in the non-pollinated ones. In accordance with current hypotheses on the mechanism involved in pollen incompatibility process, it is suggested: (1) that wall peroxidases present in the cells of the outer portion of the transmitting tissue are involved in the gametophytic self-incompatibility of Petunia; (2) that self-pollination causes an increase in the number of cells involved in the rejection process; (3) that non-pollinated styles, which are characterized by the presence of cell wall peroxidases in the outer portion of the transmitting tissue, are 'prepared' to a certain extent for the rejection of incompatible pollen tubes. The removal of peroxidase activity thus seems to be an important step in the compatible pollination process. INTRODUCTION The gametophytic self-incompatibility system of Petunia plants is characterized by a pollen tube inhibition in incompatible combinations in the upper zone of the stylar transmitting tract (Heslop-Harrison, 1983). This stylar portion was called the 'neck' by Herrero & Dickinson (1979), who stated that "the transmitting tissue contains large spherical cells with characteristic ridges in their walls, which cause them to 'key' into an adjacent cell". Compatible and incompatible intraspecific pollinations induce cytological and metabolic changes in the stylar transmitting tissue (Herrero & Dickinson, 1979; Roggen, 1967); compatible and incompatible pollen tubes growing in the stylar transmitting tissue of Petunia also show ultrastructural differences (Cresti et al. 1979; Herrero & Dickinson, 1981). Regarding the incompatibility reaction, Linskens (1975) and van der Donk (1975) have argued that the rejection of pollen tubes is the reaction for which the style is prepared, whereas unaffected pollen tube growth is related to the synthesis of special enzymes that break down the incompatibility barrier. In Nicotiana alata gametophytic self-incompatible flowers, many electrophoretic investigations have been carried out to detect the substances responsible for the recognition of the pollen tube along the style. Pandey (1967) and Bredemeijer Key words: gametophytic self-incompatibility, Petunia hybrida, stylar transmitting tissue, wall peroxidases.

