An Autoradiographic Study of the Azolla-Anabaena azollae Relationship

The symbiotic relationships between Azolla and Anabaena azollae were studied by means of autoradiography after the Azolla was administered by 3H-thymidine, 3H-uridine, 3H-leucine and 3H-glucose. The experimental results showed that the four labeled compounds mentioned above were transfered from Azolla to Anabaena azollae through cavity hairs. This indicated that there was a transfer way of substances from fern to algae. It is suggested that the symbiotic relationship between Azolla and Anabaena azollae is more complicated than we have known up to now. The fern not only get the NH3 which was formed by symbiont-blue alga, but also supplied some nitrogen-containing substances, such as amino acids (or proteins), ribonucleotides for symbiotic algae. Although the symbiont still retained photosynthetic ability, the ability of nitrogen fixation might be developed and the photosynthetic autotrophic ability might be dropped gradually in the long symbiotic life and the Anabaena azolla needed take a portion of substances from the Azolla as replenishment.     

The Effects of Light Quality on the Growth and Development of Anabaena azollae

The blue green algae Anabaena azollae has been isolated from the leaf cavity of water fern Azolla imbricata and grown aseptically in N-free medium. It was used for the study on the effects of white, red, yellow, green and blue light illuminations on the growth, acetylene reduction activity and heteroeyst formation. The results are summarized as follows: The growth rate under white, red and yellow light was greater than under green light and the yield of the former was increased nearly two folds than the latter after 7 days culture. However, the green light enhanced heterocyst formation up to 40% than the other light illuminations. Chlorophyll content was also higher under green light. Nitrogen fixation activity was proportional to the heterocyst formation. Whatever the light quality used, nitrogen fixation activity could be increased up to several folds by adding 0.2% fructose to the culture medium. When the fructose was added to the 5 days culture, green light illumination showed the highest nitrogen fixation activity. The significant reaction of Anabaena azollae to the green light seems to be a physiological feature of the symbiont.     

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.     

A Cobalt Requirement for Symbiotic Growth of Azolla filiculoides in the Absence of Combined Nitrogen

Cobalt was shown to be essential for the symbiotic growth of Azolla filiculoides and Anabacna azollac in the absence of fixed nitrogen. Addition of 0.01 μg/liter cobalt resulted in large increases in yield, chlorophyll content and nitrogen fixation as compared to control cultures without cobalt. Cobalt was not required for the growth of Azolla when nitrate nitrogen was supplied. The number of Anabaena azollae cells in the fronds of Azolla appeared to be decreased by ommission of cobalt from the culture medium containing nitrate nitrogen. It is concluded that cobalt is essential for the symbiotic growth of Azolla in the absence of combined nitrogen and it is suggested that the cobalt requirement is associated with the growth of Anabaena azollae.     

满江红鱼腥藻的异养生理

设计了一个经机械处理而获得蓝藻无菌培养物的方法,利用这一方法获得满江红鱼腥藻(Anabaena azollae)的无菌培养物。满江红鱼腥藻的无菌培养物能以果糖、葡萄糖或者蔗糖为底物,在黑暗中进行化能异养生长。将适应了光能自养生长的培养物转移至黑暗中异养生长时,以NaNO3为氮源时的生长速率比以空气中的氮气为氮源时高;然而适应了化能异养生长的培养物以空气中的氮气为氮源时生长更佳。搅拌促进生长。满江红鱼腥藻在黑暗中生长半年后,叶绿素a的含量降至光照下生长时的1/3-1/4。满江红鱼腥藻在5500勒克斯光照下生长时,添加外源果糖或葡萄糖仍能促进生长,提高固氮活性。       

镉对满江红（Azolla imbricata）-鱼腥藻（Anabaena azollae）共生体氮代谢的影响

在实验室条件下,研究了不同浓度(0、0.01、0.05、0.1、0.5mg/L)的Cd对满江红-鱼腥藻共生体异型胞频率,固氮酶、谷氨酰氨合成酶活性以及铵态氮、游离氨基酸、可溶性蛋白、总氮含量的影响.结果表明,在整个实验期间,0.01mg/L Cd处理对上述指标均没产生显著影响,说明满江红-鱼腥藻共生体对Cd具有较强的耐性.当培养液中Cd浓度≥0.05mg/L时,随溶液中Cd浓度的增加和处理时间的推移,异型胞频率、固氮酶活性、谷氨酰氨合成酶活性、可溶性蛋白含量和总氮含量逐渐下降,而铵态氮含量在处理初期显著降低,随后迅速增加,游离氨基酸含量则逐渐增加.研究结果表明高浓度的Cd处理导致满江红-鱼腥藻共生体氮代谢的紊乱,最终造成氮素积累量的下降.     

