Biomass increase, C and N content, C2H2 reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A.filiculoides Lam., A. mexicana Presl., and A. pinnata R.Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study.

SUMMARYThe nitrogen-fixing blue-green alga, Anabaena azollae, occurs as a symbiont in leaf cavities of the water fern, Azolla. Organization and morphological aspects of tbe association, with empbasis upon the cavity area, were studied by light, transmission, and scanning electron microscopy. Cleared whole mounts, sectioned, and enzymatically digested materials were employed. Leaf cavity formation, including the association of the symbiont and epidermal hairs, is shown during leaf development in A. caroliniana Willd. Enzymatic digestion of the fern leaf tissue provided preparations of algal packets whicb correspond to the leaf cavity area. The digestion process complemented otber approaches in providing insigbt into the organization of the leaf cavity. Scanning electron microscopy allowed visualization of these packets in three dimensions. The packets are surrounded by a filmy, limiting envelope of unknown composition. The envelope was isolated and sbown to remain associated with hairs which protrude into the cavity area. The hairs are multicellular and may be branched or unbranched. When nitrogen fixation by the symbiont is the sole nitrogen source, sections through some cells of hairs in mature leaf cavities exhibit the ultrastructural characteristics of transfer cells. These characteristics are not observed, however, in sections through cells of hairs associated with the early stages of leaf cavity formation in tbe shoot tip. The potential significance of the envelope and hair cells in the metabolic interaction of the host and symbiont are discussed.

Summary N2-fJxing cyanobacteria occur in symbiotic associations with fungi (ascomycetes) as lichens and with a few green plants. The associated cyanobacterium is always a species of Nostoc or Anabaena. Only a small number of plant genera are involved but there is a remarkable range of host diversity. Associations occur with several bryophytes (e.g.Anthocero& Blasia, Cavicularia), a pteridophyte (Azolla), cycads (nine genera including Macrozamia and Encephalafros) and an angiosperm (Gunnera). Except for Gunnera, where the cyanobacterium penetrates the plant cells, the cyanobacteria are extracellular with specialized morphological modifications and/or structures of the host plant organs providing an environment which facilitates interaction with the prokaryote. Salient aspects of current knowledge pertaining to the establishment, perpetuation, and functioning of the individual symbioses are summarized. Where possible this includes information concerning recognition and specificity, mode(s) of infection, morphological modifications/ adaptations of the host plant and a synopsis of morphological, physiological and biochemical changes common to the symbiotic cyanobacteria. The latter encompasses heterocyst frequencies, enzymes involved in ammonia assimilation, photosynthetic capability and metabolic interaction with the host.The Azolla-Anabaena symbioses, which have potential agronomic significance as an alternative nitrogen source and maintain continuity with the endophyte through the sexual cycle, are emphasized.

Photosynthesis in the AzoUa-Anabaena association was characterized with respect to photorespiration, early products of photosynthesis, and action spectra. Photorespiration as evidenced by an 02 inhibition of photosynthesis and an 02-dependent CO2 compensation concentration was found to occur in the association, and endophyte-free fronds, but not in the endophytic Anabaena. Analysis of the early products of photosynthesis indicated that both the fern and cyanobacterium fix CO2 via the Calvin cycle. The isolated endophytic Anabaena did not release significant amounts of amino acids synthesized from recently fixed carbon. The action spectra for photosynthesis in the Azoffa-Anabaena association indicated that the maximum quantum yield is between 650 and 670 nanometers, while in the endophyte the maximum is between 580 and 640 nanometers.Although the endophytic cyanobacterium is photosynthetically competent, any contribution it makes to photosynthesis in the intact association was not apparent in the action spectrum.Azolla is a genus of floating aquatic ferns in the Salviniaceae. An endophytic cyanobacterium, referred to as Anabaena azollae Strass., is associated with all stages of the fern's development. It colonizes cavities which are formed in the fern's dorsal aerial leaf lobes (21) and undergoes a pattern of development and differentiation which parallels that of the fern (10, 11). The endophyte can supply the association with its total N requirement by N2 fixation (18,19) and these associations have demonstrated potential as an N source for rice production (25,26).Previous studies showed that the association and filaments of Anabaena isolated from the leaf cavities could fix CO2 (16). In accord with studies on free-living N2-fixing cyanobacteria (1), these and related studies (19) have indicated the importance of photosynthesis in providing a source of reductant and ATP for nitrogenase activity. As an initial step toward understanding possible interactions of the two organisms' photosynthetic capabilities and their relationship to nitrogen fixation in the symbiotic state, this manuscript characterizes photosynthesis in the association and individual partners on the basis of products of "CO2 fixation, effects of 02 tension on CO2 fixation and CO2 compensation points, and action spectra. Preliminary accounts of some aspects of these investigations were presented previously (17,20 pooled, concentrated, and the products of CO2 fixation resolved by standard two-dimensional chromatographic methods and radioautography (2).Time course studies on "CO2 incorporation by the isolated endophyte were carried out at 29 C in small vials placed in a water-jacketed reaction vessel. A suspension of the endophyte in the N-free incubation medium (13) was maintained using a magnetic stir bar. After about 10 min of saturating illumination with white light, the vial containing the endophyte was sealed with a serum cap and 1 ml of a NaH 4CO3 solution containing 500 ,uCi of 14CO2 (58.5

