The Azolla, Anabaena azollae Relationship

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The N2-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azoila endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N2 fixation by the symbiont.The Azolla-Anabaena azollae symbiosis is an N2-fixing association between a eukaryotic fern and a prokaryotic alga, both of which exhibit a higher plant type of photosynthesis (18,19).As an approach toward the characterization of the role of the host and symbiont4 in the association, it was considered important to determine the contribution of the partners to the association's Chl, protein, and ammonia-assimilating enzymes. While the alga could be isolated free of the fern, its removal from the host was never complete. Thus it was necessary to find an independent quantitative estimate for the amount of alga in the association. While nitrogenase is unique to the alga (21) its lability makes it unsuitable for quantitation. Chl a/b ratios provided another estimate, but this was based on the absence of Chl b from the alga (20) and was therefore not as sensitive. A third method, the quantitation of phycobilins which are unique to the alga in the association, was employed. A procedure involving fluorescence emission spectroscopy at 77 K permitted quantitative measurement of phycobilins in the association, where the fern's Chl b '

Section: Acetylene Reduction Assays-nitrogenasementioning

confidence: 99%

“…Thus it was necessary to find an independent quantitative estimate for the amount of alga in the association. While nitrogenase is unique to the alga (21) its lability makes it unsuitable for quantitation. Chl a/b ratios provided another estimate, but this was based on the absence of Chl b from the alga (20) and was therefore not as sensitive.…”

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confidence: 99%

Azolla-Anabaena Relationships

Ray

Peters²,

Toia³

et al. 1978

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“…The rapid growth of this fern and its ease in handling for laboratory studies make this plant particularly attractive as experimental material for study of plant nutrition in a symbiotic system. Relatively little work had been done with this system until Peters and Mayne (4,5) recently made some initial characterizations of the relationship and showed the essential nature of the algae for growth of plants on N-free media.…”

mentioning

confidence: 99%

Altered Nitrogenous Pools Induced by the Azolla-Anabaena Azolla Symbiosis

Newton¹,

Cavins²

1976

The free amino acid and ammonia pools of Azolla caroliniana were analyzed by quantitative column chromatography on columns capable of separating all of the nitrogenous constituents normally found in physiological fluids. Comparisons were made of plants containing symbiotic algae and grown on nitrogen-free media, plants grown on media containing nitrate, and algae-free plants also grown on nitrate media. The major feature of the data was a very high level of intracellular ammonia found in plants which coutain N2-flxing algal symbionts. In addition to the more usual amino acids, serine and cystathionine were found in the free amino acid pool.The genus Azolla, small aquatic ferns of worldwide distribution, generally contains symbiotic algae resembling Anabaena sp. within their leaf cavities (3). These algae are capable of fixing N2 and consequently can supply all of the nitrogenous nutrient requirements of the fern. The rapid growth of this fern and its ease in handling for laboratory studies make this plant particularly attractive as experimental material for study of plant nutrition in a symbiotic system. Relatively little work had been done with this system until Peters and Mayne (4, 5) recently made some initial characterizations of the relationship and showed the essential nature of the algae for growth of plants on N-free media.To provide a basis for more extensive studies of the N nutrition of this symbiosis, we have recently separated and quantitatively analyzed the components of the free nitrogenous pools in these ferns with and without algal symbionts, and the results are presented in this communication. RESULTS AND DISCUSSIONTo provide as broad a base as possible for our comparisons, we have analyzed several dozen samples of different plant tissues of different ages. Plants were usually collected and immediately treated with boiling water to avoid deterioration which might alter pool contents or composition, and samples were either analyzed directly or stored frozen for subsequent analysis. In general, we found the pool size and composition to be rather uniform from one age of plant culture to another, provided the plants were grown under similar conditions. Data in Table I summarize the major effect we have noted in the pools and compares ammonia analyses obtained by microdiffusion and colorimetry and the chromatographic method. As expected, ammonia content determined by direct analysis of extracts was higher, probably due to decomposition of amides during analysis. It should be noted that the levels of ammonia found in N2-fixing plants are too high to be accounted for by hydrolysis of glutamine or any other pool constituent during sample preparation or analysis. The pool of intracellular ammonia in N2-fixing plants can rise to as much as five to 10 times that found in plants grown on media containing a N source, and can constitute as much as half of the total pool N. This effect is greatest when one compares algae-containing and algae-free plants. It should be noted that the growth rates of p...

