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Planta (1990)180:249-256 P l ~ J l l t ~9 Springer-Verlag 1990

P h y c o e r y t h r i n i s a b s e n t fr o m t h e p y re n o id o fP o r p h y r i d i u m c r u e n t u m : p h otos yn th e t i c im p l i c a t ion sR . M i c h a e l L . M c K a y * a n d S a r a h P . G i bb sD e p a r t m e n t o f B io l o g y , M c G i l l U n i v e r s i ty , M o n t r e a l , P . Q . H 3 A I B 1 , C a n a d a

Abstract. T h e t h y l a k o i d l a m e l l a e wh i c h t r a v e r s e t h e p y -reno id o f the un ice llu la r red a lga P o r p h y r i d i u m c r u e n t u m(Ag a rd h ) Nf i ge l i a p p e a r t o l a c k p h y c o b i l i s o m e s . W e h a v ec o n f ir m e d b y i m m u n o - e l e c t r o n m i c r o s c o p y t h a t p h y -c o e ry t h r i n (P E ) , a n i m p o r t a n t s t ru c t u ra l c o m p o n e n t o ft h e p h y c o b i l i s o m e s o f r e d a l ga e , i s a b s e n t f ro m t h e p y re -no id . To charac te r ize py reno id thy lako ids fu r ther , e lec-t r o n - m i c ro s c o p i c c y t o c h e m i c al m e t h o d s w e r e e m p l o y e dt o d e t e c t p h o t o s y s t e m a c t iv i t y. P h o t o s y s t e m (P S ) I a c ti v -i t y wa s d e m o n s t r a t e d i n b o t h s t ro m a l a n d p y re n o i d t h y -l a k o i d s b y t h e p h o t o o x i d a t i o n o f 3 , 3 '- d i a m i n o b e n z id i n e .In c o n t r a s t , t h e l o c a l iz a t i o n o f p h o t o r e d u c e d d i s t y ry l n i-t ro b l u e t e t ra z o l i u m d e m o n s t r a t e d t h a t P S I I a c t i v it y wa sre s t r ic t e d t o s t ro m a l t h y l a k o i d s . T h e o b s e rv e d p a r t i t io n -i n g o f P E a n d P S I I a c t i v i t y w i t h i n t h e p l a s ti d m a y b ere l a t e d t o a n o t h e r o b s e rv a t i o n , t h a t b e i n g t h e l o c a l i z a -t i o n o f n e a r l y al l r i b u l o s e - l , 5 -b i s p h o s p h a t e c a rb o x y l a s e /oxyge nase (EC 4 .1 .1 .39 ) wi th in the py re no id o f th i s a lga .I t is p o s s ib l e t h a t t h e p y re n o i d o f P . c r u e n t u m fu n c t i o n sa s a s p ec i f ic m e t a b o l i c c o m p a r t m e n t wh e re C O 2 f i x a t io ni s e n h a n c e d b y t h e a b s e n c e o f p h o t o s y n t h e t i c 0 2 e v o l u -t ion .Key words: P h y c o b i l i p ro t e i n - P h o t o s y s t e m I , I I - P o r -p h y r i d i u m - P y re n o i d - R i b u l o s e - l , 5 -b i s p h o s p h a t e c a r -b o x y l a s e /o x y g e n a s e - R h o d o p h y t a

IntroductionP h o t o s y n t h e t i c a c c e s s o ry p i g m e n t s fu n c t i o n i n t h e h a r-v e s t in g a n d t r a n s f e r o f e x c it a t io n e n e rg y f ro m a b s o rb e d* T o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r es s e dAbb r e v i a t i o n s : D A B = 3 , 3 ' - d i a m i n o b e n z i d i n e - 4 H C 1 ; D S - N B T =d i s t y r y l n i t r o b l u e t e t r a z o l i u m c h l o r i d e ; E F = e x o p l a s m i c f a c e ;L S U = l a r g e s u b u n it o f R u B i s C O ; P E = p h y c o e r y t h r i n ; P S = p h o t o -s y s t e m ; R u B i s C O - - r i b u l o s e - 1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n -a s e

