Taxonomic Status of Anabaena Azollae: an Overview

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fil;int and Soil 108. ISS-190 (1988) fflQ %f-3 ~ *I ' $lii\vcr Acadcinic Publishers PLSO BNF-48 *I

Taxonomic status of Anabaena azollae: An overview

J. PLAZINSKI, C. FRANCHE', C.-C. LIU', T. LIN', W. SHAW3, B.E.S. GUNNING and B.G. ROLFE / R~wmclrSchool oJ' Biological Sciences, Australian National Unhersity, Canberra City, ACT 2601, Airsrrcdicr. Pwt?ianent aildresses: 'ORSTOM, 213 rue Lafayette, F-75010, Paris, France, 'National Azollu Rrscurch Contre, Ftrjicrn Aíwlemy of Agriarlttiral Science, Fichoii, People's Republic of China, and .'D~irwi~iItistitirte of Technology, NT 5792, Aiistralia

Key words: Atiabuemi ~ollae,Azolla, Cyunobacterin, DNA/DNA hybridization, taxonomy

Abstract Despite the long-standing and widespread use of the symbiotic association between the aquatic fern Azolla and its cyanobacterial symbiont Anubaena azollae to augment nitrogen supplies in rice paddy soils, very little is known about taxonomic aspects of the symbiosis. The two partners normally remain associated throughout vegetative and reproductive development, limiting the opportunities for interchanges. We have used monoclonal antibodies and DNA/DNA hybridization techniques to show that the cyanobacterial partner is not uniform throughout the genus Azolla, and that substantial diversification has occurred. With these procedures it will be possible to characterize genotypes of the cyanobacterium and to monitor experiments aimed at synthesizing new combinations of Azolla species and Anabaena azollae strains.

Introduction The most remarkable feature of Azolla is its symbiotic association with the cyanobacterium Azolla is a genus of heterosporous aquatic ferns Annhaena azollae. The symbiosis can confer .high that grow on the surfaces of fresh water ponds, rates of nitrogen fixation and biomass production, lakes or streams. The genus was established by hence Azolla-Anabaena is an effective green ma- Lamarck in 1783 (Svenson, 1944) and its members nure for flooded crops and has been used as fertilizer are native to Asia (e.g., China, Vietnam), Africa in rice-growing for centuries in Vietnam and China. (cJ.~..Senegal, Zaire, Sierra Leone), the Americas Efforts are being made to improve the desirable (e.g.. southern South America to Alaska) and the agronomic attributes of this symbiotic association Antipodes. Individual species have been distributed by creating host-symbiont combinations other than by man and natural means to temperate as well as thcse available in natural populations. Under nat- tropical and subtropical areas (Lumpkin and ural conditions particular partners remain asso- Plucknett, 1982). ciated throughout cycles of vegetative and re- Most taxonomic treatments recognize seven dis- productive growth because the same symbiont is tinct species of Azolla on the basis of reproductive transmitted to successive generations by filaments and morphological features. They are grouped into which are carried in the megasporocarp: there is, in two sections (Lumpkin and Plucknett, 1982); sec- effect, maternal inheritance of the Anabaena. tion Euazolla (characterized by the presence of Production of recombinant partnership involves three floats on the megaspores) includes A. cnroli- breaking this strict association, and in order to be niuncr (Willdenow, 18IO), A.filicirloides (Lamarck, certain that a new cyanobacterial strain has been A. A, 1783), niesicanu (Presl, 1845), niicrophjdla introduced it is essential to have rigorous methods (Karlfuss. 1824), A. rirbra (R. Brown, ISIO), and of identification. section Rhizosperma (possessing nine floats on the Several problems exist. The evidence that there megaspores) includes A. pinnata (R. Brown, 1810) are in fact different strains of Anabaena azollae is and A. niloticu De Caisne (Mettenius, 1867). very limited. In the recent survey using polyclonal I -I I85

I i j 186 Pluzirish-i et al.

