7
DIVISION IN TREXTEPOHUA Sl'ORANCIA 137 Cli.ipman. R. L. 1977. Siaiining and lr:iiisnii.sMiin electron inifrtKscupic (il>sei\aUc>ns un /oosporo^etu'sis in Crphaleiirm vit'-r>rrn\ (C-liliMophyia. Clhrook-pidatcat'). J. Phyftd. I3l.):I2. ' - ('(XKI, B . H . 1977. Some comparisons mnong CrphidruTus. Phfropelti-i, Trrtitepiihlia ;inil other green algae.y. ihL l pp Hovd. C, I.. Sc Salisbury. |. 1.. 1977. (;iy(nkiu- ciizytiie.s in grceii alj^pit-.y. Phyrol. I3(suppl.):2l. Floyd. G. L.. Su-wan. K. t). Jt Mallox. K. R. 1972, Com- parali\(- (ylt)!n(^y nl l.'lothrix and Stigetnloniiim. J. Phycol. H:(iS-H(l. ' 1 if.lciitk. S. t,. C'.rulwr. I'. ]. Ik Tolljcrt. N. K. 1973. The (KiuriTnciMil ulycolatfdfhydiom-nasfinid glytolalf oxidase in giei'n |ihnH?i. An t-vtiliiLinnary Mirvey. Plaut i'hysiiil. V23 l-iillct. M.S. l«17fi. M i u » i s i n f u n R L / » ( . «m C'y/o/.-ISilkt- .'iS. ' OialKiin. I.. K. & MiBridt-. G. ¥.. 1«)~.'). The ullrasirucmrc oC niiiliilaycK'd sitiKliirt's ass<>(-ial(-<[ witli nagctlar husvs in lliohle (elK ol Trnttrpohliii nureii.J. I'hyini. 1 1:86-90. Idiida. A. A, Jt-I-nlU-r. M.S. 196M. ti|liaslriHliirc ol mJlosis ill tli('a(|uali( \\\n^\\\( .titriutJUt iingudlulae. Mycili'gia (iO:l41- il. Kuhai. 1), [. 1973. Pho evolution of tin-niitoiic spiiullr./«/. «/^^ f.v/W.-iy;l(i7-22K. ID. Kits. L. I97(i. Uiilersuchungen zur Frinstrukiur und ta- \onoini.stlien Kinordtiun^ von Glworhaetr willntckiana, einer apuplaMidalen cai>\aK'n M^v mil liliiiigriiiu-n tndosymbiim- (fii (Cvanelleii). Prvtoplasmu H7:l 19-J6. 11. LitliiU-. I... Bctm-i.S, K Silnvfinfi. H.G. 1976. Ultiasmu- linc- diirinn deveiopiTU-iu of ihe ntideus ti\ Batuphura neislritii (ChkiiopJiyia. l)asytladattat).y. Phyrol. E2;2t)l-72. 12. Manliani, II. J. it I'ickcu-Mvaps. j . D. 1973. Milosis ami k . in Culnninti-te .uutatti.J. i'iiyiol. 9;-K>]-71. 13. II. 15. 16. 17. IS. 19. 20. 21. 22. 23. 2-t. McDonald, K. L. & I'ickctt-Meaps. J. D. 1976. Ulirasirin- inre and dilfrrrnlialion in Cladophora gtomrrata. I. t!cll di- vision. Am.J. Bill. li3:r)92-601. McNiii, R. 1973. Mitosis in Phlyriochytnum irrrgiiiare. Can.J. B(./. 51:2065-74. rickctl-!!eaps. ]. D. UMi9. The evolulion of ihc niiiotit a[> ]}:ir:iltis: an aitempt al ajmparalivr iiltrastnictnral cyioltigy- in dividing plan! tflls. Cytobius 3:257-^0. 1975. Green Algae. Sintrture, Repntduflion, and t'vn- lulinn in Selected Genera. Sinauer Asstxiaics, Inc. Publishers, Snndcrland. Massachusetts. 606 pp. 197(5. C".ell divi.sion In eucaryolic algae. BioScirnre 26:415-50. & Xtartliani, H. J. 1972. The phylogenyoi the green l Cb 6256 algae: a new proposal. Cyliibiw 6:25.'»-64. I'iikftt-Ht'aps. J. D. & Ot'i. D. \V. 1974. L'lirastriicmral mor- [iliology and tell division in Pnlnwmatias. Cylolnos 11 :-l 1 -58. St<ilt. J. U. & Bullock. K. W. 197fi. Ullra'siriicture of tell division in Cladtiphuru. Pregametangial Lell divisitin in the haploid generation oi Cladophora Jirxiwsa. Can. J. Bot. Spiirr. A. R- )969. A low-visawity epoxy resin embedding medium Icn- elettroii microsco|)y, 7. Vltra.-.trHCl. Res. 26:31- 43. Slabeliau. M. 1976. Microbodie.s horn Spirogym. Organelles of a lilanieii(ou.s alga similar to It-af prroxisomes. Pi. Physiol. 5«:693-5. S^el^arl. K. D.. Mattox. K. R. & Floyd, C. L. 1973. Mitosis, cytokinesis, the dislribution of plasmmlrsinata, and other tytoiogiial characteristics in ihe L'lotrichalfs. Ulvales, and ('tiaeiopliorales: |)hylogenetic and taxonotnic coiisidera- tions.y. rhyroi. 9;I2H-41. Irieiner, R. E. &• Brown, R. M.. Jr. 1974. Cell division in .^ mimi'iwi.J. Phyrol. 10:419-33. 7. Phycoi. 1412). 197H. pp. DIVERSITY IN THE MECHANISM OK CARBON DIOXIDE EIXATION IN DUNALII-LLA TERTIOLECTA (CHLOROPHYCEAE)' D. Mukerji,^ H. E. Glover and I. Higelow LiilMiialoiy lor Oiean Stitntes. W'csl Boolhbiiy Harbor. Maine 01575 ABSTRAC:T Thr nurlutuhtn of pholmvUlu'dc anbim dhxUiv fixatum in the grvni jUigrUaic nuttjliclki IcrlioIt'Ct;) Buirhvr varies ibniug giowth in bntrh cnUure. Evidrncc fur i/ii.s clutugf amu's from i/irir .si>iirn:\: i) algae from tin- siatlon- (ity jibusr intnrfioralid a grnitcr fmifioylion of the fixed rarhon into amino urids and fmitcin than did rells from tbr inid-rxftnnentiai j>basi\ ii) tbr tuth'ily of l>ho.sf)hofnot- f>yruvaie carbitxylasi- trlativr lo thai of ribnlosr-l. 5-di- fiho\f)hate c/irbo.x\lasi' increased xvith age in batch cullure; and, iit) ct'Ii.\ from the stulionary f>hase nf)f)farvd lo utilize the bicarbonate ion a.s the substrate for ph(dosynthesis, ii'hereu\ those from mid-exftonential f)hme afi/mnrd to uti- lize fn-e nnbon dioxide. These data sugge.\t that a change ^Atrepted: /I .Wnveinher /97". * I'icMin ;«ldics!,: ••', S. R. .Mvikerji; Jaipur, Rajstlian, India. • AddiesN lor reprint re(|iirsls. 0} fihotnsynthetic mechanism can occur within a .single sf)e' cies of alga, def)ending on ils ph\siotogical siate. Key index xrords: bicarbonole: carbon dioxide; carbon dioxide fixation: cnrhoxylases: Dtiiialiclht; photosynlhetic ftrodnris The work of Calvin and co-workers (l.-l) seemed lo resolve the centnti t]uestions stirrounding the pathway ofairbon dioxide nssimiUition during pho- l(>.synthe.si.s. Although niosi oi their work had been tiiidertaken with two green algae. Chlorella ftyreuoi- dosu Chick and Seenede.smus obliquus (Tmp.) Kriig.. ihe essetitial leaitires of their proposed pathways have hccn extended lo all photosynihetic organisms. The more recent discussions ot so-called "aherna- tive" pathways in sonie photosyiithclic bacteria (II)

DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

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Page 1: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

DIVISION IN TREXTEPOHUA Sl'ORANCIA 137

Cli.ipman. R. L. 1977. Siaiining and lr:iiisnii.sMiin electroninifrtKscupic (il>sei\aUc>ns un /oosporo^etu'sis in Crphaleiirmvit'-r>rrn\ (C-liliMophyia. Clhrook-pidatcat'). J. Phyftd.I 3 l . ) : I 2 . ' •

- ('(XKI, B. H . 1977. Some comparisons mnongCrphidruTus. Phfropelti-i, Trrtitepiihlia ;inil other green algae.y.ihL lppHovd. C, I.. Sc Salisbury. |. 1.. 1977. (;iy(nkiu-ciizytiie.s in grceii alj pit-.y. Phyrol. I3(suppl.):2l.Floyd. G. L.. Su-wan. K. t). Jt Mallox. K. R. 1972, Com-parali\(- (ylt)!n(^y nl l.'lothrix and Stigetnloniiim. J. Phycol.H:(iS-H(l. • '1 if.lciitk. S. t,. C'.rulwr. I'. ]. Ik Tolljcrt. N. K. 1973. The(KiuriTnciMil ulycolatfdfhydiom-nasfinid glytolalf oxidasein giei'n |ihnH?i. An t-vtiliiLinnary Mirvey. Plaut i'hysiiil.V23l-iillct. M . S . l«17fi. M i u » i s i n f u n R L / » ( . « m C 'y /o / . - IS i lk t -.'iS. 'OialKiin. I.. K. & MiBridt-. G. ¥.. 1«)~.'). The ullrasirucmrcoC niiiliilaycK'd sitiKliirt's ass<>(-ial(-<[ witli nagctlar husvs inlliohle (elK ol Trnttrpohliii nureii.J. I'hyini. 1 1:86-90.Idiida. A. A, Jt-I-nlU-r. M.S. 196M. ti|liaslriHliirc ol mJlosisill tli('a(|uali( \\\n^\\\( .titriutJUt iingudlulae. Mycili'gia (iO:l41-

il. Kuhai. 1), [. 1973. Pho evolution of tin-niitoiic spiiullr./«/.«/^^ f.v/W.-iy;l(i7-22K.

ID. Kits. L. I97(i. Uiilersuchungen zur Frinstrukiur und ta-\onoini.stlien Kinordtiun^ von Glworhaetr willntckiana, einerapuplaMidalen cai>\aK'n M^v mil liliiiigriiiu-n tndosymbiim-(fii (Cvanelleii). Prvtoplasmu H7:l 19-J6.

11. LitliiU-. I... Bctm-i.S, K Silnvfinfi. H.G. 1976. Ultiasmu-linc- diirinn deveiopiTU-iu of ihe ntideus ti\ Batuphura neislritii(ChkiiopJiyia. l)asytladattat).y. Phyrol. E2;2t)l-72.

12. Manliani, II. J. it I'ickcu-Mvaps. j . D. 1973. Milosis amik . in Culnninti-te .uutatti.J. i'iiyiol. 9;-K>]-71.

13.

II.

15.

16.

17.

IS.

19.

20.

21.

22.

23.

2-t.