s o l a r r a d i a t i o n t o t h e r e a c t i o n -c e n t e r p i g m e n t s o f p h o t o -s y s t e m s (P S ) I a n d I I t h ro u g h i n t e rm e d i a ry c h l o ro p h y l la m o l e c u l e s . L i g h t -h a rv e s t i n g p i g m e n t s m a y i n c l u d ec h l o ro p h y l l s , c a ro t e n e s a n d x a n t h o p h y l l s ; h o we v e r , u n -i q u e t o t h e c y a n o b a c t e r i a , r e d a l g a e a n d c ry p t o m o n a d sa re t h e p h y c o b i l i p ro t e in s , a g ro u p o f wa t e r - s o l u b l e , p h o -t o s y n t h e t i c a n t e n n a p i g m e n t s wh i c h a b s o rb l i g h t o fwa v e l e n g t h s t h a t a r e p o o r l y h a rv e s t e d b y c h l o ro p h y l la ( fo r a r e c e n t r e v i e w , s e e M a c C o l l a n d Gu a rd -F r i a r1 98 7) . I n t h e c y a n o b a c t e r i a a n d r e d a l g a e , p h y c o b i l i p ro -t e in s a r e a r r a n g e d i n t o l a rge p i g m e n t -p ro t e i n c o m p l e x e s ,n a m e d p h y c o b i l i s o m e s , wh i c h a r e a t t a c h e d t o t h e o u t e rs u r f a c e o f t h e t h y l a k o i d m e m b ra n e (Ga n t t 1 9 8 0 ; G l a z e r1982) . Wi th in these s t ruc tu res , the b i l ip ro te ins a re a r -r a n g e d i n a n e n e rg e t i c a l l y f a v o u ra b l e p a t t e rn s u c h t h a te x c i t a t i o n e n e rg y c a n b e t r a n s f e r r e d b e t we e n m o l e c u l e sin a d i rec t ion o f decreas ing energy leve ls , i .e . phyco ery th -r in ~ p h y c o c y a n i n ~ a l l o p h y c o c y a n i n (Ga n t t e t a l.1 9 7 6 ) . F ro m a l l o p h y c o c y a n i n , a b s o rb e d l i g h t e n e rg y i sp a s s e d , p ro b a b l y b y wa y o f a 7 5- t o 1 2 0 -k i i o d a l to n " a n -c h o r " p o l y p e p t i d e (G a n t t e t a l . 1 9 88 ), t o t h e c h l o ro p h y l la m o l e c u l e s a s s o c i a t e d w i t h P S I I i n t h e t h y l a k o i d m e m -b ra n e .M o rp h o l o g i c a l e v i d e n ce fo r t h e c l o s e a s so c i a t i o n b e -t we e n p h y c o b i l i s o m e s a n d P S I I h a s b e e n o b t a i n e d f ro mb o t h f r e e z e - f ra c t u re m i c ro s c o p y a n d o b s e rv a t i o n s o f i s o -l a te d P S I I -p h y c o b i l i s o m e p a r t ic l e s b y t r a n s m i s s i o n e l ec -t ro n m i c ro s c o p y . E m p l o y i n g f r e e z e - f r a c t u re t e c h n i q u e s ,M 6rsche l an d M fih le tha le r (1983) and Gidd ings e t a l.( 1 9 83 ) h a v e s h o wn t h a t r o ws o f p u t a t i v e P S I I c o re p a r t i-c l es a s s o c i a t e d w i t h t h e e x o p l a s m i c f a c e (E F ) o f f r e e ze -f r a c t u re d t h y l a k o i d m e m b ra n e s a r e d i r e c t l y a l i g n ed w i t hr o w s o f p h y c o b il i s om e s l o c a t e d o n t h e o u t e r m e m b r a n es u r f a c e . P h o t o s y s t e m I I -p h y c o b i l i s o m e p a r t i c l e s h a v eb e e n i s o l a t e d f ro m c y a n o b a c t e r i a a n d t h e r e d a l g a P o r -p h y r i d i u m c r u e n t u m . In P . c r u e n t u r n , the par t i c les possessh i g h r a t e s o f 0 2 e v o l u t i o n a n d c o n s i s t o f s e v e ra l p h y c o -b i l i s o m e s a t t a c h e d t o s m a l l m e m b ra n e f r a g m e n t s (C l e m -en t -M et ra l e t a l. 1985) . These mem bran e f ragm en ts a ree n r i c h e d i n P S I I r e a c t i o n -c e n t e r p o l y p e p t i d e s , d e f i c i e n t

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2 50 R .M .L . M c K a y a n d S .P . G i b b s : A b s e n c e o f p h y c o e r y t h r i n a n d P S I I a c t i v it y i n t h e p y r e n o i di n P S I c o m p o n e n t s , a n d c o n t a i n n o d e t e c t a b l e c h l o r o -p h y l l P T o o .

T h e t h y l a k o i d s o f b o t h c y a n o b a c t e r i a a n d r e d a lg a ea r e u n s t a c k e d a n d a s a g e n e r a l r u l e w h e n c e l ls a r e g r o w -i n g i n t h e l ig h t u n d e r u n s t r e s s e d c o n d i t i o n s , p h y c o b i l -i s o m e s , i f a d e q u a t e l y p r e s e r v e d b y t h e f i x a t io n p r o c e -d u r e , a r e p r e s e n t o n e v e r y t h y l a k o i d . A n a p p a r e n t e x c ep -t i o n t o th i s o b s e r v a t i o n a r e th e t h y l a k o i d s w h i c h t r a v e r s et h e p y r e n o i d s o f r e d a lg a l c h l o r o p l a s t s . I n p u b l i s h e d m i -c r o g r a p h s o f th e p y r e n o i d s o f Porphyridium cruentum( e. g . G a n t t a n d C o n t i 1 9 6 5 ), P. aerugineum ( G a n t t e t a l.1 9 6 8 ) a n d Porphyra leucosticta ( S h e a t h e t a l . 1 9 7 7 ) , p h y -c o b i l i so m e s a p p e a r t o b e r e s t r ic t e d t o t h y l a k o i d s l o c a t e dw i t h i n th e c h l o r o p l a s t s t r o m a a n d t o b e ab s e n t f r o mt h y l a k o i d s w i t h i n t h e p y r e n o i d . H o w e v e r , i n m o s t a l g ae ,t h e p y r e n o i d m a t r i x i s v e r y e l e c t r o n - d e n s e a n d i n e l ec -t r o n m i c r o g r a p h s a p p e a r s d a r k e r t h a n t h e a d j a c e n t c h l o -r o p l a s t s t r o m a a n d i t i s p o s s i b l e , th e r e f o r e , t h a t t h e d e n s em a t r i x m a t e r i a l o f t h e p y r e n o i d o b s c u r e s a n y p h y c o b i l -i s o m e s th a t m i g h t b e p re s e n t. T h e o n l y a u t h o r s w h o ,t o o u r k n o w l e d g e , h a v e c o m m e n t e d o n w h e t h e r p h y c o -b i l is o m e s a r e p r es e n t i n t he p y r e n o i d r e p o r t t h a t p h y c o -b i l is o m e s ar e p r e se n t , b u t n o t a b u n d a n t , o n t h e c o n v o -l u t e d t h y l a k o i d s o f th e p y r e n o i d o f Rhodella cyanea ( B i l -l a r d a n d F r e s n e l 1 98 6 ). F u r t h e r m o r e , t h e p u b l i s h e d m i -c r o g r a p h s o f Nemalion ( S h e a t h e t a l. 1 9 7 9 ; B r a w l e y a n dW e t h e r b e e 1 9 81 ) i n d i c a t e t h a t p h y c o b i l i s o m e s a r e a l s op r e s e n t o n t h e p y r e n o i d t h y l a k o i d s o f th i s a lg a .

