14, 5I.3-.5I8(I978)

ON SPECIR.VL C O M ROL OK I ' I ( ; M E N T A T I O N INA.S'ACYSnS MlWL-iSS (C^•ANOPH^ CEAE)' '

Jack Myer.\,^ Jo-Ruth Graham and Richard T. WatigI)ep.irtinents of ZcM>tc>g> and Botany. I l i r Univrrsity o f Texas, Austin, Texas 78712

ABSIRACT

Growth (/Anacystis tiidulans (Riiht.) Ihourt is* Dni-h in xrairlfiigtks of li^ht fnedomiuautly itbsorbfil hy ihlo-Tophyll a laiL^es n dromntic toweriiifr in <lili)ri>f)h\ll rotitrutn'lit in ilif (htiirof>h\lllf)hya>c\unin ratio. A limit to thrfj/ert t.\ riiuhrd in far rrd (MO um) tight whnr thr ratiorhJorof}hylllf}h\rohilinogrn falh to <().3. A sprcial watrriHith for ruttinr tuhrs uti.<i illuminatnt hy tunfrstrn-halogrnl'imfi% ilnmif^h fur rrd { >(}^() nm) fillrrs; it ^nvr ihr samrfxtrrwr pigiurut ratio tngrthrr with a high sprcijir giinithrale of 2. 5 ittty '. Comfxnrd with normally fii^nrntrd frtts

Iherf me thrrr features which afcomfkitiy thr tow-chloro-

pnyll (onilitiitn of far rrd lif^ht. At ft// wm rrlativr quan-tum rj/inrniy inrrra.\r.s hut action drcrrasrs. Synthrsis oftotal fufrmrnt and total crll matrriat in far red lif^ht aL\o<lrir,;isrs. ihrsr ohsrn<ation.\ .'>uggr.\t that thr low chloro-phyll rrsfutn.\r to far rrd light rrJlrcLs an incomprtrncf^th than an adaptation.

MATKRtALS AND

^ry indrx words: Agmenellutn; .Vn.ibartia; .X"Igdl pigmrnt.\: hlurgirrn algar; chlorophyll; chromatic'idafftation; lif^ht, hlur; light, rrd; phycocyanin; pigmrnt.s;5

It) most bluegreeti algae phycocyaniti i.s a tnajor"mht-harvestitjg pigtnetit fot plu)tosvtHhesis. Kxci-'iilion etiergv is Itansfrrted fttim phvccHvanin (IH.)"I the ph\cobil isotnes to chtotoplul l a (chl) of thej""nellae with high effuienc\ and in such a way that" serves lx»ih phoiote .ut iot is of photos\titlu'sis (25).•ti askitig (jucstiotis alx>ut tho role c»f PC and the''isiribtiliotJ of ils excitatioti etu'tg\ we have used^iiacy.\ti\ iiidulans (Richt.) Drouet i*i: Daily iti which"A- and chl a te the <»t)ly significant abs(»rl)ets over'hc' spettral regioti 5.')()-7(K) titn. .\\\ aid t<) this it>-'l"ii\ is pt<ivi<led bv aliet,ttions iti the chl/lH^ tatio•"• obsetved iti pigment mutants (23.25) or in cellsRfown tituler far red illutnitiatioti (12). We have ex-?'iitned ftttther atid re|x»rt heteiti some (li .n,icter-"•lics of this sjKttrally cotitrolled pigmetit adjust-"H'tii.

^ p t t : t Jutt 197fi.l o I.uigi Ptovasoli in recifgnitinn nf ihr indrliMc imprint

*>'iich he leaves u|Hiti ph\colog> I here is a sa*ing atttihuted to'altleo: "It is the huMtu'ss of a vtriitiM to nieasuie that which is

''"'asuralile anil to make iiieaHiiieaMe that »hith is not " I.uigi.'"'"Mahlished In example his own piinciple ><hich I phrase: "ll' llie husitiess ol a phvcologist to culture the algae which arr'I'orahle iiiid to make ciiltnrahle those whuh are not."

Addrrw for reprint rec]uests.

Annf\\ti\ ni(lutiin\ is the designation fp\rn straiti Tx2fl whrti itwas iMilatcMl (I.H). It is l>elieve<l to he icientical lo .VvnnAororrus sp.li30t ol Stanier et al. (22). .\% comparative inalrrial wr alM> havrexamtned several other hluegreen algar. Synnl>ofnrru.f sp. fi30I.t')311. arul (i9<>8 were otMained from R. Slanirr. Agmrnrtlum ^uad-rupheatum straiti PR-6 was obtained from C Van Baalrn and.ifiuharna sp. strain CA from C>. Srace>.

Sttain Vx2() was tnaintainrd rouiincU in amtinuous culluirat 39 C: under tungsten illumination. I'r ( .() , in air. and at as|>ecilu giowth rate of ca. !..•> ila\ ' as reientU cles<iil>ed (19).1 hr cotititittotts culturr provided standard ituHula for Katch trsttiilircultuic-s at 39 f.. aerated with IT CX>, in air aiul ilUiiiiinatnl.1% descriU-d following. Strains 1x20. 6301. t^^\ I atul ti'.MtS werrgrowti it) a mcKlified tnedium I) (19); other siraiiu wrrr grawnin ASP-2 medium (24).

