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The role of bicarbonate ion in the mechanism of photosynthesis Jolliffe, Ethel Ann 1972

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THE ROLE OF BICARBONATE ION IN THE MECHANISM OF PHOTOSYNTHESIS  by  B.Sc,  ETHEL ANN JOLLIFFE U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1967  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n t h e Department of Botany We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1972  In p r e s e n t i n g . t h i s t h e s i s in p a r t i a l  f u l f i l m e n t o f the r e q u i r e m e n t s  an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r  for  that  r e f e r e n c e and s t u d y .  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department or by h i s r e p r e s e n t a t i v e s .  It  i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written permission.  Department of The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  ii  ABSTRACT  T h r e e a s p e c t s o f t h e r o l e o f HCO^ i o n i n p h o t o s y n t h e s i s were s t u d i e d .  The f i r s t p a r t o f t h e i n v e s t i g a t i o n i n v o l v e d  t h e s t u d y o f HCO^ i o n a s a s u b s t r a t e f o r p h o t o s y n t h e s i s i n marine benthic algae. Net  i n o r g a n i c carbon  a s s i m i l a t i o n by Sargassum muticum  was r e c o r d e d i n t h e l i g h t up t o pH 9.9 a n d a t pCC^ down t o less  t h a n 5 ppm.  C a r b o n u p t a k e was m e a s u r e d o n t h e b a s i s o f  c h a n g e s i n t h e CC^ r e l e a s e d b y a c i d f r o m 1 m l s a m p l e s o f t h e experimental sea water,  a n d a l s o c a l c u l a t e d f r o m pCC»2 a n d pH  according to standard tables. red  gas a n a l y s i s .  The pCG^ was m o n i t o r e d  by i n f r a -  T h i s a l g a a s s i m i l a t e d HCO^ i o n d i r e c t l y i n  photosynthesis. In  a c o n t i n u i n g s t u d y , c a r b o n u p t a k e was r e c o r d e d i n  the l i g h t f o r the f o l l o w i n g marine algae: Desmarestia, and  Ulva.  Alaria,  Costaria,  Enteromorpha, G i g a r t i n a , N e r e o c y s t i s , Porphyra,  The c h a n g e i n t o t a l i n o r g a n i c c a r b o n  m e n t a l w a t e r was a g a i n d e t e r m i n e d exceptions of Porphyra  i n the experi-  by a c i d r e l e a s e .  and D e s m a r e s t i a ,  With the  t h e a l g a e u s e d HCO^ i o n  as a s u b s t r a t e f o r p h o t o s y n t h e s i s . The  relationship of rate of photosynthesis to t o t a l  i n o r g a n i c carbon p r e s e n t i n seawater  was d e t e r m i n e d  f o r Ulva  f e n e s t r a t a , I r i d a e a c o r d a t a , and Sargassum mutlcum.  The  n a t u r a l l y o c c u r r i n g c a r b o n c o n c e n t r a t i o n was f o u n d t o b e l i m i t ing f o rphotosynthesis  .  In p a r t two, t h e r o l e o f c a r b o n i c anhydrase and c a r b o n i c ;acid a s a p r o t o n g e n e r a t i n g , system f o r . p h o t o p h o s p h o r y l a t i o n was i n v e s t i g a t e d . The  compound Diamox  (5-acetamido-l,3,4-thiadiazole-2-  sulphonamide) i s a s p e c i f i c i n h i b i t o r o f c a r b o n i c  anhydrase.  Diamox i n h i b i t s c a r b o n f i x a t i o n i n t h e l i g h t i n w h o l e p l a n t s and  isolated chloroplasts.  I t also inhibits  photosynthetic  e l e c t r o n t r a n s p o r t , both c y c l i c and n o n - c y c l i c .  I t was f o u n d  t h a t t h e l i g h t - i n d u c e d pH s h i f t i n u n b u f f e r e d c h l o r o p l a s t s 14 was  a l s o a f f e c t e d , b u t t h a t ATP-supported  CC> f i x a t i o n b y 2  i s o l a t e d c h l o r o p l a s t s i n t h e d a r k was n o t i n h i b i t e d .  These  f a c t s l e d t o the c o n c l u s i o n t h a t carbonic anhydrase plays i t s role i n photosynthesis  as a s u p p l i e r o f p r o t o n s  (from  carbonic  a c i d ) t o t h e h y d r o g e n i o n pump. 14 Antimycin A stimulates CC> f i x a t i o n b y c h l o r o p l a s t s 14 2  in saturating light.  Diamox i n h i b i t i o n o f  CC> f i x a t i o n i n 2  the l i g h t i s overcome b y A n t i m y c i n A a t b o t h s a t u r a t i n g and  non-saturating  light. 14  Antimycin A stimulates charge"  This i n t e r a c t i o n suggests  that  CC» f i x a t i o n b y c h a n g i n g t h e " e n e r g y 2  status of the chloroplast. During t h e f i r s t seconds o f i l l u m i n a t i o n and up t o  f o u r o r f i v e m i n u t e s t h e r e a f t e r , t h e 0^ e v o l u t i o n a s s o c i a t e d w i t h the beginning o f photosynthesis transients.  shows a number o f  The t h i r d p a r t o f t h i s i n v e s t i g a t i o n d e s c r i b e s  some s t u d i e s o f t h e s e t r a n s i e n t s a n d t h e i n t e r a c t i o n o f 0^ and i n o r g a n i c c a r b o n w i t h r e g a r d t o t h e v a r i o u s The r e s u l t s a r e d i s c u s s e d i n r e l a t i o n photosynthesis  i n higher plants.  components.  t o C» e f f e c t s o n 2  A transient heretofore  unknown i n r e d a l g a e i s a l s o r e p o r t e d .  V  TABLE OF CONTENTS Page PART I-A  Bicarbonate Ion A s s i m i l a t i o n i n P h o t o s y n t h e s i s by Sargassum muticum  INTRODUCTION MATERIALS AND METHODS RESULTS  1 .  . . . . . .  . . . . . . . . . . . . . .  2  4 .  7  DISCUSSION  12  LITERATURE CITED . . . . . . . . . . . . . . . . . . . . PART I-B S t u d i e s o n B i c a r b o n a t e I o n Uptake During Photosynthesis i n Benthic  16  Marine Algae  18  INTRODUCTION MATERIALS AND METHODS  19 . . . . . . . . .  20  RESULTS AND DISCUSSION  25  Survey f o r HCO^ I o n A s s i m i l a t i o n  .  25  The E f f e c t o f HCO^ I o n C o n c e n t r a t i o n on P h o t o s y n t h e s i s  .  LITERATURE CITED ADDENDUM - The E f f e c t o f pH on P h o t o s y n t h e s i s i n Ulva fenestrata APPENDIX - The Measurement o f I n o r g a n i c Carbon and P h o t o s y n t h e s i s i n Seawater by PCO2 and pH A n a l y s i s PART I I E v i d e n c e C o n c e r n i n g t h e Mechanism o f Diamox I n h i b i t i o n and A n t i m y c i n A Stimulation o f Photosynthesis INTRODUCTION  35 47 48 56 64 65  vi Page MATERIALS AND METHODS . Preparation of Chloroplasts C0  Production  2  The L i g h t - I n d u c e d  . .  . .  66  .  68  . . . . . . . . . . . . .  69  pH S h i f t . . . . . . . . . . . .  70  Preparation of Inhibitors •  66  F e e d i n g s : Dark and L i g h t  2  Studies of 0  RESULTS  . . . . . . . . . . .  •  •  •  •  •  o  o  B  o  e  . . . . . . . . . . . .  a  a  o  o  *  a  o  »  The E f f e c t o f Diamox on P h o t o s y n t h e s i s ^C0 14 C0  o  *  o  o  72  o  . . . . . .  2  F i x a t i o n i n the L i g h t . . . . . . . . .  2  F i x a t i o n i n t h e Dark  1  . . . . . . . . .  The E f f e c t o f Diamox on t h e H i l l R e a c t i o n  70  . . . .  72 72 __ 72 74  The E f f e c t o f Diamox on t h e L i g h t - i n d u c e d The I n t e r a c t i o n o f A n t i m y c i n A w i t h Diamox . . . .  79  The E f f e c t o f A n t i m y c i n A on t h e L i g h t i n d u c e d pH S h i f t . . . . . . . . . . . . . . . . .  83  The S t a b i l i t y o f Diamox  83  D X SOUS S ION  •  o  LITERATURE CITED PART I I I  «  *  *  o  o  a  . . . . . . . . . . . . . *  4  o  a  <  *  «  o  . . . . . . . . . .  e  *  *  «  a  o  o  . . .  INTRODUCTION  RESULTS  94  I n t e r a c t i o n o f Oxygen and I n o r g a n i c Carbon w i t h t h e Oxygen I n d u c t i o n T r a n s i e n t s i n Iridaea cordata  METHODS  84  . . . . .  97 .  . . . . . . . .  . . . . .  98 100 103  vii Page The C>2 P r o d u c t i o n I n d u c t i o n T r a n s i e n t s i n Iridaea cordata  103  The E f f e c t s o f O2 C o n c e n t r a t i o n and C. on t h e O2 P r o d u c t i o n T r a n s i e n t s . . . . . . . . .  105  The E f f e c t o f P r e v i o u s L i g h t - D a r k Regimes on (c)  113  The E f f e c t o f DCMU on 0  2  Production  Transients The Response o f t h e DISCUSSION  . . . . . . . .  LITERATURE CITED  115 Electrode  115 . . . . . . . .  118 126  L I S T OF  Results  TABLES  o f a s u r v e y f o r HCO^  uptake  The e f f e c t o f pH c h a n g e and C i c h a n g on r a t e o f p h o t o s y n t h e s i s i n I r i d a e a  ix  LIST OF FIGURES Figure Part  Page 1-A  1.  Change i n pC0 and C^ a g a i n s t t i m e f o r a 17.4 g sample o f Sargassum muticum 2  2.  3.  Part 4.  .  Change i n C^ and pH a g a i n s t time f o r a 13 g sample o f Sargassum muticum. Two r u n s s e p a r a t e d by a d a r k p e r i o d o f one hour  10  Change i n pC0~ and C-^ a g a i n s t t i m e where C^ i s c a l c u l a t e d from pH and pC02» f o r Sargassum muticum. (27 g)  11  Change i n t o t a l i n o r g a n i c c a r b o n (C^) and ppm C0 a g a i n s t time f o r a 4 g sample o f U l v a sp  29  Change i n C^ and ppm CO2 a g a i n s t time f o r a 27 g sample o f D e s m a r e s t i a munda. . . . . .  30  Change i n C^ and ppm CO2 a g a i n s t time i n U l v a f e n e s t r a t a showing dependence o f p h o t o s y n t h e t i c r a t e on C^ v a l u e r a t h e r t h a n pH o r ppm CO 2  32  Dependence o f r a t e o f p h o t o s y n t h e s i s on t o t a l i n o r g a n i c carbon f o r Ulva f e n e s t r a t a . . .  36  E f f e c t o f t o t a l i n o r g a n i c c a r b o n on r a t e o f p h o t o s y n t h e s i s by I r i d a e a c o r d a t a . . . . .  38  E f f e c t o f pH on r a t e o f p h o t o s y n t h e s i s Iridaea cordata  39  1-B  2  5. 6.  7. 8. 9. 10.  8  by  E f f e c t o f t o t a l i n o r g a n i c c a r b o n on r a t e o f p h o t o s y n t h e s i s by Sargassum muticum. ...  42  X  Page  Figure 11.  E f f e c t o f pH on r a t e o f p h o t o s y n t h e s i s by Sargassum muticum. . . . . . . . . .  43  12.  E f f e c t o f pH on r a t e o f p h o t o s y n t h e s i s i n U l v a f e n e s t r a t a a t t h r e e C\ v a l u e s  51  13.  The e f f e c t o f HCOl i o n c o n c e n t r a t i o n on r a t e o f p h o t o s y n t h e s i s a t f i v e pH v a l u e s i n Ulva f e n e s t r a t a . . . . . . . . . . . .  53  A system f o r m e a s u r i n g p h o t o s y n t h e s i s i n marine a l g a e by pCC>2 and measurement w i t h c o n t r o l o f pH and t e m p e r a t u r e . .  58  A system f o r m e a s u r i n g t o t a l i n o r g a n i c c a r b o n (C^) i n seawater by a c i d r e l e a s e and i n f i a - r e d gas a n a l y s i s . . . . . . .  61  Addendum  Appendix 14  15  Part I I 16.  17.  18 .  14 The e f f e c t o f 4 mM Diamox on CO2 f i x a t i o n by i s o l a t e d s p i n a c h c h l o r o p l a s t s : i n t h e l i g h t w i t h PPj_: i n t h e d a r k w i t h ATP . . . . . . . . . . The e f f e c t o f Diamox and s u l p h a n i l a m i d e on O2 p r o d u c t i o n by i s o l a t e d s p i n a c h and D u n a l i e l l a c h l o r o p l a s t s i n the presence o f potassium f e r r i c y a n i d e . . .  75  The e f f e c t o f pH 6.5 and time on Diamox i n h i b i t i o n o f 0 p r o d u c t i o n by s p i n a c h chloroplasts  77  The e f f e c t o f 4 mM Diamox on t h e l i g h t i n d u c e d pH s h i f t by i s o l a t e d s p i n a c h c h l o r o p l a s t s i n unbuffered sucrose medium  78  2  19.  73  xi Figure 20.  21.  Page The i n t e r a c t i o n o f i n c r e a s i n g Diamox c o n c e n t r a t i o n and 5 uM A n t i m y c i n A on 14cc>2 f i x a t i o n i n - s a t u r a t i n g l i g h t b y i s o l a t e d spinach c h l o r o p l a s t s  80  The i n t e r a c t i o n o f i n c r e a s i n g Diamox c o n c e n t r a t i o n and 5 uM A n t i m y c i n A on 1 CC>2 f i x a t i o n i n n o n - s a t u r a t i n g l i g h t by i s o l a t e d s p i n a c h c h l o r o p l a s t s . . 4  Part I I I 22.  Time c o u r s e f o r t y p i c a l p h o t o s y n t h e t i c O2 induction transients during the f i r s t minutes o f i l l u m i n a t i o n i n I r i d a e a cordata  104  23.  E f f e c t o f O2 on t h e r a t e o f s t e a d y - s t a t e photosynthesis i n Iridaea cordata .  106  24.  The e f f e c t o f h i g h and l o w O2 on (c) and (e) a t 50 m l C i p e r l i t r e  108  The e f f e c t o f h i g h and l o w 0 2 (e) a t 2 m l per l i t r e  109  25. 26. 27. 28.  on (c) and  The e f f e c t o f h i g h and l o w O2 o n (c) and (e) a t 80 ml C i p e r l i t r e The e f f e c t o f l o w C i w i t h t i m e on (c) a t h i g h and l o w 0 2  110 I l l  The e f f e c t o f t h e p r e c e d i n g d a r k p e r i o d on t h e h e i g h t o f (c)  29.  The e f f e c t o f DCMU on t h e (c) s p i k e  30.  The r e l a t i o n s h i p between e x t e r n a l ppm O2 and  the dark steady-state reading  114 116 H7  xii  ACKNOWLEDGEMENTS  I would l i k e t o e x p r e s s my g r a t i t u d e t o D r . Bruce Tregunna  f o r h i s d i r e c t i o n and encouragement d u r i n g t h e  c o u r s e o f t h e s e i n v e s t i g a t i o n s . Thanks a r e a l s o extended to D r s . J a n e t S t e i n , R.F. S c a g e l , G.H.N. Towers, and N.R. Bulley f o r h e l p f u l advice. Dr. N.J. A n t i a , F i s h e r i e s R e s e a r c h Board o f Canada supplied cultures of D u n a l i e l l a t e r t i o l e c t a .  Discussions  w i t h D r . R. Waygood, U n i v e r s i t y o f M a n i t o b a , and D r . W.E. V i d a v e r o f Simon F r a s e r U n i v e r s i t y were much a p p r e c i a t e d . Finally,  I would l i k e t o thank my husband  for his  c o n s t a n t s u p p o r t , w i t h o u t w h i c h t h i s work c o u l d n o t have been c o m p l e t e d . The a u t h o r was a r e c i p i e n t o f N a t i o n a l R e s e a r c h C o u n c i l o f Canada S c h o l a r s h i p s d u r i n g 1969-70,  and  1970-71.  to D r . Tregunna  1967-68,  1968-69,  R e s e a r c h was s u p p o r t e d by g r a n t s  from t h e N a t i o n a l R e s e a r c h C o u n c i l o f  Canada and t h e P r e s i d e n t ' s Committee on R e s e a r c h , The University of B r i t i s h  Columbia.  PART 1-A B i c a r b o n a t e I o n A s s i m i l a t i o n i n P h o t o s y n t h e s i s by Sargassum muticum.^"  T h i s a r t i c l e by E.A. Thomas ( J o l l i f f e ) and E.B. Tregunna appeared i n Canadian J o u r n a l of Botany: V o l . 46, 411-415 (1968), E.B. Tregunna s u p e r v i s e d the study.  2 INTRODUCTION  The a s s i m i l a t i o n o f HCO^ i o n d u r i n g  photosynthesis  by a q u a t i c p l a n t s has l o n g been a c o n t r o v e r s i a l t o p i c . Osterlind  (8) and Steemann N i e l s e n (11) have r e v i e w e d t h e  l i t e r a t u r e on HCO^ i o n a s s i m i l a t i o n by f r e s h w a t e r p l a n t s t o 1958.  R e c e n t l y , F e l f o l d y has s t u d i e d e x t e n s i v e l y t h e  c a r b o n s o u r c e s o f green p h y t o p l a n k t o n a l k a l i lakes (3).  i s o l a t e d from H u n g a r i a n  They have c o n c l u d e d  t h a t some phanaerogams  and a l g a e a r e a b l e t o t a k e up HCO^ i o n , w h i l e o t h e r s a r e n o t . I n c o n t r a s t t o t h e c o n s i d e r a b l e amount o f d a t a a v a i l a b l e c o n c e r n i n g HCO^ i o n a s s i m i l a t i o n b y f r e s h w a t e r  algae,  i n f o r m a t i o n about t h i s phenomenon i n m a r i n e a l g a e i s s c a n t y . This i s s u r p r i s i n g i n view o f the p o s s i b l e importance o f this  process. The  s o l u t i o n o f CO2 gas i n sea w a t e r e s t a b l i s h e s a n  e q u i l i b r i u m between f r e e C02* 2 H  follows.  C0  C 0  3 ' HCO^, and CO^ a s  (4, 16)  2  + H 0*— H C0 ^ 2  7  2  3  HCO~ + H " +  7  7 CO^ + 2 H  +  A r i s e i n pH w i l l s h i f t t h i s e q u i l i b r i u m t o t h e r i g h t , a f a l l i n pH s h i f t s i t t o t h e l e f t . are v i r t u a l l y  while  The e q u i l i b r i u m s h i f t s  instantaneous.  Normal sea w a t e r has a pH between 7.8 and 8.3. A t t h i s pH, HCO., i o n c o n s t i t u t e s most o f t h e i n o r g a n i c c a r b o n  3  (90%) under n a t u r a l c o n d i t i o n s ; o n l y 4% o f t h e c a r b o n i s i n t h e form o f f r e e CG^HCO^  Any marine p l a n t c a p a b l e o f u s i n g  i o n as w e l l as f r e e CG^/  would have a c o m p e t i t i v e  tage o v e r forms c a p a b l e o f u s i n g o n l y CO2/ for photosynthesis.  i f  advan-  i s limiting  Indeed, we would e x p e c t t o f i n d  marine  p l a n t s , p a r t i c u l a r l y the forms t h a t l i v e i n a h i g h l i g h t i n t e n s i t y , t o be c a p a b l e o f a s s i m i l a t i n g HCO^  ion directly.  The f i r s t i n v e s t i g a t i o n o f t h e a b i l i t y o f marine p h y t o p l a n k t o n t o a s s i m i l a t e HCO^ i n 1921  i o n was made by Moore e t a l . ,  ( 7 ) . He proposed t h a t any a l g a c a p a b l e o f r a i s i n g  t h e pH o f a s e a w a t e r sample t o pH 9.1 must be c a p a b l e o f a s s i m i l a t i n g HCO^  ion.  I n 1961, Hood and Park (5) s t u d i e d t h e c a r b o n s o u r c e s of the marine phytoplankton N i t z c h i a c l o s t e r i u m , s  s p . , and C h l o r e l l a s p . Nielsen  Platymonas  Watt and Paasche(18) and Steemann  (12) , however, p o i n t o u t the e r r o r o f t h e a s s u m p t i o n  o f Hood and P a r k t h a t t h e e q u i l i b r a t i o n t i m e o f C 0  2  and  HCO3 i s v e r y s l o w . The o n l y o t h e r i n f o r m a t i o n a v a i l a b l e c o n c e r n i n g  HCO^  i o n u t i l i z a t i o n by m a r i n e p h y t o p l a n k t o n has been p r e s e n t e d by Paasche, w o r k i n g w i t h C o c c o l i t h u s h u x l e y i .  The  photo-  s y n t h e t i c i m p l i c a t i o n s o f t h i s work a r e d i s c u s s e d by Steemann Nielsen  (13) .  I t was demonstrated t h a t o n l y t h o s e c o c c o l i -  t h o p h o r i d s w h i c h d e p o s i t CaCO^ use HCO^ forms a r e dependent on f r e e C0 . 0  ion, while  naked  Steemann N i e l s e n proposed  4  t h a t d e p o s i t i o n o f CaCO^ i n c o c c o l i t h f o r m a t i o n  makes p o s s i b l e  the use o f HCO^ i o n i n p h o t o s y n t h e s i s . Tseng and Sweeney (15) s t u d i e d t h e c a r b o n s o u r c e s of Gelidium  c a r t i l a g i n e u m , a p i n n a t e l y branched marine  rhodophyte.  They found t h a t t h e r a t e o f p h o t o s y n t h e s i s  creased with decreasing pC0  2  and d e c r e a s i n g  pH and, t h e r e f o r e , w i t h  HCO-j i o n .  in-  increasing  The r a t e a l s o i n c r e a s e d when  pCC>2 was i n c r e a s e d w i t h c o n s t a n t  HCO^ i o n .  They c o n c l u d e d  t h a t f o r G. c a r t i l a g i n e u m , CC^ and n o t HCO^ i s t h e f a c t o r l i m i t i n g photosynthesis.  T h e i r r e s u l t s , however, do n o t  e x c l u d e t h e p o s s i b i l i t y o f some HCO^ i o n a s s i m i l a t i o n . The  purpose o f t h i s i n v e s t i g a t i o n was t o d i s c o v e r  whether o r n o t Sargassum muticum (9) c o u l d u t i l i z e HCO^ i o n during photosynthesis.  A t t h e same t i m e , r e f e r e n c e  i s made  t o t h e i m p l i c a t i o n s o f t h e p r e s e n c e o f i n o r g a n i c i o n pumps, i n measuring photosynthesis  i n a c l o s e d aqueous s a l i n e  system.  MATERIAL AND METHODS  Samples o f Sargassum muticum were c o l l e c t e d i n t h e i n t e r t i d a l zone a t B r o c k t o n P o i n t , S t a n l e y P a r k , Vancouver, B r i t i s h C o l u m b i a , 1966 and 1967 from e a r l y May t o mid-August. The  a l g a was washed w i t h f i l t e r e d s e a w a t e r , and s t o r e d i n  s e m i - d a r k n e s s a t 5°C i n g l a s s a q u a r i a f o r n o t more t h a n three days.  5 The  two methods t h a t were used t o measure t h e concen-  t r a t i o n o f i n o r g a n i c carbon a r e d e s c r i b e d i n a previous paper ( 1 7 ) . described  A modified v e r s i o n o f the techniques i s  here.  Samples were  placed  weighing in a  anchored t o s p r e a d The  from  1000 ml  15  to  30 g  glass reaction  fresh  weight  chamber,  and  t h e m a t e r i a l f o r optimum i l l u m i n a t i o n .  chamber was f i l l e d w i t h p r e c o o l e d , f i l t e r e d s e a w a t e r  and p l a c e d i n a w a t e r b a t h , and 16°C.  2  between 12°C  The s e a l e d chamber was c o n n e c t e d i n s e r i e s w i t h  a Beckman 215 I n f r a r e d the p C 0  w h i c h was m a i n t a i n e d  Gas A n a l y s e r  (IRGA) w h i c h measured  o f t h e a i r , and a s m a l l pump w h i c h c i r c u l a t e d t h e  a i r i n t h e system a t 1 l . / m i n v i a a s c i n t e r e d g l a s s w i t h i n the r e a c t i o n f l a s k .  bubbler  A t t h e same t i m e , w a t e r was  c i r c u l a t e d a t 1 l . / m i n v i a t h e r e a c t i o n chamber t h r o u g h a w a t e r pump and a sampling  chamber c l o s e d b y a serum  t o a l l o w w a t e r samples t o be removed by s y r i n g e .  stopper  Temperature  and pH were a l s o measured as d e s c r i b e d  (17) .  i l l u m i n a t e d f r o m one s i d e by a G e n e r a l  E l e c t r i c 300 w a t t  "Cool Beam" lamp, a t 700 f t c .  The a l g a was  Darkness was p r o v i d e d by  t u r n i n g o f f t h e l i g h t and c o v e r i n g t h e a p p a r a t u s w i t h a black  cloth. The  system was m o n i t o r e d w i t h t h e IRGA and t h e pH  meter u n t i l p h o t o s y n t h e s i s pC0  2  and pH changes had r e d u c e d t h e  t o a r e a d i n g j u s t below 100 ppm.  I n some l a t e r  e x p e r i m e n t s (see F i g u r e 1) pH was a d j u s t e d w i t h s m a l l  6 i n j e c t i o n s o f c o n c e n t r a t e d NaOH i n o r d e r t o reduce pCX^ t o about 100 ppm. I n some o f t h e e x p e r i m e n t s , measurements o f the pH, pC^,  t e m p e r a t u r e and s a l i n i t y were used w i t h the t a b l e s  g i v e n by S t r i c k l a n d and P a r s o n s (14) t o c a l c u l a t e inorganic carbon.  total  From changes i n t h i s p a r a m e t e r ,  changes  i n t o t a l i n o r g a n i c c a r b o n were c a l c u l a t e d t o measure p h o t o s y n t h e s i s i n the pH range o f 7.8 t o 8.6, w h i l e a i r was c i r c u l a t i n g through the water. PCO2(atmospheres) Carbonate a l k a l i n i t y = F  P  (tabulated)  T o t a l C02(ml) = Carbonate a l k a l i n i t y x F  T  (tabulated).  