the azolla, anabaena azollae relationship - plant … of azolla caroliniana willd. free of the...

Plant Physiol. (1974) 53, 813-819

The Azolla, Anabaena Azollae RelationshipI. INITIAL CHARACTERIZATION OF THE ASSOCIATION1

Received for publication November 8, 1973 and in revised form JanLary 16, 1974

GERALD A. PETERS AND BERGER C. MAYNECharles F. Kettering Research Laboratory, Yellow Springs, Ohio 45387

ABSTRACT

Cultures of Azolla caroliniana Willd. free of the symbioticblue-green alga, Anabaena azollae, were obtained by treatmentof Azolla fronds with a regimen of antibiotics. These symbiont-free plants can be maintained only on medium containing acombined nitrogen source.

Morphological aspects of the symbiotic association show theconfinement of the Anabaena azollae within the leaf cavity ofthe Azolla. Procedures were established for the isolation ofpure preparations of Anabaena azollae and Azolla chloroplasts.It has not yet been possible to grow the isolated alga in inde-pendent culture.

Photochemical activities of the isolated alga and fern chloro-plasts were measured by spectrophotometric assays for photo-systems I and II as well as by P700-content (photosystem I)and delayed light emission (photosystem II). In the algal frac-tion, both photosystems were repressed when compared to free-living Anabaerna cylindrica, but the relative ratio of photosys-tem I to photosystem II may be appreciably greater inAnabaena azollae. Azolla chloroplasts were generally compa-rable to spinach chloroplasts.A comparison of the chlorophyll a and b content of Azolla

fronds with and without the symbiotic alga resulted in an esti-mate that in the symbiotic association, the Anabaena azollaeaccounts for from 7.5 to 15% of the total chlorophyll.

26). Nickell (23), in securing an aseptic culture of Azollacaroliniana, reported that both bacteria and algae were elimi-nated by treatment with antibiotics. Johnson et al. (18) usedantibiotics after surface sterilization to remove green algae(largely Chlamydomonas) which continually appeared in cul-tures with combined N2. They stated that these treatments alsoeliminated Anabaena azollae from the cultures since the algawas not observed in the fronds. However, neither Nickell (23)nor Johnson et al. (18) demonstrated the absence of growth andN2 fixation in N2-free medium by the fronds in which the sym-biotic alga was reportedly absent.The Azolla-Anabaena azollae association permits the study of

a symbiotic relationship between a blue-green alga and a greenplant under laboratory conditions. Previous studies on thephysiology of the symbiotic association were not well definedand were limited in scope. Various aspects of mineral nutrition,temperature, and light intensity on the growth of the organismhave been reported (22). We are not aware of any studies onthe metabolic functions of N2 fixation, respiration, and photo-synthesis in the individual organisms or their interaction in thesymbiotic association.

This manuscript is a report of initial studies on the charac-terization of the Azolla-Anabaena azollae symbiotic relation-ship. We describe the morphology, a method of freeing thefronds of the symbiotic alga, and isolation procedures devisedto fractionate Azolla-Anabaena azollae for metabolic studies.The companion publication deals with aspects of acetylene re-duction (nitrogenase activity) (25).

Azolla is a genus of small aquatic ferns with a world-widedistribution. The sporophyte consists of a pinnately branchedfloating stem. Simple roots, which hang down into the water,occur at some nodes. The branches and stem are covered withsmall, alternate, overlapping leaves. Each leaf is divided into athick, chloropyllous dorsal lobe and a thin, achlorophyllousventral lobe. The heterocyst forming blue-green alga, Anabaenaazollae, is found enclosed within a special chamber, or cavity,in the ventral side of the chlorophyllous dorsal lobe (22, 28).

