Immunocytochemical localization of nitrogenase in bacteria symbiotically associated with Azolla spp.

In situ immunogold labeling and transmission electron microscopy were used to detect nitrogenase in bacteria (bactobionts) symbiotically associated with leaf cavities of Azolla caroliniana and Azolla filiculoides. In A. caroliniana, the Fe protein of the nitrogenase complex was detected in a subset of the distinct bactobiont types present in leaf cavities of all ages. Similar results were obtained for the bactobionts of A. filiculoides with antisera against both the Fe and MoFe subunits of nitrogenase.     

Ammonium content, nitrogenase activity and heterocyst frequency within the leaf cavities of Azolla filiculoides Lam

Abstract Ion selective new microelectrodes have been used to measure the ammonium concentrations within the various leaf cavities from the apical to the basal ones, of Azolla filiculoides Lam. Ammonium is present in solution within all leaf cavities, and its concentration varies considerably from the apex to the base. Median leaf cavities, which have the highest rate of nitrogenase activity, contain 0.6–0.8 mM of ammonium and exhibit numerous cyanophycin granules accumulated within the Anabaena vegetative cells. Basal cavities contain 6 mM ammonium, the lowest nitrogenase activity and lowe cyanophycin granules in Anabaena . The mechaniems involved in ammonium accumulation in the basal leaf cavities are discussed.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

无藻萍的RAPD分析及其在三膘组满江红种间关系研究中的应用

从满江红Azolla Lam.萍-藻共生体中提取DNA进行的RAPD系统分析通常忽视了满江红样品的异质性。本研究通过获得无藻的满江红,比较有藻萍、无藻萍和离体藻之间的RAPD指纹图谱。发现从有藻萍中提取DNA的扩增反应来源于萍藻双方DNA的共同影响。依引物和植物样本的不同,共生双方对扩增产物的贡献结果不同,说明了用无藻萍进行RAPD检测的重要性。对满江红三膘组5个种的11个无藻萍样本进行了RAPD分析,由9个引物产生的127个DNA多态片段用于计算样本间的Jaccard相似系数和UPGMA树状聚类图。结果     

温度对三种满江红光合及固氮活性的影响

本文报道满江红(Azolla imbricata)、卡洲满江红(A. caroliniana)和蕨状满江红(A. filiculoides)的光合与固氮(乙炔还原)活性对温度的反应差异。根区培养液温度在7—40℃范围内满江红与卡洲满江红的光合活性在30℃达到峰值,高于或低于30℃均急剧下降。而蕨状满江红在7—25℃的根区温度范围内光合活性差异不大,高于25℃则下降(图2)。0℃24小时或40℃2小时暗预处理对三种满江红光合活性与乙炔还原活性表现出不同影响(图3,4)。本文报道的方法可用于满江红生理生态参量的比较研究。     

Occurrence and ultrastructural characterization of bacteria in association with and isolated from Azolla caroliniana.

The occurrence and ultrastructure of bacteria in leaf cavities of symbiotic Azolla caroliniana were examined by transmission electron microscopy. Bacteria were observed in all leaf cavities of Azolla cultures. Five ultrastructurally distinct types of bacteria were observed in each individual leaf cavity. Features used to characterize the bacteria included morphology, cell wall structure, and cytoplasmic organization. At least one gram-positive and as many as four gram-negative types of bacteria reside in leaf cavities of A. caroliniana. The morphological and ultrastructural characteristics of the gram-positive bacterium suggest that it is an Arthrobacter sp. The gram-negative bacteria could not be cultured; therefore, they have not been classified further. Bacterial cell shape and cell wall structure were similar in leaf cavities of different ages, but cell size and cytoplasmic composition varied. The relative contributions of each bacterial type to the total community within individual leaves was determined. Ultrastructural characteristics of bacterial isolates cultured from A. caroliniana in a free-living state were also examined.     

