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PHOTOSYNTHESIS ACTION SPECTRA IN ANACYSTIS NIDULANS AS DETERMINED BY
PHOTOACOUSTIC SPECTROSCOPY
R. Carpentier, B. Larue, R. Leblanc
To cite this version:
R. Carpentier, B. Larue, R. Leblanc. PHOTOSYNTHESIS ACTION SPECTRA IN ANACYSTIS
NIDULANS AS DETERMINED BY PHOTOACOUSTIC SPECTROSCOPY. Journal de Physique
Colloques, 1983, 44 (C6), pp.C6-355-C6-360. �10.1051/jphyscol:1983658�. �jpa-00223217�
PHOTOSYNTHESIS ACTION SPECTRA IN ANACYSTIS NIDULANS AS DETERMINED
BY
PHOTOACOUSTIC SPECTROSCOPYR. C a r p e n t i e r , B . LaRue and R.M. Leblanc
Centre de Recherche en Photobiophysique, Universitd du Qudbec d Trois-Rivie'res, Trois-Rivizres, Qudbec G9A 5H7, Canada
R6sum6
-
Le s p e c t r e d ' a c t i o n pour l e s t o c k a g e d ' b n e r g i e chimique l o r s d e l a p h o t o s y n t h S s e e s t obtenu 5 p a r t i r de mesures p h o t o a c o u s t i q u e s chez Anacybhin n i d d a a . On en c o n c l u t que l a phycocyanine r s c o l t e d e s photons au p r o f i t d e s deux p h o t o r 6 a c t i o n s t a n d i s que l a c h l o r o p h y l l e 5 achemine l a m a j e u r e p a r t i e d e l f 6 n e r g i e lumineuse q u ' e l l e c a p t e v e r s l a photo- r 6 a c t i o n I.A b s t r a c t - A c o u s t i c d a t a from Anacybhin ~ d d a a a r e used t o o b t a i n t h e a c t i o n spectrum f o r photochemical energy s t o r a g e . I t i s concluded t h a t phycocyanin h a r v e s t s l i g h t f o r b o t h p h o t o r e a c t i o n s b u t t h a t c h l o r o p h y l l g m o l e c u l e s convey most of t h e i r e x c i t a t i o n e n e r g y t o p h o t o r e a c t i o n I.
INTRODUCTION
C y a n o b a c t e r i a ( b l u e - g r e e n a l g a e ) a r e a l o n e a b l e among t h e b a c t e r i a l world t o perform a p l a n t - t y p e p h o t o s y n t h e s i s . A s shown by F i g . 1, l i n e a r p h o t o s y n t h e s i s i n p l a n t s o r c y a n o b a c t e r i a i s b a s i c a l l y a n o x y d o r e d u c t i o n r e a c t i o n s t a r t i n g w i t h t h e p h o t o l y s i s of w a t e r ( 1 ) . It i n v o l v e s two c o n s e c u t i v e p h o t o r e a c t i o n s l i n k e d t o each o t h e r by a c h a i n of e l e c t r o n c a r r i e r s ; t h e p r o c e s s l e a d s t o t h e p h o s p h o r y l a t i o n of ADP i n t o ATP w i t h a f i n a l t r a n s f e r of r e d u c i n g e q u i v a l e n t s t o NADP t. A c y c l i c
(non-reducing) p h o t o p h o s p h o r y l a t i o n pathway i s a l s o a v a i l a b l e around photosystem I.
I n each p h o t o r e a c t i o n , t h e l i g h t energy f i r s t h a r v e s t e d by a pigment a n t e n n a i s conveyed t o a s p e c i a l u n i t (P-680 o r P-700) a c t i n g a s a r e a c t i o n c e n t e r . U n l i k e p l a n t s , however, a b l u e chromoprotein, phycocyanin, r e p l a c e s Chl
k*
a s a major l i g h t - h a r v e s t i n g pigment i n c y a n o b a c t e r i a.
DCMU
P-680'-. 1
A D P
.