antibodies the similarities among 32 isolates far 1985; Gates et al., 1980; Ladha and Watanabe. outweighed thc differences (Ladha and Watanabe, 1982). Despite some inconsistency. two conclusions 1982). Isolates obtained from different fern species could bc drawn: (i) using polyclonal antibodics onc are morphologically similar (reviewed by Lumpkin can differentiate the A. rrzolluc~of Euazolla froni and Plucknett, 1980), and all belong to section IV that of Rhizosperma and (ii) laboratory cultured of Cyanobacteria, characterized by filamentous isolates of A. azollue have different antigenic heterocystous trichomes with cells dividing in one properties from those isolated freshly from the fern. plane (Rippka er al.. 1979). In Anabaena azollae the Recently, in the National Azolla Rescarch Ccn- proportion of heterocysts varies from zero in tre (Fuzhou, People's Republic of China), 13 hy- homogonia found among glands and leaf primor- bridoma cell lines secreting monoclonal antibody dia at the shoot apex of the host, up to 30-40% in against Admwa nzollue have been established those found in cavities of mature leaves, where the (Liu et (il.. 1987). Among the 13 monoclonal anti- rate of nitrogen fixation is maximal. Finally, it is bodies (MAbs), eleven MAbs reacted with all .-f. not possible to culture Anabaena azollae in the long azolhe representing seven species of Azolla, in- term as a free-living form, so physiological proper- dicating that they were detecting a common ties are not readily dctcrmined. It follows that it is antigen. Onc MAb reacted with A, ~zollu~~from necessary to develop new techniques for rigorous Euuolla but not with A. ~olltirfrom Rhizosperma characterization of Anuhaena azollae strains. (a subgroup-specific monoclonal antibody), A. In order to characterize Anabaena azollae strains, whereas the other reacted with only nzollae from techniques including immunoassays and DNA/ A. pinnuta (a species-specific MAb). None of the DNA hybridizations have recently been used (Arad MAbs reacted with two free-living N,-fixing cyan- et al.. 1985; Franche et al., 1986; 1987; Franche and obacteria Attubrirw cizoiicu and Tolypothrix. Thc Cohcn-Bazire, 1985; Gates et al., 1980; Liu et al., in authors concluded that there are at least four press). subgroups of A. azollae in Azolla species. This is the In this paper we present new data on the tax- first report showing that monoclonal antibodies onomic status of Anabaena azollae, obtained by could be used to discriminate Anuhaena azollae means of monoclonal antibodies and DNA hy- strains. bridization, and show that there are indeed substantial differences between isolates from different species of Azolla, and that these differences can be Use of DNA/DNA hybridization technique related to the taxonomy of the host plants. In order to characterize Anahaciiu azollrie strains several laboratories have begun to use a technique Use of ceil antigens of recombinant DNA research (Franche and J. Cohen-Bazire, 1985, 1987; Franche et al., 1986: Surface antigens of Anabaena azollae may be Meeks, personal communication). expected to play an active role in the establishment In nitrogen-fixing prokaryotes, including Ami- of cell-cell interactions as well as in the exchange of buena azollae, biological nitrogen fixation is cat- metabolites between Azolla and its phycobiont alysed by the nitrogenase enzyme complex. This (Peters et al., 1982). Mellor et al. (1981) have sug- complex contains two components: the nitrogenase gested that Azolla produces a lectin which recog- (called MoFe protein) and the nitrogenase reduc- nizes surface determinants on the Anabaena. Con- tase (called Fe protein) (Mortenson and Thorneley, versely it has also been suggested that in the sym- 1979). In the free-living Anubaenrz sp. PCC7 I20 biosis an Anabaena lectin recognizes the host fern these components are genetically determined by (Kobiler et al., 1981). These speculations em- three genes: nif H, nif D and tiif K (Mazur et al., phasize the potential importance of cell surface 1980; Rice et ai., 1982). Another nifgene (tifs),is composition in the development of the symbiosis. clustered with the structural nitrogenase genes and Qualitative differences were recently demon- is required for the maturation of the nitrogenase strated in antigenic structures between fresh and complex (Rice et al., 1982). cultured isolates of Ariahaem a;ollae (Arad er ai., In vegetative cells of Annhuena sp. PCC7120, niJ' Ta.uonontic status of A. azollae 187 K is separited from nifDH by an 11 kilobase (kb) conserved during evolution, presumably because of DNA fragment (Rice et al., 1982). Golden et al. stringent structural requirements in the protein (1985) have recently demonstrated that this inter- (Hennecke et al., 1985). vening region is excised and circularized during the In our hybridization studies fifteen different diferentiation of heterocysts, resulting in subse- Azolla isolates representing all known species have quent linking of the nifK and nif DH genes. Lam- so far been used (Table I), in conjunction with a mers et al. (1 986) have shown that the 11 kb DNA wide variety of heterologous gene clones as the element is excised by site-specific recombination DNA probes. The probes fell into two groups; one between directly repeated I1 base pair (bp) sequen- including DNA fragments which contain ni- ces at each of its ends, and encodes a gene, xis A trogenase genes isolated from free-living Anahaenna required for its own excision. A second rearrange- sp. PCC7120, and the second containing non- ment which leads to the excision of a circular DNA nitrogenase genes including the rRNA genes and element from vegetative-cell DNA is also observed the ribulose biphosphate carboxylase genes from near r1ifS (Haselkorn, 1986). Anacystis nidulans (Table 2). The susceptibility of Using DNA probes from the free-living Ana- Anabaena azollae DNA to 25 different en- baena sp. PCC7120 Franche and Cohen-Bazire donucleases was first determined and led to the (1985) have previously reported that the restriction selection of four restriction enzymes (e.g., Eco RI, sites within nif K, nif D and nif H genes of four HindIII, Bgl II and Kpn I) which were routinely symbiotic Anabaena azollae (freshly isolated from used. four different Euazolla species), were strongly con- 'The DNA hybridization patterns between Ana- served. The same authors also compared the re- baena azoflae isolates extracted from different striction sites in the region of the nitrogenase struc- Azolla species representing both sections Euazolla tural genes of five Rhizosperma endosymbionts to and Rhizosperma using single or combined nif thosc of the four Euazolla (Franche and Cohen- probes from the free-living Anabaena sp. PCC7120 Bazire, 1987). They presented evidence that sym- were compared. Most of the restriction sites within biotic Anabaena strains derive from a common and around the nifH and nifS genes were different ancestral Anabaena azollae and belong to two among the endosymbionts of the section Euazolla slightly divergent evolutionary lines. However, and Rhizosperma. Slight differences in the hy- from the taxonomic point of view the nifgenes that bridization patterns were observed among Ana- were investigated were not satisfactory, since the baena azollae isolates of the four species of nitrogenase structural genes have been strongly Euazolla and among a few strains of A. pinnata. As

