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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 ETHEL ANN JOLLIFFE B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia , I agree that 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 re ference and s tudy . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted 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 . I t i s understood that copying or 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 ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . 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 i i ABSTRACT Three a s p e c t s o f the 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 the 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 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 i n marine b e n t h i c a l g a e . Net i n o r g a n i c c a r b o n 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 the l i g h t up t o pH 9.9 and a t pCC^ down t o l e s s than 5 ppm. Carbon uptake was measured on the b a s i s o f changes i n t h e CC^ r e l e a s e d by a c i d from 1 ml samples o f the e x p e r i m e n t a l sea w a t e r , and a l s o c a l c u l a t e d from pCC»2 and pH a c c o r d i n g t o s t a n d a r d t a b l e s . The pCG^ was m o n i t o r e d by i n f r a -r e d gas a n a l y s i s . T his 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 p h o t o s y n t h e s i s . I n a c o n t i n u i n g s t u d y , c a r b o n uptake 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 a l g a e : A l a r i a , C o s t a r i a , D e s m a r e s t i a , Enteromorpha, G i g a r t i n a , N e r e o c y s t i s , P o r p h y r a , and U l v a . The change i n t o t a l i n o r g a n i c c a r b o n i n the e x p e r i -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 by a c i d r e l e a s e . W i t h the e x c e p t i o n s o f P o r p h y r a and D e s m a r e s t i a , t h e a l g a e used 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 r e l a t i o n s h i p o f r a t e o f p h o t o s y n t h e s i s t o t o t a l i n o r g a n i c c a r b o n p r e s e n t i n seawater was d e t e r m i n e d f o r U l v a 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 found t o be l i m i t -i n g f o r p h o t o s y n t h e s i s . 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 as 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 - a c e t a m i d o - l , 3 , 4 - t h i a d i a z o l e - 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 the l i g h t i n whole p l a n t s and i s o l a t e d c h l o r o p l a s t s . I t a l s o i n h i b i t s p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t , b o t h c y c l i c and n o n - c y c l i c . I t was found 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>2 f i x a 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 i n the 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 c a r b o n i c anhydrase p l a y s i t s r o l e i n p h o t o s y n t h e s i s as a s u p p l i e r o f p r o t o n s (from c a r b o n i c a c i d ) t o the hydrogen i o n pump. 14 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 by c h l o r o p l a s t s 14 i n s a t u r a t i n g l i g h t . Diamox i n h i b i t i o n o f CC>2 f i x a t i o n i n the l i g h t i s overcome by 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 n o n - s a t u r a t i n g l i g h t . T h i s i n t e r a c t i o n s u g g e s t s t h a t 14 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 by c h a n g i n g the "energy c h a r g e " s t a t u s o f t h e c h l o r o p l a s t . D u r i n g the 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 es 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 b e g i n n i n g o f p h o t o s y n t h e s i s shows a number o f t r a n s i e n t s . 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 and the 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 components. 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 t o C»2 e f f e c t s on p h o t o s y n t h e s i s i n h i g h e r p l a n t s . A t r a n s i e n t h e r e t o f o r e 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 Photosynthesis by Sargassum muticum 1 INTRODUCTION . 2 MATERIALS AND METHODS . . . . . . 4 RESULTS . . . . . . . . . . . . . . . 7 DISCUSSION 12 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . 16 PART I-B Studies on Bicarbonate Ion Uptake During Photosynthesis i n Benthic Marine Algae 18 INTRODUCTION 19 MATERIALS AND METHODS . . . . . . . . . 20 RESULTS AND DISCUSSION 25 Survey f o r HCO^ Ion A s s i m i l a t i o n . 25 The E f f e c t of HCO^ Ion Concentration on Photosynthesis . 35 LITERATURE CITED 47 ADDENDUM - The E f f e c t of pH on Photosynthesis i n U l v a f e n e s t r a t a 4 8 APPENDIX - The Measurement of Inorganic Carbon and Photosynthesis i n Seawater by PCO2 and pH A n a l y s i s 56 PART I I Evidence Concerning the Mechanism of Diamox I n h i b i t i o n and Antimycin A S t i m u l a t i o n o f Photosynthesis 64 INTRODUCTION 65 v i Page MATERIALS AND METHODS . . . . . . . . . . . . 66 Pr e p a r a t i o n of C h l o r o p l a s t s . . . . 66 C0 2 Feedings: Dark and L i g h t . 68 Studies of 0 2 Production . . . . . . . . . . . . . 69 The Light-Induced pH S h i f t . . . . . . . . . . . . 70 Pr e p a r a t i o n of I n h i b i t o r s . . . . . . . . . . . . 70 RESULTS • • • • • • o o B o e a a o o * a o » o * o o o 72 The E f f e c t of Diamox on Photosynthesis . . . . . . 72 1 ^ C 0 2 F i x a t i o n i n the L i g h t . . . . . . . . . 72 14 _ _ C0 2 F i x a t i o n i n the Dark . . . . . . . . . 72 The E f f e c t of Diamox on the H i l l R eaction . . . . 74 The E f f e c t of Diamox on the Lig h t - i n d u c e d The I n t e r a c t i o n o f Antimycin A w i t h Diamox . . . . 79 The E f f e c t of Antimycin A on the L i g h t -induced pH S h i f t . . . . . . . . . . . . . . . . . 83 The S t a b i l i t y of Diamox . . . . . . . . . . . . . 83 D X SOUS S ION • o « * * o o a * 4 o a < * « o e * * « a o o 84 LITERATURE CITED . . . . . . . . . . . . . 94 PART I I I I n t e r a c t i o n of Oxygen and Inorganic Carbon w i t h the Oxygen Ind 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 cordata 97 INTRODUCTION . . . . . . . . . . . 98 METHODS . . . . . . . . 100 RESULTS 103 v i i Page The C>2 Production Induction T r a n s i e n t s i n I r i d a e a cordata 103 The E f f e c t s of O2 Concentration and C. on the O2 Production Transients . . . . . . . . . 105 The E f f e c t of Previous Light-Dark Regimes on (c) 113 The E f f e c t o f DCMU on 0 2 Production T r a n s i e n t s 115 The Response o f the E l e c t r o d e 115 DISCUSSION . . . . . . . . . . . . . . . . 118 LITERATURE CITED 126 LIST OF TABLES R e s u l t s o f a s u r v e y f o r HCO^ u p t a k e The e f f e c t o f pH change and C i chang 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 i x LIST OF FIGURES Fi g u r e Page P a r t 1-A 1. Change i n pC02 and C^ a g a i n s t time f o r a 17.4 g sample of Sargassum muticum . 8 2. 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 runs separated by a dark p e r i o d of one hour 10 3. Change i n pC0~ 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 pC02» f o r Sargassum muticum. (27 g) 11 P a r t 1-B 4. Change i n t o t a l i n o r g a n i c carbon (C^) and ppm C0 2 a g a i n s t time f o r a 4 g sample of Ulv a sp 29 5. Change i n C^ and ppm CO2 a g a i n s t time f o r a 27 g sample of Desmarestia munda. . . . . . 30 6. Change i n C^ and ppm CO2 a g a i n s t time i n Ulva f e n e s t r a t a showing dependence o f ph o t o s y n t h e t i c r a t e on C^ value r a t h e r than pH or ppm CO 2 32 7. Dependence of r a t e of photosynthesis 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 8. E f f e c t of t o t a l i n o r g a n i c carbon on r a t e of photosynthesis by I r i d a e a cordata . . . . . 38 9 . E f f e c t of pH on r a t e of photosynthesis by I r i d a e a cordata 39 10. E f f e c t of t o t a l i n o r g a n i c carbon on r a t e of photosynthesis by Sargassum muticum. . . . 42 X F i g u r e Page 11. E f f e c t of pH on r a t e of photosynthesis by Sargassum muticum. . . . . . . . . . 43 Addendum 12. E f f e c t of pH on r a t e of photosynthesis i n U l v a f e n e s t r a t a at three C\ values 13. The e f f e c t of HCOl i o n c o n c e n t r a t i o n on r a t e of photosynthesis a t f i v e pH values i n Ulva f e n e s t r a t a . . . . . . . . . . . . 51 53 Appendix 14 15 A system f o r measuring photosynthesis i n marine algae by pCC>2 and measurement w i t h c o n t r o l of pH and temperature . . 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 i a - r e d gas a n a l y s i s . . . . . . . 58 61 P a r t I I 16. 17. 18 . 19. 14 The e f f e c t of 4 mM Diamox on CO2 f i x -a t i o n by i s o l a t e d spinach c h l o r o p l a s t s : i n the l i g h t w i t h PPj_: i n the dark w i t h ATP . . . . . . . . . . The e f f e c t of Diamox and sulphanilamide on O2 production by i s o l a t e d spinach 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 of potassium f e r r i c y a n i d e . . . The e f f e c t of pH 6.5 and time on Diamox i n h i b i t i o n of 0 2 p r o d u c t i o n by spinach c h l o r o p l a s t s The e f f e c t of 4 mM Diamox on the l i g h t -induced pH s h i f t by i s o l a t e d spinach c h l o r o p l a s t s i n unbuffered sucrose medium 73 75 77 78 x i F i g u r e Page 20. The i n t e r a c t i o n of 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 Antimycin 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 by i s o l a t e d spinach c h l o r o p l a s t s 80 21. The i n t e r a c t i o n of 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 Antimycin A on 14CC>2 f i x a t i o n i n non-s a t u r a t i n g l i g h t by i s o l a t e d spinach c h l o r o p l a s t s . . P a r t I I I 22. Time course 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 i n d u c t i o n t r a n s i e n t s d u r i n g 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 of O2 on the r a t e of stea d y - s t a t e photosynthesis i n I r i d a e a cordata . 106 24. The e f f e c t of high and low O2 on (c) and (e) a t 50 ml C i per l i t r e 108 25. The e f f e c t o f hi g h and low 0 2 on (c) and (e) a t 2 ml per l i t r e 109 26. The e f f e c t of high and low O2 on (c) and (e) a t 80 ml C i per l i t r e 110 27. The e f f e c t of low C i w i t h time on (c) a t high and low 0 2 I l l 28. The e f f e c t of the preceding dark p e r i o d on the he i g h t o f (c) 114 29. The e f f e c t of DCMU on the (c) spike 116 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 s t e a d y - s t a t e reading H7 x i i ACKNOWLEDGEMENTS I would l i k e to express my g r a t i t u d e to Dr. 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 the course o f these i n v e s t i g a t i o n s . Thanks are a l s o extended to Drs. Janet S t e i n , R.F. Sca g e l , G.H.N. Towers, and N.R. B u l l e y f o r h e l p f u l a d v i c e . Dr. N.J. A n t i a , F i s h e r i e s Research Board of Canada s u p p l i e d c u l t u r e s of D u n a l i e l l a t e r t i o l e c t a . D i s c u s s i o n s w i t h Dr. R. Waygood, U n i v e r s i t y of Manitoba, and Dr. W.E. Vidaver o f Simon Fr 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 . F i n a l l y , I would l i k e to thank my husband f o r h i s constant support, without which t h i s work co u l d not have been completed. The author was a r e c i p i e n t of N a t i o n a l Research C o u n c i l of Canada S c h o l a r s h i p s d u r i n g 1 9 6 7 - 6 8 , 1 9 6 8 - 6 9 , 1 9 6 9 - 7 0 , and 1 9 7 0 - 7 1 . Research was supported by grants to Dr. Tregunna from the N a t i o n a l Research C o u n c i l of Canada and the P r e s i d e n t ' s Committee on Research, The U n i v e r s i t y of B r i t i s h Columbia. PART 1-A Bicarbonate Ion As s i m i l a t i o n i n Photosynthesis by Sargassum muticum.^" This 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 Journal of Botany: V o l . 46, 411-415 (1968), E.B. Tregunna supervised the study. 2 INTRODUCTION The a s s i m i l a t i o n of HCO^ i o n d u r i n g photosynthesis by aquatic p l a n t s has long been a c o n t r o v e r s i a l t o p i c . O s t e r l i n d (8) and Steemann N i e l s e n (11) have reviewed the 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 water p l a n t s to 1958. Recently, F e l f o l d y has st u d i e d e x t e n s i v e l y the carbon sources of green phytoplankton i s o l a t e d from Hungarian a l k a l i l a k e s (3). They have concluded t h a t some phanaerogams and algae are able to take up HCO^ i o n , w h i l e others are not. In c o n t r a s t to the co n s i d e r a b l e amount of data a v a i l -able concerning HCO^ i o n a s s i m i l a t i o n by freshwater a l g a e , i n f o r m a t i o n about t h i s phenomenon i n marine algae i s scanty. 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 of t h i s process. The s o l u t i o n of CO2 gas i n sea water e s t a b l i s h e s an e q u i l i b r i u m between f r e e C02* H 2 C 0 3 ' HCO^, and CO^ as f o l l o w s . (4, 16) C0 2 + H 20*— 7H 2C0 3^ 7HCO~ + H+" 7 CO^ + 2H + 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 to the r i g h t , w h i l e a f a l l i n pH s h i f t s i t to the l e f t . The e q u i l i b r i u m s h i f t s are v i r t u a l l y instantaneous. Normal sea water 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 the i n o r g a n i c carbon 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% of the carbon i s i n the form of f r e e CG^- Any marine p l a n t capable of u s i n g HCO^ i o n as w e l l as f r e e CG^/ would have a compet i t i v e advan-tage over forms capable of u s i n g o n l y CO2/ i f i s l i m i t i n g f o r p h otosynthesis. Indeed, we would expect to 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 high l i g h t i n t e n s i t y , to be capable of a s s i m i l a t i n g HCO^ i o n d i r e c t l y . The f i r s t i n v e s t i g a t i o n of the a b i l i t y of marine phytoplankton to a s s i m i l a t e HCO^ i o n was made by Moore e t a l . , i n 1921 ( 7 ) . He proposed t h a t any a l g a capable of r a i s i n g the pH of a sea water sample to pH 9.1 must be capable o f a s s i m i l a t i n g HCO^ i o n . In 1961, Hood and Park (5) s t u d i e d the carbon sources of the marine phytoplankton N i t z s c h i a c l o s t e r i u m , Platymonas sp., and C h l o r e l l a sp. Watt and Paasche(18) and Steemann N i e l s e n (12) , however, p o i n t out the e r r o r o f the assumption of Hood and Park t h a t the e q u i l i b r a t i o n time of C 0 2 and HCO3 i s very slow. The only other i n f o r m a t i o n a v a i l a b l e concerning HCO^ i o n u t i l i z a t i o n by marine phytoplankton has been presented by Paasche, working 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 are discussed by Steemann N i e l s e n (13) . I t was demonstrated t h a t o n l y those c o c c o l i -thophorids which d e p o s i t CaCO^ use HCO^ i o n , w h i l e naked forms are dependent on f r e e C00. Steemann N i e l s e n proposed 4 t h a t d e p o s i t i o n of CaCO^ i n c o c c o l i t h formation makes p o s s i b l e the use of HCO^ i o n i n photo s y n t h e s i s . Tseng and Sweeney (15) s t u d i e d the carbon sources 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 the r a t e of photosynthesis i n -creased w i t h decreasing pH and, t h e r e f o r e , w i t h i n c r e a s i n g pC0 2 and decreasing HCO-j i o n . The r a t e a l s o increased when pCC>2 was increa s e d w i t h constant HCO^ i o n . They concluded t h a t f o r G. c a r t i l a g i n e u m , CC^ and not HCO^ i s the f a c t o r l i m i t i n g p h otosynthesis. Their r e s u l t s , however, do not exclude the p o s s i b i l i t y of some HCO^ i o n a s s i m i l a t i o n . The purpose of t h i s i n v e s t i g a t i o n was to d i s c o v e r whether o r not Sargassum muticum (9) could u t i l i z e HCO^ i o n dur i n g photosynthesis. At the same time, reference i s made to the i m p l i c a t i o n s of the presence of 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 of Sargassum muticum were c o l l e c t e d i n the i n t e r t i d a l zone a t Brockton P o i n t , Stanley Park, Vancouver, B r i t i s h Columbia, 1966 and 1967 from e a r l y May to mid-August. The a l g a was washed w i t h f i l t e r e d sea water, and stored i n semi-darkness a t 5°C i n g l a s s aquaria f o r not more than three days. 5 The two methods t h a t were used to measure the concen-t r a t i o n of i n o r g a n i c carbon are described i n a previous paper (17). A modified v e r s i o n of the techniques i s des c r i b e d here. Samples weighing from 15 to 30 g f r e s h weight were placed i n a 1000 ml g l a s s r e a c t i o n chamber, and anchored to spread the m a t e r i a l f o r optimum i l l u m i n a t i o n . The chamber was f i l l e d w i t h precooled, f i l t e r e d sea water and placed i n a waterbath, which was maintained between 12°C and 16°C. The sealed chamber was connected i n s e r i e s w i t h a Beckman 215 I n f r a r e d Gas Analyser (IRGA) which measured the pC0 2 o f the a i r , and a sm a l l pump which c i r c u l a t e d the a i r i n the system a t 1 l./min v i a a s c i n t e r e d g l a s s bubbler w i t h i n the r e a c t i o n f l a s k . A t the same time, water was c i r c u l a t e d a t 1 l./min v i a the r e a c t i o n chamber through a water pump and a sampling chamber c l o s e d by a serum stopper to a l l o w water samples to be removed by s y r i n g e . Temperature and pH were a l s o measured as de s c r i b e d (17) . The a l g a was i l l u m i n a t e d from one s i d e by a General E l e c t r i c 300 watt "Cool Beam" lamp, a t 700 f t c. Darkness was provided by t u r n i n g o f f the l i g h t and co v e r i n g the apparatus w i t h a bla c k c l o t h . The system was monitored w i t h the IRGA and the pH meter u n t i l photosynthesis and pH changes had reduced the pC0 2 to a reading j u s t below 100 ppm. In some l a t e r experiments (see F i g u r e 1) pH was adjusted w i t h small 6 i n j e c t i o n s of concentrated NaOH i n order to reduce pCX^ to about 100 ppm. In some of the experiments, measurements of the pH, p C ^ , temperature and s a l i n i t y were used w i t h the t a b l e s given by S t r i c k l a n d and Parsons (14) to c a l c u l a t e t o t a l i n o r g a n i c carbon. From changes i n t h i s parameter, changes i n t o t a l i n o r g a n i c carbon were c a l c u l a t e d to