The Azolla-Anabaenaazollae Relationship' · The Azolla caroliniana-A. azollae association was grown...

Plant Physiol. (1981) 68, 1479-14840032-0889/8 1/68/1479/06/$00.50/0

The Azolla-Anabaena azollae Relationship'XI. PHYCOBILIPROTEINS IN THE ACTION SPECTRUM FOR NITROGENASE-CATALYZEDACETYLENE REDUCTION

Received for publication March 16, 1981 and in revised form July 21, 1981

V. V. S. TYAGI2, THOMAS B. RAY', BERGER C. MAYNE, AND GERALD A. PETERS4C. F. Kettering Research Laboratory, 150 East South College Street, Yellow Springs, Ohio 45387

ABSTRACT

Visible absorption spectra are presented for the AzoUa carolinianaWilld.-Anabaena azola Strass. association and the individual partners.Although absorption by the phycobiliproteins of the endophytic cyanobac-terium clearly complements the absorption by the fern pigments, theircontribution to the absorption spectrum of the association is effectivelyconcealed by the preponderance of the Azolla pigments. Action spectra fornitrogenase-catabzed C2H2 reduction in both the AzoUa-Anabaena asso-ciation and the endophytic Anabaena demonstrate that quanta absorbed bythe phycobiliproteins is as effective as that absorbed by chlorophyll a indriving this photosystem I-linked process. Under anaerobic conditions, theinhibition of photosystem II activty by 3-(3,4-dichlorophenyl)-1,1-dimeth-ylurea, diuron did not selectively decrease the relative quantum yields inthe region of phycobiliprotein absorption. At the well-below saturating lightintensities used for the action spectra studies, the absolute rates of C2H2reduction were increased uniformly via respiratory-linked processes underaerobic conditions. The occurrence of phycobiliproteins in heterocysts ofthe endophytic Anabaena was demonstrated using fluorescence microscopyof intact filaments. Fluorescence micrographs of Anabaena cylidrica fila-ments are presented for comparison.

Specialized cavities formed in the aerial dorsal leaf lobes of theaquatic fern Azolla are occupied by an endophytic cyanobacteriumknown as Anabaena azollae Strass (9). The endophyte, whichundergoes a pattern of development and differentiation as afunction of leaf age (5, 6), can provide the association with its totalN requirement by N2 fixation.As in free-living cyanobacteria (4, 18, 20, 21) there is a close

relationship between photosynthesis and N2 fixation in the Azolla-Anabaena association (10, 11). Action spectra for photosynthesisin the Azolla-Anabaena association, Azolla freed of the Anabaena,and the Anabaena removed from the fern have been presentedpreviously (16). Although there was no readily discernible contri-bution by the endophyte to the action spectrum for photosynthesisin the association, the maximum yield per incident quantum forphotosynthesis in the isolated endophyte was quite distinct fromthat of the endophyte-free Azolla, being consistent with other

'Supported in part by National Science Foundation Grant DEB 74-11679 AOl to G.A.P.2Cuffent address: Department of Medical Biology, Memorial Research

Center, 1924 Alcoa Highway, Knoxville, Tennessee 37920.3Current address: Biochemicals Department 335/237 Experimental

Station, E.I. DuPont DeNemours and Company, Wilmington, Delaware19898.4To whom reprint requests and correspondence should be addressed.

reports for this process in cyanobacteria and green plants, respec-tively (16).

In contrast to photosynthesis in the association, N2 fixation isan attribute restricted to the endophyte. The action spectra for N2fixation (C2H2 reduction) in the association, where the green ferntissue filters the light incident on the endophyte, as well as in theisolated endophyte are reported here and compared with theirpreviously determined action spectra for photosynthesis.

MATERIALS AND METHODS

The Azolla caroliniana-A. azollae association was grown oneither a N-free 40%o Hoagland solution or a N-free modificationof a medium employed for rice culture (see ref. 13). Cultures wereroutinely grown under a 16/8 h, 26 ± 1/19 ± 1°C diurnal cycleat a light intensity of approximately 200 IAE/m2 s provided by amixture ofnormal output, cool white fluorescent and incandescentlights. When required, higher light intensities were attained byusing high output (HO) cool white fluorescent lights plus theincandescent light. Lower intensities were attained through theuse of screens. Intensities were measured as photosyntheticallyactive radiation using a Lambda Ll-1905 quantum sensor.