Zrhh 1. A:ollu specics uscd for extracting cyanobacterial endosymbiont . - A~OlIUspecies Collection site Origin or reference EUU:Q~~U 1, A. curnliniunu United States Franche and Cohen-Bazire (1985) 7. A.filiculoidi~s 2 United States H.F. Diara 3. A. $/iculoides (Shepparton) Australia w.Shaw 4. A. jilìculoides (Snowy River) Australia W. Shaw 5. A.filiculr,i(ìe..s (Canberra) Australia W. Shaw 6. A.filicirloidcs East Germany W. Shaw 7. A. fi/iculoide.s China c-c. Liu 8. A. ttticruphylla China c-c. Liu Y. A. nricroplr~llu Galapagos Franche and Cohen-Bazire (1985) 10. A. nic.sicurw United States Franche and Cohen-Bazire (1985) Rhizospmnu II. A. pinnatu var. pinnuta Sn Africa Franche and Cohen-Bazire (1987) 17. A. pinnutu var. inihricatu Ind India P. Reynaud A. 13. pinnutu var. itnbricutu ImA Africa P. Reynaud 14. A. pinnutu var. ittrbricuta (Darwin) Australia W. Shaw 15. A. pinnutu var. inrhricufa (Townsville) Australia W. Shaw o

188 Pltrzirrski et al. Tuhh 2. DNA probes used for DNA/DNA hybridization study Probe Characteristics Source/reference Artuhuettcr sp. PCC7120 pAn 154.3 1.8 kb Hiti dII1 fragment Rice ei u/. (1982) containing r$H pAn I54 I 6.0 kb Hin dI11 fragment Rice ei al. (I 982) containing riif.5 pAnZ07.3 1.8 kb Hiri dIIl subfragment Rice et (11. (198') of the 11 kb region being excised during heterotocyst formation