Illutnitiatioti W.IS provided it> M-\<-tal diiferent nuides. Fhi-orescetit and tungsten vuircen were uscnl as light lunks on holhsides of acjuaiium t\|>e water Iviths. ca. 7 cm from ihe cultures.Bands of nuHUHhromatic light (ca. 10 nm hall-band width) werrohiaitied frtitn prt>je*tors through inierlereiue and appropriatebloikitig hiters (12). I hr 43t) nm line Has isolated h% hiters frtmia tnercurv latnp. For narrow luiul Muirces lhe test tuhe cultureswere held in a four-place Kith of hiack pirxiglas appropriatelvbafiled and darkened except for the input jxirts from thrsources.

A third illumination arrangement was spetially drtisrti toachieve a high miensit> of far ie<l light. .\ It) x IH x 25 cm highUixof hl.uk plexiglascotitained two 8 x Iticin windciwsof Oirn-ing 2030 liher hiatiks on opi^nite sidc-s aiul was immertied in asomewhat Uirger glass walled water luth. Illummatutn to the win-dows was provided h\ two Shalda (iO.'i flotMllights containing 5(K)W tungMen-haloKcn lamps (Q.MMl 1 3/C:l.) in tronl of paraboliclellectors. I he bath was CCMIUXI IO 39 C b\ n>ntrolled How of lapwater deli\eied inside the box. flowing out the Itaffled bul oprniMtttom, atul escapitig via a starulpi|te iti the outer batb. I he 2030hiteis had transmissions of 2'? at (itiO, 31'{ at <>70. and M'\ atr>80 nm: the\ were not |M>lished to the thn kness nerded to giveihr somewhat lower cut-on wa\eletigths sjx-cilied for ('.orning2-64 Ihe estimated irradiance (iiom each sule) was 3 mWVrm*for thr spcx'iral region t)(>(>-70U nm: ihts is reterrrd lo as 2030Rrd.

I he culture vessels weiT 170 x 20 mm II) test tubes (13). usu-ally cotiiaining a 7 mm OV> bultbling tul>e. Iti some expernnents(as tioted) a thitnier (2 mm) la\er of suspension was obtained b\using \h mm OI) bul>t>ling tuhes. I'nder stead\-slate c^inditionss|XHific' gmwih rate was estimated from plots of log .\ v». time.For this pur|>ose altsoilMiue (.\) was measured with an Esflvncoloiinieier using a tiOO nm broad lund hiter. Pa< ketl eell s-olumr((i\ crilv'ml) was determined In centrrfugmg an alicjuoi of cellsus|x-nsion al 2r><K> f for I h in lulx-s wiih lower set tion of I mmprecision bore capillar\. c:hl was extracted in NO'} .tceione andeslimaled s|x-cttophotomelricalK using an absorption c^telfKteniof 82.0(g1) '-cm '. Wholecells[>etira wereobiatned with a Carv14 s|>ettrophotometer iisiii^ 3 mm Itghi scattering plates (2O) ofiranshHrni plexiglas (Rohm & Haas No. 7328) insrnrd in Iti mtn

513

514 JACK MYERS El AL.

TAlil.r I. Crtlirat ititurt far phtroryinin anit rhLtrnphytt anatytit fromwktitf crtt ahsoTptum \prtiTajuf Aiiacystis mdulans and .Nostoc rnus-corum.

I'. 2. Vann%x.\ tighl snurm. Ihrir frartionat ahwrplwu fry phyrnry-anin anit Ihnr rjjrcl on Ihr rht/l'C ratio for Anacystis nidujans.

Snun-rPC. Chl

Aniulvrlmi

nm

Arnon ri al. (2)* 0.04Jonrs& M v m ( l 2 ) 0.11Sigalat & dr Kouchkovsky (21) 0.040Prrsrnt work' O.(Ki2

0.256 620. 6780.244 625.6780.246 625. 6800.257 625.678

* WaoHmfih* ft'tO. (MO nm tn nnmmai. xtual w>vrlrnrh< uanj tivm in Ihirit(oluinn

>C>n S-im mmnnm Kuu ; al nahm on Amaiyatt' Valun (or fl. utajmrH fn>m nttiitr.crll ipntra <A irth J » r i l ia i int in Wl"* o r .ar « 0 t:. mu^nrnilnt in II 2 M ii>tlum Mrtj ir hultrr M pll } S Valun tot clil

m»mlit<n» t>tr|Mi«iim m iu< t>>v iniinr twllrr a) »llrr >rrmh prrwZtl.fHMf | M tniknwrij bt S K wathini jitij irmnfuKaiMtn «! Iffl.INN) f tO

cuvellrs. At>s<>rt>ances so olitairieti were ciwrected h> sutMractionof thf rrsidual small afjsorfwntr at 72.5 nm. P7(H) was rsiimatrdfrom the c haiiKr in ahs<irbance al 702 vs. 735 nm caused bv 443nm illuminanon in ihr prescncr of 2 fiSl (X^MU and usr of anabsorption corffxirnt cjf 70 (mM x t m ) ' (II). Mrasurrmrnt*were made wiih a dual wavelength mpetirfipholomeler with ap-propriate ufrguarifs afnainM nuorcvrmr ariifarts. Phoiostn-thrtic rain were measured in term* of C), evolution in flashing(2(Vs) and continuous lighl al 39 C: as previously clevril>ed (14).