measure photo-s y n t h e s i s i n the pH range of 7.8 to 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 ( t a b u l a t e d ) . A t 10 to 20 minute i n t e r v a l s , 1 ml water samples were withdrawn from the sampling chamber and stor e d i n the r e f r i g e r a t e r . At the time t h a t each sample was removed, pH, temperature, and PCO2 were recorded. At about pH 10, the system c o n t a i n i n g the a l g a l sample was disconnected from the IRGA. Now, the s t o r e d water samples were i n j e c t e d i n t o an a c i d bath v i a another serum stopper. This a c i d bath was p a r t o f a c l o s e d system i n c l u d i n g the IRGA. The amount of carbon (C^) t h a t was r e l e a s e d from the sea water was measured as a change of PCO2 i n the c l o s e d system. The change i n the carbon i n the sea water c o n t a i n i n g the algae was c a l c u l a t e d from the change i n 1 ml samples. The volume of the 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 of the IRGA, was 350 ml. Therefore, i n o r g a n i c carbon content could be c a l c u l a t e d ; ( C ) = .35 1. x ppm CQ0 r e l e a s e d x water volume i n r e a c t i o n 1 z f l a s k (ml) w i t h c o r r e c t i o n s f o r temperature, pressure, and s o l u b i l i t y of C0 o. RESULTS The data f o r F i g u r e s 1 and 2 were obtained by the modified a c i d r e l e a s e technique. The data f o r F i g u r e 3 were c a l c u l a t e d from pH and pC0 2 of the sea water. F i g u r e 1 shows changes i n pC0 2 and a g a i n s t time w i t h measurements taken a t 10 minute i n t e r v a l s . In t h i s sample the pH was adjusted i n i t i a l l y to 8.97 w i t h NaOH. as d e s c r i b e d . The r a t e of a s s i m i l a t i o n was constant d u r i n g a p e r i o d of 70 minutes w h i l e pC0 2 f e l l from 62 to 13 ppm and the pH s h i f t e d from pH 8.97 to pH 9.9. The 17.4 g f r e s h weight sample of S. muticum a s s i m i l -ated 39.5% of the carbon o r i g i n a l l y present i n the sea water a t a r a t e of 21 yg C0 2/min/g f r e s h weight. uptake d u r i n g the p e r i o d was not p r o p o r t i o n a l to pC0~ below 100 ppm. F i g u r e 1 Change i n p C 0 2 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 9 Other samples under the same c o n d i t i o n s a l s o had constant but lower r a t e s of a s s i m i l a t i o n ; 8, 11, and 14.5 ug C^/min/g f r e s h weight. F i g u r e 2 shows a second type of curve f o r C uptake, which was o c c a s i o n a l l y observed. Two runs separated by a dark p e r i o d of about one hour f o r one 13 g sample of S. muticum are shown. There was no a r t i f i c i a l adjustment of pH f o r t h i s sample. At the end of the second t r i a l the a l g a had r a i s e d the pH from an i n i t i a l ph 8.21 a t time zero, to pH 9.57. Figu r e 2 shows the observed change of pH w i t h time. Although the upper l i n e ( s o l i d l i n e ; A=C) i s s t r a i g h t , the second (broken l i n e ; A=C^) shows a tendency towards an i n -c r e a s i n g r a t e o f photo s y n t h e s i s . Over a t o t a l of two and one-h a l f hours, the p l a n t a s s i m i l a t e d 26% o f the i n i t i a l l y p resent i n the system. 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 rec o r d of t o t a l c a l c u l a t e d from pH and p C ^ p l o t t e d f o r comparison a g a i n s t pH. The experimental p e r i o d was about 45 minutes. During the i n i t i a l 18 minutes, PCO2 changed 54% from 318 ppm to 14 5 ppm w h i l e pH s h i f t e d from 7.8 to 8.12. The c a l c u l a t e d C , however, showed no change d u r i n g t h i s i n t e r v a l . A f t e r the 18 minute l a g phase, a s s i m i l a t i o n began. The r a t e of change of PCO2 i n c r e a s e d , and PCO2 f e l l to < 5 ppm at pH 8.6. As i n Fig u r e s 1 and 2, uptake was l i n e a r as PCO2 f e l l below 100 ppm. Fig u r e 3 gives an example of how F i g u r e 2 Change i n C and pH a g a i n s t time f o r a 13 g sample of Sargassum muticum Figure 3 Change i n pCC^ and C^ against time where C i s calculated from pH and pCC^, for Sargassum muticum (27 g) 11 lATd'd photosynthesis may be measured by c a l c u l a t i o n s of the carbonate a l k a l i n i t y from pC0 2 and pH. A f t e r the pC0 2 f e l l to l e s s than 5 ppm, water samples were removed from the system. The samples were analysed for C\ by acid release. uptake continued for a further 35 minutes at an average rate of 8 ug C0 2/min/g fresh wt. DISCUSSION In a l l plants that have been studied, the rate of C0 2 a s s i m i l a t i o n i n photosynthesis i s proportional to pC0 2 below 100 ppm, and i n most plants C0 2 becomes l i m i t i n g at far higher concentrations. I f Sargassum muticum were capable of using only C0 2 gas, a s i m i l a r trend would be expected i n photo-synthetic rate below 100 ppm C0 2. The r e s u l t s of t h i s i n v e s t i g a t i o n however, show that C^ a s s i m i l a t i o n continues at a constant rate below 100 ppm C0 2, while pC0 2 i t s e l f f a l l s o f f very r a p i d l y and the pH r i s e s . The continued a s s i m i l a t i o n of carbon when pC0 2 was n e g l i g i b l e (after Figure 3) represents the extreme of t h i s . The rate was four times as great as that at acid pH (1). Mainly HCO^ ion, however, was a v a i l a b l e as a substrate (At pH 9.1, CO^ ion forms 60% of C ) . I t was concluded that S. muticum must take up HCO., ion d i r e c t l y during photo-synthesis. Brown and Tregunna (1) have studied the C0 2 compensation values f o r a number of algae, including S . muticum. By d e f i n i t i o n , the C0 2 compensation point occurs when respi r a t o r y C0 2 production by the plant j u s t balances uptake i n photosynthesis. Under these conditions pC0 2 w i l l remain constant, and net photosynthesis can not occur i f free C0 2 i s r a t e - l i m i t i n g . I f , however, HCO^ ion used d i r e c t l y , net photosynthesis could occur below the CC>2 compensation l e v e l for the plant. The pC0 2 at compensation for Sargassum muticum i s rather high, 75 ppm, and therefore, much of the net a s s i m i l a t i o n which was observed below 100 ppm C0 2, was also below the C0 2 compensation l e v e l for the plant. Steemann Nielsen (11) has suggested t h i s condition as evidence of HCO^ ion a s s i m i l a t i o n . Brown and Tregunna (1) have also investigated the rate of photosynthesis for a number of algae, including S. muticum at pH 4.5 to 5.5 when v i r t u a l l y a l l the present (93 to 99%) i s i n the form of C0 2 gas. At pC0 2 of 300 ppm, the rate of photosynthesis was 1.9 ± .2 ug C0 2/min/g fresh weight. The rate values we have found, when C0 2 i s less than 3% of t o t a l C^, were 4 to 11 times greater. This may be explained by the p o s s i b i l i t y of HCO.J ion a s s i m i l a t i o n i n our studies. In a previous paper (17) Iridaea cordata showed net up-take i n the absence of free C0 2 at pH 8.5 to 9.6. The rates, as for Sargassum muticum, were much higher than those at acid pH where free C0 2 was the only a v a i l a b l e substrate f o r photosynthesis. 14 Steeman N i e l s e n (10) has proposed the p r e s e n t l y accepted mechanism of HCO^ i o n uptake. The p l a n t takes up HCO^ i o n , degrades i t to CO 2 and OH , and then excretes the OH i o n . This has been w e l l demonstrated f o r some phanerogams. Such a process w i l l r a i s e the pH of the surrounding medium. The C\ record f o r the l a g phase before the onset of photosynthesis i n Fi g u r e 3, shows t h a t S. muticum i s able to change the pH independently o f carbon a s s i m i l a t i o n . A s i m i l a r a b i l i t y has been shown by I r i d a e a cordata (16) . These pH s h i f t s occurred o n l y i n the l i g h t . Information has been accumulating r e c e n t l y concerning l i g h t a c t i v a t e d i o n pumps i n algae (eg. 2, 6 ) . The pH changes induced by Sargassum muticum and I r i d a e a cordata are i n t e r -p reted as the r e s u l t of an i n o r g a n i c i o n pump separate from HCO^ i o n uptake. pH change t o 9.0 or above, t h e r e f o r e , i s not i n i t s e l f s u f f i c i e n t evidence f o r HCO^ i o n a s s i m i l a t i o n , as Moore has suggested (7). Both the a c i d r e l e a s e technique and the pH-pC02 method f o r c a l c u l a t i o n of C have the advantage t h a t they measure t o t a l carbon, r a t h e r than depending on pH alone to estimate PCO2/ which a t normal pH i n sea water, c o n s t i t u t e s o n l y a very s m a l l p a r t of the t o t a l carbon. I f pH changes occur a p a r t from t h a t due to a b s t r a c t i o n of carbon i n photosynthesis, or i f some form of carbon other than CO2 i s taken up, measure-ments of photosynthesis based on pH become meaningless. 15 I n summary, Sargassum muticum shows n e t a s s i m i l -a t i o n : (a) a t a c o n s t a n t r a t e as pCC^ f a l l s below 100 ppm (b) i n t h e absence o f f r e e CC^, and (c) below CC>2 compensation l e v e l f o r t h e p l a n t . A l s o , t h e r a t e s a t t h e s e low pCC^ and h i g h pH v a l u e s a r e much h i g h e r 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 -s y n t h e s i s . We c o n c l u d e t h a t S. muticum 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 and we s u g g e s t t h a t CC^ gas may be o f minor i m p o r t a n c e 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 under normal m a r i n e c o n d i t i o n s . The pH changes i n d u c e d by Sargassum muticum and I r i d a e a (17) d u r i n g p h o t o s y n t h e s i s a r e i n t e r p r e t e d t o be a 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 HCO^ i o n u p t a k e , and some o t h e r i n o r g a n i c i o n pump. 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 photosynthesis by some algae. Can. J . Bot., 45: 1135-43. Dodd, W.A., Pitman, M.G., and West, K.R., 1965. Na + and K+ t r a n s p o r t i n the marine a l g a Chaetomorpha  d a r w i n i i . Aust. J . B i o l . S c i . 19: 341-54. F e l f o l d y , L.J.M., 1960. Experiments on the carbonate a s s i m i l a t i o n of some u n i c e l l u l a r algae by Ruttner's conductometric method. Acta B i o l . Acad. S c i . Hung., XI : 67-75. Harvey, H.W., 1963. The chemistry and f e r t i l i t y o f seawaters. 2nd ed. Cambridge U n i v e r s i t y P r e s s . Cambridge. 234 pp. Hood, D.W. and Park, K., 1962. Bicarbonate u t i l i z a t i o n by marine phytoplankton i n photosynthesis, P h y s i o l . P l a n t . , 15: 273-282. Hope, A.B., Simpson, A., and Walker, N.A., 1966. The e f f e c t of C l ~ from c e l l s of N i t e l l a and Chara. Aust. 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. Photo-s y n t h e s i s i n marine algae. Proc. Roy. Soc. Lond. Ser. 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 the a l g a Scenedesmus quadricauda w i t h s p e c i a l r e f e r e n c e to the i n o r g a n i c carbon sources. Symp. Bot. Up s a l . 10: 1-141. Scagel, R.F., 1967. Guide to common seaweeds of B r i t i s h Columbia, B.C. Prov. 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. Passive 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 of CC»2 by the p l a n t . In: Encyclopedia of 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 of carbon d i o x i d e , pp. 70-84. Ed. W. Ruhland. S p r i n g e r - V e r l a g , B e r l i n . Gottingen. H e i d e l b e r g . 17 12. Steemann N i e l s e n , E., 1963. On bicarbonate u t i l i z a t i o n by marine phytoplankton i n p hotosynthesis, w i t h a note on carbarn ino c a r b o x y l i c a c i d s as a carbon source. P h y s i o l . P l a n t . 16: 466-69. 13. Steemann_Nielsen, E., 1966. The uptake of f r e e C0 2 and HCO-j during photosynthesis of plankton algae w i t h s p e c i a l reference to the c o c c o l i t h o p h o r i d Cocco-l i t h u s h u x l e y i . 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 Parsons, T.R., 1965. A manual of sea water a n a l y s i s . B u l l . No. 125, F i s h e r i e s Research Board of Canada, Ottawa. 2nd ed. pp. 29, 35, 199, 200. 15. Tseng, C.K., and Sweeney, B.M., 1946. P h y s i o l o g i c a l s t u d i e s of Gelidium c a r t i l a g i n e u m . I . Photo-s 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 . Bot. 33: 706-15. 16. Svedrup, H.U., Johnson, M.W., Fleming, R.H., 1942. The Oceans. P r e n t i c e - H a l l Inc., Englewood C l i f f s , N.J. 1087 pp. 17. Tregunna, E.B., and Thomas, E. Ann, 1967. Measurement of i n o r g a n i c carbon i n sea water by two methods of pC0 2 and pH a n a l y s i s . Can. J . Bot. 46: 481-85. 18. Watt, W.D., and Paasche, E., 1963. An i n v e s t i g a t i o n of 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 C0 2 and bicarbonate u t i l i z a t i o n by algae according to the methods of Hood and Park. P h y s i o l . P l a n t . 16; 674-81. PART 1-B Studies on Bicarbonate ion Uptake During Photosynthesis 2 rn Benthic Marine Algae. This 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 Phycologia, V o l . 9: 293-303. (1970) E.B. Tregunna supervised the study. 19 INTRODUCTION In previous papers (8, 9) i t has been reported t h a t the marine algae I r i d a e a cordata (Rhodophyta) and Sargassum  muticum (Phaeophyta) use HCO^ i o n as w e l l as f r e e C0 2 gas, as a s u b s t r a t e f o r photosynthesis. Ikemori and N i s h i d a (2) have reported t h a t Ulva pertusa (Chlorophyta) a l s o uses HCO^ i o n , and Raven (5) has s t u d i e d the mechanism of HCO^ i o n uptake i n Hydrodictyon africanum (Chlorophyta). The uptake of HCO^ i o n i s , t h e r e f o r e , probably not con f i n e d to only a few marine algae, and i t seemed ap p r o p r i a t e to begin a survey of some be n t h i c algae common to Washington and B r i t i s h Columbia, f o r HCO^ i o n uptake. The v a r i o u s species of i n o r g a n i c carbon are present i n sea water at 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 from c o a s t a l areas, u n d i l u t e d by the i n f l u x of l a r g e r i v e r s , has a pH of 8.0 to 8.2. I f the t o t a l i n o r g a n i c carbon, C ( a l l species of CO,,) present, i s expressed as ml of C 0 2 gas per 1 water, seawater from such areas contains approximately 50 ml C0 2 gas per 1. At the p r e v a i l i n g pH, about 90% of t h i s carbon i s present as HCO^ i o n . Most of the remainder, about 6%, occurs as CO^ i o n . As the pH s h i f t s towards 9.0 c o n c e n t r a t i o n of CO^ i o n in c r e a s e s and becomes 50% of C a t pH 9.2 (7). A t e q u i l i b r i u m , C 0 2 should be l e s s than 1% a t pH 8.5. The s o l u t i o n of C 0 2 gas i s slow compared to the i o n i c s h i f t s of H9C0_ s p e c i e s , and t h i s 20 problem i s r e s p o n s i b l e f o r observed val u e s o f C0 2 which o f t e n d i f f e r from e q u i l i b r i u m . The c o n c e n t r a t i o n o f C0 2 gas i n a i r i s about 0.03% or 300 ppm, and C0 2 i s l i m i t i n g f o r photosynthesis i n land p l a n t s wherever l i g h t i s adequate. For marine p l a n t s able to use HC0 3 ion,the a v a i l -able carbon c o n c e n t r a t i o n i s upwards of 45,000 ppm, or 4.5% a t pH 8.0. The c o n c e n t r a t i o n of C0 2 gas i n seawater i s 300 to 320 ppm, but may vary w i t h the source of water as hig h as 500 ppm. The c o n c e n t r a t i o n of C0 2 gas i s o f t e n not i n e q u i l i b r i u m w i t h the atmosphere (7). Photosynthesis i s s a t u r a t e d i n land p l a n t s a t 0.1% or 1000 ppm, and growth i s i n h i b i t e d a t 5% CC»2 (4) . Because of these d i f f e r -ences i n form and c o n c e n t r a t i o n of 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 to i n v e s t i g a t e the frequency of occurrence of HCO^ i o n a s s i m i l a t i o n among some marine algae, and to study the e f f e c t s of HCO^ i o n , pH, and pC0 2, on the r a t e of photosynthesis i n algae 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 survey of HCO^ i o n uptake by benthic marine algae was conducted a t the U n i v e r s i t y of 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. P l a n t m a t e r i a l (6) was c o l l e c t e d i n the i n t e r t i d a l , near the 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 the 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 tanks, w i t h a constant flow of seawater pumped from the harbour. Seawater temperature a t t h a t time was 9° to 10°. A l l m a t e r i a l was used w i t h i n three days of 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 sp., 10 cm s e c t i o n s weighing 7 to 10 g ( f r e s h weight) were c u t from young t h a l l i near the t r a n s i t i o n zone: C o s t a r i a c o s t a t a , 10 cm s e c t i o n s , weighing 6 to 12 g were c u t as f o r A l a r i a : Desmarestia munda, s e v e r a l fronds 10 to 15 cm lo n g , weighing 22 to 28 g: Enteromorpha sp. samples 10 cm wide, weighing 3 to 4 g: Fucus sp., a 12 cm fro n d weighing 12 g: Laminaria  s a c c h a r i n a , a 10 by 12 cm pie c e cut near the t r a n s i t i o n zone: N e r e o c y s t i s luetkeana, 4 or 5 very young b l a d e s , (2.5 to 3 cm wide) weighing 8 to 12 g: Porphyra s c h i z o p h y l l a , 5 to 8 g samples: Ul v a sp., sm a l l fronds or pieces of f r o n d s , weighing 3 to 4 g. The technique was a modified v e r s i o n of t h a t d e s c r i b e d p r e v i o u s l y (9). P l a n t m a t e r i a l was placed i n 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 , and anchored f o r optimum i l l u m i n a t i o n by t y i n g the sample to a r e c t a n g u l a r g l a s s frame. F i l t e r e d seawater from the l a b o r a t o r y supply was used f o r the e x p e r i -ments. The p l a n t chamber was maintained a t 9° to 12° i n an i c e - c o o l e d water bath. A 3 00 watt General 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 (measured w i t h a Gossen T r i l u x f o o t - c a n d l e meter) from one s i d e . The sealed chamber was connected i n s e r i e s w i t h a Beckman 215 i n f r a -red CC»2 Gas A n a l y s e r (IRGA) and a s m a l l a i r pump which c i r c u l a t e d the a i r a t 1 l./min, v i a an a i r stone w i t h i n the r e a c t i o n f l a s k . Simultaneously water was pumped a t 1 l./min from the p l a n t chamber past a combination pH e l e c t r o d e , a sampling chamber c l o s e d w i t h a serum stopper to permit sample removal by s y r i n g e , through a water pump and back to the p l a n t chamber. The pH was monitored c o n s t a n t l y and temperature was measured w i t h a standard taper j o i n t c e n t i -grade thermometer i n s e r t e d i n the l i d