The Nostoc-Gunnera symbiosis: carbon fixation and translocation

The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4. 1. 1. 39) and the dark and light in vivo CO₂ fixation activities were determined in the cyanobiont of Gunnera . Compared to the free-living isolate Nostoc PCC 9231, the in vitro Rubisco activity was high, while the in vivo CO₂ fixation was very low. Light did not significantly influence CO₂ fixation if the cyanobiont was left in the sliced Gunnera tissues, while a small light stimulation was found for CO₂ fixation of the freshly-isolated cyanobiont. The adjacent non-infected Gunnera tissue showed a very low CO₂ fixation. A rapid translocation of fixed ¹⁴CO₂ from leaves towards apical parts of the plant was apparent, in particular to the symbiotic tissue. The ¹⁴C label appeared mainly in soluble form in this tissue and was rapidly catabolised as shown by ¹⁴C chase experiments. Also, short-term experiments revealed that maximum ¹⁴C accumulation occurred in the symbiotic tissue showing the highest rates of nitrogen fixation (Söderbäck et al. 1990), about 10–15 mm from the plant apex. The data were taken to indicate that there is a modification in the photosynthetic light reaction of the cyanobiont and that the cyanobiont lives heterotrophically in the dark on photo-synthate rapidly delivered from nearby leaves of the host plant.     

Cloning and expression of a putative cyclodextrin glucosyltransferase from the symbiotically competent cyanobacterium Nostoc sp. PCC 9229

A polymerase chain reaction-based method was used to isolate a Nostoc sp. PCC 9229 cDNA from infected glands of Gunnera chilensis. The complete gene sequence was isolated from a genomic Nostoc sp. PCC 9229 library. Sequence analysis showed 84% amino acid similarity to a putative cyclodextrin glycosyltransferase from Nostoc sp. PCC 7120 and the gene was therefore termed cgt. Southern blot revealed that the cgt gene was present in symbiotically competent cyanobacteria. The cgt gene was expressed in free-living nitrogen-fixing cultures in light or in darkness when supplemented with fructose. This is the first expression analysis of a cgt gene from a cyanobacterium.     

Comparison of photosynthesis and productivity of Gunnera tinctoria Molina (Mirbel) with and without the phycobiont Nostoc punctiforme L.

Abstract. Marked increases in growth and nitrogen content were found with Gunnera tinctoria Molina (Mirbel) plants infected (+ Nostoc ) with the cyanobacterium Nostoc punctiforme L., in comparison to uninfected (— Nostoc ) plants and this was attributed to N₂-fixation by the phycobiont. Whilst host and symbiont can be grown separately, preliminary data indicates that the host plant is reliant on the cyanobacterium to meet its nitrogen requirements because it has little capacity to assimilate nitrate. Although the maximum light-saturated rate of photosynthesis was higher in the + Nostoc plants, there was no reduction in photosynthetic efficiency under lightlimiting conditions, despite marked differences in plant nitrogen status. Differences in photosynthetic rate were implicated as the major reason for the differences in plant productivity. Stomatal conductance was insensitive to changes in plant nitrogen status and did not parallel the variation in photosynthetic rates. The ecological significance of the largely invariant stomatal response and the consequences of differences in water and nitrogen-use efficiencies between + and — Nostoc plants is discussed.     

Uninduced ammonia release by the nitrogen-fixing cyanobacterium Anabaena

Abstract Certain strains of the nitrogen-fixing cyanobacterium Anabaena were found to release varying quantities of ammonia without any induction, both in the presence and absence of combined nitrogen (nitrate) in the medium, during the different phases of their growth. In general, growth and ammonia release were comparable in both media, although there were strain differences. 3 patterns of ammonia release were observed in different strains during the growth period. They were: (1) release pattern parallel to the growth curve; (2) a continuous increase in release; and (3) release showing a bimodal curve.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Enzymes of ammonia assimilation in root nodules of Trigonella foenum-graecum

The main pathway of ammonia assimilation in the root nodules of Trigonella foenum-graecum is via nodule cytosol glutamine synthetase-glutamate synthase.     