T o d e t e r m i n e w h e t h e r o r n o t p h y c o b i l i p r o t e i n s a r ep r e s e n t i n t h e p y r e n o i d s o f r e d a l g a e , w e h a v e s t u d i e dt h e d i s t r i b u t i o n o f p h y c o e r y t h r i n ( P E ) i n t h e p l a s t id o fPorphyridium cruentum b y i m m u n o e l e c t r o n m i c r o s c op y .W e f i n d t h a t P E i s n o t p r e s e n t i n th e p y r e n o i d , a n df u r t h e r m o r e t h a t t h e i n t r a p y r e n o i d t h y l a k o i d s d i s p l a yn o r m a l P S I a c t i v i t y b u t l a c k P S I I a c t i v i ty . W e d i s c u s st h e s e f i n d i n g s w i t h r e f e r e n c e t o a n o t h e r o b s e r v a t i o n ,n a m e l y t h e l o c a l i z a t i o n o f n e a r l y a l l t h e p l a s t i d ' s r i b u -l o s e - l , 5 - b i s p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e ( R U B-i s C O , E C 4 . 1 .1 . 3 9) w i t h i n t h e p y r e n o i d .

M a t e r i a l a n d m e t h o d s

Plant material. Porphyridium cruentum (Agardh) N/ ige l i was ob-ta ined f rom the Cul tu re Col lec t ion o f Algae a t the Univers i ty o fTexas a t Aus t in (UTE X 161) and g rown in ba tch cu l tu re in anar t i f i c ia l seawate r m edium (Jones e t a l . 1963). Cu l tu res were main-ta ined a t 20 ~ C a nd were con t inuous ly ag i ta ted by use o f a ro ta ryshaker . L igh t was p rov ided by coo l -whi te f luorescen t l amps (Sy l -v a n i a F 7 2 T I 2 / C W / H O ; R o y M a r c h a n d , L a v a l , P .Q . , C a n a d a ) a ta pho ton f luence ra te o f 30 lamol, m -2 . s -1 in 16 h l igh t - 8 h da rkcycles.Immuno-electron microscopy. Logar i thmic -phase ce l l s o f P. cruen-tum were ha rves ted a t hour 3 o f the l igh t pe r iod . Ce l l pe ll e t s weref ixed a t 4 ~ C fo r 1 h in a so lu t ion con ta in ing 4 % (v /v ) pa ra fo rmal -dehyde and 0 .8% (v /v ) g lu ta ra ldeh yde in phospha te -buf fe re d sa line(PBS) , pH 7 .1 . The pe l le t s were washed w i th co ld buf fe r and thece l ls dehyd ra ted th rough a g raded e thano l se r ie s and em beddedin Lowicry l K4M (J .B . EM Serv ices, Mo nt rea l , P .Q . , Canad a) asdesc r ibed in Lac os te -Ro ya l and Gibb s (1985). Go ld-co lou red sec -t ions were cu t wi th a d iamond kn i fe and mounted on fo rmvar -

