Micron 34 (2003) 387–393 www.elsevier.com/locate/micron Cytochemical localization of calcium and X-ray microanalysis of Catharanthus roseus L. infected with phytoplasmas Rita Musettia,*, Maria Augusta Favalib a Dipartimento di Biologia Applicata alla Difesa delle Piante, Università di Udine, Via delle Scienze 208, Udine 33100, Italy b Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma, viale delle Scienze, Parma 43100, Italy Received 1 April 2003; revised 20 May 2003; accepted 12 June 2003 Abstract The potassium pyroantimonate (KPA) Ca2þ precipitation technique, X-ray microanalysis and Electron Energy Loss Spectroscopy carried out by transmission electron microscopy were used to analyze the Ca2þ distribution in Catharanthus roseus L. leaves infected with phytoplasmas belonging to different taxonomic groups, and in phytoplasma cells. The analysis revealed that the distribution of Ca2þ was different in healthy and diseased plants (where the KPA deposits were numerous) and no differences were observed in the tissues of the three types of infected C. roseus L. Since no KPA precipitates were visible in the phloem and on phytoplasma cells, it is likely that Ca2þ ions are not directly involved in phytoplasma replication, but, in infected cells is a response to the pathogen indicative of a higher Ca2þ in the plasmalemma. q 2003 Elsevier Ltd. All rights reserved. Keywords: Calcium; Transmission electron microscopy; X-ray microanalysis; Electron energy loss spectroscopy; Phytoplasma; Catharanthus roseus L 1. Introduction Pathogens, plant hormones, light receptor, and abiotic signals trigger a rise in plant leaf cytosolic calcium concentration, which mediates physiological and developmental responses. Many studies have suggested that Ca2þ might be a second messenger in a multiple stress responses in plants (Price et al., 1994; Sheen, 1996; Gelli et al., 1997; McAinsh and Hetherington, 1998; Romeis et al., 2001). In the case of pathogen attack, its recognition at the plant cell surface results in the initiation of a multicomponent defence response, and the transient influx of Ca2þ across the plasma membrane is postulated to be part of the signalling chain leading to pathogen resistance (Gelli et al., 1997; Zimmermann et al., 1997). The increase in the cytosolic Ca2þ concentration, appears to be a master regulator of many subsequent signalling steps. Active oxygen species such as O2 2 , H2O2 or nitric oxide, the activation of protein kinases, phytoalexin production, alone or in combination, were shown to be compromised by the presence of Ca2þ * Corresponding author. Tel.: þ 39-432-558521/503; fax: þ 39-432558501. E-mail address: rita.musetti@pdef.uniud.it (R. Musetti). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0968-4328(03)00082-9 chelating or Ca2þ channel inhibiting compounds (Scheel, 1998). Most of the studies on changes in cytosolic Ca2þ concern diseases causing local lesions due to fungi (Stockwell and Hanchey, 1982; Gelli et al., 1997; Romeis et al., 2000), systemic diseases caused by viruses (Favali et al., 1978, 1980; Romeis et al., 2001), or on cultured cells treated with elicitors (Sheen, 1996; Chandra and Low, 1997; Zimmermann et al., 1997); nothing has been reported for phytoplasma infections. Phytoplasmas, wallless prokaryotes, are agents of serious plant diseases (McCoy et al., 1989; Favali et al., 1990; Musetti et al., 1992, 1994); they are localized in the phloem tissue of the host and are not cultivable in vitro. Recent works have shown that in the infected tissues phytoplasmas cause changes in the concentration of important metabolites (Pertot et al., 1998) and many of these relate to defence mechanisms (Musetti et al., 1999, 2000). The aims of this work were to study the involvement of Ca2þ ions in phytoplasma diseases by analyzing the presence and distribution of Ca2þ in periwinkles infected by three phytoplasmas belonging to different taxonomic groups. Ca2þ ion distribution in the three types of 388 R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 phytoplasma cells was analyzed in situ by X-ray microanalysis. 2. Materials and methods 2.1. Plant material Leaf samples were collected from Catharanthus roseus L. plants inoculated by top-grafting (Carraro et al., 1992) with one of the three different phytoplasmas: Apple Proliferation (AP), European Stone Fruit Yellow (ESFY), belonging to the AP group (16SrX), or Clover Phyllody (CP) belonging to the Aster Yellows group (16SrI) (Lee et al., 1998). 