A T P
FIG. 1. A s i m p l i f i e d scheme of p h o t o s y n t h e s i s i n p l a n t s and c y a n o b a c t e r i a . More d e t a i l e d e x p l a n a t i o n s a r e g i v e n i n t h e t e x t .
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1983658
JOURNAL DE PHYSIQUE
We h a v e b e e n i n v o l v e d f o r some t i m e i n t h e p h o t o a c o u s t i c s p e c t r o s c o p y s t u d y o f t h e u n i c e l l u l a r b l u e - g r e e n a l g a A n a c y s t i s nidulans,. The c h o i c e o f t h i s o r g a n i s m was d i c t a t e d b o t h by t h e a b u n d a n t l i t t e r a t u r e p e r t a i n i n g t o i t s p h o t o s y n t h e t i c p r o c e s s and by i t s e a s y g r o w t h and h a n d l i n g . T h e u s e o f t h e p h o t o a c o u s t i c t e c h n i q u e r e l a t e s h e r e t o t h e f a c t t h a t h e a t p r o d u c t i o n and s t o r a g e o f p h o t o s y n t h e t i c p r o d u c t s c o m p e t e w i t h e a c h o t h e r d u r i n g t h e e v e n t s f o l l o w i n g t h e a b s o r p t i o n of a l i g h t quantum. The p h o t o s y n t h e t i c y i e l d c a n t h u s b e e s t i m a t e d by c o m p a r i n g t h e a c o u s t i c s i g n a l f r o m a c t i v e a l g a e t o t h e o n e from a n i d e n t i c a l s a m p l e w h i c h h a s been t r e a t e d w i t h a p h o t o - s y n t h e t i c i n h i b i t o r ( 2 , 3 ) . We u s e h e r e t h i s a p p r o a c h t o e v a l u a t e t h e i n v o l v e m e n t o f t h e v a r i o u s a l g a l p i g m e n t s i n t h e p h o t o s y n t h e t i c p r o c e s s .
PHOTOACOUSTIC PROPERTIES OF ALGAI. LAYERS
As d e s c r i b e d p r e v i o u s l y ( 4 ) , l a y e r s o f A . n i d u l a n s c a n b e d e p o s i t e d o n n i t r o c e l l u l o s e f i l t e r s (HA t y p e , M i l l i p o r e F i l t e r C o r p . , 2 5 nun d i a m e t e r , 0 . 4 5 um p o r e s i z e ) by f i l t e r i n g s a m p l e s f r o m a l i q u i d c u l t u r e u n d e r a g e n t l e vacuum. T h i s e n s u r e s t h e f o r m a t i o n o f a n a l g a l l a y e r h a v i n g a q u i t e u n i f o r m t h i c k n e s s w h i c h i s s i m p l y c a l c u l - a t e d a s t h e r a t i o b e t w e e n t h e d e p o s i t e d a l g a l volume and t h e f i l t e r a r e a , I n p r a c t i c e , t h i s t h i c k n e s s c a n b e v a r i e d b e t w e e n 1 and 2 0 1L.m. A f t e r c u t t i n g t o t h e a p p r o p r i a t e s i z e , t h e f i l t e r i s i n t r o d u c e d i n t o t h e p h o t o a c o u s t i c c e l l ( 5 ) w i t h o u t a n y p r e l i m i n a r y d r y i n g . P h o t o a c o u s t i c d a t a a r e c o l l e c t e d a s d e s c r i b e d e l s e w h e r e ( 4 ) . The c h o p p i n g f r e q u e n c y , s c a n n i n g r a t e and s p e c t r a l band w i d t h a r e 1 0 0 Hz, 2 5 nm min-' and 5 nm, r e s p e c t i v e l y .