McDonald, K. L. & I'ickctt-Meaps. J. D. 1976. Ulirasirin-inre and dilfrrrnlialion in Cladophora gtomrrata. I. t!cll di-vision. Am.J. Bill. li3:r)92-601.McNiii, R. 1973. Mitosis in Phlyriochytnum irrrgiiiare. Can.J.B(./. 51:2065-74.rickctl-!!eaps. ]. D. UMi9. The evolulion of ihc niiiotit a[>]}:ir:iltis: an aitempt al ajmparalivr iiltrastnictnral cyioltigy-in dividing plan! tflls. Cytobius 3:257-^0.

1975. Green Algae. Sintrture, Repntduflion, and t'vn-lulinn in Selected Genera. Sinauer Asstxiaics, Inc. Publishers,Snndcrland. Massachusetts. 606 pp.

197(5. C".ell divi.sion In eucaryolic algae. BioScirnre26:415-50.

& Xtartliani, H. J. 1972. The phylogenyoi the greenl Cb 6 2 5 6algae: a new proposal. Cyliibiw 6:25.'»-64.

I'iikftt-Ht'aps. J. D. & Ot'i. D. \V. 1974. L'lirastriicmral mor-[iliology and tell division in Pnlnwmatias. Cylolnos 11 :-l 1 -58.St<ilt. J. U. & Bullock. K. W. 197fi. Ullra'siriicture of telldivision in Cladtiphuru. Pregametangial Lell divisitin in thehaploid generation oi Cladophora Jirxiwsa. Can. J. Bot.

Spiirr. A. R- )969. A low-visawity epoxy resin embeddingmedium Icn- elettroii microsco|)y, 7. Vltra.-.trHCl. Res. 26:31-43.Slabeliau. M. 1976. Microbodie.s horn Spirogym. Organellesof a lilanieii(ou.s alga similar to It-af prroxisomes. Pi. Physiol.5«:693-5.S^el^arl. K. D.. Mattox. K. R. & Floyd, C. L. 1973. Mitosis,cytokinesis, the dislribution of plasmmlrsinata, and othertytoiogiial characteristics in ihe L'lotrichalfs. Ulvales, and('tiaeiopliorales: |)hylogenetic and taxonotnic coiisidera-tions.y. rhyroi. 9;I2H-41.Irieiner, R. E. &• Brown, R. M.. Jr. 1974. Cell division in

.^ mimi'iwi.J. Phyrol. 10:419-33.

7. Phycoi. 1412). 197H. pp.

DIVERSITY IN THE MECHANISM OK CARBON DIOXIDE EIXATIONIN DUNALII-LLA TERTIOLECTA (CHLOROPHYCEAE)'

D. Mukerji,^ H. E. Glover and I.

Higelow LiilMiialoiy lor Oiean Stitntes. W'csl Boolhbiiy Harbor. Maine 01575

ABSTRAC:T

Thr nurlutuhtn of pholmvUlu'dc anbim dhxUiv fixatumin the grvni jUigrUaic nuttjliclki IcrlioIt'Ct;) Buirhvrvaries ibniug giowth in bntrh cnUure. Evidrncc fur i/ii.sclutugf amu's from i/irir .si>iirn:\: i) algae from tin- siatlon-(ity jibusr intnrfioralid a grnitcr fmifioylion of the fixedrarhon into amino urids and fmitcin than did rells fromtbr inid-rxftnnentiai j>basi\ ii) tbr tuth'ily of l>ho.sf)hofnot-f>yruvaie carbitxylasi- trlativr lo thai of ribnlosr-l. 5-di-fiho\f)hate c/irbo.x\lasi' increased xvith age in batch cullure;and, iit) ct'Ii.\ from the stulionary f>hase nf)f)farvd lo utilizethe bicarbonate ion a.s the substrate for ph(dosynthesis,ii'hereu\ those from mid-exftonential f)hme afi/mnrd to uti-lize fn-e nnbon dioxide. These data sugge.\t that a change

^Atrepted: / I .Wnveinher /97".* I'icMin ;«ldics!,: ••', S. R. .Mvikerji; Jaipur, Rajstlian, India.• AddiesN lor reprint re(|iirsls.

0} fihotnsynthetic mechanism can occur within a .single sf)e'cies of alga, def)ending on ils ph\siotogical siate.

Key index xrords: bicarbonole: carbon dioxide; carbondioxide fixation: cnrhoxylases: Dtiiialiclht; photosynlheticftrodnris

The work of Calvin and co-workers (l.-l) seemedlo resolve the centnti t]uestions stirrounding thepathway ofai rbon dioxide nssimiUition during pho-l(>.synthe.si.s. Although niosi oi their work had beentiiidertaken with two green algae. Chlorella ftyreuoi-dosu Chick and Seenede.smus obliquus (Tmp.) Kriig..ihe essetitial leaitires of their proposed pathwayshave hccn extended lo all photosynihetic organisms.The more recent discussions ot so-called "aherna-tive" pathways in sonie photosyiithclic bacteria ( I I )

Page 2: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

138 I). MUKKRJI. II, E. GI.OVER AMI I. MORRIS

tcrtain liighei plaiiLs (15) du not alter the valid-ity ol fxtrapolating ilic cctiiral lindings oC Calvinand his cnllL-agucs to all photo.syiithclic organisins.Thcsf central ieaturcs arc: i) primary caiboxylationrt-attion catalyzed by ribuIoi.e-1, o-diphospliate car-b<jxylasc iRuDl'Case); and, ii) the tyclic rearrange-ments ol variotis plu)spliate esters lo regenerale tlicC()2 acceptor and providing interinediales for ihesyntliesis ol essential cell metaboliies.