A t 10 t o 20 m i n u t e i n t e r v a l s , 1 m l w a t e r samples were w i t h d r a w n from t h e s a m p l i n g chamber and s t o r e d i n t h e refrigerater.  A t the time t h a t each sample was removed, pH,  t e m p e r a t u r e , and PCO2 were r e c o r d e d .  A t about pH 10, t h e  system c o n t a i n i n g t h e a l g a l sample was d i s c o n n e c t e d from the IRGA.  Now, the s t o r e d w a t e r samples were i n j e c t e d i n t o an  a c i d b a t h v i a a n o t h e r serum s t o p p e r . o f a c l o s e d system i n c l u d i n g t h e IRGA.  T h i s a c i d b a t h was p a r t The amount o f c a r b o n  (C^) t h a t was r e l e a s e d from t h e sea w a t e r was measured as a change o f PCO2 i n the c l o s e d system.  The change i n t h e  c a r b o n i n t h e sea w a t e r c o n t a i n i n g the a l g a e was c a l c u l a t e d from t h e change i n 1 ml samples.  The volume o f t h e a c i d  7 r e l e a s e system i n c l u d i n g the sample chambers o f the IRGA, was  350  ml. Therefore,  i n o r g a n i c carbon content could  be  calculated; (C)  = .35 1. x ppm  1  CQ  0 z  r e l e a s e d x w a t e r volume i n r e a c t i o n f l a s k (ml)  w i t h c o r r e c t i o n s f o r t e m p e r a t u r e , p r e s s u r e , and of  solubility  C0 . o  RESULTS  The  d a t a f o r F i g u r e s 1 and  modified a c i d release technique. were c a l c u l a t e d from pH and p C 0  2 were o b t a i n e d by  The  data f o r Figure 3  o f the sea w a t e r .  2  F i g u r e 1 shows changes i n p C 0  2  and  a g a i n s t time  w i t h measurements t a k e n a t 10 m i n u t e i n t e r v a l s . sample the pH was described.  The  a d j u s t e d i n i t i a l l y t o 8.97  rate of  a s s i m i l a t i o n was  a p e r i o d o f 70 m i n u t e s w h i l e p C 0 the pH s h i f t e d from pH 8.97 The a t e d 39.5%  17.4  the  2  t o pH  In t h i s  w i t h NaOH. as constant  during  f e l l from 62 t o 13 ppm  and  9.9.  g f r e s h w e i g h t sample o f S. muticum a s s i m i l -  of t h e c a r b o n o r i g i n a l l y p r e s e n t i n the sea w a t e r  a t a r a t e o f 21 yg C0 /min/g f r e s h w e i g h t . 2  the p e r i o d was  n o t p r o p o r t i o n a l t o pC0~  uptake d u r i n g  below 100  ppm.  Figure  1  Change i n p C 0 17.4  2  and C  against time f o r a  g sample o f Sargassum  muticum  9 Other samples under t h e same c o n d i t i o n s a l s o had c o n s t a n t b u t l o w e r r a t e s o f a s s i m i l a t i o n ; 8, 1 1 , and 14.5 ug C ^ / m i n / g fresh weight. F i g u r e 2 shows a second t y p e o f c u r v e f o r C w h i c h was o c c a s i o n a l l y o b s e r v e d .  uptake,  Two r u n s s e p a r a t e d by a  d a r k p e r i o d o f about one hour f o r one 13 g sample o f S. muticum are  shown.  There was no a r t i f i c i a l  t h i s sample.  a d j u s t m e n t o f pH f o r  A t t h e end o f t h e second t r i a l t h e a l g a had  r a i s e d t h e pH from an i n i t i a l ph 8.21 a t t i m e z e r o , t o pH 9.57.  F i g u r e 2 shows t h e o b s e r v e d change o f pH w i t h t i m e .  A l t h o u g h t h e upper l i n e second  ( s o l i d l i n e ; A=C) i s s t r a i g h t , the  (broken l i n e ; A=C^) shows a tendency towards an i n -  creasing rate o f photosynthesis.  Over a t o t a l o f two and one-  h a l f h o u r s , t h e p l a n t a s s i m i l a t e d 26% o f t h e p r e s e n t i n t h e system.  initially  The average r a t e s were 21.3 and 16.7  ug -COj/min/g. F i g u r e 3 shows a r e c o r d o f t o t a l  calculated  pH and p C ^ p l o t t e d f o r comparison a g a i n s t e x p e r i m e n t a l p e r i o d was about 45 m i n u t e s . 18 m i n u t e s , PCO2 changed  pH.  from  The  During the i n i t i a l  54% from 318 ppm t o 14 5 ppm w h i l e  pH s h i f t e d from 7.8 t o 8.12. showed no change d u r i n g t h i s  The c a l c u l a t e d C , however, interval.  A f t e r t h e 18 minute l a g phase,  assimilation  began.  The r a t e o f change o f PCO2 i n c r e a s e d , and PCO2 f e l l t o < 5 ppm at  pH 8.6.  As i n F i g u r e s 1 and 2,  u p t a k e was l i n e a r as  PCO2 f e l l below 100 ppm. F i g u r e 3 g i v e s an example o f how  Figure 2  Change i n C  and pH a g a i n s t t i m e f o r a  13 g sample o f Sargassum muticum  Figure 3  Change i n pCC^ and C^ a g a i n s t time where  C  i s c a l c u l a t e d from pH and pCC^, f o r Sargassum muticum  (27 g)  11  lATd'd  photosynthesis  may  be measured by c a l c u l a t i o n s o f  carbonate a l k a l i n i t y from p C 0 to l e s s than 5 ppm, system.  The  and  2  A f t e r the p C 0  water samples were removed from  samples were analysed  uptake continued  pH.  the  f o r C\  the  by a c i d r e l e a s e .  f o r a f u r t h e r 35 minutes a t an  r a t e of 8 ug C0 /min/g f r e s h  fell  2  average  wt.  2  DISCUSSION  In a l l p l a n t s t h a t have been s t u d i e d , the r a t e of a s s i m i l a t i o n i n photosynthesis 100  ppm,  and  i n most p l a n t s C 0  concentrations. only C0  2  gas,  investigation  off  a constant  The  r a t e was  ppm  C0 .  The  2  r a t e below 100 the pH  ppm  C0 ,  this  2  while pC0  2  continues itself  a s s i m i l a t i o n of carbon when p C 0  (after Figure  3) r e p r e s e n t s  pH  i o n forms 60% o f C ) .  S. muticum must take up HCO.,  2  the extreme o f  four times as g r e a t as t h a t a t a c i d pH i o n , however, was  CO^  using  falls  rises.  M a i n l y HCO^ 9.1,  r e s u l t s of  however, show t h a t C^ a s s i m i l a t i o n  continued  negligible  higher  a s i m i l a r t r e n d would be expected i n photo-  v e r y r a p i d l y and The  becomes l i m i t i n g a t f a r  2  2  below  2  I f Sargassum muticum were c a p a b l e o f  s y n t h e t i c r a t e below 100  at  i s p r o p o r t i o n a l to p C 0  C0  was this.  (1).  a v a i l a b l e as a s u b s t r a t e I t was  concluded  ion d i r e c t l y during  (At  that  photo-  synthesis.  Brown and Tregunna  (1) have s t u d i e d the  C0  compensation v a l u e s f o r a number of a l g a e , i n c l u d i n g muticum.  By d e f i n i t i o n , the C 0  when r e s p i r a t o r y C 0  2  uptake i n p h o t o s y n t h e s i s .  balances  Under these c o n d i t i o n s p C 0  w i l l remain c o n s t a n t , and net p h o t o s y n t h e s i s can not if  free C0  2  S.  compensation p o i n t occurs  p r o d u c t i o n by the p l a n t j u s t  2  2  i s rate-limiting.  I f , however, HCO^  2  occur  i o n used  d i r e c t l y , net p h o t o s y n t h e s i s c o u l d o c c u r below the CC>  2  compensation l e v e l f o r the p l a n t .  The p C 0  2  a t compensation  f o r Sargassum muticum i s r a t h e r h i g h , 75 ppm, much of the net 100  ppm  C0 , 2  the p l a n t .  was  a s s i m i l a t i o n which was a l s o below the C 0  2  observed  below  compensation l e v e l f o r  Steemann N i e l s e n (11) has  as evidence o f HCO^  and t h e r e f o r e ,  suggested  this condition  ion assimilation.  Brown and Tregunna (1) have a l s o i n v e s t i g a t e d the r a t e o f p h o t o s y n t h e s i s f o r a number o f a l g a e , i n c l u d i n g S. muticum a t pH 4.5 99%)  to 5.5  when v i r t u a l l y a l l the  i s i n the form o f C 0  2  r a t e of p h o t o s y n t h e s i s was The r a t e v a l u e s we  gas. 1.9  present  At pC0  of 300  2  (93 to  ppm,  the  ± .2 ug C0 /min/g f r e s h 2  have found, when C 0  t o t a l C^, were 4 to 11 times g r e a t e r .  2  weight.  i s l e s s than 3% o f T h i s may  be e x p l a i n e d  by the p o s s i b i l i t y of HCO.J i o n a s s i m i l a t i o n i n our s t u d i e s . In a p r e v i o u s paper  (17) I r i d a e a c o r d a t a showed net  take i n the absence o f f r e e C 0  2  a t pH  8.5  t o 9.6.  as f o r Sargassum muticum, were much h i g h e r than a c i d pH where f r e e C 0 for  photosynthesis.  2  was  upThe  rates,  those a t  the o n l y a v a i l a b l e s u b s t r a t e  14 Steeman N i e l s e n (10) has proposed a c c e p t e d mechanism  o f HCO^ i o n u p t a k e .  the p r e s e n t l y The p l a n t t a k e s up  HCO^ i o n , degrades i t t o CO 2 and OH , and then e x c r e t e s t h e OH  i o n . T h i s has been w e l l demonstrated  f o r some phanerogams.  Such a p r o c e s s w i l l r a i s e t h e pH o f t h e s u r r o u n d i n g medium. The C\ r e c o r d f o r t h e l a g phase b e f o r e t h e o n s e t o f p h o t o s y n t h e s i s i n F i g u r e 3, shows t h a t S. muticum i s a b l e t o change t h e pH i n d e p e n d e n t l y o f c a r b o n a s s i m i l a t i o n . a b i l i t y has been shown by I r i d a e a c o r d a t a (16) .  A similar  These pH  s h i f t s occurred only i n the l i g h t . I n f o r m a t i o n has been a c c u m u l a t i n g r e c e n t l y c o n c e r n i n g l i g h t a c t i v a t e d i o n pumps i n a l g a e (eg. 2, 6 ) .  The pH changes  i n d u c e d by Sargassum muticum and I r i d a e a c o r d a t a a r e i n t e r p r e t e d a s t h e r e s u l t o f an i n o r g a n i c i o n pump s e p a r a t e from HCO^ i o n u p t a k e .  pH change t o 9.0 o r above, t h e r e f o r e , i s  n o t i n i t s e l f s u f f i c i e n t e v i d e n c e f o r HCO^ i o n a s s i m i l a t i o n , as Moore has s u g g e s t e d ( 7 ) . Both t h e a c i d r e l e a s e t e c h n i q u e and t h e pH-pC02 method for  calculation of C  have t h e advantage t h a t t h e y measure  t o t a l c a r b o n , r a t h e r t h a n depending  on pH a l o n e t o e s t i m a t e  PCO2/ w h i c h a t normal pH i n s e a w a t e r , c o n s t i t u t e s o n l y a very small p a r t o f the t o t a l carbon.  I f pH changes o c c u r  a p a r t from t h a t due t o a b s t r a c t i o n o f c a r b o n i n p h o t o s y n t h e s i s , or  i f some form o f c a r b o n o t h e r t h a n CO2 i s t a k e n up, measure-  ments o f p h o t o s y n t h e s i s based on pH become m e a n i n g l e s s .  15 I n summary, S a r g a s s u m m u t i c u m shows n e t  assimil-  ation: (a) a t a c o n s t a n t r a t e a s pCC^ f a l l s b e l o w 100 ppm (b)  i n t h e a b s e n c e o f f r e e CC^, a n d  (c) b e l o w CC>2 c o m p e n s a t i o n l e v e l Also,the higher  f o r the plant.  r a t e s a t t h e s e l o w pCC^ a n d h i g h  pH v a l u e s a r e much  t h a n a t pH 4.5 when CC^ a l o n e i s a v a i l a b l e f o r p h o t o -  synthesis.  We c o n c l u d e t h a t S. m u t i c u m a s s i m i l a t e s HCO^ i o n  i n p h o t o s y n t h e s i s a n d we s u g g e s t t h a t CC^ g a s may b e o f minor importance as a s u b s t r a t e normal marine The Iridaea  f o rphotosynthesis  conditions.  pH c h a n g e s i n d u c e d b y S a r g a s s u m m u t i c u m a n d  (17) d u r i n g  photosynthesis are interpreted  c o m p o s i t e o f OH-ion e x c r e t i o n d u r i n g and  under  some o t h e r i n o r g a n i c  i o n pump.  t o be a  HCO^ i o n u p t a k e ,  LITERATURE CITED  Brown, D.L., and Tregunna, E.B., 1967. I n h i b i t i o n o f r e s p i r a t i o n d u r i n g p h o t o s y n t h e s i s by some a l g a e . Can. J . B o t . , 45: 1135-43. Dodd, W.A., P i t m a n , M.G., and West, K.R., 1965. N a and K+ t r a n s p o r t i n t h e m a r i n e a l g a Chaetomorpha d a r w i n i i . A u s t . J . B i o l . S c i . 19: 341-54. +  F e l f o l d y , L.J.M., 1960. E x p e r i m e n t s o n t h e c a r b o n a t e a s s i m i l a t i o n o f some u n i c e l l u l a r a l g a e by R u t t n e r ' s c o n d u c t o m e t r i c method. A c t a B i o l . A c a d . S c i . Hung., X I : 67-75. Harvey, H.W., 1963. The c h e m i s t r y and f e r t i l i t y o f seawaters. 2nd e d . Cambridge U n i v e r s i t y P r e s s . Cambridge. 234 pp. Hood, D.W. and P a r k , K., 1962. B i c a r b o n a t e u t i l i z a t i o n by m a r i n e p h y t o p l a n k t o n i n p h o t o s y n t h e s i s , P h y s i o l . P l a n t . , 15: 273-282. Hope, A.B., Simpson, A., and W a l k e r , N.A., 1966. The e f f e c t o f C l ~ from c e l l s o f N i t e l l a and C h a r a . A u s t . J . B i o l . S c i . 19: 355-62. Moore, B., W h i t l e y , E., and Webster, T.A., 1921. Photos y n t h e s i s i n m a r i n e a l g a e . P r o c . Roy. S o c . Lond. S e r . B., 92: 51-60. O s t e r l i n d , S., 1949. Growth c o n d i t i o n s o f t h e a l g a Scenedesmus q u a d r i c a u d a w i t h s p e c i a l r e f e r e n c e t o the i n o r g a n i c c a r b o n s o u r c e s . Symp. B o t . U p s a l . 10: 1-141. S c a g e l , R.F., 1967. Guide t o common seaweeds o f B r i t i s h C o l u m b i a , B.C. P r o v . Museum, Handbook No. 27. The Queen's P r i n t e r , B.C. 33 0 pp. Steemann N i e l s e n , E., 1951. P a s s i v e and a c t i v e i o n t r a n s p o r t d u r i n g photosynthesis i n water p l a n t s . P h y s i o l . P l a n t . 4: 189-198. Steemann N i e l s e n , E., 1960 . Uptake o f CC»2 by t h e p l a n t . I n : E n c y c l o p e d i a o f p l a n t p h y s i o l o g y , V o l . V. The a s s i m i l a t i o n o f c a r b o n d i o x i d e , pp. 70-84. E d . W. R u h l a n d . S p r i n g e r - V e r l a g , B e r l i n . G o t t i n g e n . Heidelberg.  17 12.  Steemann N i e l s e n , E., 1963. On b i c a r b o n a t e u t i l i z a t i o n by marine p h y t o p l a n k t o n i n p h o t o s y n t h e s i s , w i t h a n o t e on carbarn i n o c a r b o x y l i c a c i d s as a c a r b o n s o u r c e . P h y s i o l . P l a n t . 16: 466-69.  13.  S t e e m a n n _ N i e l s e n , E., 1966. The u p t a k e o f f r e e C 0 and HCO-j d u r i n g p h o t o s y n t h e s i s o f p l a n k t o n a l g a e w i t h s p e c i a l r e f e r e n c e t o the c o c c o l i t h o p h o r i d Coccolithus huxleyi. P h y s i o l . P l a n t . 19: 232-40"!  14.  S t r i c k l a n d , J.D.H., and P a r s o n s , T.R., 1965. A manual o f sea w a t e r a n a l y s i s . B u l l . No. 125, F i s h e r i e s R e s e a r c h Board o f Canada, Ottawa. 2nd ed. pp. 29, 35, 199, 200.  15.  Tseng, C.K., and Sweeney, B.M., 1946. Physiological studies of Gelidium c a r t i l a g i n e u m . I . Photos y n t h e s i s w i t h s p e c i a l r e f e r e n c e to the CO2 f a c t o r , Amer. J . B o t . 33: 706-15.  16.  Svedrup, H.U., Johnson, M.W., F l e m i n g , R.H., 1942. Oceans. P r e n t i c e - H a l l I n c . , Englewood C l i f f s , N.J. 1087 pp.  17.  Tregunna, E.B., and Thomas, E. Ann, 1967. Measurement o f i n o r g a n i c c a r b o n i n sea w a t e r by two methods o f p C 0 and pH a n a l y s i s . Can. J . B o t . 46: 481-85.  2  The  2  18.  W a t t , W.D., and Paasche, E., 1963. An i n v e s t i g a t i o n o f the c o n d i t i o n s f o r d i s t i n g u i s h i n g between C 0 and b i c a r b o n a t e u t i l i z a t i o n by a l g a e a c c o r d i n g to the methods o f Hood and P a r k . P h y s i o l . P l a n t . 16; 674-81. 2  PART  1-B  S t u d i e s on B i c a r b o n a t e i o n Uptake D u r i n g P h o t o s y n t h e s i s r n B e n t h i c Marine  Algae.  2  T h i s a r t i c l e by E.A. (Thomas) J o l l i f f e and E.B. Tregunna appeared i n P h y c o l o g i a , V o l . 9: 293-303. (1970) E.B. Tregunna s u p e r v i s e d the study.  19 INTRODUCTION  I n p r e v i o u s papers ( 8 , 9) i t has been r e p o r t e d the m a r i n e a l g a e I r i d a e a c o r d a t a  that  (Rhodophyta) and Sargassum  muticum (Phaeophyta) use HCO^ i o n as w e l l as f r e e C 0 g a s , 2  as a s u b s t r a t e f o r p h o t o s y n t h e s i s . have r e p o r t e d t h a t U l v a p e r t u s a  Ikemori  and N i s h i d a (2)  (Chlorophyta)  a l s o uses  HCO^ i o n , and Raven (5) has s t u d i e d t h e mechanism o f HCO^ i o n uptake i n Hydrodictyon  africanum  (Chlorophyta).  o f HCO^ i o n i s , t h e r e f o r e , p r o b a b l y  The u p t a k e  not confined to only a  few m a r i n e a l g a e , and i t seemed a p p r o p r i a t e t o b e g i n a s u r v e y o f some b e n t h i c a l g a e common t o Washington and B r i t i s h C o l u m b i a , f o r HCO^ i o n u p t a k e . The v a r i o u s s p e c i e s o f i n o r g a n i c c a r b o n a r e p r e s e n t i n sea water a t v a s t l y d i f f e r e n t c o n c e n t r a t i o n s .  Seawater  f r o m c o a s t a l a r e a s , u n d i l u t e d by t h e i n f l u x o f l a r g e r i v e r s , has a pH o f 8.0 t o 8.2. C gas  ( a l l s p e c i e s o f CO,,) p r e s e n t , i s e x p r e s s e d p e r 1 water, seawater  approximately pH,  I f the t o t a l inorganic carbon,  50 ml  C0  2  from  such  gas p e r 1.  as ml o f C 0  areas  2  contains  A t the p r e v a i l i n g  about 90% o f t h i s c a r b o n i s p r e s e n t as HCO^ i o n .  o f t h e r e m a i n d e r , about 6%, o c c u r s as CO^ i o n .  Most  As t h e pH  s h i f t s towards 9.0 c o n c e n t r a t i o n o f CO^ i o n i n c r e a s e s and becomes 50% o f C  a t pH 9.2 ( 7 ) .  be l e s s t h a n 1% a t pH 8.5.  At equilibrium, C0  The s o l u t i o n o f C 0  2  2  should  gas i s slow  compared t o t h e i o n i c s h i f t s o f H C0_ s p e c i e s , and t h i s 9  20 problem i s r e s p o n s i b l e f o r o b s e r v e d v a l u e s o f C 0 o f t e n d i f f e r from e q u i l i b r i u m .  adequate.  which  The c o n c e n t r a t i o n o f C 0  gas i n a i r i s about 0.03% o r 300 ppm, and C 0 for photosynthesis  2  i n l a n d p l a n t s wherever  2  2  i s limiting  light i s  F o r m a r i n e p l a n t s a b l e t o use HC0  3  ion,the  avail-  a b l e c a r b o n c o n c e n t r a t i o n i s upwards o f 45,000 ppm, o r 4.5% a t pH 8.0.  The c o n c e n t r a t i o n o f C 0  2  gas i n s e a w a t e r  i s 300 t o 320 ppm, b u t may v a r y w i t h t h e source o f w a t e r as h i g h as 500 ppm.  The c o n c e n t r a t i o n o f C 0  n o t i n e q u i l i b r i u m w i t h t h e atmosphere ( 7 ) .  2  gas i s o f t e n Photosynthesis  i s s a t u r a t e d i n l a n d p l a n t s a t 0.1% o r 1000 ppm, and growth i s i n h i b i t e d a t 5% CC»  2  (4) .  Because o f t h e s e  differ-  ences i n form and c o n c e n t r a t i o n o f a v a i l a b l e s u b s t r a t e f o r photosynthesis  i n marine c o n t r a s t e d w i t h land p l a n t s , i t  was o f i n t e r e s t t o i n v e s t i g a t e t h e f r e q u e n c y  of occurrence  o f HCO^ i o n a s s i m i l a t i o n among some m a r i n e a l g a e , and t o s t u d y t h e e f f e c t s o f HCO^ i o n , pH, and p C 0 , on t h e r a t e 2  of photosynthesis  i n a l g a e showing a dependence on HCO^ i o n  as a s u b s t r a t e f o r photosynthesis„  MATERIALS AND METHODS  A s u r v e y o f HCO^ i o n u p t a k e by b e n t h i c m a r i n e a l g a e was c o n d u c t e d a t t h e U n i v e r s i t y o f Washington, F r i d a y Harbor L a b o r a t o r i e s , San Juan I s l a n d , Washington, U.S.A.,  21 i n A p r i l and May o f 1968.  Plant material  (6) was c o l l e c t e d  i n t h e i n t e r t i d a l , near t h e l a b o r a t o r y a t " C o l l i n ' s Cove," and  a t "Deadman's Bay," San Juan I s l a n d .  The p l a n t s were  h e l d i n t h e l a b o r a t o r y a t 11° i n P l e x i g l a s s t a n k s , w i t h a constant  f l o w o f seawater pumped from t h e h a r b o u r .  t e m p e r a t u r e a t t h a t time was 9° t o 10°.  Seawater  A l l m a t e r i a l was  used w i t h i n t h r e e days o f c o l l e c t i o n . The f o l l o w i n g p l a n t s were t e s t e d :  A l a r i a s p . , 10  cm s e c t i o n s w e i g h i n g 7 t o 10 g ( f r e s h w e i g h t ) were c u t from young t h a l l i  near t h e t r a n s i t i o n zone:  Costaria costata,  10 cm s e c t i o n s , w e i g h i n g 6 t o 12 g were c u t as f o r A l a r i a : D e s m a r e s t i a munda, s e v e r a l f r o n d s 22 t o 28 g: 3 t o 4 g:  10 t o 15 cm l o n g , w e i g h i n g  Enteromorpha s p . samples 10 cm w i d e , w e i g h i n g Fucus s p . , a 12 cm f r o n d w e i g h i n g 12 g:  Laminaria  s a c c h a r i n a , a 10 by 12 cm p i e c e c u t n e a r t h e t r a n s i t i o n zone:  Nereocystis  luetkeana,  4 o r 5 v e r y young  (2.5 t o 3 cm wide) w e i g h i n g 8 t o 12 g:  blades,  Porphyra s c h i z o p h y l l a ,  5 t o 8 g samples: U l v a s p . , s m a l l f r o n d s o r p i e c e s o f f r o n d s , w e i g h i n g 3 t o 4 g. The t e c h n i q u e was a m o d i f i e d previously  version of that  ( 9 ) . P l a n t m a t e r i a l was p l a c e d  described  ina 1 litre  glass  r e a c t i o n f l a s k , and anchored f o r optimum i l l u m i n a t i o n by t y i n g t h e sample t o a r e c t a n g u l a r g l a s s frame.  Filtered  s e a w a t e r from t h e l a b o r a t o r y s u p p l y was used f o r t h e e x p e r i ments.  The p l a n t chamber was m a i n t a i n e d a t 9° t o 12° i n  an i c e - c o o l e d w a t e r b a t h .  A 3 00 w a t t G e n e r a l E l e c t r i c  "Cool  Beam" lamp i l l u m i n a t e d the p l a n t a t 2000 f t c a Gossen T r i l u x f o o t - c a n d l e meter) from one chamber was  (measured w i t h  side.  c o n n e c t e d i n s e r i e s w i t h a Beckman 215  r e d CC»2 Gas A n a l y s e r  (IRGA) and  The  sealed  infra-  a s m a l l a i r pump w h i c h  c i r c u l a t e d the a i r a t 1 l . / m i n , v i a an a i r s t o n e w i t h i n reaction flask.  Simultaneously  w a t e r was  the  pumped a t 1 l . / m i n  from the p l a n t chamber p a s t a c o m b i n a t i o n pH e l e c t r o d e ,  a  s a m p l i n g chamber c l o s e d w i t h a serum s t o p p e r to p e r m i t sample r e m o v a l by s y r i n g e , t h r o u g h a w a t e r pump and the p l a n t chamber. t e m p e r a t u r e was  The  pH was  back t o  monitored c o n s t a n t l y  measured w i t h a s t a n d a r d  taper  and  joint  centi-  grade thermometer i n s e r t e d i n the l i d o f the chamber. The  concentration  t o l e s s t h a n 25 ppm;  was  1 ml w a t e r samples were w i t h d r a w n a t  10 t o 15 m i n u t e i n t e r v a l s and each s a m p l i n g t i m e , pH, Each p l a n t was  m o n i t o r e d u n t i l i t dropped  stored i n a r e f r i g e r a t o r .  