Indirect evidence has indicated that the algal-containingAzolla is capable of assimilating atmospheric N2 (7, 18, 22, 24).Correspondingly, it has been reported that Azolla freed of thesymbiotic alga requires a source of combined N2 (26).Anabaena-free Azolla has reportedly been obtained by suchdiverse methods as cold treatment, very dilute culture medium,low light intensity, and more recently, by antibiotics (18, 23,

1 Contribution No. 509 from the Charles F. Kettering ResearchLaboratory.

MATERIALS AND METHODS

Azolia Cultures. Plants of Azolla caroliniana Willd. wereobtained from a local greenhouse pond. Cultures free of con-taminating epiphytic microorganisms, especially green andblue-green algae, were obtained by washing fronds 20 timeswith vigorous agitation in large volumes of distilled water, fol-lowed by surface sterilizing with a solution containing 2%Clorox (0.12% sodium hypochlorite) and 0.01% Triton X-100as a wetting agent for 20 to 30 min. The fronds were removed,washed a minimum of six times with large volumes of steriledistilled water, and transferred to sterilized nutrient medium inErlenmeyer or Fernbach flasks, with and without a combinedN2 source. Although this treatment resulted in the death of alarge number of fronds, it was possible to obtain subculturesof small, healthy fronds after 1 week. Subsequent culturesgrew well in the absence of a combined N2 source and wereshown to reduce acetylene. Cultures grown on a combined N2source remained free of contaminating green and blue-greenalgae. Procedures employed to obtain Azolla fronds free of thesymbiotic alga are presented under "Results."

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84EEADMNPlantPhysiol. Vol. 53, 1974

nutrient solution with added micronutrients was employed.TheNr-free nutrient solution contained 2mm KCI, 2 mmCaCl2, 0.4 mmKHO PO,, and 0.8 mm MgSO,. Each liter con-tained 1 ml of a stock solution of Sequestrene NaFe (FeEDTA)containing 5 mg Fe/mi and of a micronutrient solution (1). Inthe nutrient solution with combined N2 as nitrate, the KCI andCaCl2 were replaced with equivalent concentrations of KNOsand Ca(N02),. Nutrient solutions containing combined N2 asurea or ammonium were prepared by adding urea or NH4Cl tothe N2 free nutrient solution. The total N2 was equivalent to thatpresent in the solution with nitrate. Urea was filter-sterilizedand added to the nutrient solution after autoclaving. In all casesthe nutrient solutions were pH 5.4 to 5.6.Growth Conditions. Fronds were grown in Erlenmeyer or

Fernbach flasks in a growth chamber. The day temperaturewas 23 C and night 18 C. A 16-hr light, 8-hr dark cycle wasemployed. Illumination was provided by a mixture of cool-white fluorescent, Gro-lux, and incandescent lights. Cultureswere constantly maintained at either 200 or 400 ft-c.

Chemicals. DCMU was obtained as Diuron from the E.I.duPont de Nemours Company. Na penicillin G and polyvinyl-pyrrolidone (PVP-40) were obtained from the Sigma ChemicalCompany. Polymyxin B sulfate, chlorotetracycline-HCl(Aureomycin), tetracycline-HCl, bacitracin, and streptomycinsulfate were obtained from the Nutritional BiochemicalsCompany. All other chemicals were reagent grade.

Cholorphyll Determinations. Azolla fronds from individualflasks were removed, blotted onfilter paper and ground in 80%acetone using a motor-driven Teflon homogenizer. The sampleswere placed in darkness for 15 min to allow further extractionand then centrifuged. Similar procedures were employed forchloroplasts and blue-green algal fractions. Chl was determinedaccording to Arnon (2). In addition, the Chl a of the blue-greenalga was routinely determined by the method of Biggins (5).