Sitperinipo.sed on tlie.se basic leaitire.s is a seriesol metabolic reactions which can vaty between oi-gani.stns. with changing physiological state and al-tered environmental conditions. Ol' pariictilar im-portatue ha.s been tlie so-called "C^4-pa(luvay" inceiiain higher plants (see 14 ior review). The jjho-tosynthelic assimilation of COj by siuh plants in-eludes an active phosphoenolpyruvate carboxylase(PKPC^ase) leading to ihe rapid synthesis ol C4 com-potinds (notably oxaloacetate, malate and aspar-tate). Although the reductive pentose ])lK)S])hatecycle is requited Tor regeneration ni the COz accep-tor and conse(|uent net CO2 fixation, imporlanlphysiological diflerence.s re.snit IVom ihe operaiiotiof the C4 series ol reactions. In particular, the mech-anism allows ior greater efficiency of photosynthesisunder conditions oi high oxygen levels, low CO2conceniraiions and high light iniciisities which re-sult in high rates of photoiespiiation in so-called"C3 plants."

Most ol the work on the diversity of phoiosyn-ihelic pathways and on the jjossible significance ol(.4-photosynliiesis has been confined to higherplant.*i. Recently, we have <|iiestioiied whether com-parable tliversiiy might exist in marine algae (2.12).Measurements ol the .short-term pattern of C()2 lix-ation. the ratio oi RiiDPC.asc/PEPCase and ihe War-btirg effect (oxygen inhibition of photo.synthe.sis)stiggested that the diatoms Skilftonema to.slutum(Grev.) Cleve, and Pharodarlyluin Irirornutiim Bohlinresembled "C^ plants" and the green flagellate Dun-alu'lla tcrtwUrta Butcher more closely resembled a"da plant." The dinoflagellate Cvriyaiilax tamarvmi.sLebotir atul the chrysophyle l.sothrysis galhana Pat keappeared to show a mixed Ci and C phoiosynthesis(2). H(twe\er. we now recogni/e tliat this proposeddistinction between different algal cla.s.ses may nolbe ab.soluie. It appears that, within a single speciesof alga, the pathway of phoiosynthetic COj fixationmay alter with thanging environmental conditionsand with allered physiological states.

MATKRIALS AND MKTHOD.S

Axcnit cultures of Dunaliflla trrtiulrcta (origiiiully oinailiedfrom Wimds Hole Oicanoyraphi* Instiuilton) were grown dt I.TC in batcfi culiure in inediiiiii (13) undi-r coniuiiious illuminalionh\ warni-wbite IhmieMfni ttilx's with 10 Klx incident liglii inteii-sitv. Measurcmenis ol [>hotnsynthcsis nitf has tH-en Hcvsi ribctl byMorris and (;iover (I**). ;is has the shorl-Ierin pattern of carbondioxide Itxiition (11). and the long-lerm "^iciss" patlern ol phn-tosynthcsis (19). Rii l) l ' ( jsp and PKPCise aciiviiirs were inca-stufd uiili (lie tree/e-lhdw ie<hni(|ueoi Miikerji and ^^lltIi^(2()),

Kxpcriinentslo determine the spec les <if"t in f>on dioxide" usedin ijlicitcwynlhe-iis were based on tliose ol Coupe i iiTicI c(iwurkei>i(li.7. see also 5). Cells weie lwn\c-sle(l on 3 firn 1 nipoie" lillerslesuspended in fresh giowili nucliinn ;IIHI the cell deiisiiy ad-justefl to 3 X 10^ cells/iiil. I'oiiileen ml ol lliit; snspeiisiofi wereilliimiiialed in a 20 ml sample IUIK- viitli It) Klx iticidt-nt lightintensity. Allei liO niin. 1.0 ml I f C O , " or '*CO, was iiijeuedtliroiigh Ihe viucine stopper and 1 ml saniple.s ienioved ill 30 sinlerviils. Radioaciive NiiMCO, (\aH'*COa) w,i.s added In gi\e ;ifinal cnncfiitralioii ol' 5 iiiM anil a specilic activity of 0.3 /iCi/Minole. The '*COj was prcpaied by injetiiiig I ml .'iti mMNaH'*CO:, (sprcilu adiviiy 0,1 ^Ci/^rnol) into I'l inl .SN IICI tna L'O ml sealed vessel. A gas light syiinge was used to transferI ml t)I f(a.% III Ihe lell sit.spensic»n. Ilu- pnKednre was suitidiii i/cdMl that the mixtures were vigorously shaken iiiimediately loHon-ing U'^COa and 'H:Oj additions and throiiglioiit the- iiiculMlinilperil Hi.

Radiftiutivity was crmiitetl with a Beckniiiii US 10t)C sciiitilla-ticin cniinte: using a M iniillant coniiiintng fi g 2-(l t butyl plieityl)--•>•{•] bt |>henyl) .1.3.1. oxadia/cde (Butyl l'IU)l in 1 I oltc>liiene:tnetliano|(3:l vA). Elficiencies were ticiei mined witli anexteiiial standaid.

RKSIILTS

Cfuhoxylase ucHvilii's. The ratio ol RuDPCa.se/PKP-Ca.se activities changed during batch growth. Thetatio was highest dtiring the mid-exponential phase(maxitnal iihotosynthesis). Larlier and later in thegrowih. Pl-'.PCase appeared more active thanRuDPCa.se (Fig. 1). Ihis change in the relative ini-portatice of the two carboxyiase.s was mainly causedby ihc variation in RuDPCase activity, which wasmarkedly reduced during slationary phase. Al-ihough PKPCa.se aciivity was also greittesl duringmid-exponential phase, ihe difleteiuc betweenmaxiniiiin and minitnum values was less thanRuDPCase activity.

