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Polypeptide composition of light-harvesting complexes from some brown algae and diatoms
Volume 229, number 1, 11-15
FEB 05604
February 1988
Polypeptide composition of light-harvesting complexes from some brown algae and diatoms L. C a r o n , R. R e m y * a n d C. B e r k a l o f f + Laboratoire Arago, UA C N R S 117, F-66 650 Banyuls sur mer, *Equipe de Gbn~tique Molbculaire des Plantes, UA C N R S 115, Bat. 360, Univ. Paris XI, F-91405 Orsay Cbdex and +Laboratoire des Biomembranes et Surfaces Cellulaires V~g~tales, UA C N R S 311, Ecole Normale Sup~rieure, 46, rue d'Ulm, F- 75230 Paris Cbdex 05, France
Received 28 December 1987 The polypeptide composition of some Chromophyte light-harvestingcomplexeswas investigated by SDS-PAGE and compared to LHCP of higher plants. Accordingto the species, one or two major polypeptideswere found in the range 17-25 kDa, which is noticeablylower than for higher plant LHCP polypeptides.Evidenceis provided for three species LH polypeptidespossessing differentmolecular masses that they all possess some immunologicalanalogy with a maize LHCP. In addition, Fucus serratus LH was proved to be able to phosphorylate. Immunodetection;Light-harvestingcomplex;Phosphorylation;Polypeptide;(Brownalga; Diatom)
1. I N T R O D U C T I O N The Chromophytes (or chlorophyll c-containing algae) play a major role in ocean productivity. Their photosynthetic apparatus differs from that o f green plants, in both ultrastructure and pigment composition. Besides chlorophyll a and c, their chloroplasts contain carotenoids in greater abundance than in green plants. The light-harvesting (LH) function o f these carotenoids has been proved for at least two o f them, peridinin in Dinophyceae [1] and fucoxanthin in brown algae [2,3] and diatoms [4]. The major L H pigment protein complex o f Dinophyceae, extrinsic to thylakoid membranes, has been rather extensively studied [1,5]. In contrast, although L H fractions containing fucoxanthin, Chl a and Chl c have been isolated from different materials by means o f density gradient or electrophoresis (reviews [6-8]) Correspondence address: C. Berkaloff, Laboratoire des Biomembranes et Surfaces Cellulaires V6g6tales, Ecole Normale Sup6rieure, 46 rue d'Ulm, F-75230 Paris Cedex, France Abbreviations: LH, light-harvesting complex; LHCP, light-
harvesting complexof higherplants; PAGE, polyacrylamidegel electrophoresis; PS, photosystem
some confusion remains about their number and pigment composition, probably owing to their high sensitivity to detergents. Their polypeptide composition has been studied in two species, the diatom P h a e o d a c t y l u m t r i c o r n u t u m and the Prymnesiophyte P a v l o v a g y r a n s [9]. For t}. tricorn u t u m , several reports exist [9-12] that do not describe the same number or exactly the same molecular mass o f the constituent polypeptides. In addition, polyclonal antibodies against this last L H complex have been raised [11,12] and tested against total membrane polypeptides of a large variety o f diatoms [11]. In green plants, the L H C P complex has been proven to be implicated in the regulation o f the energy distribution between the two photosystems. The overreduction of the plastoquinone pool triggers the phosphorylation o f L H C P , which modifies the Charge o f L H C P molecules and leads to disconnection o f a part of them from the PS II centers (review [13]). This kind of state transition has not been demonstrated in brown algae or diatoms and, to date, it appears rather that L H of these algae normally transfers energy to both photosystems [4,14]. Furthermore, their chloroplasts do not possess the granal structure needed
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/88/$3.50 © 1988 Federation of European BiochemicalSocieties
11
Volume 229, number 1
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f o r this r e g u l a t i o n process. H o w e v e r , the v a r i a b i l i t y o f light i n t e n s i t y a n d q u a l i t y in the n a t u r a l h a b i t a t o f these o r g a n i s m s suggests t h a t s o m e k i n d of equilibration of energy distribution must o p e r a t e in their p h o t o s y n t h e t i c a p p a r a t u s . I n C y a n o b a c t e r i a , in w h i c h t h e t h y l a k o i d a r r a n g e m e n t is also a g r a n a l a n o t h e r k i n d o f r e g u l a t i o n occurs, which seems to implicate protein p h o s p h o r y l a t i o n processes [131. H e r e , we s h o w t h a t the m o l e c u l a r m a s s o f m a j o r polypeptides of LH from different brown algae a n d d i a t o m s m a y v a r y b e t w e e n 17 a n d 22 k D a , t h a t t h e y exhibit i m m u n o l o g i c a l c r o s s - r e a c t i o n s w i t h a n t i b o d i e s o f green p l a n t L H C P a n d m o r e o v e r t h a t b r o w n algae L H a r e c a p a b l e o f light-driven phosphorylation.