PCO2/ and  tested 3  (C^)  described  e x p r e s s e d as ml CO2 (9).  s i d e arm  t e m p e r a t u r e were r e c o r d e d .  times.  Water samples were a n a l y s e d carbon  for total  was  used as an a c i d r e l e a s e chamber.  readings  a  a t the base j u s t above the s c i n t e r e d d i s c  s i d e arm  was  T h i s column  contained  c l o s e d w i t h a r u b b e r serum s t o p p e r  f o r c o n v e n i e n t sample i n j e c t i o n . increased  previously  A 20 cm g l a s s chromatography column w i t h  cm  The  inorganic  p e r l i t r e w a t e r , as  2.5  IN HC1.  At  Use  o f t h i s column  the e f f i c i e n c y o f m i x i n g i n the system and CC>2 reached e q u i l i b r i u m i n 3 to 5 minutes i n s t e a d  of  23 15 t o 20 minutes, as was f o r m e r l y the case ( 9 ) . A t a l l times a s h o r t g l a s s tube f i l l e d w i t h MgClO^ was  p l a c e d i n the gas f l o w system j u s t b e f o r e the IRGA t o  p r e v e n t m i c r o d r o p l e t s o f seawater  o r a c i d from e n t e r i n g the  i n s t r u m e n t and damaging the sample t u b e s .  Some e t c h i n g o f  the l a t t e r by seawater has been o b s e r v e d .  Any measurement  e r r o r due to i n f r a r e d a b s o r p t i o n by water was a l s o a v o i d e d . These s t u d i e s were c o n t i n u e d a t t h e U n i v e r s i t y o f B r i t i s h Columbia, Vancouver, B r i t i s h Columbia. c o l l e c t e d from A p r i l t o mid-August  P l a n t s were  i n the i n t e r t i d a l  zone  a t Brockton P o i n t , S t a n l e y Park, Vancouver, i n 1968 and 1969. The f o l l o w i n g m a t e r i a l was s t o r e d a t 9° i n p l e x i g l a s s tanks i n a c o n t r o l l e d environment room a t the U n i v e r s i t y , and used w i t h i n two days o f c o l l e c t i o n . G i g a r t i n a s p . ( c r i s t a t a ? ) was added t o the survey list,  11 t o 12 g samples: Sargassum muticum and I r i d a e a c o r d a t a which have  a l r e a d y been r e p o r t e d t o use HCO^ i o n s (8,9) U l v a f e n e s t r a t a samples weighing 3 to 5 g, were a l s o examined. S t u d i e s o f the e f f e c t o f HCO^ i o n c o n c e n t r a t i o n on the r a t e o f p h o t o s y n t h e s i s were conducted w i t h U l v a f e n e s t r a t a , Sargassum  muticum, and I r i d a e a c o r d a t a .  I n i t i a l l y , measurements made w i t h U. f e n e s t r a t a were done as above, m o n i t o r i n g b o t h CC»2 and C^. in April  from the west c o a s t o f Vancouver  Seawater  collected  I s l a n d was used i n  24 t h e s e e x p e r i m e n t s ; w a t e r from t h i s s o u r c e i s c l e a n e r and has a h i g h e r s a l i n i t y t h a n t h a t from B u r r a r d I n l e t .  When  pCC»2 f e l l below 15 ppm and pH r e a c h e d 9.0 t o 9.5, t h e pH o f the  e x p e r i m e n t a l w a t e r was a c i d i f i e d t o pH 8.0 t o 8.2, by  injecting  s m a l l q u a n t i t i e s o f IN HC1 v i a t h e s a m p l i n g chamber.  The pH was t h e n a g a i n a l l o w e d t o r i s e t o about pH 9.0, w h i l e pCC^ was m o n i t o r e d w i t h t h e IRGA.  Samples o f w a t e r were  w i t h d r a w n e v e r y 10 m i n u t e s and a n a l y s e d l a t e r .  This routine  was r e p e a t e d , u s u a l l y 3 o r 4 t i m e s u n t i l  i n t h e system  was r e d u c e d from about 50  than  Thus t h e e f f e c t o f  ml/1 t o l e s s  5 ml/1.  on t h e r a t e o f p h o t o s y n t h e s i s c o u l d  be o b s e r v e d under c o n d i t i o n s o f known pCG^ and pH. I n l a t e r e x p e r i m e n t s PCO2 was n o t m o n i t o r e d because i t d i d n o t appear t o s i g n i f i c a n t l y i n f l u e n c e t h e r a t e o f photosynthesis.  A f l a t P l e x i g l a s s chamber, 27 cm by 17 cm  by 2.5 cm deep was used t o h o l d t h e p l a n t , f a s t e n e d t o a g l a s s frame.  T h i s system, i n c l u d i n g t h e tygon w a t e r  circu-  l a t i o n l i n e s , sample chamber, and water pump, had a volume o f 1300 ml and c o n t a i n e d a t l e a s t 1200 ml o f w a t e r d u r i n g the  experiments.  Temperature was m a i n t a i n e d a t 13° t o 16°.  The p l a n t s were i n i t i a l l y a l l o w e d t o p h o t o s y n t h e s i z e f o r about 40 m i n u t e s t o p e r m i t i n d u c t i o n o f p h o t o s y n t h e s i s . A s o l u t i o n o f NaHCO^ was t h e n added t o t h e system t o r a i s e the  c o n c e n t r a t i o n t o about 50 m l / 1 .  s y n t h e s i s was t h e n f o l l o w e d as  The c o u r s e o f p h o t o -  was r e d u c e d .  When a  s e r i e s o f measurements were made w i t h one sample, i n t h e  vicinity  o f a g i v e n C\ v a l u e , s a t u r a t e d NaHCO^ was u s e d t o  adjust C .  The p l a n t s w e r e t e s t e d a t t h e e n d o f e a c h  experiment f o r adverse e f f e c t s o f t h e treatment by measuring p h o t o s y n t h e s i s a t normal  v a l u e s , f o r 40 t o 60 m i n u t e s .  The r a t e s o f p h o t o s y n t h e s i s w e r e c a l c u l a t e d o v e r 10 minute  i n t e r v a l s a s ug C 0  2  gas/min/g  f r e s h w e i g h t , and  p l o t t e d a g a i n s t C., e x p r e s s e d a s m l Cc^ g a s / 1 s e a w a t e r The  free C0  2  g a s i n a i r pumped t h r o u g h t h e s e a w a t e r i s  e x p r e s s e d a s ppm CO-  ( y l C0 /1 o f a i r ) . 9  RESULTS AND DISCUSSION  S u r v e y f o r HCO^ i o n A s s i m i l a t i o n T a b l e 1 shows t h e r e s u l t s o f t h e s u r v e y f o r HCO^ i o n u p t a k e , made a t F r i d a y The  Harbor.  r a t e o f p h o t o s y n t h e s i s a b o v e 100 ppm C 0 g a s 2  e x p r e s s e d a s y g C 0 / m i n / g f r e s h w e i g h t , R^, i s c o m p a r e d f o r 2  e a c h a l g a , w i t h R , t h e r a t e o f p h o t o s y n t h e s i s b e l o w 100 2  ppm C 0 . 2  termed total  y.  The c h a n g e i n p h o t o s y n t h e t i c r a t e , R The v a l u e s C^ a n d C  inorganic carbon, C ,  calculated, respectively. is  2  - 2_/ l R  2  R  ^  s  a r e t h e median v a l u e s o f  a b o u t w h i c h R^ a n d R The c h a n g e i n C ,  were  2  or C  2  - C,/C^  e x p r e s s e d as x, and y/x r e p r e s e n t s t h e r a t i o o f change  i n p h o t o s y n t h e t i c r a t e t o change i n s u b s t r a t e f o r photo-  Table I  R e s u l t s o f a s u r v e y f o r HCO-j u p t a k e . R^  r a t e o f p h o t o s y n t h e s i s when pCC^ exceeds 100 ppm  R  r a t e o f p h o t o s y n t h e s i s below 100 ppm  2  y  t h e change i n r a t e o f p h o t o s y n t h e s i s ,  median v a l u e o f t o t a l i n o r g a n i c c a r b o n (C^) about w h i c h R^ was c a l c u l a t e d C„2  median v a l u e o f C.1 about w h i c h R~2 was calculated  x  t h e change i n C.  y/x  r e p r e s e n t s t h e r a t i o o f change i n p h o t o s y n t h e t i c r a t e t o change i n s u b s t r a t e concentration  26 TABLE  I  Rate of P h o t o s y n t h e s i s ug/min/g.fr.  wt R -R 2  Plant  Ulva  R  fenestrata  Alaria  sp.  Nereocystis Luetkeana  Costaria costata  En teromorpha linza  Porphyra schizophylla  Desmarestia munda  l  15.4 16.1 14.0  R  2  12.6 12.6 12.6  R  1  - y C-  l  2  = x  Cl  C  y/x  l  -.18 -.10 -.22  -.22 -.25 -.24  .8 •4(.7) .9  5.9 4.5 7.8  4 .3 4.5 4.1  -.27 -.00 -.47  -.17 -.20 -.23  1.6 0 2.0  5.4 2.8 2.4  3.6 2.1 2.1  -.28 -.23 -.09  -.17 -.12 -.19  1.6 1.9 1.3 .47  6.0 7.5 4.6  4.1 5.4 2.8  -.29 -.27 -.30  -.18 -.20 -.20  1.6 1.4 1.5  1.5  15.4 8.4 11.9  10 .8 5.4 6.6  -.30 -.36 -.35  -.16 -.13 -.22  1.9 2.8 1.6  2.3  7.0 16 .6 10.9  3.3 5.8 5.1  -.55 -.65 -.53  -.20 -.18 -.21  2.8 4.0 2.5  3.1  .56 1.0 .75  -.63 -.54 -.50  -.16 -.17 -.14  3.9 3.2 3.6  3.5  2.1 2.0 1.4  1.2  27 s y n t h e s i s , a n d i f C\ v a l u e s r e p r e s e n t a v a i l a b l e for  p h o t o s y n t h e s i s , y/x w i l l  becomes l i m i t i n g .  substrate  i n c r e a s e t o 1 as s u b s t r a t e  Where n o t a l l t h e C\ i s a v a i l a b l e  p l a n t , o r some p a r t o f i t i s i n h i b i t o r y y / x w i l l  to the  exceed  one. The  a l g a e t e s t e d g e n e r a l l y have a l o w e r  r a t e b e l o w 100 ppm CC» . 2  see  I f we e x a m i n e T a b l e  t h a t the algae appear t o f a l l  values f o r y/x. y/x  equal  and  Laminaria  A t one e x t r e m e i s U l v a f e n e s t r a t a , w i t h  t o 0.7, l e s s t h a n 1.  l e s s than  1, h o w e v e r , we  i n t o a graded s e r i e s o f  S i n g l e samples o f Fucus s p .  s a c c h a r i n a showed y / x e q u a l  showed no c h a n g e i n r a t e o f p h o t o s y n t h e s i s to  photosynthetic  25 ppm C 0 . 2  used, they are not l i s t e d  t o 0, t h a t i s , t h e y f r o m 400 ppm C 0  S i n c e o n l y o n e s a m p l e o f e a c h was i n Table  1.  U l v a showed a d r o p  o f 10 t o 2 0 % i n - p h o t o s y n t h e t i c r a t e , w i t h a f a l l 25%  i n C . i  2  o f 22 t o  28 A t t h e o p p o s i t e extreme, P o r p h y r a and D e s m a r e s t i a have y/x v a l u e s g r e a t e r than 3.0.  Both e x h i b i t e d d e c l i n e s  o f over 50% i n p h o t o s y n t h e t i c r a t e , w h i l e the change i n  for  P o r p h y r a , 18 to 21%, i s o n l y s l i g h t l y l e s s than t h a t f o r U l v a . O b v i o u s l y the d e c l i n e i n r a t e i s n o t d i r e c t l y r e l a t e d t o d e c l i n e i n t o t a l inorganic carbon. Between t h e extremes, N e r e o c y s t i s , C o s t a r i a and perhaps A l a r i a appear t o be more a l l i e d w i t h U l v a than w i t h Porphyra or Desmarestia.  These l a m i n a r i a n s show y/x v a l u e s  o f 1.3, 1.5, 1.2 r e s p e c t i v e l y .  The changes i n C^ f o r t h e  t h r e e k e l p genera a r e s i m i l a r t o each o t h e r , and s l i g h t l y l o w e r than f o r U l v a .  Enteromorpha appears  t o be i n t h e m i d d l e  o f t h e s e r i e s , w i t h y/x e q u a l t o 2.3, and a change i n C^ s i m i l a r to Desmarestia. On t h e b a s i s o f the d a t a p r e s e n t e d i n Table 1, and t o s i m p l i f y the d i s c u s s i o n , we have d i v i d e d the a l g a e t e s t e d i n t o two groups,:  t h e " U l v a - t y p e " w i t h y/x e q u a l t o o r l e s s  t h a n 1.5, and the " D e s m a r e s t i a - t y p e " w i t h y/x e q u a l t o , o r g r e a t e r than 2.3. F i g u r e s 4 and 5 show p l o t t e d r e s u l t s f o r U l v a and Desmarestia.  The C^ and ppm C 0  f o r both algae.  2  a r e p l o t t e d a g a i n s t time  The s l o p e o f t h e l i n e f o r C^ ( s o l i d  i s the r a t e o f p h o t o s y n t h e s i s .  Since, f o r a l l  line)  the a l g a e  t e s t e d , pH r o s e from a b o u t 8.0 t o above 9.5, d u r i n g t h e c o u r s e o f the e x p e r i m e n t , t h e o b s e r v e d change i n ppm C 0 may be l a r g e l y a t t r i b u t e d t o a s h i f t o f the H_CO_  2  equilibrium  Figure  4  Change i n t o t a l i n o r g a n i c c a r b o n  (C)  and  ppm  CC>2 a g a i n s t t i m e f o r a 4 g s a m p l e o f U l v a  sp.  29  TIME  MINUTES  Figure  5  Change i n C  and ppm  CC^  a g a i n s t time f o r a  27 g s a m p l e o f D e s m a r e s t i a munda  0  20 '40 60 TIME MINUTES  80  100  120  140  160  180  w i t h pH.  From t h e g r a p h s , i t i s a p p a r e n t t h a t w h i l e t h e r a t e  of photosynthesis  f a l l s o f f m a r k e d l y i n D e s m a r e s t i a as pCC>2  d r o p s below 100 ppm, t h e r a t e f o r U l v a f a l l s o f f much l e s s . (See  Table 1 ) .  described  These d a t a a r e s i m i l a r t o those p r e v i o u s l y  f o r Sargassum muticum ( 8 ) .  F u r t h e r a c i d i f i c a t i o n s t u d i e s made w i t h U l v a i n d i c a t e t h a t f o r t h i s a l g a , and, t h e r e f o r e , p o s s i b l y f o r those o f other algae i n the "Ulva-group", the d e c l i n e observed i n photosynthetic  r a t e i s caused by a r e d u c t i o n i n C\ r a t h e r  t h a n by a d r o p i n p C 0  2  o r an i n h i b i t o r y r i s e i n pH.  shows t h e r e s u l t s o f a r e p r e s e n t a t i v e sample o f U l v a , w i t h C  and ppm C 0  2  Figure 6  t e s t made on an 8.6 g  plotted against  time.  A t i n t e r v a l s , when pH r o s e from a b o u t 8.0 t o 10.0 and p C 0 f e l l t o l e s s than 5 ppm, t h e e x p e r i m e n t a l w i t h I N HC1 t o pH 8.8. From F i g u r e  2  w a t e r was a c i d i f i e d  The system was n o t opened.  6, i t i s a p p a r e n t t h a t t h e sudden  i n c r e a s e i n f r e e CC» gas r e l e a s e d by a c i d i f i c a t i o n d i d n o t 2  restore the rates of photosynthesis the b e g i n n i n g  o f the experiment.  s t e a d i l y w i t h the drop i n  .  t o those o b s e r v e d a t The r a t e o f u p t a k e d e c l i n e s  During the experiment, pC0 ,  C^, pH, and temperature were r e c o r d e d . o f pH i s a p p a r e n t . values o f was  The p h o t o s y n t h e t i c  below 1 ml/1.  No s i g n i f i c a n t e f f e c t r a t e r e a c h e d zero a t  When a s a t u r a t e d NaHCO^ s o l u t i o n  i n j e c t e d i n t o t h e c l o s e d system near t h e end o f t h e  experiment, the r a t e of photosynthesis was  2  immediately r e s t o r e d .  initially  observed  The d e c l i n e i n p h o t o s y n t h e t i c  Figure  6  Change  i n  C  and  ppm  fenestrata  showing  rate  value  on  C.  CC^  against  dependence  rather  than  of pH  time  i n  Ulva  photosynthetic or  ppm  C0_  32  48 460 44 0 40 380 36  32  28  v  24  pH 8.5  120  16 H 9.8 12  o P  H 10.2 P  CvJ  O  H 8.8  18  u  ^A \ \ pH 8.8  o  >H 10.0 ^ DH  0 0  40 TIME  80  120  MINUTES  160  200  240  9.4 280  320  0  r a t e was, t h e r e f o r e , r e v e r s e d by r e s t o r i n g t h e i n o r g a n i c carbon.  S i n c e o n l y HCO^ i o n was added t o t h e system, one  may c o n c l u d e  t h a t t h e d e c l i n e i n p h o t o s y n t h e s i s was n o t  caused by i n c r e a s e d Incomplete  concentration. s t u d i e s on Fucus s p . , L a m i n a r i a s a c c h a r i n a  and G i g a r t i n a s p . ( c r i s t a t a ? ) , i n d i c a t e t h a t t h e s e belong to the "Ulva-type".  algae  C o n s i d e r a b l e d i f f i c u l t y was  encountered  i n m e a s u r i n g p h o t o s y n t h e s i s i n Fucus and  Laminaria.  The l a t t e r  of mucilage  from c u t s u r f a c e s , w h i c h may i n h i b i t  synthesis.  These t h r e e a l g a e , however, showed y/x v a l u e s  of zero.  particularly,  exudes l a r g e q u a n t i t i e s photo-  U l v a , A l a r i a , and N e r e o c y s t i s a l s o o c c a s i o n a l l y  show y/x v a l u e s o f z e r o . From graphs o f t h e t y p e s shown i n F i g u r e s 4 and 6, i t i s concluded  t h a t p l a n t s o f t h e " U l v a - t y p e " have  little  dependence on CO2 gas as a s u b s t r a t e f o r p h o t o s y n t h e s i s and a r e a b l e t o use HCO^ i o n as t h e i r major s o u r c e o f c a r b o n . I n a d d i t i o n , U l v a shows a r a t e o f p h o t o s y n t h e s i s dependent on C^ as t h e c o n c e n t r a t i o n o f HCO^ i o n changes from more t h a n 80% a t pH 8.0 t o l e s s than 20% a t pH 10, and CO^ i o n c o n c e n t r a t i o n changes from l e s s than 20% a t pH 8.0 t o more than 80% a t pH 10.0.  U l v a may be a b l e t o use b o t h HCO~ i o n  and CO~ i o n . It  i s n o t c l e a r why t h e r a t e s o f p h o t o s y n t h e s i s  o f f more r a p i d l y i n D e s m a r e s t i a  and P o r p h y r a  fall  than i n t h e o t h e r  genera.  V a l u e s o f y/x l e s s t h a n 2.5 o r a d r o p i n p h o t o -  s y n t h e t i c r a t e o f l e s s t h a n 50% were n o t found i n P o r p h y r a or Desmarestia.  D e s m a r e s t i a i s w e l l known f o r i t s extreme  s e n s i t i v i t y to s t r e s s .  I t has a v e r y low v a c u o l a r pH,  when o n l y s l i g h t l y d e s i c c a t e d o r warmed, t h e c e l l s b r e a k down r e l e a s i n g H^SO^ .  Possibly  readily  Desmarestia i s  s e n s i t i v e t o pH change, so t h a t a s h i f t i n pH photosynthesis.  and  inhibits  I n T a b l e 1, the second and t h i r d  rates  g i v e n f o r D e s m a r e s t i a were measured on the same p l a n t sample. Whatever the cause o f t h e r a t e d e c l i n e , the f i g u r e s show t h a t i t was  at least partly  reversible.  The s u g g e s t i o n t h a t a r i s e i n pH i n h i b i t s  photo-  s y n t h e s i s i s l e s s s a t i s f a c t o r y f o r P o r p h y r a t h a n f o r Desmarestia .  P o r p h y r a r o u t i n e l y t o l e r a t e s s e v e r e d e s i c c a t i o n as  i t d r i e s t o a b r i t t l e c o n d i t i o n on the r o c k s d u r i n g low periods.  tide  As t h i s o c c u r s , i t p r o b a b l y s u r v i v e s q u i t e l a r g e  pH s h i f t s a t the p l a n t s u r f a c e . A l t e r n a t i v e l y , o r perhaps a d d i t i o n a l l y , p l a n t s showi n g the same.shaped c u r v e as D e s m a r e s t i a and P o r p h y r a r e q u i r e both C0  2  gas and HCO-j i o n , o r o n l y the f o r m e r , as  s u b s t r a t e f o r p h o t o s y n t h e s i s ; o r , t h e y may o r be i n h i b i t e d by CO^ the pH range employed.  t o 100 ppm,  be u n a b l e t o u s e ,  i o n , which i n c r e a s e s r a p i d l y over The r a t e o f p h o t o s y n t h e s i s  s h a r p l y i n the r e g i o n pH 8.0 400 ppm  may  and CO^  t o 8.5 where pCC^ r i s e s from 6% o f C  falls  drops from  t o 15%.  P o r p h y r a , l i k e D e s m a r e s t i a shows a t l e a s t 60% r e c o v e r y when  35 p l a c e d i n f r e s h seawater f o r a second t e s t ,  (see T a b l e 1,  samples 2 and 3 f o r Porphyra) so t h a t t h e i n h i b i t i o n may be considered e s s e n t i a l l y r e v e r s i b l e .  The E f f e c t o f HCO^ I o n C o n c e n t r a t i o n on P h o t o s y n t h e s i s F i g u r e 7 shows t h e e f f e c t o f C synthesis i n Ulva f e n e s t r a t a .  on t h e r a t e o f p h o t o -  I n t h e pH range 7.8 t o 9.0,  i n w h i c h t h e measurements were made, HCO^ i o n i s t h e dominant form o f C , and a l l C  (50% o f C  a t pH 9.0 i s i n t h e form o f HC0~ i o n ) ,  i s e f f e c t i v e l y ionized; therefore, i n the f o l l o w -  ing discussion, C  i s synonomous w i t h " i o n i z e d C^".  In  F i g u r e 7, r a t e o f p h o t o s y n t h e s i s i n ug CC^/min/g f r e s h w e i g h t i s plotted against C  i n ml C C ^ / l s e a w a t e r .  Under t h e  e x p e r i m e n t a l c o n d i t i o n s o f 5000 f t c i l l u m i n a t i o n , p h o t o s y n t h e s i s i n U l v a appears t o be n e a r l y s a t u r a t e d a t 4 5 t o 50 m l C ^ / l w a t e r , w i t h a b r e a k a t about 30 ml C^ where t h e r a t e o f i n c r e a s e i n p h o t o s y n t h e s i s w i t h C^ b e g i n s t o d e c l i n e . From 5 ml  t o 30 m l C^, t h e dependence o f r a t e o f p h o t o -  s y n t h e s i s on C  i s essentially linear.  A t 45 t o 50 ml C ,  the v a l u e s o r d i n a r i l y measured i n s e a w a t e r , t h e r a t e o f p h o t o s y n t h e s i s i n U l v a i s 200 ug CC^/min/g f r e s h w e i g h t o r 12 mg C 0 / h r / g . 2  Figure 4 i s r e p r e s e n t a t i v e of the general order. T h i s i s s a t i s f a c t o r y , s i n c e t h e a l g a e used came from t h e v i c i n i t y o f F r i d a y Harbour (Figure 6 ) .  ( F i g u r e 4) and from B u r r a r d  I n F i g u r e 4 t h e v a l u e s o f R, and R_ were  Inlet  Figure  7  Dependence o f r a t e o f p h o t o s y n t h e s i s inorganic  carbon f o r Ulva  fenestrata  on  total  RATE  O 4^U  OF  PHOTOSYNTHESIS  pG.  C 0 ^ M IN^G. 2  FR. WT.XlO  calculated at seawater,  v a l u e s o f 38 ml/1  respectively.  seawater and a t 25  The v a l u e o f y was  p h o t o s y n t h e s i s d e c l i n e s by 18%.  -.18,  ml/1  that i s ,  From the c o m p o s i t e r a t e  v e r s u s c o n c e n t r a t i o n c u r v e i n F i g u r e 4, the p r e d i c t e d v a l u e f o r y, when C\ a d e c l i n e of  f a l l s from 3 8 ml/1  t o 29.5 m l / 1 ,  i s -.22  or  22%.  I r i d a e a c o r d a t a samples o f 5 t o 10 g were i l l u m i n a t e d a t 2500 f t c. versus  The c o m p o s i t e g r a p h o f r a t e o f p h o t o s y n t h e s i s  i s shown i n F i g u r e Some d i f f i c u l t y was  8. experienced i n i l l u m i n a t i n g  p l a n t because o f v a r i a b i l i t y i n t h i c k n e s s o f the  this  thallus.  S e c t i o n s o f t h a l l u s o f the same a r e a o f t e n v a r y c o n s i d e r a b l y i n t h i c k n e s s and, t h e r e f o r e , i n w e i g h t .  Thus, the  thicker  p i e c e s o f m a t e r i a l i l l u m i n a t e d a t 2500 f t c f r o m one tended  t o e x h i b i t reduced  rates of photosynthesis.  side When  h i g h e r l i g h t i n t e n s i t i e s , such as 5000 f t c were u s e d , I r i d a e a showed an i r r e v e r s i b l e r e d u c t i o n i n p h o t o s y n t h e t i c r a t e i n s h o r t e r t i m e s t h a n when under 2500 f t c. I r i d a e a a l s o showed a g r e a t e r s e n s i t i v i t y t o pH e i t h e r U l v a o r Sargassum. observed  than  I n F i g u r e 9 some tendency i s  f o r rates of photosynthesis to d e c l i n e w i t h i n c r e a s -  i n g pH as w e l l as d e c r e a s i n g C\. t a k e n from F i g u r e  Table 2 shows some examples  9.  I t can be seen t h a t a 0.5 c o n c e n t r a t i o n reduces  change i n pH a t the same  the r a t e of p h o t o s y n t h e s i s by  When the pH i s c o n s t a n t , however, and C. i s reduced  15%.  a large  Figure  8  E f f e c t o f t o t a l i n o r g a n i c carbon on r a t e o f p h o t o s y n t h e s i s by  Iridaea  cordata  RATE  8€  OF  PHOTOSYNTHESIS  (JG. C0 JM\H./ 2  G. FR. WT.  