Gross pattern.^ of CO3 fixation. Kigure 2 describes theway in which the- di.stribution ol asslmllatecl ' ClOjamongst the major prodttcls of phi.tosynthesischanged during growih. The ptoptH tion of '*C in-corporated into protein was greatest at each end oftlie growth curve (when the PKPClase/RuDPCase ra-tio was highest) and was least dtiritig tnid-expotien-tial growth (when RuDPt^a.se was more active thanPKPCasc). These changes in the labelling of proteinwere paralleled by changes (in the reverse dit ection)in the labelling ofenthanol-solublecompoutids. Theproportion ol' ' C assimilated into polysac( haride(lid not vary in a way which could be related to thechanges in the carlx.xylases.

Short-term COjftxathu. More pieci.sc infbrmatiotits provided by .shorl-term experitnents which detectthe rapid labellhig nf tfie jirimary prodtuts ol COafixation. Table 1 contrasts the short-term labellingpatterns taken Itotii ihe mid-exponential ])ha.se withtho.se frotn cells in the stationary ])liasc. I'he lattercells did not show exclusive fabelling of :i-Pr.A(phosphoglyceiic acid), even after a period as shortas 2 s. Additional labelling of atnitio acids (notablyaspattate) and intertnediates i)f the tricarb(jxylicacid cycle (notably malate atid citrate) were ptomi-iieiit features of the photosynthetic inechatiisni of

Page 3: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

l-HOrOSYNrHESIS IS DUXAUELM 139

60r

-2 1 r

CULTURE AGE(DAYS)

Kit:. 1. Cllan^irl^ nite& ot pbotosyntbesis and ratio ol Rii-Dl'Case/l'Kl'Case aclivilies during batcb growth ttiDnuatiillti tei-

l

cells IVom iht* siationiny ])hase. li is interesting, too.thai the heavy labelling of glycerol at longer times(also reported by Wegniann. 29) was observed inexponcniially-growing cells whereas aniino acidsand amides were heavily labelled in cells irom tliestalionaiy phase.

Sftecies of "CO2" used. Coiiiparison of the "CO2"species tised by cells Irom diOereiit stages in baichculture (Fig. ?>) depends on the fact that at lemper-attnes below l.> C ihe e(|iiilibntini between CO; andMCO.-i~ is attained in several mintitcs. Thns. initi-ating photosynihesis wilh the radioactive "CO2 spe-cies" not tised directly, wotild be followed by a lagperiod before assimilation t)f '*C begins. No stublag is observed when the reaction is initiated withihe stibstrate u.sed directly in photosynthesis. Kigtire3 shows ihe way in which lree ("O2 appeared to beused by cells irom the mid-exponential phasewhereas HCOn" appeare<l to be titili/ed hv cellshoin ilie stationary phase. The lag when H'^COa"was used i(> initiate the reaction in exponential cellswas most market!. No stich lag was ohsei ved wheti'\X)2 was added to slalionary-phase cells stiggestingihat stich cells utili/ed both COj :i"(l IKXV. i' P'H'-nomenon consistent witb botb PEI'Case andRuDI'Case being active in such cells.

nisc:tissioNKour types of measurements have been made in

the work reported here: the relative activities of twocarboxylating en/ymes. the long-term pattern olcarbon assimilation into the major end-products ()lphotosynlhe.sis. the shoit-lenn ]jalhway of CO2 (ix-aiion into ihe primary piodtuts and the "COa" spe-cies used as the substrate in ])boU)syntbesis. All (bur

CULTURE AGE (DAYS )Fit.. 2. I'hotosyiuhctic CX)i assiinilaiion diiriiij; hatdi growth,

alter -1 h iiuutKUion ))ov in^ proportion ol ^*C assiinilaEcd intoclliaiiol-solulilc tompouiids, |>ttly^aallaridc inaleiial and pro-tein.

measurements suggest that the pliotosyntheticmechanism in D. tertiolecta alters dtn'ing growth inbatch ctilturc. At the time when the phoiosyntheticcapacity is maximal, the cells exhibit an activeRuDPCase ttsing free CO2 as the stibstrate and pro-dticing ,S-PGA as the primary ptt>duct ol CO2 assim-ilation. Later in the growth curve many of the pho-tosyntiieiic characteristics appear to include ihelixation of HCOa" by an active PEPCase and theai)pearance of intermediates of the tricarboxylicacid c\cle atid associated aniino acids as pritnaryproducts o( CO2 hxaiion. It is apparent, also, thatthis change in the primary reactions involved inphotosynlTietic COa assimilation is paralleled by analtered flow of carbon into the encl products. Theappearance of "'C4-characteristics" is accompaniedhy an increa.sing proportion of '*C entering protein.This occurs at the expense ol' the synthesis of alco-hol-soltible compotinds—notably glycetol.

It must be recognized that st»me ttf the data areopen to alternative interpretation. For example,measurements of the short-ierni patterns of fixationhave not been accomjianied by appropriate pulse-chase experiments. Thus, any analogies with theconcepts ol "C;t" and "CV photosyntliesis are ten-

Page 4: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

HO IX MUKKRJI. H. K. CLOVER AM) I. .MORRIS

2 9 0A5S1MIIATI0N TIM E 1 MINUTES)

J 1 1 L

Klti. 3. Tinu'-ioiiise ol pluHti.vynihesis m III C when ihr re-i i i is iiiiliated wiili H' CO^^ and with "CO,. A »i-IK Iiniii tlir

mid fxpinu-niiiil [iluist- (clyy 2) and B tells from llic siaiionary|»lias<- (day 9).

uitivc. Kurtlier. llie changes in tlte patterns of plio-losynihfsis ob.st-rvfd tuuards t l i f end ni hatchgrowth mi^Hii reflect increased iniporiance ol darkC.O2 (ixiilion. That i.s, a marked decrease in phoio-syntheiic rate combined with a constant dark fixa-tion ol C'O2 might he expected to yield the sameresults. However, in our experiments, the tate ol'dark CO2 fixation is tarely greater than b'/t ol pho-losynihesis and woidd contrihute little to the patternol hxation. Also, the apparent iitili/ation ol bothCO2 and HCO.r by cells in the stationary phaseniiglu reflect increased carbonic anhydrase at tivityand not changes in llie carboxylating en/yme.