2 2 L. Carraro, G. Lombardo and F. M. Gerola (Bredemeijer & Blaas, 1975) have reported the presence of peroxidase isoenzymes in style extracts; however, the actual role of these enzymes in the gametophytic selfincompatibility mechanism is still an open question. Pandey (1967) has regarded peroxidases as a direct expression of the 5 gene; Bredemeijer (Bredemeijer & Blaas, 1980) has reported an indirect non-specific action of peroxidases in the incompatibility reactions. This author also showed that pollination and pollen tube growth caused an increase in total peroxidase activity (Bredemeijer, 1974) and that there is a positive correlation between peroxidase isoenzyme 10 and intensity of the rejection reaction (Bredemeijer & Blaas, 1975; Bredemeijer, 1976). In subsequent works Bredemeijer (1977, 1979) noted an even distribution of peroxidase 10 across the style, and he also proposed that only the fraction of peroxidase 10 present in the intercellular spaces and on the cell walls is responsible for pollen tube inhibition. In a previous ultrastructural investigation on self-pollinated 'pin' flowers of Primula acaulis treated with 3-3'-diaminobenzidine (DAB) Carraro et al. (1985) reported the presence of peroxidases on the cell walls and intercellular spaces of the stylar transmitting tissue, whereas treatment with DAB did not provide evidence of the presence of peroxidases in cross-pollinated flowers. To obtain further information supporting the hypothesis of the involvement of peroxidases in the incompatibility reaction, we repeated the DAB treatment on self-, cross- and non-pollinated styles of Petunia hybrida. MATERIALS AND METHODS Seedlings of P. hybrida were raised from seeds of clones Ka3 and T2U (genotypes 22 and 33, respectively), known to be self-incompatible and cross-compatible (Herrero & Dickinson, 1979). Just before anthesis, the buds of T2U plants were detached and placed in small glass tubes containing fresh water in a well-lit air-conditioned chamber, maintained at a constant temperature of 25 C. Stamens and corolla were then removed and the pistils were divided into three groups. The first was self-pollinated by rubbing mature T2U anthers gently on the stigmas; the second was cross-pollinated with Ka3 anthers, the third was not pollinated. After 40 h the stigmas and upper 2 mm of the style (the neck region) were cut away with a razor blade, prefixed in 3 % glutaraldehyde in 0-05 M-cacodylate buffer, ph7-4 for 30min, at 4 C. Afterwards, a few pistils were postfixed in cacodylate-buffered 1% osmium tetroxide, ph7-4, for 2h, at 4 C, dehydrated in ethanol and embedded in Araldite. Other style samples were used for the histochemical determination of peroxidase. They were first rinsed in the same cacodilate buffer for 30min and then in 0-05 M- 2-amino-2-methyl-l,3'-propandiol buffer, ph7-4, for 30min. Incubation with DAB (Sigma Chemical Co., St Louis, MO) followed, for 60min, at 37 C. The incubation medium was always prepared immediately before use: it contained lomg DAB, 4-9ml of 0-05M-propandiol buffer, ph7-4, and 0-1 ml H 2 O2. The following procedures served as control to determine whether any observed precipitate could be reasonably ascribed to peroxidase: (1) preincubation for 30min at 37 C with propandiol buffer, ph7-4, containing 0-02M-aminotriazole (3-amino-l,2,4-triazole, Aldrich Chemical Co., Inc., Milwaukee, WI), followed by incubation in DAB medium, also containing 0-02M-aminotriazole (Rechcigl & Warren, 1963; Frederick & Newcomb, 1969); (2) preincubation for 30min at 37 C in propandiol buffer, ph7-4, containing O01 M-KCN, followed by incubation in DAB medium containing 0-01 M-KCN (Frederick & Newcomb, 1969; Burns & Little, 1949). After incubation, the segments were rinsed in 0-05 M-propandiol buffer, ph 7-4, and postfixed in 1 % cacodylate-buffered osmium tetroxide, ph 7-4, for 2h at 4 C. The samples were then dehydrated in ethanol and embedded in Araldite. Ultrathin sections of the neck region were obtained with an LKB Ultrotome III and examined in a Philips TM 400 transmission electron microscope (TEM). Semithin sections were cut with a glass

3 Pervxidases and incompatibility in Petunia 3 knife and examined with a light microscope. The sections were not stained, to avoid any overlap with reaction products. RESULTS Self-pollinated styles By light microscopy of semithin cross-sections of the style neck, the transmitting tissue was seen to be characterized by spherical cells, whose size increased towards the periphery. After DAB treatment the cell walls of the outer portion of the transmitting tissue appeared much darker than those of the central portion (Fig. 1). By electron microscopy of ultrathin sections, DAB treatment was seen to result in pronounced deposit of electron-opaque material only on the wall of the cells in the outer portion of the transmitting tissue (Figs 2, 4). No deposit was present in the central portion of the transmitting tissue (Fig. 3) or on the wall of pollen tubes (Fig. 4). The cells of the outer portion of the transmitting tissue (Fig. 2) were spherical and defined large intercellular spaces containing a granular electron-dense matrix. The cells possessed a thin wall, evident nuclei, starch-containing plastids, small mitochondria and small vacuoles or a single large central vacuole. Pollen tubes were observed in the outer portion of the transmitting tissue (Fig. 1); they appeared infolded, close to the transmitting cells, and were characterized by degenerated cytoplasm and a thick multilayered wall with fibrillar inclusions (Fig. 4). In the central portion of the transmitting tissue (Fig. 3) the cells were spherical and possessed characteristic 'key junctions' as described by Herrero & Dickinson (1979). Chloroplasts containing large starch grains were particularly numerous. When the DAB treatment was omitted, the deposition of electron-opaque material on the cell walls of the transmitting tissue was absent and no difference was observed between the central and the outer portion of the transmitting tissue (data not shown). When aminotriazole was added to the DAB medium, deposit of electron-opaque material on the cell walls of the outer portion of the transmitting tissue was still present, even if less evident than in samples treated with DAB only (Fig. 5). This control confirmed that the dark deposit was related to peroxidase activity, since aminotriazole is a specific inhibitor of catalase but not peroxidase (Carraro et al. 1985). When KCN was added to the DAB medium, the electron-opaque deposit on the cell walls of the outer transmitting tissue was completely absent, since KCN inhibits both catalase and peroxidase (Fig. 6) (Carraro et al. 1985). Non-pollinated styles Light microscopic observation of DAB-treated non-pollinated styles revealed the presence of cell wall peroxidases in the outer portion of the transmitting tissue, However, the layer of cells characterized by the presence of peroxidase activity appeared to be reduced more than that in the self-pollinated styles (Fig. 7). Electron microscopic examination of the outer transmitting tissue after DAB treatment revealed electron-opaque deposits on the cell walls similar to those