U i t h o u r e x p e r i m e n t a l s y s t e m t h e f o l l o w i n g a s s u m p t i o n s c a n b e made: 1 ) t h e t h e r m a l p r o p e r t i e s o f t h e g a s p h a s e ( a i r ) a r e much l o w e r t h a n t h o s e o f t h e s a m p l e and 2) b o t h t h e s a m p l e and i t s b a c k i n g m a t e r i a l ( f i l t e r ) a r e h e a v i l y h y d r a t e d and h a v e t h e same t h e r m a l p a r a m e t e r s a s w a t e r . As shown p r e v i o u s l y ( 4 ) , t h i s l e a d s t o a s i m p l i f i c a t i o n o f t h e m a t h e m a t i c a l t r e a t m e n t d e v e l o p p e d by Rosencwaig ( 6 ) , s u c h t h a t t h e n o r m a l i z e d i n t e n s i t y o f t h e a c o u s t i c s i g n a l , Q , becomes e x p r e s s e d by:
w h e r e K i s c o n s t a n t a t a g i v e n c h o p p i n g f r e q u e n c y . L , a a n d a a r e t h e t h i c k n e s s , t h e o p t i c a l a b s o r p t i o n a n d t h e r m a l d i f f u s i o n c o e f f i c i e n t s o f t h e a l g a l l a y e r , r e s p e c t - i v e l y . Eq. 1 shows t h a t Q a p p r o a c h e s a p l a t e a u a s e i t h e r a o r a g e t s l a r g e r t h a n 1 / L . As shown by F i g . 2 , t h e h i g h a b s o r p t i v i t y of t h e a l g a l l a y e r u s u a l l y c o n s t i t u - t e s t h e l i m i t i n g f a c t o r s i n c e t h e o p t i c a l a b s o r p t i o n l e n g t h i s a t l e a s t t w i c e s m a l l - e r t h a n i t s t h e r m a l d i f f u s i o n l e n g t h ( 2 1 Ilm a t 1 0 0 Hz) f o r most w a v e l e n g t h s . A s
i n d i c a t e d by a c o m p a r i s o n o f F i g . 3 and 4 , t h i s o p t i c a l a b s o r p t i o n s a t u r a t i o n phenomenon p r o d u c e s a f l a t t e n i n g o f t h e p h o t o a c o u s t i c s p e c t r u m ( 7 ) when t h i c k ( > 1 0 um) s a m p l e s a r e u s e d . As a c o u n t e r p a r t t o t h e s e s p e c t r a l d i s t o r s i o n s , t h e e x p e r i m e n t a l f l u c t u a t i o n of t h e a c o u s t i c s i g n a l r e m a i n s l e s s t h a n 2 p e r c e n t u n d e r t h e s e c o n d i t i o n s . T h i s r e p r o d u c i b i l i t y , u s u a l l y n o t a t t a i n a b l e w i t h most b i o l o g i c a l m a t e r i a l s , p e r m i t s h e r e a c c u r a t e m e a s u r e m e n t s o f t h e e f f e c t s o f p h o t o s y n t h e t i c i n h i b i t o r s ( s e e b e l o w ) .
IDENTIFICATION OF PIGEEKT llOLOCHROMS
P h o t o a c o u s t i c s p e c t r a f r o n t h i n l a y e r s o f A. n i d u Z a n s ( F i g . 3) r e v e a l n o t o n l y t h e same g e n e r a l f e a t u r e s a s a b s o r p t i o n s p e c t r a r e c o r d e d a t room t e m p e r a t u r e , b u t a l s o e x h i b i t i n t h e i r m i n o r d e t a i l s a v a r i e t y o f p i g m e n t s holochrorns. T h e i r i d e n t i f i c a - t i o n by t h e p h o t o a c o u s t i c t e c h n i q u e i s s r o n g l y s u p p o r t e d by t h e i r c o r r e s p o n d e n c e w i t h t h e p e a k s a n d s h o u l d e r s d e t e c t e d i n low t e m p e r a t u r e m e a s u r e m e n t s o f a b s o r p t i o n o r f l u o r e s c e n c e e x c i t a t i o n s p e c t r a ( T a b l e 1 a n d r e f . ( 8 ) ) . I n p a r t i c u l a r , o n e n o t i c e s : t h e d o u b l e t n a t u r e o f t h e p h y c o c y a n i n peak, t h e 670 nm Chl g * h o l o c h r o m a n d
*