Fractionation and Gradients. Azolla fronds were groundwith a Polytron (type PT20ST/ OD, Kinematica Gmbh, LuzerSchweiz) in an isolation mix containing 0.4M sorbitol, 0.1MTricine (pH 7.8), 10 mm KCl, 1 % 2-mercaptoethanol (v/v), 1%Na ascorbate (w/v), 1% PVP-40 (w/v), and bovine serum al-bumin at 2.5 mg/ml. The grinding was carried out at 0 to 4 C for30 sec at 60 v and 1 min at 100 v. The brei was passed through4, then 8 layers of cheesecloth and centrifuged for 7 min at1000g. The pellets were resuspended in 0.4M sorbitol, 20 mMTricine (pH 7.8) and 10 mm KCl. The Chl content was adjustedfrom 0.25 to 0.5 mg/ml, and 2 ml of this mixture were layeredonto sucrose density gradients. Continuous gradients werefrom 1.0 to 2.5M sucrose in 32 ml of 20 mm Tricine (pH 7.8).Discontinuous gradients had 9 ml each of the following bands in20 mM Tricine (pH 7.8): 2.5 M, 2.0M, 1.5 M and 1.0 M.Centrifugation was for 60 min at 25,000 rpm in a Spinco SW-27 rotor. Following centrifugation on discontinuous gradients,Azolla chloroplasts are found at the 1.0 to 1.5M sucrose inter-face and the blue-green alga at the 2.0 to 2.5M sucrose interface(Fig. 4). The band at the 1.5 to 2.0M interface was a mixtureof chloroplast fragments, algal cells, and other debris. Thebands were removed from the gradients with a cannula, dilutedto approximately 0.5M sucrose with 20 mm Tricine (pH 7.8) and10 mm KCl and centrifuged 15 min at 27,000g. The chloro-plast pellets were resuspended in a minimal volume of 0.4 Msorbitol, 20 mm Tricine (pH 7.8), 10 mm KCl and used directlyor frozen in liquid N2 and stored until use. The algal fractionswere treated in the same way except that frozen fractions couldnot be used for acetylene reduction assays (25).

"Gentle" Anabaena azollae Isolation. A single layer ofAzolla fronds was placed in a shallow porcelain pan, and N2-

free BG 1 1 medium (1, 30) supplemented with 1% PVP-40 wasadded until the fronds began to float. A Teflon roller was usedas a rolling pin to gently squash the fronds. The resulting mix-ture was passed through four, then eight layers of cheeseclothand centrifuged at room temperature in a clinical centrifugefor 2min. The pellets were resuspended in N2-free BG I1medium, passed through two layers of 100,um nylon mesh,pelleted by centrifugation, and resuspended in a small volumeof the N2-free medium. The entire procedure was carried outin a positive pressure room utilizing sterile techniques.

Absorption and Fluorescence Emission Spectra. A CaryModel 14 spectrophotometer equipped with a scatter trans-mission apparatus was used to obtain absorption spectra.Fluorescence spectra were obtained as described previously(21) except that a 0.25-i B & L monochromater was used toobtain monochromatic actinic light.

Delayed Light Emission. Delayed light was measured witha phosphoroscope as described by Clayton (8, 9) except thatthe delayed light was measured approximately 0.5 msec afterillumination.

Cell-free Extracts and P700 Determinations. The chloroplastsand A nabaena azollae cells were subjected to two passesthrough a French Pressure Cell at 14,000 psi and centrifuged toremove whole cells and debris. In some cases the Anabaenaazollae was obtained by the "roller" method and in others fromgradients. Cell-free extracts of vegetative cells and heterocystswere prepared by the method of Fay and Walsby (14) exceptthat heterocysts were broken by two or more passes through thepressure cell at 18,000 psi. P700 was measured by photochemi-cal oxidation (6) using an absorbance coefficient of 64 mmole1cm-'1 (16).

Thin Layer Chromatography. Whole fronds and samplesfrom the discontinuous gradients were exhaustively ground in80% acetone to extract the Chl. In each case, the extract wascentrifuged and transferred to one-half its volume of petroleumether (30-60 C) by washing with a 10% saline solution. Afterseparation, the lower aqueous-acetone layer was discarded andthe petroleum ether-extract was washed five times with salinesolution. The extract was concentrated under a stream of dryN2 and dried over anhydrous sodium sulfate. Approximatelyequivalent amounts of each extract, based on the initial Chl,were applied to Gelman-type SG TLC sheets, and dried with astream of dry N2. The sheets were developed with a solvent sys-tem of 1 % n-propanol (v/v) in petroleum ether. Developmentwas complete within 15 min and pictures were taken im-mediately.