Absorption Spectra of Azoffa Leaves and Endophyte. Azollastem segments bearing leaves were mounted in a single layerbetween two pieces of glass. Glass spacers prevented crushing. Asimilar procedure was employed with the isolated endophyteexcept that the inserts were omitted and edges of the glass weresealed with dental wax. The glass holders were masked with blacktape so that only the light beam passing through the sample wasmeasured. To minimize the effect of scattering, the samples werepositioned adjacent to the detector of a Perkin-Elmer 557 spectro-photometer.

Action Spectra. Acetylene reduction was used as a measure ofnitrogenase activity. For the association, sufficient plant materialwas added to uniformly cover the liquid surface in 10-ml roundbottom flasks containing 3 or 5 ml of the growth medium. Theflasks were sealed with serum caps, evacuated and filled with 10%3oC2H2:0.03% CO2 in Ar in the dark using an automated system,and then inverted and lightly shaken to distribute the associationuniformly right side up again. Groups of three inverted flaskswere placed in a metal cylinder (7 cm diameter, 9 cm length withblackened internal walls) which was positioned in a 25°C water-bath. The water depth was approximately 3 cm, enabling the threeinverted flasks to float within the confines of the cylinder. Lightwas isolated from a Sylvania DXM Tungsten-halogen lamp withinterference filters. The light was passed through a 10-cm waterfilter and an IR reflection filter prior to the interference filters.Light intensity was controlled with a Variac and measured as ergs.cm .s at the level of the plant material with a KetteringRadiometer.The endophytic Anabaena was isolated using the roller method

(9) and suspended in a N-free growth medium (12). For C2H21479

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Plant Physiol. Vol. 68, 1981

reduction assays in monochromatic light isolated with interferencefilters, 0.25 ml of the endophyte suspension at approximately 20Ag Chl a/ml was placed in 2-ml crimp-capped vials. The vialswere evacuated and filled with Ar: 1%o C2H2:0.03% CO2 using anautomated system for anaerobic assays. For aerobic assays the gasphase was 10%/0 C2H2 in air. Using the illumination system de-scribed above, four vials were placed horizontally in a gray plasticcontainer equipped with an opening for the radiometer probe.The container was affixed to a reciprocal shaker in the bottom ofa 300C water bath. Bath water passed through holes in the sidesof the container which was just wide enough to grip the ends ofthe submerged, horizontal vials. The filaments of the endophytewere maintained in suspension by shaking at 50 cycles/min andthe four vials remained in the monochromatic light throughoutthe throw of the shaker. C2H2 reduction assays with the endophytewere also carried out using single samples with light isolated witha Bausch and Lomb 0.25 m monochromator. A 5-ml serum-capped vial containing 1.5 ml of the endophyte suspension and asmall stir bar was placed in water in the center well of a water-jacketed all-glass reaction vessel maintained at 300C. Light inten-sity was measured at the position of the endophyte suspension inthe center well.The rate of acetylene reduction was measured using at least

three intensities at each wavelength and the assays were conductedat intensities within the linear portion of the response curves. Theincubation period was either 15 or 20 min except for the studieswith the monochromator where 30 min was used. When used,DCMU was added prior to illumination at a final concentrationof 12 ,U. At the end of the incubation period, the light was shutoffand, in the case ofthe association, gas samples were withdrawnas quickly as feasible for analysis. In the case of the isolatedendophyte, immediately after shutting off the light 100 pl 25%trichloroacetic acid was injected into the cell suspension. The C2H4formed was quantitated as described previously (10, 12). Chl wasmeasured after extraction with 95% ethanol (25). Rates wereinitially determined in nmol C2H4/mg Chl.min and subsequentlyexpressed as relative rates per incident quantum.

Isolation and Quantitation of Phycobiliproteins. Filaments ofthe endophyte were broken by passage through a French pressurecell at 20,000 p.s.i. and centrifuged at 80,000g for 30 min. Thepellet was resuspended, centrifuged again, and the supernatantscombined. The total PBP5 concentration was estimated using theprocedures of Bennett and Bogorad (2) employing the extinctioncoefficient for phycoerythrin to estimate the phycoerythrocyanincontribution from the endophyte (23). For the purpose of com-paring the total PBP in individual preparations of the endophyte,it was assumed that this would not lead to any serious error.

