Immunocytochemical localization of Fe-SOD in different cells of Anabaena cylindrica Lemm. grown at two different photon irradiances

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1 New PhytoL (1993), 125, Immunocytochemical localization of Fe-SOD in different cells of Anabaena cylindrica Lemm. grown at two different photon irradiances BY MARIA GRILLI CAIOLA* AND ANTONELLA CANINI Department of Biology, University of Rome,' Tor Vergata', Via della Ricerca Scientifica Rome, Italy (Received 28 July 1992; accepted 15 May 1993) SUMMARY Superoxide dismutase (SOD) converts superoxide radicals into hydrogen peroxide and molecular oxygen and therefore represents the primary defence against oxygen damage. Using antibodies against Anabaena cylindrica Lemm. iron-sod, the isoenzyme was localized by immunogold labelling in vegetative cells, heterocysts and akinetes of the cyanobacterium Anabaena cylindrica grown at different light intensities. At a low photon irradiance (25 //mol photons m"^ s~'), the density of iron-sod labelling was similar in vegetative cells and in heterocysts, whereas in akinetes only 60 % of the gold particles were present as compared with the other cell types. At a higher photon irradiance (140 //mol photons m~'^ s"'), iron-sod labelling increased by 100 'o i" both vegetative cells and in heterocysts but did not change in akinetes. The cultures grown at the photon irradiance of 140/«mol photons m"^ s"' showed a rate of photosynthetic oxygen evolution 40 % higher than that of cultures grown at the lower photon irradiance. The increase of SOD labelling in vegetative cells is in line with the increased photosynthetic oxygen evolution induced by light. The unchanged SOD labelling observed in the akinetes suggests that light intensity does not seem to affect the oxidative metabolism due to respiratory activity. Key words: Anabaena cylindrica, superoxide dismutase, akinetes, heterocysts, cyanobacteria. amounts. The localization of Fe-SOD in heterocvsts INTRODUCTION r /i ; j T U or A. cyundrtca Lemm., where nitrogenase is Superoxide dismutase (SOD) converts superoxide present, led us to hypothesize a role for the SOD radicals, produced hy oxidative metabolism, into enzyme in the mechanism of nitrogenase protection hydrogen peroxide and molecular oxygen. For this against the toxic effect of superoxide radicals (Canini reaction the enzyme SOD represents the primary et al., 1992a). defence against oxygen damage. Based on the metals In Anabaena cylindrica, as well as in the cyanobiont present at the active site, the following four classes of Anabaena azollae, Fe-SOD activity increased when SOD have been described: copper/zinc (Cu, Zn- the cells were exposed to high light intensities SOD), iron (Fe-SOD), manganese (Mn-SOD) and (Canini et al., 1991; Grilli Caiola et al., 1991). This the hybrid, iron/manganese (Hy-SOD) (Bannister, increase of Fe-SOD could arise from an increase of Bannister & Rotilio, 1987). oxygenic photosynthesis or oxidative metabolism. Fe-SOD and Mn-SOD isoenzymes have been However, the true mechanism by which SOD detected in different cells of free-living cyanobacteria activity increases is not known. The increase of Feof the genus Anabaena (Grilli Caiola et al., 1991) SOD may be derived either from arfe >7Ofo synthesis whereas Hy-SOd, Fe-SOD and Mn-SOD, are found of the isoenzyme or from an increased activity of the in the symbiotic cyanobacterium Anabaena azollae pre-existent protein levels. Strasb. (Canini et al., 1991), and also in free-living Anabaena cylindrica appears to provide a suitable and symbiotic species of Nostoc (Canini et al., model for studying the role of SOD in cell 19926). In all the cyanobacteria examined, Fe-SOD metabolism since it is formed by cells with different is the class of enzyme which is present in highest metabolic activity. The vegetative cells carry aerobic respiration and oxygen-evolving photosynthesis. * To whom correspondence should be addressed. The heterocysts are capable of both nitrogen fixation