2. M A T E R I A L S A N D M E T H O D S Thalli of different species of brown algae were collected at the seashore near the marine laboratories of Roscoff (for Fucus serratus L. Dictyota dichotoma (Hudson) Lamouroux, Laminaria saccharina (L.), Lamouroux, or Banyuls (for Cystoseira mediterranea (Sauvageau) (France). Chloroplasts were prepared according to [15]. Diatom species (P. tricornutum Bohlin, Skeletonema costatum (Greville) Cleve) were cultivated as in [16] and harvested by centrifugation. Chloroplasts or diatom cells, resuspended in a 10 mM HepesNa buffer (pH 7.4), 2 mM MgC12, 2 mM MnC12, 10 mM KCI, 1 M sorbitol, were fragmented with a French pressure cell (136 MPa) in the presence of digitonin at a detergent/Chl ratio of 100 and incubated for 1 h in the same medium. The homogenate was loaded on the top of a sucrose gradient and centrifuged at 140000 x g for 15 h. The LH complexes obtained in the upper part of the gradient were diluted with 0.5 M Tris-HCl buffer, pH 8.0, with protease inhibitors (1 mM benzamidine and phenylmethanesulfonyl fluoride), then concentrated by centrifugation overnight at 200000 x g and 4°C. The pellets were incubated in dissolution buffer, containing 0.06 M Tris, 5% SDS, 1 M mercaptoethanol, for 1 h at room temperature. Samples were loaded on electrophoresis slabs to determine polypeptide composition. The stacking gel was 5°/o acrylamide with 0.1o70 SDS, pH 6.8, the running gel comprising a 10-22% acrylamide gel gradient with 0.1 o70SDS, pH 8.8. The buffer system of Laemmli [17] was used and 0.1 SDS was added to the upper reservoir. Proteins were stained with Coomassie brilliant blue R250. Immunological detection of electroblotted LH apoproteins was based on techniques described by Towbin et al. [18] and Burnette [19]. A polyclonal antibody raised in rabbits against maize LHCP apoproteins was used [20]. A horseradish peroxidase-conjugated IgG (Biosys) was used as antibody directed against the immunoglobulins of the first antiserum. Revelation of the peroxidase activity was performed using 0.015°/0 H202, 0.05°/o 4-chloronaphthol, in 0.02 M Tris, pH 8.2, and 0.9o70 NaCI. 12
February 1988
For thylakoid phosphorylation, the method in [21] was followed except that chlorophyll concentration in the incubation medium was 150/~g/ml and ATP was 100/~M. Illumination was provided by a white light (500/~E. m -2. s-t). 10/~Ci/ml of [y-32P]ATP (Amersham, spec. act. 3000 Ci/mM) was added to the reaction mixture. Dark phosphorylation was performed by adding 20 mM Ha dithionite. To inhibit phosphatases, 20 mM NaF was added to the reaction mixture. Following phosphorylation (15 min), the membranes were immediately pelleted at 20000 x g for 5 min and solubilized for SDS-PAGE [17]. Phosphoproteins were located after 15 days of autoradiography of the stained and dried gel, using X-ray film (Agfa-Gevaert Curix).
