Host glutamine synthetase activity decreases in Pocillopora damicornis
(Linnaeus) following exposure of the coral to seawater containing elevated
ammonium (20 uM). Zooxanthellae isolated from these corals exhibited
lower ammonium uptake capacity and glutamine synthetase activity compared
with those from the control corals. Ammonium concentration of the surrounding
seawater had no effect on the NADPH-dependent glutamate dehydrogenase
activity in the host

Fitt, W.K.

Rees, TAV

Yellowlees, D.

Pacific Science

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Pacific Science (1994), vol. 48, no. 3: 291-295© 1994 by University of Hawaii Press. All rights reservedEffect of Ammonium-supplemented Seawater on Glutamine Synthetaseand Glutamate Dehydrogenase Activities in Host Tissue andZooxanthellae of Pocillopora damicornis andon Ammonium Uptake Rates of the Zooxanthellae1D. YELLOWLEES,2 T. A. V. REES,3 AND W. K. FITI4ABSTRACT: Host glutamine synthetase activity decreases in Pocillopora dami-cornis (Linnaeus) following exposure of the coral to seawater containing ele-vated ammonium (20 jlM). Zooxanthellae isolated from these corals exhibitedlower ammonium uptake capacity and glutamine synthetase activity comparedwith those from the control corals. Ammonium concentration of the sur-rounding seawater had no effect on the NADPH-dependent glutamate dehy-drogenase activity in the host.INTRACELLULAR AMMONIUM in corals can bederived both from normal catabolic pro-cesses within coral tissue and by uptake ofammonium from seawater. The latter wasfirst observed by Kawaguti (1953). Subse-quently, Muscatine and D'Elia (1978) dem-onstrated that only corals symbiotic withzooxanthellae can take up ammonium andalso retain it. They assumed that ammoniumcan be assimilated within the symbiotic asso-ciation and that zooxanthellae are essentialto this process.Few enzymes directly utilize ammoniumas a substrate. Apart from carbamoyl phos-phate synthetase I, which is present in organ-isms that possess the urea cycle, glutaminesynthetase (GS) is the principal enzyme usedin ammonium utilization, although NADPH-dependent glutamate dehydrogenase (GDH)has also been implicated in ammonium as-similation despite its low affinity for ammo-nium. Both GS and NADPH-GDH have1 Manuscript accepted 10 September 1993.2 Department of Molecular Sciences, James CookUniversity of North Queensland, Townsville, Queens-land 4811, Australia.3 Leigh Marine Laboratory, University of Auckland,R.D. 5, Warkworth, New Zealand.4Department of Zoology, University of Georgia,Athens, Georgia 30602.been detected in corals and other zooxan-thellate symbioses. GS in Acropora formosa(Dana) (D.Y., unpubl. data) could not bedetected in host tissue, although its presenceis assumed because GS is the sole enzymecapable of synthesizing glutamine. Its unlike-ly absence would indicate either that zooxan-thellae must act as the source of glutamine orthat it is acquired from heterotrophic feed-ing. High NADPH-GDH activity has beendetected in host tissue of A.formosa (Catmullet al. 1987) and Stylophora pistillata Esper(Rahav et al. 1989).GS has been found in zooxanthellae(Wilkerson and Muscatine 1984, Andersonand Burris 1987), although no activity mea-surements were presented. The presence ofGS and GOGAT (glutamate synthetase) infreshly isolated zooxanthellae from coralscan also be concluded from the data pre-sented by Summons et al. (1986). GOGATcatalyzes the reductive transfer of the amide-amino group of glutamine to a-ketoglutarateto produce two glutamates. NADPH-GDHis present in freshly isolated zooxanthellaefrom corals, although its specific activity isan order of magnitude higher after inductionwith ammonium (Dudler and Miller 1988).This report examines two of the centralaspects of ammonium assimilation in marinealga-invertebrate associations: the relation-291292ship of GS and GDH from both Pocilloporadamicornis (Linnaeus) and its freshly isolatedzooxanthellae to the seawater ammoniumconcentration; and whether the uptake