Azolla--a model organism for plant genomic studies

The aquatic ferns of the genus Azolla are nitrogen-fixing plants that have great potentials in agricultural production and environmental conservation. Azolla in many aspects is qualified to serve as a model organism for genomic studies because of its importance in agriculture, its unique position in plant evolution, its symbiotic relationship with the N2-fixing cyanobacterium, Anabaena azollae, and its moderate-sized genome. The goals of this genome project are not only to understand the biology of the Azolla genome to promote its applications in biological research and agriculture practice but also to gain critical insights about evolution of plant genomes. Together with the strategic and technical improvement as well as cost reduction of DNA sequencing, the deciphering of their genetic code is imminent.     

结合态氮对满江红内生细菌群落组成和结构的影响

研究从超微结构和分子水平上探讨结合态氮对满江红(Azolla)叶腔中以共生藻占优势的微生物群落的影响。为排除外源污染并保留其内生菌的多样性, 采用茎尖组织培养并添加结合态氮等方法, 取得了表面无菌的含藻满江红(AmA)和无藻满江红(AmB)。电镜观察揭示, AmB较AmA的萍体表型有某种程度的修饰。AmA的微生物群落结构以共生藻、内生菌和宿主腺毛及其分泌物组成的生物被膜和藻囊为主要特征, 而AmB的叶腔几乎中空。基于16S rRNA基因和nifH基因的高通量测序结果显示, 生长于无氮培养液的AmA样品的微生物群落有相当高的多样性, 共有17个可操作分类单元(OTU), 分属4个细菌门, 并有一个以Nostoc azollae为优势的固氮微生物亚群包括草螺菌、根瘤菌和Niveispirillum等。而生长于富含结合态氮培养液的AmB样品的群落多样性明显降低, 仅有8个OTU, 且以Nostoc azollae为优势的上述固氮微生物亚群全部消失。研究结果表明, 通过调整氮素营养, 可改变宿主植物的微生物群落组成与结构, 进而改良植物的生长发育。     

满江红鱼腥藻与其宿主的遗传多样性和协同性的RAPD分析

从16个代表不同种属或地域来源的满江红样本中分离出共生藻并通过处理获得无藻的满江红宿主,对二者同步进行了RAPD扩增,分别得到了大量DNA多态片段。通过建立满江红鱼腥藻及其宿主的UPGMA聚类关系图,看出二者在遗传分支上存在着一定程度的协同对应关系。但在种内的不同品系间,这种协同性有所减少,发现有的品系的共生藻发生了明显的变异。 Abstract:Symbiotic Anabeana azollae and its host plant Anabeana-free Azolla were isolated from 16 Azolla accessions representing different Azolla species or geographic origins.DNA polymorphic fragments were obtained by simultaneous RAPD amplification of both symbiont and host.The UPGMA clusters of Anabeana azollae and its host Azolla were established separately based on Dice coefficient caculation and a coordinated relationship was shown between Anabeana azollae and its Azolla host along both individual genetic divergence,but this genetic homology was reduced among different strains within Azolla species while the obvious mutants of Anabeana azollae were detected in some Azolla tested strains collected from different geographic area in the same host species.     

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     

Electron Microscopic Observation of the Azolla-Anabaena azollae Relationship

The structure of Azolla pinnate leaves was examined by means of light and transmission electron microscopy. Emphasis was put on the ultra structural cytology of leaf cavity hairs both in association with Anabaena and in Anabaena-free cultures of Azolla and on the roles of the hair in substance change between the symbionts. The cavity hairs were multicultural and branched. There were numerous mitochondria, plastids, endoplasmic reticular and ribosome’s in the cytoplasm of the hair. A. marked characteristic of the hair was the cell wall ingrowths; There were large electron-transparent area between the in growing cell wall and the plasmolemma. Some vesicles were found in this area. It was suggested that these vesicles as transporters played the role in transporting substances. Electron microscopy revealed that some differences were present between the basal cell and the terminal branched cell of the hairs. In the latter, the cytoplasma, organelles, growing wall and vesicles were richer in the electron-transparent area than in the former This feature of the terminal branched cell showed that the terminal cell of the hair was more active in absorption and/or secretion of metabolites than that of the basal cell. Some hairs were found near the stem apex of Azolla. It was suggested that these hairs functioned in supplying the nitric compounds for the algae living on the stem apex of Azolla. These algae had no ability to fix nitrogen because of lacking heterocyst. In the absence of Anabaena azollae, the leaf cavity hairs were still present in Azolla pinnata. However, a lot of osmiophilic substances can often be seen in the vacuole of this hair.     

Megaspore germination, embryo development and maintenance of the symbiotic association in Azolla filiculoides Lam.