Photochemical Assays. These were conducted as describedpreviously (3) and as briefly set forth in the text and tablelegends.

Blue-Green Alga. Anabaena cylindrica, strain 1403/2a, wasgrown on N2-free BGI 1 medium (1, 30) at 400 ft-c, 23 C withconstant shaking and bubbling with 1 % C02 in air.

RESULTS

Morphology and Anatomy. An Azolla caroliniana frondgrown on N2-free medium is shown in Figure 1. Figure 2, aand b, shows sections of a frond which was fixed with 2%glutaraldehyde in 50 mm phosphate buffer (pH 7) and 1%osmium tetroxide for 1 hr each, embedded in plastic (29),sectioned on a Porter-Blum ultramicrotome and stained with1% basic Fuschin in 50% acetone for 10 min. These sectionsillustrate the morphology of the leaf and cavity. The presenceof secretory cells and the relative numbers of the symbioticblue-green alga in two planes through the same leaf can beseen. A photomicrograph of the symbiotic blue-green alga

814 PETERS AND MAYNE

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AZOLLA, ANABAENA AZOLLAE RELATIONSHIP

isolated from the leaf cavity with a micronshown in Figure 3.

Azolla Free of the Symbiotic Alga. In our initobtain Azolla free of the symbiotic alga, we (

light, dilute culture media, prolonged growth atand the use of a graduated series of the algiciparts CuSO, to 1 part AgNO3). None of these alsuccessful. The use of antibiotics, however, e)alga-free Azolla. Antibiotics were employed in(in various combinations and concentrations. Inboth N2-free medium and medium containing asource were employed. Cultures were left in nuwith antibiotics for 1 week with one change to fr

!lb~~~~~~

~~..~.

FIG. 1. Azolla frond from culture grown for sevnutrient solution without combined N2. Individuacavity containing symbiotic alga.

nanipulator is containing medium, transferred to antibiotic-free media for 1week, and then transferred to media containing different anti-

ial attempts to biotics. This procedure was continued until four differentemployed dim combinations or individual antibiotics had been employed.4 C (22, 26), Three of the sequential treatments were successful and gave

ide CA350 (6 fronds which have remained free of the symbiont and incapa-pproaches was ble of acetylene reduction. These were: (a) Aureomycin 2ventually gave ,ug/ml to Na penicillin G 50 ,ug/ml to streptomycin sulfate 10dividually and ytg/ml to bacitracin 10 jug/ml and polymyxon B sulfate 12.5each instance, jug/ml; (b) polymyxin sulfate 25 ,ug/rnl to tetracycline 10combined N2 jug/ml to bacitracin 20 jug/ml to streptomycin sulfate 20 jug/ml

trient medium and Na penicillin G 100 ,ug/ml; (c) tetracycline 10 ,tg/ml,*esh antibiotic- polymyxin B sulfate 25 jug/ml, bacitracin 20 ,ug/ml to Na peni-

cillin G 100 ,ug/ml, streptomycin sulfate 20 /tg/ml, Aureo-mycin 4 jug/ml to tetracycline-HCl 10 ,tg/ml, polymyxin Bsulfate 25 ,ug/ml, bacitracin 20 ,ug/ml to Na penicillin G 100yg/ml, streptomycin sulfate 20 ,ug/ml, Aureomycin 4 [ktg/ml.Azolla treated with these antibiotic regimens in a medium freeof combined nitrogen regressed. When small fragments of thefronds were transferred to a medium containing combined N2they grew and, during a 6-month period, they have not regainedtheir ability to reduce acetylene. Furthermore, there is nomorphological evidence that they contain the microsymbiont.These fronds are much more compact than those which containthe symbiotic alga and grow more slowly. The fronds whichwere maintained in medium with a combined N2 source re-mained relatively healthy during the course of the same treat-ments and gradually reverted to the symbiotic state upon con-