Figure 9  E f f e c t of pH on rate of photosynthesis by Iridaea cordata  R A T E OF  PHOTOSYNTHESIS  U G./M I N./G.FR. WT.  Table  2  The e f f e c t o f pH change and C  i  change on r a t e  of photosynthesis i n I r i d a e a cordata  40  TABLE 2 Rate o f P h o t o s y n t h e s i s a t : C.(ml/1)  pH 8.0  pH 8.5  1  44  57 yg/min/g  43  Change i n Rate of Photosynthesis  -15% 49 ug/min/g  47  57 yg/min/g  18  41 yg/min/g  7  34 yg/min/g  -45%  f a l l i n photosynthetic r a t e i s observed.  D e s p i t e the  effect  o f pH i n d e p r e s s i n g p h o t o s y n t h e t i c r a t e s , the major f a c t o r c a u s i n g a d e c l i n e i n r a t e i n F i g u r e s 5 and to a f a l l i n substrate concentration.  2  gas  be  attributed  As has been shown  p r e v i o u s l y , I r i d a e a can p h o t o s y n t h e s i z e free C0  6 may  i n the absence o f  ( 9 ) , as measured by t h e IRGA, and t h e r e f o r e ,  must be c a p a b l e o f u s i n g HCO^  ion.  The r a t e s o f p h o t o s y n t h e s i s Iridaea at  from 45 t o 50 ml/1  shown i n F i g u r e 8 f o r  w a t e r , a r e low compared t o  t h o s e f o r U l v a ; 54 ug/min/g f r e s h w e i g h t o r 3.2 I r i d a e a compared t o 12 mg/hr/g f o r U l v a .  mg/hr/g f o r  The g r a p h i n  F i g u r e 8 i n d i c a t e s t h a t under the e x p e r i m e n t a l c o n d i t i o n s , HCO^  i o n was  l i m i t i n g ; the r a t e of photosynthesis  from 10 ml linear  t o 50 ml C^.  The g r a p h shown i s e s s e n t i a l l y  b u t we must assume a s h a r p downward c u r v e  a t about 12 ml 0.0 ml C,.  increased  beginning  and e x t r a p o l a t i n g a t o r s l i g h t l y above  I t i s i n t e r e s t i n g t o n o t e t h a t a t 12 ml C\  rate of photosynthesis 50% o f the r a t e r e a c h e d  the  i s a l r e a d y about 30 ug/min/g, a b o u t a t o v e r 50 ml C^.  the p l a n t t o time e f f e c t s p r e v e n t e d f o r p h o t o s y n t h e t i c r a t e below 10 ml  S e n s i t i v i t y of  us from o b t a i n i n g v a l u e s C^.  F i g u r e 10 shows the e f f e c t o f C\ on the r a t e o f photosynthesis  f o r Sargassum muticum.  r e s u l t s o f a t y p i c a l r u n f o r Sargassum.  F i g u r e 11 shows t h e T h i s p l a n t has  p r e v i o u s l y been shown t o be dependent on HCO^ photosynthesis.  ion for  The pH v a l u e a t w h i c h each p l o t t e d r a t e o f  F i g u r e 10  E f f e c t o f t o t a l i n o r g a n i c c a r b o n on r a t e o f p h o t o s y n t h e s i s by Sargassum muticum  pG. C 0 / M 1 N.y" G.FR. WT.  RATE OF P H O T O S Y N T H E S I S  2  ro o  NO  oo  CO  ro  F i g u r e 11  E f f e c t o f pH o n r a t e o f p h o t o s y n t h e s i s Sargassum muticum  by  36 32. 8,6  *28J  O O o  8.45 A.  13  8  of  d  Q.25\  8.8  24j  87  9.1  20J 812  GO  A  ao  LU ^ >-  CO  o o x  CL  83A  i o  A  "  8.6  9.0i  8J 85*^8.3  <  4J  92*^9.1  CC  01 0  8 Cj  12  ML. COo/L. )o/L  16  20  24  28  32  36  40  44  48  52  44 p h o t o s y n t h e s i s was d i f f i c u l t y was  measured i s g i v e n on the graph.  experienced  i n Sargassum w i t h s e n s i t i v i t y  r a t e of p h o t o s y n t h e s i s to pH between pH Rates measured a t pH 19 ml/1  8.25  and  be observed  examples we may r i s e i n pH; due  8.3/  when  and pH was  9.0.  52 ml/1  and  a t pH 9.0.  i n that order.  (See F i g u r e 11).  Similar data From these  conclude t h a t the r a t e change i s not due  to a  the d e c l i n e i n p h o t o s y n t h e t i c r a t e i n s t e a d i s  to the f a l l  t h a t Sargassum,  i n c o n c e n t r a t i o n o f HCO^ l i k e U l v a , may  ion.  It i s possible  be a b l e to use COj i o n .  As f o r I r i d a e a c o r d a t a , HCO^ ing  8.0  of  r e s p e c t i v e l y , have v a l u e s of 30 yg/min/g f r e s h weight  and 15 yg/min/g f r e s h weight, may  Little  ion i s apparently l i m i t -  f o r p h o t o s y n t h e s i s i n Sargassum muticum, under the  experimental c o n d i t i o n s .  A t 45 to 50 ml C\ , the r a t e o f  p h o t o s y n t h e s i s i s about 30 yg/min/g f r e s h weight o r mg/hr/g f r e s h  1.8  weight.  Except f o r the work e s t a b l i s h i n g the dependence o f r a t e o f p h o t o s y n t h e s i s i n U l v a on C^,  a l l the r a t e s o f  carbon a s s i m i l a t i o n measured f o r marine b e n t h i c a l g a e i n t h i s study are extremely for  low compared t o those commonly  such l a n d p l a n t s as wheat and c o r n .  observed  F u r t h e r study i s  n e c e s s a r y to f i n d out whether these low r a t e s are a f u n c t i o n of  the e x p e r i m e n t a l o r growing c o n d i t i o n s and i f so, what  p h o t o s y n t h e t i c r a t e s can be a c h i e v e d by these p l a n t s under ideal  c o n d i t i o n s o f l i g h t and s u b s t r a t e c o n c e n t r a t i o n .  The r a t e s o b s e r v e d f o r U l v a f e n e s t r a t a o f 12 mg/hr/g f r e s h w e i g h t are much h i g h e r t h a n t h o s e f o r I r i d a e a c o r d a t a and  Sargassum muticum;  3.2  mg/hr/g f r e s h w e i g h t . than those obtained weight. (1).  mg/hr/g f r e s h w e i g h t and  1.8  In a d d i t i o n , these r a t e s are  a t F r i d a y Harbor; 0.9  higher  mg/hr/g f r e s h  They a r e s i m i l a r t o p h o t o s y n t h e t i c  r a t e s f o r wheat  I t has been s u g g e s t e d t h a t the w o r l d f o o d p r o b l e m m i g h t  be s o l v e d by  "farming  the s e a " .  L a t i e s (3) has  considered  the p o s s i b l e p r o d u c t i v i t y o f the oceans w i t h r e g a r d  to  c o n c e n t r a t i o n s o f n i t r o g e n and phosphorus w h i c h a r e  limiting  i n nature.  the  He s u g g e s t s t h a t e s t i m a t e s o f p o s s i b l e p r o d u c 14  t x v i t y based on s h o r t term r a t e s o f C»2 p r o d u c t i o n , a r e f a r t o o h i g h .  CC^  a s s i m i l a t i o n or  C a l c u l a t i o n s of p r o d u c t i v i t y  based on the a v a i l a b l e phosphorus and n i t r o g e n i n d i c a t e t h a t a 1% e f f i c i e n t c o n v e r s i o n o f p h y t o p l a n k t o n a y i e l d o n l y 0.1  t o 0.2%  t o f i s h would g i v e  o f the e d i b l e l a n d c r o p .  On  this  b a s i s , " m a r i c u l t u r e " i s o b v i o u s l y not a s o l u t i o n f o r world f o o d needs.  The  algae c o l l e c t e d at Brockton P o i n t i n  Vancouver Harbour u n d o u b t e d l y had a h i g h e r n i t r a t e  and  phosphate c o n c e n t r a t i o n a v a i l a b l e t o them (because o f sewage o u t f a l l ) t h a n the a l g a e c o l l e c t e d a t F r i d a y H a r b o r , i n c r e a s e d r a t e s o b s e r v e d a t Vancouver f o r U l v a may  and  the  reflect  this nutritional status. The  low r a t e s o f p h o t o s y n t h e s i s  h e r e s u p p o r t the s u g g e s t i o n  g e n e r a l l y observed  t h a t more measurements are needed  o f a c t u a l marine p r o d u c t i v i t y .  I n v i e w o f the e f f e c t s  d e s c r i b e d h e r e o f pH a n d pCC^ o n p h o t o s y n t h e s i s i n some a l g a e , c a r e s h o u l d be taken t o m o n i t o r parameters  when u s i n g s u c h  b o t t l e method f o r m e a s u r i n g  or control  these  techniques as t h e l i g h t - d a r k productivity.  47  LITERATURE CITED  1.  Hesketh, J.D. 1967. Enhancement o f p h o t o s y n t h e t i c CC^ a s s i m i l a t i o n i n t h e absence o f oxygen, as depend e n t upon s p e c i e s and t e m p e r a t u r e . P l a n t a 76s 371-374.  2.  I k e m o r i , M. and N i s h i d a , K. 1966. I n o r g a n i c c a r b o n s o u r c e and t h e i n h i b i t i o n o f Diamox on t h e photosynthesis o f marine algae - U l v a p e r t u s a . Ann. Rep. Noto M a r i n e Lab. 7: 1-5.  3.  L a t i e s , G.G. 1969. M e t a b o l i c S i n k s - D i s c u s s i o n . I n P h y s i o l o g i c a l a s p e c t s o f c r o p y i e l d . E d i t e d by J.D. E a s t i n , F.A. H a s k i n s , C.Y. S u l l i v a n , and G.H.M. v a n B a v e l . A m e r i c a n S o c i e t y o f Agronomy, Crop S c i e n c e S o c i e t y o f A m e r i c a . Madison, W i s c o n s i n , U.S.A. pp. 182-184.  4.  R a b i n o w i t c h , E . I . 1945. P h o t o s y n t h e s i s and r e l a t e d processes. V o l . I . Interscience P u b l i s h e r s , I n c . , New Y o r k , N.Y. 2088 pp.  5.  Raven, J.A. 1968. The mechanism o f p h o t o s y n t h e t i c u s e o f b i c a r b o n a t e by H y d r o d i c t y o n a f r i c a n u m . J . E x p t , Botany, 19: 193-206.  6.  S c a g e l , R.F. 1967. Guide t o common seaweeds o f B r i t i s h Columbia. B.C. P r o v . Museum, Handbook No. 27. The Queen's P r i n t e r , B.C. 330 pp.  7.  S k i r r o w , G. 1965. The d i s s o l v e d gases - c a r b o n d i o x i d e , I n C h e m i c a l oceanography V o l . I . E d i t e d by J . P . R i l e y and G. S k i r r o w . Academic P r e s s , London and New Y o r k . pp. 227-322.  8.  Thomas, E.A. and Tregunna, E.B. 1968. B i c a r b o n a t e i o n a s s i m i l a t i o n i n p h o t o s y n t h e s i s by Sargassum muticum. Can. J . Botany, 46: 411-415.  9.  Tregunna, E.B. and Thomas, E.A. 1968. Measurement o f i n o r g a n i c c a r b o n and p h o t o s y n t h e s i s i n seawater by pCO~ and pH a n a l y s i s . Can. J . B o t a n y , 46: 481-485.  PART 1 - ADDENDUM The e f f e c t of pH on p h o t o s y n t h e s i s by U l v a  fenestrata  49 ADDENDUM The E f f e c t o f pH on P h o t o s y n t h e s i s by U l v a f e n e s t r a t a (4)  I t has been r e p o r t e d p r e v i o u s l y (2) t h a t between pH 8.0  and pH 9.5 changes i n t h e t o t a l i n o r g a n i c c a r b o n  available  f o r p h o t o s y n t h e s i s had a g r e a t e r e f f e c t on r a t e o f p h o t o s y n t h e s i s t h a n d i d pH changes.  T h i s was p a r t i c u l a r l y t r u e  o f U l v a f e n e s t r a t a . The f o l l o w i n g s t u d y was u n d e r t a k e n t o f u r t h e r examine t h e e f f e c t o f pH on p h o t o s y n t h e s i s i n U l v a .  MATERIALS AND METHODS  U l v a f e n e s t r a t a was c o l l e c t e d from the i n t e r t i d a l  zone  a t Brockton P o i n t , S t a n l e y Park, Vancouver, B r i t i s h Columbia, from June 8 t o 26 i n 1971. of low t i d e s .  This p e r i o d covered  two s e r i e s  I t was hoped t h a t p l a n t s c o l l e c t e d d u r i n g t h i s  t i m e , a l l o f s i m i l a r s i z e , would be o f s i m i l a r m a t u r i t y and physiogical condition.  P l a n t m a t e r i a l was s t o r e d a t 12°C a s  p r e v i o u s l y d e s c r i b e d (2) and used w i t h i n 48 h o u r s . P h o t o s y n t h e s i s was measured as i n p r e v i o u s  experiments  (7) w i t h one m o d i f i c a t i o n ; t h e pH was h e l d c o n s t a n t w i t h 20mM T r i s b u f f e r d u r i n g t h e c o u r s e o f e a c h s e r i e s o f measurements. Three s e p a r a t e t e s t s , on d i f f e r e n t p l a n t samples were r u n a t pH 7.5,  8.0, 8.5, 9.0 and 9.5. The pH was checked a t t h e  end o f t h e e x p e r i m e n t ,  and b e f o r e t h e a d d i t i o n o f HCO_  50 when t e s t i n g f o r time e f f e c t s ;  (See r e f e r e n c e 2 ) .  The  pH  a d j u s t m e n t s were made i n c o n j u n c t i o n w i t h the t a b l e s g i v e n by Sigma C h e m i c a l C o r p o r a t i o n , showing the e f f e c t o f temperat u r e on T r i s b u f f e r . (5)  RESULTS AND  DISCUSSION  The r a t e o f p h o t o s y n t h e s i s p l o t t e d a g a i n s t C^,  f o r U l v a a t each pH  was  and l i n e a r r e g r e s s i o n s c a l c u l a t e d .  Values  were t a k e n by i n t e r p o l a t i o n from the l i n e a r r e g r e s s i o n s t o produce F i g u r e 12, w h i c h shows the a p p a r e n t e f f e c t o f pH  on  r a t e o f p h o t o s y n t h e s i s a t f i v e pH v a l u e s and t h r e e c a r b o n concentrations.  The v a l u e s a t pH 8.0  and  8.5  are not  signifi-  cantly different.  These r a t e s were t a k e n from the a p p r o p r i a t e  regression lines.  I t i s apparent t h a t a p r o g r e s s i v e increase  i n pH i s accompanied by a r e d u c t i o n i n the r a t e o f p h o t o synthesis.  This i s apparent a t a l l three carbon c o n c e n t r a t i o n s .  The r a t e i s a l s o d e c r e a s e d  as C. i s r e d u c e d . i  A t f i r s t i t m i g h t appear t h a t h i g h pH i t s e l f has  an  i n h i b i t o r y e f f e c t on p h o t o s y n t h e s i s o r on some f a c t o r i n fluencing  photosynthesis.  As pH changes from 7.5  t o 9.5,  s h i f t i n the dominant form o f H^CO^ to  35% HC0.J i o n .  ( 6 ) , from 90% HCO^  A t the same time CO~  changes from 1% t o 65% o f C .  there i s a massive ion  ion concentration  The r e m a i n i n g C. a t pH  7.5  F i g u r e 12  E f f e c t o f pH on r a t e o f p h o t o s y n t h e s i s i n U l v a f e n e s t r a t a a t t h r e e C.  values  i s CC>2 gas and t h a t U l v a may  undissociated use C0~  I^CO^.  I t has been s u g g e s t e d  i o n as a s u b s t r a t e f o r  on the b a s i s o f such o b s e r v a t i o n s  photosynthesis  as are p r e s e n t e d h e r e .  (2)  F i g u r e 13 r a t h e r d r a m a t i c a l l y r e i n f o r c e s t h i s hypothesis.  Rate o f p h o t o s y n t h e s i s  a l l f i v e pH v a l u e s was ion  concentration,  at three  values  and  p l o t t e d a g a i n s t the c a l c u l a t e d  i n s t e a d o f a g a i n s t C^,  which  HCO^  includes  a l l inorganic carbon.  I t appears from F i g u r e 13, t h a t  pH i n c r e a s e s  ion concentration  (and HCO^  the p l a n t u s e s HCO^  i o n more e f f i c i e n t l y .  t h a t the r a t e a c h i e v e d a t pH 9.5, c l o s e t o t h a t a c h i e v e d a t pH 7.5 to increased  falls  and and  The  as  rapidly) result i s  20 ml HCO~,  i s very  40 ml HCO^.  A tendency  e f f i c i e n c y i s a l s o a p p a r e n t a t pH  9.0.  The most l i k e l y i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s t h a t the p l a n t i s a l s o c a p a b l e o f u s i n g CO"^ a t pH 9.0,  t h a t CO~  concentration  ion.  r e a c h e s 40% o f C ,  thence becomes an i m p o r t a n t s o u r c e o f c a r b o n . concentration  i s i n c l u d e d i n C^,  amazing a f f i n i t y f o r HCO^  It is  If  CO^  as i n e a r l i e r work, t h i s  i o n a t h i g h pH v a l u e s  disappears.  There i s a d e p r e s s a n t e f f e c t o f i n c r e a s e d pH rate of photosynthesis, the above e f f e c t s .  on  however, w h i c h i s superimposed on  O t h e r w i s e , use o f HCO~  i o n and CO~  p r o b a b l y r e q u i r e s an e n e r g e t i c p r o c e s s f o r i m p o r t i n g s i n c e t h e y a r e not l i p i d  and  soluble  ions them,  ( t h a t i s , can n o t d i f f u s e  f r e e l y t h r o u g h the l i p i d c e l l u l a r and c h l o r o p l a s t membranes)  F i g u r e 13  The  e f f e c t o f HCO^  photosynthesis at fenestrata  i o n c o n c e n t r a t i o n on r a t e o f f i v e pH v a l u e s i n U l v a  unlike C0  2  gas* Raven (3) i n d i c a t e d l i n k a g e o f HCO^ i o n  u p t a k e t o Photosystem I I .  P o s s i b l y an enzyme i s i n v o l v e d ,  w i t h a g r e a t e r a f f i n i t y f o r HCO^ i o n than f o r C0~ i o n . I f t h i s were s o , C0~ m i g h t have t o r e a c h a f a i r l y h i g h c o n c e n tration  (40%?) b e f o r e c o m p e t i t i o n f o r t h e enzymes makes i t  a v a i l a b l e to the carbon f i x i n g apparatus.  I f the a f f i n i t y  f o r CO^ were l e s s t h a n f o r HCO^,  r a t e o f photo-  a reduced  s y n t h e s i s would n o t be unexpected as CO^ became t h e dominant substrate.  Much h i g h e r  v a l u e s where a l l C\ was COj would  be r e q u i r e d t o s a t u r a t e p h o t o s y n t h e s i s than when HCO^ composed most o f C.. x  Some o f t h e r e d u c t i o n o f r a t e between pH 7.5 and 8.0 c o u l d be caused by t h e d i s a p p e a r a n c e source.  About 10% o f  i s C0  2  of C0  2  as a c a r b o n  gas a t pH 7.5.  A t pH v a l u e s  o f about 5, U l v a w i l l p h o t o s y n t h e s i z e a t a l o w r a t e 0.05% o f t h e v a l u e a t 7.5)  ( 1 ) . A t t h i s pH, C  i  (about  i s over  90% C 0 , w h i c h c a n p r o b a b l y e n t e r t h e c e l l by p a s s i v e 2  d i f f u s i o n , since i t i s l i p i d soluble. F i n a l l y , i t i s l i k e l y t h a t e x t e r n a l pH s t r e s s p l a c e s a s t r a i n on t h e i n t e r n a l pH r e g u l a t i n g mechanisms o f t h e plant.  T h i s may be p a r t l y r e s p o n s i b l e f o r t h e d e c r e a s e i n  r a t e o f p h o t o s y n t h e s i s , when s u b s t r a t e c o n c e n t r a t i o n r e m a i n s the same, b u t pH i n c r e a s e s .  LITERATURE CITED  Brown, D.L. and Tregunna, E.B. 1967. I n h i b i t i o n of r e s p i r a t i o n d u r i n g p h o t o s y n t h e s i s by some a l g a e . Can. J . B o t . , 45: 1135-1143. J o l l i f f e , (Thomas) E.A. and Tregunna, E.B. 1970. S t u d i e s on HCO^ i o n u p t a k e d u r i n g p h o t o s y n t h e s i s i n b e n t h i c marine a l g a e . P h y c o l o g i a , 9: 293303. Raven, J.A. 1968. The mechanism o f p h o t o s y n t h e t i c use o f b i c a r b o n a t e by H y d r o d i c t y o n a f r i c a n u m . J . Expt. Botany, 19: 193-206"; S c a g e l , R.F., 1967. Guide t o common sea-weeds o f B r i t i s h C o l u m b i a . B.C. P r o v . Museum, Handbook No. 27. The Queen's P r i n t e r , B.C. 330 pp. Sigma C h e m i c a l Company, 1971.  P r i c e l i s t , p.  299.  S k i r r o w , G. 1965. The d i s s o l v e d g a s e s - c a r b o n d i o x i d e . I n : C h e m i c a l Oceanography V o l . I . Ed. J.P. R i l e y and G. S k i r r o w . Academic P r e s s , London and New Y o r k . pp. 227-322. Tregunna, E.B. and Thomas, E.A. 1967. Measurement o f i n o r g a n i c c a r b o n i n sea w a t e r by two methods o f pC02 and pH a n a l y s i s . Can. J . B o t . , 46: 481-485.  PART 1 - Appendix  Measurement o f i n o r g a n i c c a r b o n and p h o t o s y n t h e s i s i n s e a w a t e r by pC0  9  and pH a n a l y s i s  The q u e s t i o n o f whether marine a l g a e c a n d i r e c t l y a s s i m i l a t e HCO^ i o n was l o n g a c o n t r o v e r s i a l one, l a r g e l y because o f t h e t e c h n i c a l d i f f i c u l t i e s i n v o l v e d i n d i f f e r e n t i a t i n g between use o f d i s s o l v e d CC^ gas and d i r e c t u p t a k e o f HCO^ i o n .  The methods d e s c r i b e d here were d e s i g n e d t o  a l l o w d i f f e r e n t i a t i o n between use o f CC^ gas and HCO^ i o n (or o t h e r i o n i c s p e c i e s o f H^CO^). F i g u r e 14 shows t h e apparatus s y n t h e s i s i n marine a l g a e .  used t o measure p h o t o -  Samples o f a l g a e were p l a c e d i n  a g l a s s o r p l e x i g l a s s chamber and anchored t o a g l a s s frame or weight,  t o spread t h e m a t e r i a l f o r optimum i l l u m i n a t i o n .  I n i t i a l l y a 1 l i t r e g l a s s r e a c t i o n f l a s k was u s e d , w i t h f o u r standard taper j o i n t s i n the l i d .  ( I n l a t e r work, a p l e x i -  g l a s s chamber was s u b s t i t u t e d , as d e s c r i b e d i n p a r t I-B.) Temperature and pH s e n s o r s  ( F i g u r e 14h,j) e n t e r e d  through  the l i d , as d i d t u b i n g f o r c i r c u l a t i n g gas and/or w a t e r . The  gas passed t h r o u g h a s c i n t e r e d g l a s s d i s c ,  o r an a i r s t o n e , t o e n t e r t h e w a t e r . a f l o w o f 1 l . / m i n was m a i n t a i n e d . by pumping a t 1.4 1/min.  ( F i g u r e 14b)  During a i r c i r c u l a t i o n , Water was c i r c u l a t e d  The pCC»2 was measured w i t h a  Beckman I n f r a r e d Gas A n a l y s e r  ( F i g u r e 1 4 e ) , Model 215,  w i t h f u l l s c a l e e q u a l t o 600 ppm. c a r b o n d i o x i d e .  The  i n s t r u m e n t was c a l i b r a t e d from a c y l i n d e r o f compressed gas s t a n d a r d i z e d by Matheson o f Canada, L t d . t a i n e d 180 ppm c a r b o n d i o x i d e .  T h i s gas c o n -  The a n a l y s e r was s e t t o  z e r o w i t h wet C C u - f r e e a i r o r N~, and t h e r e a d i n g s  recorded  F i g u r e 14  A system f o r m e a s u r i n g p h o t o s y n t h e s i s i n marine a l g a e by pCC^ and C\ measurement, w i t h c o n t r o l o f pH and t e m p e r a t u r e (a. (b (c (d (e (f (g (h (i (j (k (1 (m (n (o  reaction flask scintered glass bubbler a i r pump tube o f d r i e r i t e  (magnesium p e r c h l o r a t e )  I n f r a r e d gas a n a l y s e r (IRGA) w a t e r s a m p l i n g chamber w a t e r pump pH e l e c t r o d e pH meter t e l e t h e r m o m e t e r probe Telethermometer paper f i l t e r water bath search l i g h t water bath  58  59 on a s t r i p c h a r t r e c o r d e r . A S a r g e n t p o r t a b l e pH meter ( F i g u r e 1 4 i ) was used t o c o n t i n u o u s l y measure pH, w h i c h was r e c o r d e d by s t r i p c h a r t r e c o r d e r , o r noted e v e r y 5 m i n u t e s .  The c o m b i n a t i o n pH  e l e c t r o d e had a low sodium e r r o r , and a s t a n d a r d t a p e r j o i n t f o r i n s e r t i o n i n t o the chamber c o n t a i n i n g s e a w a t e r . i z a t i o n , u s u a l l y a t pH 7.4  Standard-  and 10.0 was done w i t h c o m m e r c i a l  b u f f e r s w h i c h a r e a c c u r a t e t o ± 0.01  pH.  Slight  temperature  c o r r e c t i o n s were made when a p p r o p r i a t e . Diaphragm pumps ( F i g u r e 14c,g) were used t o c i r c u l a t e t h e a i r and  seawater  t h r o u g h Tygon and g l a s s t u b i n g . Water temperature was measured w i t h a  telethermometer  ( F i g u r e 14k) h a v i n g a s t a n d a r d t a p e r j o i n t and a g l a s s - c o a t e d sensor.  