RESULTS

Analysis of in vivo pigmrnt ahsorption. Since PC arulchl provide the only significant absorptiotis at 550-700 ntn. analyses for lhe two pigttiettis <,itt l>e tnadefrom absorbances at two discriminating wave-lengths. I he infbrmatioti needed iticludes the ratiosof absorbances between the chosen wavelengths foreach pigment. Values of the critical ratios frotn fottrsources are given it> Fable I. We shall use valuesobtained in the presetu w«)rk by methixis sutnma-rized iti fcHHtiote c and tratislated itito Fcjttations Iatid 2, following. Small variatiotis in estimaie ol theratios arise from iticomplete separatioti of pigmentsin the preparations used or iti alteration of the pig-ments in separation. One likely Mjurce of error re-centlv recogni/ed (9) can arise by disvxiation of PCfrom lhe presumetl in vivo hexamer to the motio-mer; dis.s(xiation is minimi/ed on 0.2 Nf acetatebuffer at pH 5.5 (9). I able I emphasizes the agree-ment rather than the differetues fx-tweeti estimatesof dif feretit lalx>ratories. Except for otie high valueof 0. II for A,r,/A«s of PC, the variatiotis are ofdoubtful signiBcance. Further, the choice of wave-lengths is not very critical: there is <\'7f differencein absorfxitue l)etween 620 and 625 tint for PC orbetween 678 and 680 nm for chl. Relative alisor-bances for PC atul chl were obtained by the follow-ing set of rquations:

(I)(2)

Values of A^"^ and A. ,'*" are compU-trfv .iclc-c|iiateas indices of relative amounts of PC atid thl. Fur-ther conversion t»» numliers of af>s4>rf)ing moleculesre(|uircs estimates of in vivo abvirption COC-HK ients.

ttj*^ - 1.0162 Aw» - 0.2612 A, , ,/ " - 1.0162 A,r, - 0.0630 A«s

Snunr* re

Crnwihriir'

(lold fluorescent 78 2.4 0.85Warm white fluorescent 57 2.0 0.85Rc-d fluorescent 50 2.5 0.9I uiigsten (2.VH)°K) 48 1.5 0.85Blue fluorescent 18 3.0 0.6Neon + 2-(i-» filter 13 0.6 0.4BCI Safe-Umps 10 0.5 0.2I ungsten (3(HM)»K) + 2030 6 2.4 0.22

436 (Mercury + 3-73 + 5-57) 6 1.6 0.45«i88(l2.8)nni • 4 0.4 0.22686(35)nm ' 7 2.1 0.23677 (11.9) nm 6 0.9 0.22672 (9.0) nm 9 l.l 0.3657 (11.2) nm 44 4.6 0.6650 (10.9) nm 64 3.3 0.7638 (12.1) nm 78 4.1 0.7632 (11.5) nm 80 4.1 0.7621(10.9) nm HI 3.9 0.7

'fmmtr XMircn tnrtudr h l l m i p n l h n l by (>>rninK n u m h m . In n<inuilin( »•*»-Imfth •liurihu<ti>«>. nunufacturrt • lUia IC;rm-ral Klnttk) utrd €%tryx lot Ihf Noman<l nCJUm|n t.H chrUllrt i r b l r t l l h t t H h ioan<l nC.JUm|n. t.H chrUllrt i rbl i tr 4>ut|>iil « » mr»u ln l wilh < ciititH.ilrHnuiin plHUomiilimlirr t.i« t linht •oiiiin jrr inlrifririKr filirn uml in iji«m»nptiifr«lf«t with VMl W Ump. nti inhm ttr«i]|tulr irnlrr ul itir iMniltMM (numlirl* Hi(Mlrnlhr««-« 4tr«iBii«tr \\M iMfitl WHithat

'AhMtipfHin •» bt H. >•« lakuUlrit tnt ihr iripiHi •KHI 7IHI nm ai tiilli»>« <*"aliviliMHm i | m t i u m liic n . irat lalrn Irnni SigaUl ami ilr Kiiwhkinilv iVIl AnataiHiXKMi uarttium liK ihr iwmhfanr pi|(mrnl> l<hl • •armrniiuUl oa* lairn t<*IrattMMi y M AiitiMi rl al {'i) \\w twii •|tr<tia wrrr |M>|mallfnl l.» 1 II al Ihrir tr«l|>rak> A ihird (prilrum <tr«iitnl ihr rrlaiirr rnrixv rHiilNil ut ihc «>ul<r Al H> nmirMirmmii ihr ituipui «pr<itum wa> nmliiplirrt ht ihr 1*1. tiirtiiuin and alio h< ih<mrmbraiK tpmrutn Simimalion of ihr iHitfnii « alMoilMiur pristiMU gjvr lwo «atiir«priilMHiiiinal In nftiKalNhiir* ot ali*.H|Ht<i*i Uy PC: ami b> n»rtttli<anr (NKmriiu Ihrtaliir otr«l tor Pt: It '( ot loial

' ^1"^'*** K'"wlh ralr illr<i onlv lo »l>ow valiir otwivrtt uiHtrt lr»l tonttitHMit' rtgmrni laiKi in irtU|[rown umlri ra< h wniiir M »i»rn in irrmi ol ralui »l ah««-

iMmn itmrttirA m ihr al>«il|M«Hi •|>rilia of wluilr irtti Whrn AW^aa - " "'*• ''*'amr al fi2^ nm \n Vi. nniaii ati«ortHtmr al f>7N nm tn iht i 'w ot tmt^ »lMfh i hnuir implm ih«t cibtrrvrd «aluo vrrr vartatrfr or Ins crrtain.

For PC we take the absorption c<K'fficient tepotlcd(9) for the hexatneric form (but referred to themotiomer): .S.33 X !()» M ' cm ' at 621 titti. We alsoaccept the estimate of .S chtomophores/motiomer (9)-Hence, we take the in vivo absorptioti c<H."fficictUas 111 tnM ' c m ' at 621 (or 625) nm for thephycol)ilitiogen (htomophoie. This may be com-pared lo a value of 89 tiiM '-ctn ' calcttlaled frottithe PC; specific absorptioti coefficient reported hyCraig atid Carr (5) and used by Artioti et al. (2).

