Isolation of cyanobacteria from the aquatic fern,Azolla

A procedure has been developed to isolate cyanobacteria from the aquatic fern Azolla. The method is based upon recovery of cyanobacterial bundles from digests of plants and use of this material as a massive inoculum for nitrogen-free media, followed by prolonged incubation in light. The procedure appears to select for those cells capable of growth in vitro. Isolated cyanobacteria were found to resemble Anabaena sp. morphologically but were capable of heterotrophic growth and had high nitrogenase activity when grown on fructose in the dark.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Paleoazolla,a new heterosporous fern from the Upper Cretaceous of Argentina

Megaspore and microspore massulae of Paleoazolla patagonica gen. et sp. nov. are described from the Upper Cretaceous of La Colonia Formation, Chubut Province, Argentina. The new fern possesses megaspore complexes with three to four glochidiate floats attached directly to the megaspore; a columella-like structure appears to be absent. The megaspore wall consists of a two-layered exine that is smooth to irregularly perforate, a two-layered perine with a spongy, densely packed endoperine, and a loosely organized exoperine. Infrafilosum hairs cover the exoperine. Microspore massulae are irregular in size and shape and bear multibarbed glochidia that generally have anchor-shaped tips. A comparison with other azollaceous and salviniaceous genera, particularly Azolla, is provided, together with a discussion of some evolutionary trends within the family.     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent     

Tolerance and phytoaccumulation of Chromium by three Azolla species

Azolla, an aquatic fern is ideal candidate for exploitation in constructed wetlands for treating metal-contaminated wastewaters. This study demonstrates the potential of Azolla spp. namely A. microphylla, A. pinnata and A.␣filiculoides to tolerate Cr ions in the growth environment and bioconcentrate them. These species could grow in presence of up to 10 μg ml⁻¹ Cr and showed biomass production 30–70% as compared to controls. Nitrogenase activity was not affected at 1–5 μg ml⁻¹ but at higher concentrations it diminished. There was no growth at higher concentrations of chromium. However, the necrosed biomass harvested from treatments containing higher concentrations of chromium, accumulated Cr to the levels 5000–15,000 μg g⁻¹. At increased levels of Cr, the metal was accumulated in higher amount in dry biomass. Bioconcentration Factor (BCF) ranged between 243 and 4617 for the three species. A. microphylla showed highest bioconcentration potential. Thus, these Azolla spp. can be exploited for treatment of tannery and other Cr contaminated wastewaters.     

Influence of urea on biological N2 fixation and N transfer from Azolla intercropped with rice

The N₂ fixed by Azolla before and after urea application during the rice cycle, the mineralisation of Azolla-N as well as its availability to rice was studied in two greenhouse experiments conducted in 1996 and 1997 and in June 1998 in Goettingen (Germany). Dry matter production of the various rice parts of experiment 1 showed a clear positive synergism between treatment with Azolla and urea with a resulting apparent N recovery by rice increasing from 40% (without Azolla) to 57% in the presence of Azolla. Part of this increase may be due to N fixed biologically by Azolla and transferred to the rice. The second experiment shed some light on the role of BNF. Using an iterative method of estimation, the daily rate of N fixation was estimated at 0.6 – 0.7 kg N ha^–1. The rate was not so much affected by the age of the Azolla crop. At this rate, the BNF would amount to up to 100 kg N ha^–1 over a 130-day season. Assuming that BNF may be inhibited for a period of 5 – 10 days following urea application due to high levels of N in the floodwater, this might reduce the BNF by between 6 and 14 kg N ha over the season. Using the mean-pool-abundance concept, it was estimated that around 75 – 80% of the Azolla-N mineralized during the growth period was actually absorbed by the rice plants. Of the N taken up by rice around 28% was derived from the biologically fixed Azolla N, the remainder was urea N cycled through the Azolla. Azolla also seems to help sustain the soil N supply by returning N to the soil in quantities roughly equal to those extracted from the soil by the rice plant.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

The role of Azolla cover in improving the nitrogen use efficiency of lowland rice