ofammonium by the zooxanthellae is influ-enced by the environmental ammoniumconcentration.MATERIALS AND METHODSCoralsCoral branches of P. damicornis were ob-tained as previously described in this issue(Stambler et al. 1994). Samples of control(ambient) corals and corals in seawater sup-plemented with 20-IlM and 50-IlM concen-trations of ammonium incubated for 2 weeksand 8 weeks were used. Animal tissue wasremoved from small coral branches with a jetof compressed air from a scuba tank. Thetank was connected to a plastic tube and theair delivered through a wide-bore plasticpipette tip inserted in the end of the plastictube. During this procedure (air piking), thecoral branches were wetted with extractionbuffer (50 mM Tris-HCI buffer, pH 8.0, con-taining 1 mM reduced glutathione, 1 mMDTT, 20 mM mercaptoethanol, 1 mMEDTA, and 10 mM MgS04) as required.Zooxanthellae were removed from air-pikedsamples by centrifugation for 5 min at roomtemperature in a microfuge (Beckman). Ani-mal tissue was used for assay of both GDHand GS.ZooxanthellaeZooxanthellae were prepared by removingthe coral tissue from the skeleton of P. dami-cornis colonies with filtered seawater usingthe Water Pik technique (Johannes andWiebe 1970). The zooxanthellae were washedthree times with filtered seawater and thenresuspended in extraction buffer (containing5 mM mercaptoethanol) before lysing thecells in a precooled (4°C) Veda Press at 9000kPa (1200 psi). The lysate was centrifuged ina microfuge for 5 min to remove unbrokencells and cell debris. The supernatant wasused for assay of GDH and GS.PACIFIC SCIENCE, Volume 48, July 1994Enzyme AssaysGDH was assayed in 50 mM HEPES-KOH buffer (pH 7.4) containing 100 mM(NH4hS04, 0.2 mM NAD(P)H, 10 mM rx-ketoglutarate, and enzyme extract (total vol-ume = 1.05 ml). The reaction was monitoredat 340 nm in a diode array spectrophotom-eter (Hewlett Packard 8452) at 30°C. AnyNAD(P)H oxidase activity was measured for2 min before initiating the GDH reactionwith rx-ketoglutarate. The GDH activity wasadjusted for any NAD(P)H oxidase activitypresent. The oxidase activity was always<20% of the GDH activity.The transferase and synthetase activitiesof GS were determined as described byRhodes et al. (1975) and Guiz et al. (1979),respectively. Protein was measured by theBradford (1976) method using reagents sup-plied by BioRad. Bovine serum albumin wasused as a calibration standard.Ammonium UptakeZooxanthellae (0.5-1 x 106 cells ml-1 ),prepared by the Water Pik technique andwashing three times in filtered seawater, wereincubated in seawater containing 20 IlMNH4CI. The samples were incubated at 30°Cunder a fluorescent light source (200 IlE m- 2sec-I) and mixed regularly. Aliquots (5 ml)were removed at regular intervals, vacuum-filtered on GFjC filters (Whatman) and theammonium content of the sample measured(Liddicoat et al. 1975).RESULTS AND DISCUSSIONZooxanthellae, freshly isolated from cor-als exposed to either 20- or 50-IlM concentra-tions of ammonium, incubated in an elevatedammonium concentration took up ammo-nium ions at a slower rate than zooxanthellaeisolated from control corals (Figure I). Theuptake rate for zooxanthellae from controlcorals (0.042 pmol NH4 hr-1 cell-I) wasroughly equivalent to that for freshly iso-lated zooxanthellae from Acropora formosa(Gunnersen et al. 1988). However, a five-fold"M Wlh",!l/, 4 ¥tk.¥$!Ammonium Metabolism in Coral and Zooxanthellae-YELLOWLEES ET AL. 293FIGURE I. The depletion of ammonium from sea-water by freshly isolated zooxanthellae from Pocilloporadamicornis. The corals were maintained in seawater con-taining either 20-JlM (n = 3) or 50-JlM (n = 2) concen-trations of ammonium. The ambient ammonium con-centration of seawater used for the control corals (n = 3)was 0.1 JlM.decrease in the mean uptake rate was ob-served in zooxanthellae isolated from P. da-micornis grown in 20-,uM-supplemented sea-water (8 fmol NH4 hr-1 cell-I). The uptakerate observed with zooxanthellae isolatedfrom corals grown in 50-,uM ammonium-supplemented