Interactions of H2 and carbon metabolism in moderating nitrogenase activity of the Gunnera/Nostoc symbiosis

Nitrogenase activity in the Gunnera Nostoc symbiosis is shown to respond dramatically to the addition of glucose. H₂ can replace glucose in stimulating nitrogenase activity, but there is no H₂ stimulation in the presence of excess glucose. Net hydrogen evolution is strongly stimulated by addition of glucose. We postulate that carbohydrate supply and uptake hydrogenase can moderate the apparent activity of nitrogenase by supplying reductant and/or ATP. The recycling of a large proportion of the electron flux in nitrogenase through uptake hydrogenase maintains a high level of potential nitrogenase ready to take advantage of an influx of carbohydrate.     

The α-amylase inhibitor of bean seed: two-step proteolytic maturation in the protein storage vacuoles of the developing cotyledon

In the nitrogen fixing symbiosis between Nostoc and the angiosperm Gunnera , the cyanobiont is found in stem glands and is thought to have a heterotrophic mode of nutrition. To investigate whether the photosynthetic machinery in the cyanobiont is down-regulated in the symbiosis, the presence of the phycobiliproteins, phycoerythrin and phycocyanin, and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) in cyanobionts of Gunnera magellanica Lam. and in a free-living (cultured) isolate of the cyanobacterium was studied by immunoelectron microscopy. Carboxysomes were numerous in all vegetative cells (ca 3.5 per cell section), and on an area basis they showed a high Rubisco label compared to the cytoplasm; but recalculation on a volume basis demonstrated that the carboxysomal fraction of Rubisco decreased in the cyanobiont along the plant stem. Along the whole Gunnera stem both types of phycobiliproteins were present in the symbiotic Nostoc and in amounts equivalent to or above those detected in the free-living isolate. As the symbiotic Nostoc is located intracellularly, out of reach of light in the plant stem, the findings indicate a lack of regulation of the photosynthetic protein synthesis in the symbiotic state.     

Ammonium assimilation and amino acid metabolism in conifers

Conifers are the most important group of gymnosperms, which include tree species of great ecological and economic importance that dominate large ecosystems and play an essential role in global carbon fixation. Nitrogen (N) economy has a special importance in these woody plants that are able to cope with seasonal periods of growth and development over a large number of years. As N availability in the forest soil is extremely low, efficient mechanisms are required for the assimilation, storage, mobilization, and recycling of inorganic and organic forms of N. The cyclic interconversion of arginine and the amides glutamine and asparagine plays a central role in the N metabolism of conifers and the regulation of these pathways is of major relevance to the N economy of the plant. In this paper, details of recent progress in our understanding of the metabolism of arginine and the other major amino acids glutamine, glutamate, aspartate, and asparagine in pine, a conifer model tree, are presented and discussed.     

Pathway of ammonia assimilation in illuminated Lemna minor

Plants of duckweed (Lemna minor) were grown under constant illumination and with a controlled supply of ammonium-N so as to maintain a constant low concentration. In two kinetic experiments (differing in illumination and N level) with ¹⁵N-ammonia, plants were periodically harvested and their free amino acids analysed for ¹⁵N abundance. Attempts were then made to fit the data by computer simulation models. Only models which had at least two or more intracellular compartments gave adequate fits. Two two-compartment models were tested fully. Both had in compartment 1 the glutamine synthetase-glutamate synthase cycle and in compartment 2 a second site of glutamine synthesis. In one model the glutamate for compartment 2 was derived by transport from compartment 1; in the second model it was synthesized from ammonia by glutamate dehydrogenase at a rate equivalent to 10% of the total N uptake. This second model was rejected after it was found that plants previously treated with methionine sulphoximine and aza-serine (inhibitors of the glutamate synthase cycle) were unable to incorporate ¹⁵N. In spite of wide differences in labelling pattern between the two experiments the first model gave acceptable fits to both when different pool sizes were allowed for. Operation of the glutamate synthase cycle was confirmed by the correspondence between model and data for labelling of glutamine amide, glutamine amino and glutamic acid. Consideration of enzyme distributions suggested that compartment 1 (the glutamate synthase system) is the chloroplasts and compartment 2 the cytosol. Analysis of asparagine and neutral amino acids made it possible to construct balance sheets for N uptake in the two experiments. They suggest that all glutamine synthesized in the chloroplast is used for glutamate and asparagine synthesis and that the cytosol enzyme meets the need of the cell for glutamine per se. The high turnover rates for asparagine indicate that this compound is an important intermediate even under steady state conditions, and carries between 20 and 50% of the products of N assimilation.     