The development of management techniques to improve the poor N use efficiency by lowland rice (Oryza sativa L.) and reduce the high N losses has been an important focus of agronomic research. The potential of an Azolla cover in combination with urea was assessed under field conditions in Laguna, Philippines. Two on-station field experiments were established in the 1998–1999 dry season and eight on-farm experiments per season were carried out in the 2000–2001 wet and dry seasons. Treatment combinations consisting of N levels applied alone or combined with Azolla were evaluated with respect to floodwater chemistry, ¹⁵N recovery, crop growth, and grain yield. A full Azolla cover on the floodwater surface at the time of urea application prevented the rapid and large increase in floodwater pH and floodwater temperature. As a consequence, the partial pressure of ammonia (NH₃), which is an indicator of potential NH₃ volatilization, was significantly depressed. ¹⁵N recovery was higher in plots covered with Azolla where the total ¹⁵N recovery ranged between 77 and 99%, and the aboveground (grain and straw) recovery by rice ranged between 32 and 61%. The tiller count in Azolla-covered plots was significantly increased by 50% more than the uncovered plots at all urea levels. Consequently, the grain yield was likewise improved. Grain yields from the 16 on-farm trials increased by as much as 40% at lower N rates (40 and 50 kg N ha^–1) and by as much as 29% at higher N rates (80 and 100 kg N ha^–1). In addition, response of rice to treatments with lower N rates with an Azolla cover was comparable to that obtained with the higher N rates without a cover. Thus, using Azolla as a surface cover in combination with urea can be an alternative management practice worth considering as a means to reduce NH₃ volatilization losses and improve N use efficiency.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Extraction of RAPD-friendly DNA from Laminaria japonica (Phaeophyta) after enzymatic dissociation of the frozen sporophyte tissues

A new extraction protocol has been developed to obtain high quality DNAfrom Laminaria japonica, which involves enzymatic dissociation ofsporophyte tissues and subsequent elimination of the remainingpolysaccharides with cetyltrimethyl ammonium bromide. Unicells isolatedfrom frozen kelp tissues with alginate lyase prepared from the abalone Haliotis diversicolor were used to extract total DNA; the yield wasapproximately 13 to 22.5 g DNA g^-1 (wet sporophyteweight). The average size of genomic DNA was around 23 kb estimated byagarose gel electrophoresis, and the purity of total DNA determinedspectrophotometrically as the ratio of OD₂₆₀/OD₂₈₀ wasabout 1.7. The extracted kelp DNA (20–40 ng) could be usedsuccessfully as a template for polymerase chain reaction (PCR) under theoptimized conditions (100 M dNTP, 0.2 M primer, 1.0unit Taq DNA polymerase). The random amplified polymorphicDNA (RAPD) patterns were highly reproducible. These results suggest thatthe contamination by soluble polysaccharides which interferes with RAPDreproducibility was largely controlled. This RAPD-suited method for DNAextraction from kelp sporophytes using enzyme treatment providedsufficient material, and was inexpensive and convenient to carry out.     

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5–3-m-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.     

Response of 19 Azolla strains to a high concentration of ammonium ions

The response of 19 Azolla strains from five species to 20 mM ammonium ions was examined. The response varied even among strains of the same species. The tolerance indexes of both relative growth rate and nitrogen fixation [(values for plants grown in the presence of ammonium ions)/(values for plants grown in the absence of ammonium ions)] showed that ammonium ions did not always simultaneously inhibit growth and nitrogen fixation of individual strains. The tolerance of each Azolla–Anabaena association to ammonium ions is assumed to be determined separately by both the host fern and the symbiotic Anabaena. The inhibitory effects of ammonium appeared predominantly in the mature region of Azolla–Anabaena associations. In the sensitive strains, both chlorophyll content and the number of cyanobionts were reduced only in the mature region when ammonium ions were present. Possible strains for treatment of wastewater, which contains a high concentration of ammonium ions, are discussed.     

The supraspecific taxonomy and evolution of the fern genusAzolla (Azollaceae)

The systematics of the aquatic fern genusAzolla Lam. is revised at the supraspecific level. Published morphological data are reviewed, and cytological and cladistic analyses of the genus conducted, enabling a discussion of the putative evolutionary trends in the genus, and a revision of the current taxonomy. A phylogenetically more acceptable subgeneric and sectional classification is proposed, viz. subg.Azolla sect.Azolla (comprisingA. filiculoides Lam.,A. rubra R. Br.,A caroliniana auct. nonWilld.,A. microphylla auct. nonKaulf. andA. mexicana Presl), subg.Azolla sect.Rhizosperma (Mey.)Mett. (comprisingA. pinnata R. Br.), and subg.Tetrasporocarpia subg. nov. (comprisingA. nilotica Decne. exMett.).     