A b b r e v i a t i o n s u s e d a r e : C h l , c h l o r o p h y l l ; PSI and P S I I , p h o t o s y s t e m s I and I1 r e s p e c t i v e l y ; DCMU, 3-(3,4-dichloropheny1)-1, 1 - d i m e t h y l u r e a ; TMPD, N , N , N 1 , N 1 - t e t r a m e t h y l - p - p h e n y l e n e d i a m i n e .THICKNESS. p m WAVELENGTH
,
nmF I G . 2 . E f f e c t of s a m p l e t h i c k n e s s o n t h e i n t e n s i t y o f t h e a c o u s t i c s i g n a l a t 4 d i f f e r e n t w a v e l e n g t h s . -
- -,
t h e o r e t i c a l c u r v e s a c c o r d i n g t o Eq. (1) and -e-e-, e x p e r i m e n t a l d a t a . T h e o r e t i c a l and e x p e r i m e n t a l c u r v e s w e r e n o r m a l i z e d w i t h e a c h o t h e r a t a 1 5 pm t h i c k n e s s . a i s t h e o p t i c a l a b s o r p t i o n c o e f f i c i e n t a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g .ua
i s t h e o p t i c a l p e n e t r a t i o n l e n g t h , a s g i v e n by p = I/,.FIG. 3 . Carbon b l a c k n o r m a l i z e d p h o t o a c o u s t i c s p e c t r a o f A . NiduZans l a y e r s . L , s a m p l e t h i c k n e s s i n pm.
TABLE 1
PHOTOACOUSTIC A N D ABSORPTION PEAKS I N SPECTRA OF ANACYSTIS NIDULANS
Pigment Absomtion s w c t r u m ' Photoacoustic spectrum 298-306 K 7 7 K 298 K
Fhloro~hyll a 670 1s) 670 1SI'
(red reg&) 680 (P) 679
i ~ i
678iPj
686 (S) 690 (S:weak)' 705 (S)
Carotenoids 460-465 (S) 465-470 (S) 465 (P/S) 480 (P) 490 (P) 490-505 (S) 502 (PI 500 (PI
Phycocyanin 580 (S) 580 (S) 550-610 (Several shoulders) 625 (P) 625 (P) 625 (P)'
635 (S;weak) 634 (P) 635 (P)'
Allophycocyanin 650 (S) 650 (%weak)
S = s h o u l d e r , P = p e a k . V a l u e s i n nm.
D a t a f r o m r e f . (8) and F i g . 5
B e s t s e e n w i t h s a m p l e t h i c k n e s s > 1 0 pm
JOURNAL DE PHYSIQUE
ACTIOK SPECTRA
S i n c e t h e quantum f l u o r e s c e n c e y i e l d o f a l g a l p i g m e n t s a t room t e m p e r a t u r e i s n e g l i g i b l e
( a ) ,
t h e a b s o r b e d l i g h t e n e r g y i s d i s s i p a t e d b e t w e e n h e a t p r o d u c t i o n and c h e m i c a l e n e r g y s t o r a g e . A c c o r d i n g t o M a l k i n and Cahen ( 2 ) , t h e f r a c t i o n o f t h e a b s o r b e d e n e r g y w h i c h i s s t o r e d a s p h o t o s y n t h e t i c p r o d u c t s ,+;,
i s t h e n g i v e n by:Qc i s t h e a c o u s t i c s i g n a l . from a c t i v e a l g a e and Q i t h e o n e from a n i d e n t i c a l s a m p l e t r e a t e d w i t h a p h o t o s y n t h e t i c i n h i b i t o r a n d w h i c h , c o n s e q u e n t l y , c o n v e r t s t o t a l l y i n t o h e a t t h e e n e r g y n o r m a l l y u s e d f o r t h e i n h i b i t e d p r o c e s s . S i n c e p h o t o n s d i f f e r - i n g by t h e i r e n e r g y c a n i n i t i a t e t h e same p h o t o c h e m i c a l p r o c e s s , t h e quantum
y i e l d f o r p h o t o s y n t h e s i s w i l l b e p r o p o r t i o n a l t o w h e r e X is t h e w a v e l e n g t h f o r l i g h t a b s o r p t i o n . The a c t i o n s p e c t r u m , F, w h i c h m e a s u r e s t h e p h o t o s y n t h e t i c e f f e c t a s a f u n c t i o n o f w a v e l e n g t h f o r i n c i d e n t p h o t o n s , w i l l t h u s b e e x p r e s s e d by:
The f o l l o w i n g c h e m i c a l s w e r e u s e d , a l o n e o r i n c o m b i n a t i o n , f o r m e a s u r e m e n t s o f
6;
and F:- DCMU: 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 b e t w e e n t h e two p h o t o s y s t e m s a n d b l o c k s a l l l i n e a r p h o t o s y n t h e s i s when u s e d a l o n e .