seawater was not significantlydifferent from that of corals grown in 20-,uM-supplemented seawater. This indicates thatthe N status of the zooxanthellae was influ-enced by the seawater concentration; the am-~-I~ 0.06oNI..c:'0S 0.04-8Cll.!o:ol....Po;:lS 0.02;:l'2oS~0.1 J.LM 20 J.LMmonium uptake system was repressed withincreased concentrations of ammonium. Thisresponse has not been previously reported inzooxanthellate symbioses, but Rees (1990)observed a similar result in symbiotic Chlo-rella and used this to argue for a low ambientconcentration of ammonium in the perialgalspace.GS activity in host tissue was measuredusing both the transferase and synthetase as-say; however, the measurement of synthetaseactivity proved unreliable and only the re-sults for transferase are reported in Table I.The transferase/synthetase ratios for GS inplants (15-100) and giant clam (Tridacnagigas) animal tissue (36 [Rees et al. 1994])indicate that at the transferase levels ob-served, synthetase activity measurementswould have been low. A decrease (> 50%) inGS was observed after incubation of corals inseawater supplemented with 20 ,uM ammo-nium ions for 2 weeks. Only one sample wasmeasured after 8 weeks exposure, and the GSlevel in that was further reduced. Althoughthe presence of GS has been assumed, thispaper represents the first report of GS activ-ity in coral tissue.In contrast, no evidence was obtainedfor changes in GDH in host tissue, indicat-ing that any intracellular change in ammo-nium concentration resulting from an in-crease in seawater concentration did not in-fluence the level of GDH. In all instances,> 95% of GDH activity measured wasNADP-dependent, and there was no evi-dence for any change in the coenzyme ratioTABLE IGLUTAMINE SYNTHETASE AND GLUTAMATE DEHYDROGENASE ACTIVITIES IN THE CORAL Pocillopora damicornis AND ITSZooXANTHELLAEGLUTAMINE SYNTHETASE GLUTAMATE DEHYDROGENASEFRESHLY ISOLATED FRESHLY ISOLATEDTREATMENT HOST ZOOXANTHELLAE HOST ZOOXANTHELLAEControl 186 (±23) 15 34 (± 3) ND2 weeks 77 (±5) 7 31 (±4) ND8 weeks 55 29NOTE: Units are expressed as nmoles min-t (mg protein)-t. Each assay was repeated and the mean taken. The results presentedare the means of separate experiments (n = 3) with their standard errors. NO denotes not detected.294of dependence. Deaminating activity of GDHwas not measured. Aminating activity was,however, 12-fold greater than that reportedfor Stylophora pistil/ata (Rahav et ai. 1989),but six-fold less than that of Acropora for-mosa (Catmull et ai. 1987).Assuming that the concentration of am-monium in seawater influences the intracel-lular concentration, the results indicate thatGS activity must be regulated either directlyor indirectly by cytoplasmic ammonium ionconcentration. However, without knowingthe intracellular concentration of ammoniumions or metabolites derived from its assimila-tion, it is not possible to speculate further onthe control of GS levels in host tissues.The specific activity of both GS and GDHin zooxanthellae was always < 10% of that inthe host tissue. Indeed, in the case of GDH itwas impossible to reliably detect any oxida-tion ofeither NADPH or NADH above back-ground, indicating that GDH levels were verylow. However, GS was detected in zooxan-thellae using the transferase assay, indicatingthat it must be the major means of ammo-nium assimilation in zooxanthellae.From these results, we propose that thehost has a role in the assimilation of ammo-nium ions in the symbiosis. It has been ar-gued that the zooxanthellae are the drivingforce behind the acquisition and retention ofammonium in the symbiosis (Muscatine andD'Elia 1978). However, uptake of ammoni-um by zooxanthellae relies on diffusion ofammonium through the host cell and into theperialgal space. With relatively high concen-trations of ammonium-assimilating enzymesin the cytoplasm of the control hosts, thereis a likelihood that much of the ammoniumwhen it is present at natural seawater con-centrations will be assimilated before it candiffuse to the perialgal membrane. The re-pression of the ammonium uptake rate ofzooxanthellae in corals exposed to elevatedammonium ions supports this conclusion.ACKNOWLEDGMENTSWe are grateful to the U.S.