3. R E S U L T S A N D D I S C U S S I O N
3.1. Isolation o f the L H fractions A f t e r d i g i t o n i n t r e a t m e n t , similar g r a d i e n t p a t terns were o b t a i n e d f r o m all t h e screened species. A s a l r e a d y d e s c r i b e d for Fucus [3], t h r e e m a j o r t y p e s o f b a n d s c o u l d be o b s e r v e d : (i) o n e o r t w o h e a v y green, P S I - e n r i c h e d f r a c t i o n s ; (ii) in s o m e cases a few a b u n d a n t m e d i a n f r a c t i o n s w i t h P S I I characteristics; (iii) in the u p p e r p a r t o f the grad i e n t a large z o n e with a n intense o r a n g e - b r o w n c o l o r : in s o m e e x p e r i m e n t s t w o d i f f e r e n t c o l o r e d layers c o u l d b e d i s t i n g u i s h e d in this zone. I n this case, o n l y the l o w e r o f t h e t w o layers was used for t h e p r e s e n t s t u d y , since e x c i t a t i o n fluorescence s p e c t r a h a d a l r e a d y [3] d e m o n s t r a t e d t h e a c t u a l l i g h t - h a r v e s t i n g role o f this f r a c t i o n . Its p i g m e n t c o m p o s i t i o n h a d been a n a l y s e d b y m e a n s o f H P L C . It c o n t a i n e d all t h e m a i n p i g m e n t s p r e s e n t in the w h o l e c h l o r o p l a s t b u t with a n e n r i c h m e n t in c h l o r o p h y l l c a n d f u c o x a n t h i n , a n d conversely a d e p l e t i o n in c h l o r o p h y l l a a n d ~ - c a r o t e n e [22].
3.2. Polypeptide composition o f L H fractions T h e p o l y p e p t i d e c o m p o s i t i o n s o b s e r v e d in L H f r a c t i o n s f r o m t h e 6 s t u d i e d species a r e listed in t a b l e 1. T h e L H f r a c t i o n s o f all the tested species exhibited one or two major polypeptide comp o n e n t s . E l e c t r o p h o r e s i s o f the d i f f e r e n t s a m p l e s ( f i g . l ) clearly s h o w e d t h a t the m o l e c u l a r m a s s o f these p o l y p e p t i d e s v a r i e d f r o m 17 t o 21 k D a acc o r d i n g t o t h e species. T h e n u m b e r o f b a n d s in this molecular mass range varied from one to three and t h e i r relative a b u n d a n c e d i f f e r e d . S o m e o t h e r p o l y p e p t i d e s were p r e s e n t b e y o n d this r a n g e , b u t in m u c h l o w e r quantities; in b r o w n a l g a e t h e y were o n l y seen o n o v e r l o a d e d gels. T h e y were relatively m o r e a b u n d a n t a n d also m o r e
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February 1988
Table 1 Presently reported polypeptide compositions of LH complexes from brown algae and diatoms Species
Major LH polypeptides (kDa) This work Other reports
Brown algae Fucus serratus Cystoseira mediterranea Dictyota dichotoma Laminaria saccharina
21.5, 23 21, 22 20, 19 20.5, 21.5
Diatoms Phaeodactylum tricornutum
18.5, 18
16, 16.4, 17,
17.5 [9] 16.4, 16.9 [10]
17.5, 18 [11] 19, 19.5 [12] Skeletonema costatum
22, 19.5
Fig.2. (A) Coomassie blue-stained gel. (B) Western blot and immunodetection using LHCP antiserum. Lanes: (1) protein markers, (2) Fucus serratus LH, O) Dictyota dichotoma LH, (4) Phaedactylum tricornutum LH, (5) polypeptides from pea thylakoids. In order to obtain clear immunodetection, lane 5 in B was loaded with 5-times less protein than the corresponding lane in A.
Fig. 1. Coomassiebine-stained gel loaded with 2/~g chlorophyll. LH from 0anes): (1) Cystoseira mediterranea, (2) Skeletonema costatum, (3) Laminaria saccharina, (4) Fucus serratus, (5) Phaedactylum tricornutum, (6) Dictyota dichotoma.