Fluorescence Microscopy. Anabaena filaments were mountedon glass slides in water under a cover glass sealed to the slide withdental wax. Specimens were photographed on a Leitz Ortholux IIequipped with a Ploemopak epifluorescence illuminator. A Ploe-mopak filter block M provided excitation at 546 nm, dichroicbeam splitting at 580 nm, and a 580 nm suppression filter. ThePBP fluorescence was observed through a Ditric Optics short passfilter and the Chl a fluorescence through a Corning 2030 filter(Fig. 1). Fluorescence images were recorded on Kodak Profes-sional Ektachrome 200 daylight film. The black and white printswere obtained from internegatives made from composite colortransparencies.Timed exposures were used to record the relative intensities of

the fluorescence emissions. The image produced with the totalfluorescence was recorded on film with the camera's automaticexposure system and the duration of exposure noted. The selectedfilters were then introduced in the light path within the microscopeand the image recorded using the same exposure.

5 Abbreviation: PBP, phycobiliprotein.

600X in nm

FIG. 1. Transmission spectra of filters used in conjunction with Ploe-mopak epifluorescence illuminator and filter block M. Ditric Optics shortpass filter ( ) and Corning 2030 filter (---). Filters were 18 x 18 mmand mounted in a specially constructed three-position holder in the lightpath of the Leitz Ortholux II.

F>o8<~~~~~~1.1.6

A0.5 /

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ct: 500 600 7000

<0.8 N

w

w

400 500 600 700X in nm

FIG. 2. Absorption spectra of whole leaves of Azolla with (---) andwithout ( ) the endophyte and filaments of the endophytic Anabaenaazollae (--). The spectra have been normalized at 680 nm. (Inset),absorption spectra of purified phycobiliproteins from the endophyte sim-ulating their quantitative distribution in vivo; (--) phycoerythrocyanin,( ) phycocyanin, and (---) allophycocyanin.

RESULTS

Absorption Spectra. The in vivo absorption of Azolla with andwithout the endophyte and intact filaments of the endophyticAnabaena are shown in Figure 2. Since these spectra have been

1480 TYAGI ET AL.



AZOLLA-ANABAENA RELATIONSHIP

normalized at 680 nm, it is important to note that the endophyteaccounts for 15 to 20%o of the association's Chl (9, 17). In theassociation, absorption by the endophyte's PBP is masked by thefern pigments. This is apparent from the similarities in the ab-sorption spectra of the association and the endophyte-free Azollabetween 550 and 650 nm. In contrast, absorption by the PBP isobvious in the spectrum of the endophyte filaments, as is theabsence of Chl b. The endophytes' PBP complement, comprisedof phycocyanin, phycoerythrocyanin, and allophycocyanin (23),is simulated in the absorption spectra of the purified PBP (Fig. 2,inset). These in vivo absorption spectra show the complementarypigmentation of the fern and endophyte noted previously withcell-free extracts and are consistent with the action spectra ofphotosynthesis obtained for the association and individual part-ners (16).

Action Spectra for C2H2 Reduction. Action spectra for N2fixation (C2H2 reduction) by the Azolla-Anabaena association andthe isolated endophytic Anabaena are shown in Figure 3, a and b,respectively. The spectrum of the association is based on triplicatesamples at each wavelength in each of seven experiments exceptfor the values at 620 and 630 nm which are based on five and twoexperiments, respectively. Each value at each wavelength wasnormalized to the mean at 690 nm in the individual experimentsprior to determining the mean ± SD from the seven experiments.The spectrum for the isolated endophyte is based on quadruplicatesamples at each wavelength in each of four experiments withinterference filters and five experiments using individual samplesat each wavelength with the monochromator. The data obtainedfor the endophyte was treated the same as that from the associationexcept the monochromator data was normalized at 680 nm. Al-though the variation among replicates within an experiment was

small, the response at individual wavelengths did vary among theexperiments with both the association and the endophyte. Thelatter resulted in the relatively high standard deviations of thenormalized composite spectra. These spectra show the relative rateof C2H2 reduction per incident quantum, as opposed 'o the relativerate per unit of incident energy (3) at each wavelength.The action spectra of photosynthesis by the association and

isolated endophyte are also shown in Figure 3, a and b, respec-

tively, using the mean values of data presented previously (16). Acomparison of the action spectra for the two processes in the

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a plot of the mean values derived from Ray et al. (16).

association and endophyte clearly illustrates that C2H2 reductionoccurs at a high efficiency after photosynthesis has undergone itscharacteristic red drop. This result is consistent with C2H2 reduc-tion being a PSI-linked process only indirectly dependent on PSII(4, 11, 18, 21).