For « hi most of our analyses were b.ised upon 80%acelotte extracts atid did iioi depetid ti|M)n in vivos|)ectra. However, we did estimate an in vivi) af>-sorption coefficietit obtaitied frotn dala on cells ofwi<lely varied chl/PC ratios from 38 different cttl-tures. Values of A,;,"" cottipared to thi atial\ses ot>the same cells via 8O'l acetotie extracts yielded atiin vivo absorptioti c«K'fficietit of 68 ± 3 ttiM ' cm''al 678 nm. I his ma\ IK' cottipared to a value of <>*'niM"''cm"' estimated from the data of Arnon et al.(2).

Comparison oj ttght simrcrs. Table 2 shows lhe resultsof spectral calculations for fraction.il absorption ititoI*C irtmi variotts lighl sources. It also shows observedpigmetit ratios in terttis «»f the ( rude whole cell ;tl)-sorfutice ratif> \t^J\n:,• I fif tesiilts a te similar to

PU.MEN 1 VARI.X I ION IN AX.ICYSriS 515

TABU- ^. CnmpnriMm of \ . nidul;m«, strain Tx20, with othrrUraitujor pigmrni mpoiuf to tUuminatwn.

TABLE 4. CJutraflmstics if tells groum utuirr GM fluarnrmt and

undfT 2031) Rfd lUummatum at 39 C far A. nidulatu.

StrainWiirtn whiirfliHirrtrmi ltr<l

Tx206301"

.Igmrtiettum quattruhttcatHm

PR-Ji

Anaharna up. CA

(gruwn withoul fixed N)

0.850.80.81.0

1.0

1.3

0.80.S0.4

0.7

0.9' Vr liil>tr 'i. fommiird'KtiHtl R Sl^llirt 111 22).

those previously rejxirted. Ihe ie is liule variationin pi^tnciit ratio witli s|H*(tral c|tialii\ so loti^ as alarge ir.ution i>i tlu- li^lii is .il>s<>il>c(l l>\ PC. liow-cvft. ilu* (lil/l'C; ratio i.s not an cxticrnciv rigid thai-iiileiistii lor .-1. uiitiitany. even ior a pariiciilar linlnSource. It is a l la trd Mxnculial (l.'i). and in one to-{Jort severely (18). In linlil itiiensiix. as well as mi-iricnt dcli<ictuifs: i.e.. iturrased In nitrojjen (I) andcarl)on dioxide (7) limitation: tierrea.setl hy nian-Raiieso and cohalt limitations (4: Myers et al.. un-ptihl). In (act. the (hfl'C tatit>. lor reast»ns sou^lithut not f ouiul. has varied from 0.8 lo ().*.) in dilTerentcultutes in our lalMiratory growti under tutigsten il-lumination.

For light sources |XM>rIv ahsotln-d hv PC (and ah-VirlK'd piedoinin.uilh by (hi) there is a decreasedilil/r(; ratio. What appears lo IK* a limit is seen v\ het ethe Aa7VA«2 ratio = 0.2, coi t c"s|H)nding to a ratio olIhcchland P(;al>sorption |K-aks .\«.«""/.\rtj,*'' = 0.13.With n.irrow h.nid soutces the entire v.niation inpigtnetit ratio cHcuts within the stnall specttal tange65()-{J77 nni. A lowered chl/I*C ratio is develo|H-dalso In growth in the hitie (l.Sn tun atid Blue Klu-orescetit) wliere l'(". is .l piK)r absorln-r.

Otie objective ol our exploration ol light souices^as the search lor an experimetiiallv convenietitSource giving i ise to large vat iation in piginetit ratio,f oth the IU!| .Sa(e-I.;nnpsand the useol interferencehiteis luive pt.ictic.il ex|K'timent,il limitatiotis. Weconsidered hy s|K'ctral calculations and rejected anutnlK-r ol <»ther comhitiatiotis (not shown) of pos-»il)le sotirces atid hiters. iinallv. we adopted tlie'Ungsieti-halogen lamp with 'J030 filter.

ComfHirisoin utth othrr hlurfprrii algar. Table 3 prc-»cnts sotne data fr«)tii our limited observations ono'her algal sttaitis. Kroni these we derive two con-flusiotis: i) the three strains (iVMU. IVMI, (")<»0S) ofStrain Cluster 4 of Stanier et al. (2'2) all show the

y reduced chl/I'C tatio characteristic of I x20grown in far red light; ii) the same dratiiatic

i i s e to (.11 led light is not showti e(|U.iIl\ b\ s<ime'>ihc-r hluegieen alg.ie. W ith strains l*R-li atid C.\ we"hscrved only tnitior decreases iti the clil/P(' ratio.^ studies ot> other hluegreetjs usitig blue or far

A. Afaanrptmm and pi||innM concrmnuora.

Gold

2030 Red

0.85

0.22

* ~

0.509

0.115

0.505

0.674

Chl*

7.75(0.29)1.67

(0.08)

B PhcMfMvnthnir rhjiracirrnDci ()9 CI

Gold

2030 Red

P700

150

200

RCS

350250

Aciion'

0.74

0.31

Rrl «

0.031

0.045

nt»ii*lcTU>

4.55(0.21)6.08

(0.031)

S.t«a.«.