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 '

The water ferm, Azolla caroliniana Willd., containing the symbiotic, heterocystous blue-green alga, Anabaena azollae, has been studied uder various growth conditions to characterize its light-dependent production of H2. The response of H2 production to N2 and C2H2 and the absence of a differential effect of m-chlorocarbonyl cyanide phenylbydrazone on H2 production and C2H2 reduction, coupled with the paralel ihibition of both processes by DCMU imply that the production of H2 is nitrogenase-catalyzed and ATP-dependent.H2 was produced by fronds grown under air-CO2 in the presence or absence of combined nitrogen. When cultured under argon-02-C02, only those fronds provided with combined nitrogen remained viable and produced H2. Fronds grown on nitrate under air plus 2% CO also produced H2. In comparison to fronds grown on N2 alone, fronds grown on nitrate had an increased rate of H2 production reltive to C2H2 reduction, and the inhibition of H2 production by air was less.CO in argon ± CO2 resulted in a partial inhibition of H2 production, whereas CO in argon-CO2-C2H2 enhanced H2 production in fronds grown without combined nitrogen. Our studies strongly indicate that H2 production is nitrogenase-catalyzed but the possibility that the symbiont contains a hydrogenase cannot be totally exduded. This paper reports on H2 production by the water fern, Azolla caroliniana Willd. which contains the symbiotic, heterocystous blue-green alga, Anabaena azollae within specialized leaf cavities (29). The symbiont is capable of N2 fixation when isolated from the fern and, in the intact association, the alga can supply the host plant with its total nitrogen requirement (23,24,26). Azolla freed of the symbiont is incapable of N2 fixation (C2H2 reduction) and must be provided with combined nitrogen (26,30).Azolla plants containing the symbiont and grown under air on nitrate or urea for 35 days retained 70% or more of their capacity for C2H2 reduction and appreciable nitrogenase activity was still present after 6 to 7 months of such growth (26). When fronds were grown on nitrate or urea under 1 % C02, 20% 02 in argon, growth and the capability for C2H2 reduction were comparable to those of controls grown under air. Preliminary studies indicated that H2 production followed the same pattern as C2H2 reduction (23,24 Growth Conditions. Fronds were usually grown under air with or without combined nitrogen. In experiments in which atmospheres of 1% Co2, 20% 02 in Ar, or 1% CO2 in air + 2% CO were employed, the growth flasks were vigorously purged at the onset of the experiment and then daily. Between purgings, the medium was continuously bubbled to maintain the desired gas atmosphere. The cultures were maintained at 24 C under either continuous illumination or, in the case of routine maintenance, with a 16-hr light, 8-hr dark period. Illumination at 400 ft-c was provided by a mixture of cool white and Gro-lux fluorescent lamps.C2H4, H2 and 02 Determinations. C2H2 reduction assays were carried out essentially as described previously (...

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...

Biomass increase, C and N content, C2H2 reduction, percentage dry weight and chlorophyll a/b ratios were determined for clones of Azolla caroliniana Willd., A.filiculoides Lam., A. mexicana Presl., and A. pinnata R.Br. as a function of nutrient solution, pH, temperature, photoperiod, and light intensity in controlled environment studies. These studies were supplemented by a glasshouse study.