t tinued culture in antibiotic-free media.Fractionation Studies. Azolla fronds in an isolation medium

were ground in a "polytron," filtered through cheesecloth andlayered on discontinuous sucrose gradients. The separation ob-tained is shown in Figure 4, which compares the fractionationof N2-fixing fronds containing the symbiotic algae and non-

0 25 cm N2-fixing fronds freed of the symbiotic algae.The purity of the fractions was demonstrated by light

microscopy, absorption spectra, low temperature (77 K)fluorescence emission spectra and TLC. Light micrographs of

eral months on the chloroplast fraction, band 1, and the blue-green algal frac-1 leaflets have tion, band 3, are shown in Figure 5, a and b. Band 2 in both

cases is composed of broken chloroplast, fragments of stems,

* Ot4*;.;^_ t

FIG. 2, a and b. Sequential sections from a serial series through young Azolla leaves showing morphology of the leaf and cavity and relativeamount of symbiotic alga in two planes through the same leaf. Glutaraldehyde-osmium fixation, stained with 1%- basic Fuschin. sc: secretory cells;c: cavity; AA: Aniabaenia azollae.

Plant Physiol. Vol. 53, 1974 815



Plant Physiol. Vol. 53, 1974

FIG. 3. Anabaena azollae isolated from leaf cavity with micromanipulator.

FIG. 4. Discontinuous sucrose gradients showing separation ob-tained after fractionating fronds with (left) and without (right) thesymbiotic algae. Band 1: chloroplast fraction; band 2: mixture ofbroken chloroplasts, cellular debris, and when present in fronds,short chains and pieces of the symbiotic alga; band 3: the symbi-otic alga, Anabaena azollae.

and in the case of the blue-green-containing fronds, some algalcells. Absorption spectra of bands 1 and 3 were obtained in60% glycerol at approximately equal Chl concentrations. Incontrast to the chlorop.ast fraction, the blue-green algal frac-

tion (band 3) showed a pronounced maximum at about 620 nmarising from phycocyanin (10, 19). In addition, the strongfluorescence emission from phycocyanin at 640 nm, observedwhen the algal fraction was illuminated with 560 nm light at77 K, was not detected in the chloroplast fraction.

Another criterion of purity was the isolation and chromato-graphic separation of the pigments from the gradient fractions.Figure 6 compares the pigment extract of a whole frond, andthe three bands from a gradient as shown in Figure 4. Theabsence of Chl b in the isolated algal fractions also demon-strates the absence of any contaminating chloroplasts.

Photochemical Activity, Chl/P700 Ratios and DelayedLight Emission. Chloroplasts and Anabaena azollae fractionsobtained from gradients and Anabaena azollae obtained viathe roller method were assayed utilizing the diphenyl carba-zide-DCIP (DCMU-sensitive portion) assay for PSII2 andNADP+ reduction using the ascorbate-DCIPH, assay for PSI(3). The rates obtained from several experiments along withChl/P700 ratios are shown in Table I. Photochemical activity,especially PSI, was dependent upon the inclusion of 1%PVP-40 in the isolation mixture and with chloroplasts, was in-creased further by 1% 2-mercaptoethanol, 1% sodium as-corbate, and 0.25% bovine serum albumin. With the exceptionof the results obtained from continuous gradients, the rates ob-tained for chloroplast PSII activity were relatively low. Wecan offer no explanation for the difference.