of the chamber. The c o n c e n t r a t i o n was monitored u n t i l i t dropped to l e s s than 25 ppm; 1 ml water samples were withdrawn a t 10 to 15 minute i n t e r v a l s and s t o r e d i n a r e f r i g e r a t o r . A t each sampling time, pH, PCO2/ and temperature were recorded. Each p l a n t was t e s t e d 3 times. Water samples were analysed f o r t o t a l i n o r g a n i c carbon (C^) expressed as ml CO2 per l i t r e water, as p r e v i o u s l y d e s c r i b e d ( 9 ) . A 20 cm g l a s s chromatography column w i t h a 2.5 cm s i d e arm a t the base j u s t above the s c i n t e r e d d i s c was used as an a c i d r e l e a s e chamber. This column contained IN HC1. The s i d e arm was c l o s e d w i t h a rubber serum stopper f o r convenient sample i n j e c t i o n . Use o f t h i s column inc r e a s e d the e f f i c i e n c y of mixing i n the system and CC>2 readings 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 to 20 minutes, as was formerly the case (9). At a l l times a short glass tube f i l l e d with MgClO^ was placed i n the gas flow system just before the IRGA to prevent microdroplets of seawater or acid from entering the instrument and damaging the sample tubes. Some etching of the l a t t e r by seawater has been observed. Any measurement error due to inf r a r e d absorption by water was also avoided. These studies were continued at the Unive r s i t y of B r i t i s h Columbia, Vancouver, B r i t i s h Columbia. Plants were c o l l e c t e d from A p r i l to mid-August i n the i n t e r t i d a l zone at Brockton Point, Stanley Park, Vancouver, i n 1968 and 1969. The following material was stored at 9° i n pl e x i g l a s s tanks i n a co n t r o l l e d environment room at the Unive r s i t y , and used within two days of c o l l e c t i o n . Gigartina sp. (cristata?) was added to the survey l i s t , 11 to 12 g samples: Sargassum muticum and Iridaea cordata which have already been reported to use HCO^ ions (8,9) Ulva fenestrata samples weighing 3 to 5 g, were also examined. Studies of the e f f e c t of HCO^ ion concentration on the rate of photosynthesis were conducted with Ulva fenestrata, Sargassum muticum, and Iridaea cordata. I n i t i a l l y , measurements made with U. fenestrata were done as above, monitoring both CC»2 and C^. Seawater c o l l e c t e d i n A p r i l from the west coast of Vancouver Island was used i n 24 these experiments; water from t h i s source i s c l e a n e r and has a higher s a l i n i t y than t h a t from Burrard I n l e t . When pCC»2 f e l l below 15 ppm and pH reached 9.0 to 9.5, the pH o f the experimental water was a c i d i f i e d to pH 8.0 to 8.2, by i n j e c t i n g small q u a n t i t i e s of IN HC1 v i a the sampling chamber. The pH was then again allowed to r i s e to about pH 9.0, w h i l e pCC^ was monitored w i t h the IRGA. Samples of water were withdrawn every 10 minutes and analysed l a t e r . This r o u t i n e was repeated, u s u a l l y 3 or 4 times u n t i l i n the system was reduced from about 50 ml/1 to l e s s than 5 ml/1. Thus the e f f e c t o f on the r a t e of photosynthesis c o u l d be observed under c o n d i t i o n s of known pCG^ and pH. In l a t e r experiments PCO2 was not monitored because i t d i d not appear to s i g n i f i c a n t l y i n f l u e n c e the r a t e o f phot o s y n t h e s i s . 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 to ho l d the p l a n t , fastened to a g l a s s frame. This system, i n c l u d i n g the tygon water c i r c u -l a t i o n l i n e s , sample chamber, and water pump, had a volume of 1300 ml and contained a t l e a s t 1200 ml of water d u r i n g the experiments. Temperature was maintained a t 13° to 16°. The p l a n t s were i n i t i a l l y allowed to photosynthesize f o r about 40 minutes to permit i n d u c t i o n of photosynthesis. A s o l u t i o n of NaHCO^ was then added to the system t o r a i s e the c o n c e n t r a t i o n to about 50 ml/1. The course o f photo-s y n t h e s i s was then f o l l o w e d as was reduced. When a s e r i e s of measurements were made w i t h one sample, i n the v i c i n i t y 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 used t o a d j u s t C . The p l a n t s were t e s t e d a t t h e end o f each e x p e r i m e n t f o r a d v e r s e e f f e c t s o f t h e t r e a t m e n t by m e a s u r i n g 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 were 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 as 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 as ml Cc^ gas/1 se a w a t e r The f r e e C 0 2 gas i n a i r pumped t h r o u g h t h e seaw a t e r i s e x p r e s s e d as ppm CO- ( y l C 0 9 / 1 o f a i r ) . RESULTS AND DISCUSSION Su 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 H a r b o r . The r a t e o f p h o t o s y n t h e s i s above 100 ppm C 0 2 gas e x p r e s s e d as yg C0 2/min/g f r e s h w e i g h t , R^, i s compared f o r each a l g a , w i t h R 2, t h e r a t e o f p h o t o s y n t h e s i s below 100 ppm C 0 2 . The change i n photo s y n t h e t i c r a t e , R 2 - R 2 _ / R l ^ s termed y. The v a l u e s C^ and C 2 a r e t h e median v a l u e s 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^ and R 2 were c a l c u l a t e d , r e s p e c t i v e l y . The change i n C , o r C 2 - C,/C^ i s 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 p h o t o -Table I R e s u l t s of a survey f o r HCO-j uptake. R^ r a t e o f photosynthesis when pCC^ exceeds 100 ppm R 2 r a t e of photosynthesis below 100 ppm y the change i n r a t e of photo s y n t h e s i s , median value of t o t a l i n o r g a n i c carbon (C^) about which R^ was c a l c u l a t e d C„ median value of C. about which R~ was 2 1 2 c a l c u l a t e d x the change i n C. y/x represents the r a t i o of change i n photo-s y n t h e t i c r a t e to change i n s u b s t r a t e c o n c e n t r a t i o n 26 Plant Ulva fenestrata A l a r i a sp. Nereocystis Luetkeana Costaria costata En teromorpha l i n z a Porphyra  schizophylla Desmarestia munda TABLE I Rate of Photosynthesis ug/min/g.fr. wt R 2-R 1 - y C2-Cl = x y/x R l R 2 R l C l 15.4 12.6 -.18 -.22 .8 16.1 12.6 -.10 -.25 •4(.7) 14.0 12.6 -.22 -.24 .9 5.9 4 .3 -.27 -.17 1.6 4.5 4.5 -.00 -.20 0 1.2 7.8 4.1 -.47 -.23 2.0 5.4 3.6 -.28 -.17 1.6 2.8 2.1 -.23 -.12 1.9 1.3 2.4 2.1 -.09 -.19 .47 6.0 4.1 -.29 -.18 1.6 7.5 5.4 -.27 -.20 1.4 1.5 4.6 2.8 -.30 -.20 1.5 15.4 10 .8 -.30 -.16 1.9 8.4 5.4 -.36 -.13 2.8 2.3 11.9 6.6 -.35 -.22 1.6 7.0 3.3 -.55 -.20 2.8 16 .6 5.8 -.65 -.18 4.0 3.1 10.9 5.1 -.53 -.21 2.5 2.1 2.0 1.4 .56 1.0 .75 -.63 -.54 -.50 -.16 -.17 -.14 3.9 3.2 3.5 3.6 27 s y n t h e s i s , and 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 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 , y/x w i l l i n c r e a s e t o 1 as s u b s t r a t e becomes l i m i t i n g . Where n o t a l l t h e C\ i s a v a i l a b l e t o t h 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 e x c e ed 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 p h o t o s y n t h e t i c r a t e below 100 ppm CC»2. I f we examine T a b l e 1, however, we see t h a t t h e a l g a e appear t o f a l l i n t o a graded s e r i e s o f v a l u e s f o r y/x. A t one extreme i s U l v a f e n e s t r a t a , w i t h y/x e q u a l t o 0.7, l e s s t h a n 1. S i n g l e samples o f Fucus s p . and L a m i n a r i a s a c c h a r i n a showed y/x e q u a l t o 0, t h a t i s , t h e y showed no change i n r a t e o f p h o t o s y n t h e s i s from 400 ppm C 0 2 t o l e s s t h a n 25 ppm C 0 2 . S i n c e o n l y one sample o f each was u s e d , t h e y a r e n o t l i s t e d i n T a b l e 1. U l v a showed a d r o p o f 10 t o 20% 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 o f 22 t o 25% i n C i . 28 At the opposite extreme, Porphyra and Desmarestia have y/x values 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 of over 50% i n photosynthetic r a t e , w h i l e the change i n f o r Porphyra, 18 to 21%, i s only s l i g h t l y l e s s than t h a t f o r U l v a . Obviously the d e c l i n e i n r a t e i s not d i r e c t l y r e l a t e d to d e c l i n e i n t o t a l i n o r g a n i c carbon. Between the 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 to be more a l l i e d w i t h Ulva than w i t h Porphyra or Desmarestia. These la m i n a r i a n s show y/x values of 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 the three k e l p genera are s i m i l a r to each o t h e r , and s l i g h t l y lower than f o r Ul v a . Enteromorpha appears to be i n the middle of the s e r i e s , w i t h y/x equal to 2.3, and a change i n C^ s i m i l a r to Desmarestia. On the b a s i s o f the data presented i n Table 1, and to 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 algae t e s t e d i n t o two groups,: the "Ulva-type" w i t h y/x equal to or l e s s than 1.5, and the "Desmarestia-type" w i t h y/x equal t o , or gr e a t e r than 2.3. Figures 4 and 5 show p l o t t e d r e s u l t s f o r Ulva and Desmarestia. The C^ and ppm C0 2 are p l o t t e d a g a i n s t time f o r both algae. The slope o f the l i n e f o r C^ ( s o l i d l i n e ) i s the r a t e of photosynthesis. Since, f o r a l l the algae t e s t e d , pH rose from about 8.0 to above 9.5, duri n g the course of the experiment, the observed change i n ppm C0 2 may be l a r g e l y a t t r i b u t e d to a s h i f t of the H_CO_ e q u i l i b r i u m F i g u r e 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 sample o f U l v a s p . 29 T I M E M I N U T E S F i g u r e 5 Change i n C and ppm CC^ 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 0 20 '40 6 0 80 100 120 140 160 180 T I M E M I N U T E S w i t h pH. From the graphs, i t i s apparent t h a t w h i l e the r a t e of photosynthesis f a l l s o f f markedly i n Desmarestia as pCC>2 drops below 100 ppm, the r a t e f o r Ulva f a l l s o f f much l e s s . (See Table 1 ) . These data are s i m i l a r to those p r e v i o u s l y d e s c r i b e d 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 Ulva 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 of other algae i n the "Ulva-group", the d e c l i n e observed i n phot o s y n t h e t i c 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 than by a drop i n pC0 2 or an i n h i b i t o r y r i s e i n pH. Fi g u r e 6 shows the r e s u l t s of a r e p r e s e n t a t i v e t e s t made on an 8.6 g sample of U l v a , w i t h C and ppm C0 2 p l o t t e d a g a i n s t time. At i n t e r v a l s , when pH rose from about 8.0 to 10.0 and pC0 2 f e l l to l e s s than 5 ppm, the experimental water was a c i d i f i e d w i t h IN HC1 to pH 8.8. The system was not opened. From Fi g u r e 6, i t i s apparent t h a t the sudden in c r e a s e i n f r e e CC»2 gas re l e a s e d by a c i d i f i c a t i o n d i d not r e s t o r e the r a t e s of photosynthesis to those observed a t the beginning of the experiment. The r a t e of uptake d e c l i n e s s t e a d i l y w i t h the drop i n . During the experiment, pC0 2, C^, pH, and temperature were recorded. No s i g n i f i c a n t e f f e c t o f pH i s apparent. The photo s y n t h e t i c r a t e reached zero a t values of below 1 ml/1. When a sa t u r a t e d NaHCO^ s o l u t i o n was i n j e c t e d i n t o the cl o s e d system near the end of the experiment, the r a t e of photosynthesis i n i t i a l l y observed was immediately r e s t o r e d . The d e c l i n e i n pho t o s y n t h e t i c F i g u r e 6 C h a n g e i n C a n d ppm C C ^ a g a i n s t t i m e i n U l v a f e n e s t r a t a s h o w i n g d e p e n d e n c e o f p h o t o s y n t h e t i c r a t e o n C. v a l u e r a t h e r t h a n p H o r ppm C0_ 32 460 0 380 0 o P H 10.2 PH 8.8 \ A^ 0 40 80 120 TIME MINUTES \ pH 8.8 ^ 160 >H 10.0 vpH 8.5 H 9.8 DH 9.4 200 240 280 320 48 44 40 36 32 28 24 120 16 12 CvJ O 18 u o 0 r a t e was, t h e r e f o r e , reversed by r e s t o r i n g the i n o r g a n i c carbon. Since only HCO^ i o n was added to the system, one may conclude t h a t the d e c l i n e i n photosynthesis was not caused by increased c o n c e n t r a t i o n . Incomplete s t u d i e s on Fucus sp., Laminaria saccharina and G i g a r t i n a sp. ( c r i s t a t a ? ) , i n d i c a t e t h a t these algae belong to the "Ulva-type". Considerable d i f f i c u l t y was encountered i n measuring photosynthesis i n Fucus and Laminaria. The l a t t e r p a r t i c u l a r l y , exudes l a r g e q u a n t i t i e s of mucilage from c u t s u r f a c e s , which may i n h i b i t photo-s y n t h e s i s . These three algae, however, showed y/x values of zero. 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 values o f zero. From graphs of the types shown i n Figures 4 and 6, i t i s concluded t h a t p l a n t s of the "Ulva-type" have l i t t l e dependence on CO2 gas as a s u b s t r a t e f o r photosynthesis and are able to use HCO^ i o n as t h e i r major source of carbon. In a d d i t i o n , Ulva shows a r a t e o f photosynthesis dependent on C^ as the c o n c e n t r a t i o n of HCO^ i o n changes from more than 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 to more than 80% a t pH 10.0. Ulva may be able to use both HCO~ i o n and CO~ i o n . I t i s not c l e a r why the r a t e s of photosynthesis f a l l o f f more r a p i d l y i n Desmarestia and Porphyra than i n the other genera. Values of y/x l e s s than 2.5 or a drop i n photo-s y n t h e t i c r a t e of l e s s than 50% were not found i n Porphyra or Desmarestia. Desmarestia 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 very low v a c u o l a r pH, and when o n l y s l i g h t l y d e s i c c a t e d or warmed, the c e l l s r e a d i l y break down r e l e a s i n g H^SO^ . P o s s i b l y Desmarestia i s s e n s i t i v e to pH change, so t h a t a s h i f t i n pH i n h i b i t s p h o t o s y n t h e s i s . In Table 1, the second and t h i r d r a t e s given f o r Desmarestia were measured on the same p l a n t sample. Whatever the cause o f the 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 a t l e a s t p a r t l y r e v e r s i b l e . The suggestion 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 Porphyra than f o r Desmar- e s t i a . Porphyra r o u t i n e l y t o l e r a t e s severe d e s i c c a t i o n as i t d r i e s to a b r i t t l e c o n d i t i o n on the rocks d u r i n g low t i d e p e r i o d s . As t h i s occurs, i t probably 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 , or perhaps a d d i t i o n a l l y , p l a n t s show-i n g the same.shaped curve as Desmarestia and Porphyra may r e q u i r e both C0 2 gas and HCO-j i o n , or only the former, as s u b s t r a t e f o r photosynthesis; o r , they may be unable to use, or be i n h i b i t e d by CO^ i o n , which i n c r e a s e s r a p i d l y over the pH range employed. The r a t e o f photosynthesis drops s h a r p l y i n the r e g i o n pH 8.0 to 8.5 where pCC^ f a l l s from 400 ppm t o 100 ppm, and CO^ r i s e s from 6% of C to 15%. Porphyra, l i k e Desmarestia shows a t l e a s t 60% recovery when 35 placed i n f r e s h seawater f o r a second t e s t , (see Table 1, samples 2 and 3 f o r Porphyra) so t h a t the 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 of HCO^ Ion Concentration on Photosynthesis F i g u r e 7 shows the e f f e c t of C on the r a t e o f photo-s y n t h e s i s i n Ulva f e n e s t r a t a . In the pH range 7.8 to 9.0, i n which the measurements were made, HCO^ i o n i s the dominant form of C , (50% of C a t pH 9.0 i s i n the form of HC0~ i o n ) , and a l l C i s e f f e c t i v e l y i o n i z e d ; t h e r e f o r e , i n the f o l l o w -i n g d i s c u s s i o n , C i s synonomous w i t h " i o n i z e d C^". In Fi g u r e 7, r a t e of photosynthesis i n ug CC^/min/g f r e s h weight i s p l o t t e d a g a i n s t C i n ml CC^/l seawater. Under the experimental c o n d i t i o n s of 5000 f t c i l l u m i n a t i o n , photo-s y n t h e s i s i n Ulva appears to be n e a r l y s a t u r a t e d a t 4 5 t o 50 ml C ^ / l water, w i t h a break a t about 30 ml C^ where the r a t e of i n c r e a s e i n photosynthesis w i t h C^ begins to d e c l i n e . From 5 ml to 30 ml C^, the dependence of r a t e o f photo-s y n t h e s i s on C i s e s s e n t i a l l y l i n e a r . A t 45 to 50 ml C , the values o r d i n a r i l y measured i n seawater, the r a t e of photosynthesis i n Ulva i s 200 ug CC^/min/g f r e s h weight or 12 mg C0 2/hr/g. Fig u r e 4 i s r e p r e s e n t a t i v e of the general order. This i s s a t i s f a c t o r y , s i n c e the algae used came from the v i c i n i t y of F r i d a y Harbour (Figure 4) and from Burrard I n l e t (Figure 6 ) . In Figu r e 4 the values of R, and R_ were F i g u r e 7 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 c a r b o n f o r U l v a f e n e s t r a t a R A T E O F P H O T O S Y N T H E S I S pG. C0 2^M IN^G. FR. WT.XlO O 4^U c a l c u l a t e d a t values of 38 ml/1 seawater and a t 25 ml/1 seawater, r e s p e c t i v e l y . The value of y was -.18, t h a t i s , photosynthesis d e c l i n e s by 18%. From the composite r a t e versus c o n c e n t r a t i o n curve i n F i g u r e 4, the p r e d i c t e d value f o r y, when C\ f a l l s from 3 8 ml/1 to 29.5 ml/1, i s -.22 or a d e c l i n e of 22%. I r i d a e a cordata samples o f 5 to 10 g were i l l u m i n a t e d a t 2500 f t c. The composite graph o f r a t e of photosynthesis versus i s shown i n Figure 8. Some d i f f i c u l t y was experienced i n i l l u m i n a t i n g t h i s p l a n t because of v a r i a b i l i t y i n t h i c k n e s s of the t h a l l u s . S e c t i o n s of t h a l l u s of the same area o f t e n vary 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 weight. Thus, the t h i c k e r p i e c e s of 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 from one s i d e tended to e x h i b i t reduced r a t e s o f photosynthesis. When higher l i g h t i n t e n s i t i e s , such as 5000 f t c were used, 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 times than 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 to pH than e i t h e r U l v a or Sargassum. In Figure 9 some tendency i s observed f o r r a t e s 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 decreasing C\. Table 2 shows some examples taken from Fi g u r e 9. I t can be seen t h a t a 0.5 change i n pH a t the same co n c e n t r a t i o n reduces the r a t e of photosynthesis by 15%. When the pH i s constant, however, and C. i s reduced a l a r g e Figure 8 E f f e c t of t o t a l inorganic carbon on rate of photosynthesis by Iridaea cordata RATE OF PHOTOSYNTHESIS (JG. C02JM\H./ G. FR. WT. 8€ Figure 9 Effect of pH on rate of photosynthesis by Iridaea cordata RATE OF PHOTOSYNTHESIS U G./M I N./G. FR. WT. Table 2 The e f f e c t of 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 of Photosynthesis at: C.