Sub-cellular distribution of nitrogen compounds inAzolla andAnabaena by ESI and EELS analysis

Summary The sub-cellular localization of some nitrogen compounds within the leaf cavities ofAzolla filiculoides Lam. was obtained by means of electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The analyses were performed on ultrathin unstained sections of differentAzolla leaf cavities which contain epidermal hairs,Anabaena azollae Strasb. and bacteria. Net nitrogen distributions were visualized by image analysis, and nitrogen peaks were evidenced in spectra recorded in the same areas. Different distributions of nitrogen compounds were observed within the leaf cavities along the stem, in particular inside the epidermal hairs ofAzolla and the vegetative cells and heterocysts ofA. azollae.     

Biomass and Pigment Production in Three Isolates of Azolla I. Response to Water Stress

Two isolates of Azolla Lam. from Colombia, South America, andone from the USA (Florida) were selected for characterizationof their growth potentials in certain defined stress environments.These isolates were Amazonian (RAR) and Atlantic coastal (M-3)specimens of Azolla caroliniana Willd and a Floridian variety(LA) of Azolla filiculoides Lam. Osmotic stress (from polyethylene glycol-amended media) andosmotic/salt stress (from seawater-amended media) were the chosendeterminants in these trials. Their effects on growth responseswere monitored. Initial growth rates of LA and M-3 were stimulatedat 1 and 5 ppt seawater salts, relative to growth under controlconditions. RAR, however, was generally the most stable isolatein the experiments with seawater-induced ionic stress. LA wasmost stable in its biomass growth in experiments with polyethyleneglycol 6000, but chlorophyll accumulation data were again leastaffected in RAR. LA survived the longest of the three isolatesat a water potential of approximately —10 bars with eitherseawater (13 ppt salts) or polyethylene glycol (26 per cent).The latter amendment was usually more inhibitory to acetylenereduction rates in RAR and consistently more inhibitory in LA.RAR was tentatively judged by its reactions to water stressto be the superior isolate of the three. Azolla caroliniana Willd., Azolla filiculoides Lam., biomass and chlorophyll accumulation, acetylene reduction, osmotic/salt stress     

Genetic diversity and classification of cyanobacteria in different Azolla species by the use of PCR fingerprinting

Symbiotically associated cyanobacteria from 18 accessions within all known species in the genus Azolla were examined and classified by the use of polymerase chain reaction (PCR)-fingerprinting. A repetitive sequence specific for cyanobacteria, the short tandemly repeated repetitive (STRR) sequence, was used as a primer in the reaction. Cyanobacterial filaments isolated directly from the Azolla leaf cavity or contained within homogenised symbiotic Azolla tissue were used as templates. Based on the fingerprint pattern, distinct differences were demonstrated between cyanobacteria isolated from the Euazolla and Rhizosperma sections. In addition, individual fingerprints were obtained from all cyanobacteria isolated from the different Azolla species. The fingerprints were used to generate a phylogenetic tree. Three clusters were distinguished: one contained the four isolates from the section Euazolla, a second the isolate from Azolla filiculoides, and a third the three isolates from the section Rhizosperma. By the use of STRR-PCR fingerprinting, new data on the taxonomy of cyanobacteria in Azolla were obtained, which have been difficult to generate by other classification methods. PCR-fingerprinting may, therefore, be a valuable tool for diversity and classification studies of symbiotic cyanobateria from Azolla and, as co-evolution between the cyanobacteria and its corresponding host exists the method may also be useful for the taxonomy of Azolla. Received: 19 December 1998 / Accepted: 31 May 1999     

Herbarium survey on the genus Azolla (Salviniaceae) in Italy: distributive and taxonomic implications