Production of polysaccharides by Arthrobacter globiformis associated with Anabaena azollae in Azolla leaf cavity

Abstract The composition of the capsular polysaccharides (CPS) and exopolysaccharides (EPS) of three strains of Arthrobacter globiformis , isolated from the leaf cavities of Azolla caroliniana (strain B1), A. filiculoides (strains A3 and L1) and A. globiformis ATCC 8010 have been analysed by HPLC and enzymatic assays. Glucose and galactose were detected in the EPS of all the strains, while rhamnose was present only in the EPS of the strain L1 and uronic acids in B1 and ATCC 8010. Traces of fructose were detected by enzymatic assays in all the strains. The CPS contained glucose, galactose and rhamnose, while uronic acids were present only in strain B1. In all the strains the amount of EPS was higher than CPS. The reactivity to different dyes and lectins of the mucilagineous matrix of the algal packets extracted from the fern and of the bacterial mucilage were similar.     

The taxonomy and distribution of Azolla species in southern Africa

The three Azolla species which occur in southern Africa, A. pinnata var. pinnata, A. nilotica and A. filiculoides are identified and their distributions illustrated. The endosymbiotic cyanobacterium Anabaena azollae occurred in dorsal leaf lobe cavities of all three species. The first two species are indigenous to southern Africa, whilst the third, A. filiculoides , is an introduced species from North America. Possible means whereby this species could have been introduced are discussed.     

Scanning Electron Microscopic Observation of Symbiotic Relationship of Azolla-Anabaena AzoUae During the Vegetative Growth

The occurrence and development of the hair ceils on the shoot tips and in the leaf cavities of A. filiculoides, A. microphylla, A. pinnata and their algae-free cultures were examined by means of scanning electron microscopy with microdissect technique. The patterns of Anabacna moving into the leave cavities from the shoot tips were investigated on three species of Azolla during their vegetative growth. The results showed that the patterns of symbiotic Anabaena infecting the leaf cavities are similarity among three species of Azolla and may be divided to the four phases which are summarized as follows: 1. occurrence of primary branched hair and adhesion of Anabaena; 2. development of primary branched hair and spreding of Anabaena; 3. building of hair bridge and entrance of Anabaena into the cavities; 4. formation of secondary simple hair and transference of Anabaena within the cavity. These observations resulted in a hypothesis that hair induces and leads its partner. It is suggested that the hair cell is likely to be a structure of Azolla for attracting and recognizing its symbiont in addition to transport substance between fern and algae.     

Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp

Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C(2)H(2) --> C(2)H(4)) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed.     

Genetic diversity among and within cultured cyanobionts of diverse species of <Emphasis Type="Italic">Azolla</Emphasis>

The cyanobionts isolated from 10 Azolla accessions belonging to 6 species (Azolla mexicana, A. microphylla, A. rubra, A. caroliniana, A. filiculoides, A. pinnata) were cultured under laboratory conditions and analyzed on the basis of whole cell protein profiles and molecular marker dataset generated using repeat sequence primers (STRR(mod) and HipTG). The biochemical and molecular marker profiles of the cyanobionts were compared with those of the free-living cyanobacteria and symbiotic Nostoc strains from Anthoceros sp., Cycas sp. and Gunnera monoika. Cluster analysis revealed the genetic diversity among the selected strains, and identified 3 distinct clusters. Group 1 included cyanobionts from all the 10 accessions of Azolla, group 2 comprised all the symbiotic Nostoc strains, while group 3 included the free-living cyanobacteria belonging to the genera Nostoc and Anabaena. The interrelationships among the Azolla cyanobionts were further revealed by principal component analysis. Cyanobionts from A. caroliniana-A. microphylla grouped together while cyanobionts associated with A. mexicana-A. filiculoides along with A. pinnata formed another group. A. rubra cyanobionts had intermediate relationship with both the subgroups. This is the first study analyzing the diversity existing among the cultured cyanobionts of diverse Azolla species through the use of biochemical and molecular profiles and also the genetic distinctness of these free-living cyanobionts as compared to cyanobacterial strains of the genera Anabaena and Nostoc.     