410 3.3

2000 2.9

Sum

12.30(0.57)7.75

(0.36)

r M r

RCS/>'

160550

. ..............IV ............-^ to < hi al tVTB af>d to PC at ftHh nm aA caliulalc^ Imnt wKu4rirll ahMHiiaiirr \ia ti)ualii>m I. if. Un rrll cnncrnmiHin I ^ rrlh/ml. palh Icm "^

' ( Mimaml fmni w n i m r r»rai-u (valun m narrn ihnn *n SRI• I>imi4inl IriMn A» ," and ataorpatn «irlh>arnl III mM ' u n ' .* 4 i h l ( 4 « itnuilO,) • prr Ibth.

JfrM« X h) '-dnW/tin')"'.nxUrhl « hi '

. . J crtt. « hi •> Maximum wrmnTT rate; nkuUird u PM " chJRCa x JH x 10 ^ t h • x

ted light ((>. 10, 16) did not re|X)rt ativ marked re-duction in chl compared to phycobilins.

I'arialion in crltular rhnractnistics of Tx20 f^ownundrr tirn chosen li^ht snurcrs. Of the several lightsources cotisidered in 1 able 2 we explored the dif-ferences Ixnween two: the 2030 Red and the (.oldfUioiescetit. These provided extretnes of values forthe cliL'I'C ratio and supjxirted alxun ecjual steadystate growth rates; fx = 2.") day ' or 3.6 divisiotisday-' at 39 C.

Steady-state cell characteristics are presented inTable 4. T he chl and VV. absorbances atui contetitsshow n for (iold ate also reasotuibh characteristic forcells gtown utider white fluotescetit or tungsten il-lumitiation.

Of the several photosMithetic characteiistics ex-amined (Table 4B) we c«)nsider first the estimates ofreaction cetiters. C'ells gn)wt> in (iohl have almut thesatne values found geneiallv (or Tx20 grown undertungsteti or (luorescent lights: a ratio of ca. twoP7OOs (RCl s) to one reaction cetiter II (RC2). Kc»rA. nuiuliins and some other bluegreeti algae a P7(K)cotitetit of ca. l/L'>Ochl iscotnmonlv reported (e.g.,8. 17. 18). It has been suggested bv Fujita (8. pcrs.conitii.) that the RC;2/chl isca. half as great. Our ownestimates of RC?2/chl made from rejx'titive flashyields have varied from I/3.iO to 1/250 in sattiplestaken from different cultures of 1x20 grown underdifferetit cotiditions. There are well ktiinvti sourcesof ertxir in measuremetits of P700 and in measure-ments of flash yield. We considered these in sufR-cietit detail to f>elieve that svstetnatic errors in ourmc-asurements ate small for normallv grown cells.We ate less confident of our results on 2030 Redcells. ttiainU Ixxause the \er\ 1cm chl content (andlow reaction cetiter ruimlKM/cell) makes the tec hnicalproblems of tueasuremeni more difiieuh. However,

516 JACK MYERS El AL.

o

ocro

UJ

TIMEFIG. I. TinMT course ol iviiilir»» li>r I'C, (A), chl (B). »uiii ol

chl ••• Pt: (C) and crll viilumc (O) %(Mnniiig speitntl iranMiionit inAnatyitu Hutuiam.

we consider the measuretrients adetjuaie lo assertthat reaction centers |x-r chl in 2030 Red are noimuch chatigi>d from their usual values in spile of ihevery much rcrduted chl content.

In a sectmd set ol phoiosvnihetic measuretnentswe of>taincd actions as initial SIO|K-S of the ligfit in-tensitv tiirve in ti77 nm light (IIIUV I \.\i nm), I he2030 Red cells have a iniic h teduced ()77 nm at tion.From the valuc-s ft>r action and estimates oi Iractionalal>s4irptioii hasttl u|>on whole tell ahsorption spettrawe estimated relative (|uaiitum \iel<Ls, As ex|H'ctedfrom their Itiwereti thI and increased PC absorption(cf. 25), the 2030 Red cells have higher t|uaiitumyield at •>77 ntn.

Saturated rates of photosvnthesis are presentedcm several different bases. Rates attainahle hy 2030Ret! cells are remarkahly high when referred to unitchltir reactioti centers. Kvidently the smaller titunU-rol reat tion centers tan turn over more rapi<ily anddo not significantly Itiwer the light-saturated rateattaitiafile.

Time four\f,\ of pifrmrut ailftL\tmrut\. I t ) t>l>serve t h etime course of transition in pigment tontetus follow-ing a spectral chatige in illumitiation we adopted thefollowing protcNol. Use of 13 mm ODfniiiiiling tulx-sinserted in 20 mm II) test tulxrs gave thin annular

titltures. Duplicate cultures, itUKtilated Irom a coti-tiniious cultuie cli.nnlK'r, vvete statted in (iold atidin 20:^0 Red, Multiple tulx-s of each culture werepooled and tneastirt nients made at chosen time iti-tervals. I wice daily the tttltiues were (juantitativelydiluted iti order to hold the whole cell absorbancel>etweet) values o ld . I and (),<) ((i'* .'> nin, 0,7 nun pathlength). This arrangement solved the problem oiohtaitiing sutficieni cell densitv ior measurement hulalso maintaining an opticallv thin cultuie, .Aitcr 2days the cultuies wete swa|)|)ed between (iold atul2030 Red, thus providing both transitions. In a iiistseries oi ex|H'rittients oiilv whole cell absoiptiotis|X'Ctra were measured and thI atid PCI contetitta-tions (JKT ml culture) were caUulated, In a secoiulseries of ex|K-rimetits mote replicate IUIK'S were{MM)led/culture, sampling was less iief|uent, and cellvolumes, (hi hv exit act and whole cell absorbaticcspectra were olxaiiiecl.