2 362 M. Grilli Caiola and A. Canini and aerobic respiration, but they lack oxygenevolving photosynthesis. The akinetes possess low levels of oxygen-evolving photosynthesis and lack nitrogenase activity (Fay, 1988). Akinetes diflferentiate from vegetative cells through modifications in structure and macromolecular composition, becoming more resistant to desiccation (Yamamoto, 1975) and to higher temperatures than vegetative cells (Reddy, 1983). However, until now few data are available on akinete differentiation and their metabolism. Some authors consider akinetes as quiescent cells (Nichols & Carr, 1978) whereas others have shown that they are metabolically active (Grilh Caiola & Favali, 1982; Thiel & Wolk, 1983). We therefore investigated SOD by immunogold labelling in these three different cell types of Anabaena cylindrica grown at different light intensities to obtain information on the synthesis of this enzyme and its role in the different metabolic processes. MATERIAL AND METHODS Organism Anabaena cylindrica Lemm. from the National Institute for Environmental Studies-Collection (Onogawa, Tsukuba, Japan) was grown aerobically in nitrogen-free BGll medium (Stanier et al, 1971) in a New Brunswick incubator (Edison, New Jersey, U.S.A.), at 25 C and at photon irradiances of 25 and 140/(mol photons m"^ s"^ Cells were harvested in exponential phase growth, and when akinetes were present. Photosynthetic oxygen evolution Photosynthetic oxygen evolution by 3 ml of exponentially growing cultures of A. cylindrica with the same optical density was measured polarographically at 25 C in a Clark-type Oj electrode (YSI model 5300, Yellow Springs Instrument Co., Yellow Springs, Ohio). The electrode was calibrated with air-saturated water. The cultures were continuously mixed with a magnetic stirring bar. Photon irradiance incident upon the chamber was 90 /tmol photons m~^ s"'. Respiratory oxygen consumption was monitored maintaining the chamber of the electrode in the dark. Immunogold labelling at TEM (transmission electron microscopy) A. cylindrica cells were fixed in 2-5 % glutaraldehyde in 0-2 M Na-cacodylate buffer, ph 7-2, overnight at 4 C. Cells were enclosed in 1 % agar and embedded in Epon 812 resin (Agar Aids Ltd, England) after dehydration ofthe samples in a graded ethanol series and propylene oxide. Thin sections were mounted on 200-mesh nickel grids. Immunogold-labelling was carried out according to Canini et al. (1992a). The primary antibody was a rabbit anti-fe-sod antibody diluted 1:500, and the secondary was a goat anti-rabbit IgG antibody conjugated with 10-nm colloidal gold particles at a dilution 1:25. The grids were observed with a Zeiss CEM 902 operating at 80 kv. Controls were performed by omitting the primary Fe-SOD antibody, and using preimmune serum instead ofthe Fe-SOD antibody. The density of the gold particles was counted on at least 5 electronmicrographs of each cell type obtained at the same magnification. RESULTS Photosynthetic oxygen evolution Photosynthetic oxygen evolution values for A. cylindrica cultures grown at photon irradiances of 25 and 140 //mol photons m '^ s ' are shown in Table 1. When the cyanobacterium was grown at a photon irradiance of 140//.mol photons m~'^ s"', the rate of oxygen evolution increased by 40 %. Fe-SOD immunogold labelling on A. cylindrica Cells grown at a photon irradiance of 25 /J,mol photons m'^s~^. Fe-SOD was located in vegetative cells, in heterocysts and in akinetes. Gold particle density was similar in heterocysts and in vegetative cells (Table 2). In heterocysts, Fe-SOD was located through the cytoplasm (Fig. 1), whereas in vegetative Table 1. Photosynthetic oxygen evolution in cultures of Anabaena cylmdrica grown at photon irradiances of 25 and 140 fimol photons m~^ s~^ Photon irradiances* * fimox photons m ^ s '. t //I Oj ml"' culture h~'. The values are the means+ SD. Oxygenf ±1 Table 2. Gold particle density of Fe-SOD isoenzyme localized in vegetative cells, heterocysts and akinetes of Anabaena cylindrica.^rown at photon irradiances of 25 and 140 fimol photons w" s"* Vegetative cells Heterocysts Akinetes Fe-SOD* (Gold particle 25t * Gold particle density //m"^. t //.mol photons m~^ s~'. The values are the means+ SD. density /im ') 140t