Temperature c o n t r o l was  a c h i e v e d by p l a c i n g t h e  f l a s k c o n t a i n i n g t h e seawater i n a g l a s s aquarium w a t e r b a t h ( F i g u r e 14o) whose t e m p e r a t u r e was  c o o l e d by a B r o n w i l l  T h e r m o r e g u l a t o r , o r by s i m p l y a d d i n g i c e t o the b a t h . A l l e x p e r i m e n t s were c a r r i e d o u t i n t h e range 12° t o I l l u m i n a t i o n f o r p h o t o s y n t h e s i s was  16°C.  p r o v i d e d by a  G e n e r a l E l e c t r i c "Cool-Beam" lamp; 300 w a t t s  (Figure 14n).  The l i g h t passed t h r o u g h a p p r o x i m a t e l y 8 i n c h e s o f w a t e r and an i n c h o f g l a s s b e f o r e r e a c h i n g the a l g a e .  In early  e x p e r i m e n t s the l i g h t i n t e n s i t y o f 7000 l u x was  controlled  w i t h s h e e t s o f w h i t e f i l t e r paper and measured w i t h a Weston model 756 l i g h t meter. 25,000 t o 50,000 l u x .  I n l a t e r work, l i g h t i n t e n s i t y  was  To measure p C 0 , gas c i r c u l a t e d through the seawater 2  and through the i n f r a r e d gas a n a l y s e r . constant  2  became  w i t h i n one minute o f c i r c u l a t i n g the gas through  the seawater. t o t a l C0  The p C 0  2  To remove water samples f o r r e l e a s e o f the  by a c i d i f i c a t i o n , a water pumping system w i t h a  sampling chamber was In o r d e r  added t o the system.  to r e l e a s e the t o t a l C 0  o f the seawater,  2  water samples were removed w i t h a s y r i n g e v i a a rubber serum stopper.  Any a i r bubbles i n the s y r i n g e were removed  immediately, and 0.5 o r 1.0 ml samples were i n j e c t e d through a second serum s t o p p e r i n t o a 60 ml g l a s s column c o n t a i n i n g acid.  This column  c i r c u l a t i n g system  25 ml of IN h y d r o c h l o r i c o r s u l p h u r i c (Figure 15b) was  part of a closed,  (Figure 15) i n c l u d i n g a pump ( F i g u r e 15f)  and i n f r a r e d gas a n a l y s e r o f t h i s system was  chromatography  (Figure 15e).  250 ml.  The t o t a l volume  The a c i d was bubbled v i g o r o u s l y  by the c i r c u l a t i n g gas p a s s i n g  upwards through a s c i n t e r e d  glass d i s c .  o f CC»  system was  The c o n c e n t r a t i o n reduced b e f o r e  The CC»  2  was  i n the a c i d - r e l e a s e  each i n j e c t i o n by f l u s h i n g the  system w i t h C 0 - f r e e a i r o r 2  2  N. 2  r e l e a s e d by a c i d from 0.5  o r 1.0 ml  samples  used to c a l c u l a t e the t o t a l amount o f i n o r g a n i c carbon  i n the system, expressed as ml o f C 0  2 >  This c a l c u l a t i o n  r e q u i r e d the water volume i n the chamber c o n t a i n i n g  the a l g a ,  the volume o f the a c i d r e l e a s e system, and c o r r e c t i o n s f o r temperature, p r e s s u r e ,  and s o l u b i l i t y o f CO-  i n the a c i d .  •  Figure  15  A system f o r measuring t o t a l i n o r g a n i c  carbon  ( C ) i n seawater by a c i d r e l e a s e and i n f r a r e d gas a n a l y s i s (a) serum  stopper f o r sample i n j e c t i o n  (b) column w i t h HCl (c) manometer (d) tube o f d r i e r i t e  (Magnesium  (e) I n f r a r e d gas a n a l y s e r (f) a i r pump  (IRGA)  perchlorate)  61  The and  system allowed  simultaneous  t o t a l i n o r g a n i c carbon o r C^„  measurements o f  Photosynthetic  pCO  rates  c o u l d then be compared w i t h the c o n c e n t r a t i o n s o f gaseous or i o n i c CC^,  i n o r d e r to determine the s u b s t r a t e o f photo  synthesis. F u r t h e r t h e o r e t i c a l d i s c u s s i o n o f these may  be found  i n the r e f e r e n c e  (1).  techniques  LITERATURE CITED  Tregunna, E.B. and E. Ann Thomas 1968. Measurement of inorganic carbon and photosynthesis i n seawater by pCC"2 and pH a n a l y s i s . Can. J . Bot. 46: 481-485.  PART I I  Evidence  Concerning  t h e Mechanism o f Diamox I n h i b i t i o n  Antimycin A S t i m u l a t i o n of  and  Photosynthesis.^  T h i s a r t i c l e by E.A. (Thomas) J o l l i f f e and E.B. Tregunna has been s u b m i t t e d t o P l a n t P h y s i o l o g y ; ( r e c e i v e d A u g u s t 3/ 1 9 7 2 ) . E.B. Tregunna s u p e r v i s e d the s t u d y .  65  INTRODUCTION  There has been a r e c e n t surge o f i n t e r e s t  concerning  t h e r o l e o f t h e enzyme c a r b o n i c anhydrase (E.C. No. i n photosynthesis.  4.2.1.1)  T h i s enzyme i s p r e s e n t i n c h l o r o p l a s t s  (9,16,22) and i s p r o b a b l y l o o s e l y bound t o the membranes (9,22).  There have been s u g g e s t i o n s t h a t c a r b o n i c anhydrase  f a c i l i t a t e s t r a n s p o r t o f CO 2 a c r o s s the c h l o r o p l a s t membrane. A l t e r n a t i v e l y , o r perhaps a d d i t i o n a l l y , i t may c o r r e c t s u b s t r a t e , CO2, boxylase  supply  (9,22) t o r i b u l o s e d i p h o s p h a t e  (E.C. 4 . 1 . I f ) , f o r i n c o r p o r a t i o n i n t o t h e  synthetic product.  (22).  car-  photo-  The r e c e n t r e p o r t t h a t c a r b o n i c anhydrase  i s d i s t r i b u t e d w i t h r i b u l o s e diphosphate this role  the  carboxylase  favours  The r e p o r t s (9,16,19,22) t h a t Diamox (5-  acetamido-l,3,4-thiadiazole-2-sulphonamide),  a specific  i n h i b i t o r o f a n i m a l c a r b o n i c anhydrase (20) , i n h i b i t s p l a n t c a r b o n i c a n h y d r a s e , i n c o n t r a s t t o e a r l i e r work (7,2) v i d e s a new in  pro-  t o o l f o r s t u d y i n g the r o l e o f c a r b o n i c anhydrase  photosynthesis. Data w i l l be p r e s e n t e d c o n c e r n i n g Diamox e f f e c t s  on  c h l o r o p l a s t s o f s p i n a c h and the m a r i n e u n i c e l l u l a r g r e e n a l g a Dunaliella tertiolecta.  The l a t t e r o r g a n i s m was  originally  s t u d i e d because the f i r s t r e p o r t s o f Diamox i n h i b i t i o n o f p h o t o s y n t h e s i s concerned  the marine green a l g a , U l v a  (16).  66  The  a c t i o n of carbonic  anhydrase might a l s o be to p r o v i d e  protons f o r the l i g h t - i n d u c e d pH s h i f t observed i n c h l o r o plasts  (8,15,31)  .  Recent work by Champigny and o t h e r s cated  t h a t low c o n c e n t r a t i o n s  Antimycin A stimulate chloroplasts. but  ( 5 , 6 ) has i n d i -  ( 0 . 5 x 1 0 ^ to 1 0 ^ M) o f  photosynthetic  CC^ f i x a t i o n i n i s o l a t e d  The mechanism o f t h i s s t i m u l a t i o n i s unknown,  appears to i n v o l v e the c h l o r o p l a s t membrane.  Investi-  g a t i o n o f the p o s s i b l e r e l a t i o n s h i p between A n t i m y c i n A s t i m u l a t i o n and Diamox i n h i b i t i o n o f p h o t o s y n t h e s i s i s a l s o reported  here.  MATERIALS AND METHODS  Preparation  of Chloroplasts  Except where o t h e r w i s e s t a t e d , s p i n a c h were prepared  chloroplasts  (18) from commercially grown spinach;  30 g  chopped l e a v e s were blended 30 seconds i n an O s t e r i z e r a t high  speed i n c o l d 100 ml sucrose T r i s b u f f e r  b u f f e r , 0.4 M s u c r o s e , 0.01M K C l , pH 7.8).  (50 mM  The b r e i s was  s t r a i n e d through 4 l a y e r s o f c h i l l e d c h e e s e c l o t h centrifuged The  supernatant was c e n t r i f u g e d  a t i o n was used f o r s t u d i e s o f 0 14 o  and  one minute a t 200 g_ i n a r e f r i g e r a t e d c e n t r i f u g e . f o r 5 minutes a t 600 g_ and  the p e l l e t suspended i n s u c r o s e - T r i s  C0  Tris  fixation  studies.  2  buffer.  production  This  prepar-  and f o r some  D u n a l i e l l a t e r t i o l e c t a , obtained i n axenic c u l t u r e from Dr. N . J . A n t i a , F i s h e r i e s R e s e a r c h Board o f Canada, Vancouver L a b o r a t o r y , was grown a t 20°C i n e n r i c h e d seawater medium ( 1 ) . Four l i t r e b a t c h e s were b u b b l e d w i t h a i r i n m o d i f i e d Erlenmeyer f l a s k s  (1) on l i g h t e d g l a s s s h e l v e s .  By  a d j u s t i n g t h e i n o c u l u m volume c u l t u r e s were produced i n 3 t o 4 days. A l g a l c e l l s were c o l l e c t e d a t 200 g_ f o r 7 m i n u t e s , a t room t e m p e r a t u r e , washed i n g r i n d i n g medium (100 mM  Tris,  0.001 EDTA, 0.1 M s u c r o s e , pH 7.8) r e c o l l e c t e d , and suspended i n 5 ml g r i n d i n g medium.  The c e l l s were c h i l l e d and r u p t u r e d 2  by 20 seconds s o n i c a t i o n a t 52.5 watts/cm Biosonic I I I . 200 g_.  with a Bronwill  The b r e i s was c e n t r i f u g e d 10 m i n u t e s a t  The s u p e r n a t a n t was c e n t r i f u g e d f o r 10 m i n u t e s a t  600 g_, and t h e p e l l e t resuspended and used t o s t u d y 0 14 p r o d u c t i o n and d a r k  C0  2  assimilation.  2  F o r e x p e r i m e n t s i n t h e p r e s e n c e o f A n t i m y c i n A, c h l o r o p l a s t s were p r e p a r e d as d e s c r i b e d by Champigny and M i g i n i a c Maslow (6) i n an i s o l a t i o n medium c o n t a i n i n g 10 mM  sodium  p y r r o p h o s p h a t e , 330 mM s o r b i t o l , 2 mM NaNO^, 1 mM M n C l , 2  1 mM M g C l , 0.15 mM K H P 0 , 2 mM EDTA, 20 mM N a C l , pH 7.62  2  4  C h l o r o p l a s t s used t o s t u d y t h e l i g h t - i n d u c e d pH s h i f t were p r e p a r e d i n 0.4 m s u c r o s e , 20 mM T r i s , 40 mM KC1, 4 mM MgCl  2  pH 7.8.  The b r e i s was c e n t r i f u g e d f o r one m i n u t e a t  200 g_ t o remove c e l l d e b r i s , t h e n 5 m i n u t e s a t 3000 g_. p e l l e t was resuspended i n washing medium, pH 7.8,  The  (similar  t o t h e i s o l a t i o n medium b u t l a c k i n g T r i s ) and washed 3 t i m e s by c e n t r i f u g i n g a t 3000 g_.  The f i n a l p e l l e t was  suspended  and s t o r e d i n washing medium.  _CO_2  Feedings:  Dark  F o r d a r k f e e d i n g s , .3 ml d i l u t e d c h l o r o p l a s t s were added a t time z e r o t o a h y p o t o n i c , N  scrubbed  2  suspending  medium, w i t h o r w i t h o u t 4 mM Diamox (0.04 M HEPES, 0.5  mM  EDTA, 0.5 mM d i t h i o t h r e i t o l , pH 7.5,  ATP,  20 mM M g C l , 7 mM 14 2  3.3 mM r i b o s e - 5 - p h o s p h a t e , and 2 mM NaH  CO^  [.5  uCi])to  g i v e a f i n a l volume o f 3 m l . 14 C h l o r o p l a s t s were f e d  CC» w i t h o r w i t h o u t Diamox 2  i n the l i g h t i n 3 ml N ~ s c r u b b e d  i s o t o n i c medium adapted  2  from E v e r s o n  ( 9 ) ; (50 mM HEPES, 330 mM  sorbitol,  20 mM  NaCl  1 mM M n C l , 2 mM EDTA, 500 uM K H P 0 ) o r from Champigny and 14 2  2  Miginiac-Maslow (6).  4  For c h l o r o p l a s t  C0  2  feedings i n  Champigny's medium, NaCl was o m i t t e d from, and 50 mM pH 8.1, was  Tris,  added t o the i s o l a t i o n medium, d e s c r i b e d above.  C h l o r o p l a s t c o n c e n t r a t i o n was  a d j u s t e d t o g i v e 30 t o 60 ug  c h l o r o p h y l l per ml. 14 The light.  C0  2  f e e d i n g s were s t a r t e d by t u r n i n g on t h e  A f t e r 20 m i n u t e s , t h e l i g h t was  t u r n e d o f f , and i n  b o t h l i g h t and d a r k f e e d i n g s , t h e r e a c t i o n was  s t o p p e d by  a d d i n g 0.2 ml 10% h y p o c h l o r i c a c i d .  accomplished  by s p o t t i n g 0.25 ml o f a c i d i f i e d  A s s a y was  s u s p e n s i o n on 1 x 2 cm  s t r i p s o f f i l t e r paper, w h i c h were d r i e d i n the fumhood,  69 then c o u n t e d by l i q u i d s c i n t i l l a t i o n i n a t o l u e n e fluor"  "Spectra-  (Amersham/Searle) m i x t u r e as suggested by t h e  manufac t u r e r . L i g h t was p r o v i d e d by t h r e e 300 w a t t G e n e r a l  Electric  "Cool Beam" f l o o d l i g h t s , g i v i n g an average 20,000 l u x . l a r g e experiments,  s m a l l t e s t t u b e s , c l o s e d w i t h serum  In stoppers,  were h e l d i n h o l e s on a l a r g e " P l e x i g l a s s " w h e e l , r o t a t i n g i n the l i g h t .  Up t o 98 samples c o u l d be i l l u m i n a t e d s i m u l -  taneously with t h i s  S t u d i e s o f 02  apparatus.  Production  Oxygen p r o d u c t i o n by i s o l a t e d c h l o r o p l a s t s a t pH 7.8 o r 6.5 was f o l l o w e d w i t h a C l a r k - t y p e Y e l l o w S p r i n g s I n s t r u ment Co. oxygen e l e c t r o d e i n a c l e a r , i c e - c o o l e d , " P l e x i g l a s s " chamber, and r e c o r d e d  on a R i k a d e n k i  recorder.  P o t a s s i u m f e r r i c y a n i d e (0.2 M s t o c k s o l u t i o n ) was used as a H i l l reagent. and  The r e a c t i o n m i x t u r e was s t i r r e d  l i g h t e d a t 30,000 l u x w i t h a G e n e r a l  magnetically,  Electric  "Cool-  Beam" l i g h t . For s t u d i e s o f Diamox o r S u l p h a n i l a m i d e  effects,  the i n h i b i t o r s were d i s s o l v e d d i r e c t l y i n t h e s u s p e n d i n g medium a t t h e a p p r o p r i a t e  concentration.  The L i g h t - i n d u c e d pH S h i f t C h l o r o p l a s t s prepared the l i g h t - i n d u c e d pH s h i f t .  as d e s c r i b e d were used to study A Radiometer PHM 53 S p e c i f i c  Ion Meter, was used on expanded s c a l e ,  (one pH u n i t  full  s c a l e ) to measure pH, which was r e c o r d e d on a R i k a d e n k i r e c o r d e r , 100 mV f u l l initial  s c a l e , 0.2 seconds response  pH o f a 15 ml c h l o r o p l a s t suspension  time.  The  350 ug c h l / m l ,  was a d j u s t e d t o 6.0 to 6.1 i n the dark, and allowed t o s t a b i l i z e a t about 10°C. the s o - c a l l e d  The l i g h t was then turned on and  " l i g h t s t e a d y - s t a t e " pH allowed  A f t e r 2 to 3 minutes the l i g h t  to s t a b i l i z e .  (two 300 watt G.E. C o o l Beam  lamps) was turned o f f , and the suspension was darkened f o r at  l e a s t 5 minutes t o a l l o w the suspension  previous  "dark steady s t a t e " pH v a l u e .  p h o r y l a t i o n was induced by FMN (10  t o r e a c h the  C y c l i c photophos-  M).  Preparation of Inhibitors 14 For s t u d i e s o f 0  2  production or  C0  2  f i x a t i o n a t pH  7.8, Diamox was d i s s o l v e d i n the a p p r o p r i a t e b u f f e r e d medium at  the r e q u i r e d s t r e n g t h and then used t o suspend the  chloroplasts.  F o r s t u d i e s of 0  2  p r o d u c t i o n a t pH 6.5, the  Diamox s o l u t i o n was a c i d i f i e d a few minutes b e f o r e use. Sulphanilamide  was handled  i n the same manner.  When Diamox was used t o study the pH s h i f t i n unbuffered  sucrose medium, i t was d i s s o l v e d by magnetic  s t i r r i n g , w i t h a c o n t i n u a l slow a d d i t i o n o f 0.1 N NaOH, s u f f i c i e n t t o keep t h e pH a t 7.5. A n t i m y c i n A, t o p r o v i d e a f i n a l c o n c e n t r a t i o n o f 5 uM, was d i s s o l v e d i n a b s o l u t e e t h a n o l j u s t p r i o r t o u s e . The  f i n a l a l c o h o l c o n c e n t r a t i o n was never h i g h e r t h a n 0.3  percent.  C o n t r o l s were r u n w i t h t h i s c o n c e n t r a t i o n o f a l -  c o h o l i n t h e absence o f A n t i m y c i n A. The i n h i b i t o r s were  always f r e s h l y p r e p a r e d  use and k e p t i n t h e r e f r i g e r a t o r o r on i c e u n t i l  before actually  used. A n t i m y c i n A was o b t a i n e d from N u t r i t i o n a l C o r p o r a t i o n ; ATP, r i b o s e - 5 - p h o s p h a t e ,  Biochemical  and FMN from Sigma,  and HEPES from C a l b i o c h e m i c a l Co. K o c h - L i g h t L a b o r a t o r i e s L t d . s u p p l i e d t h e Diamox and S u l p h a n i l a m i d e came f r o m J.T. B a k e r C h e m i c a l Co.  ATP -  adenosine-5'-triphosphate  Diamox - (Acetazolamide) sulphonamide  5-acetamido-l,3,4-thiadiazole-2-  FMN - r i b o f l a v i n monophosphate n u c l e o t i d e EDTA - d i s o d i u m  (Ethylene d i n i t r i l o ) - t e t r a c e t a t e  HEPES N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 e t h a n e S u l p h a n i l a m i d e - p - a m i n o b e n z e n e sulphonamide  sulphonic  acid  RESULTS The E f f e c t of_ Diamox on P h o t o s y n t h e s i s ; the L i g h t  14  C0  Fixation in  9  14 F i g u r e 16 shows the e f f e c t o f 4 mM  Diamox on  C0  2  f i x a t i o n by i l l u m i n a t e d i n t a c t s p i n a c h c h l o r o p l a s t s under l i m i t i n g C0  2  concentration  pyrrophosphate. to the  (2 mM)  Photosynthesis  and i n the p r e s e n c e o f i s i n h i b i t e d by 80%  compared  control. I n F i g u r e 20, the l o w e r l i n e shows the e f f e c t  on  s p i n a c h c h l o r o p l a s t s of a p r o g r e s s i v e i n c r e a s e i n Diamox concentration. above 2 mM;  L i t t l e increase i n i n h i b i t i o n i s apparent  a t t h i s p o i n t about 70% i n h i b i t i o n was  As p r e v i o u s l y m e n t i o n e d , 4 mM i n h i b i t i o n t o 80%. a t 1 mM,  and 0.4  photosynthesis  Diamox may  increase  observed. the  U s u a l l y , above 60% i n h i b i t i o n i s o b s e r v e d  t o 0.5  mM  gave 50% i n h i b i t i o n .  appeared i n s e n s i t i v e t o the  The r e s i d u a l  inhibitor.  Under b o t h l i g h t l i m i t i n g c o n d i t i o n s , and n e a r l i g h t s a t u r a t i o n , 50% i n h i b i t i o n was  o b s e r v e d a t about the same  Diamox c o n c e n t r a t i o n , t h a t i s 0.4 was  2  t o 0.5  mM,  when NaHCO^  mM.  14 CO2  F i x a t i o n i n the Dark F i g u r e 16 a l s o shows the e f f e c t o f 4 mM  Diamox on  14 C0  f i x a t i o n by i n t a c t s p i n a c h c h l o r o p l a s t s f e d i n the d a r k 14 and p r o v i d e d w i t h ATP, r i b o s e - 5 - p h o s p h a t e , and NaH CO^. 2  .  Figure  16  The by  e f f e c t o f 4mM isolated  w i t h PP^:  Diamox on  spinach  CC^  chloroplasts:  i n the dark w i t h  ATP  fixation i n the  light  vj  CO  Under t h e s e c o n d i t i o n s On t h e c o n t r a r y ,  (2 mM NaH  CO3), Diamox d i d n o t i n h i b i t .  i t somehow i n d u c e d a l a r g e i n c r e a s e i n  14 CO2 f i x a t i o n , w h i c h was as h i g h a s 170% o f t h e c o n t r o l . This increase v a r i e d i n absolute repeatably.  value, but occurred  A s i m i l a r s t i m u l a t i o n was a l s o o b s e r v e d i n  Dunaliella tertiolecta chloroplasts The E f f e c t o f Diamox on t h e H i l l  (not shown).  Reaction  F i g u r e 17 shows t h e e f f e c t o f 4 mM Diamox on O2 production  by i n t a c t s p i n a c h c h l o r o p l a s t s i n t h e p r e s e n c e o f  potassium f e r r i c y a n i d e .  An i n h i b i t i o n o f 70% i s shown. 14  i s comparable t o t h e i n h i b i t i o n o f The c o n c e n t r a t i o n  o f C0  2  CO2 f i x a t i o n  This  (Figure 1 6 ) .  i s l o w , 0 t o 2 mM; no NaHCO^ was  added, and f r e s h l y d i s t i l l e d w a t e r was used t o p r e p a r e t h e buffer.  The e f f e c t i s l a r g e l y overcome by 10 mM NaHCO^/  under s a t u r a t i n g l i g h t c o n d i t i o n s .  The c o n t r o l r a t e was  about 20 uM oxygen/hr/mg c h l o r o p h y l l . S u l p h a n i l a m i d e , a t 0.5 mM and 1 mM, d i d n o t a f f e c t O2 p r o d u c t i o n  i n spinach.  A t 4 mM, Diamox i n h i b i t e d t h e H i l l r e a c t i o n by 70 t o 80%  i nDunaliella tertiolecta chloroplasts.  i n h i b i t e d 50% by 0.5 mM s u l p h a n i l a m i d e . described 7.8.  They were  The e x p e r i m e n t s  above were c o n d u c t e d i n 50 mM T r i s b u f f e r a t pH  We a l s o s t u d i e d t h e e f f e c t o f Diamox on t h e H i l l  r e a c t i o n o f s p i n a c h c h l o r o p l a s t s a t pH 6.5, because t h e l i g h t - i n d u c e d pH s h i f t i s b e s t s t u d i e d  a t t h i s pH v a l u e .  F i g u r e 17  The  e f f e c t o f D i a m o x and  p r o d u c t i o n by  isolated  s u l p h a n i l a m i d e on  s p i n a c h and  c h l o r o p l a s t s , i n the presence ferricyanide  of  0  2  Dunaliella potassium  76 The  r e s u l t s  not  i n h i b i t .  i n h i b i t i o n 70  usual The  pH  s h i f t  sucrose 6.0  a  by  19  f e l l  The  i n i t i a l t h e pH  and by  The  lower  on  t h e pH  inhibitor, is  The a c t u a l  When  the l i g h t  reaching  T h e FMN  curve  had a  i n Figure  s h i f t .  Except  apparent was  that  turned  i t s maximum,  19  constant turned  was  shows  o n , t h e pH  the  higher  .15  was  to  action. o f  o f t h e  the c o n t r o l .  a f f e c t s  t o f a l l  to depend  the e f f e c t  as  rose  constant.  buffering  f o r the presence t h e same  turned o f f  the s h i f t , s h i f t  t h e pH  maintained  appeared  pH  a t  on  and remained  strong  Diamox  i tbegan  and  unbuffered  o f fand  i n the dark  a l lc o n d i t i o n s were  immediately  value  o f decay.  i n the c o n t r o l .  concentration.  Shift  was  the l i g h t  the greater  .17  the  control  pH  was  leveled  When dark  than  an  o f the l i g h t - i n d u c e d  The pH  achieved;  i n i t i a l  Diamox  r a t e  o f both  the l i g h t  one minute,  o f decrease  the  4 mM  a t a  course  M).  When  to the previous  change  less  c h l o r o p l a s t s i n an  (10  FMN  Diamox d i d  6.5.  to  t h e time  i n the l i g h t .  rate  was  the Light-induced  i n t h e dark.  state  pH  on  This  mixture  6.4  2 mM,  A t  employed, however,  observed  spinach  with  was  achieved.  shows  i n t a c t  rapidly,  the  on  Diamox  i n h i b i t i o n  medium,  steady  was  o f Diamox  t o 6.1,  rose  mM  c h l o r o p l a s t s was  E f f e c t  18.  i n Figure  o f the reaction  Figure pH  50%  t o 80%  i n h i b i t e d  4  When  o f  f i n a l  The  a r e shown  t h e pH  r a p i d l y ,  o f fr a p i d l y ,  I t  s h i f t . b u t on and  F i g u r e 18  The  e f f e c t o f pH  h i b i t i o n o f 0^ chloroplasts  6.5  and  time on  p r o d u c t i o n by  Diamox  Spinach  vj  Figure  19  The pH  e f f e c t of s h i f t by  4 mM  Diamox on  light-induced  i s o l a t e d spinach chloroplasts  u n b u f f e r e d s u c r o s e medium  r  the  in  78  P  H 6.30  6.28  6.26 .LIGHT  OFF  6.24  6.22  6.20  6.18  6.16  6.14  6.12  6.10  LIGHT  I  6.08  p  H 6.06L  •LIGHT  0  OFF  ON  2  3 TIME  4 MINUTES  5  7  8  r e t u r n e d t o t h e d a r k v a l u e , w h i c h was t h e n m a i n t a i n e d . A f t e r t h e d a r k pH v a l u e was a c h i e v e d i n t h e l i g h t , t u r n i n g o f f t h e l i g h t d i d n o t a f f e c t t h e pH.  