Figure I shows ihelittuMriinscorsviitheNesofchl.PC;, sum of till + I'O and cc-ll volume. D.ita are pre-sfiiled as sciiii-log plois so that slopes tiieastiir SJM*-cific rates of increase, I be stead\-state slopes oicurves of A, 11, atid C (Fig, I) all irom ihe iitsi ex-t)erimetilal series, correspond losin-ciiic rates oi II,O'lh"'. Curve I) (Fig. I) is taken itoni the second seriesiti which eiiective illumin,itions were slightly greaterand steady-slate rates were 0. |() h '.

Foll«»wing the ( io ld-» 2050 Red transitioti thesloi>e ior chl synthesis drtips tt> ca. half t>f its fitialsteatly-state value. Concurrently, though with ashort lag, the SIOJK' for PC synthesis is ca, twice thesieady-stale valtte. .Slo|>es for IKMII pigments are al-most symtiietrically reversed iollowing the 20!<0 Re<l—•(iold iransiiion. Following IMIMI tratisitions pig-ment adjustnienis are nearU cc>in|>lc-te(l and newsleady-slale conditions approxittiated within 21 h orwithin < 4 divisicms.

In spite of lix changes in the chl/PC ratio whichgive rise to the excursions of curves A and \\, curve(; ii>r the Sinn of chl + PC shows much smaller de-parttires from sieadv-state slopes. Following the(iold —• 2O;iO Red transition, the slope ior total pig-ment sMithesis remains constant over ihe fitsi 7 hduring which the chl/PC ratio is changing f»y I.'.>'<•A suhsetjuent small offset in SIO[K- will IK.- considere<llater. Perturb,itions in curve C following the 2030Red—» (iold tt.uisiiion aie even smaller, (iompari-son of curve C vs. curves A and \\ (Fig. I) sup|M)ttsthe tiotion that totitrols leading to adjustmetit iti the*cfil/PC ratio o|M-tatc- u|M)n rates of synthesis from atonunon prettitsor.

Curve I) (Fig, I) descril>es increase in cell volumewhich is almost invariatit with dry weight undct' awide ratige of totiditiotis for •rx2() (I.'V, 2.3); l,<> M'cells - 0.27 mgdrv wt = 2.H x IO"cells (2.3), Curves(not shown) ior .ipp,uent absoriiance (scattering) i"725 titti were close fits to curve I). (Icmsider first llic20S0 R e d - • CHIICI transition. There is an initial lagor decrea.sed slope iti curve I) implving that cells

rUlMKM VARIA I ION IN A 517

adjusted t«) 2():U) Rt-d piguioiitation arc impaired incoMvt'ision o t Ciold li^llI lor <cll s\iitlu*sis. We ex-p<M(*d a >iinilar iiiilial l.i^ l(>ll()\vin^ tiie (told —»'2{)'M) Red (laiisitioii. Instead we foutid an itiitial rateo l t e l l synthesis ^t eater tlian the rate sustained alterpignietit adjustinetit to 20:^0 Red, In order to ex-atiiitie this ;is|)ett we addtessed the simpler (|ues-lioti: what is the arithmetic i iurement in cell mate-rial fortned duiitig llie lirst vs. the secotid dav altereach specttal transition? I'ahle .') ptesents the re-sultitl^ data in tertns of amoutit ol material lortnedin each peri<Kt from an initial cell c|uantity <>i I M'cells.

InsjH'ction ol Table .*> shows ihc following. After2():U) Red—•('•old transition the second day showsimproved perJortnatue. (lells with pij^metits char-aaerisi i t of Gold perform In-tter than cells with pig-metils characteristic ol L'O.SO Red light. However,cxactU the opjxisite happens alter tlie CioUl —• 2():U)Ked ttatisiiion. IVrlortnatue is tiol itnptoved cltiiitigthe setoiid da\ when pi^^u'nIs have adjusted tosic;i(ly-siatc v.ilucs (luuiKicri.siic of L'O.'iO Red.

BLC 5. Growth incrrmmti fMmnng sprrlral Iranatums: tnnrrmmtscalrutatrH as adual ifuanltttn^lmt nf ruiturr s\nlhntzftl during first andSffond day ajin IranMtion: inrrrmmt.\ for snond day apprnximalf ihitsrjar ilrady-ilalr. Earh 24 k prrtnd ilaiint unlh 1.0 /J trlUlml.

During growth iti illumination limited to far red•i. uululaus shows two kitids of trsponses. The fitst'Hcuts rapidly, ap|KMts to he tic\nlv completc-d itiJour ^etierations. atid is evident as a drop it» c hi/PC,fatio to a value as low as V« oi that .seeti tnuler whiteor jrold light (Table •!. Tig, 1). Our cotuern iti thepiesetit tepott is limited to I his earix res|H)nse. Weshou|<| tiote also a sc-cond kind ol respotise whichoccurs alter tnatiy (L'O-IOO) generations, is charac-•cri/ed hv inciease in chl/I*c; ratio evetJtiialK to ini-"Miallv high levels, aticl a|)|H'ars to result from se-lection ol mutatus with mote la\otal)le pigmentai'iatigemetus. The secotid res|)c)ti.se is uttder coti-tituiitig study. It is tioted hete only Ixxause lack ofreccigtiitioti and ttse olct i l tures tuaititained lor lotigpc't iods under lar red le,i<ls to cotilusioti ol the two

ects. We tiote, tor exatnple, a previous rc{>ort ('J5)action spectra lor A. tiululaus gtcmti in cotitittii-citltuie undet lar tvd light ol \\C] lamps, I he

i7i A«j., ol the cells used was (t,-l(). Reexaminatiot)tecotcls showed that the cultute earlier had had

A«j., = O.UO atid had IKHMI tnaititaiticci for 48ati<l ptobabU >2l) getieratiotis at the time of

*«>inpling. Hence the te|M»rted actioti spectra onl\partly descrilx- the parent sttain alter adjustmetu c»rP'gnietus iti the itiitial tc-sponse lo lar red liglil.

