Gunnera is the only genus of angiosperms known to host cyanobacteria and the only group of land plants that hosts cyanobacteria intracellularly. Motile filaments of cyanobacteria, known as hormogonia, colonize Gunnera plants through cells in the plant's specialized stem glands. It is commonly held that Gunnera plants always possess functional glands for symbiosis. We found, however, that stem gland development did not occur when Gunnera manicata plants were grown on nitrogen (N)-replete medium but, rather, was initiated at predetermined positions when plants were deprived of combined N. While N status was the main determinant for gland development, an exogenous carbon source (sucrose) accelerated the process. Furthermore, a high level of sucrose stimulated the formation of callus-like tissue in place of the gland under N-replete conditions. Treatment of plants with the auxin transport inhibitor 1-naphthylphthalamic acid prevented gland development on N-limited medium, most likely by preventing resource reallocation from leaves to the stem. Optimized conditions were found for in vitro establishment of the Nostoc-Gunnera symbiosis by inoculating mature glands with hormogonia from Nostoc punctiforme, a cyanobacterium strain for which the full genome sequence is available. In contrast to uninoculated plants, G. manicata plants colonized by N. punctiforme were able to continue their growth on N-limited medium. Understanding the nature of the Gunnera plant's unusual adaptation to an N-limited environment may shed light on the evolution of plant-cyanobacterium symbioses and may suggest a route to establish productive associations between N-fixing cyanobacteria and crop plants.Nitrogen (N)-fixing cyanobacteria enter into symbiotic associations with a phylogenetically diverse group of eukaryotes ranging from fungi to vascular plants (Rai et al., 2000). Gunnera is the only flowering plant genus known to host cyanobacteria (Bonnett, 1990;Bergman et al., 1992;Bergman and Osborne, 2002). Plants in the genus Gunnera are mainly distributed in the southern hemisphere with about 40 species, which includes small, stoloniferous species (e.g. Gunnera magellanica) and plants 6 to 8 ft in height (e.g. Gunnera manicata). Even in the latter case, N 2 fixed by the symbiotic cyanobacteria, most likely in the form of NH 3 (Silvester et al., 1996), is able to fulfill the N needs of the plant (Osborne et al., 1991(Osborne et al., , 1992.In nature, Gunnera plants form symbioses with cyanobacteria belonging to the genus Nostoc (Bergman et al., 1992). Unlike other multicellular plant hosts, Gunnera harbors Nostoc intracellularly, surrounding the Nostoc filaments with a membrane most likely of plasma membrane origin (Silvester and McNamara, 1976;Johansson and Bergman, 1992). The specialized glands on the Gunnera stem serve as entry points for Nostoc filaments. Gunnera glands are conspicuously red due to the accumulation of anthocyanins such as pelargonidin and cyanidin (G. Lanigan, K.G. Black, and B. Osborne, unpublished data). Shortly after ge...