Geosiphon pyriforme,an Endosymbiotic Consortium of a Fungus and a Cyanobacterium (Nostoc), Fixes Nitrogen

The bladders of Geosiphon pyriforme, an endosymbiotic consortium of a fungus and the cyanobacterium Nostoc punctiforme, show nitrogenase activity. This suggests that the organism is capable of nitrogen fixation.     

Effects of nitrogen source on the synthesis of the UV-screening compound, scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102

The effects of nitrogen source (N(2), NO(3)(-) and NH(4)(+)) on scytonemin synthesis were investigated in the heterocystous cyanobacterium Nostoc punctiforme PCC 73102. With the required UVA radiation included, Nostoc synthesized three to seven times more scytonemin while fixing nitrogen than when utilizing nitrate or ammonium. A similar increase in scytonemin synthesis occurred when nitrate or ammonium became depleted by growth and Nostoc switched to diazotrophic metabolism with the differentiation of heterocysts. In addition, UVA-exposed cultures grown in medium with both NO(3)(-) and NH(4)(+) synthesized some scytonemin but synthesis increased when NH(4)(+) was depleted and growth had become dependent on NO(3)(-) reduction. Although the mechanism is unclear, these results suggest that the greater the restriction in nitrogen accessibility, the greater the production of scytonemin. Perhaps the entire response may be an interaction between this restriction and a resultant sensitivity to UV radiation that acts as a cue for determining the level of scytonemin synthesis. Scytonemin is a stable UVR screening compound and appears to be synthesized by cyanobacteria as a long-term solution for reducing UVR exposure and damage, but mainly or solely, when metabolic activity is absent. It is likely that during metabolic resurgence, the presence of a dense scytonemin sheath would facilitate the recovery process without the need for active defenses against UV radiation.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Nitrogen nutrition and pathway of ammonia assimilation in brown Rhodospirillum species

Abstract Nine strains of brown rhodospirilla, i.e. Rhodospirillum photometricum, R. molischianum and R. fulvum were examined with respect to nitrogen nutrition and the pathway of ammonia assimilation. R. photometricum strains were nutritionally more versatile than strains of the other two species; glutamate, aspartate, and several other amino acids supported good growth of R. photometricum but were poorly utilized by R. molischianum and R. fulvum . Glutamine and N₂ supported excellent growth of strains of all species. The glutamine synthetase/glutamate synthase pathway served as the major means of ammonia assimilation in brown rhodospirilla; no evidence for glutamate dehydrogenase was obtained from any species. NADPH was required as coenzyme for glutamate synthase activity in R. photometricum strain while only NADH served in this connection in R. molischianum and R. fulvum .     

Azolla and other plant-cyanobacteria symbioses: Aspects of form and function

Nostoc, a genus of filamentous, heterocystous, cyanobacteria, is widely distributed in the free-living state. It is also the most common phycobiont in N₂-fixing lichens and occurs as the N₂-fixing symbiont in a small and diverse group of green plants. These include several bryophyte genera (e.g. Anthoceros and Blasia), a pteridophyte genus (Azolla; while the symbiont is referred to asAnabaena azollae, it may be aNostoc spp.), a division of gymnosperms (the 10 cycad genera) and one angiosperm genus (Gunnera). In Gunnera the Nostoc apparently penetrates into the cells of the host. In the other associations Nostoc is extracellular but specific morphological modifications and/or structures of the host plant organs create an environment which fosters interaction and metabolite interchange.The individual group of Nostoc-green plant symbioses other than Azolla are summarized in regard to the current understanding of their establishment, perpetuation, and host-symbiont interaction. This includes available information on recognition and specificity, mode(s) of infection if applicable, and a synopsis of morphological modifications of the partners. The symbiosis withAzolla is then addressed separately with a more indepth account of the foregoing areas. In addition, the concept ofAzolla harboring a dominant, obiligately symbiotic Nostoc which has not been cultured as well as minor symbionts capable of free-living growth, the distinction between re-constituting and simply re-establishing the symbiosis, and current approaches to improving the symbiosis and to authenticating the establishment of new associations are considered.