- TMPD: d o n a t e s e l e c t r o n s t o P S I and r e s t o r e s i t s a c t i v i t y when DCMU a l s o is p r e s e n t .
- NHGC1: u n c o u p l e s ADP p h o s p h o r y l a t i o n f r o m e l e c t r o n t r a n s p o r t , s u c h t h a t t h e e n e r g y n o r m a l l y s t o r e d a s ATP i s t h e n w a s t e d a s h c a t . A t h i g h c o n c e n t r a t i o n s , NH C1 a l s o i n h i b i t s O2 e v o l u t i o n i n A . PJidxZans ( u n p u b l i s h e d r e s u l t s ) .
4
As e x p e c t e d , t h e c o m p a r i s o n b e t w e e n DCMU-inhibited and a c t i v e a l g a e i n d i c a t e s t h a t DCMU i n d u c e s a s i g n i f i c a n t i n c r e a s e o f t h e a c o u s t i c , s i g n a l . b e t w e e n 4 0 0 a n d 700 nm ( F i g . 4A o r 4B). T h i s e f f e c t , a s q u a n t i t a t e d by '$r ( F i g . 5A), shows a b r o a d maxi- mum b e t w e e n 550 and 650 nm. The c o i n c i d e n c e b e t w e e n t h e a b s o r p t i o n and a c t i o n p e a k s i n t h i s s p e c t r a l r a n g e ( F i g . 5B) a l s o p o i n t o u t t h a t p h y c o c y a n i n i s t h e most e f f i c i e n t l i g h t - h a r v e s t i n g p i g m e n t i n l i n e a r p h o t o s y n t h e s i s . On t h e o t h e r hand, t h e r e l a t i v e d e c r e a s e w h i c h f o l l o w s t h e a d d i t i o n o f TNPD t o DCMU-treated a l g a e ( F i g . L A ) Is l a r r t a r o u n d t h e r e d a b s o r p t i o n maximum o f Chl 2. I n t h i s c a s e , t h e s p e c t r a o f r a n d I: ( F i g . 5 ) , wtiich r e f l e c t t h e r e a c t i v a t i o n o f l i n e a r P S I i n t h e p r e s e n c e o f a p o i s o n e d P S I I , a l s o i n d i c a t e t h a t Chl 5 a c t s a s t h e most e f f i c i e n t a n t e n n a f o r p h o t o r e a c t i o n I. Ilowever, p h y c o c y a n i n s t i l l r e t a i n s a b o u t two t h i r d s o f t h e quantum y i e l d of Chl
a.