I f t h e l i g h t was  t u r n e d o f f b e f o r e t h e pH had r e t u r n e d t o t h e d a r k v a l u e the decay r a t e was a c c e l e r a t e d ; however, t h e f i n a l pH reached was t h e same.  T h i s time c o u r s e was r e p e a t e d s e v e r a l  t i m e s by t u r n i n g t h e l i g h t on and o f f a t a p p r o p r i a t e i n t e r v a l s , always a l l o w i n g a t l e a s t 5 m i n u t e s d a r k r e c o v e r y t i m e . Because Diamox has a b u f f e r i n g e f f e c t , t h e a c t u a l i n i t i a l number o f hydrogen i o n s pumped appeared v e r y n e a r l y equal to the c o n t r o l ; the i n h i b i t o r a f f e c t s not the extent b u t t h e c o u r s e o f t h e phenomenon.  The I n t e r a c t i o n o f A n t i m y c i n A w i t h Diamox Under c o n d i t i o n s a p p r o a c h i n g  l i g h t saturation 14  15,000 l u x ) 5 uM A n t i m y c i n A s t i m u l a t e s  (above  CC^ f i x a t i o n by  i n t a c t s p i n a c h c h l o r o p l a s t s by 100%, as shown i n F i g u r e 20. T h i s f i g u r e a l s o shows t h a t as t h e Diamox c o n c e n t r a t i o n i n c r e a s e d from 0.0 t o 10 mM, achieved.  50 t o 60% i n h i b i t i o n was  Over t h i s range o f Diamox c o n c e n t r a t i o n , a d d i t i o n  o f A n t i m y c i n A c o m p l e t e l y overcame t h e Diamox i n h i b i t i o n . The r e s u l t was t h a t samples c o n t a i n i n g 0.1 mM t o 1 mM Diamox p l u s 5 uM A n t i m y c i n A, f i x e d t h e same a b s o l u t e amount o f 14 CO2 as t h e c o n t r o l p l u s A n t i m y c i n A.  Not o n l y d i d  A n t i m y c i n A overcome Diamox i n h i b i t i o n b u t i t was c a p a b l e o f  The  i n t e r a c t i o n of  and  5  i n c r e a s i n g Diamox 14  A n t i m y c i n A on  saturating light,  by  CC^  concentration  . . . fixation in  i s o l a t e d spinach  chloroplasts  80  480  0.0  .1  .2  .3  4  .5  DIAMOX mM  .6  .7  .8  ,.9  1.0  81 s t i m u l a t i n g f i x a t i o n to double the u n i n h i b i t e d c o n t r o l  The  c o n t r o l r a t e was 6.0 uM CC>2 f i x e d / h r / m g c h l o r o p h y l l . concentration  Ethanol  was l e s s t h a n 0.3% and had no e f f e c t on t h e  control. I t i s a l s o n o t e w o r t h y t h a t c h l o r o p l a s t s i n h i b i t e d by various  amounts o f Diamox were s t i m u l a t e d  t o t h e same a b s o l u t e  value.  The r e s u l t i s t h a t t h e e f f e c t i v e s t i m u l a t i o n i n t h e  p r e s e n c e o f 1 mM Diamox, was c l o s e t o 500%, compared t o 100% i n the c o n t r o l .  Diamox i n h i b i t i o n appears t o s e n s i t i z e t h e  c h l o r o p l a s t s t o s t i m u l a t i o n by A n t i m y c i n A, i t s e l f an i n h i b i t o r of c y c l i c  photophosphorylation.  When t h e l i g h t i n t e n s i t y was n o t s u f f i c i e n t t o s a t u r a t e the c o n t r o l r a t e o f p h o t o s y n t h e s i s as i n F i g u r e 14 did not stimulate  21, A n t i m y c i n A  CC»2 f i x a t i o n o v e r t h e c o n t r o l r a t e .  The  p e r c e n t a g e i n h i b i t i o n by Diamox, however, was about t h e same as when l i g h t was n e a r l y s a t u r a t i n g , t h a t i s , about 50% b y 0.5 mM.  The e f f e c t o f A n t i m y c i n A i n c o m b i n a t i o n w i t h  Diamox, under c o n d i t i o n s where i t d i d n o t n o r m a l l y was t o r a i s e t h e f i x a t i o n r a t e t o t h e c o n t r o l r a t e . Diamox c o n c e n t r a t i o n  increased  stimulate, As  t o 0.9 mM, t h e e f f e c t i v e  s t i m u l a t i o n by A n t i m y c i n A r o s e r a p i d l y from n o t h i n g t o a peak o f a l m o s t 170%. and  The e f f e c t t h e n d i m i n i s h e d r a p i d l y  as t h e degree o f i n h i b i t i o n l e v e l e d o f f around 2.0 mM  Diamox, t h e s t i m u l a t i o n a l s o began t o l e v e l o f f a t 90 t o 100%.  Once more Diamox i n h i b i t i o n appeared t o s e n s i t i z e  the system t o A n t i m y c i n A.  Figure  21  The  i n t e r a c t i o n o f i n c r e a s i n g Diamox  concentration  14 and  5 uM A n t i m y c i n  A on  CC>2 f i x a t i o n  s a t u r a t i n g l i g h t by. i s o l a t e d  in  non-  spinach c h l o r o p l a s t s  82  200  180  0.0  .4  .8  1.2 DIAMOX  1.6 mM  2.0  2.4  83  The  E f f e c t o f A n t i m y c i n A ori t h e L i g h t - i n d u c e d pH S h i f t A c h l o r o p l a s t p r e p a r a t i o n was i l l u m i n a t e d and a l l o w e d  to a c h i e v e a steady pH v a l u e .  A n t i m y c i n A was t h e n added  to g i v e a c o n c e n t r a t i o n o f 10 yM. The r e s p o n s e was an immediate steady  s t a t e pH v a l u e .  Antimycin A  r a p i d decay t o t h e d a r k added i n t h e d a r k d i d  n o t a f f e c t t h e pH v a l u e .  The  S t a b i l i t y o f Diamox Diamox d i s s o l v e d i n T r i s - b u f f e r e d s u c r o s e m e d i a , a t  pH 7.6 i s s a t u r a t e d a t 4 t o 5 mM, and l o s e s i t s a c t i v i t y w i t h i n 24 hours under r e f r i g e r a t i o n . d i s s o l v e d i n unbuffered  I f t h e compound i s  s u c r o s e media i n i t i a l l y  adjusted to  pH 7.5, t h e s o l u t i o n r a p i d l y d r o p s below pH 6, and l o s e s i t s a b i l i t y to i n h i b i t .  We have found t h a t i f t h e pH i s  maintained  above pH 7.0 by c o n t i n u a l l y a d d i n g 0.1 N NaOH w h i l e t h e compound d i s s o l v e s , a c t i v i t y i s p r e s e r v e d , and t h e s o l u t i o n may be s t o r e d i n i c e f o r s e v e r a l hours and used t o suspend chloroplasts.  The pH was a d j u s t e d t o 6.0 - 6.1 j u s t  t u r n i n g on t h e l i g h t  before  when s t u d y i n g t h e pH s h i f t .  When t h e f e r r i c y a n i d e - s u p p o r t e d H i l l r e a c t i o n was s t u d i e d , 4 mM Diamox began t o show some l o s s o f i t s i n h i b i t o r y p r o p e r t i e s a t t h e end o f one hour a f t e r i t was acidified 20 mM  t o pH 6.1. T h i s s o l u t i o n was b u f f e r e d w i t h  Tris.  84 DISCUSSION  The o b s e r v a t i o n  r e p o r t e d h e r e t h a t Diamox i n h i b i t s  the p o t a s s i u m f e r r i c y a n i d e - m e d i a t e d  H i l l r e a c t i o n , b u t does  not i n h i b i t c a r b o n f i x a t i o n by c h l o r o p l a s t s p r o v i d e d  with  ATP i n t h e d a r k , i n d i c a t e s t h a t t h i s compound somehow i n h i b i t s the l i g h t r e a c t i o n of photosynthesis.  This r e s u l t  s u p p o r t s t h e work o f Swader and J a c o b s e n ( 3 0 ) . They r e p o r t t h a t Diamox ( A c e t a z o l a m i d e ) i n h i b i t s p h o t o s y s t e m I I near the w a t e r s p l i t t i n g r e a c t i o n . The f a i l u r e o f Diamox t o i n h i b i t ATP s u p p o r t e d d a r k 14 f i x a t i o n of carbonic  CO2 i s s i g n i f i c a n t  anhydrase  that  w h i c h a s s o c i a t e s w i t h r i b u l o s e - 1 , 5-  diphosphate carboxylase, photosynthetic  i n l i g h t o f proposals  i s t h e r a t e l i m i t i n g enzyme i n  carbon f i x a t i o n .  Diamox has no a p p a r e n t i n h i b i t o r y  When ATP i s made a v a i l a b l e , e f f e c t on t h e c a r b o n f i x i n g  system. The g r o s s e f f e c t o f a Diamox i n h i b i t i o n on CO2 a t i o n by c h l o r o p l a s t s (9) and whole p l a n t s  fix-  (16) i n t h e l i g h t  would a l s o a r i s e from a b l o c k a g e o f t h e l i g h t r e a c t i o n . 14 o b s e r v e up t o 80% i n h i b i t i o n o f l i g h t - s u p p o r t e d in  spinach  chloroplasts.  CO2  We  fixation  Swader and J a c o b s e n (30) s u g g e s t  t h a t Diamox may be an e l e c t r o n - t r a n s p o r t i n h i b i t o r l i k e CMU and DCMU because i t has a m e t h y l a t e d c a r b o n y l a m i d e t o an u n s a t u r a t e d in a similar  ring  fashion.  attached  s t r u c t u r e ; w i t h such i t may behave He i n d i c a t e s t h a t Diamox  inhibition  85 o f p h o t o s y n t h e s i s may have l i t t l e  t o do w i t h i n h i b i t i o n o f  c a r b o n i c a n h y d r a s e , u n t i l now i t s assumed r o l e . C o n s i d e r i n g the r e s u l t s we have p r e s e n t e d , however, we s u g g e s t t h a t the i n t e r p r e t a t i o n t h a t Diamox  inhibits  e l e c t r o n t r a n s p o r t , e x c l u s i v e o f i t s e f f e c t on c a r b o n i c a n h y d r a s e , i s n o t n e c e s s a r i l y the o n l y v a l i d e x p l a n a t i o n o f the known  facts.  Diamox o b v i o u s l y i n h i b i t s t h e l i g h t r e a c t i o n ; i t i s also considered  t o be a v e r y s p e c i f i c i n h i b i t o r o f c a r b o n i c 14  anhydrase ( 2 0 ) . F i x a t i o n o f  C0  2  c h l o r o p l a s t s i s markedly i n h i b i t e d .  i n t h e l i g h t by i s o l a t e d Our r e s u l t s  those o f E v e r s o n ( 9 ) . We may c o n c l u d e  support  t h a t Diamox has two  s i t e s o f a c t i o n , one a t t h e l i g h t r e a c t i o n and a n o t h e r  a t the  enzyme l e v e l , o r t h a t c a r b o n i c anhydrase has two r o l e s ; t h e f i r s t i n f a c i l i t a t i n g C0  2  second i n t h e p h o t o c h e m i c a l  t r a n s p o r t and s t o r a g e , and the act itself.  The f a c t t h a t 50%  i n h i b i t i o n o f the p a r t i a l l y p u r i f i e d enzyme i s a c h i e v e d by one t w e n t y - f i f t h (0.02 mM)  t h a t r e q u i r e d f o r 50% i n h i b i t i o n  o f p h o t o s y n t h e s i s as p o i n t e d o u t by Swader and Jacobsen ( 3 0 ) , i s inadequate for  reason  t o assume two d i f f e r e n t s i t e s o f a c t i v i t y  Diamox. C a r b o n i c anhydrase may p l a y a r o l e i n r e g u l a t i n g  p r o t o n g r a d i e n t s i n c h l o r o p l a s t s ( 1 4 ) . There i s c o n s i d e r a b l e evidence  t h a t the l i g h t - i n d i c e d g e n e r a t i o n o f protons  c h l o r o p l a s t membranes i s c l o s e l y l i n k e d t o  across  photophosphorylation  (15,31) and may compete f o r a h i g h energy i n t e r m e d i a t e o r  a c t u a l l y be how  that intermediate  (15,17).  This would  explain  Diamox, a s p e c i f i c i n h i b i t o r of the enzyme somehow  b l o c k s ATP  formation  state value  and  causes a breakdown i n the  of the l i g h t - i n d u c e d pH  In the presence o f FMN, plasts i s cyclic.  The  steady  shift.  electron transport i n chloro-  e f f e c t o f 5 uM A n t i m y c i n A,  which  p r e f e r e n t i a l l y i n h i b i t s c y c l i c photophosphorylation  (2) i n  causing  a r a p i d decay o f the pH  thesis  t h a t we  are o b s e r v i n g  electron transport. induced pH was  a p r o t o n f l u x a t t r i b u t a b l e to c y c l i c t h a t the  light-  s h i f t i n chromatophores o f R h o d o s p i r i l i u m  rubrum  i n h i b i t e d by I f ATP  s h i f t s , and  Stedingk  2 x 10  production  i f carbonic  anhydrase should  (29) r e p o r t e d  ^ (2uM)  Antimycin  i s inseparably  (or t h a t l i n k e d to any  associated proton transport.  electron transport.  l i n k e d to p r o t o n  then an i n h i b i t o r o f  a f f e c t both c y c l i c and  e l e c t r o n t r a n s p o r t and  A.  anhydrase i s r e q u i r e d  a c t i v i t y o f these s h i f t s ,  transport  change supports the  for  optimal  carbonic  non-cyclic  electron  p h o s p h o r y l a t i o n ) and  Diamox i n h i b i t s  the  non-cyclic  the p r o t o n s h i f t supported by  Although an e f f e c t on p r o t o n  cyclic  shifts  o c c u r r i n g as a r e s u l t o f n o n - c y c l i c e l e c t r o n t r a n s p o r t not been demonstrated, these would be expected from e f f e c t s on oxygen The (Figure 19)  has  the  production.  shape o f the pH  change i n the presence o f Diamox  i n d i c a t e s t h a t an i n i t i a l  c o u l d not be s u s t a i n e d .  s h i f t occurred,  but  Is t h i s because o f a p r o t o n shortage  87  or i s the passive increased?  l e a k a g e r a t e back a c r o s s t h e membrane  D u r i n g t h e decay i n t h e l i g h t and b e f o r e t h e d a r k  v a l u e i s reached, removal o f l i g h t i n c r e a s e s  the r a t e o f  decay, so some i n w a r d f l u x was o c c u r r i n g i n t h e l i g h t . recovery occurred i n the dark. a c c o u n t s f o r t h e phenomenon. i n f l u x i s apparently  Also,  Probably a shortage of protons The a c t u a l s i t e o f p r o t o n  t h e t h y l a k o i d membrane (15); changes  w i t h whole c h l o r o p l a s t s r e f l e c t t h i s .  C a r b o n i c anhydrase  o c c u r s i n t h e stroma (22) o r a t t a c h e d  probably l o o s e l y to  the membranes  (9). I f a continuing  supply o f protons i s  c u r t a i l e d by i n h i b i t i o n o f t h e enzyme, t h e s t e a d y s t a t e  will  decay, and t h e l i g h t - d r i v e n i n f l u x w i l l f a i l , w h i l e t h e normal p a s s i v e  l e a k remains  unaffected.  Graham, A t k i n s , Reed, P a t t e r s o n , reported  and S m i l l i e ( 1 3 )  no e f f e c t o f Diamox on l i g h t - i n d u c e d pH  gradients  i n c h l o r o p l a s t s i s o l a t e d from Chlamydomonas o r Pisum.  On  the o t h e r hand, E v e r s o n (10) and E v e r s o n and Graham (11) reported  f i r s t an i n h i b i t i o n , b u t on f u r t h e r s t u d i e s , a  s t i m u l a t i o n o f t h e i n i t i a l r a t e and f i n a l magnitude o f t h e pH change i n i n t a c t c h l o r o p l a s t s , by E t h o x y z o l a m i d e , a l s o a s p e c i f i c i n h i b i t o r of carbonic  anhydrase.  The e f f e c t was  overcome by 2 mM NaHCO^, and d i d n o t o c c u r i n b r o k e n c h l o r o plasts.  I t was suggested t h a t t h e s e c o n t r a d i c t o r y r e s u l t s  depend on t h e c o n d i t i o n o f t h e c h l o r o p l a s t p r e p a r a t i o n . any  event, our observations  I t i s probable that not only  In  a r e somewhat d i f f e r e n t a g a i n . the c o n d i t i o n of the c h l o r o -  88  p l a s t s , b u t the s t a b i l i t y o f the i n h i b i t o r may  account f o r  these v a r i a b l e r e s u l t s . The  l i n k a g e o f Diamox w i t h CMU  b a s i s can be c o u n t e r e d sulphanilamide.  and DCMU on a s t r u c t u r a l  by a c o m p a r i s o n o f Diamox w i t h  Both Diamox and s u l p h a n i l a m i d e s h a r e  a 14  sulphonamide g r o u p i n g .  Furthermore, although spinach  CC>2  f i x a t i o n , H i l l r e a c t i o n , and c a r b o n i c anhydrase a r e u n a f f e c t e d by s u l p h a n i l a m i d e , t h e s e compounds a r e known t o i n h i b i t c a r b o n i c anhydrase (20)/from a n i m a l s and (16) and o t h e r m a r i n e a l g a e inhibited i n Ulva.  Photosynthesis  Sulphanilamide  i n the m a r i n e p h y t o p l a n k t o n we have i n d i c a t e d .  (4).  from U l v a  pertusa  i s also  i n h i b i t s the H i l l r e a c t i o n  alga Dunaliella t e r t i o l e c t a  as  Presumably the r e s u l t s r e f l e c t some  d i f f e r e n c e s i n the s t r u c t u r e o f the enzyme.  At l e a s t  two  forms o f c a r b o n i c anhydrase e x i s t e d i n t w e l v e p l a n t s p e c i e s tested  (21).  M i n o r d i f f e r e n c e s m i g h t n o t be u n e x p e c t e d  between d i f f e r e n t p l a n t g r o u p s , such as l a n d p l a n t s and marine a l g a e . I f our i n t e r p r e t a t i o n i s c o r r e c t , some c u r i o u s r e q u i r e m e n t s o f c h l o r o p l a s t s m i g h t be e x p l a i n e d . w e l l documented t h a t C 0  2  o r HCO~  I t has been  i s required for e f f i c i e n t  f u n c t i o n i n g o f the H i l l r e a c t i o n (12,32).  I f carbonic  anhydrase f u n c t i o n s to r e g u l a t e p r o t o n s u p p l y , through i o n i z a t i o n of carbonic a c i d ,  the  89 H 0 + C0 * 2  2  the requirement  7  H C0 ^—-7H 2  3  +  + HCO~  would be e x p l a i n e d , and h e r e i n would be an  e x p l a n a t i o n o f Good's r e p o r t no e f f e c t i n " C 0 - d e p l e t e d "  (12) t h a t u n c o u p l i n g chloroplasts.  2  a g e n t s have  I f protons  from  R" CO.j a r e r e q u i r e d f o r e l e c t r o n t r a n s p o r t , t h e r e w o u l d b e 2  nothing  to uncouple i n t h e i r  absence.  I t i s a l s o known t h a t Diamox i n h i b i t i o n  c a n be o v e r -  come b y i n c r e a s i n g HCO^ c o n c e n t r a t i o n , u s u a l l y a b o v e 5 t o 10 mM. (20), by  The i n h i b i t i o n  affects hydration of C0  s o we may s i m p l y b e l o o k i n g a t a n e g a t i o n  substrate competition.  Swader and J a c o b s o n  2  competitively of inhibition (30) f o u n d  t h i s r e l i e f o f i n h i b i t i o n b y HCO^ p u z z l i n g i n t e r m s o f i n h i b i t i o n o f the l i g h t r e a c t i o n . exists  The c o n f u s i o n no l o n g e r  i f carbonic anhydrase i s t h e s i t e o f i n h i b i t i o n . One o t h e r i m p o r t a n t  f a c t must n o t be o v e r l o o k e d .  are d e a l i n g w i t h a system which has a c o n s i d e r a b l e rate of reaction.  pump t o o p e r a t e .  uncatalyzed  A s HCO^ c o n c e n t r a t i o n i s i n c r e a s e d  spontaneous r e a c t i o n might supply Conceivably,  sufficient  this  protons  could occur  Diamox i n h i b i t i o n were n o n - c o m p e t i t i v e .  We  the f o r the  even i f  This l i n e o f thought  m i g h t a l s o e x p l a i n t h e i n h i b i t i o n o f growth and p h o t o s y n t h e s i s observed  i n very high C0  2  concentrations  (24) a b o v e 5%.  The  c h l o r o p l a s t may " o v e r l o a d " , t h e " p r o t o n m o t i v e f o r c e " d i s s i p a t e d  o r overwhelmed by h i g h I^CO-j w i t h i n the t h y l a k o i d making the maintenance o f an e f f e c t i v e p r o t o n g r a d i e n t i m p o s s i b l e . A f u r t h e r f a c t l i n k i n g c a r b o n i c anhydrase t o Diamox i n h i b i t i o n i s the e f f e c t o f C l c e n t r a t i o n o f 10 mM,  ion.  This anion a t a con-  causes 50% i n h i b i t i o n o f  partially  p u r i f i e d c a r b o n i c anhydrase from s p i n a c h c h l o r o p l a s t s ( 9 ) . Good (12) r e p o r t e d t h a t "C0  2  depleted" chloroplasts i n  w h i c h the H i l l r e a c t i o n i s s u p p r e s s e d , (30%) by 70 mM ion  NaCl.  are f u r t h e r i n h i b i t e d  Are t h e s e e f f e c t s r e l a t e d ?  The C l "  c o n c e n t r a t i o n s a r e d i f f e r e n t , b u t we m i g h t e x p e c t  the  i n v i t r o o r i s o l a t e d enzyme t o be more e a s i l y a v a i l a b l e t o the i o n than when i n v i v o . f u r t h e r by the r e q u i r e m e n t  The  s i t u a t i o n i s complicated  o f the H i l l r e a c t i o n f o r C l  somewhere near the w a t e r s p l i t t i n g r e a c t i o n . compatible ion  ion,  This i s  a t l e a s t , w i t h the a p p a r e n t r e q u i r e m e n t  for Cl  i n the p r o t o n pump; H . i o n uptake i s accompanied +  s t o i c h i o m e t r i c a l l y by C l  i o n uptake (15).  o f the H i l l r e a c t i o n f o r c h l o r i d e may  The  requirement  stem from the r o l e  o f c h l o r i d e i n the p r o t o n pump. The r e p o r t s (5,26) o f an i n c r e a s e d a f f i n i t y Km)  o f c h l o r o p l a s t s f o r CC^/  (lower  i n the p r e s e n c e o f A n t i m y c i n  were i n t r i g u i n g . A n t i m y c i n A i s a b l e t o overcome o r b l o c k by Diamox.  inhibition  This occurs not o n l y i n l i g h t s a t u r a t i n g con-  d i t i o n s , where A n t i m y c i n A produces a s t r o n g s t i m u l a t i o n  A  o v e r t h e Diamox-free c o n t r o l  (Figure 19), but i n l i g h t  l i m i t i n g c o n d i t i o n s , where A n t i m y c i n A does n o t s t i m u l a t e the c o n t r o l .  Diamox " s e n s i t i z e s " t h e c h l o r o p l a s t t o  stimulation. More r e c e n t work (25) t h a n  (5) and (26) i n d i c a t e s  t h a t t h e mechanism o f A n t i m y c i n A s t i m u l a t i o n i s n o t t o i n c r e a s e HCO^ o r CO2 c o n c e n t r a t i o n w i t h i n t h e c h l o r o p l a s t . A d d i t i o n o f A n t i m y c i n A caused an i n c r e a s e r a t h e r t h a n a decrease  i n t h e c o n c e n t r a t i o n o f t h e a c c e p t o r r i b u l o s e - 1 , 5-  d i p h o s p h a t e ; when t h e HCO^ c o n c e n t r a t i o n was i n c r e a s e d d i r e c t l y , the s i z e of the acceptor pool f e l l . Bassham (25) suggested  S h a c t e r and  t h a t A n t i m y c i n A somehow s t i m u l a t e d  carboxydismutase  and hexosemonophosphatase ( f r u c t o s e - 1 ,  6-diphosphatase,  sedoheptulose-1,  7-diphosphatase,  6-phosphatase) s i n c e PGA a c c u m u l a t e d , f r u c t o s e - 1 , 6-diphosphate,  and t h e p o o l s i z e s o f  fructose-6-phosphate,  h e p t u l o s e - 1 , 7-diphosphate d e c r e a s e d .  fructose-  and sedo-  The e f f e c t was n o t  c o n s i d e r e d t o be v i a an e f f e c t on ATP l e v e l s , as t h e s e 32 a u t h o r s f e l t t h a t t h e observed  25% a p p a r e n t d r o p i n  P  f i x e d c o u l d be due t o i n c r e a s e d t u r n o v e r d u r i n g CC^ f i x a t i o n . Diamox a c t i v i t y and i t s i n t e r a c t i o n w i t h A n t i m y c i n A p r o v i d e e v i d e n c e f o r a mechanism o f t h e s t i m u l a t i o n . S i n c e Diamox i n h i b i t s t h e l i g h t r e a c t i o n , i t must l i m i t ATP p r o d u c t i o n  c o n c u r r e n t w i t h any e f f e c t on CC^  s u p p l i e d t o carboxydismutase. feedings  (Figure 16).  T h i s i s s u p p o r t e d by dark  The l i m i t a t i o n on ATP p r o d u c t i o n  92 would r e d u c e CC>2 f i x a t i o n a t s a t u r a t i n g and l i m i t i n g light. I n t h e l a s t few y e a r s , A t k i n s o n (3) has e l a b o r a t e d the c o n c e p t o f "energy-charge" i n t h e c e l l .  