4 L. Carraro, G. Lombardo and F. M. Gerola Figs 1-4. For legends see p. 7

5 Peroxidases and incompatibility in Petunia cp Figs 5-8. For legends see p. 7

6 L. Carraro, G. Lombardo and F. M. Gerola

7 Peroxidases and incompatibility in Petunia Fig. 1. Semithin cross-section through the neck of a self-pollinated style, after DAB treatment; the outer portion of the transmitting tissue (op) is characterized by cell walls much darker than those of the central portion (cp); incompatible pollen tubes are detectable in the outer transmitting tissue (arrows). Light micrograph. X300. Fig. 2. TEM of the outer portion of the transmitting tissue in a self-pollinated style, treated with DAB; a pronounced deposit of electron-opaque material is clearly visible on the cell walls. The cells appear spherical, bound by a thin wall and defining large intercellular spaces filled by an electron-dense matrix. The cells possess large nuclei (n), starch-containing plastids (/>), small mitochondria (m), small vacuoles or a single large central vacuole (v). X3600. Fig. 3. TEM of the central transmitting portion of a self-pollinated style, treated with DAB. Electron-opaque material on the cell walls is absent. The cells have key junctions with contiguous cells (arrows)./), starch-containing plastids. X4200. Fig. 4. Detail of the outer portion of the transmitting tissue of a self-pollinated style, treated with DAB. An incompatible pollen tube (it) is clearly visible. Deposits of electron-opaque material are present on the transmitting cell walls (arrows). The incompatible pollen tube possesses a thick multilayered wall (tv) with fibrillar inclusions (/) and lacks electron-opaque deposit. X7800. Fig. 5. TEM of the outer transmitting portion of a self-pollinated style treated with DAB in the presence of aminotriazole. A light deposit of electron-opaque material due to peroxidase activity is still visible on the cell walls of the transmitting tissue (arrows), it, incompatible pollen tube. X7800. Fig. 6. TEM of the outer transmitting portion of a self-pollinated style treated with DAB in the presence of KCN. Electron-opaque material on the cell walls of the transmitting tissue is absent, it, incompatible pollen tube; p, starch-containing plastids. XS000. Fig. 7. Semithin cross-section through the neck of a non-pollinated style, treated with DAB. Only the outermost cell layer of the outer transmitting tissue (op) shows wall peroxidase activity. The remaining outer portion and the central portion (cp) of the transmitting tissue contain cells with an almost indistinguishable cell wall devoid of contrast. Light micrograph. x400. Fig. 8. TEM of the outer transmitting portion of a non-pollinated style after DAB treatment; only the outermost cell layer of this portion shows the electron-dense deposit on the wall. The deposit disappears gradually in the inner cell layers, visible on the lower left side of the micrograph, p, starch-containing plastids; v, vacuoles. X3300. Fig. 9. Semithin cross-section through the neck of a cross-pollinated style, after DAB treatment: central (cp) and outer portions (op) of the transmitting tissue possess cell walls devoid of contrast that did not react with the DAB medium. Light micrograph. X400. Fig. 10. TEM of the outer transmitting portion of a cross-pollinated style, after DAB treatment. There is no electron-opaque deposit on the cell walls. The cells contain a dense cytoplasm rich in organelles. The compatible pollen tube (ct) present among these cells appears collapsed but not yet 'empty', n, nuclei; p, starch-containing plastids; m, mitochondria; u, vacuoles. X6000. Fig. 11. TEM of the outer transmitting portion of a cross-pollinated style, after DAB treatment. Many compatible pollen tubes (ct) are detectable among the transmitting cells. The pollen tubes appear empty, flattened and collapsed. The transmitting cells possess a large central vacuole and clear cell walls and lack any electron-opaque deposit. X330O.