Studies on the Microstructure and Ultrastructure of Photosynthetic Apparatuses in Azolla

The structures of photosynthetic apparatuses such as leaves, chloroplasts and symbiotic cyanobacterum (blue-green algae) in Azolla-Anabaena azollae associations (Azolla imbricata (Roxb) Nakai) which occur in paddy fields of China were examined using light, scanning and transmission electrn microscopy. Some comparisons were made with A. filiculoides, A. japonica, A. caroliniana, A. pinnata and A. mexicana. Cross sections of A. imbricata were observed by light microscopy and the symbiotic association between the eukaryotic water fern and its prokaryotic blue-green algal symbiont, an Anabaena, was studied. The symbiotic cyanobacterum cells occur not only in a mature leaf cavity, but also in early stages of leaf development, around leaf primordia, and even in macrospores. Under scanning electron microscopy (SEM) it is possible to see stomata and nipples on the surface of dorsal lobes of the fern. The species in the subgenus Euazolla (i.e.A. filiculoides, A. japonica, A. caroliniana and A. mexicana) have rounded nipples, but those in the subgenus Rizosperma (i.e.A. imbricata and A. pinnta) prolate ones. This morphological character is first reported to be related to the taxonomic system. The result of the observation with transmission electron microscopy (TEM) shows that A. filiculoides contains more thylakoides in chloroplasts than A. imbricata does, and the grana lamellae have more stacks in the former than in the latter. The differences are in agreement with the differentiation of the two species in photosynthetic capacity. This may be one of the differences between the two subgenera. The ultrastructures of the symbiotic cyanobacterum are similar to those of free-living Anabaena. The vegetative cells show a typical bilayered cell wall and the heterocysts have a thikened wall. The thylakoid membranes in both heterocysts and vegetative cells are oftenseen forming whirls. During the division of vegetative cells, their contents aggregate and then redistribute.     

Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla.

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.     

Preliminary Studies on Cytoplasmic Inheritance and Variation of Azolla

By investigating the variance of Azolla leaf colour of F1 generation obtained from negative and positive crossing of Azolla between two species (Azolla filiculoides × A. microphylla, A. filiculoides × A. mescicana) and two subgenus (A. filiculoides × A. imbricata), it was revealed that the albinism of the hybrid F1 generation was variation resulting from maternal cytoplasmic inheritance, when A. filiculoide was used as female parent. Electron microscopic observation demonstrated abnormal development of plastid in the albino sporeling. The cell of light green seedling contained both normal and abnormal plastid. Both were probably related to variation in the plastid genotype. Significant difference occurred in the degree and freguency of albinism from various crossing forms, and such change had its vegularity in accord with the variation of the nuclear genotype. The results speculated that albinism were also closely related to the nuclear genotype.     

Quantitative and qualitative DNA variations in Anabaena azollae Strasb. living in Azolla filiculoides lam.

Heterocysts and vegetative cells of the filamentous nitrogen-fixing Anabaena azollae isolated from the apex to the basal leaf cavities of Azolla filiculoides were examined by epifluorescent microscope after fluorochrome staining. Acridine orange (AO), DAPI, and chromomycin fluorochromes were used in order to evidence total DNA content and respectively, A + T and G + C bases. Measurements of fluorescence intensities were made on photographic prints by the automatic image analysis system Quantimet 970. Heterocysts contained higher amounts of DNA than did vegetative cells, and their content strongly increased in the basal leaf cavities. The heterocyst DAPI brightness was quite uniform, whereas in vegetative cells DAPI brightness increased from the apex to the basal groups. In vegetative cells from the apex to the median group, the percentage of DAPI brightness was 60-85% with respect to AO brightness, whereas in heterocysts of the same groups DAPI brightness was 40-50% with respect to AO brightness. In the basal group, brightness due to DAPI staining was comparable with those of previous group both in heterocysts and in vegetative cells, whereas chromomycin brightness increased strongly in heterocysts. These data show that heterocyst changes its DNA content and composition in the basal leaf cavities, suggesting that its lifetime is not completely over.     