Atruc~siistticlirlutis 630 I pAn4 6.5 kb PSI Ifragment Tomioka and Sugiura rm of entire A operon (I983) (16S, 23s and 5s rRNA genes) pANPl I55 2.3 kb Psi Ifragment Shinozaki and Sugiura of the genes for the large (1985) (LS) and smull (SS) subunits. of ribulose- I ,5-bisphosphate carboxylaseloxygenase (ruBisCo) -

previously reported (Franche and Cohen-Bazire, ABCD 1987) no hybridization was found between DNA from the A. azollae symbionts and the probe pAn207.3, carrying a part of the 11 kb region that separates ti(f K from ti(f DH in vegetative cells of Atiahaciza sp. PCC7120. This observation is in agreement with a contiguous nifH, D, K organization in A. azollae, as suggested by mRNA studies (Nierzwicki-Bauer and Haselkorn, 1985) and by hybridization studies (J. Meeks, personal communication). DNA polymorphism was observed among Ana- B baena endosymbionts when a 2.3 kb Pst l fragment of the RuBisCO gene cluster was used (Fig. 1). The .. .'k use of rRNA cloned genes as hybridization probe also revealed polymorphism within these genes isolated from different symbiotic Anabaena (Fig. 2). Subclones of these genes, while used as DNA -4 probes, were found to be very useful in differentia- ting amongst endosymbionts extracted from different isolates of the same Azofla species (e.g.. when I isolated from different A.piririata and A.Jiliculoitkis strains). Fi.q. 1. Autoradiogram of '?P-labelled RuBisCo probe hy- On the basis of our present data, combined with bridized to A. DNA extracted from: (A) A. fil~ciiloide.~ u:ol/oe A. results prescnted by Franche and Cohen-Bazire (East Germany), (B) A./ilicir/uirles (Shepparton). (C) pitinofa (1985, 1987), we were able to draw a simple phy- var. piririuiu and A. DNA digested with (D) coroliniuna. The was logenetic tree symbiotic Anabaena strains (Fig. the cndonuclcase Eco RI. Arroiw indicate unique hybridizing of bends that distinguish between A. a:ollue strains. 3). At least nine different strains of Ariahacwa ozoll- Taxonomic status of A. azollae 189 ’, Q ABCD E’ different hybridization patterns were observed in the Anabaena azollae DNA digests. Interestingly, -œ all the Australian Azolla strains so far studied ap- pear to contain a symbiont which has phylogenetic- ally diverged from the Azolla strains collected on the other continents.