(ml/1) pH 8.0 pH 8.5 Change i n Rate 1 of Photosynthesis 44 57 yg/min/g -15% 43 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. Despite the e f f e c t o f pH i n depressing 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 causing 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 6 may be a t t r i b u t e d t o a f a l l i n s u b s t r a t e c o n c e n t r a t i o n . As has been shown p r e v i o u s l y , I r i d a e a can photosynthesize i n the absence of f r e e C 0 2 gas (9), as measured by the IRGA, and t h e r e f o r e , must be capable o f u s i n g HCO^ i o n . The r a t e s of photosynthesis shown i n F i g u r e 8 f o r I r i d a e a a t from 45 to 50 ml/1 water, are low compared to those f o r Ulva; 54 ug/min/g f r e s h weight or 3.2 mg/hr/g f o r I r i d a e a compared to 12 mg/hr/g f o r U l v a . The graph i n F i g u r e 8 i n d i c a t e s t h a t under the experimental 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 i n c r e a s e d from 10 ml to 50 ml C^. The graph shown i s e s s e n t i a l l y l i n e a r but we must assume a sharp downward curve beginning a t about 12 ml and e x t r a p o l a t i n g a t or s l i g h t l y above 0.0 ml C,. I t i s i n t e r e s t i n g t o note t h a t a t 12 ml C\ the r a t e of photosynthesis i s a l r e a d y about 30 ug/min/g, about 50% of the r a t e reached a t over 50 ml C^. S e n s i t i v i t y of the p l a n t to time e f f e c t s prevented us from o b t a i n i n g values f o r p h o t o s y n t h e t i c r a t e below 10 ml C^. F i g u r e 10 shows the e f f e c t of C\ on the r a t e of photosynthesis f o r Sargassum muticum. Fig u r e 11 shows the r e s u l t s o f a t y p i c a l run f o r Sargassum. This p l a n t has p r e v i o u s l y been shown to be dependent on HCO^ i o n f o r p h o t o s y n t h e s i s . The pH value a t which each p l o t t e d r a t e of F i g u r e 10 E f f e c t of t o t a l i n o r g a n i c carbon on r a t e of photosynthesis by Sargassum muticum RATE OF PHOTOSYNTHESIS pG. C 0 2 / M 1 N.y" G.FR. WT. ro o NO CO oo ro F i g u r e 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 36 32. of *28J d 24j O O o 20J GO LU >- i o CO o o x CL < CC 8J 4J 83A 9.0i 8 5 * ^ 8 . 3 92*^9.1 812 ^ A " 8.6 A ao 8.8 87 9.1 8,6 Q.25\ 8.45 A. 813 01 0 8 12 16 20 24 28 32 36 40 44 48 52 Cj ML. COo/L. ) /L 44 photosynthesis was measured i s given on the graph. L i t t l e d i f f i c u l t y was experienced i n Sargassum with s e n s i t i v i t y of rate of photosynthesis to pH between pH 8.0 and pH 9.0. Rates measured at pH 8.25 and 8.3/ when was 52 ml/1 and 19 ml/1 respectively, have values of 30 yg/min/g fresh weight and 15 yg/min/g fresh weight, i n that order. Similar data may be observed at pH 9.0. (See Figure 11). From these examples we may conclude that the rate change i s not due to a r i s e i n pH; the decline i n photosynthetic rate instead i s due to the f a l l i n concentration of HCO^ ion. I t i s possible that Sargassum, l i k e Ulva, may be able to use COj i o n . As f o r Iridaea cordata, HCO^ ion i s apparently l i m i t -ing f o r photosynthesis i n Sargassum muticum, under the experimental conditions. At 45 to 50 ml C\ , the rate of photosynthesis i s about 30 yg/min/g fresh weight or 1.8 mg/hr/g fresh weight. Except f o r the work e s t a b l i s h i n g the dependence of rate of photosynthesis i n Ulva on C^, a l l the rates of carbon a s s i m i l a t i o n measured f o r marine benthic algae i n t h i s study are extremely low compared to those commonly observed for such land plants as wheat and corn. Further study i s necessary to f i n d out whether these low rates are a function of the experimental or growing conditions and i f so, what photosynthetic rates can be achieved by these plants under i d e a l conditions of l i g h t and substrate concentration. The r a t e s observed f o r U l v a f e n e s t r a t a of 12 mg/hr/g f r e s h weight are much higher than those f o r I r i d a e a cordata and Sargassum muticum; 3.2 mg/hr/g f r e s h weight and 1.8 mg/hr/g f r e s h weight. In a d d i t i o n , these r a t e s are higher than those obtained a t F r i d a y Harbor; 0.9 mg/hr/g f r e s h weight. They are s i m i l a r to p h o t o s y n t h e t i c r a t e s f o r wheat (1). I t has been suggested t h a t the world food problem might be solved by "farming the sea". 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 of the oceans w i t h regard to the 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 which are l i m i t i n g i n nature. He suggests t h a t estimates of p o s s i b l e produc-14 t x v i t y based on s h o r t term r a t e s of CC^ a s s i m i l a t i o n o r C»2 p r o d u c t i o n , are f a r too h i g h . 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 nversion of phytoplankton to f i s h would g i v e a y i e l d o n l y 0.1 to 0.2% o f the e d i b l e land crop. On t h i s 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 food needs. The algae c o l l e c t e d a t Brockton P o i n t i n Vancouver Harbour undoubtedly had a higher 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 to them (because of sewage o u t f a l l ) than the algae c o l l e c t e d a t F r i d a y Harbor, and the i n c r e a s e d r a t e s observed a t Vancouver f o r Ulva may r e f l e c t t h i s n u t r i t i o n a l s t a t u s . The low r a t e s of photosynthesis g e n e r a l l y observed here support the suggestion t h a t more measurements are needed of a c t u a l marine p r o d u c t i v i t y . In view of the e f f e c t s d e s c r i b e d h e r e o f pH and pCC^ on 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 t a k e n t o m o n i t o r o r c o n t r o l t h e s e p a r a m e t e r s when u s i n g such t e c h n i q u e s as t h e l i g h t - d a r k b o t t l e method f o r m e a s u r i n g p r o d u c t i v i t y . 47 LITERATURE CITED 1. Hesketh, J.D. 1967. Enhancement of phot 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 the absence of oxygen, as depen-dent upon species and temperature. P l a n t a 76s 371-374. 2. Ikemori, M. and N i s h i d a , K. 1966. Inorganic carbon source and the i n h i b i t i o n of Diamox on the photosynthesis of marine algae - Ulva p e r t u s a . Ann. Rep. Noto Marine Lab. 7: 1-5. 3. L a t i e s , G.G. 1969. Metab o l i c Sinks - D i s c u s s i o n . In P h y s i o l o g i c a l aspects of crop y i e l d . E d i t e d by J.D. E a s t i n , F.A. Haskins, C.Y. S u l l i v a n , and G.H.M. van B a v e l . American S o c i e t y of Agronomy, Crop Science S o c i e t y of America. Madison, Wisconsin, U.S.A. pp. 182-184. 4. Rabinowitch, E . I . 1945. Photosynthesis and r e l a t e d processes. V o l . I . I n t e r s c i e n c e P u b l i s h e r s , Inc., New York, N.Y. 2088 pp. 5. Raven, J.A. 1968. The mechanism of phot o s y n t h e t i c use of bicarbonate by Hydrodictyon africanum. J . Expt, Botany, 19: 193-206. 6. Scagel, R.F. 1967. Guide to common seaweeds of B r i t i s h Columbia. B.C. Prov. Museum, Handbook No. 27. The Queen's P r i n t e r , B.C. 330 pp. 7. Skirrow, G. 1965. The d i s s o l v e d gases - carbon d i o x i d e , In Chemical oceanography V o l . I . E d i t e d by J.P. R i l e y and G. Skirrow. Academic P r e s s , London and New York. pp. 227-322. 8. Thomas, E.A. and Tregunna, E.B. 1968. Bicarbonate i o n a s s i m i l a t i o n i n photosynthesis by Sargassum  muticum. Can. J . Botany, 46: 411-415. 9. Tregunna, E.B. and Thomas, E.A. 1968. Measurement of i n o r g a n i c carbon and photosynthesis i n seawater by pCO~ and pH a n a l y s i s . Can. J . Botany, 46: 481-485. PART 1 - ADDENDUM The e f f e c t of pH on photosynthesis by U l v a fenestrata 49 ADDENDUM The E f f e c t of pH on Photosynthesis by Ulva f e n e s t r a t a (4) I t has been reporte 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 the t o t a l i n o r g a n i c carbon a v a i l a b l e f o r photosynthesis had a greater e f f e c t on r a t e of photo-s y n t h e s i s than d i d pH changes. This was p a r t i c u l a r l y t r u e of Ulva f e n e s t r a t a . The f o l l o w i n g study was undertaken t o f u r t h e r examine the e f f e c t of pH on photosynthesis i n U l v a . MATERIALS AND METHODS Ulva 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 to 26 i n 1971. This p e r i o d covered two s e r i e s of low t i d 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 time, a l l of s i m i l a r s i z e , would be of s i m i l a r m a t u r i t y and p h y s i o g i c a l c o n d i t i o n . P l a n t m a t e r i a l was s t o r e d a t 12°C as 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 hours. Photosynthesis was measured as i n previous experiments (7) w i t h one m o d i f i c a t i o n ; the pH was he l d constant w i t h 20mM T r i s b u f f e r d u r i n g the course o f each s e r i e s o f measurements. Three separate t e s t s , on d i f f e r e n t p l a n t samples were run at pH 7.5, 8.0, 8.5, 9.0 and 9.5. The pH was checked a t the end of the experiment, and before the a d d i t i o n of 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 adjustments 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 given by Sigma Chemical C o r p o r a t i o n , showing the e f f e c t of tempera-tur e on T r i s b u f f e r . (5) RESULTS AND DISCUSSION The r a t e of photosynthesis f o r Ulva a t each pH was p l o t t e d a g a i n s t C^, 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 taken 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 to produce F i g u r e 12, which shows the apparent e f f e c t o f pH on r a t e of photosynthesis a t f i v e pH values and three carbon c o n c e n t r a t i o n s . The v a l u e s a t pH 8.0 and 8.5 are not s i g n i f i -c a n t l y d i f f e r e n t . These r a t e s were taken from the a p p r o p r i a t e r e g r e s s i o n l i n e s . I t i s apparent t h a t a p r o g r e s s i v e i n c r e a s e i n pH i s accompanied by a r e d u c t i o n i n the r a t e of photo-s y n t h e s i s . 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 decreased as C. i s reduced. i A t f i r s t i t might 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 photosynthesis or on some f a c t o r i n -f l u e n c i n g p h o t o s y n t h e s i s . As pH changes from 7.5 t o 9.5, there i s a massive s h i f t i n the dominant form of H^CO^ ( 6 ) , from 90% HCO^ i o n to 35% HC0.J i o n . At the same time CO~ i o n c o n c e n t r a t i o n changes from 1% to 65% of C . The remaining C. a t pH 7.5 Figure 12 E f f e c t of pH on rate of photosynthesis i n Ulva fenestrata at three C. values i s CC>2 gas and u n d i s s o c i a t e d I^CO^. I t has been suggested t h a t Ulva may use C0~ i o n as a s u b s t r a t e f o r photosynthesis on the b a s i s of such obs e r v a t i o n s as are presented here. (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 h y p o t h e s i s . Rate of photosynthesis a t three values and a l l f i v e pH values was 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 HCO^ i o n c o n c e n t r a t i o n , i n s t e a d o f a g a i n s t C^, which i n c l u d e s a l l i n o r g a n i c carbon. I t appears from F i g u r e 13, t h a t as pH i n c r e a s e s (and HCO^ i o n c o n c e n t r a t i o n f a l l s r a p i d l y ) the p l a n t uses HCO^ i o n more e f f i c i e n t l y . The r e s u l t i s t h a t the r a t e achieved a t pH 9.5, and 20 ml HCO~, i s very c l o s e to t h a t achieved a t pH 7.5 and 40 ml HCO^. A tendency to in c r e a s e d e f f i c i e n c y i s a l s o apparent 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 of these 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 capable of u s i n g CO"^  i o n . I t i s a t pH 9.0, t h a t CO~ c o n c e n t r a t i o n reaches 40% of C , and thence becomes an important source of carbon. I f CO^ c o n c e n t r a t i o n i s i n c l u d e d i n C^, as i n e a r l i e r work, t h i s amazing a f f i n i t y f o r HCO^ i o n a t high pH values d i s a p p e a r s . There i s a depressant e f f e c t of i n c r e a s e d pH on r a t e o f photosynthesis, however, which i s superimposed on the above e f f e c t s . Otherwise, use of HCO~ i o n and CO~ ions probably r e q u i r e s an e n e r g e t i c process f o r importing them, s i n c e they are not l i p i d s o l u b l e (that i s , can not d i f f u s e f r e e l y through 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^ 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 U l v a f e n e s t r a t a u n l i k e 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 uptake to 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 so, C0~ might have to reach a f a i r l y h i g h concen-t r a t i o n (40%?) before competition f o r the 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 than f o r HCO^, a reduced r a t e of photo-s y n t h e s i s would not be unexpected as CO^ became the dominant s u b s t r a t e . Much higher values where a l l C\ was COj would be r e q u i r e d to s a t u r a t e photosynthesis than when HCO^ composed most o f C.. x Some of the r e d u c t i o n of r a t e between pH 7.5 and 8.0 could be caused by the disappearance of C 0 2 as a carbon source. About 10% of i s C 0 2 gas a t pH 7.5. At pH values of about 5, Ulva w i l l photosynthesize a t a low r a t e (about 0.05% o f the value a t 7.5) (1). At t h i s pH, C i i s over 90% C0 2, which can probably enter the c e l l by pas s i v e d i f f u s i o n , s i n c e i t i s l i p i d s o l u b l e . 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 places a s t r a i n on the i n t e r n a l pH r e g u l a t i n g mechanisms of the p l a n t . This may be p a r t l y r e s p o n s i b l e f o r the decrease i n r a t e of photosynthesis, when su b s t r a t e c o n c e n t r a t i o n remains the same, but 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 photosynthesis by some algae. Can. J . Bot., 45: 1135-1143. J o l l i f f e , (Thomas) E.A. and Tregunna, E.B. 1970. Studies on HCO^ i o n uptake d u r i n g photosynthesis i n benthic marine algae. P h y c o l o g i a , 9: 293-303. Raven, J.A. 1968. The mechanism o f p h o t o s y n t h e t i c use of bicarbonate by Hydrodictyon africanum. J . Expt. Botany, 19: 193-206"; Sca g e l , R.F., 1967. Guide to common sea-weeds of B r i t i s h Columbia. B.C. Prov. Museum, Handbook No. 27. The Queen's P r i n t e r , B.C. 330 pp. Sigma Chemical Company, 1971. P r i c e l i s t , p. 299. Skirrow, G. 1965. The d i s s o l v e d gases-carbon d i o x i d e . I n : Chemical Oceanography V o l . I . Ed. J.P. R i l e y and G. Skirrow. Academic Pr e s s , London and New York. pp. 227-322. Tregunna, E.B. and Thomas, E.A. 1967. Measurement of i n o r g a n i c carbon i n sea water by two methods o f pC02 and pH a n a l y s i s . Can. J . Bot., 46: 481-485. PART 1 - Appendix Measurement of i n o r g a n i c carbon and photosynthesis i n sea-water by pC0 9 and pH a n a l y s i s The question of whether marine algae can d i r e c t l y a s s i m i l a t e HCO^ i o n was long a c o n t r o v e r s i a l one, l a r g e l y because of the 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 of d i s s o l v e d CC^ gas and d i r e c t uptake of HCO^ i o n . The methods d e s c r i b e d here were designed to all o w d i f f e r e n t i a t i o n between use of CC^ gas and HCO^ i o n (or other i o n i c species of H^CO^). Fig u r e 14 shows the apparatus used to measure photo-s y n t h e s i s i n marine algae. Samples o f algae were placed i n a g l a s s or p l e x i g l a s s chamber and anchored to a g l a s s frame or weight, to spread the 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 used, w i t h four standard taper j o i n t s i n the l i d . (In 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 de s c r i b e d i n p a r t I-B.) Temperature and pH sensors (Figure 14h,j) entered through the l i d , as d i d tu b i n g f o r c i r c u l a t i n g gas and/or water. The gas passed through a s c i n t e r e d g l a s s d i s c , (Figure 14b) or an a i r s t o n e , to enter the water. During a i r c i r c u l a t i o n , a f l o w o f 1 l./min was maintained. Water was c i r c u l a t e d by pumping a t 1.4 1/min. The pCC»2 was measured w i t h a Beckman I n f r a r e d Gas Analyser (Figure 14e), Model 215, w i t h f u l l s c a l e equal to 600 ppm. carbon d i o x i d e . The instrument was c a l i b r a t e d from a c y l i n d e r of compressed gas standardized by Matheson of Canada, L t d . This gas con-t a i n e d 180 ppm carbon d i o x i d e . The an a l y s e r was s e t to zero w i t h wet CCu-free a i r o r N~, and the readings recorded F i g u r e 14 A system f o r measuring photosynthesis i n marine algae by pCC^ and C\ measurement, w i t h c o n t r o l o f pH and temperature (a. (b (c (d (e (f (g (h ( i ( j (k (1 (m (n (o r e a c t i o n f l a s k s c i n t e r e d g l a s s bubbler a i r pump tube of 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 analyser (IRGA) water sampling chamber water pump pH e l e c t r o d e pH meter telethermometer 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 Sargent p o r t a b l e pH meter (Figure 14i) was used to c o n t i n u o u s l y measure pH, which was recorded by s t r i p c h a r t r e c o r d e r , or noted every 5 minutes. The combination pH e l e c t r o d e had a low sodium e r r o r , and a standard taper 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 seawater. Standard-i z a t i o n , u s u a l l y a t pH 7.4 and 10.0 was done w i t h commercial b u f f e r s which are accurate to ± 0.01 pH. S l i g h t 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 (Figure 14c,g) were used to c i r c u l a t e the a i r and seawater through Tygon and g l a s s t u b i n g . Water temperature was measured w i t h a telethermometer (Figure 14k) having a standard taper 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 achieved by p l a c i n g the f l a s k c o n t a i n i n g the seawater i n a g l a s s aquarium water bath (Figure 14o) whose temperature was cooled by a B r o n w i l l Thermoregulator, or by simply adding i c e to the bath. A l l experiments