Chromosome numbers and taxonomic implications in the fern genusAzolla (Azollaceae)

Investigation of chromosome numbers of allAzolla species, and for the first time of hybrids, has been undertaken. Removal of wax from the leaf surface proved invaluable in achieving clear cytological preparations and providing unambiguous chromosome numbers. In contrast to previous records, the speciesA. pinnata, A. filiculoides, A. filiculoides var.rubra, A. caroliniana, A. microphylla, andA. mexicana were found to be 2n=44, andA. nilotica to be 2n=52. Several triploids (2n=66) and one tetraploid (2n=88) were identified. No geographical pattern could be observed in the distribution of triploids which probably derive from the function of unreduced gametes. The chromosome number of hybrids occasionally deviates from the diploid chromosome number (2n=44). The small chromosome size limits karyotypic analysis and only differences in overall chromosome size can be observed. Taxonomic implications of chromosome numbers and sizes are discussed.     

Formative and proliferative cell divisions,cell differentiation,and developmental changes in the meristem of Azolla roots

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.     

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells,and an hypothesis on the development of cortical arrays of microtubules

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are not confined to edges made by the junction of new cell plates with parental walls, but occur also along older edges. Similar matrices and vesicles are seen amongst phragmoplast microtubules and where pre-prophase bands intersect the edges of cells. It is suggested that the complexes participate in the development of cortical arrays of microtubules. The observations are combined with others, made on pre-prophase bands and on the substructure of cortical arrays lying against the faces of cells, to develop an hypothesis on the development of cortical microtubules, summarised below: Microtubules are nucleated along the edges of cells, at first growing in unspecified orientations and then becoming bridged to the plasma membrane. Parallelism of microtubules in the arrays arises by inter-tubule cross-bridging. Lengths of microtubule are released from, or break off, the nucleating centres and are moved out onto the face of the cell by intertubule and tubule-membrane sliding, thus accounting for the presence there of short tubules with randomly placed terminations. The nucleating zones along cell edges might have vectorial properties, and thus be able to control the orientation of the microtubules on the different faces of the cell. Also, localised activation could generate localised arrays, especially pre-prophase bands in specified sites and planes. Two possible reasons for the spatial restriction of nucleation to cell edges are considered. One is that the geometry of an edge is itself important; the other is that along most cell edges there is a persistent specialised zone, inherited at cytokinesis by the daughter cells when the cell plate bisects the former pre-prophase-band zone.     

Reiterative growth in the complex adaptive architecture of the Paleozoic (Pennsylvanian) filicalean fern Kaplanopteris clavata

Reiteration is a widespread component of plant growth whose evolutionary importance in ferns is not recognized widely. We introduce and discuss the growth architecture of Kaplanopteris clavata, a fossil filicalean fern from the Pennsylvanian (ca. 305 million yeas old), focusing on types of reiteration exhibited by this species and on the adaptive and phylogenetic significance of reiteration for ferns in general. Kaplanopteris clavata combines two types of reiterative growth where growth modules are borne on fronds: (1) entire fronds derived from primary pinnae, and (2) epiphyllous plantlets. This combination of reiterative pathways, unique among fossil and living ferns, allowed K. clavata to explore ecospace through an opportunistic combination of scrambling, climbing and epiphytic growth. Kaplanopteris clavata underscores the organographic importance of fronds (as opposed to stems) in the adaptive architecture of ferns, emphasizing functional convergences between the different Baupla̋ne of ferns and angiosperms. This unique combination of reiterative pathways is interpreted as a derived condition illustrating the structural and developmental complexity achieved by some filicaleans during the first major evolutionary radiation of leptosporangiate ferns.     

Age-related and origin-related control of the numbers of plasmodesmata in cell walls of developing Azolla roots

Plasmodesmata were counted in the longitudinal and transverse walls in developmental sequences of merophytes in roots of Azolla pinnata R.Br. The differences between certain categories of longitudinal wall were traced to factors that govern the surface area of the cell plates, the density of plasmodesmata (number per unit area of cell plate), and the amount by which each type of plate expands. No evidence for secondary augmentation of plasmodesmatal numbers after the cell-plate stage of development was found, but plasmodesmata are lost from the walls of sieve and xylem elements during their differentiation. Losses caused by cell separation occur in other tissues. The relatively high density of plasmodesmata in transverse walls is based not so much on a high density in the cell plates as on the relatively low expansion that these walls undergo. There appears to be a compensatory mechanism that relates initial plasmodesmatal density to the future expansion of the cell plate. The root shows determinate growth, the apical cell dividing about 55 times. Beginning at about the 35th division there is a progressive failure to maintain the plasmodesmatal frequencies that were developed in earlier cell divisions in the apical cell. The divisions that occur within the later-produced merophytes also show progressive diminution of plasmodesmatal numbers. The result is that the apex of the root, and particularly the apical cell, becomes more and more isolated symplastically, a phenomenon which could account for its limited lifespan and the determinate growth pattern of the root.