Conclusions

In recent years molecular biology has provided important information in the taxonomic study of Azolla endosymbionts. At the present time it seems that Southern blot hybridization analysis using heterdogous DNA probes gives finer discrimina- tion with respect to the taxonomy of symbiotic Anabaena than do immunological analyses. How- Fig. 2. Autoradiogran 3f ’*P-labelled rR probe hybridized ever, the use of monoclonal antibody techniques to to A. molhe DNA extracted from (A) A. caroliniana. (B) A. study surface antigens of A. azollae strains will also pinnara var. pinnata, (C) A. filiculoides (Shepparton), (D) A. provide a powerful tool, alone or in combination filiculoides (East Germany) and (E) Anabaena pos-aquae (free- with molecular techniques in future characteriza- living algae). The DNA was digested with Hin dIII. Arrows tion of symbiotic Anabaena. indicate unique hybridizing bands. The current results distinguish at lest nine dif- A. &ann ferent strains of Anabaena azollae among symbiotic A. A. caroliniana (10) microphylla cyanobacteria associated with Azolla. The results are repeatable, and we have obtained reproducible blot patterns over several years. Most interestingly, using the ‘fingerprinting’ method one can now distinguish between Anabaena strains extracted from \I/ different biotypes of the same Azolla filiculoides RHIWSPERMA EUAZOLLA species; several strains of Anabaena azollae were also identified in the species A. pinnata. The study of more intra-specific isolates of Azolla, in par- \/ ticular with Azolla caroliniana, A. mexicana and A. SYMBIOnC microphylla, together with the use of more test AMhlCMUWk probes, will be necessary to confirm that the symbiotic cyanobacterium is represented by many different genotypes. The symbiotic relationship between Azolla and Anabaena could therefore be as Fig. 3. Schematic presentation of the relationship between sym- host, and/or strain-specific as in the case of the biotic Anabaena strains isolated from different Azolla isolates. Rhizobium-legume symbiosis. as deduced from hybridization patterns. Strain numbers corres- pond to the numbers used in Table 1. *According to the present hybridization studies strain number 8 (,+olla mirrophylla, China) seems to be similar to the strain no. Acknowledgments 9 (see Table I), however, more data is required before final conclusion can be drawn. We thank Professors G Stanier and R Haselkorn ae are associated with Azolla plants. Among five as well as Dr K Shinozaki for providing us with isolates of A. filiculoides, we were able to distin- their plasmid clones, Drs P Reynaud, H F Diara guish three different genotypes of Anabaena azol- and Professor Van Hove for gifts of Azolla species lae; and among five isolates of Azolla pinnata, three and Rona Taylor for technical assistance. Prof G 190 Ttrsononiic siutus of’A. azollae Stanier, Drs R Rippka and J Meeks are thanked for Hennecke H, Kaluza K. Thony B, Furhmann M, Ludwig W and helpful discussions. Stackebrandt E 1985 Concurrent evolution of nitrogenase C Franche was supported by the French Institute gcncs and 16s rRNA in Rliizohitmr species and othcr nitrogen fixing bacteria. Arch. Microbiol. 142, 342-348. of Scientific Research for Development Through Kobiler D. Cohen-Sharon A and Tel-Or E 1981 Recognition a Cooperation and was Visiting Fellow in the Re- between the N,-fixing Anabaena and the water fern Azolla. FEBS Lett. 133. 157-160. search School of Biological Sciences, Australian J National University. Lltdha K and Watannbe I 1982 Antigenic similarity among dnuhaenn crcohe separated from different species of Azolla. This program of collaboration between the Na- Biochem. Biophys. Res. Comm. 109, 675-682. J tional Azolla Research Centre, Fuzhou, and the Lammen P J, Golden Wand Haselkorn R 1986 Identification Research School of Biological Sciences, ANU, and sequence of a gene required for a developmentally re- Canberra, was developed under the auspices of the gulated DNA excision in Anabaena. Cell 44, 905-91 I. Australian Centre for International Agricultural Liu C-C, Chen Y, Tang L. Zheng Q. Song T. Chcn M, Li Y and Research, project 850 Lin T 1987 Studies of monoclonal antibodies against Ana- 1, burnu u:ol/ae. Acta Botanica Sinica (In press). Lumpkin T A and Plucknctt C 1982 Azolla as a grcen manure: use and management in crop production. West View Press. References Boulder. Mazur B J, Rice D and Hasclkorn R 1980 Identification of blue-green algae nitrogen fixation genes by using heterolo- Arad H, Keysari A. Tel-Or E and Kobiler D 1985 A comparison gous DNA hybridization probes. Proc. Natl. Arad. Sci. between cell antigens in different isolates of Anabaena azollae. (USA) 77, 186-190. 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Shinozaki K and Sugiura M 1985 Genes for the large and small Gates E, Fisher R W, Goggin W and Azrolan N 1980 J T I subunits of ribulose-I ,S-biphosphate carboxylaseloxygenase Antigenic differences between Anabaena azollae fresh from constitute a single operon in a cyanobacterium .4nacystis the Azolla fern leaf cavity and the free-living cyanobacteria. nidulans 6301. Mol. Gen. Genet. 200, 27-32. Arch. Microbiol. 128, 126-129. Svenson H K 1944 The new world species of Azolla. Am. Fern Golden J J W, Robinson S and Haselkorn R 1985 Rearrange- J. 34, 69-84. ment of nitrogen fixation genes during heterocyst differentia- Tomioka N and Sugiura M 1983 The complete nucleotide tion in the cyanobllcterium Anabaena. Nature 314,419-423. sequence of a ribosomal RNA gene from a blue-green Haselkorn K 1986 Organization of the genes for nitrogen fixa- 16s alga, Anacysfis nidulans. Mol. Gen. Genet. 191: 46-50. tion in photosynthetic bacteria and cyanobacteria. Ann. Rev. Microbiol. 40, 525-547.