coa ted n icke l g r ids ( J.B . EM Serv ices ). For imm unolabe l l ing , thegr ids were p laced sec t ion-side down on d rops o f the fo l lowing so lu -t ions : PBS, 15 min ; 1% (w /v) bov ine se rum a lbum in ( f rac t ion V)(BSA) in PBS, 15 ra in ; an t i se rum, 30 min ; PBS r inse ; 1% BSAin PBS, 15 min ; p ro te in A -go ld d i lu ted 1 :10 , 30 min ; PBS r inse ;de - ion ized wa te r r inse . Rabb i t an t i se rum ag a ins t PE 545 pur i f i edf r o m t h e c r y p t o m o n a d a l g a Rhodomonas lens w a s k i n d l y p r o v i d e db y R o b e r t M a c C o l l ( N e w Y o r k S t a t e D e p a r t m e n t o f H e a l t h , A l -bany , N .Y. , USA) and was used a t a d i lu t ion o f 1 :5000 . Ant i se rato sod ium-dodecy l - su l fa te -d i s soc ia ted a rge (LSU) and smal l sub-un i t s o f tobacco RuBisCO were k ind ly p rov ided by Jacque l ineF leck ( lns t i tu t de b io log ie mol6cu la i re e t ce l lu la ire , CN RS, S t ras -bourg , F rance) and were used a t a d i lu t ion o f 1 :2000 . Ant i se raand p ro te in A-go ld were d i lu ted in 1% BSA in PBS. Col lo id a lgo ld was p repared accord ing to the method o f F rens (1973) andw a s c o n j u g a t e d t o p r o t e i n A ( P h a r m a c i a , D o r v a l , P .Q . , C a n a d a )as desc r ibed p rev ious ly (Roth e t a l . 1978). Imm unolabe l led sec t ionswere pos t - s ta ined wi th 2% (w/v) aqueous u rany l ace ta te andv iewed in a Ph i l ips (E indhoven , The N e ther lands ) EM 410 e lec t ronmicroscop e a t an ope ra t ing vo l tage o f 80 kV.In con t ro l exper iments , the an t ibody was rep laced wi th rabb i tn o n - i m m u n e i m m u n o g l o b u l i n G , o r w i t h P B S a l o n e , p r i o r t o p r o -te in A-go ld incuba t ion .Quantitative evaluation. The dens i ty o f l abe l l ing over va r ious ce l lc o m p a r t m e n t s w a s o b t a i n e d b y d e t e r m i n i n g th e n u m b e r o f g o l dp a r t ic l e s p e r s q u a r e m i c r o m e t e r o f c o m p a r t m e n t s e c ti o n e d . A r e ade te rm ina t ions were mad e us ing a Ze iss (New York , N .Y. , USA )MO P-3 d ig i t a l ana lyse r.Cytochemical detection o,f PS activity. Log-p hase ce l l s o f P. cruen-turn were ha rves ted a t hours 4 o r 6 o f the da i ly l igh t pe r iod andt rea ted to de tec t PS ac t iv i ty essen t ia l ly as desc r ibed in Vaughnand Ou tlaw (1983) and Vaughn (1987). Cell pel lets were f ixed in2% (v /v ) pa ra fo rmald ehyd e in 0 .1 M sod ium -phosp ha te buf fe r , pH7.4, con ta in ing 0 .2 M sucrose , fo r 20 min a t 4 ~ C in da rkn ess (d a rk -ness and low tempera tu re were main ta ined in a l l s t eps p r io r tores in in f i l t r a t ion excep t a s no ted) . Th e pe l le t s were washed in buf fe rth rough a dec reas ing suc rose g rad ien t and then incuba ted wi tha reagen t so lu t ion spec i f i c fo r each PS . Pho tosys tem -! ac t iv i ty wasd e t e c t e d b y t h e p h o t o o x i d a t i o n o f 3 ,3 ' - d i a m i n o b e n z i d i n e .4 H C l(DA B) (Po lysc iences , Wa r r ing ton , Penn . , US A) , whereas PSI I ac -t i v it y w a s d e m o n s t r a t e d b y t h e p h o t o r e d u c t i o n o f d i s t y r y l n i t r o b l u et e t r a z o l i u m c h l o r i d e ( D S - N B T ) ( A n a l y c h e m C o r p . , M a r k h a m ,Ont . , Can ada) . Bo th reagen ts were used a t a concen t ra t ion o f I r ag /ml in 0 .1 M sod ium-phospha te buf fe r , pH 7 .4 . The DS-NBT so lu -t ion con ta ined , in add i t ion , 0 .5% (v /v ) d imethy l su l fox ide to a idin so lub i l i za t ion o f the t e t razo l ium sa l t . Fo r PS loc a l iza t ion , ce l lssuspended in f resh ly p repared reagen t so lu t ion were incuba ted ona ro ta ry shaker a t 20 DC under coo l -whi te f luorescen t l amps p rov id -ing a ph o ton f luence ra te o f 30 p -mol .m -2 . s - 1 fo r e i the r 1 h (DA Boxida t ion) o r 45 min (D S-NB T reduc t ion) . Con t ro l s w ere pe r -fo rmed by e i the r incuba t ing the ce l l s in da rkness a t 20~ 'C o r , in

F ig . 1 . Log-ph ase ce l l o f P. cruentum l abe l led by an t i -PE 545. Go ldp a r t ic l e s ar e a b u n d a n t t h r o u g h o u t t h e c h l o r o p l a s t s tr o m a , u s u a l lyassoc ia ted wi th the ou te r su r face o f the thy lako ids . The pyreno id(py) , which i s t r aversed by numerous thy lako id l amel lae , r emainsvir tua l ly label-free, x 27000; bar = 0.5 lamFig . 2 . Log-phase ce l l o f P. cruentum l a b e ll e d b y a n t i - L S U o f R u B -i sCO. Gold pa r t i c les a re h igh ly concen t ra ted over the pyreno id(py) whereas the ch lo rop las t s t rom a d i sp lays a ve ry low leve l o flabe l l ing , s imi la r to tha t o bse rved over the nuc leus (n ) and cy to -p lasm. A smal l , e lec t ron-dense reg ion loca ted a t the ch lo rop las t ' sp e r i p h e r y (arrowhead) i s a l so modera te ly l abe l led and may repre -sent a satel l i te pyren oid, m, mitoc hon drio n, x 28 000; ba r = 0.5 ~tm

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252 R.M.L. M cKay and S.P. Gibbs: Absence of phycoerythrin and PSII activity in the pyrenoidthe case of DS-NBT treatment, by pretreating and incubating w iththe PSII inhibitor atrazine (gift from Ciba-Geigy, Dorval, P.Q.,Canada; provided under the commercial name Aatrex| and usedat a concentration of 5.4 gg/ml). Following incubation, the pelletswere washed with buffer and then postfixed in either 1% (v/v)OsO4 in 0.1 M sodium cacodylate, pH 7.4, for 70 min at 4 ~(DAB-treated cells) or in cacodylate-buffered 2% OsO4 for 90 m inat ro om temperature (DS-NB T-treated cells). The ce ll pellets weredehydrated through a graded ethanol series and then infiltratedand embedded in low-viscosity epoxy resin (Spurr 1969). Gold-and silver-coloured sections were mounted on formvar-coated cop -per grids and unstained sections were viewed in either a PhilipsEM200 o r a Philips EM 410 electron microscope at operating volt-ages of 60 and 80 kV, respectively.