In norm,ill\ pigmented cells a large Iractioti ol'•H- (III is cottntiitled to the atitetitia ol pholoreactioti' and only a small lt,ictioti {'/«) is accessible to pho-•oreactioti II ('Ib). The detreased chl in far red light"^ay IK- accoitipanied bv a stnall itictease iti Itaction"'ctessible to phototeactioti II, ,is evi<lenced b\ ap-patent sliilt iti tcictioti center ratios ( 1 able \\\).•lowever, redisttibutioti ol chl Ix'tween the plioto-

ioii atiteiuKis CI<H*S not seem to IH- a major partthe phetiomenon even ihough lamella-phvcobi-

PrrioH

.Vflcr CK>II1 - • 20.10 Red

Kir<«l <la\

S<*ii>i)<l (l.iv

Alter 20.10 Re«l — C-.MKirM (ld>Sefcind AA\

c>uvnlumr

ml

2.5II

7.512

CJtlnmtd

ml

3118

.50

nmc4

ml

14165

32.54

Chl• «

nnvfl

mi

17283

82144

lisome arrangements must Ix" rather drnsticiillv al-tered. The loweti-d absorption In chl leads one toex|H'ct the dcntc-ase in action observed at <>77 inn( t a b l e W\). At tlie s.ime lime an increased iractiotiof llie liglit absotlx-d at 677 is absorlx'd b\ p h v o vc:yanin. .\ result is a more nearly ec]ual distributionof fxd la t io t t cnetgv lo (he two pliototractions andan improved <|uantum \icld, . \ s observed also in ablue (low chl) ttiutant (L\S, 2")), the red drop it) (juan-tum \ ield is less severe than in normally pigmentedcells.

We have no romiorting explanation of mecha-nism which conttols the cliange to low chl in far reellight. Our evaluation of light sources confirms andextends a previous obset vatioti (12): the low chl con-dition is observed under light sources providingmost of their energy in the lar red (Xi'H) nm) or inthe blue (<.')()() ntn). The sjKttral transition in thered is abrupt. The low chl condition is develoiH'dcompletel\ within a wavelength span probabK evennarrower tlian the tange tir>(i-()77 nm observed withour 10 nm hall-band width hiters (Table 2). Wehave considered the jntssible involvetnent ol an al-loph\C(K\atiiti-like cotitrol pigmetit which mighthave severeK dec reasing absorption l>eyond (»'>(> inn(6). However, we are not satisfied that this couldexplain the effec t at I.Sti nm. (i5(M>8() nm is also thesjHHttal range in which the action spectra lor pho-toieactions I and II cross to favor photoreaction Ionly at longer wavelengths (25). In short we can s;iythat growth in a light preferentiallv absorlx^l bv chl,and therelcue photosvntheticalU a light 1. yyill giverise to the low chl condition: hut we have no furtherclue as to iiuHhanism.

In the origitial rej)ott (12) and iti a suhse(|ucntreview treatment (.S) the pigment adjustment of .-1.tiidultnis to lar l ed light h,is Ix'en viewtnl as a six'c-tralh controllcMl adaptation. In the present reixirtwe have carefullv avoided the term adaptation. Weptestimo that adaptation is pro|K'rl\ applied to aies|Kinsive change b\ virtue ol which an organismcan Ix'tter COJH- yviih an alteted environment. Weate tunessariU skeptical whether the low chl condi-tion develojXHl in lar red light makes A. niHulamIx'tter able to grow in far red light. Three o b s c n a -

518 JACK MYERS El AL.

tions arc pcrtincni. Following ihe pigment adjusi-incnl, there is an improvcfl cfficicncv in the use olfar red light which is absorbed (< . «)77 nm. Table4B). However, ability to absorb and use a low inten-sity of far red light is decreased (action. »i77 nm.Table 4B). .Xs may fx- deduced from Fig. I, andd<xumenie<l in Table 5, prcKluction of cell materialand total pigment in far red light is not improvedby the low chl condition.

Far red (>fK5() nm) illumination is an exotic con-diiioii not likely to IK* met in nature. We can imaginethat the low chl condition arises as an incompetenceand should be judged as a failure. Whether it turnsout to be a failure or a lx»na fide adaptation, thecausative chain of events remains a significant ques-

rhi» Mucl> was supported by Grant CM 11300 from the NationalInstitute* of Mrallh. We ihank (; Sia<cy. R. .Slanirr aiul C. VanBaatcn for t u t i u m kiiKlty priiviilni. We ihank V. tie Kourhkov-iky f«>r kindly providing u* the «|)ectral data of refcretice (21) ind i i l iorm.