were c a r r i e d out i n the range 12° t o 16°C. I l l u m i n a t i o n f o r photosynthesis was provided by a General E l e c t r i c "Cool-Beam" lamp; 300 watts (Figure 14n). The l i g h t passed through approximately 8 inches of water and an i n c h o f g l a s s before reaching the algae. In e a r l y experiments the l i g h t i n t e n s i t y of 7000 l u x was c o n t r o l l e d w i t h sheets of white f i l t e r paper and measured w i t h a Weston model 756 l i g h t meter. In l a t e r work, l i g h t i n t e n s i t y was 25,000 to 50,000 l u x . To measure pC0 2, gas c i r c u l a t e d through the seawater and through the i n f r a r e d gas analyser. The pC0 2 became constant within one minute of c i r c u l a t i n g the gas through the seawater. To remove water samples f o r release of the t o t a l C0 2 by a c i d i f i c a t i o n , a water pumping system with a sampling chamber was added to the system. In order to release the t o t a l C0 2 of the seawater, water samples were removed with a syringe v i a a rubber serum stopper. Any a i r bubbles i n the syringe were removed immediately, and 0.5 or 1.0 ml samples were injected through a second serum stopper into a 60 ml glass chromatography column containing 25 ml of IN hydrochloric or sulphuric a c i d . This column (Figure 15b) was part of a closed, c i r c u l a t i n g system (Figure 15) including a pump (Figure 15f) and i n f r a r e d gas analyser (Figure 15e). The t o t a l volume of t h i s system was 250 ml. The acid was bubbled vigorously by the c i r c u l a t i n g gas passing upwards through a scintered glass d i s c . The concentration of CC»2 i n the acid-release system was reduced before each i n j e c t i o n by f l u s h i n g the system with C0 2-free a i r or N 2. The CC»2 released by acid from 0.5 or 1.0 ml samples was used to c a l c u l a t e the t o t a l amount of inorganic carbon i n the system, expressed as ml of C 0 2 > This c a l c u l a t i o n required the water volume i n the chamber containing the alga, the volume of the acid release system, and corrections f o r temperature, pressure, and s o l u b i l i t y of CO- i n the a c i d . • Figure 15 A system f o r measuring t o t a l inorganic carbon ( C ) i n seawater by acid release and i n f r a r e d gas analysis (a) serum stopper for sample i n j e c t i o n (b) column with HCl (c) manometer (d) tube of d r i e r i t e (Magnesium perchlorate) (e) Infrared gas analyser (IRGA) (f) a i r pump 61 The system allowed simultaneous measurements of pCO and t o t a l inorganic carbon or C^„ Photosynthetic rates could then be compared with the concentrations of gaseous or i o n i c CC^, i n order to determine the substrate of photo synthesis. Further t h e o r e t i c a l discussion of these techniques may be found i n the reference (1). LITERATURE CITED Tregunna, E.B. and E. Ann Thomas 1968. Measurement of inorganic carbon and photosynthesis in sea-water by pCC"2 and pH analysis. Can. J. Bot. 46: 481-485. PART I I Evidence Concerning the Mechanism of Diamox I n h i b i t i o n and Antimycin A S t i m u l a t i o n of Photosynthesis.^ This 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 submitted to P l a n t Physiology; (received August 3/ 1972). E.B. Tregunna supervised the study. 65 INTRODUCTION There has been a recent surge of i n t e r e s t concerning the r o l e of the enzyme carbonic anhydrase (E.C. No. 4.2.1.1) i n p h otosynthesis. This enzyme i s present i n c h l o r o p l a s t s (9,16,22) and i s probably l o o s e l y bound to the membranes (9,22). There have been suggestions t h a t carbonic anhydrase f a c i l i t a t e s t r a n s p o r t of CO 2 across 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 , or perhaps a d d i t i o n a l l y , i t may supply the c o r r e c t s u b s t r a t e , CO2, (9,22) to r i b u l o s e diphosphate c a r -boxylase (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 the photo-s y n t h e t i c product. 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 carboxylase favours t h i s r o l e (22). 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 s p e c i f i c i n h i b i t o r of animal carbonic anhydrase (20) , i n h i b i t s p l a n t c a r b o n i c anhydrase, i n c o n t r a s t t o e a r l i e r work (7,2) pro-v i d e s a new t o o l f o r s t u d y i n g the r o l e of carbonic anhydrase i n p h o t o s y n t h e s i s . Data w i l l be presented concerning Diamox e f f e c t s on c h l o r o p l a s t s of spinach and the marine u n i c e l l u l a r green a l g a D u n a l i e l l a t e r t i o l e c t a . The l a t t e r organism was o r i g i n a l l y s t u d i e d because the f i r s t r e p o r t s of Diamox i n h i b i t i o n of photosynthesis concerned the marine green a l g a , Ulva (16). 6 6 The action of carbonic anhydrase might also be to provide protons for the light-induced pH s h i f t observed i n chloro-plasts ( 8 , 1 5 , 3 1 ) . Recent work by Champigny and others ( 5 , 6 ) has i n d i -cated that low concentrations ( 0 . 5 x 1 0 ^ to 1 0 ^ M) of Antimycin A stimulate photosynthetic CC^ 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 . The mechanism of t h i s stimulation i s unknown, but appears to involve the chl o r o p l a s t membrane. I n v e s t i -gation of the possible r e l a t i o n s h i p between Antimycin A stimulation and Diamox i n h i b i t i o n of photosynthesis i s also reported here. MATERIALS AND METHODS Preparation of Chloroplasts Except where otherwise stated, spinach chloroplasts were prepared (18) from commercially grown spinach; 30 g chopped leaves were blended 30 seconds i n an Osterizer a t high speed i n cold 100 ml sucrose T r i s buffer (50 mM T r i s buffer, 0.4 M sucrose, 0.01M KCl, pH 7.8). The b r e i s was strained through 4 layers of c h i l l e d cheesecloth and centrifuged one minute at 200 g_ i n a r e f r i g e r a t e d centrifuge. The supernatant was centrifuged for 5 minutes at 600 g_ and the p e l l e t suspended i n sucrose-Tris b u f f e r . This prepar-ation was used for studies of 0 2 production and f o r some 14 C0 o f i x a t i o n studies. 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 Research Board o f Canada, Vancouver Laboratory, was grown a t 20°C i n enriched seawater medium (1). Four l i t r e batches were bubbled w i t h a i r i n modified 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 the inoculum volume c u l t u r e s were produced i n 3 to 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 minutes, a t room temperature, washed i n g r i n d i n g medium (100 mM T r i s , 0.001 EDTA, 0.1 M sucrose, 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 ruptured 2 by 20 seconds s o n i c a t i o n a t 52.5 watts/cm w i t h a B r o n w i l l B i o s o n i c I I I . The b r e i s was c e n t r i f u g e d 10 minutes a t 200 g_. The supernatant was c e n t r i f u g e d f o r 10 minutes a t 600 g_, and the p e l l e t resuspended and used to study 0 2 14 pro d u c t i o n and dark C0 2 a s s i m i l a t i o n . For experiments i n the presence of Antimycin A, c h l o r o -p l a s t s were prepared as des 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 pyrrophosphate, 330 mM s o r b i t o l , 2 mM NaNO^, 1 mM MnCl 2, 1 mM MgCl 2, 0.15 mM K 2HP0 4, 2 mM EDTA, 20 mM NaCl, pH 7.6-C h l o r o p l a s t s used to study the l i g h t - i n d u c e d pH s h i f t were prepared i n 0.4 m sucrose, 20 mM T r i s , 40 mM KC1, 4 mM Mg C l 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 minute a t 200 g_ to remove c e l l d e b r i s , then 5 minutes a t 3000 g_. The p e l l e t was resuspended i n washing medium, pH 7.8, ( s i m i l a r to the i s o l a t i o n medium but l a c k i n g T r i s ) and washed 3 times 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 For dark 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 zero to a hypotonic, N 2 scrubbed suspending medium, w i t h or without 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, 20 mM MgCl 2, 7 mM ATP, 14 3.3 mM ribose-5-phosphate, and 2 mM NaH CO^ [.5 u C i ] ) t o g i v e a f i n a l volume of 3 ml. 14 C h l o r o p l a s t s were fed CC»2 w i t h or without Diamox i n the l i g h t i n 3 ml N 2~scrubbed i s o t o n i c medium adapted from Everson (9); (50 mM HEPES, 330 mM s o r b i t o l , 20 mM NaCl 1 mM MnCl 2, 2 mM EDTA, 500 uM KH 2P0 4) o r from Champigny and 14 Miginiac-Maslow (6). For c h l o r o p l a s t C 0 2 feedings i n Champigny's medium, NaCl was omitted from, and 50 mM T r i s , pH 8.1, was added to 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 adjusted to give 30 to 60 ug c h l o r o p h y l l per ml. 14 The C0 2 feedings were s t a r t e d by t u r n i n g on the l i g h t . A f t e r 20 minutes, the l i g h t was turned o f f , and i n both l i g h t and dark f e e d i n g s , the r e a c t i o n was stopped by adding 0.2 ml 10% h y p o c h l o r i c a c i d . Assay was accomplished by s p o t t i n g 0.25 ml of a c i d i f i e d suspension on 1 x 2 cm s t r i p s of f i l t e r paper, which were d r i e d i n the fumhood, 69 then counted by l i q u i d s c i n t i l l a t i o n i n a toluene "Spectra-f l u o r " (Amersham/Searle) mixture as suggested by the manufac t u r e r . L i g h t was provided by three 300 watt General E l e c t r i c "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 . In l a r g e experiments, small t e s t tubes, c l o s e d w i t h serum stoppers, were h e l d i n holes on a l a r g e " P l e x i g l a s s " wheel, r o t a t i n g i n the l i g h t . Up to 98 samples could be i l l u m i n a t e d s i m u l -taneously w i t h t h i s apparatus. Studies o f 02 Production Oxygen production 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 or 6.5 was fo l l o w e d w i t h a C l a r k - t y p e Yellow Springs 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 recorded on a Rikadenki r e c o r d e r . Potassium f e r r i c y a n i d e (0.2 M stock s o l u t i o n ) was used as a H i l l reagent. The r e a c t i o n mixture was s t i r r e d m a g n e t i c a l l y , and l i g h t e d a t 30,000 l u x w i t h a General E l e c t r i c "Cool-Beam" l i g h t . For s t u d i e s of Diamox or Sulphanilamide e f f e c t s , 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 the suspending medium at the app r o p r i a t e c o n c e n t r a t i o n . The Light-induced pH S h i f t Chloroplasts prepared as described were used to study the light-induced pH s h i f t . A Radiometer PHM 53 S p e c i f i c Ion Meter, was used on expanded scale, (one pH u n i t f u l l scale) to measure pH, which was recorded on a Rikadenki recorder, 100 mV f u l l scale, 0.2 seconds response time. The i n i t i a l pH of a 15 ml chloroplast suspension 350 ug chl/ml, was adjusted to 6.0 to 6.1 i n the dark, and allowed to s t a b i l i z e at about 10°C. The l i g h t was then turned on and the so-called " l i g h t steady-state" pH allowed to s t a b i l i z e . A f t e r 2 to 3 minutes the l i g h t (two 300 watt G.E. Cool Beam lamps) was turned o f f , and the suspension was darkened f o r at l e a s t 5 minutes to allow the suspension to reach the previous "dark steady state" pH value. C y c l i c photophos-phorylation was induced by FMN (10 M ) . Preparation of In h i b i t o r s 14 For studies of 0 2 production or C0 2 f i x a t i o n at pH 7.8, Diamox was dissolved i n the appropriate buffered medium at the required strength and then used to suspend the chlo r o p l a s t s . For studies of 0 2 production at pH 6.5, the Diamox so l u t i o n was a c i d i f i e d a few minutes before use. Sulphanilamide was handled i n the same manner. When Diamox was used to study the pH s h i f t i n unbuffered sucrose medium, i t was dissolved 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 to keep the pH a t 7.5. Antimycin A, to provide a f i n a l c o n c e n t r a t i o n of 5 uM, was d i s s o l v e d i n absolute ethanol j u s t p r i o r to use. 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 higher than 0.3 percent. C o n t r o l s were run 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 -cohol i n the absence of Antimycin A. The i n h i b i t o r s were always f r e s h l y prepared before use and kept i n the r e f r i g e r a t o r o r on i c e u n t i l a c t u a l l y used. Antimycin A was obtained from N u t r i t i o n a l Biochemical C o r p o r a t i o n ; ATP, ribose-5-phosphate, and FMN from Sigma, and HEPES from C a l b i o c h e m i c a l Co. Koch-Light L a b o r a t o r i e s L t d . s u p p l i e d the Diamox and Sulphanilamide came from J.T. Baker Chemical Co. ATP - adenosine-5'-triphosphate Diamox - (Acetazolamide) 5 - a c e t a m i d o - l , 3 , 4 - t h i a d i a z o l e - 2 -sulphonamide FMN - r i b o f l a v i n monophosphate n u c l e o t i d e EDTA - disodium (Ethylene d i n i t r i l o ) - t e t r a c e t a t e HEPES N-2-hydroxyethylpiperazine-N'-2ethane sulphonic a c i d Sulphanilamide-p-aminobenzene sulphonamide RESULTS 14 The E f f e c t of_ Diamox on Photosynthesis; C0 9 F i x a t i o n i n  the L i g h t 14 F i g u r e 16 shows the e f f e c t of 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 spinach c h l o r o p l a s t s under l i m i t i n g C0 2 c o n c e n t r a t i o n (2 mM) and i n the presence of pyrrophosphate. Photosynthesis i s i n h i b i t e d by 80% compared to the c o n t r o l . In F i g u r e 20, the lower l i n e shows the e f f e c t on spinach 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 c o n c e n t r a t i o n . L i t t l e i n c r e a s e i n i n h i b i t i o n i s apparent above 2 mM; a t t h i s p o i n t about 70% i n h i b i t i o n was observed. As p r e v i o u s l y mentioned, 4 mM Diamox may i n c r e a s e the i n h i b i t i o n to 80%. U s u a l l y , above 60% i n h i b i t i o n i s observed a t 1 mM, and 0.4 to 0.5 mM gave 50% i n h i b i t i o n . The r e s i d u a l photosynthesis appeared i n s e n s i t i v e to the i n h i b i t o r . Under both l i g h t l i m i t i n g c o n d i t i o n s , and near 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 observed 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 to 0.5 mM, when NaHCO^ was 2 mM. 14 CO2 F i x a t i o n i n the Dark Fi g u r e 16 a l s o shows the e f f e c t of 4 mM Diamox on 14 C0 2 f i x a t i o n by i n t a c t spinach c h l o r o p l a s t s fed i n the dark 14 and provided w i t h ATP, ribose-5-phosphate, and NaH CO^. . F i g u r e 16 The e f f e c t o f 4mM Diamox on CC^ 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 PP^: i n t h e d a r k w i t h ATP vj CO Under these c o n d i t i o n s (2 mM NaH CO3), Diamox d i d not i n h i b i t . On the c o n t r a r y , i t somehow induced 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 , which was as high as 170% of the c o n t r o l . This i n c r e a s e v a r i e d i n absolute v a l u e , but occurred r e p e a t a b l y . A s i m i l a r s t i m u l a t i o n was a l s o observed i n D u n a l i e l l a t e r t i o l e c t a c h l o r o p l a s t s (not shown). The E f f e c t of Diamox on the H i l l Reaction Fig u r e 17 shows the e f f e c t of 4 mM Diamox on O2 p r o d u c t i o n by i n t a c t spinach c h l o r o p l a s t s i n the presence of potassium f e r r i c y a n i d e . An i n h i b i t i o n of 70% i s shown. This 14 i s comparable to the i n h i b i t i o n of CO2 f i x a t i o n (Figure 16). The c o n c e n t r a t i o n of C0 2 i s low, 0 to 2 mM; no NaHCO^ was added, and f r e s h l y d i s t i l l e d water was used to prepare the b u f f e r . 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 . Sulphanilamide, a t 0.5 mM and 1 mM, d i d not a f f e c t O2 p r o d u c t i o n i n spinach. At 4 mM, Diamox i n h i b i t e d the H i l l r e a c t i o n by 70 to 80% i n D u n a l i e l l a t e r t i o l e c t a c h l o r o p l a s t s . They were i n h i b i t e d 50% by 0.5 mM sulphanilamide. The experiments d e s c r i b e d above were conducted i n 50 mM T r i s b u f f e r a t pH 7.8. We a l s o s t u d i e d the e f f e c t of Diamox on the H i l l r e a c t i o n of spinach c h l o r o p l a s t s a t pH 6.5, because the l i g h t - i n d u c e d pH s h i f t i s best studied 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 Diamox and s u l p h a n i l a m i d e on 0 2 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 t h e p r e s e n c e o f p o t a s s i u m f e r r i c y a n i d e 76 T h e r e s u l t s a r e s h o w n i n F i g u r e 18. A t 2 mM, D i a m o x d i d n o t i n h i b i t . When 4 mM D i a m o x w a s e m p l o y e d , h o w e v e r , a n i n h i b i t i o n o f 50% w a s a c h i e v e d . T h i s w a s l e s s t h a n t h e u s u a l 70 t o 80% i n h i b i t i o n o b s e r v e d a t a 4 mM c o n c e n t r a t i o n . T h e f i n a l p H o f t h e r e a c t i o n m i x t u r e o f b o t h c o n t r o l a n d i n h i b i t e d c h l o r o p l a s t s w a s 6 .4 t o 6 . 5 . T h e E f f e c t o f D i a m o x o n t h e L i g h t - i n d u c e d p H S h i f t F i g u r e 19 s h o w s t h e t i m e c o u r s e o f t h e l i g h t - i n d u c e d p H s h i f t b y 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 a n u n b u f f e r e d s u c r o s e m e d i u m , w i t h FMN (10 M ) . T h e p H w a s c o n s t a n t a t 6 . 0 t o 6 . 1 , i n t h e d a r k . When t h e l i g h t w a s t u r n e d o n t h e p H r o s e r a p i d l y , a n d b y o n e m i n u t e , l e v e l e d o f f a n d m a i n t a i n e d a s t e a d y s t a t e i n t h e l i g h t . When t h e l i g h t w a s t u r n e d o f f t h e p H f e l l t o t h e p r e v i o u s d a r k v a l u e a n d r e m a i n e d c o n s t a n t . T h e i n i t i a l r a t e o f d e c r e a s e i n t h e d a r k a p p e a r e d t o d e p e n d o n t h e p H c h a n g e a c h i e v e d ; t h e g r e a t e r t h e s h i f t , t h e h i g h e r t h e i n i t i a l r a t e o f d e c a y . T h e a c t u a l p H s h i f t w a s .15 t o .17 i n t h e c o n t r o l . T h e FMN h a d a s t r o n g b u f f e r i n g a c t i o n . T h e l o w e r c u r v e i n F i g u r e 19 s h o w s t h e e f f e c t o f D i a m o x o n t h e pH s h i f t . E x c e p t f o r t h e p r e s e n c e o f t h e i n h i b i t o r , a l l c o n d i t i o n s w e r e t h e sam e a s t h e c o n t r o l . I t i s i m m e d i a t e l y a p p a r e n t t h a t D i a m o x a f f e c t s t h e p H s h i f t . When t h e l i g h t w a s t u r n e d o n , t h e pH r o s e r a p i d l y , b u t o n r e a c h i n g i t s maximum, i t b e g a n t o f a l l o f f r a p i d l y , a n d F i g u r e 18 The e f f e c t o f pH 6 . 