Localization of iron-superoxide dismutase in the cyanobiont ofAzolla filiculoides Lam

Summary Immunogold labeling and transmission electron microscopy were used to localize iron-superoxide dismutase (Fe-SOD) in the different cells of nitrogen-fixing cyanobacterial symbiont present within different leaf cavity groups ofAzolla filiculoides Lam. As evidenced by Western blotting and immunoprecipitation, Fe-SOD antibody fromAnabaena cylindrica recognized Fe-SOD in extracts of the cyanobiont and showed the same electrophoretic mobility and pattern as purifiedA. cylindrica Fe-SOD. In vegetative cells of the cyanobiont, Fe-SOD was mainly localized in the thylakoidal membranes and in the outer membrane. The labeling pattern was similar in vegetative cells of the various groups of leaf cavities examined except at the apex where a lower gold particle density was seen. In heterocysts of the leaf cavity groups containing high nitrogenase activity, Fe-SOD labeling was most pronounced and more intense than in vegetative cells. The Fe-SOD label was preferentially located throughout the heterocyst cytoplasm and in the honeycomb regions. In accordance with the decline in nitrogenase activity, the Fe-SOD gold particle density decreased significantly in heterocysts of basal leaf cavity group. The presence of Fe-SOD in regions of high nitrogenase protein levels, and the fact that the pattern of Fe-SOD label parallels that of nitrogenase activity support a role of Fe-SOD in the protection of nitrogenase against superoxide radicals.     

北方农家屋顶养绿萍技术及生态经济效益

北方农家屋顶养绿萍技术及生态经济效益安淑苹（中国科学院石家庄农业现代化研究所,０５００２１）ＴｈｅＥｃｏｌｏｇｉｃａｌａｎｄＥｃｏｎｏｍｉｃａｌＢｅｎｅｆｉｔｓｏｆＣｕｌｔｉｖａｔｉｎｇＡｚｏｌａｆｉｌｉｃｕｌｏｉｄｅｓｏｎｔｈｅＲｏｏｆｔｏｐｉｎｔ...     

Ultrastructural characterization of eubacteria residing within leaf cavities of symbiotic and cyanobiont-freeAzolla mexicana

The occurrence and ultrastructure of eubacteria within leaf cavities of symbiotic and cyanobiont-freeAzolla mexicana were examined with transmission electron microscopy. Bacteria were observed in all leaf cavities of bothAzolla cultures, showing increasing numbers concomitant with leaf age. In symbioticAzolla mexicana two ultrastructurally distinct types of bacteria were found; a helical bacterium and a slightly curved, rod-shaped bacterium. Both had typical Gram-negative cell wall ultrastructure. The helical bacteria comprised 60% of the total population in young leaf cavities (1–5) and 74–80% of the population in older cavities. The mode of cell division for the rod-shaped bacterium was binary fission. In cyanobiont-freeAzolla mexicana at least three distinct types of bacteria were observed; two were ultrastructurally similar to, if not identical, with the types present in symbioticAzolla mexicana.     

Studies on Nitrogen Fixation and Photosynthesis in Azolla imbricata Roxb and Azolla fiticuloides Lam.

Azolla imbricata and Azolla filieuloides were studied in regard to the nitrogenase- catalyzed reactions of C2H2 reduction and H2 production, employing gas chromatography; and photosynthetic CO2 uptake as well as simultaneous determinations of C2H2 reduction, photosynthesis and respiration in a closed system. Photosynthetic CO2 uptake and respiratory C02 production were determined using an infrared gas analyzer. These studies have indicated the following 1. Nitrogenase-catalyzed C2H2 reduction is largely light dependent. About 10,000 lux were required for saturation in A imbricata. A concentration of 10% C2H2 in the gas phase is saturating for C2H2 reduction and 1% CO inhibits C2H2 reduction with concomitant H2 production. 2. A determination of C2H2 reduction activity as a function of leaf age established a develop mental gradient in both A. imbricata and A. fgliculaides. In both species activity is negligible in the apex, increases markedly in progressively older leaves, plateaus, and decreases as leaves senesce. The developmental gradient of activity is much steeper in A. imbricate than in A. filiculoieds due to differences in their gross morphology. 3. Nitrogenase-eatalyzed H2 production in A. imbricata was not detectable under Ar but was appreciable under Ar containing 15% C2H2 and 2% CO. H2 production was also determined under the latter gas phase as a function of leaf. These studies implicate the occurrence of an uptake hydrogenase. 4. The photosynthetic compensation points in air are approximately 30 ppm CO2 for A. imbricata and A. filiculoides. 5. Simultaneous measurements of photosynthesis, respiration and C2H2 reduction in A. imbricata demonstrated the immediate dependence of nitrogenase on photosynthetically captured radiation for energy but an indirect dependence on CO2 fixation.