Nitrogen Deprivation Stimulates Symbiotic Gland Development in Gunnera manicata

Abstract

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...

Multiple Roles of Soluble Sugars in the Establishment of Gunnera-Nostoc Endosymbiosis

“…The Nostoc-Gunnera association is an ideal system with which to study plant-cyanobacteria symbioses, not only because Gunnera is the only genus of angiosperms known to form endosymbioses with N 2 -fixing cyanobacteria but also because the association between the two can be readily established in the laboratory (Bergman et al, 1992;Chiu et al, 2005). Nostoc hormogonia enter Gunnera plants through specialized glands located on the stem.…”

“…Nostoc hormogonia enter Gunnera plants through specialized glands located on the stem. As the gland matures, it secretes polysacchariderich mucilage that attracts cyanobacteria (Nilsson et al, 2006), supports their growth on the gland surface (Towata, 1985;Chiu et al, 2005), and permits further hormogonia differentiation (Rasmussen et al, 1994). From there, hormogonia enter the gland and penetrate cells near the base of the gland in the stem (Bonnett, 1990;Bergman et al, 1992).…”

Plastid origin: who, when and why?

“…The vertically transmitted endosymbiont (Anabaena) of the pteridophyte Azolla, which was reduced to an organism devoted to nitrogen fixation, is a multicellular cyanobacterium [89]. Another example is the angiosperm Gunnera manicata that uses the nitrogen provided by the filamentous symbiont (Nostoc punctiforme) and continues growing under N-limited conditions in the presence of the symbiont [90]. In gymnosperms, a nitrogen-fixing cyanobacterial symbiont (Nostoc) was found in the roots of most known cycad species [91,92].…”

“…It has been shown that nitrogen deprivation stimulates modern symbiotic associations [86,90] and that nitrogenrich conditions on the contrary facilitate the dissipation of pre-established symbiotic relationships [86]. If nitrogen was the key factor for the host and symbiont to form the initial association, it is congruent with the finding that present-day members of filamentous, heterocyst-forming and N 2 -fixing cyanobacterial sections IV and V have a collection of genes most similar to that possessed by the plastid ancestor [39,46].…”

“…Another example is the angiosperm Gunnera manicata that uses the nitrogen provided by the filamentous symbiont (Nostoc punctiforme) and continues growing under N-limited conditions in the presence of the symbiont [90]. In gymnosperms, a nitrogen-fixing cyanobacterial symbiont (Nostoc) was found in the roots of most known cycad species [91,92].It has been shown that nitrogen deprivation stimulates modern symbiotic associations [86,90] and that nitrogenrich conditions on the contrary facilitate the dissipation of pre-established symbiotic relationships [86]. If nitrogen was the key factor for the host and symbiont to form the initial association, it is congruent with the finding that present-day members of filamentous, heterocyst-forming and N 2 -fixing cyanobacterial sections IV and V have a collection of genes most similar to that possessed by the plastid ancestor [39,46].…”