Several plant  enzymes have been t e s t e d i n t h i s c o n t e x t , and t h e e v i d e n c e supports Atkinson's theory.  L i t t l e work has been done c o n -  c e r n i n g "energy c h a r g e " , however, on t h e C a l v i n c y c l e enzymes.  I t has been demonstrated t h a t c h l o r o p l a s t FDPase  i s n o t i n h i b i t e d by AMP. ferredoxin  I t i s s t i m u l a t e d by r e d u c e d  ( 2 3 ) . FDPase o f c a s t o r bean endosperm, a g l u c o n -  e o g e n i c t i s s u e , i s c o n t r o l l e d by energy c h a r g e .  Since the  C a l v i n c y c l e i s d i r e c t e d towards energy s t o r a g e , a h i g h energy charge s h o u l d s t i m u l a t e i t s r e g u l a t o r y enzymes, o f w h i c h FDPase and c a r b o x y d i s m u t a s e a r e members. We have i n d i c a t e d t h a t Diamox d e c r e a s e s energy c h a r g e ( l i m i t s ATP) .  A n t i m y c i n A s t i m u l a t e s CC»2 f i x a t i o n b u t a l s o  a p p a r e n t l y d e c r e a s e s energy charge photophosphorylation).  ( l i m i t s ATP from c y c l i c  The "energy-charge" c o n c e p t does  n o t seem t o e x p l a i n t h e s e r e s u l t s . F e r r e d o x i n i n t h e reduced s t a t e , s t i m u l a t e s FDPase (23).  T h i s compound c o u l d be t h o u g h t o f a s a s i g n a l  energy i s a v a i l a b l e .  that  By l i m i t i n g 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 ,  A n t i m y c i n A c o u l d maximize p r o d u c t i o n o f NADPH (or r e d u c e d f e r r e d o x i n ) and s t i m u l a t e FDPase, and t h e o t h e r phosphatases of the C a l v i n c y c l e .  S i n c e ATP s u p p l y , however, i s now  somewhat l i m i t e d , such phenomena a s PGA a c c u m u l a t i o n a r e observed.  When A n t i m y c i n A i s f e d under l i g h t l i m i t i n g  conditions,  i n the presence of Diamox, i t would behave i n the same manner. The apparent  " s e n s i t i z a t i o n " would r e s u l t from i n c r e a s e d  e f f i c i e n c y o f use o f a v a i l a b l e l i g h t ; none i s now c y c l i c photophosphorylation.  "wasted" i n  N o t i c e t h a t under these  con-  d i t i o n s , A n t i m y c i n A cannot s t i m u l a t e the c o n t r o l , and does not r a i s e the i n h i b i t e d r a t e above t h a t of the c o n t r o l . F i n a l l y i t should be noted t h a t 5 uM A n t i m y c i n A can o n l y r e v e r s e the Diamox i n h i b i t i o n completely up to a p o i n t , about 1  mM.  9 4  LITERATURE CITED  A n t i a , N . J . , and J . K a l m a k o f f . 1 9 6 5 . Growth r a t e s and and c e l l y i e l d s from a x e n i c mass c u l t u r e o f f o u r t e e n s p e c i e s o f marine p h y t o p l a n k t e r s . F i s h . Res. Bd. Can., M a n u s c r i p t R e p o r t S e r i e s ; 2 0 3 . 2 4 pp. A r n o n , D.I. 1 9 6 7 . P h o t o s y n t h e t i c a c t i v i t y o f i s o l a t e d c h l o r o p l a s t s . P h y s i o l . Rev. 4 7 : 3 1 7 - 3 5 8 . A t k i n s o n , D.E. 1 9 6 8 . The energy charge o f t h e a d e n y l a t e p o o l as a r e g u l a t o r y p a r a m e t e r . Interaction with feedback m o d i f i e r s . B i o c h e m i s t r y , 7 : 4 0 3 0 - 4 0 3 4 . Bowes, G.W. 1 9 6 9 . C a r b o n i c anhydrase i n m a r i n e a l g a e . Plant Physiol. 4 4 : 7 2 6 - 7 3 2 . Champigny, M.L. and M. G i b b s . 1 9 6 9 . P h o t o s y n t h e s i s by i s o l a t e d c h l o r o p l a s t s i n the presence o f A n t i m y c i n A and a s c o r b i c a c i d . I n : H. M e t z n e r , e d . , P r o g r e s s i n P h o t o s y n t h e s i s Research, V o l . I I I . Tubingen, pp.  1 5 3 4 - 1 5 3 7 .  Champigny, M.L. and M. M i g i n i a c - M a s l o w . 1 9 7 1 . R e l a t i o n s e n t r e 1 ' a s s i m i l a t i o n p h o t o s y n t h e t i q u e de CO2 e t l a p h o t o p h o s p h o r y l a t i o n des c h l o r o p l a s t e s i s o l e s . I . S t i m u l a t i o n de l a f i x a t i o n de CO2 p a r 1 ' A n t i m y c i n e A, a n t a g o n i s t e de s o n i n h i b i t i o n p a r l e p h o s p h a t e : Biochim. Biophys. Acta. 2 3 4 : 3 3 5 - 3 4 3 . Day, R., and J . F r a n k l i n . 1 9 4 6 . P l a n t c a r b o n i c Science 1 0 4 : 3 6 3 - 3 6 5 .  anhydrase.  D i l l e y , R.A. 1 9 7 1 . C o u p l i n g o f i o n and e l e c t r o n t r a n s port i n chloroplasts. I n : D. Rao S a n a d i , e d . , C u r r e n t T o p i c s i n B i o e n e r g e t i c s , V o l . 4 . Academic P r e s s , New Y o r k . pp. 2 3 7 - 2 4 1 . E v e r s o n , R.G. 1 9 7 0 . C a r b o n i c anhydrase and CO2 f i x a t i o n i n i s o l a t e d c h l o r o p l a s t s . Phytochemistry 9 : 2 5 - 3 2 . E v e r s o n , R.G. 1 9 7 0 . C a r b o n i c anhydrase i n p h o t o s y n t h e s i s . I n : M.D. H a t c h , C.B. Osmond, and R.O. S l a t y e r , e d . , P h o t o s y n t h e s i s and P h o t o r e s p i r a t i o n . 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Reed. 1971. C a r b o n i c anhydrase and the r e g u l a t i o n o f p h o t o s y n t h e s i s . N a t u r e , New B i o l . , 231: 81-83.  15.  G r e v i l l e , G.D. 1969. A s c r u t i n y o f M i t c h e l l ' s chemio s m o t i c h y p o t h e s i s of r e s p i r a t o r y c h a i n and photosynthetic phosphorylation. I n : D. Rao S a n a d i , ed., C u r r e n t T o p i c s i n B i o e n e r g e t i c s , V o l . 3. Academic P r e s s , New Y o r k . pp. 1-78.  16.  I k e m o r i , M., and K. N i s h i d a . 1968. C a r b o n i c anhydrase i n the m a r i n e a l g a U l v a p e r t u s a . P h y s i o l . P l a n t . 21: 292-297.  17.  J a g e n d o r f , A.T., and J . Neumann. 1965. E f f e c t o f unc o u p l e r s on the l i g h t - i n d u c e d pH r i s e w i t h s p i n a c h c h l o r o p l a s t s . J . B i o l . Chem. 204: 3210-3214.  18.  Johnson, E . J . , and B.S. B r u f f . 1967. Chloroplast i n t e g r i t y and ATP-dependent C 0 f i x a t i o n i n Spinacia oleracea. P l a n t P h y s i o l . 42: 1321-1328.  the H i l l  reaction.  2  19.  K i s i e l , W., and E. G r a f . 1972. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f c a r b o n i c anhydrase from Pisum s a t i v u m . Phytochem. 11: 113-117.  20.  Maren, T.H. 1967. Carbonic anhydrase: c h e m i s t r y , p h y s i o l o g y , and i n h i b i t i o n . P h y s i o l . Rev. 47: 595-781.  21.  P a t t e r s o n , B.D., C A . A t k i n s , and D. Graham. 1971. C a r b o n i c a n h y d r a s e : m u l t i p l e forms i n p l a n t s . Biochem. J . 125: 34P-35P.  96  22.  P o i n c e l o t , R.P. 1972. 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 c a r b o n i c anhydrase i n s p i n a c h l e a v e s . Biochim. B i o p h y s . A c t a . 258: 637-642.  23.  P r e i s s , J . and T. Kosuge. 1970. R e g u l a t i o n o f enzyme a c t i v i t y i n p h o t o s y n t h e t i c systems. Ann. Rev. P l a n t P h y s i o l . 21: 433-466.  24.  R a b i n o w i t c h , E . I . 1945. P h o t o s y n t h e s i s and R e l a t e d Processes. V o l . I . Interscience Publishers, Inc., New Y o r k . N.Y. 2088 pp.  25.  S c h a c t e r , B.Z. and J.A. Bassham. 1972. A n t i m y c i n A s t i m u l a t i o n of r a t e - l i m i t i n g steps of photosynthesis i n i s o l a t e d spinach c h l o r o p l a s t s . P l a n t P h y s i o l . 49: 411-416.  26.  S c h a c t e r , B.Z., J.H. E l e y , and M. G i b b s . 1971. Involvement o f p h o t o s y n t h e t i c c a r b o n r e d u c t i o n c y c l e i n t e r m e d i a t e s i n CO2 f i x a t i o n and O2 e v o l u t i o n by i s o l a t e d c h l o r o p l a s t s . P l a n t P h y s i o l . 48: 707711.  27.  S c h a c t e r , B.Z., M. G i b b s , and M.L. Champigny. 1971. E f f e c t o f A n t i m y c i n A on p h o t o s y n t h e s i s o f i n t a c t s p i n a c h c h l o r o p l a s t s . P l a n t P h y s i o l . 48: 443-446.  28.  S i b l y , P.M. and J.G. Wood. 1951. The n a t u r e o f c a r b o n i c anhydrase from p l a n t s o u r c e s . A u s t . J . S c i . Res. B4: 500-510.  29.  S t e d i n g k , L.-V. Von, 1969. I o n t r a n s p o r t and energy c o n s e r v a t i o n i n chromatophores from R h o d o s p i r i l l u m rubrum. I n : H.. M e t z n e r e d . , P r o g r e s s i n P h o t o s y n t h e s i s R e s e a r c h , V o l . I I I . Tubingen, pp. 1410-1419.  30.  Swader, J.A. and B.S. J a c o b s o n , 1972. A c e t a z o l a m i d e i n h i b i t i o n of photosystem I I i n i s o l a t e d spinach c h l o r o p l a s t s . Phytochem. 11: 65-70.  31.  T e l f e r , A., and M.C.W. Evans. 1972. E v i d e n c e f o r chemiosmotic c o u p l i n g o f e l e c t r o n t r a n s p o r t t o ATP s y n t h e s i s i n s p i n a c h c h l o r o p l a s t s . B i o c h i m . B i o p h y s . A c t a . 256: 625-637.  32.  V e n n e s l a n d , B., E. O l s o n , R.N. Ammeraal. 1965. R o l e of carbon d i o x i d e i n the H i l l r e a c t i o n . Fed. P r o c . 24: 873-880.  PART I I I  I n t e r a c t i o n o f Oxygen and I n o r g a n i c Induction Transients  i n Iridaea  Carbon w i t h t h e Oxygen  cordata  98 INTRODUCTION  I t has been known f o r many y e a r s t h a t 0 p h o t o s y n t h e s i s i n a v a r i e t y o f t e r r e s t r i a l and plants  (18).  normal 21% 0  T h i s i n h i b i t i o n may 2  inhibits  2  aquatic  even be a p p a r e n t  a t the  c o n c e n t r a t i o n o f a i r . Wheat and some o t h e r  temperate-zone g r a s s e s , a l o n g w i t h most d i c o t y l e d o n o u s p l a n t s , p h o t o s y n t h e s i z e about 30% f a s t e r i n 2% 0 t o a i r . T h i s i n h i b i t i o n v a r i e s w i t h temperature  compared  2  and  carbon  d i o x i d e c o n c e n t r a t i o n . (13) The 0  2  i n h i b i t i o n i s a s s o c i a t e d w i t h the  o f p h o t o r e s p i r a t i o n , and the a p p a r e n t  occurrence  i n h i b i t i o n of  photo-  s y n t h e s i s i s c o n s i d e r e d t o be l a r g e l y a s t i m u l a t i o n o f photorespiration. d i s a p p e a r s a t 2% 0  (17)  Photorespiration essentially  (at which dark r e s p i r a t i o n i s saturated) ,  2  c a u s i n g an a p p a r e n t  i n c r e a s e i n carbon uptake.  Other  phenomena thought t o be a s s o c i a t e d w i t h p h o t o r e s p i r a t i o n a r e a f f e c t e d by low 0 ; 2  the c a r b o n d i o x i d e compensation p o i n t  d r o p s a l m o s t t o zero and the s o - c a l l e d burst" disappears  "post-illumination  (17).  A number o f m a r i n e a l g a e t e s t e d f o r 0  2  i n h i b i t i o n of '  p h o t o s y n t h e s i s showed i n s i g n i f i c a n t i n h i b i t i o n between 5% and  21% 0 , 2  b u t were i n h i b i t e d up t o 60% by a change from  5% t o 100% 0 . 2  (19)  The  i n h i b i t i o n was  reversible.  Very l i t t l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g occurrence o f p h o t o r e s p i r a t i o n i n marine a l g a e .  the  Several  w o r k e r s (2,10) r e p o r t e d t h e e x i s t e n c e o f a l i g h t s t i m u l a t e d r e s p i r a t i o n i n C h l o r e l l a , and A n a c y s t i s n i d u l a n s and Scenedesmus.  The r a t e o f l i g h t - i n d u c e d r e s p i r a t i o n was  a p p a r e n t l y i n t e n s i t y dependent t o h i g h v a l u e s , u n a f f e c t e d by g l u c o s e o r i n h i b i t e d by i t , and i n h i b i t e d by DCMU. i t appeared c l o s e l y r e l a t e d t o p h o t o s y n t h e s i s . Brown and Tregunna  (3)  Thus  A s t u d y by  i n d i c a t e d t h a t the marine r e d algae  I r i d a e a and G i g a r t i n a have e l e v a t e d CC>2 compensation p o i n t s i n a c i d i f i e d seawater  and may, t h e r e f o r e , have some form  of p h o t o r e s p i r a t i o n . The s t u d y d e s c r i b e d here o r i g i n a t e d i n t h e o b s e r v a t i o n t h a t I r i d a e a c o r d a t a showed an " C ^ - b u r s t " d u r i n g t h e f i r s t minutes o f i l l u m i n a t i o n .  T h i s b u r s t w h i c h i s comparable t o  i n d u c t i o n t r a n s i e n t s r e p o r t e d by o t h e r w o r k e r s (4) marked r e s p o n s e t o  showed a  c o n c e n t r a t i o n , as d i d t h e subsequent  steady-state rate of photosynthesis. The purpose o f t h i s s t u d y was t o i n v e s t i g a t e t h e interactions of  and CC»2 on t h e 0  2  sensitive  photosynthetic  C>2 i n d u c t i o n t r a n s i e n t s . A component o f t h e i n d u c t i o n t i m e course  not p r e v i o u s l y reported i n r e d algae w i l l  discussed.  a l s o be  100  METHODS  The  t e c h n i q u e used t o measure 0  simple v e r s i o n of t h a t pioneered (20).  2  p r o d u c t i o n was  a t the C a r n e g i e  A s m a l l p i e c e o f I r i d a e a c o r d a t a was  d i r e c t l y o v e r the end Instrument  Co.,  heavy c o t t o n .  Institute  stretched  (1 cm d i a m e t e r ) o f a Y e l l o w  gold Clark-type 0 T h i s assembly was  a  Springs  e l e c t r o d e and s e c u r e d  2  with  suspended i n a 200 ml beaker  o f f i l t e r e d s e a w a t e r , c o o l e d and a e r a t e d b e f o r e u s e , a t 9 t o 10°C.  The  t e m p e r a t u r e was  ments w i t h an i c e b a t h .  The  An aluminum r e f l e c t o r was  maintained  d u r i n g the e x p e r i -  seawater was  stirred constantly.  p l a c e d b e n e a t h the b e a k e r and  system l i g h t e d from above by a 300 w a t t G e n e r a l "Cool Beam" s p o t l i g h t ; d a r k n e s s was  the  Electric  o b t a i n e d by c o v e r i n g  a m e t a l frame w i t h a d o u b l e t h i c k n e s s o f heavy b l a c k The o u t p u t from the oxygen e l e c t r o d e was  cloth.  r e c o r d e d on a  S a r g e n t Model s t r i p c h a r t r e c o r d e r . The 0 N; 2  N  2  2  c o n c e n t r a t i o n s were v a r i e d u s i n g 0.09%  .alone; 0.09%  and 80% 0  in N .  2  2  C0  2  w i t h 21% 0  2  in N ; 2  21% 0  2  C0  in  2  in  N ' 2#  These gas m i x t u r e s were s u p p l i e d and  a n a l y z e d by Matheson o f Canada. P h o t o s y n t h e s i s was measured on a r e l a t i v e s c a l e . constant temperature was  achieved  external 0  2  a steady reading of 0  i n the d a r k .  2  concentration  This reading represents  c o n c e n t r a t i o n minus 0  2  At  the  used i n d a r k r e s p i r a t i o n ,  and w i l l be r e f e r r e d t o as t h e "dark s t e a d y - s t a t e " .  I n the  l i g h t , a second s t e a d y r e a d i n g , t h e " l i g h t i s u l t i m a t e l y achieved.  T h i s v a l u e was c o n s i d e r a b l y e l e v a t e d  above t h e d a r k v a l u e , and r e p r e s e n t e d photosynthesis  steady-state"  the 0  2  p r o d u c t i o n by  above t h e c o n s t a n t l e v e l e x t e r n a l l y a v a i l a b l e ,  p l u s C>2 used i n t h e d a r k , p l u s C> use by any l i g h t - i n d u c e d 2  respiration. S i n c e i t i s i m p o s s i b l e t o c a l i b r a t e a b s o l u t e l y an oxygen e l e c t r o d e w h i c h employs a l i v i n g membrane as a b a r r i e r t o oxygen d i f f u s i o n , a second C> e l e c t r o d e o f t h e 2  same t y p e , b u t c o v e r e d w i t h a T e f l o n membrane, was used t o record the a c t u a l 0 ing the a l g a .  2  c o n c e n t r a t i o n i n t h e seawater  surround-  A c o m p a r i s o n c o u l d t h e n be made between t h e  actual external 0  2  c o n c e n t r a t i o n and t h e d a r k  steady-state  reading. For s t u d i e s o f the 0  2  response, the experimental  w a t e r was f l u s h e d w i t h an a p p r o p r i a t e gas m i x t u r e . (See preceding  list).  A s o l u t i o n o f NaHCO^ was used t o a d j u s t  the i n o r g a n i c c a r b o n c o n c e n t r a t i o n o r C\ i n o r g a n i c carbon expressed of seawater).  ( d e f i n e d as t o t a l  as ml o f c a r b o n d i o x i d e per l i t r e  was t h e n checked by i n f r a r e d gas a n a l y s i s  as p r e v i o u s l y d e s c r i b e d .  (16)  V e r y low  was  achieved  by a c i d i f y i n g seawater t o l e s s t h a n pH 1, p u r g i n g w i t h 21% 0  2  inN  2  f o r 3 0 m i n u t e s , and then a d j u s t i n g t h e pH t o 8 . 0 ±  0.2 under t h e same gas m i x t u r e .  T h i s gas o r N  2  a l o n e was  then r e l e a s e d a t the water surface during experiments t o  102 prevent CC^ being  absorbed from the a i r .  Experiments were  always conducted a t pH 8.0 ± 0.2. U s i n g 150 t o 200 ml o f water and about 100 mg o f a l g a e , v e r y  l i t t l e change was  observed over the c o u r s e o f a two t o three hour experiment i n e i t h e r pH o r C^, so t h a t the system c o n d i t i o n s were constant  and may be d e s c r i b e d  as open.  Unless o t h e r w i s e  noted, l i g h t - d a r k c y c l e s c o n s i s t e d o f 5 minute dark followed  by 5 minute l i g h t  periods  periods.  When DCMU was a p p l i e d i t was added d i r e c t l y t o the beaker a f t e r completion o f a c o n t r o l p e r i o d , o r the e l e c t r o d e a l g a assembly was t r a n s f e r r e d i n the dark, from c o n t r o l t o treatment water w i t h i n two t o t h r e e  seconds.  The f i n a l  —6  concentration The  was 10  M DCMU.  p l a n t s were t e s t e d w i t h and w i t h o u t a T e f l o n  membrane between a l g a and e l e c t r o d e .  Half-saturated KCl  s o l u t i o n , as s u p p l i e d by YSI, was r o u t i n e l y used as t h e electrolyte.  The a l g a was a l s o t e s t e d u s i n g  half-saturated  NaCl; performance o f the e l e c t r o d e was s a t i s f a c t o r y w i t h NaCl.  The K C l d i d n o t appear t o damage t h e a l g a o v e r t h e  experimental period; that i s , rates o f photosynthesis no  serious decline. Because the algae  physiological condition  seemed t o vary  showed  considerably i n  (they a r e c o l l e c t e d from the sea-  shore) , each sample was used as i t s own c o n t r o l . r e l a t i v e comparisons may be made between d i f f e r e n t  Only samples.  103 The a l g a e were c o l l e c t e d from A p r i l t o August a t P o i n t , S t a n l e y P a r k , Vancouver, i n 1970 The oxygen e l e c t r o d e was o f response was  using N  2  and  Brockton  1971.  checked f o r i t s l i n e a r i t y  and 100% 0 ; 2  a Matheson gas p r o p o r t i o n e r  used t o mix i n t e r m e d i a t e C> c o n c e n t r a t i o n s . 2  RESULTS The 0_ P r o d u c t i o n I n d u c t i o n T r a n s i e n t s i n I r i d a e a c o r d a t a 2  F i g u r e 22 shows a t y p i c a l 0  2  time c o u r s e r e s p o n s e  p r o d u c t i o n by I r i d a e a i n t h e i n i t i a l m i n u t e s o f  a t i o n , and j u s t a f t e r the l i g h t was  turned o f f .  for illumin-  For r e f e r e n c e ,  the v a r i o u s t r a n s i e n t s have been l e t t e r e d as i n F i g u r e F o r the most p a r t , t h e s e l e t t e r s c o r r e s p o n d designation  by  Chandler  and  Vidaver  to  22.  the  (4) and  will  be  used i n d i s c u s s i o n . When the l i g h t i s t u r n e d on, t h e r e i s an immediate rapid rise i n 0 (a).  2  p r o d u c t i o n , w h i c h peaks a t 1 t o 2 seconds  There f o l l o w s a d e c l i n e i n 0  production  2  ( b ) , succeeded  by a somewhat s l o w e r r i s e t o a second peak ( c ) , h i g h e r the f i r s t , w h i c h r e a c h e s  i t s maximum i n 25 t o 30  From t h i s second peak t h e r a t e o f 0 f o r some t i m e .  2  a f t e r the l i g h t i s t u r n e d on.  After  seconds.  production f a l l s  A minimum ( d ) , i s reached  than  again  from 1 t o 3 m i n u t e s  (d),  the r a t e o f Cu  Figure  22  Time c o u r s e f o r t y p i c a l p h o t o s y n t h e t i c induction  t r a n s i e n t s d u r i n g the  first  of i l l u m i n a t i o n i n , I r i d a e a cordata . text for explanation.  oxygen minutes See  104  6  0  1  2  3 TIME  4 MINUTES  5  6  7  8  105 p r o d u c t i o n u s u a l l y r i s e s a g a i n , and t h e s t e a d y - s t a t e , (e) i s achieved.  This steady-state i s maintained  hours i f l i g h t , C\ , 0 , and temperature 2  for several  remain  constant.  When t h e l i g h t i s t u r n e d o f f , t h e r a t e o f C> p r o 2  d u c t i o n f a l l s o f f extremely r a p i d l y  (f) .  The f a l l  i n C»  2  p r o d u c t i o n appears q u i t e smooth; w i t h i n 1 minute a f t e r d a r k e n i n g a minimum i s reached  (g) w h i c h i s below t h e  p r e v i o u s d a r k r e a d i n g a t t h e s t a r t o f t h e sequence. t r a c e then r i s e s again  The  ( i ) , e i t h e r t o the previous dark  level,  o r t o a s l i g h t l y higher steady r e a d i n g which i s then maintained. The g r e a t e r p a r t o f t h e s e e x p e r i m e n t s  concerned  f a c t o r s w h i c h appeared t o a f f e c t t h e peak ( c ) , and ( e ) , t h e steady-state of photosynthesis.  The E f f e c t s o f 0_ C o n c e n t r a t i o n and C. on t h e 0_ P r o d u c t i o n Transients 2  2  The most marked and c o n s i s t e n t r e s p o n s e s  t o C» were 2  shown by t h e t r a n s i e n t s (c) and ( e ) . F i g u r e 23 shows t h e r e s p o n s e  o f (e) t o i n c r e a s i n g 0  a t normal t o moderate C. (30 t o 50 ml C . / l ) .  2  In this  s e r i e s o f measurements, t h e i n h i b i t i o n a t 9 ppm ( h i g h 0 ) 2  compared t o 1 ppm (low 0 ) was 65%. 2  t o 1 t o 5 ml/1 t h e i n h i b i t i o n by 0 20% from 65%. high C  The r e s p o n s e  (80 ml/1)  values.  to 0  2  2  When C^ was reduced a t 9 ppm was reduced t o  a l s o decreased  a t very  A t 80 ml C / l t h e r e was a 30%  F i g u r e 23  E f f e c t o f o x y g e n on  the r a t e of  steady-state  photosynthesis i n Iridaea cordata  o ZD Q  o cc  5  ° u_  4  Q_  O  < CC  UJ  Q •0.  -  ^  A C T U A L  "  2  PPM  —  6  —  OXYGEN  -  f IN  -j  10  12  E X P E R I M E N T A L  1_  14 WATER  107 i n h i b i t i o n i n (e) on c h a n g i n g from low t o h i g h 0 . 