An analysis of Italian herbarium specimens belonging to the genus Azolla was carried out using optical microscope and SEM techniques. According to recent international reviews, three micro-morphological characters have been considered for the specimen analysis: (i) number of leaf trichome cells, (ii) septation of glochidia and (iii) features of the macrospore perine. The analysis of the patterns concerning both vegetative and reproductive characters was complicated by the relatively low number of sporulated specimens stored in the Italian Herbaria. The analysis of the selected specimens showed the presence of two well distinct systematic groups. One group was characterized by bicellular leaf trichomes, pluriseptate glochidia and not-warty perine, traits that permitted to taxonomically attribute it to A. cristata. The second group was characterized by monocellular leaf trichomes, non-septate or uniseptate glochidia and warty perine, so that it could be taxonomically attributed to A. filiculoides Lam. This study confirmed the large use in the past of the name ‘A. caroliniana’ to indicate the species actually named A. cristata. In Italy, this species was found to be collected only in the past, while A. filiculoides was found to be present with both ancient and recent herbarium specimens.     

Differential responses ofAzolla microphylla Kaulf andAzolla filiculoides Lam. to lead nitrate

The aquatic fernAzolla is used as a green manure for rice production systems in warm temperate and tropical regions throughout the world. We used lead nitrate in nutrient media to assess the tolerance/response of twoAzolla microphylla andAzolla filiculoides to heavy metals. While both species showed negative responses in growth with an increase in lead concentrations, They were distinctly different in their tolerance for higher concentration of the heavy metal. This was apparent in their growth and biochemical characteristics. Amicrophylla was more tolerant of the higher concentrations of lead nitrate (25 and 50 ppm), whereasA. filiculoides had a higher rate of lead uptake.     

Differential response of antioxidant enzymes to salinity stress in two varieties of Azolla (Azolla pinnata and Azolla filiculoides)

The activity and modulation of antioxidant components were comparatively analyzed in two varieties of Azolla (Azolla pinnata and Azolla filiculoides) under different concentrations of NaCl. The total superoxide dismutase (SOD) and ascorbate peroxidase (APX) activity increased in A. pinnata, whereas both enzyme activities decreased in A. filiculoides. The plants of A. pinnata exposed to 30 mM NaCl contained a lower amount of Na⁺ ions and showed a lower electrolyte leakage than A. filiculoides. Our studies indicate a differential response of antioxidant enzymes in relation to salt tolerance in Azolla plants. On the basis of our comparative analysis, A. pinnata has been ranked salt tolerant as compared with A. filiculoides, which is salt sensitive.     

Effects of sodium dodecyl sulphate on the aquatic macrophytes Azolla and Lemna

Abstract

In this work, we determined the tolerance to sodium dodecyl sulphate (SDS) of Azolla filiculoides and of Lemna minor. The presence of the detergent in the media affected growth more than the chlorophyll content. On the basis of the effect indices, Lemna is more tolerant to SDS than Azolla. The fern had a better capacity, compared with duckweed, to remove and accumulate the pollutant.     

DIVERSITY AND HOST SPECIFICITY OF AZOLLA CYANOBIONTS1

A unique, hereditary symbiosis exists between the water fern Azolla and cyanobacteria that reside within a cavity in the dorsal leaf‐lobe of the plant. This association has been studied extensively, and questions have frequently been raised regarding the number and diversity of cyanobionts (cyanobacterial symbionts) among the different Azolla strains and species. In this work, denaturating gradient gel electrophoresis (DGGE) and a clone library based on the 16S rRNA gene were used to study the genetic diversity and host specificity of the cyanobionts in 35 Azolla strains covering a wide taxonomic and geographic range. DNA was extracted directly from the cyanobacterial packets, isolated after enzymatic digestion of the Azolla leaves. Our results indicated the existence of different cyanobiont strains among Azolla species, and diversity within a single Azolla species, independent of the geographic origin of the host. Furthermore, the cyanobiont exhibited host‐species specificity and showed most divergence between the two sections of genus Azolla, Azolla and Rhizosperma. These findings are in agreement with the recent redefinition of the taxon Azolla cristata within the section Azolla. With regard to the taxonomic status of the cyanobiont, the genus Anabaena of the Nostocaceae family was identified as the closest relative by this work.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp.     