Since the response being measured is restricted to the endophytein the leaf cavities, the light incident on the endophyte in theassociation is filtered by the leaf tissue. Thus, the number ofquanta reaching the endophyte would presumably be greater inthe region of minimum absorption by the fern tissue, about 550 to650 nm, corresponding to the region of absorption by the endo-phytes PBP (Fig. 2). Therefore, we thought that the higher quan-tum yield in this region was probably due to less absorption bythe fern tissue. However, in the isolated endophyte, where screen-ing by the fern tissue is excluded, the rate of C2H2 reduction perincident quantum in the region of PBP absorption was found toremain high, being essentially equivalent to that in the Chl aabsorption region (Fig. 3b). The high rates of C2H2 reduction perincident quantum in the region ofPBP absorption with the isolatedendophyte were unexpected. Additional studies were undertakento further substantiate their involvement in a PSI-linked process.

Effect of 02 and Effect of DCMU. In the action spectrum forphotosynthesis, 02 production by the endophyte is maximal in theregion of PBP absorption (Fig. 3b) (21). Therefore, it was possiblethat under an atmosphere of Ar:C2H2:C02, the high rates of C2H2reduction at wavelengths absorbed by the PBP could arise fromphotosynthetically-mediated C2H2 reduction being augmented byrespiratory-driven C2H2 reduction, the latter using photosyntheti-cally generated 02. To determine if this was the case, the spectrumfor C2H2 reduction by the endophyte was determined under 10%oC2H2 in air to minimize the effect of photosynthetically produced02 at specific wavelengths on any 02-driven process. While therewas an appreciable contribution from respiratory-driven acetylenereduction at the low light intensities used in these studies, theshape of the action spectrum under an atmosphere containing 20%02 was essentially the same as that obtained under microaerobicconditions. In addition, the action spectrum was determined undermicroaerobic conditions (Ar:C2H2:C02) with and without DCMU.A DCMU concentration resulting in greater than 95% inhibitionof 02 production did not selectively inhibit C2H2 reduction in theregion of PBP absorption (Table I). Although we did not attemptto measure the 02 in flasks evacuated, flushed, and filled with Ar:C2H2:C02, our studies showed that this gas phase had less than0.1% 02 (limits of detection by our thermal conductivity gaschromatograph) and substrate reduction in the dark with this gasphase was negligible. Thus, we conclude that in the assays withDCMU there was inadequate 02 to support any respiratory-drivenprocess and that the high rates of C2H2 reduction in the region ofPBP absorption are photosynthetically mediated. Moreover, inaccord with previous studies (10, 11) the nitrogenase activity was

Table I. Effect ofDCMU on Acetylene Reduction by the IsolatedEndophyte

Values are based on the relative rate/incident quantum in duplicates± DCMU in four experiments. Data are expressed as the mean ± SD ofthe 12,M DCMU treated as a percentage of the control at each wavelengthfrom the four experiments.

DCMU Treated as % of Control ±SD

590 89+26610 108±37630 108 + 39650 124 ± 16670 97 29680 118+5690 90± 18

Plant Physiol. Vol. 68,1981 1481




clearly not directly dependent upon PSII for a source of ATP orreductant during the short-term assays.

Alteration of PBP/Chl Ratio in the Endophyte. Previous studies(17, 23), as well as unpublished observations, have shown that thePBP content of the endophyte is dependent on the light intensityused for growth of the association. The endophyte isolated fromAzolla plants grown at 55 and 500 ,gE/m2.s, yielded preparationsin which the PBP/Chl a ratios were 17.14 and 6.36, respectively.The action spectra for C2H2 reduction by both preparations weresimilar to Figure 3b and are not shown. Although based on asingle experiment with each preparation, the contribution fromlight absorbed in the PBP region was actually somewhat greaterin the action spectrum obtained for the endophyte preparationwith the lower PBP/Chl a ratio. Inasmuch as about 80%o of theendophyte filaments are vegetative cells, these results were inter-preted to suggest that large variations in PBP content associatedwith them was possible without affecting the fraction involvedwith N2 fixation and possibly localized in heterocysts as reportedfor Anabaena variabilis (14).