Resul ts

Immuno-electron microscopy. T h e i n t r a c e l l u l a r d i s t r i b u -t i o n o f b o t h t h e p h o t o s y n t h e t i c a c c e s so r y p ig m e n t P Ea n d t h e C a l v i n -c y c l e e n z y m e R u B i s C O i n ce ll s o f Por-phyridium cruentum i s s h o w n i n F i g s . 1 a n d 2 . F i g u r eI s h o w s a s e c t io n t h r o u g h a l o g - p h a s e c e ll w h i c h h a sb e e n l a b e l l e d w i t h a n t i - P E . G o l d p a r t i c l e s a r e a b u n d a n tt h r o u g h o u t t h e c h l o r o p l a s t s t r o m a a n d a r e u s u a l l y a s s o -c i a t e d w i t h t h e o u t e r s u r f a c e s o f th e t h y l a k o i d s . I n c o n -t r a s t , t h e p y r e n o i d r e g i o n i s v i r t u a l l y l a b e l - f r e e . T h u s ,P E , a s v i s u a l i z e d b y p r o t e i n A - g o l d l a b e l l i n g , d o e s n o ta p p e a r t o b e a s s o c i a t e d w i t h i n t r a p y r e n o i d t h y l a k o i d s .C e l l s e c t i o n s i n c u b a t e d w i t h a n t i s e r u m a g a i n s t t h eL S U o f R u B i s C O e x h i b i t a d i f fe r e n t p a t t e r n o f la b e l li n g( F ig . 2 ). G o l d p a r t i c l e s a r e h i g h l y c o n c e n t r a t e d o v e r t h ep y r e n o i d r e g i o n w h e r e a s th e r e m a i n d e r o f th e c h l o r o -p l a s t s h o w s o n l y a f e w d i s p e r s e d p a r t i c le s . O n l y a s m a l la m o u n t o f b a c k g r o u n d l a b e l l in g is p r e s e n t o v e r t h e n u -c l e u s a n d c y t o p l a s m . S e c t i o n s la b e l le d b y a n t i b o d ya g a i n st t h e s m a l l s u b u n it o f R u B i s C O d i d n o t s h o w t h ep r e s e n c e o f a n y s p e c if i c l a b e l li n g ( n o t s h o w n ) , i n d i c a t i n gt h a t o u r a n t i s e r u m a g a i n s t t h e s m a l l s u b u n i t , p r o d u c e df r o m a t o b a c c o a n t i g e n , d o e s n o t c r o s s - r e a c t w i t h t h es m a l l s u b u n i t o f R u B i s C O i n P. cruentum.A q u a n t i t a t i v e a n a l y s is o f t h e d e n s i t y o f l a b el l in gb y e a c h a n t i b o d y o v e r d i f f e r e n t c e ll a n d p l a s t i d c o m p a r t -m e n t s i n P. cruentum s h o w e d t h a t l a b e l l i n g b y a n t i - P E

w a s h i g h o v e r t h e c h l o r o p l a s t s t r o m a ( 8 7 . 2 _ + 3 . 0 p a r t i -c l e s /g i n z ) b u t o v e r t h e p y r e n o i d r e g i o n w a s n o t a b o v eb a c k g r o u n d c y t o p l a s m i c l a be l l in g ( T a b l e 1). I n c o n t r a s t ,t h e d e n s i t y o f la b e l li n g b y a n t i - L S U w a s v e r y h i g h o v e rt h e p y r e n o i d m a t r i x ( 1 9 6 .6 + 7 . 2 / g m 2 ) b u t v e r y l o w o v e rt h e s t r o m a ( 2 . 9 - t - 0 . 2 p a r t i c l e s / g m Z ) . S t r o m a l l a b e l l i n g ,h o w e v e r , w a s s i g n if i c a n tl y h i g h e r t h a n b a c k g r o u n d c y t o -p l a s m i c l a b e l l i n g ( o n e t a i l e d p a i r e d t - t e s t , P < 0 . 0 0 5 ) .C o n t r o l e x p e r i m e n t s w e r e p e r f o r m e d b y r e p l a c i n gt h e a n t i s e r u m w i t h e i t h e r n o n - i m m u n e r a b b i t i m m u n o -g l o b u l i n G o r p h o s p h a t e - b u f f e r e d s a l i n e a l o n e , f o l l o w e db y p r o t e i n A - g o l d ( T a b l e 1). F o r e a c h c o n t r o l , l a b e l l in gd e n s i t i e s w e r e l o w o v e r e a c h c e l l u l a r c o m p a r t m e n t a n a -l y z ed . I n a d d i t i o n , t h e s p e c i f ic i ty o f t h e a n t i - L S U a n da n t i - P E 5 4 5 u s e d i n t h i s s t u d y , t o t h e i r c o r r e s p o n d i n ga n t ig e n s, h a s b e e n d e m o n s t r a t e d b y M a n g e n e y a n dG i b b s ( 1 9 8 7 ) a n d L u d w i g a n d G i b b s ( 1 9 8 9 ) .Cytochemical detection of PSI. U s in g p h o t o o x i d a t i o n o fD A B t o d e m o n s t r a t e P S I a c t i v i ty w i th i n t h e c h l o r o p l a s to f P. cruentum, w e f o u n d a n e l e c t r o n -d e n s e p o l y m e r t ob e a s s o c i a t e d w i t h a l l o f t h e t h y l a k o i d s o f t h e p l a s ti d ,i n c l u d i n g t h o s e w h i c h t r a v e r s e t h e p y r e n o i d ( F ig . 3 a ).T h e d e n s e m a t e r i a l w a s d e p o s i t e d w i t h i n t h e l u m e n s o fm o s t t h y l a k o i d s ; h o w e v e r , w h e r e s t r o m a l t h y l a k o i d sw e r e s w o l le n , t h e p o l y m e r a p p e a r e d a s s o c i a t e d o n l y w i t ht h e m e m b r a n e .I n a d d i t i o n t o t h y l a k o i d s , t h e m i t o c h o n d r i a l c r i s t a ea n d e n v e l o p e w e r e o f t e n s t a i n e d ( F i g . 3 a ). T h i s h a s b e e no b s e r v e d p r e v i o u s l y ( S e l i g m a n e t a l . 1 9 6 8 ; M a r t y 1 9 7 7 ;V a u g h n a n d O u t l a w 1 9 8 3) a n d i s i n d i c a t iv e o f c y t o -c h r o m e - o x i d a s e a c t i v i t y .I n c o n t r o l c e ll s i n c u b a t e d i n D A B i n d a r k n e s s , m i t o -c h o n d r i a r e t a i n e d t h e d e n s e st a i n i n g w h e r e a s c h l o r o p l a s tt h y l a k o i d s w e r e l a r g e l y u n s t a i n e d ( F i g . 3 b ) . In t h e c h l o -r o p l a s t , e l e c t r o n - d e n s e d e p o s i t s w e r e r e s t ri c t e d t o s m a l ls e g m e n t s o f o n l y a f e w t h y l a k o i d s .Cytochemical detection of PSII. P h o t o s y s t e m - I I a c t i v i t yw a s d e t ec t e d b y t h e p h o t o r e d u c t i o n o f D S - N B T t o d i f o r-m a z a n . R e d u c t i o n o f th e t e t r a z o l i u m s a l t w a s v i s u a ll ym o n i t o r e d t h r o u g h a c h a n g e i n c o l o u r o f th e c e ll su s p e n -