Only very limited PSI and PSII activities have been obtainedusing these assay systems for the intact blue-green alga. Al-though it is possible that this is the result of an osmotic effect(15), it is doubtful that this alone is responsible, since we didnot obtain significant rates when the symbiont was isolated ina medium with or without an osmotic agent. Therefore, cell-free extracts of the alga were employed. As seen in Table I,cell-free extracts of vegetative cells have appreciable PSI ac-tivitv and a lower but easily detectable PSII activity.The Chl/P700 ratio of Azolla chloroplasts is slightly higher

2 Abbreviations: PSII: photosystem II; PSI: photosystem I; DCIP:dichlorophenol indophenol.

816 AND MAYNE

,"e,




FiG. 5, a and b. Photomicrographs of (a) the chioroplast fraction (band 1) and (b) the symbiotic algal fraction (land 3) from gradientsas shown in Figure 4 illustrating purity of the individual fractions.

than the average but still within the range observed with spin-ach chloroplasts (3). In the case of the Anabaena azollae theChl/ P700 ratio of the vegetative cells was always slightlyhigher than that of the heterocyst fraction. Since the alga inthe cavities was heterogeneous, the heterocyst fraction alwayscontained some akinetes. Donze et al. (11) found Anabaenacylindrica heterocysts contained about twice as much P700, ona Chl basis, as vegetative cells. When corrected for the differ-ences in the molar extinction coefficient of P700 employed,their re-ults would be 58 Chl/P700 in heterocysts and 109Chl/P700 in vegetative cells. We have obtained comparablevalues for Anabaena cylindrica. This would indicate thatAnabaena azoltae has a lower concentration of reaction centerP700 than Anabaena cylindrica grown under N2-fixing condi-tions in air. With respect to P700 concentration, the heterocystfraction of Anabaena azollae is more similar to the vegetativecells of Anabaena cylindrica.

Just as the P700 content is an index of PSI, delayed lightfrom chloroplast has been shown to arise, at least predomi-nately, from PSII (4, 20). Using the same algal preparation asemployed for P700 assays, we found that on a Chl basis thedelayed light emission from intact filaments of Anabaenacylindrica was approximately 8 times greater than fromAnabaena azollae. In a comparison of the delayed light emis-sion from cell-free extracts of vegetative cells and of hetero-cysts from Anabaena azollae, we found the delayed light fromthe vegetative cells was 10 times greater than that from theheterocysts. In conjunction with the effect of akinetes on theP700 ratio of the heterocyst fraction noted above, their pres--nce in this fraction would presumably increase the amount of

delayed light. These assumptions are based on the reportedpigment composition (12) and metabolic activities (13) of iso-lated vegetative cells, spores, and heterocysts from Anabaenacylindrica.Chkwophyl a/b Ratios; Estimate of Albl orophyll. As

mentioned previously we have obtained estimates of the algalcontri'bution to the total Chl. This has been done using the Chla/b ratio of fronds containing the symbiotic blue-green algaeand the a/b ratio of fronds without the blue-green alga. Thesedata are summarized in Table II. The a/b ratio in frondswithout the symbiotic alga is nearly identical to that of the iso-lated chloroplasts end all are lower than that obtained fromfronds with the symbiotic alga. This is to be expected if theratio in the chloroplast is relatively constant Since the blue-green alga has no Chl b, its presence would be expected to in-crease the overall a/b ratio. By emp oying the equation

Chi a (total) Chl a (Azolla) + Chi a (Anabaena)Chl b (total) Chl b (total = Azolla)

and solving for the Chl a Anabaena, utilizing the informationshown in Table II, a value of 14% of the Chi a is obtained forthe algal contribution. This corresponds to about 0.06 mgblue-green algal Chl a/gram fresh weight of fronds. We feelthat it is reasonable to assign 10 to 20% of the Chi a, or from7.5 to 15% of the total Chl to the blue-green algal fraction.