-Israel Bina-tional Science Foundation and the UniversityPACIFIC SCIENCE, Volume 48, July 1994of Hawaii Sea Grant Program for support ofthis research and to Paul Falkowski for theprovision of the Yeda Press.LITERATURE CITEDANDERSON, S. L., and J. E. BURRIS. 1987.Role of glutamine synthetase in ammoniaassimilation by symbiotic marine dinofla-gellates (zooxanthellae). Mar. BioI. (Beri.)94: 451-458.BRADFORD, M. M. 1976. A rapid sensitivemethod for quantitation of microgramquantities of protein utilising the principleof protein dye binding. Anal. Biochem.72: 248-254.CATMULL, J., D. YELLOWLEES, and D. J.MILLER. 1987. NADP+ -dependent gluta-mate dehydrogenase from Acropora for-mosa: Purification and properties. Mar.BioI. (Beri.) 95: 559-563.DUDLER, N., and D. J. MILLER. 1988. Char-acterisation of two glutamate dehydroge-nases from the symbiotic microalga Sym-biodinium microadriaticum isolated fromthe coral Acropora formosa. Mar. BioI.(Beri.) 97: 427-430.Gurz, C. D., B. HIREL, G. SHEDLOFSKY, andP. GADAL. 1979. Occurrence and influenceof light on the relative proportion of twoglutamine synthetase in rice leaves. PlantSci. Lett. 15:271-277.GUNNERSEN, J., D. YELLOWLEES, and D. J.MILLER. 1988. The ammonium/methyl-ammonium uptake system of Symbiodi-nium microadriaticum. Mar. BioI. (Beri.)97: 593-596.JOHANNES, R. E., and W. J. WIEBE. 1970. Amethod for determination of coral tissuebiomass and composition. Limnoi. Ocean-ogr. 15: 822-824.KAWAGUTI, S. 1953. Ammonium metabolismof the reef corals. BioI. J. Okayama Univ.I: 151-156.LIDDICOAT, M. 1., S. TIBBITS, and E. 1. BUT-LER. 1975. The determination of ammoniain sea water. Limnol. Oceanogr. 20: 131-132.MUSCATINE, L., and C. F. D'ELIA. 1978. Theuptake, retention, and release of ammo-ZW" ;419$& a&g iIiJ2Ed&jjAmmonium Metabolism in Coral and Zooxanthellae-YELLOWLEES ET AL. 295nium by reef corals. LimnoI. Oceanogr.23: 725-734.RAHAV, 0., Z. DUBINSKY, Y. ACHITUV, andP. G. FALKOWSKI. 1989. Ammoniummetabolism in the zooxanthellate coral,Stylophora pistillata. Proc. R. Soc. Lond.B BioI. Sci. 236: 325-337.REES, T. A. V. 1990. The green hydra symbio-sis and ammonium IV. Methylammoniumuptake by freshly isolated symbiontsand cultured algae; evidence for low-ammonium concentrations in the perialgalspace. Proc. R. Soc. Lond. B BioI. Sci.242: 255-259.REES, T. A. V., W. K. FITT, and D. YELLOW-LEES. 1994. Host glutamine synthetaseactivities in the giant clam-zooxanthellaesymbiosis: Effects of clam size, elevatedammonium and continuous darkness. Mar.BioI. (BerI.) (in press).RHODES, D., G. A. RENDON, and G. R. STEW-ART. 1975. The control of glutaminesynthetase level in Lemna minor L. Planta(BerI.) 125: 203-210.STAMBLER, N., P. L. JOKIEL, and Z. DUBIN-SKY. 1994. Nutrient limitation in the sym-biotic association between zooxanthellaeand reef-building corals: The experimentaldesign. Pac. Sci. 48: 219-223.SUMMONS, R. E., T. S. BOAG, and C. B.OSMOND. 1986. The effect of ammoniumon photosynthesis and the pathway ofammonium assimilation in Gymnodiniummicroadriaticum in vitro and in symbiosiswith tridacnid clams and corals. Proc. R.Soc. Lond. B BioI. Sci. 227: 147-159.WILKERSON, F. P., and L. MUSCATINE. 1984.Uptake and assimilation of dissolved inor-ganic nitrogen by a symbiotic sea anem-one. Proc. R. Soc. Lond. B BioI. Sci. 221:71-86.

Effect of Ammonium-supplemented Seawater on Glutamine Synthetase and Glutamate Dehydrogenase Activities in Host Tissue and Zooxanthellae of Pocillopora damicornis and on Ammonium Uptake Rates of the Zooxanthellae

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Effect of Ammonium-supplemented Seawater on Glutamine Synthetase and Glutamate Dehydrogenase Activities in Host Tissue and Zooxanthellae of Pocillopora damicornis and on Ammonium Uptake Rates of the Zooxanthellae

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