Use of Azolla as a test organism in a growth chamber of simple design

Summary The construction and application of a new type of growth chamber, in which different growth conditionsi.e.: temperature, humidity, pH, light intensity, light colour, change in nutrient composition and gas exchange can easily be controlled, are presented. The method has previously been applied to twoAzolla speciesviz. Azolla filiculoides, which is cold tolerant andAzolla pinnata (distinguished in Vietnam as the form Xanh), which is heat tolerant. In the growth chamber natural growth conditions of the Azolla —Anabaena azollae symbiotic association were imitated as much as possible. For testing the system, methods discussed earlier^8,14 and some previously presented data, concerning photosynthetic activities, such as oxygen evolution and nitrogen fixation (acetylene reduction) of twoAzolla species³⁹, were partially used. Biomass ofA. filiculoides was measured and reactions to its environment at conditions when grown in the field and in the growth chamber, were studied. Growth and photosynthesis measurements were performed under special light conditions and with whole plants grown under laboratory conditions. Anthocyanin synthesis was studied in relation with humidity. Anthocyanin spectra were analyzed by means of a spectrum-deconvolution method. On leave from the Department of Plant Physiology of the University of Hanoi, Vietnam.     

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.).     

Proteomic analysis of the cyanobacterium of the Azolla symbiosis: identity, adaptation, and NifH modification

Cyanobacteria are able to form stable nitrogen-fixing symbioseswith diverse eukaryotes. To extend our understanding of adaptationsimposed by plant hosts, two-dimensional gel electrophoresisand mass spectrometry (MS) were used for comparative proteinexpression profiling of a cyanobacterium (cyanobiont) dwellingin leaf cavities of the water-fern Azolla filiculoides. Homology-basedprotein identification using peptide mass fingerprinting [matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF-MS)],tandem MS analyses, and sequence homology searches resultedin an identification success rate of 79% of proteins analysedin the unsequenced cyanobiont. Compared with a free-living strain,processes related to energy production, nitrogen and carbonmetabolism, and stress-related functions were up-regulated inthe cyanobiont while photosynthesis and metabolic turnover rateswere down-regulated, stressing a slow heterotrophic mode ofgrowth, as well as high heterocyst frequencies and nitrogen-fixingcapacities. The first molecular data set on the nature of theNifH post-translational modification in cyanobacteria was alsoobtained: peptide mass spectra of the protein demonstrated thepresence of a 300–400 Da protein modification localizedto a specific 13 amino acid sequence, within the part of theprotein that is ADP-ribosylated in other bacteria and closeto the active site of nitrogenase. Furthermore, the distributionof the highest scoring database hits for the identified proteinspoints to the possibility of using proteomic data in taxonomy. Key words: Azolla, cyanobacteria, NifH modification, proteomics, symbiosis, taxonomy Received 24 June 2007; Revised 21 October 2007 Accepted 22 October 2007     

Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

Interacting effects of atmospheric CO2 enrichment and solar radiation on growth of the aquatic fern Azolla filiculoides

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Free-living culturable bacteria and protozoa from the rhizoplanes of three floating aquatic plant species

Terrestrial plant roots exude compounds that promote the proliferation of microorganisms, a phenomenon called the rhizosphere effect. However, little is known about the influence of roots of aquatic plants on microbial populations. We compared cultivable bacteria and protozoa from the rhizoplanes of Azolla filiculoides Lam., Lemna gibba L., and Ricciocarpus natans L., collected at the Tecocomulco Lagoon (Hidalgo, Mexico). The functional bacterial groups isolated from A. filiculoides, L. gibba and R. natans, were macro- and microscopically characterized, and phylogenetically identified using the 16S rDNA gen. About 96% of isolates corresponded to Gram-negative bacteria, and potential N-fixing free-living bacteria (diazotrophic bacteria) were the most abundant. Molecular analysis detected 15 bacterial genera in the rhizoplane of R. natans, whereas A. filiculoides and L. gibba only yielded five genera, among which Aeromonas was predominant. Twenty-five genera of flagellated and 20 genera of ciliated protozoa were identified. Bodo was the more abundant flagellated, whereas Halteria was the most frequent ciliate. All three aquatic plants showed the rhizospheric effects. The most abundant and diverse community of protozoa was found in A. filiculoides, which also had the most abundant bacterial community, but the highest bacterial diversity was found in R. natans.     

