Developmental patterns in the Nostoc-Gunnera symbiosis

Abstract: Gunneraceae is an angiosperm family comprising about 60 species scattered in the southern hemisphere. All members of the family develop a nitrogen-fixing symbiosis with intracellularly located Nostoc. The aim of the present study was to characterize the symbiosis, structurally and physiologically, with emphasis on G. magellanica and G. chilensis.Activity of nitrogenase, the nitrogen fixing enzyme, showed a distinct developmental sequence along the plant stem, with peak activity in intermediately aged symbiotic tissues. Heterocysts, the Nostoc cells carrying the nitrogenase protein, showed extremely high frequencies in the cyanobiont (60% compared to 5% in free-living Nostoc). However, nitrogenase activity and heterocyst frequency were coordinated only in young parts. Maximal nitrogenase activity occurred before maximal heterocyst frequencies were reached. After the peak, the nitrogenase activity rapidly dropped, even though nitrogenase protein was largely retained.Compared to free-living isolates, the glutamine synthetase (GS) activities of the cyanobiont were lowered and GS protein levels of the heterocysts reduced to the levels seen in vegetative cells. Gunnera cell extracts showed considerable GS activity as well as GS protein levels, with peak activities coinciding with that of the nitrogenase. These results indicate that ammonia is the nitrogenous compound released by the cyanobiont and subsequently assimilated by plant GS.The photosynthetic ability was diminished in the cyanobiont, although pigments (light capturing) and ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) were present and at similar levels as in free-living cyanobacteria. An impaired photosynthetic electron transport is proposed to cause this inactivation. Without a light-dependent CO2 fixation the cyanobiont is obliged to live heterotrophically on plant-derived photosynthates to maintain e.g. a nitrogenase activity. A fast translocation of fixed carbon from leaves to symbiotic tissues was demonstrated, possibly reaching the cyanobiont via the polystelic vascular strands and the non-infected Gunnera cells interspersing the symbiotic tissue. Carbon loading into symbiotic tissues showed a similar developmental sequence as nitrogenase and GS activities, with a peak in intermediately aged tissues. The decline in symbiosis-related activities noted coincided with senescence and shedding of nearby leaves. This was taken to indicate that the plant controls the symbiotic activities through the delivery of photosynthates. Symbiotic cells also showed structures indicative of a high metabolite exchange between the symbionts. These included highly convoluted membranes surrounding the cyanobiont and high numbers of mitochondria and Golgi bodies. Structurally, the symbiotic system in many respects resembled that of other intracellular nitrogen-fixing symbiosis (e.g. Rhizobium and Franlda symbiosis). Compatibility is restricted to Nostoc, but the specificity whithin the genus is low as evidenced by differences in pigmentation and colony morphology among the numerous isolates obtained from Gunnera spp. growing in various parts of the world.