Section: Introductionmentioning

confidence: 99%

“…In the intact association, the alga can provide the Azolla plant with its total nitrogen requirement. Previous studies showed that the symbiont contained nitrogenase and was capable of C2H2 reduction, ATP-dependent H2 evolution, and excretion of ammonia (10,11,13).Although C2H2 reduction is a simple and sensitive assay of nitrogenase activity, C2H2 is not the biologically important substrate, and the assay is an indirect measurement of nitrogen fixation. Since the reduction of N2 to 2NH3 requires six electrons while reduction of C2H2 to C2H4 requires two electrons, a theoretical conversion factor of 3C2H2 reduced per N2 fixed is frequently used in estimating nitrogen fixation from C2H2 reduction assays.…”

mentioning

confidence: 99%

Azolla-Anabaena azollae Relationship

Peters

Toia²,

Lough³

1977

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In order to characterize the reactions catalyzed by nitrogenase in the Azolla-Anabaena association, '5N2 fixation, C2H2 reduction, and ATPdependent H2 production were measured in both the Azolla-Anabaena complex and in the alga isolated from the complex.The rate of reduction of substrates and of ATP-dependent H2 evolution was determined at varous partial pressures of C2H2 and N2. A pC2H2 of 0.1 atm was nearly optimal for C2H4 production and inhibited H2 production by 95%. The ratio of C2H2 reduced to N2 fixed was determined as a function of constant pC2H2 (0.1 atm) and variable pN2. This ratio decreased with increasing pN2 and the decrease was correlated with less H2 production. Ratios obtained at N2 partial pressures of approximately 0.3, 0.6, and 0.8 atm, respectively, were 3.2, 2.0, and 1.7 for the association and 4.4, 3.0, and 2.5 for the isolated symbiont.Rates obtained for C2H2 reduction, N2 fixation, and H2 production were used to obtain an expression of the electron balance in vivo.Azolla is a genus of small aquatic ferns which, under natural conditions, invariably contain the heterocystous blue-green alga, Anabaena azollae, as a symbiont in an enclosed chamber in the dorsal leaf lobes (10,12). In the intact association, the alga can provide the Azolla plant with its total nitrogen requirement. Previous studies showed that the symbiont contained nitrogenase and was capable of C2H2 reduction, ATP-dependent H2 evolution, and excretion of ammonia (10,11,13).Although C2H2 reduction is a simple and sensitive assay of nitrogenase activity, C2H2 is not the biologically important substrate, and the assay is an indirect measurement of nitrogen fixation. Since the reduction of N2 to 2NH3 requires six electrons while reduction of C2H2 to C2H4 requires two electrons, a theoretical conversion factor of 3C2H2 reduced per N2 fixed is frequently used in estimating nitrogen fixation from C2H2 reduction assays. Studies on isolated nitrogenase have shown that when provided with a source of ATP and reductant the rate of electron flow through the enzyme is independent of the substrate (7,20). Moreover, while normal assay levels of C2H2 almost totally suppress H2 production, some electrons continue to be utilized in reducing protons to H2 when N2 is the substrate and employed at saturating levels (16,20). Thus, the C2H2/N2 ratio is usually closer to 4 for nitrogenase in vitro. However, in vivo studies have produced a range of ratios from less than 2 to greater than 8 (8), and it would appear that the ratio may be dependent upon both the organism and experimental conditions employed. This is a report on 15N2 fixation by the fern-algal association and the symbiont isolated directly from the leaf cavities. In order to determine whether a specific conversion factor can be used, and to assess some factors capable of affecting it, the effect of pN2 on N2 fixation and ATP-dependent H2 evolution was determined in parallel with measurements of C2H2 reduction. Ratios of C2H2 reduction to N2 fixation and the effect of H2 evoluti...