5 and t i m e on Diamox 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 c h l o r o p l a s t s vj F i g u r e 19 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 u n b u f f e r e d s u c r o s e medium r 78 PH 6.30 6.28 6.26 6.24 6.22 6.20 6.18 6.16 6.14 6.12 6.10 6.08 .LIGHT OFF LIGHT OFF I •LIGHT ON pH 6.06L 0 2 3 4 5 TIME MINUTES 7 8 returned to the dark v a l u e , which was then maintained. A f t e r the dark pH value was achieved i n the l i g h t , t u r n i n g o f f the l i g h t d i d not a f f e c t the pH. I f the l i g h t was turned o f f before the pH had returned to the dark value the decay r a t e was a c c e l e r a t e d ; however, the f i n a l pH reached was the same. This time course was repeated s e v e r a l times by t u r n i n g the 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 at l e a s t 5 minutes dark recovery time. Because Diamox has a b u f f e r i n g e f f e c t , the a c t u a l i n i t i a l number of hydrogen i o n s pumped appeared very 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 but the course of the phenomenon. The I n t e r a c t i o n of Antimycin A w i t h Diamox Under c o n d i t i o n s approaching l i g h t s a t u r a t i o n (above 14 15,000 lux) 5 uM Antimycin A s t i m u l a t e s CC^ f i x a t i o n by i n t a c t spinach 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. This f i g u r e a l s o shows t h a t as the Diamox c o n c e n t r a t i o n increased from 0.0 to 10 mM, 50 to 60% i n h i b i t i o n was achieved. Over t h i s range of Diamox c o n c e n t r a t i o n , a d d i t i o n of Antimycin A completely overcame the 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 to 1 mM Diamox plus 5 uM Antimycin A, f i x e d the same absolute amount o f 14 CO2 as the c o n t r o l plus Antimycin A. Not only d i d Antimycin A overcome Diamox i n h i b i t i o n but i t was capable of 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 14 . . . and 5 A n t i m y c i n A on CC^ 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 , 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 80 480 0.0 .1 .2 .3 4 .5 .6 .7 .8 ,.9 1.0 DIAMOX mM 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 fixed/hr/mg c h l o r o p h y l l . Ethanol c o n c e n t r a t i o n was l e s s than 0.3% and had no e f f e c t on the c o n t r o l . I t i s a l s o noteworthy 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 va r i o u s amounts of Diamox were s t i m u l a t e d to the same absolute v a l u e . The r e s u l t i s t h a t the e f f e c t i v e s t i m u l a t i o n i n the presence of 1 mM Diamox, was c l o s e to 500%, compared to 100% i n the c o n t r o l . Diamox i n h i b i t i o n appears to s e n s i t i z e the c h l o r o p l a s t s to s t i m u l a t i o n by Antimycin 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 the l i g h t i n t e n s i t y was not s u f f i c i e n t to s a t u r a t e the c o n t r o l r a t e of photosynthesis as i n Figur e 21, Antimycin A 14 d i d not s t i m u l a t e CC»2 f i x a t i o n over the c o n t r o l r a t e . The percentage i n h i b i t i o n by Diamox, however, was about the 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% by 0.5 mM. The e f f e c t of Antimycin A i n combination w i t h Diamox, under c o n d i t i o n s where i t d i d not normally s t i m u l a t e , was to r a i s e the f i x a t i o n r a t e to the c o n t r o l r a t e . As Diamox c o n c e n t r a t i o n i n c r e a s e d to 0.9 mM, the e f f e c t i v e s t i m u l a t i o n by Antimycin A rose r a p i d l y from nothing to a peak of almost 170%. The e f f e c t then diminished r a p i d l y and as the degree of 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, the s t i m u l a t i o n a l s o began to l e v e l o f f a t 90 to 100%. Once more Diamox i n h i b i t i o n appeared to s e n s i t i z e the system t o Antimycin A. F i g u r e 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 c o n c e n t r a t i o n 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 i n non-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 82 200 180 0.0 .4 .8 1.2 1.6 2.0 2.4 DIAMOX mM 8 3 The E f f e c t of Antimycin A ori the Light - 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 allowed to achieve a steady pH v a l u e . Antimycin A was then 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 response was an immediate r a p i d decay to the dark steady s t a t e pH v a l u e . Antimycin A added i n the dark d i d not a f f e c t the pH v a l u e . The S t a b i l i t y of 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 sucrose media, a t pH 7.6 i s satu r a t e d a t 4 to 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 . I f the compound i s d i s s o l v e d i n unbuffered sucrose media i n i t i a l l y adjusted to pH 7.5, the s o l u t i o n r a p i d l y drops 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 the pH i s maintained above pH 7.0 by c o n t i n u a l l y adding 0.1 N NaOH w h i l e the compound d i s s o l v e s , a c t i v i t y i s preserved, and the s o l u t i o n may be sto r e d i n i c e f o r s e v e r a l hours and used to suspend c h l o r o p l a s t s . The pH was adjusted to 6.0 - 6.1 j u s t before t u r n i n g on the l i g h t when studying the pH s h i f t . When the 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 to show some l o s s of 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 the end o f one hour a f t e r i t was a c i d i f i e d to pH 6.1. This s o l u t i o n was b u f f e r e d w i t h 20 mM T r i s . 84 DISCUSSION The o b s e r v a t i o n reported here t h a t Diamox i n h i b i t s the potassium 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 , but does not i n h i b i t carbon f i x a t i o n by c h l o r o p l a s t s provided w i t h ATP i n the dark, 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 supports the work of Swader and Jacobsen (30). They r e p o r t t h a t Diamox (Acetazolamide) i n h i b i t s photosystem I I near the water 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 of Diamox t o i n h i b i t ATP supported dark 14 f i x a t i o n of CO2 i s s i g n i f i c a n t i n l i g h t of proposals t h a t carbonic anhydrase which 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 c a r b o x y l a s e , i s the r a t e l i m i t i n g enzyme i n phot o s y n t h e t i c carbon f i x a t i o n . When ATP i s made a v a i l a b l e , Diamox has no apparent i n h i b i t o r y e f f e c t on the carbon f i x i n g system. The gross e f f e c t of a Diamox i n h i b i t i o n on CO2 f i x -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 (16) i n the l i g h t would a l s o a r i s e from a blockage o f the l i g h t r e a c t i o n . We 14 observe up to 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 CO2 f i x a t i o n i n spinach c h l o r o p l a s t s . Swader and Jacobsen (30) suggest 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 methylated carbonylamide attached to an unsaturated r i n g s t r u c t u r e ; w i t h such i t may behave i n a s i m i l a r f a s h i o n . He i n d i c a t e s t h a t Diamox i n h i b i t i o n 85 of photosynthesis may have l i t t l e to do w i t h i n h i b i t i o n o f carbonic anhydrase, u n t i l now i t s assumed r o l e . C onsidering the r e s u l t s we have presented, however, we suggest t h a t the i n t e r p r e t a t i o n t h a t Diamox i n h i b i t s 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 of i t s e f f e c t on carbonic anhydrase, i s not n e c e s s a r i l y the only v a l i d e x p l a n a t i o n of the known f a c t s . Diamox o b v i o u s l y i n h i b i t s the l i g h t r e a c t i o n ; i t i s a l s o considered to be a very s p e c i f i c i n h i b i t o r of carbonic 14 anhydrase (20). F i x a t i o n of C0 2 i n the l i g h t by i s o l a t e d c h l o r o p l a s t s i s markedly i n h i b i t e d . Our r e s u l t s support those of Everson (9). We may conclude t h a t Diamox has two s i t e s of a c t i o n , one a t the l i g h t r e a c t i o n and another a t the enzyme l e v e l , or t h a t carbonic anhydrase has two r o l e s ; the f i r s t i n f a c i l i t a t i n g C0 2 t r a n s p o r t and storage, and the second i n the photochemical a c t i t s e l f . 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 achieved 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 of photosynthesis as pointed out by Swader and Jacobsen (30), i s inadequate reason to assume two d i f f e r e n t s i t e s of a c t i v i t y f o r Diamox. Carbonic anhydrase may p l a y a r o l e i n r e g u l a t i n g proton g r a d i e n t s i n c h l o r o p l a s t s (14). 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 generation of protons across 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 to photophosphorylation (15,31) and may compete f o r a high energy intermediate or a c t u a l l y be that intermediate (15,17). This would explain how Diamox, a s p e c i f i c i n h i b i t o r of the enzyme somehow blocks ATP formation and causes a breakdown i n the steady state value of the light-induced pH s h i f t . In the presence of FMN, electron transport i n chloro-p l a s t s i s c y c l i c . The e f f e c t of 5 uM Antimycin 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 rapid decay of the pH change supports the thesis that we are observing a proton flux a t t r i b u t a b l e to c y c l i c electron transport. Stedingk (29) reported that the l i g h t -induced pH s h i f t i n chromatophores of Rhodospirilium rubrum was i n h i b i t e d by 2 x 10 ^ (2uM) Antimycin A. I f ATP production i s inseparably linked to proton s h i f t s , and i f carbonic anhydrase i s required for optimal a c t i v i t y of these s h i f t s , then an i n h i b i t o r of carbonic anhydrase should a f f e c t both c y c l i c and non-cyclic electron transport (or that linked to any phosphorylation) and the associated proton transport. Diamox i n h i b i t s non-cyclic electron transport and the proton s h i f t supported by c y c l i c electron transport. Although an e f f e c t on proton s h i f t s occurring as a r e s u l t of non-cyclic electron transport has not been demonstrated, these would be expected from the e f f e c t s on oxygen production. The shape of the pH change i n the presence of Diamox (Figure 19) indicates that an i n i t i a l s h i f t occurred, but could not be sustained. Is t h i s because of a proton shortage 87 or i s the passive leakage r a t e back across the membrane increased? During the decay i n the l i g h t and before the dark value 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 inward f l u x was o c c u r r i n g i n the l i g h t . A l s o , recovery occurred i n the dark. Probably a shortage of protons accounts f o r the phenomenon. The a c t u a l s i t e of proton i n f l u x i s apparently the 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 . Carbonic anhydrase occurs i n the stroma (22) or attached probably l o o s e l y to the membranes (9). I f a c o n t i n u i n g supply of protons i s c u r t a i l e d by i n h i b i t i o n of the enzyme, the steady s t a t e w i l l decay, and the 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 the normal pas s i v e l e a k remains u n a f f e c t e d . Graham, A t k i n s , Reed, P a t t e r s o n , and S m i l l i e ( 1 3 ) r e p o r t e d no e f f e c t of Diamox on l i g h t - i n d u c e d pH gr a d i e n t s i n c h l o r o p l a s t s i s o l a t e d from Chlamydomonas or Pisum. On the other hand, Everson (10) and Everson and Graham (11) re p o r t e d f i r s t an i n h i b i t i o n , but 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 the i n i t i a l r a t e and f i n a l magnitude of the pH change i n i n t a c t c h l o r o p l a s t s , by Ethoxyzolamide, 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 not occur i n broken c h l o r o -p l a s t s . I t was suggested t h a t these c o n t r a d i c t o r y r e s u l t s depend on the c o n d i t i o n of the c h l o r o p l a s t p r e p a r a t i o n . In any event, our observations are somewhat d i f f e r e n t again. I t i s probable t h a t not only 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 , but the s t a b i l i t y of 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 of Diamox w i t h CMU and DCMU on a s t r u c t u r a l b a s i s can be countered by a comparison of Diamox w i t h sulphanilamide. Both Diamox and sulphanilamide share a 14 sulphonamide grouping. 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 carbonic anhydrase are u n a f f e c t e d by sulphanilamide, these compounds are known to i n h i b i t c arbonic anhydrase (20)/from animals and from Ulva pertusa (16) and other marine algae (4). Photosynthesis i s a l s o i n h i b i t e d i n U l v a . Sulphanilamide i n h i b i t s the H i l l r e a c t i o n i n the marine phytoplankton a l g a D u n a l i e l l a t e r t i o l e c t a as we have i n d i c a t e d . 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 of the enzyme. At l e a s t two forms of carbonic anhydrase e x i s t e d i n twelve p l a n t species t e s t e d (21). Minor d i f f e r e n c e s might not be unexpected between d i f f e r e n t p l a n t groups, such as land p l a n t s and marine algae. 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 -quirements of c h l o r o p l a s t s might be e x p l a i n e d . I t has been w e l l documented t h a t C0 2 or HCO~ i s r e q u i r e d f o r e f f i c i e n t f u n c t i o n i n g of 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 proton supply, through the i o n i z a t i o n o f carbonic a c i d , 89 H 20 + C 0 2 * 7 H 2 C 0 3 ^ — - 7 H + + HCO~ the r e q u i r e m e n t 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 (12) t h a t u n c o u p l i n g a g e n t s have no e f f e c t i n " C 0 2 - d e p l e t e d " c h l o r o p l a s t s . I f p r o t o n s from R"2CO.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 would be n o t h i n g t o u n c o u p l e i n t h e i r a bsence. I t i s a l s o known t h a t Diamox i n h i b i t i o n can be o v e r -come by 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 above 5 t o 10 mM. The i n h i b i t i o n a f f e c t s h y d r a t i o n o f C 0 2 c o m p e t i t i v e l y ( 2 0 ) , so we may s i m p l y be l o o k i n g a t a n e g a t i o n o f i n h i b i t i o n by s u b s t r a t e c o m p e t i t i o n . Swader and J a c o b s o n (30) found t h i s r e l i e f o f i n h i b i t i o n by HCO^ p u z z l i n g i n terms 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 . The c o n f u s i o n no l o n g e r e x i s t s i f c a r b o n i c 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 . We a r e d e a l i n g w i t h a system w h i c h has a c o n s i d e r a b l e u n c a t a l y z e d r a t e o f r e a c t i o n . As 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 t h e spontaneous r e a c t i o n m i g h t s u p p l y s u f f i c i e n t p r o t o n s f o r t h e pump t o o p e r a t e . C o n c e i v a b l y , t h i s c o u l d o c c u r even i f 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 . T h i s l i n e o f t h o u g h t 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 o b s e r v e d i n v e r y h i g h C 0 2 c o n c e n t r a t i o n s (24) above 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 or overwhelmed by high I^CO-j w i t h i n the t h y l a k o i d making the maintenance of an e f f e c t i v e proton 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 carbonic anhydrase to Diamox i n h i b i t i o n i s the e f f e c t of C l i o n . This anion a t a con-c e n t r a t i o n of 10 mM, causes 50% i n h i b i t i o n o f p a r t i a l l y p u r i f i e d carbonic anhydrase from spinach c h l o r o p l a s t s (9). Good (12) reported t h a t "C0 2 d e p l e t e d " c h l o r o p l a s t s i n which the H i l l r e a c t i o n i s suppressed, are f u r t h e r i n h i b i t e d (30%) by 70 mM NaCl. Are these e f f e c t s r e l a t e d ? The C l " i o n c o n c entrations are d i f f e r e n t , but we might expect the i n v i t r o or i s o l a t e d enzyme to 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 . The s i t u a t i o n i s complicated f u r t h e r by the requirement of the H i l l r e a c t i o n f o r C l i o n , somewhere near the water s p l i t t i n g r e a c t i o n . This i s compatible a t l e a s t , w i t h the apparent requirement f o r C l i o n i n the proton 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). The requirement of 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 stem from the r o l e of c h l o r i d e i n the proton pump. The r e p o r t s (5,26) of an i n c r e a s e d a f f i n i t y (lower Km) o f c h l o r o p l a s t s f o r CC^/ i n the presence of Antimycin A were i n t r i g u i n g . Antimycin A i s able to overcome or block i n h i b i t i o n by Diamox. 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 Antimycin A produces a strong s t i m u l a t i o n over the 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 Antimycin A does not 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 " the c h l o r o p l a s t to s t i m u l a t i o n . More re c e n t work (25) than (5) and (26) i n d i c a t e s t h a t the mechanism of Antimycin A s t i m u l a t i o n i s not to i n -crease HCO^ or CO2 c o n c e n t r a t i o n w i t h i n the c h l o r o p l a s t . A d d i t i o n of Antimycin A caused an inc r e a s e r a t h e r than a decrease i n the c o n c e n t r a t i o n of the acceptor r i b u l o s e - 1 , 5-diphosphate; when the HCO^ c o n c e n t r a t i o n was in 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 . Shacter and Bassham (25) suggested t h a t Antimycin 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, f r u c t o s e -6-phosphatase) s i n c e PGA accumulated, and the pool s i z e s o f f r u c t o s e - 1 , 6-diphosphate, fructose-6-phosphate, and sedo-heptulose-1, 7-diphosphate decreased. The e f f e c t was not considered to be v i a an e f f e c t on ATP l e v e l s , as these 32 authors f e l t t h a t the observed 25% apparent drop i n P f i x e d c o u l d be due to increased turnover 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 Antimycin A provide evidence f o r a mechanism of the s t i m u l a t i o n . Since Diamox i n h i b i t s the l i g h t r e a c t i o n , i t must l i m i t ATP production concurrent w i t h any e f f e c t on CC^ s u p p l i e d to carboxydismutase. This i s supported by dark feedings (Figure 16). The l i m i t a t i o n on ATP pr o d u c t i o n 92 would reduce 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 l i g h t . In the l a s t few yea r s , A t k i n s o n (3) has ela b o r a t e d the concept o f "energy-charge" i n the c e l l . S e v e r a l p l a n t enzymes have been t e s t e d i n t h i s c o n t e x t , and the evidence supports Atkinson's theory. L i t t l e work has been done con-ce r n i n g "energy charge", however, on the 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 not i n h i b i t e d by AMP. I t i s s t i m u l a t e d by reduced f e r r e d o x i n (23). FDPase o f c a s t o r bean endosperm, a glucon-eogenic t i s s u e , i s c o n t r o l l e d by energy charge. 