Immunocytochemical localization of cytokinin in Azolla filiculoides A. CHIAPPETTA & A. M. INNOCENTI Laboratorio di Botanica, Dipartimento di Ecologia, Universita ` degli Studi della Calabria, Arcavacata di Rende, Cosenza, Italy Abstract In the small aquatic fern Azolla filiculoides, cytokinin immunolocalization was performed in longitudinal axial sections of plantlet shoots. The reaction was detected: (i) in the contiguous cell sheet which encircles vascular tissues, (ii) in shoot and root meristem target cells, and (iii) in the teat cells of the leaf cavity pore. Our results demonstrate, for the first time, that in ferns the cytokinin translocation pattern can be different to that described in seed plants. Thus, this class of hormones is translocated, via vascular tissues in seed plants, whereas in Azolla it depends upon a sheet layer of cells encircling the conducting tissues. In shoot and root meristems, cytokinin distribution widely differs; in fact, in the shoot apex, the signal is present only in a few target cells, whereas in the root the signal is localized in numerous contiguous cells. Another finding concerns the clear signal observed at the level of the teat cells delimiting the pore which connect the leaf cavity with the exterior. This result provides indication that cytokinins, which are known to be involved also in light perception, might play a key role in the control of Anabaena movement into and out of the leaf cavity. This is the first report concerning cytokinin distribution in fern cells and tissues. Our results suggest that these hormones are implicated in the different plant organs in very different and specific functions. Key words: Cytokinin immunolocalization, zeatin, Azolla filiculoides, leaf cavity pore Introduction The water fern Azolla is the only genus within the Pteridophyta known to be able to form an association with a diazotrophic cyanobacterium. In fact, it maintains an association with the cyanobacterium Anabaena azollae living in its leaf cavities. This symbiosis is of agronomic interest as the nitrogen, fixed as ammonium (Meeks et al., 1987) by the cyanobacterium heterocysts, is released in the cavity of the fern leaf. Consequently, the Azolla – Anabaena association has been used, as a source of organic nitrogen, in flooded paddy fields to increase rice production (Nierzwicki-Bauer, 1990). The sub- cellular distribution of nitrogen compounds within the leaf cavities was investigated by ESI (Electron Spectroscopic Imaging) and EELS (Electron Energy Loss Spectroscopy) both in Anabaena cells and in epidermal hairs of Azolla (Albertano et al., 1993). Since surface antigens were found interacting with Anabaena filaments on the cell walls of epidermal hairs, a cell – cell interaction was postulated to trigger the translocation of fixed nitrogen products from the cyanobiont to the host (Tel-Or et al., 1991). The leaf cavities also contain heterotrophic eubacteria (Grilli Caiola et al., 1988; Plazinski et al., 1990) including Arthrobacter which, when isolated from the leaf cavities, is able to produce indole-3-acetic acid (IAA) in the presence of tryptophan (Forni et al., 1992) as does Anabaena (Forni, 1996). Thus, in the tripartite association Azolla, Anabaena and bacteria, all three partners have the ability to produce IAA. Inspite of the numerous studies concerning auxin, no information is available regarding cytokinins in Azolla, although these hormones, which influence fundamental processes of plant development, in- cluding cell division and morphogenesis (Deikman & Ulrich, 1995), have been widely studied in higher plants. The aim of the present work was to focus on cytokinin immunolocalization in the vascular cryp- togam Azolla filiculoides, and to compare the observed distribution of this class of hormones with the data available in higher plants. Correspondence: Adriana Chiappetta, Dipartimento di Ecologia, Universita ` della Calabria, Cubo 6B, 87030 Arcavacata di Rende (CS), Italy. Tel.: þ39 0984492963. Fax: þ39 0984492986. E-mail: a.chiappetta@unical.it Plant Biosystems, Vol. 140, No. 3, November 2006, pp. 229 – 233 ISSN 1126-3504 print/ISSN 1724-5575 online ª 2006 Societa ` Botanica Italiana DOI: 10.1080/11263500600756736