Despite the alternative interpretations, the (actthat changes in all four criteria examined in thispresent study parallel each other suggests that themost imtbable interpretati<tn is a changing mecha-nism ol photosynthetic CO2 hxation during batchgrowth oi 0. trrtwlrcta. We suggest, theielore. thatour earlier proposal {2) be modified. It is nol pos-sible to make a clear-cut distinction between thephoiosyntbetic mechanism in marine diatoms andthat in greeti flagellates. At least one species ofgreen flagellate is capahle of chatiging the relativeimportance o( ihe various reactions involved in CX)alixation. Zelitch (31) has also raised doulns on the

question of an absolute disiinction between "<:;( atid(V* plants and Khanna and Sinha (Hi) have ob-served "C;.," characteristics at certain developmentalstages of ihe "CM-pIant" Sorghum.

Althtjugh the pholosynthetic mechanism ofVJ. trr-tiolrcta changes during growth in hatch culture, thepreviously reported (2) dilTererues between thisalga and certain mat ine diatoms remaiti oi interest.That is, tit thr timr of ma.xiuium fthutosyuthrsis, theie isa real dilferente between the diatoms we investi-gate<i and the green flagellate. The ptKssible ecolog-ical significance of siuli a difTetence has not beendetermined, nor have the preci.se environmental(onditions which cause atiy change in the |)hotosyn-thetic mechanism. We need \o translate ihe super-ficial statement of "age in batch culture" into |)reciseterms. In some of our experiments, the pH of thegrowth mediiun increased from 8.2 to 9.1 duringhatch culture. Gullet and Benson (21) earlier le-ported increased labelling til malate and asparlatewhen the green •d\^\\ Scrnrdrsmus assimilated carbondioxide at higber pH values. However, tlie changeswe report here camiot be related simply to changingp u . Kor example, for measurements of the C sub-strate and the patterns ol j^hotosynthesis. the <ellsare harvested and tesuspended inVresh growth me-dium (pH H.2) lor the experimeni. Also, many olthe "CM chatacteristics" ohserved in statitnuny phasecould also be seen ou tbe (n st <lay olgrouth—beforephotosyntfiesis heconies maximal. Smh an obset-vation also makes it difficult tn relate ihe cli;mge>i tothe availability of certain nutiients. Controlled ex-periments with (ontinuoiis cultures wilf be net e.ssarybefore ilie precise environmental control of thephotosynthetic mechanism can be understood.

In this and in a previous paper (2) we reportedattempts to apply ilie techni<|tte o(" Ctn.per and co-workers (6.7) to the elucidation ol the "CCV speciesu.sed hi |>hotosynthesis. We realize the difliculties inapplying a techni(|ue from jjurilied en/ymes to sus-pensions of intact algae. Kor example, the pre.sencet>l an active carbonic anhydrase would make suchan approach invalid. Despite such dillitulties wesuggest that the presence of an active I'KPCiase incertain species of marine phyto|)l;uikton tinder cer-tam conditions, and the direct experiments utilizingthe techni(|ue of Cooper and colleagues (().7) indi-cate that lixatifHi of the HCO;," if>n might be morecommun than is generally supposed. Although alunnber ol <lillereru algae appear to be able to uti-lize HCOa" (see 2: lor review), the weight of evi-dence (H-I0.22.2r).2H) suggests that free CO., is gen-erally the substrate used in phytoplanktot ipliot()syntliesis. This evidence comes fVom tfireety|>es ol nieastireinents: i) ellect ol pM on plioto-synthesis: ii) elfect of purging the algal suspensionoi COa; and iii) raiio ni '•X::'='C in phyUiplaiikton.In es.sence, tlie fiist two methods depetid on alteringthe relative conientrations of tfie various " (XV ' spe-cies. The way in which this manipulatit)n might itself

Page 5: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

PHOTOSYNTHESIS IN DUNAUELLA 141

1AIU» 1. Shiirt-trnn fmUeni of WCOs inrmporaUnn(ri-M4lt.s exprti.\etl «.v 7, total "C nicorporatedf.

Inlriinrilidtrk

asparaginegl u tain i TICglycine + scrincihrconiiicalaniiu-vuliiu-leu cine-isolciKiiifKliitamatfaNpiirtati-Tola! aiiiinci acids, amides:

MICcilLilf

'urnaralfiiiahiu-tit rail-

a-kciOKlutaratcTotal TCA iiilcinicdiiites

siicro.se3-)jlic>spliiiylYcc'ratt'oihtT sii^ar phcisphiiicsphcisplin(ciiol)pyruvate + pvnivateRlya-iau-Klycfiolphospliii^lvtulate + (>lyTnliitciinknijwii—1uiikiKnvn—2tinkiHmn—.1

2

00000000000

d000•P0

0100

0000io000

by D. trniolccta tin ttay of

D..V3AuimiUimn iimr »

i d

06.200

7.0000000

000000

d24.13I.S

00

30.600o:0

maximum pliDlnsynthesU (day 3i and

AO

01.400

10.45.60003.1

20.500000i.i06.2

lO.S00

61.50Q00

2

0\.^4.053.261.93000

2.16•ts.se56.06

1.760.530.56

14.941.05

23.S4

00.7<l9.4900

1.642.462.161.92..S

in statiottary phase (day 6)

Dav 6

60

06.689.851.4

26.30.530.15O,3j

1^290;72

64.230.4-10.9S0^42?.0S

d2.47

00.048

25.4700

9.50l i s00 -0

determine the inorganic carbon substrate used inphotosynthesis makes interpretation of the restiltsdiflicult (sec 23).