2.2. Trasmission electron microscopy Potassium pyroantimonate (KPA) was used as a cytochemical agent for the localization of Ca2þ ions (Hayat, 1993). Immediately before use, potassium hexahydroxyantimonate [KSb(OH)6], was added to give 2% (w/v) in a solution of 3% glutaraldehyde in 0.1 M potassium phosphate buffer pH 6.9: leaf samples of healthy and infected C. roseus were fixed in this solution for 2 h at 4 8C. The samples were then rinsed in buffer, postfixed in 1% osmium tetroxide in 0.1 M potassium phosphate for 1 h at 4 8C, dehydrated in ethanol and embedded in Epon-Araldite according to the method described by Musetti et al. (1999). For morphological studies, ultrathin sections were stained with uranyl acetate and lead citrate and observed in a PHILIPS CM 10 electron microscope, operated at 80 kV. 2.3. Analysis of antimonate precipitates and TEM X-ray microanalysis Fig. 1. Phloem in C. roseus L. leaf tissue infected with Apple Proliferation (AP) phytoplasma and prepared by the KPA method. AP agents are numerous and pleomorphic in shape, some are organized into short chains (arrows), No KPA deposits are visible in the phloem or in phytoplasma cells. Bar: 1 mm. They are numerous, well preserved and pleomorphic in shape (Fig. 1). Some of the ultrastructural alterations caused by all three types of phytoplasmas in the host tissues were similar, i.e. callose accumulation in the sieve plates and near the plasmodesmata, distortion and thickening of the cell walls, as reported by Musetti et al. (2000). In AP and ESFY infected leaf tissues the cell organelles and nuclei appeared significantly altered, as previously reported (Musetti et al., 1992, 1994). Many KPA Ca2þ precipitates are visible in the tissues of infected plants (Figs. 2– 5). The ultrastructural localization of these deposits is the same in all the three types of infected periwinkles. The main site of KPA precipitates is the plasmalemma of mesophyll cells (Figs. 2 and 3, arrows) and the secondary thickenings of the tracheid walls (Figs. 4 The nature of KPA precipitates was determined in two different ways. Unstained ultrathin sections, previously checked for KPA deposits, were floated on a 5 mM solution of EDTA at 60 8C for 1 h. Control sections were incubated in EDTA free medium. Unstained ultrathin sections of the same material were analyzed using a Jeol JEM 2010 electron microscope operated at 200 kV, equipped with an X-ray microanalysis system (Link Inca 100), a Gatan Imaging Filter and Multiscan Camera 794 (Software Digital Micrograph 3.1/ Win NT). The calcium maps were obtained by Electron Energy Loss Spectroscopy (EELS). 3. Results The presence of phytoplasmas in the phloem cells of all infected C. roseus was demonstrated by TEM observation. Fig. 2. Mesophyll cells in C. roseus leaf tissue infected with AP phytoplasmas and treated by the KPA method. Note that the precipitates are associated with the plasmalemma in mesophyll cells (arrows; Chl ¼ chloroplast). Bar: 1 mm. R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 Fig. 3. Mesophyll cells in C. roseus leaf tissue infected with European Stone Fruit Yellows (ESFY) phytoplasmas and treated by the KPA method. Note that the precipitates are associated with the plasmalemma in mesophyll cells (arrows; Chl ¼ chloroplast). Bar: 0.1 mm. and 5, arrows). No precipitates are found in the phloem and in phytoplasma cells (Figs. 6 and 7). In the chloroplasts rare deposits are found, while nuclei and organelles of the companion and mesophyll cells are KPA deposit free. In the tissues of healthy C. roseus plants no precipitates were observed in the phloem (Fig. 8), scanty in the xylem on the secondary thickenings of the tracheid walls (Fig. 9, arrows). No precipitates were observed in the control mesophyll cells, the chloroplast membranes and mitochondria (Fig. 10). The analysis of unstained ultrathin sections floated on EDTA and in EDTA-free medium revealed that the KPA precipitates disappeared after the treatment with EDTA (Figs. 11 and 12), but remained essentially unaltered in Fig. 4. Xylem cells in KPA treated C. roseus tissues infected with Clover Phyllody (CP). In vein cells many KPA deposits are visible on the secondary thickenings of the tracheid walls (arrows). Bar: 1 mm. 389 Fig. 5. Xylem cells in KPA treated C. roseus tissues infected with AP. In vein cells many KPA deposits are visible on the secondary thickenings of the tracheid walls (arrows). Bar: 1 mm. Fig. 