Attempts to Culture the Symbiotic Algae. Thus far all at-tempts to culture the symbiont have been fruitless. A largevariety of solid and liquid media normally employed for thecultivation of free-living blue-green algae, with and without

Plant Physiol. Vol. 53, 1974 817

I



Plant Physiol. Vol. 53, 1974

9..*

j** i

W9*{}^ ---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Band IChlo roplast

Band 2Mixture

Band 3Anab. Azollae

FIG. 6. Thin layer chromatograph of pigment extracts from whole Azolla fronds containing the symbiotic blue-green alga and the three bandsobtained after fractionation and density gradient centrifugation as shown in Figure 4. Note the absence of any contaminating Chl b in the Aln-abaeiia azollae extract.

Origin

Chloroplasts from fronds con-

taining algae

Chloroplasts from fronds freedof algae

Aniabaenza azollae3

Vegetative cells3Heterocysts3

PS IIActivity'

,u??zoleslpng(hi hr

454232

1504

3228

28

PS I Chl/P700Activity2 Ratio

;stJoles/mgChli- hr

2461591402044

160137

128

5803

158138108

'Assayed with diphenyl carbazide DCIP (DCMU-sensitive

portion).2 Assayed with ascorbate + DCIPH2- NADP+ (plastocyanin,

ferredoxin, and ferredoxin-NADP reductase and Triton X-100were included).

3Cell-free extracts.4Continuous gradient fractions, all other chloroplasts are from

discontinuous.

Table II. Chlorophyll a/b Ralios

Source Chl a/b SD No. ofReplicates

Whole fronds with algae 3.32 0.15 13

Chloroplasts from fronds with 2.82 0.06 3algae

Whole fronds free of algae 2.75 0.01 3

Chloroplast from fronds free of 2.78 0.03 2algae

carbon sources and frond extracts, has been employed as havevarious temperatures and aerobic and anaerobic gassing. Asnoted by Holm-Hansen (17) and strengthened by our findings,this may reflect structural or biochemical alterations of thesymbiotic alga which have led to the dependence of the alga onthe fern.

DISCUSSION

The study of a symbiotic relationship between two photo-synthetic organisms presents certain basic problems. As shown

Chl a .v.

Chl b

WholeFrond

Table I. Photochemical Activities anid Chl/P700 Ratios

818 PETERS AND MAYNE




in Figure 2, a and b, the alga is in an enclosed cavity in thefrond leaf. The cavity is filled with a mucilaginous substanceof unknown composition and is penetrated by secretory cellsor glandular hairs which arise from the Azolla epidermis.Therefore, the algal cells are not in direct contact with the ex-ternal environment. Rather they are in a microenvironmentcreated within and subject to parameters imposed by the fernleaf. Thus, while the alga is provided physical protection bythe fern, it is also dependent upon the fern for mineral uptake.Furthermore, it seems apparent that the quality and intensityof light incident on the algae and possibly the gas phase withinthe cavity are modified.

In order to gain an initial insight into the type of interac-tions involved in the symbiotic association it was deemed de-sirable to obtain viable cultures of Anabaena-free Azolla forcomparative purposes. This was accomplished with antibiotics.Subsequently, we devised methods for obtaining clean prepara-tions of the two photosynthetic systems, i.e., Azolla chloroplastand Anabaena azollae. The purity of the two components wasestablished by several methods.A comparison of the Chl a/b ratios of whole fronds with

algae, whole fronds without algae, and pure chloroplastpreparations from each, resulted in a reasonable estimate ofthe algal contribution to the total Chl in the fronds. This allowsus to obtain a comparison of rates, on a Chl basis, of specificmetabolic functions in the intact association and with the iso-lated alga.The pure chloroplast preparations were characterized by

absorption spectra and low temperature fluorescence emissionspectra. Metabolic activity was demonstrated by PSI andPSII assays. The rates were usually low, but this was not un-reasonable since they were obtained under conditions whichare optimal for spinach chloroplasts and the plastocyanin,ferredoxin, and ferredoxin-NADP+ reductase added were fromspinach.The major difference in the absorption spectra of chloro-

plasts and algal cells is due to the absorption of phycobilins inthe blue-green algae. However, from the fluorescence emissionspectrum (not shown) it appears that the phycobilins ofAnabaena azollae do not transfer energy as efficiently as inAnabaena cylindrica. This is in contrast to what one wouldexpect. Since the major Chl a absorption and fluorescenceemission maxima occur at virtually identical wavelengths inboth the chloroplast and algae, the light energy available tothe alga is first screened by the fern. Therefore, it would seemthat to be competitive the energy absorbed by the phycobilinswould be more effective in transferring energy.