8 8 L. Carraro, G. Lombardo and F. M. Gerola observed in the self-pollinated samples (Fig. 8). The photograph was taken of the border of the DAB-reacting cell layer; cells lacking the characteristic electron-opaque material in the wall can thus be observed. Treatment with aminotriazole and KCN revealed that even in non-pollinated styles the electron-opaque deposit observed following DAB treatment was due to peroxidase activity (data not shown). Cross-pollinated styles In semithin cross-sections observed by light microscopy after DAB treatment, no dark deposit was observed on the cell walls of any portion of the transmitting tissue in the neck region, resulting in an appearance devoid of contrast (Fig. 9). When observed by electron microscopy, cell walls always appeared to be without electronopaque deposit (Figs 10, 11). By comparison of these two figures the different ultrastructure of the transmitting cells is clearly evident. As previously reported (Herrero & Dickinson, 1979), probably owing to the elongation stage of the pollen tube, the cells of the surrounding transmitting tissue appear to undergo large modifications leading to cells characterized by large vacuoles and a thin peripheral layer of cytoplasm, and no longer rich in reserves (Fig. 11). At this level the intercellular spaces were filled with pollen tubes, which were almost empty; they appeared flattened and collapsed and were characterized by a thin wall lacking any fibrillar inclusion (Fig. 11). The absence of fibrillar inclusions in compatible pollen tubes has also been previously reported (Herrero & Dickinson, 1981). DISCUSSION Whereas wall-peroxidase activity was detectable in both self- and non-pollinated flowers, the 'peroxidase characterized' cell layer at the outer portion of the transmitting tissue was thicker in the self-pollinated than in the non-pollinated sample. Preliminary results might indicate a correlation between elongation of the incompatible pollen tubes and increase in thickness of the cell layer presenting peroxidase activity. These data are in agreement with previous observations (Pandey, 1967; Bredemeijer & Blaas, 1975; Bredemeijer, 1976) of an increase in peroxidase content of the transmitting tissue following self-pollination. Our results indicating the presence of peroxidases on the cell wall of non-pollinated and self-pollinated styles support the hypothesis of Bredemeijer (1977) that only the fraction of extracellular peroxidases is responsible for pollen tube growth inhibition. Also, our observation of an even distribution of peroxidases between the central and the outer portion of the transmitting tissue is reminiscent of the even distribution of peroxidase 10 observed by Bredemeijer (1979) between cortex and transmitting tissue of Nicotiana. Of particular importance is the observation that compatible pollination does not cause the increase in the number of cells showing peroxidase activity, but does cause the disappearance of the peroxidase activity observed in non-pollinated samples. This finding is basically in agreement with the hypothesis of Linskens (1975) and van