Table 1. Density of labelling ove r various cell comp artments of Porphyridium cruentum.Values= N o. gold particles-Bm-2+_ SEChloroplast Cytoplasm" Nucleus b n cPyrenoid Stromal region

PhycoerythrinAnti-PE 545 1.6+ 0.3 87.2+_3.0RuBisCOAn ti-LSU 196.6 +_ 7.2 2.9 +_0.2ControlsNon immu ne IgG 0.3 _+0.2 0.9 _+0.2Protein A-gold alone 0.8 _+0.4 0.7 _ 0.1

1.6 +0 .2 1.1 -I-0.6 (9) 240.9+_0.2 2.0+_0.9 (4) 200.8_+0.2 1.9+-0 .9 (6) 140.3+ 0.1 1.1 +-0.7 (6) 14

Includes mitochondria and starchb No. of nuclei examined in ( )No. of cell sections analyzed

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R.M.L. McKa y and S.P. Gibbs: Absence of phycoerythrin and PSII activity in the pyrenoid 253

Fig. 3a, b. Cytochemical localization ofPSI activity in log -phase cells of P.cruentum. 18 500 ; b ars = 1 I-tm. a Cellincubated in DAB in the light. Theelectron-dense deposits produ ced by thephotooxidation of DA B are associatedwith all chloroplast thylakoids includingthose which traverse the pyrenoid.Mitochondrial membranes are also stained(arrowhead). b Control cell incubated inDAB in the dark. The chloroplastthylakoids are largely unstained whereasmitochondrial membranes retain theirelectron-dense deposits (arrowheads)

s i o n f r o m r e d t o d a r k p u r p l e o r b l a c k . T h i s c o l o u rc h a n g e is c h a r a c t e ri s t i c o f t h e p h o t o r e d u c t i o n o f t e t r a zo -l i u m s a lt s a n d w a s i n i ti a t e d w i t h i n 1 - 2 m i n o f i n c u b a t i o ni n t h e l ig h t . I n c e ll s e c t io n s , d i f o r m a z a n w a s l o c a l i z e di n th e l u m e n s o f s t r o m a l t h y l a k o i d s b u t w a s a b s e n t f r o m

i n t r a p y r e n o i d t h y l a k o i d s ( F i g . 4 a ) . I n t h y l a k o i d s w h i c he x t e n d f r o m t h e c h l o r o p l a s t s t r o m a i n t o t h e p y r e n o i dr e g i o n t h e l u m e n a l e l e c t r o n - d e n s e d e p o s i t s s t o p p e da b r u p t l y a t t h e p y r e n o i d b o r d e r ( F i g . 4 a , a r r o w h e a d s ) .

C e l l s u s p e n s i o n s i n c u b a t e d i n d a r k n e s s o r i n t h e p r e s -

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254 R.M.L. McK ay and S.P. Gibbs: Absence of phycoerythrin and PSII activity in the pyrenoid

Fig. 4a, b. Cytochemical localization of PSII activity in log-phasecells of P. eruentum, x 32500; ba rs= 0.5 gm. a Cell incubated inDS-NBT in the light. Electron-dense deposits resulting from pho-toreduction of DS-NBT are found in the lumens of most stromalthylakoids but not in the pyrenoid thylakoids. In thylakoids whichextend from the chloroplast stroma into the pyrenoid, the lumenal

electron-dense deposits end abruptly at the pyrenoid bo rder (arrow-heads), b Control cell incubated in DS-NBT in the light in thepresence of atrazine. Dense produ ct is not present in the thylakoids,many of which are greatly swollen. However, som e regions of thechloroplast envelope are stained (arrows)

e n c e o f t h e P S I I i n h ib i t o r a t r a z in e d id n o t c h a n g e c o lo u rdur ing in cuba t ion . In the a t raz ine - t rea ted ce l l in F ig . 4b ,the thy lakoids do not possess e lec t ron-dense depos i t s .Dark ly s ta ined reg ions a re l imi ted ma in ly to a reas ofthe ch loroplas t enve lope (F ig . 4b , a r rows) . In addi t ion ,sca t te red osmiophi l ic g lobules a re present in the p las t id .These hav e been o bse rve d previous ly (Se l igman e t a l .1 9 71 ) a n d a r e n o t c o n s id e r e d t o r e p r e se n t p h o to r e d u c e dD S - N B T .

