The Gunnera manicata: Nostoc Symbiosis: Is the Red Stipulate Tissue Symbiogenetic?

Abstract

THE GUNNERA MANICATA-NOSTOC SYMBIOSIS: IS THE RED STIPULATE TISSUE SYMBIOGENETIC? Jennifer Benson and Lynn Margulis Jennifer Benson, Department of Biology, Smith College, Northampton, Massachusetts 01063, UnitedStatesof America, andLynn Margulis(corresponding author), Organismic and Evolutionary Biology Program, Department of Geosciences, University ofMassachusetts Amherst, Amherst, MA 01002,UnitedStatesof America. THE GUNNERA-NOSTOC SYMBIOSIS All species of the angiosperm genus Gunnera form an intracellular symbiosis with hormogonium-pro ducing strains of Nostoc cyanobacteria. Gunnera is the only angiosperm genus known to enter symbi otic associations with cyanobacteria capable of fixing nitrogen. Unlike most symbioses, the Gun nera cyanobacterial associations are intracellular (Bergman et al. 1992). Of the 45 species of Gun nera, the smaller species are typically aquatic, whereas the larger species tend to occupy high-al titude cliff faces and stream banks (Osborne et al. 1991). Free-living Nostoc cyanobacteria live in the moist soils of the Gunnera habitats. They form long photosynthetic filaments that bear nitrogen-fixing heterocysts interspersed among the cells. The port of entry for Nostoc into Gunnera is via the unique glands present at the base of each leaf petiole in all Gunnera species (Bergman et al. 1992). Aseriate photoautotrophic Nostoc filaments that bear heterocysts are induced to differentiate into motile, heterocyst-free hormogonia filaments by various compounds present in the mucus secreted by the Gunnera gland tissue. The Nostoc hormogonia respond to a chemical signal and glide down the extracellular channels of the Gunnera gland, against the direction of both gland mucilage production and their usual positive phototaxis, to enter the Gunnera cortical cells. In the dark, Nostoc cells become heterotrophic, intracellular sym bionts. They rely on Gunnera for photosynthates, and the fixed nitrogen from their now re-differen tiated heterocysts is assimilated by the plant tissue. 'Whereas all Gunnera species, small and large, bear Nostoc-infecting glands, only the larger Gunnera species possess clusters of large red stipules that abundantly surround glands at the base of each leaf petiole (P1. IA-C). They are more distinc tively distributed, pigmented, mucous-laden and conspicuous than other plant stipules. We concur that the term 'stipule' (rather than bract or leaf bud) is the most accurate one for these structures. The focus of Gunnera-Nostoc symbiosis research is on the process by which Nostoc infects the Gunnera gland, not on the presence of Nostoc on the plant tissue surrounding the gland. Such an emphasis causes the stipulate tissue on the larger Gunnera species to be ignored in the literature, as it is often removed from the plant by researchers who have studied the gland tissue buried beneath. STIPULE MORPHOLOGY AND PATTERNS OF NOSTOC PRESENCE ON G. MANICATA STIPULE TISSUE Using phase-contrast and epifluorescence mi croscopy, we observed the morphology of the stipules and characterised the distribution and the form of Nostoc cyanobacteria on four clusters of stipules at various heights above the soil and stages of development. A group of stipules at the base of one developed leaf petiole or surrounding one developing leaf is referred to as a 'cluster'. Each cluster surrounded different glands, leaf petioles and inflorescences of one Gunnera manicata plant. We related the presence of Nostoc to the intensity of pigmentation and abundance of mucus on each respective stipule. A positive correlation between Nostoc presence and both intensity of red pigmentation and mucus abundance on the stipule can be inferred from our preliminary data (Chapman et al. 2002). Nostoc presence was un ambiguously correlated with mucus abundance as determined by phase-contrast and fluorescence mi croscopy (Table 1). Nostoc cells and gland mucus were absent on older, white stipule-tissue clusters that surrounded older, larger developed leaves, whereas the younger, bnrght-red, mucus-covered stipule clusters that surrounded and touched im mature leaves and inflorescences possessed Nostoc cyanobacteria either as single cells, aseriate fiaments or heterocyst-bearing seriate filaments. The correlation is evident even within a given cluster. Brighter-red and abundantly mucus covered stipules near the centre of a given cluster had a relatively higher presence of Nostoc cells than the less intensely coloured and lessmucus-covered outer stipules of the same cluster. Motile hor mogonia filaments were absent on all stipule tissue. The red pigmentation isvacuolar, present only in the vascular...