6. Phytoplasmas in the phloem of ESFY infected C. roseus treated with KPA. No deposits are visible on the plasmalemma or on phytoplasmas. Bar: 0.1 mm. Fig. 7. High magnification of ESFY cells. The simple ultrastructure of the phytoplasmas is clearly visible: three layered membrane (closed arrow), ribosomes (R) and nucleic acid (arrow). One phytoplasma is budding (B). Bar: 0.1 mm. 390 R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 Fig. 8. Healthy C. roseus leaf tissue treated with KPA. No deposits are present in the phloem, where P-protein (P) is recognizable. Bar: 1 mm. Fig. 11. Ultrathin sections of CP infected C. roseus showing xylem cells after EDTA treatment. The KPA deposits visible in Fig. 4 are absent after floating in the EDTA calcium-chelating agent. Bar: 1 mm. the uninfected controls. The spectrum of KPA precipitates revealed a high Ca2þ peak (Chart 1). The Ca2þ map made by the EELS on the same point of the section, showed many scintillant dots corresponding to KPA deposits (Figs.13 and 14). X-ray microanalysis performed on the phytoplasmas revealed that an insignificant amount of Ca2þ is present in the cell of these prokaryotes. 4. Discussion Fig. 9. Healthy C. roseus leaf tissue treated with KPA. Deposits are rare in the xylem on the secondary thickenings of the tracheid walls (arrows). Bar: 1 mm. The movement and accumulation of ions in diseased tissues are of importance to understand the metabolism of both pathogen and host cell at the point where they interact. In phytoplasma diseases, our knowledge of the ions implicated in cell metabolism is limited because of Fig. 10. Healthy C. roseus leaf tissue treated with KPA. No precipitates are localized on the chloroplast membranes (Chl), on mitochondria (m) and on the wall of parenchymal cells. Bar: 1 mm. Fig. 12. Ultrathin sections of CP infected C. roseus showing xylem cells after EDTA treatment. The KPA deposits visible in Fig. 4 are absent after floating in the EDTA calcium-chelating agent. Bar: 1 mm. R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 391 Chart 1. Spectra obtained by TEM X-ray microanalysis performed on the deposits visible in Fig. 13. Note the presence of Ca peaks. the chemical and physical complexity of phloem, and the site of phytoplasma replication. The KPA technique and EELS demonstrated the precise localization and distribution of Ca2þ ions in periwinkle infected leaf tissues. Comparing observations in diseased and healthy plants, it appears that there is a significant difference in the amount of Ca2þ in infected and healthy tissues. In fact, an accumulation of KPA precipitates is found in infected tissue compared to healthy tissue: on the plasmalemma of the mesophyll cells and on the secondary thickenings of the tracheid walls. This result is in accordance with several studies on the involvement of Ca2þ in the response to abiotic and biotic 392 R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 Fig. 13. High magnification images of KPA deposits (a) and maps of the Ca2þ made by Electron Energy Loss Spectroscopy (EELS) (b). Many scintillant dots are visible corresponding to precipitates. Bar: 100 nm. stress (Bush, 1993; Plieth, 2001). Moreover, it seems that Ca2þ ions are not directly involved in the phytoplasma replication, as no KPA precipitates were visible in the phloem and in the phytoplasma cells. Regarding phytoplasma cells, a few studies on the chemical composition of their membranes have been carried out (Sinha, 1979; Sinha and Madhosingh, 1980; Poggi Pollini and Giunchedi, 1992), using purified preparations; ours is the first attempt to analyze phytoplasmas in situ using X-ray microanalysis and the EELS. Fig. 14. High magnification images of KPA deposits (a) and maps of the Ca2þ made by Electron Energy Loss Spectroscopy (EELS) (b). Many scintillant dots are visible corresponding to precipitates. Bar: 0.2 mm. Acknowledgements This research was supported by funds from MIUR 2002, Italy. The authors are grateful to CIGS (Cento Interdipartimentale Grandi Strumenti), University of Modena, and to Prof. Alberto Bianchi of the Department of Evolutionary and Functional Biology, University of Parma, for their helpful technical assistance during X-ray microanalysis performance. R. Musetti, M.A. Favali / Micron 34 (2003) 387–393 References Bush, D.S., 1993. Regulation of cytosolic calcium in plants. Plant Physiol. 