3 Fe-SOD localization in Anabaena cells Figures 1-3 Figure 1. Fe-SOD labelling in a heterocyst of Anabaena cylindrica grown at a photon inadiance of 25 //mol photons m"" s"'. Gold particles (10 nm) are located within the cytoplasm, c = cytoplasm; e = envelope. Bar = 1 iim. Figure 2. Fe-SOD labelling in a vegetative cell of Anabaena cylindrica grown at a photon irradiance of 25 //mol photons m"^ s"'. Fe..SOD is localized in the nucleoplasm (n) and associated with the thylakoids (t). Bar = 1 iim. Figure 3. Localization of Fe-SOD in a transverse section of an akinete from Anabaena cylindrica grown at a photon irradiance of 25 //mol photons ni'^ s"'. Fe-SOD labelling occurs mainly within the cytoplasm. Some particles are localised on the envelope (e). Bar = 1 //m. 363

4 364 M. Grilli Caiola and A. Canini Figures 4-6 Figure 4. Localization of F'e-SOD in a heterocyst of Anabaena cylindrica grown at a photon iriadiance of 140//mol photons m"^ s '. Gold particles have increased in the cytoplasm. Bar = 1 //m. Figure 5. Fe-SOD labelling in a vegetative cell of Anabaena cylindrica grown at a photon irradiance of 140 //mol photons m"^ s"'. Bar 1 fim. Figure 6. Fe-SOD labelling in a longitudinal section of an akinete from Anabaena cylindrica grown at a photon irradiance of 140 //mol photons m"^ s~'. The distribution of gold particles is the same as in Figure 3, Bar = 1 //.m.