These studies have provided an initial characterization ofthe symbiotic association and some of its photochemicalparameters. In the following paper, we present the results ofour studies on acetylene reduction (nitrogen fixation).

Acknowledgments-The authors wish to acknowledge the excellent over-alltechnical assistance of 'Mrs. Joan Schnieders. We thank MTr. Elwood Shaw forthe photochemical assays, 'Mrs. N-orma Jean Rav-eed for assistance with themicrographs, and Mr. Steve Dunbar, Mfr. Joel Sinks, and 'Mrs. Donna Currentin the photography unit.

LITERATURE CITED

1. ALLEN-, AM. Mr. 1968. Simple conditions for growth of unicellular blue-greenalgae on plates. J. Phycol. 4: 1-3.

2. ARNON, D. I. 1949. Copper enzyme in isolated chloroplast. Polyphenoloxidasein Beta vulgaris. Plant Physiol. 24: 1-15.

3. ARNTZEN, C. J., R. A. DILLEY, G. A. PETERS, AN-,D E. R. SHAW. 1972. Photo-chemical activity and structural studies of photosystems derived fromchloroplast grana and stroma lamellae. Biochim. Biophys. Acta 256: 85-107.

4. BERTSCH, W. F., J. R. Azzi, AND J. B. DAVIDSON. 1967. Delayed light studieson photosynthetic energy conversion. I. Identification of the oxygen-evolv-ing photoreaction as the delayed light emitter in mutants of Scenedesmusobliquus. Biochim. Biophys. Acta 143: 129-143.

5. BIGGINS, J. 1967. Photosynthetic reactions by lysed protoplasts and particlepreparations from the blue-green alga, Phormidium lucidum. Plant Physiol.42: 1447-1456.

6. BLACK, C. C., JR. A-ND B. C. MAYNE. 1970. P700 activity andl chllorophyllcontent of plants with different photosynthetic carbon dioxide fixationcycles. Plant Physiol. 45: 738741.

7. BORTELS, H. 1940. Uber die Bedeutung des 'Molybdiins fur die stickstoff-bindende Nostocaceen. Arch. 'Mikrobiol. 11: 155-186.

8. CLAYTON, R. K. 1965. Characteristics of fluorescence and delayed light emissionfrom green photosynthetic bacteria and algae. J. Gen. Physiol. 48: 633-646.

9. CLAYTON, R. K. 1970. Light and Living Matter. The Physical Part, Vlol. I..McGraw-Hill Book Company, New York.

10. COHEN-BAZIRE, G. AND M. LEFORT-TRAN. 1970. Fixation of phycobiliproteinsto photosynthetic membranes by glutaraldehyde. Arch. Mlikrobiol. 71: 245-257.

11. DONZE, 'M., J. HAVEMAN, AND P. SCHIERECE. 1972. Absence of photosystem 2in heterocysts of the blue-green alga Anabaena. Biochim. Biophys. Acta256: 157-161.

12. FAY, P. 1969. Cell differentiation and pigment composition in 4nabaenacylindrica. Arch. 'Mikrobiol. 67: 62-70.

13. FAY, P. 1969. 'Metabolic activities of isolatedl spores of A.4abaenia cylindrica.J. Exp. Bot. 20: 100-109.

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819Plant Physiol. Vol. 53, 1974



the azolla, anabaena azollae relationship - plant … of azolla caroliniana willd. free of the...

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