At lirst sight, the '•'C:' C ratio method might ap-pear to be a powerful techni(|ue lor determining thecarbon suhsiiate for photosynthesis. Using thismethod, Degetis, Deuser and colleagties (8-10) ob-tained evidence for the utilization of free C'Oa bymarine phytophtnkttm. However, we question\vluHher such a conclusion can indeed be drawnhoni the '•'C:'^C data. In doitig so, we also relate the''C:"'C work with marine phyioplankton (8-10) to

the extensive informalion now available Irom com-parable measurements witb higher plants (e.g.

In essence the '"'Cli' C method depends on mea-suring a parameter known as 8^\'.. I'liis parameterallows the '•'(;:"(; ratio in a particular satnple to becompared with the same ratio in a standard soitrce.

Oegens, Deuser and co-workers have applied thisU'chni(|ue to the probletn of the catbon substrate inphoiosynthesis by coniparitig the 6'"'C of phyio-plankton cells wilii that of the IICX):,"- A differencebetween these two values is inter])ieted as evidencelor the I KX);," not heitig utilized directly in )>hoto-synthesis. Conliiniation is gained Irotn expetimeniswhich exatnine the elTect of temperature and thet'lfect ol changing the availabjliiy of C()2 (8). I'hai

is. the difference between S' C of cell-C and that ofHCO3" in the medium increases when tlie supply offree COz is increased.

However, the work on fi'^C values amongst higherplants of botb tbe "CV and "C/' type makes onedoubt whether such values can be used to elucidatetbe catbon stibslrate for photosynthesis. It is appar-ent thai there is isotopic IVactionaiion during carbonfixation by carboxylating enzymes. That is. a differ-ence between fi'^C of HC.O;r and that of a productol a carboxylation teaction does ntU necessarilytnean that 1 ICO.i~ is not used directly in tbe carbox-yladon reaction. Rather, it simply means that tlieenzyme discriminates again.st the heavy isotope (' C)within the substrate. It is clear, also, (3.30) that PEP-C'ase discriminates less than does RuDPCiise. Be-cause of this, the S'"'C values lor C4 plants are gen-erally lower than those of C3 plants (3,24.26,28).Interestingly, Deuser and colleagues (9.10) report8' C values resembling "C4 plants" for natural pop-ulations of marine phytoplankton. It is apparent.thetefore, that data oti the 6 ' \ : values in marinephytoplankton might afford valuable insight intothe tnechanism of phoiosyntliesis, btit we agree withRaven (23) in suggesting tbat "experiments on iso-lopic discritnitiation are more helpful in investigat-ing HCOa" ti.se in kno^xn HCOa" users than as amethod of deciding which plants can ttse

Page 6: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

142 D, MUKKRJl, H. E. GLOVER AND r. MORRIS

Ihis worL was siippmicd in par! hy NSI- gtiinis DKS 75-131(1)and Di-S 75-2112« ami hv a griiiii lu the Bi^clow Uilmr;ilo)yIrnni llie Stalf nl Miiine Depamnetil cil Maiinc ReMJitrct-s.

9.

10.

II.

12.

13.

14.

. J. & C;alviti. M. 1957. Thf Path of Carhon w P/wlo-Prcntirc-lhitl. FnRlcKcH)d Clills. New Jtrscy. 101

pp.Be;ii(lall. J.. Muki-iji. D.. Clnwt. II. F. & Monis. 1, 197(1.The paih nl larboii in phoiosyntlu-sis liy marine phyiopliiiik-inn.y./ViyiW. I2:4()<l-17.Bender, M. M. 1971, Viiriiiliiin.s in the "CV"C liilios of

in ri-lattnn lo the |niihwiiy of [ihniosynihetit tailinti(ixation. Ptiylw hrmi.Ury 10; I239—tl.

Calvin, .M. & Bii5.sham. J. 19(52. Tfir Phutm-nllirm of CurbimCiintpiiumii. W. A. Betijainiti, Im.. New York. 127 pp.CfMnnhs. J.. Maw. S. L. 8c Bitldiy. C. W. 197r). Metaholiiref{tilatinn in i'.* phnt()synilie<tis; ilic inorganii tarbiMi stili-Mratc l<.r I'El* carlx.xylasc. Plnnt Sn. Lrll. 4:97-11)2.CVrnpt-r. T. i',. & FilmtT. D. I9(i9. The aclivc spcties oi"COt" iilili/etl by ribuloNc dipliiLspluiu- nirboxylasc. y, Biol.Chrm. 24-1:1 OH 1-3.C:ooper, T. C-. Tchfii, T. T.. Woml. H. C, &- Benedict. C.R. i96S. The caibtixylalitxi ul pho.sph(K-iiol [lyruvate andpyriivatc. The active species i>f "tXV" utilized by pliospho-cnolpyitiv;itc larlxtxylasc. (arlMixytransjiluwplmrylase. andpynivate carlioxylase.y. Itiot. Chrm. 24S::iH57-(>H.Di-gcns, t . r., c;iiillard. R. R. L.. Sarkel. W. M. &- HelUbtiM.J. A. I96H. Mc-talH)lii frudionalirin nf CHTIIOII isotopes inmarine planklon. 1. Teniperdltire and reMpiriition expcri-inents. Prrp-Sm Rn. 15:1-9.Ut'user. W. G- 1970. Isotopic evidence for dimii)ishiii[5 sup-ply of available larhon during diutoin bltioiii in ihe Blat kSea. .Valure London 22.1:1069-71.