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 storage, a high energy charge should s t i m u l a t e i t s r e g u l a t o r y enzymes, of which FDPase and carboxydismutase are members. We have i n d i c a t e d t h a t Diamox decreases energy charge ( l i m i t s ATP) . Antimycin A s t i m u l a t e s CC»2 f i x a t i o n but a l s o apparently decreases energy charge ( l i m i t s ATP from c y c l i c p hotophosphorylation). The "energy-charge" concept does not seem to e x p l a i n these r e s u l t s . F e r r e d o x i n i n the reduced s t a t e , s t i m u l a t e s FDPase (23). This compound could be thought o f as a s i g n a l t h a t energy i s a v a i l a b l e . By l i m i t i n g c y c l i c photophosphorylation, Antimycin A could maximize production of NADPH (or reduced f e r r e d o x i n ) and s t i m u l a t e FDPase, and the other phosphatases of the C a l v i n c y c l e . Since ATP supply, however, i s now somewhat l i m i t e d , such phenomena as PGA accumulation are observed. When Antimycin A i s fed 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 increased e f f i c i e n c y of use of avai l a b l e l i g h t ; none i s now "wasted" i n c y c l i c photophosphorylation. Notice that under these con-d i t i o n s , Antimycin A cannot stimulate 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 rate above that of the c o n t r o l . F i n a l l y i t should be noted that 5 uM Antimycin A can only reverse the Diamox i n h i b i t i o n completely up to a point, about 1 mM. 9 4 LITERATURE CITED A n t i a , N.J., and J . Kalmakoff. 1 9 6 5 . Growth r a t e s and and c e l l y i e l d s from axenic mass c u l t u r e o f fourteen species of marine p h y t o p l a n k t e r s . F i s h . Res. Bd. Can., Manuscript Report S e r i e s ; 2 0 3 . 2 4 pp. Arnon, D.I. 1 9 6 7 . Photosynthetic a c t i v i t y of 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 of the adenylate pool as a r e g u l a t o r y parameter. I n t e r a c t i o n w i t h feedback m o d i f i e r s . Biochemistry, 7 : 4 0 3 0 - 4 0 3 4 . Bowes, G.W. 1 9 6 9 . Carbonic anhydrase i n marine a l g a e . P l a n t P h y s i o l . 4 4 : 7 2 6 - 7 3 2 . Champigny, M.L. and M. Gibbs. 1 9 6 9 . 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Role 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. Proc. 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 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 I r i d a e a c o r d a t a INTRODUCTION 98 I t has been known f o r many years t h a t 0 2 i n h i b i t s photosynthesis i n a v a r i e t y of t e r r e s t r i a l and a q u a t i c p l a n t s (18). This i n h i b i t i o n may even be apparent a t the normal 21% 0 2 c o n c e n t r a t i o n of a i r . Wheat and some other temperate-zone grasses, along w i t h most dicotyledonous p l a n t s , photosynthesize about 30% f a s t e r i n 2% 0 2 compared to a i r . This i n h i b i t i o n v a r i e s w i t h temperature 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 occurrence of p h o t o r e s p i r a t i o n , and the apparent i n h i b i t i o n of photo-s y n t h e s i s i s considered to be l a r g e l y a s t i m u l a t i o n of p h o t o r e s p i r a t i o n . (17) P h o t o r e s p i r a t i o n e s s e n t i a l l y disappears a t 2% 0 2 (at which dark r e s p i r a t i o n i s saturated) , causing an apparent i n c r e a s e i n carbon uptake. Other phenomena thought to 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 are a f f e c t e d by low 0 2; the carbon d i o x i d e compensation p o i n t drops almost to zero and the s o - c a l l e d " p o s t - i l l u m i n a t i o n b u r s t " disappears (17). A number of marine algae t e s t e d f o r 0 2 i n h i b i t i o n o f ' photosynthesis 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, but were i n h i b i t e d up to 60% by a change from 5% to 100% 0 2. (19) The i n h i b i t i o n was r e v e r s i b l e . 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 concerning the occurrence o f p h o t o r e s p i r a t i o n i n marine algae. Several workers (2,10) reported the 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 of l i g h t - i n d u c e d r e s p i r a t i o n was apparently i n t e n s i t y dependent to high v a l u e s , unaffected by glucose or i n h i b i t e d by i t , and i n h i b i t e d by DCMU. Thus i t appeared c l o s e l y r e l a t e d to photosynthesis. A study by Brown and Tregunna (3) i n d i c a t e d t h a t the marine red algae I r i d a e a and G i g a r t i n a have elevated 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 study described here o r i g i n a t e d i n the o b s e r v a t i o n t h a t I r i d a e a cordata showed an "C^-burst" d u r i n g the f i r s t minutes of i l l u m i n a t i o n . This b u r s t which i s comparable to 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 other workers (4) showed a marked response to c o n c e n t r a t i o n , as d i d the subsequent st e a d y - s t a t e r a t e o f photo s y n t h e s i s . The purpose of t h i s study was to i n v e s t i g a t e the i n t e r a c t i o n s of and CC»2 on the 0 2 s e n s i t i v e p h o t o s y n t h e t i c C>2 i n d u c t i o n t r a n s i e n t s . A component of the i n d u c t i o n time course not p r e v i o u s l y r e p o r t e d i n red algae w i l l a l s o be d i s c u s s e d . 100 METHODS The technique used to measure 0 2 production was a simple v e r s i o n of t h a t pioneered a t the Carnegie I n s t i t u t e (20). A small piece of I r i d a e a cordata was s t r e t c h e d d i r e c t l y over the end (1 cm diameter) of a Yellow Springs Instrument Co., gold C l a r k - t y p e 0 2 e l e c t r o d e and secured w i t h heavy c o t t o n . This assembly was suspended i n a 200 ml beaker of f i l t e r e d seawater, cooled and aerated before use, a t 9 to 10°C. The temperature was maintained d u r i n g the e x p e r i -ments w i t h an i c e bath. The seawater was s t i r r e d c o n s t a n t l y . An aluminum r e f l e c t o r was placed beneath the beaker and the system l i g h t e d from above by a 300 watt General E l e c t r i c "Cool Beam" spot l i g h t ; darkness was obtained by c o v e r i n g a metal frame w i t h a double t h i c k n e s s of heavy b l a c k c l o t h . The output from the oxygen e l e c t r o d e was recorded on a Sargent Model s t r i p c h a r t r e c o r d e r . The 0 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% C 0 2 i n N 2; N 2 .alone; 0.09% C0 2 w i t h 21% 0 2 i n N 2; 21% 0 2 i n N2#' and 80% 0 2 i n N 2. These gas mixtures were s u p p l i e d and analyzed by Matheson of Canada. Photosynthesis was measured on a r e l a t i v e s c a l e . A t constant temperature a steady reading of 0 2 c o n c e n t r a t i o n was achieved i n the dark. This reading represents the e x t e r n a l 0 2 c o n c e n t r a t i o n minus 0 2 used i n dark 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 to as the "dark s t e a d y - s t a t e " . In the l i g h t , a second steady r e a d i n g , the " l i g h t s t e a d y - s t a t e " i s u l t i m a t e l y achieved. This value was c o n s i d e r a b l y e l e v a t e d above the dark v a l u e , and represented the 0 2 p r o d u c t i o n by photosynthesis above the constant l e v e l e x t e r n a l l y a v a i l a b l e , pl u s C>2 used i n the dark, p l u s C>2 use by any l i g h t - i n d u c e d r e s p i r a t i o n . Since i t i s impos s i b l e to 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 which employs a l i v i n g membrane as a b a r r i e r to oxygen d i f f u s i o n , a second C>2 e l e c t r o d e of the same type, but covered w i t h a T e f l o n membrane, was used t o rec o r d the a c t u a l 0 2 c o n c e n t r a t i o n i n the seawater surround-i n g the a l g a . A comparison could then be made between the a c t u a l e x t e r n a l 0 2 c o n c e n t r a t i o n and the dark s t e a d y - s t a t e r e a d i n g . For s t u d i e s o f the 0 2 response, the experimental water was f l u s h e d w i t h an app r o p r i a t e gas mixture. (See preceding l i s t ) . A s o l u t i o n of NaHCO^ was used to a d j u s t the i n o r g a n i c carbon c o n c e n t r a t i o n o r C\ (defined as t o t a l i n o r g a n i c carbon expressed as ml of carbon d i o x i d e per l i t r e of seawater). was then 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) Very low was achieved by a c i d i f y i n g seawater to l e s s than pH 1, purging w i t h 21% 0 2 i n N 2 f o r 3 0 minutes, and then a d j u s t i n g the pH to 8 . 0 ± 0.2 under the same gas mixture. This gas or N 2 alone was then r e l e a s e d a t the water surface during experiments to 102 prevent CC^ being absorbed from the a i r . Experiments were always conducted at pH 8.0 ± 0.2. Using 150 to 200 ml of water and about 100 mg of algae, very l i t t l e change was observed over the course of a two to three hour experiment i n e i t h e r pH or C^, so that the system conditions were constant and may be described as open. Unless otherwise noted, light-dark cycles consisted of 5 minute dark periods followed by 5 minute l i g h t periods. When DCMU was applied i t was added d i r e c t l y to the beaker a f t e r completion of a control period, or the electrode-alga assembly was transferred i n the dark, from control to treatment water within two to three seconds. The f i n a l — 6 concentration was 10 M DCMU. The plants were tested with and without a Teflon membrane between alga and electrode. Half-saturated KCl solut i o n , as supplied by YSI, was rou t i n e l y used as the e l e c t r o l y t e . The alga was also tested using half-saturated NaCl; performance of the electrode was s a t i s f a c t o r y with NaCl. The KCl did not appear to damage the alga over the experimental period; that i s , rates of photosynthesis showed no serious d e c l i n e . Because the algae seemed to vary considerably i n phy s i o l o g i c a l condition (they are 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 . Only r e l a t i v e comparisons may be made between d i f f e r e n t samples. 103 The algae were c o l l e c t e d from A p r i l to August a t Brockton P o i n t , Stanley Park, Vancouver, i n 1970 and 1971. The oxygen e l e c t r o d e was checked f o r i t s l i n e a r i t y of response u s i n g N 2 and 100% 0 2; a Matheson gas p r o p o r t i o n e r was used to mix intermediate C>2 c o n c e n t r a t i o n s . RESULTS The 0_2 Production Induction T r a n s i e n t s i n I r i d a e a cordata F i g u r e 22 shows a t y p i c a l time course response f o r 0 2 p r o d u c t i o n by I r i d a e a i n the i n i t i a l minutes of i l l u m i n -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 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 22. For the most p a r t , these l e t t e r s correspond to the d e s i g n a t i o n by Chandler and Vidaver (4) and w i l l be used i n d i s c u s s i o n . When the l i g h t i s turned on, there i s an immediate r a p i d r i s e i n 0 2 p r o d u c t i o n , which peaks a t 1 t o 2 seconds (a). There f o l l o w s a d e c l i n e i n 0 2 p r o d u c t i o n (b), succeeded by a somewhat slower r i s e to a second peak ( c ) , higher than the f i r s t , which reaches i t s maximum i n 25 to 30 seconds. From t h i s second peak the r a t e of 0 2 production f a l l s again f o r some time. A minimum (d), i s reached from 1 to 3 minutes a f t e r the l i g h t i s turned on. A f t e r (d), the r a t e of Cu F i g u r e 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 oxygen i n d u c t i o n t r a n s i e n t s d u r i n g t h e f i r s t m i n u t e s o f i l l u m i n a t i o n i n , I r i d a e a c o r d a t a . See t e x t f o r e x p l a n a t i o n . 104 6 0 1 2 3 4 5 6 7 8 TIME MINUTES 105 production u s u a l l y r i s e s again, and the s t e a d y - s t a t e , (e) i s achieved. This steady-state i s maintained f o r s e v e r a l hours i f l i g h t , C\ , 0 2, and temperature remain constant. When the l i g h t i s turned o f f , the r a t e of C>2 pro-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 darkening a minimum i s reached (g) which i s below the previous dark reading at the s t a r t of the sequence. The t r a c e then r i s e s again ( i ) , e i t h e r to the previous dark l e v e l , o r to a s l i g h t l y higher steady reading which i s then maintained. The g r e a t e r p a r t of these experiments concerned f a c t o r s which appeared to a f f e c t the peak ( c ) , and ( e ) , the stea d y - s t a t e of pho t o s y n t h e s i s . The E f f e c t s o f 0_2 Concentration and C. on the 0_2 P r o d u c t i o n  Transients The most marked and c o n s i s t e n t responses to C»2 were shown by the t r a n s i e n t s (c) and ( e ) . F i g u r e 23 shows the response of (e) to i n c r e a s i n g 0 2 a t normal to moderate C. (30 to 50 ml C . / l ) . In t h i s s e r i e s o f measurements, the i n h i b i t i o n a t 9 ppm (high 0 2) compared to 1 ppm (low 0 2) was 65%. When C^ was reduced to 1 to 5 ml/1 the i n h i b i t i o n by 0 2 a t 9 ppm was reduced to 20% from 65%. The response to 0 2 a l s o decreased a t very h i g h C (80 ml/1) v a l u e s . At 80 ml C / l there was a 30% F i g u r e 23 E f f e c t o f oxygen on t h e r a t e o f 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 i n I r i d a e a c o r d a t a o ZD Q o 5 cc Q_ ° 4 u_ O < CC UJ Q •0. - ^ " 2 — 6 — - f -j 1_ 10 1 2 14 A C T U A L P P M O X Y G E N IN E X P E R I M E N T A L W A T E R 107 i n h i b i t i o n i n (e) on changing from low to high 0 2 . Fi g u r e s 24 to 26 show the response o f (e) on chang-i n g from hi g h to low to high 0 2, a t moderate, low, and very-h i g h C^. Note t h a t (e) responds r e v e r s i b l y and immediately to the changes i n C^. There i s a tendency f o r a slow down-ward d r i f t i n the st e a d y - s t a t e r a t e of photosynthesis d u r i n g the course of the experiment. As shown i n F i g u r e s 24 to 26 the spike ( c ) , a l s o responded to a change i n 0 9 c o n c e n t r a t i o n . A t 50 ml/1 C , (c) i s apparently i n h i b i t e d 60% by a change from 1 to 9 ppm 0 2. As shown i n Fig u r e s 24 to 26, however, (c) r e q u i r e s longer than (e) to respond to a change i n C>2. T y p i c a l l y the f i r s t (c) peak i n low 0 2 i s l e s s than 40% of the peak h e i g h t reached 2 o r 3 c y c l e s l a t e r , w h i l e the (e) value reaches a t l e a s t 80% of i t s u l t i m a t e value d u r i n g the f i r s t l i g h t - d a r k c y c l e . At t h i s p o i n t , (beginning o f the f i r s t low C»2 l i g h t period) the a l g a would have been subjected to low C»2 f o r not more than 5 minutes. When the G*2 i s again i n c r e a s e d to 9 ppm, (c) once more r e q u i r e s time to a d j u s t , w h i l e (e) responds immediately. A t v e r y low C^, F i g u r e 25, (c) shows almost no i n c r e a s e or even a decrease a t low 0 2. F i g u r e 27 shows a second aspect of the response of (c) to low 0 2. At 9 ppm 0 2 , the peak i s i n i t i a l l y 5 to 8 times the height of the st e a d y - s t a t e photosynthesis. The peak h e i g h t decreases r a p i d l y w i t h s u c c e s s i v e c y c l e s over 25 minutes, ( i , i i , i i i ) and as al r e a d y F i g u r e 24 The e f f e c t of high and low oxygen on (c) and (e) a t 50 ml per l i t r e 108 oi ; -0 10 20 30 40 50 ~60 70 80 90 TIME MINUTES F i g u r e 25 The e f f e c t o f h i g h and low oxygen on (c) and (e) a t 2 ml C per l i t r e Figure 26 The e f f e c t of high and low oxygen on (c) and (e) at 80 ml C per l i t r e 160 140 O120 HIGH 02 8-10 RRM. o Q O CC 100 -^ 801 o o 60 < cc S oi ^20 0 c -V— LOW Oo < 1 PRlvf HIGH 0o 8 - 1 0 P F T M . 