5 Fe-SOD localization in Anabaena cells cells the enzyme was present in the nucleoplasm and associated with the thylakoids (Fig. 2). In contrast, akinetes showed a low level of Fe-SOD labelling with respect to the other cell types (Table 2). FeSOD labelling of akinetes was mainly within the cytoplasm (Fig. 3). However, a few gold particles were also localized on the envelope of the akinetes. As demonstrated in our previous work (Canini et al., 1992a) no significant gold labelling was observed on the cell sections when the primary antibody was omitted or when rabbit serum was used instead of the Fe-SOD antibody. Cells grown at a photon irradiance of 140 /itnol photons m^^ s~^. In Anabaena cylindrica cells grown in the same nutritional conditions and temperature but at a higher irradiance, Fe-SOD labelling increased 100 % in both vegetative cells and in heterocysts (Table 2). In heterocysts the increase was throughout the cytoplasm (Fig. 4) whereas in vegetative cells it was mainly over the thylakoids (Fig. 5). In contrast, the gold particle density in akinetes showed no change between low and high irradiances (Table 2; Fig. 6). DISCUSSION 365 bolically active, and should not be considered as entirely quiescent cells (Fay, 1969; Grilli Caiola & Favali, 1982). Within these cells, superoxide radicals are mainly generated from respiration. Since gold particle density in akinetes is approximately 60 % that in vegetative cells and heterocysts, aerobic respiration in these cells may generate oxygen-active species. Whereas the increase of superoxide radicals in vegetative cells could be explained by an increased photosynthetic oxygen evolution, the enhancement of Fe-SOD in heterocysts is more likely due to a higher rate of respiration in order to provide more reductant for nitrogen-fixation which was found to increase with the light intensity (Grilli Caiola et al., 1991). In akinetes the increased light intensity does not affect the Fe-SOD content and localization. This could be due to the composition of the thick cell envelope or to a steady basic aerobic metabolism which is unaffected by light. In isolated akinetes of Anabaena circinalis Lemm. exposed to light. Fay (1988) reported an increased rate of respiratory oxygen uptake rather than photosynthetic oxygen evolution. He showed that stimulation of respiratory activity was more pronounced at lower light intensities, whereas at higher light intensities the oxygen uptake rate became saturated. However, akinetes of A. cylindrica differentiate from vegetative cells which contain twice the amount of SOD content with respect to mature akinetes. Thus degradation of the amount of SOD synthesized or a loss in SOD activity must be taking place during akinete differentiation. The present results are different from those obtained by biochemical assays which demonstrated increased amounts of Fe-SOD in akinetes grown at higher light intensities (Grilli Caiola et al., 1991). However, immunolabelling is a more specific assay of Fe-SOD in akinetes which are at the same developmental stage. The in situ labelling of FeSOD appears to provide clear indications of the metabolic activity of different cell types in Anabaena cvlindrica. Further information on the synthesis and role of Fe-SOD in the akinetes would be obtained by assaying Fe-SOD in germinating akinetes. The data presented here indicate that Fe-SOD labelling density is similar in vegetative cells and heterocysts. The present results differ from the previous ones obtained by biochemical assay (Grilli Caiola et al., 1991) in which heterocysts contained only 20% of the SOD activity found in vegetative cells. However, the immunogold labelling was carried out on whole filaments whereas the biochemical assay was made on isolated heterocysts. It is therefore possible that during the isolation procedure preceding the biochemical assay, not all heterocysts remained completely healthy. On the otber hand, a high SOD protein content is not always correlated with a high SOD activity. Immunogold labelling detects only the presence of an antigen which in vivo may be either active or nonactive. In vegetative cells, Fe-SOD may be related to the presence of superoxide radicals produced from both oxygen-evolving photosynthesis as well as by aerobic respiration. The increased rate of oxygen evolution measured in cultures grown at the higher photon ACKNOWLEDGEMENTS irradiance (140/(.mol photons m~^ s^') affects the production of superoxide radicals in these cells. In This work was supported by the Ministry of University Scientific and Technological Research (MURST). contrast, the high content of Fe-SOD in the heterocysts, which lack PSII, and are surrounded by a thick envelope, correlates with superoxide radicals REFERENCES mainly produced from aerobic respiration. Bannister JV, Bannister WH, Rotilio G Aspects of the.structure, function, and.npplication of superoxide dismutase. Akinetes do not carry out nitrogen fixation and CRC Critical Revie^cs in Biochemistry 22: oxygen-evolving photosynthesis but they appear Canini A, Civitareale P, Marini S, Grilli Caiola M, Rotilio G. capable of endogenous respiration as recorded for 1992a. Purification of iron-superoxide dismutase from the cyanohacterium of Anabaena cylindrica Letntn. and localization the mature spores (Fay, 1969). Although they show of the enz\me in heterocysts hy immunogold laheling. Planta low labelling in comparison with that recorded in 187; vegetative cells and heterocysts, they appear meta- Canini A, Galiazzo F, Rofilio G, Grilli Caiola M

6 366 M. Grilli Caiola and A. Canini Superoxide dismutase in the symbiont Anabaena azollae Stiasb. Plant Physiology 97: Canini A, Grilli Caiola M, Civitareale P, Galiazzo F Superoxide dismutase in symbiont, free-living and wild Anahaena and Nostoc (Nostocales, Cyanophyta). Phycolo/^ia 31: Fay P Metabolic activities of isolated spores of Anabaena cylindrica. Journal of Experimental Botany 20: Fay P Viability of akinetes ofthe planktonic cyanobacterium Anabaena circinalis. Proceedings of the Royal Society of London B 234: Grilli Caiola M, Canini A, Galiazzo F, Rotilio G Superoxide dismutase in vegetative cells, heterocysts and akinetes of Anabaena cylindrica Lemm. FEMS Microbiology Letters 80: Grilli Caiola M, Favali A Electron microscope autoradiography of Anabaena sp. and Nostoc sp. akinetes labelled with ''H-uridine. Microbiologica 5: Nichols JM, Carr NG Akinetes of cyanobacteria. Spores 7: Reddy PM Effects of temperatlire pretreatment, desiccation and aging on the viability of spores of lialophylic bluegreen algae. Hydrobiologia 106: Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriological Reviews 35: Thiel T, Wolk CP Metabolic activities of isolated akinetes of the cyanobacterium. Nostoc spoitgiacforme. Journal of Bac.- teriology 156: Yamamoto Y Effect of desiccation on the germination of akinetes of Anabaena cvlindrica. Plant and Cell Physiology 16:

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