9 Peroxidases and incompatibility in Petunia 9 der Donk (1975) that the style is prepared by nature for the rejection reaction of the pollen tube, whereas the unaffected growth of compatible pollen tubes is due to the break in the incompatibility barrier. At this time we cannot distinguish between different mechanisms involved in the disappearance of peroxidase activity in cross-pollinated styles, i.e. inhibition, sequestration or degradation of the enzyme. Further studies will be carried on this aspect. As previously reported by Herrero & Dickinson (1979), we also observed a considerable ultrastructural modification in the transmitting cells in the crosspollinated samples, probably related to the elongation and growth of the pollen tube. However, in cross-pollinated styles the peroxidase activity was absent also in the wall of the cells that had not yet undergone extensive ultrastructural changes (see Fig. 10), indicating that the removal of peroxidase activity is a preceding process. All these results suggest that peroxidases are some of the components responsible for incompatibility reactions. To examine this hypothesis, the absence or presence of peroxidases in very young buds of Petunia, which seem to lack the incompatibility barrier (Ascher, 1984), will be investigated. The authors thank Professor Linskens, who kindly supplied the Petunia seeds. The authors are indebted to Dr P. D. Gerola for his critical reading and correction of the manuscript. This work was also supported by M.P.I. (40%). REFERENCES ASCHER, P. D. (1984). Self-incompatibility. In Petunia (ed. K. C. Sink), pp Berlin, Heidelberg, New York, Tokyo: Springer-Verlag. BREDEMEIJER, G. M. M. (1974). Peroxidase activity and peroxidase isoenzyme composition in selfpollinated, cross-pollinated and unpollinated styles of Nicotiana alata. Acta bot. neerl. 23, BREDEMEIJER, G. M. M. (1976). Effect on bud-pollination and delayed self-pollination on the induction of a possible rejection peroxidase in styles of Nicotiana alata. Acta bot. need. 25, BREDEMEIJER, G. M. M. (1977). Peroxidase leakage and pollen tube growth inhibition in aged Nicotiana alata styles. Acta bot. neerl. 26, BREDEMEIJER, G. M. M. (1979). The distribution of peroxidase isoenzymes, chlorogenic acid oxidase and glucose-6-phosphate dehydrogenase in transmitting tissue and cortex of Nicotiana alata styles. Acta bot. need. 28, BREDEMEIJER, G. M. M. & BLAAS, J. (1975). A possible role of a stylar peroxidase gradient in the rejection of incompatible growing pollen tubes. Acta bot. need. 24, BREDEMEIJER, G. M. M. & BLAAS, J. (1980). Do S allele-specific peroxidase isoenzymes exist in self-incompatible Nicotiana alata? Theor. appl. Genet. 57, BURRIS, R. H. & LITTLE, H. N. (1949). Oxidases, peroxidases, and catalase. In Respiratory Enzymes (ed. H. Lardy), pp Minneapolis: Burgess. CARRARO, L., LOMBARDO, G. & GEROLA, F. M. (1985). Electron-cytochemical localization of peroxidase in self- and cross-pollinated styles of Primula acaulis. Caryologia 38, CRESTI, M., CIAMPOUNI, F., PACINI, E., SARFATTI, G., VAN WENT, J. L. & WILLEMSE, M. T. M. (1979). Ultrastructural differences between compatible and incompatible pollen tubes in the stylar transmitting tissue of Petunia hybrida.j. submicrosc. Cytol. 11, FREDERICK, S. E. & NEWCOMB, E. M. (1969). Cytochemical localization of catalase in leaf microbodies (peroxisomes). J. Cell Biol. 43,

10 10 L. Carraro, G. Lombardo and F. M. Gervla HERRERO, M. & DICKINSON, H. G. (1979). Pollen-pistil incompatibility in Petunia hybrida: changes in the pistil following compatible and incompatible intraspecific crosses, J. Cell Sci. 36, HERRERO, M. & DICKINSON, H. G. (1981). Pollen tube development in Petunia hybrida following compatible and incompatible intraspecific matings.,7. Cell Sci. 47, HESLOP-HARRISON, J. (1983). Self-incompatibility: phenomenology and physiology. Proc. R. Soc. B 218, LlNSKENS, H. F. (1975). Incompatibility mpetunia. Proc. R. Soc. B 188, PANDEY, K. K. (1967). Origin of genetic variability: Combinations of peroxidase isozymes determine multiple allelism of the 5 gene. Nature, Land. 18, RECHCIGL, M. & WARREN, H. E. (1963). Role of catalase and peroxidase in the metabolism of leucocytes. Nature, Land. 199, ROGGEN, H. P. J. R. (1967). Changes in enzyme activities during the progame phase in Petunia hybrida. Ada bot. neerl. 16, VAN DER DONK, J. A. W. M. (1975). Recognition and gene expression during the incompatibility reaction in Petunia hybrida L. Molec. gen. Genet. 141, (Received 8 July Accepted, in revised form, 8 November 1985)