10 • 20 30 40 50 60 70 80 90 100 110 TIME MINUTES F i g u r e 27 The e f f e c t o f low C\ w i t h t i m e on (c) a t h i g h and low oxygen mentioned, responds very l i t t l e to a lowering of the C»2 c o n c e n t r a t i o n ( i v ) . These were consecutive c y c l e s w i t h the same sample, separated by 5 minute dark p e r i o d s . The r e l a t i o n s h i p of (c) to (e) appeared f a i r l y c o n s t a n t . U s u a l l y (c) was 2 or 3 times the h e i g h t of (e) except a t low (Figure 27) where, as s t a t e d , (c) decreases w i t h time from 5 to 8 times (e) to the usual v a l u e . At low C\ (Figure 27) (c) became lower than (a) . The (a) spike was about 50% o f (c) a t moderate and low 0 2 and about 8% of (c) a t 9 ppm 0 2. At high C^, a s i m i l a r r e l a t i o n -s h i p i s e v i d e n t . The (a) s p i k e (Figure 22) occurs extremely r a p i d l y and i s complete i n 4 t o 5 seconds. I t i s o c c a s i o n a l l y apparently swamped by the massive 0 2 p r o d u c t i o n o f (c) p a r t i c u l a r l y a t low C»2, and consequently may appear as a shoulder on (c) o r be masked completely. The b r i e f d i p which f o l l o w s (a) was r e a d i l y observed a t 9 to 10° C, i n moderate l i g h t , a f t e r 3 to 5 minutes darkness. The h e i g h t of (b) the lowest p o i n t of t h i s d i p , shows some tendency to be constant, r e g a r d l e s s o f the maximum values o f (a) and (c) . The (d) component of F i g u r e 22, t h a t i s a d i p before (e), d i d not always appear a t 9 ppm oxygen, but was u s u a l l y revealed a t low 0 2 c o n c e n t r a t i o n s . The component (d) always i n t e n s i f i e d somewhat a t low 0 2, but t h i s was p a r t i c u -l a r l y marked a t 50 ml C , where (d) c o n s i s t e n t l y increased 113 4 or 5 times when 0 2 was reduced from 9 to 1 ppm. The f i n a l component to which attention was directed was (g). This appears as a "post-illumination" stimulation of r e s p i r a t i o n . The e f f e c t of 0 2 on (g) i s uncertain, but we observed some tendency for i t s magnitude to be reduced at low 0 2. The E f f e c t of Previous Light-dark Regimes on (c) Figure 28 shows the e f f e c t of l i m i t i n g the previous dark period on the (c) spike. When the dark period preceding i l l u m i n a t i o n i s very short, the burst i s reduced i n s i z e . In a b r i e f experiment, the dark steady-state was achieved with a sample of Iridaea. The l i g h t was turned on. Immediately the height of (c) was reached, the l i g h t was turned o f f f o r 30 seconds, then turned on again u n t i l the burst height was reached. This was repeated several times. In the second l i g h t period, the burst height was reduced by 25%, and i n the following 4 l i g h t periods, reached a nearly constant value. The s i x t h l i g h t period l a s t ed 2 minutes and showed only a very s l i g h t f a l l from (c) to a l i g h t steady-state equal to that of the c o n t r o l . When a single 4 minute dark period was then given, (c) reached about 85% of i t s o r i g i n a l height. If the dark time i s decreased i n succeeding cycles from 8 minutes to 15 seconds the r e s u l t i s s i m i l a r to that j u s t described. A p l o t of the values of (c) against previous F i g u r e 28 The e f f e c t o f t h e p r e c e 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) 115 dark time would show (e) n e a r l y constant, w h i l e (c) f a l l s p r o g r e s s i v e l y u n t i l i t meets (e ) . Changing the l e n g t h of time i n the l i g h t had l i t t l e s p e c i f i c e f f e c t on the value of ( c ) , but caused a p a r a l l e l downward d r i f t i n the heig h t of a l l components. The E f f e c t of DCMU on 0 2 Production T r a n s i e n t s F i g u r e 29 shows the e f f e c t of 10~ 6 M DCMU on the C»2 p r o d u c t i o n t r a n s i e n t s . This experiment was performed a t 3 ppm 0 2« The (c) spike appears to be p r e f e r e n t i a l l y attacked by the i n h i b i t o r . Both (a) and (e) show l i t t l e e f f e c t i n i t i a l l y . In the next l i g h t p e r i o d a marked depression o f a l l t r a n s i e n t s and the f i n a l s t e a d y - s t a t e r a t e of photo-s y n t h e s i s occurred. The Response of the 0_2 E l e c t r o d e When checked w i t h v a r i o u s known co n c e n t r a t i o n s o f 0 2, the e l e c t r o d e was found to respond l i n e a r l y to C»2 c o n c e n t r a t i o n . Using values taken from one study o f the e f f e c t of 0 2 on stea d y - s t a t e photosynthesis, we attempted to determine whether the p e r m e a b i l i t y of the a l g a l t h a l l u s to 0 2 remained constant, as 0 2 changed. Fig u r e 30 shows a p l o t of a c t u a l ppm 0 2 i n the experimental water versus the dark steady 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 on t h e (c) s p i k e R E L A T I V E R A T E O F 0 2 P R O D U C T I O N m m co 911 F i g u r 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 oxygen and t h e d a r k s t e a d y - s t a t e r e a d i n g 117 0 1 2 3 4 5 6 7 8 9 0 11 12 13 14 DARK STEADY STATE APPARENT PPM 0 2 118 DISCUSSION The components of the 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 cordata are s i m i l a r to those published by Vidaver (21) f o r Ulva l o b a t a , I l e a f a s c i a , and Ankistrodesmus f a l c a t u s , and by Chandler and Vidaver (4) f o r Ulva l a c t u c a L. The spike marked (a) i n Figu r e 22 appears to be i d e n t i c a l to the "pre-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) reported t h a t he was unable to d e t e c t the "pre-a" spike i n the red a l g a Porphyra, Govindjee and Govindjee, (8) reported a r a p i d s p i k e i n Porphyridium, a u n i c e l l u l a r red a l g a . I t i s i n t e r e s t i n g , however, t h a t t h i s spike appeared i n Porphyridium o n l y a f t e r a long dark p e r i o d of 15 minutes to s e v e r a l hours and a t 1 to 5°C. I t disappeared a f t e r s e v e r a l exposures to l i g h t . (8) The "pre-a" spike of U l v a , I l e a , Ankistrodesmus and I r i d a e a does not disappear i n t h i s manner, nor does i t r e q u i r e very long dark p e r i o d s . The e a r l y s p i k e r e p o r t e d by Govindjee, t h e r e f o r e , may not be the same as t h a t r e p o r t e d here o r by Vi d a v e r . Vidaver (21) a l s o rep o r t e d t h a t low (4°C) o r h i g h (30°C) temperatures were r e q u i r e d to i s o l a t e the "pre-a" t r a n s i e n t i n Ulva and I l e a . In I r i d a e a (as i n Ankistrodesmus) the "pre-a" spike 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 or 4 minutes dark; our experiments were conducted a t 9 to 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 to r e v e a l the 119 "pre-a" t r a n s i e n t i n I r i d a e a . This appears to be the f i r s t r e p o r t of a "pre-a" t r a n s i e n t i n the red algae. To our knowledge, only the Bangiophycidae i n the red algae 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 of 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 the "pre-a" spike repeatedly under a p p r o p r i a t e c o n d i t i o n s , but P o r p h y r i d -ium d i d not, Govindjee and Govindjee (8), suggested some d i f f e r e n c e i n the o r i g i n of the "pre-a" i n red versus green algae. Our r e s u l t s suggest t h a t t h i s i s not the case. I t has been noted i n these experiments t h a t 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 , the second 0 2 s p i k e , (c) i s always c o n s i d e r a b l y higher than the "pre-a" or (a), and (e) the ste a d y - s t a t e r a t e of phot o s y n t h e s i s . The (c) spike appears to be the same as the a-spike r e f e r r e d to by Vidaver (21). In h i s published r e p o r t s , the f i n a l steady r a t e of photosynthesis i s always higher than the va r i o u s i n d u c t i o n t r a n s i e n t s . This d i f f e r e n c e may be caused by an extremely slow r i s e to the maximum steady s t a t e i n I r i d a e a , or by some d i f f e r e n c e i n p o o l - s i z e s of o x i d i z a b l e components. The time course o f oxygen production t r a n s i e n t s published f o r Ulva by Chandler and Vidaver (4) rev e a l e d a secondary 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 0 2 p r o d u c t i o n . This 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 of 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 French and Fork (6) always appeared. The 50% i n h i b i t i o n of photosynthesis i n I r i d a e a by 9 ppm 0 2, 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 other marine algae ( i n c l u d i n g some reds) by Turner, Todd, and B r i t t a i n (19). They noted l i t t l e s i g n i f i c a n t i n h i b i t i o n of photo-s y n t h e s i s by 20% C»2 a t h i g h l i g h t i n t e n s i t y (near s a t u r a t i o n ) and 36 u M carbon d i o x i d e . Seawater sa t u r a t e d w i t h 0 2 i n a i r a t 10°C, has about 9 ppm 0 2. The carbon c o n c e n t r a t i o n s r e p o r t e d as moderate, 50 ml C ^ / l are equal to j u s t over 2 mM. There i s no evidence f o r an e f f e c t of l i g h t i n t e n s i t y on the percent i n h i b i t i o n by 0 2 (11) . 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 i n c r e a s e s w i t h photosynthesis. Various r e p o r t s i n c l u d i n g (12) and (18) i n d i c a t e t h a t h i g h C 0 2 tends to e r a d i c a t e the 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 . In l i n e w i t h t h i s , we have observed a r e d u c t i o n (to 20%) of C»2 i n h i b i t i o n a t 80 ml/1 C . On the other hand, at low C 0 2 most organisms have shown an increased 0 2 e f f e c t . J o l l i f f e and Tregunna (12) showed t h a t C\ i s l i m i t i n g f o r photosynthesis i n I r i d a e a up to 50 to 60 ml C\ (2 to 2.5 mM). Apparently the very low a v a i l a b i l i t y of carbon a t 2 to 5 ml/1 so l i m i t s photosynthesis t h a t the 0 2 e f f e c t i s not 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 have a l s o noted the s e n s i t i v i t y of the second l a r g e 0 2 spike (c) to 0 2 c o n c e n t r a t i o n . Since the i n h i b i t i o n k i n e t i c s d i f f e r , the i n h i b i t i o n of t h i s s p i k e may not occur 121 by t h e same mechanism as 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 p h o t o s y n t h e s i s . I t a p p e a r s t h a t d u r i n g t h e 0 2 p r o d u c t i o n i n d u c t i o n p e r i o d o f p h o t o s y n t h e s i s , C>2 i s t a k e n up by a t l e a s t 2 d i f f e r e n t p r o c e s s e s . The " p r e - a " s p i k e i s s e p a r a t e d from (c) t h e second l a r g e s p i k e by a marked d e p r e s s i o n . F l a s h i n g l i g h t e x p e r i -ments by C h a n d l e r and V i d a v e r (4) and R e i d (15) i n d i c a t e t h a t t h i s d e p r e s s i o n r e p r e s e n t s an a c t i v e u p t a k e o f 0 2 w h i c h o c c u r s 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 2 i s pr o d u c e d . 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 r e s i s t a n c e o f t h i s b r i e f 0 2 u p t a k e t o DCMU. 0 2 e v o l u t i o n was s u p p r e s s e d , b u t a " p r e - a " s p i k e a p p e a r e d . S i n c e R e i d (15) showed t h a t t h e f i r s t 0 2 b u r s t was i n h i b i t e d by DCMU, the 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 due t o s u r v i v a l o f an 0 2 u p t a k e t r a n s i e n t superimposed on a p a r t i a l l y i n h i b i t e d C»2 p r o d u c t i o n . Heber and F r e n c h (9) and Kouchkovsky and J o l i o t (14) have measured an 0 2 u p t a k e i n i s o l a t e d c h l o r o p l a s t s , w h i c h i s a t t r i b u t e d t o o x i d a t i o n o f a p r o d u c t o f p h o t o s y s t e m I . T h i s system I - l i n k e d 0 2 u p t a k e i s c o n s i d e r e d by them t o be a r e q u i s i t e f o r a c t i v a t i o n o f the p h o t o c h e m i c a l complex o f system I I . The second 0 2 u p t a k e 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 a s s o c i a t e d w i t h t h e 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 r e p o r t e d (2,10) i n some u n i c e l l u l a r algae and higher p l a n t s (5). I t may be t h a t the c-spike i n I r i d a e a i s not d i r e c t l y a f f e c t e d by 0 2 c o n c e n t r a t i o n . The spike 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 to DCMU, 7 07 nm l i g h t ) which i t s e l f produces massive amounts of 0 2. Rather, the apparent i n h i b i t i o n of (c) a t high C»2 may be the r e s u l t of a stimu-l a t i o n of one or both l i g h t - i n d u c e d C>2 uptake r e a c t i o n s . Except a t low C , the (c) peak r e a c t s s i m i l a r l y to (e) , the stead y - s t a t e of photosynthesis, when C»2 i s changed. I t i s probable, t h e r e f o r e , t h a t a continuous l i g h t - i n d u c e d r e s p i r -a t i o n i s r e s p o n s i b l e f o r the 0 2 e f f e c t on both (c) and ( e ) , r a t h e r than the very b r i e f 0 2 use by photosystem I . A t reduced 0 2 c o n c e n t r a t i o n s , t h i s " p h o t o r e s p i r a t i o n " i s i n -h i b i t e d f o r l a c k o f 0 2 and the 0 2 b u r s t appears l a r g e r , as does the 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 re c e n t photosynthate, as we assume, i t would not begin u n t i l a few seconds a f t e r carbon f i x a t i o n . A "bur s t " of 0 2 uptake as r e s p i r a t i o n began, fo l l o w e d by a s e t t l i n g to the steady-s t a t e could e x p l a i n the d i p a f t e r (c) fo l l o w e d by a r i s e to the steady s t a t e . This "undershoot" might be emphasized a t low C»2, when l i m i t e d 0 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 to t h a t a t high 0 2. The l a c k of response to low 0 2 by (c) when C was very low i n i t i a l l y presented a problem of i n t e r p r e t a t i o n 123 i n terms of the above model. B a n n i s t e r (1), however, reporte 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 poisoning of carbon f i x a t i o n or i n the absence of CC^/ i n d i c a t i n g some i n f l u e n c e of carbon f i x a t i o n on these 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 of the C a l v i n c y c l e i n I r i d a e a by very low C causes a r e s t r i c t i o n of 0 2 p r o d u c t i o n d u r i n g i n d u c t i o n , thus reducing the peak h e i g h t of ( c ) . A f u r t h e r aspect o f the (c) s p i k e , i s the e f f e c t on i t of the l e n g t h o f the dark p e r i o d preceding i l l u m i n a t i o n , upon which i t i s dependent. Apparently some component which i s reduced i n the l i g h t i s stored or produced i n the dark. Govindjee and Govindjee (8) reported t h a t (c) was s t i m u l a t e d by repeated exposures to l i g h t , and unaffe c t e d by dark i n t e r v a l s ranging from 1 to 8 minutes. We found t h a t the h e i g h t of the (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 dark p e r i o d s of l e s s than 2 minutes. No reasonable 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 response of the stea d y - s t a t e i s immediate. The e x i s t e n c e of 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 o f r e s p i r a t i o n i n algae has been reported p r e v i o u s l y (6). Such a response a l s o occurs i n higher p l a n t s having photo-r e s p i r a t i o n , where the 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 , the 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 a s s o c i a t e d w i t h l i g h t - s t i m u l a t e d r e s p i r -124 a t i o n . 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 of p h o t o r e s p i r a t i o n i n algae i s u n c l e a r . In higher 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 metab-o l i s m of g l y c o l i c a c i d by the C»2-using enzyme g l y c o l i c a c i d oxidase. The algae l a c k g l y c o l i c a c i d oxidase and have g l y c o l i c a c i d dehydrogenase i n s t e a d . Furthermore/ under c o n d i t i o n s of high G^, high l i g h t i n t e n s i t y , and l i m i t i n g CC>2, which 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 higher p l a n t s , the algae excrete g l y c o l i c a c i d . A more recent r e p o r t by Gibbs (7) i n d i c a t e s t h a t formation o f g l y c o l a t e i n spinach c h l o r o p l a s t s i s dependent on production of H 2 0 2 i n the photo-a c t . The peroxide r e l e a s e s g l y c o l i c a c i d from the two-carbon t r a n s k e t o l a s e a d d i t i o n - p r o d u c t . This r e a c t i o n i s C»2 s e n s i t i v e , and i s s t i m u l a t e d by c o n d i t i o n s which 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 higher p l a n t s , both production and metabolism of g l y c o l a t e are G ^ - s e n s i t i v e , ( ^ - u t i l i z i n g r e a c t i o n s . I t has already been noted t h a t some algae have a photosystem-I-linked 0 2 uptake. Work by Wilson and C a l v i n (22) 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 p r o d u c t i o n could be l i n k e d to f e r r i c y a n i d e r e d u c t i o n , thus l i n k i n g the photoact w i t h g l y c o l a t e production i n the algae. Glyco-l a t e p r o d u c t i o n by an 0 2 ~ s e n s i t i v e process i s probably common to algae and higher p l a n t s . Heber and French (9) concluded t h a t t h i s i n i t i a l 0- uptake was not s u f f i c i e n t to explain the quantity of 0^ apparently used in photorespiration. The results presented here confirm that the algae as well as higher plants have a second C^-using process which occurs in the light and is linked to photosynthesis. 126 LITERATURE CITED B a n n i s t e r , T.T. 1965. Simple o s c i l l a t i o n s i n photo-s y n t h e t i c oxygen p r o d u c t i o n . Biochim. Biophys. A c t a . 109: 97-107. B r a c k e t t , F.S., Olson, R.A., and C r i c k a r d , R.G. 1953. Photosynthesis and r e s p i r a t i o n . Arch. Biochem. Biophys., 101: 171-180. 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 photosynthesis by some algae. Can. J . Bot., 45: 1135-43. Chandler, M.T. and Vid a v e r , W.E. 1970. P h o t o s y n t h e t i c oxygen i n d u c t i o n t r a n s i e n t s i n the a l g a Ulva  l a c t u c a L. Phycolog., 9: 133-142. Decker, J.P. and Wien, J.D. 1958. Carbon d i o x i d e surges i n green l e a v e s . I I CO2 uptake versus l i g h t i n t e n -s i t y and CO- c o n c e n t r a t i o n . J . S o l . Energy S c i . Eng., 2: 39-41. French, C.S. and Fork, D.C. 1961. Two primary photo-chemical r e a c t i o n s i n photosynthesis d r i v e n by d i f f e r e n t pigments. Carnegie I n s t , of Washington, Yearbook 60: 351-357. Gibbs, M. 1971. B i o s y n t h e s i s o f g l y c o l i c a c i d i n M.D. Hatch, C.B. Osmond, and R.O. S l a y t e r , ed. Photo-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 . W i l e y - I n t e r s c i e n c e . pp. 433-441. Govindjee, and Govindjee, R. 1964. I n d u c t i o n t r a n s i e n t s i n O2 e v o l u t i o n by Porphyridium cruentum i n mono-chromatic l i g h t . Carnegie I n s t . Wash. Year Book, 63: 468-472. Heber, U. and French, C.S., 1968. E f f e c t s of oxygen on the e l e c t r o n t r a n s p o r t chain of pho t o s y n t h e s i s . P l a n t a , 79: 99-112. Hoch, G., Owens, 0. v. H., and Kok, B. 1963. Photo-s y n t h e s i s and r e s p i r a t i o n . Arch. Biochem. Biophys., 101: 171-180. 127 Jackson, W.A. and Volk, R.J. 1 9 7 0 . Photorespiration i n L. Machlis, ed. Ann. Rev. Plant Physiol. 2 1 : 3 8 5 - 4 3 2 . J o l l i f f e , E.A. and Tregunna, E.B. 1 9 7 0 . Studies on HCO^ ion uptake during photosynthesis i n benthic marine algae. Phycologia, 9 : 2 9 3 - 3 0 3 . J o l l i f f e , P.A. and Tregunna, E.B. 1 9 6 8 . E f f e c t of temperature, CO2 concentration and l i g h t i n t e n s i t y on O2 i n h i b i t i o n of photosynthesis i n wheat leaves. Plant Physiol., 4 3 : 9 0 2 - 9 0 6 . Kouchovsky, Y. de, and J o l i o t , P. 1 9 6 7 . Cinetique des exhcanges d'oxygene et de l a fluorescence des chloroplastes i s o l e s . Photochem. Photobiol. 6 : 5 6 7 - 5 8 7 . Reid, A. 1 9 6 8 . Interactions between photosynthesis and r e s p i r a t i o n i n C h l o r e l l a . I Types of transients of oxygen exchange a f t e r short l i g h t exposures. Biochim. Biophys. 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Hydrobiol. , 5 4 : 6 9 7 - 7 4 7 . Wilson,A.T. and Calvin, M . 1 9 5 5 . Photosynthetic c y c l e -CO2 dependent transients. J . Amer. Chem. S o c , 7 7 : 5 9 4 8 . 

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