I t f o l l o w s f r o m t h e c o m p a r i s o n w i t h t h e p r e v i o u s d a t a t h a t p h y c o c y a n i n c a n d i s t r i b u t e i t s e x c i t a t i o n e n e r g y t o b o t h t y p e s o f p h o t o - r e a c t i o n s c e n t e r s , w h i l e C h l a i s a s s i g n e d a l m o s t e x c l u s i v e l y t o t h e P S I a n t e n n a . S i m i l a r c o n c l u s i o n s w e r e r e a c h e d by o t h e r s f r o m i n v i v o m e a s u r e m e n t s o f Chl 5 f l u o r e s c e n c e e m i s s i o n ( 9 ) o r a c t i o n s p e c t r a f o r O2 e v o l u t i o n ( 1 0 ) .I n o r d e r t o d e t e c t c y c l i c p h o t o p h o s p h o r y l a t i o n , i t is n e c e s s a r y h e r e t o f i r s t i n h i b i t l i n e a r p h o t o s y n t h e s i s w i t h DCMU and t o add n e x t NH4C1, w h i c h u n c o u p l e s ADP p h o s p h o r y l a t i o n f r o m c y c l i c e l e c t r o n t r a n s p o r t by c o 1 , l a p s i n g t h e p r o t o n g r a d i e n t a c r o s s t h c t h y l a k o i d membranes ( 1 1 ) . Q u i t e s u r p r i s i n g l y , t h e f i n a l i n c r e a s e o f t h e a c o u s t i c s i g n a l i s n e a r l y a s l a r g e as t h e o n e i n d u c e d by DCMU a l o n e ( F i g . 4B). T h i s would i n d i c a t e t h e p r e s e n c e o f a much l a r g e r c y c l i c a c t i v i t y t h a n u s u a l l y t h o u g h t .
WAVELENGTH,
nmFIG. 4 . E f f e c t o f p h o t o s y n t h e t i c i n h i b i t o r s o n t h e p h o t o a c o u s t i c s p e c t r u m o f A . NiduZans. D a t a from F i g s . 4A and 4B r e s p e c t i v e l y were o b t a i n e d f r o m
two d i f f e r e n t b a t c h e s o f a l g a e and c a n n o t b e d i r e c t l y compared t o e a c h o t h e r . DCMU, TMPD and NH CI c o n c e n t r a t i o n s a r e 5 x 2 x and 3 x M, r e ~ ~ e c t i v e l y . ~ Sample t h i c k n e s s i s 20 pm. S e e t e x t f o r e x p l a n a t i o n .
FIG. 5 . 5A. S p e c t r a f o r t h e p h o t o c h e m i c a l e n e r g y s t o r a g e y i e l d
(d)\r).
5B. A c t i o n s p e c t r a .
The symbols u s e d a r e -0-o-, c o u p l e d p h o t o s y s t e m s -0-0-, l i n e a r PSI
a l o n e , a n d
.... ,
a b s o r p t i o n s p e c t r u m from t h e l i q u i d c u l t u r e . C a l c u l a t i o n s o f +r and F a r e performed a c c o r d i n g t o Eqs. (2) and ( 3 ) by u s i n g t h e d a t a from F i g . 4A. D e t a i l s a r e g i v e n i n t h e t e x t .JOURNAL DE PHYSIQUE
CONCLUSION
The p r e s e n t s t u d y shows t h a t p h o t o a c o u s t i c s p e c t r o s c o p y o n l a y e r e d s a m p l e s o f A. NiduZans c a n s u p p l y s p e c t r o s c o p i c i n f o r m a t i o n c o m p a r a b l e t o l o w t e m p e r a t u r e
a b s o r p t i o n o r f l u o r e s c e n c e d a t a . The m o s t p r o m i s i n g a s p e c t o f t h i s work l i e s i n t h e h i g h r e p r o d u c i b i l i t y o f a c o u s t i c s i g n a l i n t e n s i t y w h i c h i s o b t a i n e d w i t h s a m p l e s t e n s o f m i c r o n s i n t h i c k n e s s . T h i s p e c u l i a r i t y e n a b l e s u s t o m e a s u r e p h o t o s y n t h e t i c a c t i v i t i e s u n d e r a w i d e v a r i e t y o f c o n d i t i o n s . T h e c o m p a r i s o n b e t w e e n o u r d a t a a n d a c t i o n s p e c t r a o b t a i n e d by c o n v e n t i o n a l t e c h n i q u e s c o n f i r m s t h e v a l i d i t y o f o u r a p p r o a c h .
R.C. was s u p p o r t e d by a p o s t g r a d u a t e s c h o l a r s h i p f r o m t h e N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada (NSERC). The a u t h o r s a r e g r a t e f u l f o r f i n a n c i a l s u p p o r t f r o m t h e NSERC a n d t h e M i n i s t ' e r e d e 1 ' E d u c a t i o n du Qusbec.
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