4

1. Alien. M. M. & Sttiiih. A. J. 1969. Nitrojjen chlorosis inblue-ip-een algae. Arch. .Mikr.Jnul. »><»: 1 |4-V!(1.

2. Arnon. I). I., McSwaiti. B. I ) . 1 «ujirnoto, H. Y. k Wad.i,K. |y74, Photothemical artiviiv and components of IIMMII-biane ptr|Mrati<>rM Irom btue-^rrt-n algae. 1. CoexlMeiite oltwo photosvsieins in rcLiiiiin i<i (hlorophyll a and removalof phstiKVaiiin. Hunhim. limphyy. Aila 3.'»7:?3t-4r>.

3. Bogorad. L. XSilb. Hbycobiliproirint and roiiipldneniarychrotnatic a<laptation. Ann. fin: I'timl I'hwint. 2():.1(i9-4()|.

4 Cheniae, G. W. &• Martin. I. F. t MiH. Site of inangaiirsclunttion in photimnthesis. Hiorhim. ttuiphyf. Aita I5.1HI9-37.

5. Craig. I. W. & Carr. N. G. 1968. C-phycocyanin and allo-phycoryanin in two species of blue-ureen alifae. Biorhrm. I

' 106:361-6. » » 76. Dtakoff. S. &S<beibe.J. 1973. Action spedra for chromatic

adaptation in Tiiiypnthnx Imuu. Ptani Phytutt. h\.m2.-h.7. i.lev.J. M. 1971. Kf feel of cailMin dmxiile concent ration on

pigmentation in the blue-greeti alga Aiuuystu nutulans. I'tantCetiPhytwl. 12:311-6.

8. Fujita. Y. 1976. The C . O phoioresponse al reK>m temper-ature observed in membrane fragmetits of the blue-greenalga Anabama vanabtiu. Plant Crtl PhjiioL 17;187.-91.

10.

Gla/er. A. N.. Fang. S. gc Brown. I). M. 1973. S|>ectroMopicpr(i|MMlies of G-pbyttKyanin and of its a anti fi subunils. y.Hwi. Ctirm. 24H:.')(>79-H.Vllaurv. J. F. & Bogorad. I.. 1977. Aclitm spectra for phy-(obiliprttiein s\nlbesis in a chromalicallv a<la{>ting cyano-phvle, Frrmyrttti (tiptostphun. Ptatil Physwt. (')(l:H3.'j-9.

11. Hiv.iina. I. & Ke. H. I')71.'. Oiflereiue sjM-ctra and extinc-tion ctK-ltuieiii!) ol 1*700. tiimhim. Hiiif)h\\. Aria 2()7: lt>0-71.

12. Jones. I.. W. ik Mvert. J 19<>.5. Tiginenl v.iriations iti Ana-nulls nututans itidu<ed by ligbt t»f self* ted wavelengtbs. J.

13. Krarz. W. A. & Myeri. J. 1955. Nutrition aiul grt.wth t)fseveral blue-green algae, .-fm. /. lint. 42:2M2-7.

14. Myers. J. Jfc(;raham.J. R. 1971. 1 he photosynihetic unit inCMorftta measured by repetitive short flashes. PtnnI Phy.iiol.4H:2«2-<).

15. Myers. J. &• Krai/. W. A. 19.'i.'). Relations Ix-tween |>igintntcttnieni and pboi<is\niheiic characteristics in a blue-grc-enalga, y (,rn. Phyunt 39:11-22.

16. I'ulit h. W. M. & Van Baaleti, G. 1974. Growth reijuirementst)f blue-greeti algae untler blue light ctindititins. Arrh. Afirro-biot. 97:303-12.

1 7 . O g a w a . I . . V e i i i o i i . I . . I*, j t M i i l l t - n h a u c r , I f . M . I<M><».Properties and Mruciute ol fractions prepared from /ffw-txirna varvttntft by the action of Triloti X-IOO. Bimhim. Bio-phy^. Ada M'i.'iU^'i'.t.

IH. ()(M|uisi. G. 1974. Disitibiiiion of chlorophyll iH'twccn twophtilorea< lions in phtiiosvnihesis of the blue.green alga An-a<y\ti.\ nulultins grown at Iwoilifletent Ijgbt inientilies. I'hysiot.Ptanl. .10:38-14.

I'.) R.io. V. S. K.. Brantl. ) J Jt- Mvers. |. 1977. Gold shocks\ndt<)me iti Anacy\lii nuiuians. PUinl Physuit. 59:9«»5-9.

20. Shiluita. K. 1958. .S|>ectrophotonietry <if intact biologicalmaterial, y. Hwrhrm. 45:599-623.

21. Sigalal. G. & tie Kotuhkovsky, Y. 1974. Preparation atidpro|)erlies of pholt>sytithelic fragments tif the utiicelliilarblue-green alga Anaryslu nulutnm. PriK. Inl. C.ongr, I'holmyiilh.

22. Slanier. R. Y.. Kunisawa. R.. Mandel. M. & Gt>hen-Ba/ire,(i. 1971. Purihcaiion and properties of unicelluUr blue-green algae. Bad. Rn\ 35:171-205.Slevens. G. I.. R. &- Nfvers. J. 1976. Characterisation of pig-ment mutants in a bluegteen alga Anacy\lu nututans. j . Ph\eot.

24. Vati Baalen. G. 1965. Quanlilalive stirface plaling of coc-coid blue-green algae, y. Phytnt. 1:19-22.

25. Wang. R. I,, Slevens. C. L. R. Sc Myers. J. 1977. Actions^tectra for pbotoreaclions I and II of pholo«Mithe<iis in theblue-green alga Anaryilit nulutam. PhoUuhrm. Pholntnot.25:103-8.

23.

:u J ._.:„