103, 7 –13. Carraro, L., Osler, R., Refatti, E., Favali, M.A., 1992. Natural diffusion and experimental transmission of plum leptonecrosis. Acta Horticulturae 309, 285 –290. Chandra, S., Low, P.S., 1997. Measurament of Ca2þ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells. J. Biol. Chem. 272 (45), 28274–28280. Favali, M.A., Barbieri, N., Bonecchi, R., 1978. Electron microscopy and Xray microanalysis of antimonate precipitates in healthy and virus infected leaves of tabacco. Caryologia 31 (3), 331–342. Favali, M.A., Barbieri, N., Bianchi, A., Bonecchi, R., Conti, M., 1980. Xray microanalysis of leaf tumors from maize plants experimentally infected with maize rough dwarf virus: scanning and transmission electron microscopic study. Virology 103, 357 –368. Favali, M.A., Musetti, R., Ragozzino, A., Camele, I., 1990. A study on peach decline. II. Transmission electron microscopy. Riv. Pat. Veg. S. IV 26, 11–22. Gelli, A., Higgins, V.J., Blumwald, E., 1997. Activation of plant plasma membrane Ca2þ permeable channels by race specific fungal elicitors. Plant Physiol. 113, 269 –279. Hayat, M., 1993. Stains and cytochemical methods, Plenum Press, New York. Lee, I., Gundersen-Rindal, D.E., Bertaccini, A., 1998. Phytoplasma: ecology and genomic diversity. Phytopathology 88 (12), 1359–1366. McAinsh, M.R., Hetherington, A.M., 1998. Encoding specificity in Ca2þ signaling systems. Trends Plant Sci. 3, 32–36. McCoy, R.E., Caudwell, A., Chang, C.J., Chen, T.A., Chiykowsky, L.N., Cousin, N.T., Dale, J.L., De Leeuw, G.T.N., Golino, E., Hackett, K.J., Kirkpatrick, B.C., Marwitz, R., Petzold, H., Sinha, R.C., Sugiura, M., Whitcomb, R.F., Yang, L.L., Zhu, B.M., Seemuller, E., 1989. Plant diseases associated with mycoplasma-like organisms. In: Whitcomb, R.F., Tully, G.J. (Eds.), The Mycoplasmas, vol. V. Academic Press, San Diego, CA, pp. 546– 562. Musetti, R., Favali, M.A., Carraro, L., Osler, R., 1992. An attempt to differentiate by microscopic methods two plant mycoplasma-like organisms. Cytobios 72, 71– 82. Musetti, R., Favali, M.A., Carraro, L., Osler, R., 1994. Histological detection of mycoplasma-like organisms causing leptonecrosis in plum trees. Cytobios 78, 81– 90. 393 Musetti, R., Scaramagli, S., Vighi, C., Pressacco, L., Torrigiani, P., Favali, M.A., 1999. The involvement of polyamines in phytoplasma-infected periwinkle (Catharanthus roseus L.) plants. Plant Biosystems 133 (1), 37– 45. Musetti, R., Favali, M.A., Pressacco, L., 2000. Histopathology and polyphenol content in plants infected by phytoplasmas. Cytobios 102, 133– 147. Pertot, I., Musetti, R., Pressacco, L., Osler, R., 1998. Changes in indole-3acetic acid level in micropropagated tissues of Catharanthus roseus infected by the agent of the clover phyllody and effect of exogenus auxins on phytoplasma morphology. Cytobios 95, 13–23. Plieth, C., 2001. Plant calcium signalling and monitoring: pros and cons and recent experimental approaches. Protoplasma 218, 1–23. Poggi Pollini, C., Giunchedi, L., 1992. Ultrastructural visualization of glucide determinants in membranes of mycoplasma-like organisms. J. Phytopathol. 136, 67–72. Price, A.H., Taylor, A., Ripley, S.J., Griffiths, A., Trewavas, A.J., Knight, M.R., 1994. Oxidative signals in tobacco increase cytosolic calcium. Plant Cell 6, 1301– 1310. Romeis, T., Piedras, P., Jones, J.D.G., 2000. Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defence response. The Plant Cell 12, 803– 816. Romeis, T., Ludwig, A.A., Martin, R., Jones, J.D.G., 2001. Calciumdependent protein kinase play an essential role in a plant defence response. EMBO J. 20 (20), 5556–5567. Scheel, D., 1998. Resistance response physiology and signal trasduction. Curr. Opin. Plant Biol. 1, 305– 310. Sinha, R.C., 1979. Lipid composition of mycoplasma-like organisms purified from clover phyllody and aster Yellows-affected plants. Phytopath. Z. 96, 132–139. Sinha, R.C., Madhosingh, C., 1980. Proteins of mycoplasma-like organisms purified from clover phyllody and aster yellows-affected plants. Phytopath. Z. 99, 294–300. Sheen, J., 1996. Ca2þ-dependent protein kinases and stress signal trasduction in plants. Science 274 (5294), 1900–1902. Stockwell, V., Hanchey, P., 1982. Cytohistochemical techniques for calcium localization and their application to diseased plants. Plant Physiol. 70, 244 –251. Zimmermann, S., Nurnberger, T., Frachisse, J.M., Wirtz, W., Guern, J., Hedrich, R., Sheel, D., 1997. Receptor-mediated activation of a plant Ca2þ-permeable ion channel involved in pathogen defense. Proc. Natl. Acad. Sci. USA 94, 2751–2755.