A CONTRIBUTION TO THE EARLY TERTIARY FOSSIL RECORD OF THE SALVINIACEAE

Megaspores and massulae of Azolla, Azollopsis, and Salvinia are recorded from the Paleocene and Eocene of Montana and the Dakotas. Three new species of Azolla are described and two species of Azolla are redescribed. The columella of the floating apparatus of the megaspore apparatuses of Azolla appears to be a phylogenetic localization of the perispore around the megaspore. A new section of Azolla (section Kremastospora) is created for species whose megaspore apparatuses have many floats and whose massulae have hooked (anchor-shaped) glochidia. Salvinia preauriculata is based only on leaf remains from the Eocene of North America. Megaspores and massulae which probably are conspecific with the leaf remains are described. The megaspores and massulae of the fossil species are much smaller than those of the living species, S. auriculata, with which S. preauriculata has been compared. The similar size of the megaspores and massulae of the fossil species suggests a less specialized condition than in living species, where these two structures differ greatly in size. Azollopsis tomentosa, previously known from the late Cretaceous, has been found in the Eocene.     

The effects of floating mats of Azolla filiculoides Lam. and Lemna minuta Kunth on the growth of submerged macrophytes

In laboratory experiments, the growth characteristics of the submerged species Elodea nuttallii (Planch.) St. John and Potamogeton crispus L. were assessed in the presence and absence of floating mats of Azolla filiculoides Lam. and Lemna minuta Kunth. Light penetration and the development of pH and dissolved oxygen differences were monitored. The growth of P. crispus was suppressed much more than that of E. nuttallii and the effects of A. filiculoides were more severe than those of L. minuta. Findings are related to possible field responses of submerged plants under floating mats, especially their abilities to compensate for the potential suppressive effects of floating mats under natural conditions.     

MITOTIC ACTIVITY IN THE ROOT APICAL MERISTEM OF AZOLLA FILICULOIDES LAM., WITH SPECIAL REFERENCE TO THE APICAL CELL

The meristematic activity of the apical cell and its derivatives (merophytes) in the unbranched, determinate roots of Azolla filiculoides Lam. was investigated. The plane of division of the apical cell indicates that it is the initial of each merophyte. The division plane of each newly formed merophyte is strictly periclinal to the root surface and provides confirmation that the immediate derivatives of the apical cell cannot be the ultimate root initials. The frequency of cell division as determined by the mitotic index, and by the duration of the cell cycle as determined by the colchicine method, confirmed the meristematic activity of the apical cell. As roots increase in length, the duration of the cell cycle in the total meristem increases, with the apical cell possessing the longest cell cycle, whereas the immediate derivatives maintain approximately the same cycle duration as in shorter roots. In determinate Azolla roots, cell division appears to play a major role up to a certain root length, then increase in length is produced mainly by cell elongation.     

Differential response ofAzolla microphylla kaulf. andAzolla filiculoides Lam. to sodium fluoride

We assessed the differential response ofAzolla microphylla andAzolla filiculoides to fluoride stress by growing them in culture media containing 1 to 50 ppm sodium fluoride (NaF).A microphylla had a higher total chlorophyll content thanA. filiculoides. Both species showed gradual decreases in protein content as the concentration of NaF increased.A. microphylla accumulated less proline thanA. filiculoides when more NaF was added to the culture medium. For all concentrations tested, the amount of residual fluoride in the nutrient media that supportedA. microphylla was higher than in the nutrient media used for growingA. filiculoides.