Discuss ionAbsence of PE and PSII activity in the pyrenoid. W eh a v e d e m o n s t r a t e d t h a t t h e i n t r a p y r e n o id t h y l a k o id s o fthe red a lga Porphyridum cruentum a re not labe l led byant i -PE a l though a h igh leve l of labe l l ing i s a ssoc ia tedwi th the thy lakoid lamel lae outs ide the pyrenoid . Phy-c o e r y th r in is th e m a jo r c o m p o n e n t o f t h e p h y c o b i l iso m e so f P. cruentum, a c c o u n t in g f o r m o r e t h a n 7 0 % o f th e i rt o t a l p r o t e in ( G a n t t 1 9 8 6 ) . T h i s p ig m e n t - p r o t e in c o m -plex occupies a pe r iphera l pos i t ion in the numerous rodsw h ic h r a d i a t e o u t f r o m th e c en t r a l a l l o p h y c o c y a n in c o r eo f P. cruentum p h y c o b i l i so m e s ( G a n t t a n d L ip sc h u l t z1977) . Our da ta do not ru le out the poss ib i l i ty of o the rp h y c o b i l i p r o t e in s b e in g p r e se n t i n t h e p y r e n o id o f P .cruentum; however , s ince phycobi l i somes of th is spec iesa lw a y s co n t a in P E , w e c o n c lu d e t h a t t h e r e a re n o p h y c o -

bi l i somes a t tached to the thy lakoids which t r ave rse thepyren oid o f th is a lga . This con f i rms the v isua l impres-s ion g iven by the publ ished m ic rog raphs of th is spec ies(e .g . Gant t and Cont i 1965) .I n a d d i t i o n , w e o b se r v e d t h a t d i f o r m a z a n r e su l t i n gf r o m th e p h o to r e d u c t i o n o f D S - N B T w a s r e s t ri c t ed t othe s t romal thy lakoids . This ind ica tes tha t PSI I ac t iv i tyin t h e i n t r a p y r e n o id t h y l a k o id s i s e i t h e r m a r k e d ly r e -duced o r en t i r e ly absent . L ey and But le r (1977) haver e p o r t e d t h a t u p w a r d s o f 9 5 % o f t he l i g h t e x c i t a t io ne n e r g y t r a p p e d b y t h e p h y c o b i l i so m e s o f P. cruentumi s in i t ia l ly t r ansfe r red to PSI I . Thus , s ince phycobi l -i so m e s i n t h e p y r e n o id o f P. cruentum a re absent , theo n ly e n e r g y t h a t w o u ld b e t r a n s f e r r e d t o P S I I c o m p le x e sin t h e t h y l a k o id m e m b r a n e s w o u ld b e t h a t t r a p p e d b ythe smal l numb er o f ch loro phyl l a molecules a ssoc ia tedwi th these reac t ion cente r s . Thus one would expec t PSI Ia c t iv i t y i n t h e p y r e n o id t o b e m u c h r e d u c e d .A fur the r cont r ibut ing fac tor to the low PSI I ac t iv i tyobse rve d in the in t rap yreno id thylakoi ds might be a def i -c iency of PSI I complexe s a ssoc ia ted wi th the exoplasm icf ac e s ( E F ) o f t h ese t h y l a k o id s . I n t h e c r y p to m o n a d a l-gae , where PE is a t tache d to the lumena l sur face of thethylak oid me mb rane (Licht l6 et a l. 1987; Ludw ig andGibbs 1989) , Rhiel e t a l . (1985) and Lichtl6 e t a l . (1986)h a v e sh o w n th a t w h e n th e p h y c o b i l i p r o t e in c o n t e n t o fce l l s grown in h igh l igh t i s r educed by 90% or 50%,th e r e i s a c o r r e sp o n d in g r e d u c t io n i n t h e n u m b e r o f E F

