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 '
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