Megaspore germination and embryo development in Azolla filiculoides was examined using SEM and thin–sectioning. Within the released megaspore apparatus, resting cells of the endosymbiont Anabaena azollae Stras. arc located distally to the outside of the mcgasporangial wall and adhering to the inside of the megasporocarp wall. Growth of the female gametophyte displaces the floats pushing this part of the wall (the indusial cap) upwards, so providing access to the archegonia for the multifiagellalc spermalozoids. Embryo development and its inoculation with Anabaena involves a subtly–timed sequence of events resulting in the perpetuation of the symbiosis. Growth of the lunnel–shaped cotyledon leaf ruptures the mcgasporangial wall to provide access and a channelled route between the Anabaena and embryo shoot apex; subsequent leaf development severely restricts such access. During this process, the Anabaena is dislodged by the cotyledon leaf and growth of the first leaf traps the now actively–dividing Anabaena colonv; this becomes established around subapical trichomes from where filaments become incorporated into the cavities of developing leaves. The voung sporophyte rises vertically to the water surface as a result of gas accumulation in intercellular spaces; at no stage do floats endow buoyancy.     

Use of DNA/DNA hybridization techniques to authenticate the production of new Azolla-Anabaena symbiotic associations

Abstract The establishment of Azolla-Anabaena 'recombination' symbioses by grafting the Anabaena -containing indusium (cap) from the donor megasporocarp onto a decapitated megasporocarp from an Anabaena -free recipient has been recently reported. We have generated strain- and species-specific DNA probes for unequivocally establishing the identity of Anabaena azollae strains isolated from Azolla-Anabaena symbioses (whether new, i.e. heterosymbioses or natural i.e. homosymbioses). Eight out of nine heterosymbioses tested proved to contain the expected heterosymbiont while one recombined association was shown to contain both its own homosymbiont and a heterosymbiotic Anabaena strain. In addition, a species-specific plant probe was isolated from the total DNA of Azolla microphylla roots and used to establish the identity of a host plant used for recombination experiments.     

The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.     

A cyanobacterial recombination study, involving an efficient N2-fixing non-heterocystous partner

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7+/-0.2 n moles ethylene microg Chl. a(-1) h(-1) when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het- nif+ (non-heterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25+/-1 degrees C. This het- nif+ strain is an effective donor of the nif+ marker to a het+ nif- strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.     

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.     

Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces

Abstract The surfaces of cells of several species of cyanobacteria have been studied using low-temperature scanning electron microscopy (SEM), and have been shown to be covered in a layer of hydrated mucilage. This mucilage is observed in specimens of Anabaena azollae adhering to plant cells in their natural symbiotic niche (the cavity of the fronds of Azolla species) and in samples of the various species of cyanobacteria immobilised on polyurethane and polyvinyl support matrices. The mucilage appears to maintain the close contact observed between the cyanobacteria and these surfaces. Comparable films observed surrounding plant cells immobilised on similar polymeric surfaces are considered to be performing a similar function.     

黄进华 Electron Microscopic Observation of Symbiotic Relationship Between Azolla and Anabaena During the Megaspore Germination and the Sporeling Development of Azolla

The process of establishing symbiotic relationship of Anabaena azollae with its host during the megaspore germination and sporeling development was examined using electron microscope. The observations revealed that most of the Anabaena spores were primarily adhered to the hair cells arisen from the sporeling subsequently introduced into the cavity of the 1st true leaf by the hair ceils. Following the sporeling development, the Anabaena spores were migrated to the newly developing cavities and the branch apex in the same way. The pattern of germination of the Anabaena spore is similar to that of free-living cyanobacteria. Germinating Anabaena spores were only found at shoot apex region and the cavities of the sporeling, 92% of them being onto or near the hair cells which exhibit the ultrastructural characteristics of the transfer cell. The results suggested that Anabaena spore might get the chemical signal stimulating germination or the substance supporting cell multiplication from the host. Some of vegetative cells derived from the Anabaena spore were differentiated in to nitrogen-fixing heterocysts within the cavity. This means that the new generation of the symbiosis between Anabaena and Azolla has begun.     

Promiscuity of hosting nitrogen fixation in rice: an overview from the legume perspective

The subject area of this review provides extraordinary challenges and opportunities. The challenges relate to the fact that the integration of various fields such as microbiology, biochemistry, plant physiology, eukaryotic as well as bacterial genetics, and applied plant sciences are required to assess the disposition of rice, an alien host, for establishing such a unique phenomenon as biological nitrogen fixation. The opportunities signify that, if successful, the breakthrough will have a significant impact on the global economy and will help improve the environment. This review highlights the literature related to the area of legume-rhizobia interactions, particularly those aspects whose understanding is of particular interest in the perspective of rice. This review also discusses the progress achieved so far in this area of rice research and the possibility of built-in nitrogen fixation in rice in the future. However, it is to be borne in mind that such research does not ensure any success at this point. It provides a unique opportunity to broaden our knowledge and understanding about many aspects of plant growth regulation in general.