2  F i g u r e s 24 ing  from h i g h t o low t o h i g h 0 , a t moderate, l o w , and very2  h i g h C^. to  t o 26 show t h e r e s p o n s e o f (e) on chang-  Note t h a t (e) responds r e v e r s i b l y and  t h e changes i n C^.  ward  immediately  There i s a tendency f o r a slow down-  d r i f t i n the steady-state r a t e of photosynthesis  the c o u r s e o f t h e  during  experiment.  As shown i n F i g u r e s 24 t o 26 t h e s p i k e ( c ) , a l s o responded t o a change i n 0  concentration.  9  A t 50 ml/1  C,  (c) i s a p p a r e n t l y i n h i b i t e d 60% by a change from 1 t o 9 ppm 0 . 2  As shown i n F i g u r e s 24 t o 26, however, (c) r e q u i r e s  longer than first reached  (e) t o respond t o a change i n C> . 2  (c) peak i n low 0  2  T y p i c a l l y the  i s l e s s than 40% o f t h e peak h e i g h t  2 o r 3 c y c l e s l a t e r , w h i l e t h e (e) v a l u e r e a c h e s a t  l e a s t 80% o f i t s u l t i m a t e v a l u e d u r i n g t h e f i r s t cycle.  light-dark  A t t h i s p o i n t , ( b e g i n n i n g o f t h e f i r s t low C»  2  light  p e r i o d ) t h e a l g a would have been s u b j e c t e d t o low C» f o r n o t 2  more t h a n 5 m i n u t e s .  When t h e G* i s a g a i n i n c r e a s e d t o 9 ppm, 2  (c) once more r e q u i r e s time t o a d j u s t , w h i l e  (e) r e s p o n d s  immediately. A t v e r y low C^, F i g u r e 25, (c) shows a l m o s t no i n c r e a s e o r even a d e c r e a s e a t low 0 . 2  F i g u r e 27 shows a second  a s p e c t o f t h e r e s p o n s e o f (c) t o low 0 . 2  A t 9 ppm 0 , t h e 2  peak i s i n i t i a l l y 5 t o 8 times t h e h e i g h t o f t h e s t e a d y - s t a t e photosynthesis.  The peak h e i g h t d e c r e a s e s  rapidly with  s u c c e s s i v e c y c l e s o v e r 25 m i n u t e s , ( i , i i , i i i ) and as a l r e a d y  F i g u r e 24  The  e f f e c t o f h i g h and low oxygen on  (e) a t 50 ml  per  litre  (c)  and  108  ;  oi 0  10 TIME  20  30  MINUTES  40  50  ~60  70  80  90  F i g u r e 25  The e f f e c t o f h i g h and (e) a t 2 ml C  per  low oxygen on  litre  (c)  and  F i g u r e 26  The e f f e c t o f h i g h and (e) a t 80 ml C  per  low oxygen on  litre  (c)  and  160  HIGH 02 8-10 RRM.  HIGH 0o 8-10PFTM.  LOW Oo < 1 PRlvf  140  O120  o  Q O 100 CC  o-^801 o  60  <  c  cc  S oi ^20  0  -V— 10 TIME  • 20  30  MINUTES  40  50  60  70  80  90  100  110  Figure  27  The  e f f e c t o f l o w C\  and  low  oxygen  w i t h t i m e o n (c) a t  high  mentioned, responds v e r y l i t t l e t o a l o w e r i n g o f the concentration  C»  2  ( i v ) . These were c o n s e c u t i v e c y c l e s w i t h  the  same sample, s e p a r a t e d by 5 m i n u t e d a r k p e r i o d s . The Usually  r e l a t i o n s h i p of  (c) was  (c) to  (e) appeared f a i r l y  2 o r 3 times the h e i g h t o f  constant.  (e) e x c e p t a t  low  ( F i g u r e 27) where, as s t a t e d , (c) d e c r e a s e s w i t h time from 5 t o 8 times  (e) t o the u s u a l v a l u e .  A t low C\ (a) s p i k e was about 8% o f  ( F i g u r e 27)  about 50% o f (c) a t 9 ppm  (c) became lower t h a n (a) . (c) a t moderate  0 . 2  A t h i g h C^,  and low 0 a similar  2  The and  relation-  ship i s evident. The and  (a) s p i k e ( F i g u r e 22) o c c u r s e x t r e m e l y r a p i d l y  i s complete i n 4 t o 5 seconds.  a p p a r e n t l y swamped by the m a s s i v e 0 p a r t i c u l a r l y a t low C» , 2  s h o u l d e r on  I t i s occasionally production of  2  and c o n s e q u e n t l y  (c) o r be masked c o m p l e t e l y .  w h i c h f o l l o w s (a) was  may  appear as a  The b r i e f d i p  r e a d i l y o b s e r v e d a t 9 t o 10° C, i n  moderate l i g h t , a f t e r 3 t o 5 m i n u t e s d a r k n e s s . of  (c)  The  height  (b) the l o w e s t p o i n t o f t h i s d i p , shows some tendency  to be c o n s t a n t , r e g a r d l e s s o f the maximum v a l u e s o f and  (a)  (c) . The  (d) component o f F i g u r e 22, t h a t i s a d i p  ( e ) , d i d n o t always appear a t 9 ppm  oxygen, b u t was  r e v e a l e d a t low 0  The  2  concentrations.  a l w a y s i n t e n s i f i e d somewhat a t low 0 , 2  l a r l y marked a t 50 ml C ,  component b u t t h i s was  before usually  (d) particu-  where (d) c o n s i s t e n t l y i n c r e a s e d  113 4 o r 5 times when 0 The was ( g ) .  2  was reduced from 9 t o 1 ppm.  f i n a l component t o which a t t e n t i o n was d i r e c t e d T h i s appears as a " p o s t - i l l u m i n a t i o n " s t i m u l a t i o n  of r e s p i r a t i o n .  The e f f e c t o f 0  2  on (g) i s u n c e r t a i n , b u t  we observed some tendency f o r i t s magnitude t o be reduced a t low 0 . 2  The  E f f e c t o f P r e v i o u s L i g h t - d a r k Regimes on (c) F i g u r e 28 shows the e f f e c t o f l i m i t i n g the p r e v i o u s  dark p e r i o d on the (c) s p i k e .  When the dark p e r i o d  preceding  i l l u m i n a t i o n i s v e r y s h o r t , the b u r s t i s reduced i n s i z e . a b r i e f experiment, the dark s t e a d y - s t a t e was achieved a sample o f I r i d a e a .  The l i g h t was turned o n .  In  with  Immediately  the h e i g h t o f (c) was reached, the l i g h t was turned o f f f o r 30 seconds, then turned on a g a i n u n t i l the b u r s t h e i g h t was r e a c h e d .  T h i s was repeated  s e v e r a l times.  In t h e second  l i g h t p e r i o d , the b u r s t h e i g h t was reduced by 25%, and i n the f o l l o w i n g 4 l i g h t p e r i o d s , reached a n e a r l y value.  The s i x t h l i g h t p e r i o d l a s t e d 2 minutes and showed  only a very s l i g h t f a l l equal  constant  from  t o t h a t o f the c o n t r o l .  p e r i o d was then g i v e n , original  (c) t o a l i g h t  steady-state  When a s i n g l e 4 minute dark  (c) reached about 85% o f i t s  height.  I f the dark time i s decreased  i n succeeding  cycles  from 8 minutes t o 15 seconds the r e s u l t i s s i m i l a r t o t h a t just described.  A p l o t o f t h e v a l u e s o f (c) a g a i n s t  previous  Figure  28  The  e f f e c t of the preceeding  the h e i g h t of  (c)  dark period  on  115 d a r k time would show (e) n e a r l y c o n s t a n t , w h i l e p r o g r e s s i v e l y u n t i l i t meets ( e ) . o f time i n the l i g h t had l i t t l e  (c)  falls  Changing the l e n g t h  s p e c i f i c e f f e c t on t h e  v a l u e o f ( c ) , b u t caused a p a r a l l e l downward d r i f t i n t h e h e i g h t o f a l l components.  The E f f e c t o f DCMU on 0  2  Production Transients  F i g u r e 29 shows the e f f e c t o f 1 0 ~ M DCMU on the C» 6  2  p r o d u c t i o n t r a n s i e n t s . T h i s e x p e r i m e n t was performed a t 3 ppm  0 «  The (c) s p i k e appears t o be p r e f e r e n t i a l l y  2  by the i n h i b i t o r . initially. all  effect  I n the n e x t l i g h t p e r i o d a marked d e p r e s s i o n o f  t r a n s i e n t s and t h e f i n a l s t e a d y - s t a t e r a t e o f p h o t o -  synthesis  The  B o t h (a) and (e) show l i t t l e  attacked  occurred.  Response o f the 0_ E l e c t r o d e 2  When checked w i t h v a r i o u s known c o n c e n t r a t i o n s o f 0 ,  the e l e c t r o d e was found t o respond l i n e a r l y t o C»  2  2  concentration. effect of 0  2  U s i n g v a l u e s t a k e n from one s t u d y o f t h e  on s t e a d y - s t a t e p h o t o s y n t h e s i s , we a t t e m p t e d  to  d e t e r m i n e whether the p e r m e a b i l i t y o f the a l g a l  to  0  2  remained c o n s t a n t , as 0  a p l o t o f a c t u a l ppm 0  2  2  changed.  thallus  F i g u r e 30 shows  i n the e x p e r i m e n t a l w a t e r v e r s u s  the d a r k s t e a d y s t a t e v a l u e .  The r e l a t i o n s h i p i s l i n e a r .  F i g u r e 29  The  e f f e c t o f DCMU o n  the  (c)  spike  RELATIVE  m  m co  911  RATE  OF  0  2  PRODUCTION  F i g u r e 30  The r e l a t i o n s h i p b e t w e e n e x t e r n a l ppm and t h e d a r k s t e a d y - s t a t e  reading  oxygen  117  0  1  2  DARK  3  4  5  6  STEADY STATE  7  8  9  0 11  APPARENT  12 13 14  PPM  0  2  118 DISCUSSION  The components o f t h e i n d u c t i o n p e r i o d f o r I r i d a e a c o r d a t a a r e s i m i l a r t o those p u b l i s h e d by V i d a v e r  (21) f o r  U l v a l o b a t a , I l e a f a s c i a , and A n k i s t r o d e s m u s f a l c a t u s , and by C h a n d l e r and V i d a v e r  (4) f o r U l v a l a c t u c a L.  The s p i k e marked (a) i n F i g u r e 22 appears t o be i d e n t i c a l t o t h e " p r e - a " o r a^ t r a n s i e n t i d e n t i f i e d by Vidaver  (21). Although  Vidaver  (21) r e p o r t e d t h a t he was  unable t o d e t e c t the "pre-a" spike i n the r e d a l g a G o v i n d j e e and G o v i n d j e e , Porphyridium,  Porphyra,  (8) r e p o r t e d a r a p i d s p i k e i n  a unicellular red alga.  I t i s interesting,  however, t h a t t h i s s p i k e appeared i n P o r p h y r i d i u m  only  after  a l o n g d a r k p e r i o d o f 15 m i n u t e s t o s e v e r a l hours and a t 1 to 5°C. (8)  I t disappeared  a f t e r s e v e r a l exposures to l i g h t .  The " p r e - a " s p i k e o f U l v a , I l e a , A n k i s t r o d e s m u s and  I r i d a e a does n o t d i s a p p e a r  i n t h i s manner, n o r does i t  r e q u i r e very long dark p e r i o d s . by G o v i n d j e e , here  The e a r l y s p i k e r e p o r t e d  t h e r e f o r e , may n o t be t h e same as t h a t r e p o r t e d  o r by V i d a v e r . Vidaver  (21) a l s o r e p o r t e d t h a t low (4°C) o r h i g h  (30°C) t e m p e r a t u r e s were r e q u i r e d t o i s o l a t e t h e " p r e - a " t r a n s i e n t i n U l v a and I l e a .  I n I r i d a e a (as i n A n k i s t r o d e s m u s )  the " p r e - a " s p i k e appears r e g u l a r l y and r e p e a t e d l y , a f t e r 3 o r 4 m i n u t e s d a r k ; o u r e x p e r i m e n t s were conducted a t 9 t o 10°C  and no s p e c i a l e f f o r t s were r e q u i r e d t o r e v e a l t h e  119 T h i s appears t o be t h e f i r s t  "pre-a" t r a n s i e n t i n I r i d a e a .  r e p o r t o f a "pre-a" t r a n s i e n t i n the r e d a l g a e . knowledge, o n l y t h e B a n g i o p h y c i d a e  To o u r  i n t h e r e d a l g a e have been  examined p r e v i o u s l y i n t h i s r e s p e c t ;  I r i d a e a i s a member o f  the more complex F l o r i d e o p h y c i d a e . Since the green algae  (21) w i l l produce t h e " p r e - a "  s p i k e r e p e a t e d l y under a p p r o p r i a t e c o n d i t i o n s , b u t P o r p h y r i d ium d i d n o t , G o v i n d j e e  and G o v i n d j e e  ( 8 ) , suggested  some  d i f f e r e n c e i n the o r i g i n o f the "pre-a" i n r e d versus green algae.  Our r e s u l t s s u g g e s t t h a t t h i s i s n o t t h e c a s e . I t has been noted i n these experiments  that except  under s p e c i a l c o n d i t i o n s , d e s c r i b e d p r e v i o u s l y , t h e second 0  2  or  s p i k e , (c) i s always c o n s i d e r a b l y h i g h e r than t h e " p r e - a " ( a ) , and (e) t h e s t e a d y - s t a t e r a t e o f p h o t o s y n t h e s i s .  The  (c) s p i k e appears t o be t h e same as t h e a - s p i k e r e f e r r e d t o by V i d a v e r  (21).  I n h i s published r e p o r t s , the f i n a l  s t e a d y r a t e o f p h o t o s y n t h e s i s i s always h i g h e r than t h e various induction transients.  T h i s d i f f e r e n c e may be caused  by an e x t r e m e l y slow r i s e t o t h e maximum s t e a d y s t a t e i n I r i d a e a , o r by some d i f f e r e n c e i n p o o l - s i z e s o f o x i d i z a b l e components. The time c o u r s e o f oxygen p r o d u c t i o n t r a n s i e n t s p u b l i s h e d f o r U l v a by C h a n d l e r secondary 0  2  and V i d a v e r  (4) r e v e a l e d a  t r a n s i e n t i n the " p o s t - i l l u m i n a t i o n " d e c l i n e i n  production.  T h i s never appeared i n I r i d a e a .  A "post-  i l l u m i n a t i o n " s t i m u l a t i o n o f r e s p i r a t i o n , however, s i m i l a r  120 to t h a t d e s c r i b e d by F r e n c h and F o r k The  (6) always a p p e a r e d .  50% i n h i b i t i o n of p h o t o s y n t h e s i s  i n I r i d a e a by  9 ppm  0 ,  i s a t v a r i a n c e w i t h the r e p o r t s f o r o t h e r m a r i n e  algae  ( i n c l u d i n g some r e d s ) by T u r n e r , Todd, and  2  (19).  They noted l i t t l e s i g n i f i c a n t  s y n t h e s i s by 20% C»  2  and  a i r a t 10°C,  has  i n h i b i t i o n of photo-  at high l i g h t intensity  36 u M c a r b o n d i o x i d e .  Brittain  (near s a t u r a t i o n )  Seawater s a t u r a t e d w i t h 0  about 9 ppm  0 . 2  The c a r b o n  in  2  concentrations  r e p o r t e d as moderate, 50 ml C ^ / l are e q u a l t o j u s t o v e r 2 There i s no e v i d e n c e f o r an e f f e c t the p e r c e n t i n h i b i t i o n by 0  2  (11) .  of l i g h t i n t e n s i t y  photosynthesis.  V a r i o u s r e p o r t s i n c l u d i n g (12) and 2  t e n d s t o e r a d i c a t e the 0  photosynthesis. reduction  2  (to 20%)  effect.  o f C» i n h i b i t i o n 2  of  a t 80 ml/1  C .  On  Apparently  c a r b o n a t 2 to 5 ml/1  the  increased  J o l l i f f e and Tregunna (12) showed t h a t C\  (2 t o 2.5 mM).  is  i n I r i d a e a up t o 50 t o 60 ml  C\  the v e r y low a v a i l a b i l i t y o f  so l i m i t s p h o t o s y n t h e s i s  t h a t the  0  2  i s n o t r e v e a l e d f o r l a c k of s u b s t r a t e . We  large 0  inhibition  most organisms have shown an  2  l i m i t i n g f o r photosynthesis  effect  2  (18) i n d i c a t e  I n l i n e w i t h t h i s , we have o b s e r v e d a  o t h e r hand, a t low C 0 0  on  As l i g h t i n t e n s i t y i n -  creases p h o t o r e s p i r a t i o n increases with  that high C0  mM.  2  kinetics  have a l s o noted the s e n s i t i v i t y o f the second spike  (c) t o 0  2  concentration.  S i n c e the  d i f f e r , the i n h i b i t i o n o f t h i s s p i k e may  inhibition not  occur  121 b y t h e same m e c h a n i s m a s t h e i n h i b i t i o n o f s t e a d y - s t a t e photosynthesis. I t appears t h a t d u r i n g t h e 0  2  production induction  p e r i o d o f p h o t o s y n t h e s i s , C> i s t a k e n up b y a t l e a s t 2 2  different  processes.  The  "pre-a"  spike i s separated  l a r g e s p i k e by a marked d e p r e s s i o n . ments b y C h a n d l e r and V i d a v e r that this depression occurs  (4) a n d R e i d  2  u p t a k e t o DCMU.  2  0  spike appeared.  which  2  2  evolution  Since  Reid  b u r s t was i n h i b i t e d b y DCMU,  t h e p a t t e r n i n F i g u r e 29 i s p r o b a b l y uptake t r a n s i e n t superimposed  due t o s u r v i v a l o f a n  on a p a r t i a l l y  inhibited  production. Heber and F r e n c h  have measured an 0 is  indicate  The r e s u l t s i n F i g u r e 29 a l s o i n d i c a t e t h e  (15) showed t h a t t h e f i r s t 0  2  (15)  2  was s u p p r e s s e d , b u t a " p r e - a "  C»  Flashing light experi-  represents an a c t i v e uptake o f 0  resistance of this brief 0  2  (c) t h e second  i n t h e p r e s e n c e o f DCMU o r a t 707 nm, when no 0 i s  produced.  0  from  attributed  This  uptake i n i s o l a t e d  2  a n d J o l i o t (14)  c h l o r o p l a s t s , which  t o o x i d a t i o n o f a product o f photosystem I .  system I - l i n k e d 0  a requisite  (9) a n d K o u c h k o v s k y  2  uptake i sconsidered  b y them t o b e  f o r a c t i v a t i o n o f t h e p h o t o c h e m i c a l complex  of system I I . The  second 0  2  uptake i n t h e i n d u c t i o n p e r i o d i s t h a t  associated with the light-stimulated respiration  reported  (2,10) i n some u n i c e l l u l a r a l g a e and h i g h e r p l a n t s ( 5 ) . I t may be t h a t t h e c - s p i k e i n I r i d a e a i s n o t d i r e c t l y a f f e c t e d by 0  2  concentration.  The s p i k e i s a s s o c i a t e d w i t h  system I I ( s e n s i t i v e t o DCMU, 7 07 nm l i g h t ) w h i c h produces m a s s i v e amounts o f 0 .  Rather,  2  itself  the apparent  i n h i b i t i o n o f (c) a t h i g h C» may be t h e r e s u l t o f a s t i m u 2  l a t i o n o f one o r b o t h l i g h t - i n d u c e d C> u p t a k e r e a c t i o n s . 2  Except a t low C ,  t h e (c) peak r e a c t s s i m i l a r l y t o (e) , t h e  s t e a d y - s t a t e o f p h o t o s y n t h e s i s , when C» i s changed. 2  probable,  t h e r e f o r e , t h a t a continuous  a t i o n i s responsible f o r the 0 r a t h e r than the very b r i e f 0 reduced 0  2  2  2  It is  light-induced respir-  e f f e c t on b o t h (c) and ( e ) ,  use by p h o t o s y s t e m I . A t  concentrations, this "photorespiration" i s i n -  hibited f o r lack of 0  2  and t h e 0  2  b u r s t appears l a r g e r , as  does t h e s t e a d y - s t a t e . I f t h i s p h o t o r e s p i r a t i o n i s dependent on r e c e n t photosynthate,  as we assume, i t would n o t b e g i n u n t i l a  few seconds a f t e r c a r b o n f i x a t i o n .  A "burst" of 0  2  uptake  as r e s p i r a t i o n began, f o l l o w e d by a s e t t l i n g t o t h e s t e a d y s t a t e could e x p l a i n the d i p a f t e r to t h e s t e a d y  state.  T h i s "undershoot" m i g h t be emphasized  a t l o w C» , when l i m i t e d 0 2  (c) f o l l o w e d by a r i s e  2  permits only a r e s t r i c t e d  steady  r a t e o f r e s p i r a t i o n compared t o t h a t a t h i g h 0 . 2  The  l a c k o f response t o low 0  very low i n i t i a l l y presented  2  by (c) when C  was  a problem o f i n t e r p r e t a t i o n  123 i n terms o f t h e above model.  Bannister  ( 1 ) , however, r e p o r t e d  o s c i l l a t i o n s i n 0^ i n d u c t i o n t r a n s i e n t s under KCN p o i s o n i n g o f c a r b o n f i x a t i o n o r i n t h e absence o f CC^/ i n d i c a t i n g some i n f l u e n c e o f c a r b o n f i x a t i o n on t h e s e e a r l y t r a n s i e n t s . P o s s i b l y , severe l i m i t a t i o n o f the C a l v i n c y c l e i n I r i d a e a by v e r y l o w C  causes a r e s t r i c t i o n o f 0  2  production  during  i n d u c t i o n , thus r e d u c i n g t h e peak h e i g h t o f ( c ) . A f u r t h e r a s p e c t o f t h e (c) s p i k e , i s t h e e f f e c t on i t o f the l e n g t h o f the dark p e r i o d preceding upon w h i c h i t i s dependent.  Apparently  illumination,  some component w h i c h  i s r e d u c e d i n t h e l i g h t i s s t o r e d o r produced i n t h e d a r k . G o v i n d j e e and G o v i n d j e e (8) r e p o r t e d t h a t (c) was s t i m u l a t e d by r e p e a t e d  e x p o s u r e s t o l i g h t , and u n a f f e c t e d by  d a r k i n t e r v a l s r a n g i n g from 1 t o 8 m i n u t e s .  We found t h a t  the h e i g h t o f t h e (c) b u r s t was s e v e r e l y r e s t r i c t e d by d a r k p e r i o d s o f l e s s than 2 minutes. No r e a s o n a b l e  e x p l a n a t i o n i s apparent f o r the adaption  time r e q u i r e d by (c) when 0  2  c o n c e n t r a t i o n i s changed, s i n c e  the r e s p o n s e o f t h e s t e a d y - s t a t e i s immediate. The  existence of a "post-illumination" stimulation  o f r e s p i r a t i o n i n a l g a e has been r e p o r t e d p r e v i o u s l y ( 6 ) . Such a r e s p o n s e a l s o o c c u r s i n h i g h e r p l a n t s h a v i n g  photo-  r e s p i r a t i o n , where t h e s o - c a l l e d "PIB" i s i n h i b i t e d under c o n d i t i o n s r e s t r i c t i n g p h o t o r e s p i r a t i o n , i n c l u d i n g low 0 . 2  As i n d i c a t e d , t h e PIB i n I r i d a e a may be r e s t r i c t e d a t low 0_.  Probably  i t i s associated with light-stimulated respir-  124 ation.  F u r t h e r work i s r e q u i r e d on t h i s p a r t i c u l a r t r a n s i e n t . The mechanism o f p h o t o r e s p i r a t i o n i n a l g a e i s u n c l e a r .  I n h i g h e r p l a n t s the phenomenon i s a s s o c i a t e d w i t h the metabo l i s m o f g l y c o l i c a c i d by the C» -using enzyme g l y c o l i c a c i d 2  oxidase.  The  a l g a e l a c k g l y c o l i c a c i d o x i d a s e and  g l y c o l i c a c i d dehydrogenase i n s t e a d . c o n d i t i o n s o f h i g h G^,  have  F u r t h e r m o r e / under  h i g h l i g h t i n t e n s i t y , and  limiting  CC>2, w h i c h s t i m u l a t e p h o t o r e s p i r a t i o n i n h i g h e r p l a n t s , algae excrete g l y c o l i c a c i d . (7) i n d i c a t e s t h a t f o r m a t i o n  A more r e c e n t r e p o r t by of glycolate i n  c h l o r o p l a s t s i s dependent on p r o d u c t i o n act.  The  and  Gibbs  spinach  of H 0 2  2  i n the p h o t o -  p e r o x i d e r e l e a s e s g l y c o l i c a c i d from the  transketolase addition-product.  the  two-carbon  T h i s r e a c t i o n i s C» s e n s i t i v e , 2  i s s t i m u l a t e d by c o n d i t i o n s w h i c h promote p h o t o r e s p i r a t i o n .  Thus i t appears t h a t i n h i g h e r p l a n t s , b o t h p r o d u c t i o n metabolism of g l y c o l a t e are G ^ - s e n s i t i v e ,  and  (^-utilizing  reactions. I t has  a l r e a d y been noted t h a t some a l g a e have a  photosystem-I-linked (22)  0  uptake.  2  Work by W i l s o n and  i n d i c a t e s t h a t i n Scenedesmus g l y c o l a t e  Calvin  production  c o u l d be l i n k e d t o f e r r i c y a n i d e r e d u c t i o n , t h u s l i n k i n g the p h o t o a c t w i t h g l y c o l a t e p r o d u c t i o n l a t e production  i n the a l g a e .  Glyco-  by an 0 ~ s e n s i t i v e p r o c e s s i s p r o b a b l y 2  common t o a l g a e and h i g h e r p l a n t s .  Heber and F r e n c h  c o n c l u d e d t h a t t h i s i n i t i a l 0- u p t a k e was  (9)  not s u f f i c i e n t t o  explain the quantity of 0^ apparently used i n photorespiration. 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