Evidence for Phycobiliproteins in Heterocysts. Above findingsimply that some of the energy absorbed by PBP is used in PSI.There is abundant evidence from free-living cyanobacteria thatheterocysts contain the nitrogenase (4, 18, 20, 21), and inasmuchas nitrogenase activity parallels heterocyst differentiation in theendophytic Anabaena (5-7, 11), the results suggested that the PBPeffective in driving C2H2 reduction were also located in theheterocysts. Attempts to isolate PBP from pure heterocyst prepa-rations of the endophyte were negated by the large variation insize and age of the heterocysts (see 6, 11) and the presence ofakinetes in the preparations (8). Evidence for the occurrence ofPBP in heterocysts of the endophyte was obtained by fluorescencemicroscopy. Figure 4a shows the total fluorescence of filaments ofthe endophyte using 546 nm excitation and a 580 nm dichroicfilter. Figure 4b shows the fluorescence from the PBP, using aDitric short pass cut-on filter and Figure 4c the Chl fluorescence,using a Corning 2030 filter. (The transmission spectra of thesefilters are presented in Fig. 1.) The A. azollae filaments were fromthe cavity of the seventh leaf on the Azolla main stem axis, thearea of maximal nitrogenase activity (7). Figure 5a-c shows thesame series for filaments from an actively growing, N2-fixingculture of Anabaena cylindrica. Whereas the heterocysts of A.cylindrica exhibit very little fluorescence, the fluorescence fromheterocysts of the endophyte is as intense as that of the vegetativecells, even when only PBP fluorescence is being recorded (cf. Fig.4b). Since the micrographs might be interpreted to indicate thatmost of the fluorescence recorded in Figures 4a and 5a is from thePBP, it is important to note that the spectral sensitivity of the filmis maximal at about 650 nm and decreases at longer wavelengths.However, it can be stated that the heterocysts exhibit less Chlfluorescence than the vegetative cells with this being more pro-nounced in A. cylindrica (Fig. Sc) than in the endophyte (Fig. 4c).Heterocysts of A. cylindrica have been reported to contain onlyone-half the Chl content of the vegetative cells (1).

DISCUSSION

In the Azolla-Anabaena association, the pigmentation of theendophyte complements that of the fern, absorbing light energyin that portion of the visible spectrum where absorption by thefern pigments is minimal. Because of the morphology of theassociation, light energy reaching the endophyte is attenuated andfiltered by the leaf tissue. Thus, more of the light incident on thefern reaches the endophyte at those wavelengths least effectivelyabsorbed by the fern pigments. It was initially assumed that thehigh relative quantum yield for C2H2 reduction by the associationat wavelengths corresponding to the minimum absorption by thefern and the region of absorption by the endophytes' PBP simplyreflected these factors. However, in contrast to the expected result

FIG. 4. Fluorescence micrographs ofA. azollae filaments removed froma leaf cavity known to exhibit high nitrogenase activity. a, Total fluores-cence; b, PBP fluorescence; and c, Chl fluorescence. Heterocysts are readilydistinguishable by their larger size and polar bodies.

of a distinct maximum for the quantum yield in the region of Chla absorption by the endophyte removed from the leaf tissue, therelative rate of C2H2 reduction per incident quantum in the regionof PBP absorption was equivalent to that in the region of Chl aabsorption.

In free-living cyanobacteria, there is abundant evidence thatheterocysts are the sites of nitrogenase activity under aerobicgrowth conditions (4, 18, 20, 21). PBP have been reported to beabsent or to occur at low levels in the heterocysts and this hasgenerally been correlated with the absence of PSII activity in thesespecialized cells (1, 3, 4, 18, 22). In fact, the PBP complexes, whichcan account for up to 40%o of the soluble cell protein, have beenstated to serve as the light harvesting pigments exclusively forPSII (4). We had previously found that the PBP levels in theendophyte were only 15 to 35% of those reported for free-livingcyanobacteria and stated that the diminished PBP content wasconsistent with its high heterocyst frequency (17). Previous studieshad also shown that nitrogenase activity paralleled heterocystdifferentiation as a function of leaf age, that heterocyst frequenciesof the endophyte could reach 30Yo, and that the heterocysts infilaments did not fix 14Co2 (6, 8, 11). Therefore, the finding thatenergy absorbed by PBP was effective in driving C2H2 reductionin heterocysts, as well as CO2 fixation in vegetative cells, was