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R.M.L. Mc Kay and S.P. Gibbs: Absence of phycoerythrin and PSII activity in the pyrenoid 255p a r t i c l e s ( t h e p u t a t i v e P S I I c o m p l e x e s ) l o c a t e d o n t h et h y l a k o i d m e m b r a n e s . I n f r e e z e - f r a c t u r e s t u d i es o f th ec h l o r o p l a s t o f P . c r u e n t u m ( N e u s h u l 1 9 7 0 , 1 9 7 1 ) , t h eE F f a c e o f t h e p y r e n o i d t h y l a k o i d s i s n o t i d e n t if i e d .C l e a r l y , a d d i t i o n a l f r e e z e - f r a c t u r e s tu d i e s o r , a l t e r n a t i v e -l y , i m m u n o c y t o c h e m i c a l s tu d i e s u s i n g a n t i b o d i e s a g a i n s tP S I I p o l y p e p t i d e s a r e n e e d e d i n o r d e r t o d e t e r m i n ew h e t h e r p y r e n o i d t h y l a k o i d s a r e d e f i c ie n t i n P S I I c o m -p l e x e s o r w h e t h e r t h e a b s e n c e o f P S I I a c t i v i t y re s u l tss o l e ly f r o m t h e l a c k o f P S I I l i g h t - h a r v e s t i n g a n t e n n ap i g m e n t s .P h o t o s y s t e m - I a c t i v it y i n t h e p y r e n o i d . P h o t o s y s t e m - I a c -t iv i ty w a s d e te c t e d t h r o u g h t h e p h o t o o x i d a t i o n o f D A B .T h e r e s ul t in g e l e c t r o n - d e n se r e a c t i o n p r o d u c t w a s f o u n da s s o c i a t e d w i t h a l l t h y l a k o i d l a m e l l a e i n c l u d i n g t h o s ew h i c h t r a v e r s e t h e p y r e n o i d . T h e s t a i n i n g o f i n t r a p y r e n -o i d t h y l a k o i d s i s b e l i e v e d t o b e g e n u i n e . I t s e e m s u n l i k e l yt h a t r e a c t i o n p r o d u c t s h a v e s e c o n d a ri l y m i g r a t e d i n t ot h e p y r e n o i d r e g i o n s in c e t he o x i d i z e d p o l y m e r o f D A Bi s h i g h l y i n s o l u b l e a n d i s a s s u m e d t o r e m a i n c l o s e t ot h e s it e o f it s f o r m a t i o n ( F r e d e r i c k 1 9 8 7) . O a k l e y a n dD o d g e ( 1 9 7 4 ) h a v e s i m i l a r l y o b s e r v e d D A B r e a c t i o np r o d u c t a s s o c i a t e d w i t h t h e p y r e n o i d t h y l a k o i d s i n P .c r u e n t u m . T h e s e f i n d i n g s i n d i c a t e t h a t t h e p y r e n o i d t h y -l a k o id s a r e c a p a b l e o f cy c li c p h o t o p h o s p h o r y l a t i o n m e -d i a t e d b y P S I . T h e r e w o u l d b e l i tt l e n o n - c y c l i c p h o t o -p h o s p h o r y l a t i o n o r 0 2 e v o l u t i o n a s s o c i a t e d w i t h i n t r a -p y r e n o i d t h y l a k o id s , h o w e v e r .R i b u l o s e - l , 5 - b is p h o s p h a t e c a r b o x y l a s e / o x y g e n a s e a n d p y -r e n o i d s . T h e a p p a r e n t i n t r a c e l l u l a r c o m p a r t m e n t a t i o n o fb i l i p r o t e i n s a n d P S I I a c t i v i t y o b s e r v e d i n P . c r u e n t u mi n t h e p r e s e n t s t u d y m a y b e r e l a t e d t o p y r e n o i d f u n c t i o n .A t p r e s e n t , t h e r o l e o f p y r e n o i d s i s n o t e s t a b l i s h e d ; h o w -e v e r , o w i n g t o t h e i r p l a s t i d i c l o c a t i o n , t h e y a r e l i k e l yt o b e i n v o l v e d i n p h o t o s y n t h e s i s . E v i d e n c e s u p p o r t i n gt h i s c o m e s f r o m b i o c h e m i c a l a n d i m m u n o c y t o c h e m i c a lr e p o r t s a l l o f w h i c h i n d i c a t e t h a t p y r e n o i d s o f v a r i o u ss p e ci e s o f a lg a e c o n t a i n t h e b i f u n c t i o n a l C a l v i n - c y c l e e n -z y m e R u B i s C O ( f o r r e f er e n c es , s ee M c K a y a n d G i b b s1 9 8 9 ) . I n t h i s s t u d y w e h a v e e s t a b l i s h e d t h a t R u B i s C Oi s a l s o p r e s e n t i n t h e p y r e n o i d o f P . c r u e n t u m . T h e L S Uo f R u B i s C O w a s h i g h ly c o n c e n t r a t e d i n t h e p y r e n o i do f t h is a l g a w h e r e a s s t r o m a l l e v e ls o f t h e e n z y m e w e r eo n l y s l i g h t l y , a l t h o u g h s i g n i f i c a n t l y , a b o v e b a c k g r o u n dc y t o p l a s m i c l e v e l s ( T a b l e 1 ) .W h e t h e r p y r e n o i d R u B i s C O i s a c t i v e i n v i v o i s n o tk n o w n . S o m e s t u d i e s i n d i c a t e t h a t t h e p y r e n o i d r e p r e -s e nt s a s t o r a g e p o o l o f t h e e n z y m e ( L a c o s t e - R o y a l a n dG i b b s 1 9 8 7 ) , w h e r e a s o t h e r s i n d i c a t e t h a t t h e p y r e n o i df u n c t i o n s a s a s p e c if i c m e t a b o l i c c o m p a r t m e n t w h e r eC O 2 f i x a t i o n i s i n i t i a te d ( M i y a c h i e t al . 1 9 8 6 ; K u c h i t s ue t a l. 1 9 8 8 ; M c K a y a n d G i b b s 1 9 8 9 ) . T h e a b s e n c e o fp h y c o b i l i s o m e s a n d P S I I a c t i v i t y i n th e p y r e n o i d o f P .c r u e n t u m i n d i c a t e s t h a t t h e r e w o u l d b e n e g li g i b le 0 2 e v o -l u t io n w i t h i n t h e p y r e n o i d . A l t h o u g h d i f f u si o n o f 0 2i n t o t h e p y r e n o i d f r o m t h e s u r r o u n d i n g c h l o r o p l a s t s tr o -m a w o u l d o c c u r , c o n c e i v a b l y t h e 0 2 t e n s i o n i n t h e p y r e -n o i d m i g h t b e l o w e r t h a n t h a t i n t h e a d j a c e n t s t r o m a .

I n a d d i t i o n , c e l l s o f P . c r u e n t u m g r o w n i n l o w C O 2 h a v eb e e n s h o w n t o t a k e u p a n d u t i l i z e d i s s o l v e d i n o r g a n i cc a r b o n w i t h h i g h e f f i c ie n c y i n p h o t o s y n t h e s i s ( C o l m a na n d G e h l 1 9 8 3 ) . T h e c e l l s p o s s e s s a n i n t r a c e l l u l a r c a r -b o n i c a n h y d r a s e ( D i x o n e t a l . 1 9 8 7 ) w h i c h h a s b e e ns h o w n t o b e l o c a t e d m a i n l y i n t h e c h l o r o p l a s t s t r o m a( Y a g a w a e t a l. 1 9 8 7 ) . T h u s c o n c e i v a b l y , t h e m i c r o e n v i -r o n m e n t o f t h e p y r e n o i d w o u l d b e o n e o f h i g h C O 2a n d l o w 0 2 a n d t h i s i n tu r n w o u l d f a v o u r th e c a r b o x y l -a t i o n r e a c t i o n o f R u B i s C O .We thank Drs. Jacqueline Fleck (CNRS, Strasbourg) and RobertMacColl (New York State Department of Health, Albany) for pro-viding us with the antibodies use d in this study. W e also than kDr. C .E. Smith for use of the Z eiss MO P-3 digital analyser andDr. Genevi+ve Bricheux for kindly providing Lowicryl-embeddedsamples of P. cruentum. Aatre x| was kindly don ated by Ciba-Geigy. This research was supported by the Natural Sciences andEngineering Research Council of Canada (grant No. A-2921).

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Rece ived 31 M ay; acc ep ted 9 Augus t 1989