. K. r. Sc Guillard. R. R. I.. 19liH.isotope relniion^bips iK-iweeii ]>lankion and seawaler. i'lro-chem. Ciumochim. Ada 32:(J57- iO.Kvans. M. C. U'.. Buchanan. B. B. fr Arnon. O. I. 1906, Anew lcrrcdr)xin-de|K'ndfni carbon ittltiition (ytir in a pluntusyndietic biittcriiini. Prac. \'aiL Atad. Sn. U.S.A. 55:U2S-34.Clover, H. F... Beardall. J. & Morris, I. 1975. h'.itvvls oienvirontncntal lattois on pholosyntheMK patterns in Pfittni-<tactyium truornutum (Bantlaiiopbyccae). I. tflect of niirogctidclicicniy and lijtht init-nsity.y. Phyrol. 11:424-9.Gtiillard. R. R. L. & Rytlu-r. J. M. 1962. Sluclies o( marineI)lankionit diatoms. I. Cyrlnirlh miiui Htislcdl und DriiiinihfonjmHicm (CIcvcl C.ran.Cnn.J, MifTobwI. «:229-3H.Hatch. M. D.. Osmond. C. B. & Slatyer. R. O. | tds . | 1971.Photiaynlhesis and Phtitnre\piratton. Wilcy-lnttTStifntc,York. 565 pp.

15.

IG.

17.

IK.

20.

21.

24.

25.

2ti.

27.

2H.

29.

30.

31.

Haul). M. n. frSlitck. C. K. 1970. I'holosyntheUi CO, lix-allon palhway-D. Ann Hn'. I'laul Pliy\ii>i. 21: II l-()2.Kl)at)t)ii, R. Jt Sinhi), S. K. I<l7:i. Cliatige in ilie ptetlonii-naiiie from C, to ('.;, inithwiiy lollowing ariliiilie.Ni.s in Sur-ghiiin. Itnirlirm. Iiii>fi/iy\. Hr\. Comm. h'2.Vl\~-\.M:nttyani;t. II.. Kasierday, R. I... Chang. M. C. it.- l.ane. M,1). liUifi. The cn/ymalit tailmxylation of PKIV 1. Ptiiihta-lion and propeilie.Mil I'KrCase,/ Hiol Chrm. 2 11 :'i lO.'i-lii.Morris, I. it (Uovri. II, F.. 1*171. QttesiiotiN on ihe tnei ha-nistit of lenipeniluif adapMlion in marine iilivtoplatiklim.Mar. liiol. i;4;l47-.M.

*.' Yeiti.sih, C:. S. M)7-!. Ptodtnlfi ol pyby maiiile phyloplankton: ihe eflett ol eiivironinenlal f:«-tors un ibf icbiive raics ol' iirolt-iti svnthe.sis. .Mor. liiol. 27:1-9.Mukeiji. D. & Morris. 1. 1970. riioiosyiiihetit (nrboxylaiinKen/yme.s in Phmmluftylum IrniirTiiitiim: assay melliods andpropellie^. Mar. Biol. .Ifi: 199-206.Otillel. C. &- Bet)son. A. A. I9r.2. The pallt ol larlnm injilirjiosynthcsls. XIII. pll t-lletl.s in '""COi lixalion Uy Srrnr-driinu\.J. Exp. Hoi. :i:2;i7-15.I'aasche, F. 1961. A liacer stndy ol ihe inorgatiii caiimntipUiki'diiiiiiguKKinih lotnialion and pholosynllie.sis in tlifciKiolilliophoitiid C»Huitithii.\ hn.\lryi. Phy-iol. Plant. Suppl. H2pp.Riiveti. J. A. 1970. F.xoHeiimiA inorpanii carbon sotnces inplant photosynthesis./iir,/. Htf. Ifi: 167-2*21.Smiih, B. N.'& Fpstein. .S. 1971. Iwo calenories ol '•\:/"(:nilios for bit^her jdiint.s. Plunl PhyMiil. 47:3HO-1,Stet-mann Nielsen. K. 1960. Ihe iipiake of tree CO, andIICX)3~ dining plu)tcisynili(->iis4tl plankton iil)>af will) specialri'lerenie to ihe ccHColitliophoioid CorwlUhm hn.\lryi. Physiol.Plant, 19:232-10.Trt-gtinna. P. B.. Smillt. B. N.. Berry, j . A. &• Dountun. VV,J. S. 1970. Some metbods lor studying tbe pbolosyniheticlaxonotny of tbe angio.spertii\. Can. J. Bol. 4«: 1209-11.Tronghlt»n. J. II. 1971. Aspens ol ilie evoltiliim ol plmu*-synthetic c.nlxixylation reaction in plants. In llaicb. M. D.,Ostiioiul, C. B. &• Slaiyei, R. O, (Kd.v.I PhotmvHhrsi\ and I'hii-toTr\fihatiim. Wiley-lnierscietice. New York. 124 pp.Watt. W. 1). & I'aaMlie. K. 1963, Ait invesliKaiioii oi theconditions (or (lislinnitisliinj; between CO, ;nul hitatlmnait-'utili/alion by algae actnrding |i> tbe tnetbods of IIoo<I andViirk. Physiot. f'latil. I0:74«-«|.WfKmatin, K. Il)(i9. On ibe paihways iil CO, lixaiion inDunahrlta. l*t,,jrrey, in Pli,ilo.\ynlltr\i.\ lirsniiih 3:1359-04.Whelan. r.Sackftt. W. M.S.-Bi-ncdiil. C. R. 1973. Fn/y-maii( riaclionaiiim ol carbon isolopt-.s by phosiihetiolpyrii-viitecinboxylase from C:., \i\nnls. Platil Phy.yiot. 51:1051-4.Zelitch. 1. ]97.'i. l'alhw;tys olcailjon hxaiion in Rreen plants.Ann. fin: Biorhrm. 44:123—15.

Page 7: DIVERSITY IN THE MECHANISM OF CARBON DIOXIDE FIXATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1