1482 TYAGI ET AL.



AZOLLA-ANABAENA RELATIONSHIP

FIG. 5. Fluorescence micrographs of N2-fixing A. cylindrica. a, Totalfluorescence; b, PBP fluorescence; and c, Chl fluorescence. Heterocysts are

the less fluorescent cells in each case.

surprising. It necessarily implied that either some of these acces-

sory pigments were localized in heterocysts, and transferring en-

ergy to PSI, or that we were observing a secondary effect. Afterexperimentally excluding the possibility that 02 generated invegetative cells, upon illumination with light absorbed by PBP,stimulated C2H2 reduction by promoting respiratory-driven proc-

esses in the heterocysts, the occurrence of PBP in heterocysts as

well as in vegetative cells of the endophytic Anabaena was dem-onstrated by fluorescence microscopy.Although the occurrence of PBP in heterocysts ofthe endophyte

would seem to be a logical adaptation for an organism existingwithin a green leaf, neither the occurrence of PBP in heterocystsnor the transfer of excitation energy from PBP to PSI islimited tothe Azolla endophyte. Low light intensities favor synthesis ofphycocyanin and phycoerythrin, but not allophycocyanin, in het-erocysts ofAnabaena sp. L-31 (22), and Stewart et al. (19) reportedin situ measurements with filaments of A. cylindrica in which thephycocyanin content of heterocysts was 20 to 50%1o of that presentper unit area in vegetative cells. The association of PBP with PSIof cyanobacteria has likewise been reported previously. Wang et

al. (24) estimated the light harvesting pigments of PSI in Anacystisnidulans (now known as Synechococcus sp.) included 85% of theChl and 40% of the PBP and Pullin et al. (15) reported the

association of allophycocyanin with PSI particles from Chlorog-loeafritschii.The absorption spectra of heterocysts and the action spectrum

for N2 fixation (C2H2 reduction) in A. cylindrica presented by Fay(3) are frequently cited as evidence for the absence of PBP inheterocyst. However, this action spectrum, plotted as the relativeC2H2 reduction/mm3 h per incident energy versus wavelength,shows a distinct contribution in the region of PBP absorption.When Fay's data are plotted as the relative rate of C2H2 reduction/incident quantum, the 625 nm light absorbed by the PBP appearsto be at least 70%Yo as effective as light absorbed by Chl a. In regardto this, it should be noted that in our fluorescence micrographs,heterocysts of Anabaena cylindrica show much less PBP fluores-cence, relative to the vegetative cells, than the endophyte, but theynevertheless exhibit some PBP fluorescence. Moreover, heterocystsfrom A. variabilis, which lack PSII activity, not only contain PBPbut in these heterocysts the quantum efficiency of the PBP is 50to 70%o of that of Chl a in sensitizing P700 oxidation and the PBPare nearly as effective as CMl a in driving H2-supported C2H2reduction (14). In regard to the latter, we have obtained similarresults with intact filaments of A. variabilis using the assay systemdescribed for the Azolla endophyte (Tyagi, unpublished). Finally,whereas the fluorescence emission of heterocysts in A. variablisfilaments exhibit a wide range of intensity (14), their fluorescenceis closer to that observed with the endophyte than to that observedin heterocysts of A. cylindrica (H. E. Calvert, personal communi-cation).

Additional studies are required to ascertain the structural or-ganization of the PBP in heterocysts since phycobilisomes havenot been demonstrated in them. However, the results reportedhere and elsewhere (14) strongly suggest that a reassessment ofthe role of PBP in PSI-linked processes in the heterocysts ofcyanobacteria is needed. In at least these cyanobacteria, hetero-cysts contain nitrogenase, PBP, and lack PSII activities.

Acknowledgment-The authors are indebted to H. E. Calvert for the fluorescencemicroscopy, Steve Dunbar and Marsha Tootle for preparation of figures and photo-graphs, Dorothy McNeil and Debbie Patten for clerical assistance, and W. R. Evansfor comments on the manuscript.

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Anabaena variablis. Biochim Biophys Acta 634: 237-24815. PULLIN CA, RG BROWN, EH EVANS 1979 Detection of allophycocyanin in

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