numerous in the two diatoms' L H (figs 1,2). Probably, these minor bands were not constituents o f L H itself, but originated f r o m other membranes components still present in the gradient fraction. It must be noted that, whereas in P. t r i c o r n u t u m two successive detergent incubations seem to be needed to purify the 20 k D a range polypeptides [11,12], we obtained the same result after only digitonln action in the brown algal fractions. Concerning P. t r i c o r n u t u m , if we compare our results with published analyses (table 1), we note t h a t the molecular mass estimations o f the m a j o r polypeptide were closer to the value given by Friedm a n and Alberte [11] than to those reported in [9,10]. It is noteworthy that the polypeptide composition o f S. c o s t a t u m L H was very different f r o m that observed in P. t r i c o r n u t u m . Concerning brown algae L H , to our knowledge no molecular mass estimation has yet been published. All species tested here showed L H polypeptide with lower molecular mass than the current value observed for L H C P o f green plants. 13
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3.3. Immunochemical analogy with higher plant LHCP In order to determine whether there was a molecular analogy in the polypeptide composition of LH complexes from Chlorophytes and Chromophytes, an immunological study was performed using a maize LHCP polyclonal antibody. The resulting immunoblot is presented in fig.2. As a control the cross-reaction with pea LHCP polypeptides is given in lane 5. Although LH apoproteins of brown algae (lanes 2,3) and diatoms (lane 4) have noticeably lower molecular masses than higher plants, it is clear that the maize LHCP antibody recognizes the different Chromophyte LH. This indicates that at least some peptide sequences are common to Chromophyte and Chlorophyte LH proteins. However, as a polyclonal antibody was used, it cannot be ascertained whether it was the same peptide sequence that was recognized in the different LH investigated. Recently, very interesting data were published showing that a monospecific polyclonal antibody raised against the two apoproteins (17.5 and 18kDa) of the major light-harvesting pigment-protein from the diatom P. tricornutum [23] cross-reacted with neither LH polypeptides of three Chlorophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta) nor two classes of Chlorophyta algae. This probably indicated that the antibodies used in [23] recognize by chance a very sharp sequence. The maize LHCP antibody used in our work showed monospecific reactions with three constituent polypeptides of 29, 27 and 25 kDa of the maize LHCP [20] and with similar polypeptides of pea LHCP; therefore, larger sequences were probably recognized, which could explain why a cross-reaction was found here with P. tricornutum LH. As the different polypeptides of green plant LHCP are known to be coded by a multiple family of genes [24], it might be suggested that an analogous family is present in brown algae and diatoms, and that different genes of this family are expressed in the different genera or species, explaining the variability in molecular mass from one to another. 3.4. Phosphorylation o f brown alga L H Owing to this newly demonstrated homology between LH of brown algae and green plants, it 14
February 1988
A
B
Fig.3. Evidenceshowingthat Fucus serratus LH ca~ be phosphorylated. IsolatedFucusmembraneswerephosphorylatedin vitro with [-r-32p]ATP. (A) Coomassieblue-staine~dgel loaded with 5/Lg chlorophyll; lanes: 1, light-phosphorylatedmembranes; 2, dark control; 3, dark plus dithionite. (B) Autoradiogram of gel in A. seemed interesting to investigate whether brown alga LH was able to be phosphorylated and perhaps to play a role in light adaptation. Fig.3 demonstrates that Fucus LH was able to phosphorylate under light, or in darkness in the presence of dithionite, as higher plant LHCP. If photosynthetic membrane phosphorylation has now been demonstrated in organisms other than higher plants such as Cyanobacteria [25] and some purple photosynthetic bacteria [26,27], it is to our knowledge+the first time that such a possibility has been reported with regard to brown algae. At present, it is not possible to ascertain if this phosphorylation is related with modification of the light energy distribution between the two photosystems.
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REFERENCES [1] Prezelin, B.B. and Haxo, T. (1976) Planta 128, 132-141. [2] Barrett, J.A. and Anderson, J.M. (1980) Biochim. Biophys. Acta 590, 309-323. [3] Caron, L., Dubacq, J.P., Berkaloff, C. and Jupin, H. (1985) Plant Cell Physiol. 26, 131-139. [4] Owens, T.G. and Wold, E.R. (1986) Plant Physiol. 80, 732-738. [5] Song, P.S., Koka, P., Pr~zelin, B.B. and Haxo, T. (1976) Biochemistry 15, 4422-4427. [6] Dural, J.C., Jupin, H. and Berkaloff, C. (1983) Physiol. V6g. 6, 1145-1157. [7] Anderson, J.M. and Barrett, J.A. (1986)in: Encyclopedia of Plant Physiology, Photosynthesis III (Staehelin, L.A. and Arntzen, C.J. eds) pp.269-284, Springer, Berlin. [8] Caron, L. and Brown, J. (1987) Plant Cell Physiol. 28, 775-785. [9] Fawley, M.W., Morton, J.S., Stewart, K.D. and Mattox, K.R. (1987) J. Phycol. 23, 377-381. [10] Gugllelmeili, L.A. (1984) Biochim. Biophys. Acta 766, 45-50. [11] Friedman, A.L. and Alberte, R.S. (1984) Plant Physiol. 76, 483-489. [12] Fawley, M.W. and Grossman, A.R. (1986) Plant Physiol. 81, 149-155. [13] Williams, W.P. and Allen, J.F. (1987) Photosynth. Res. 13, 19-45. [14] Caron, L., Jupin, H. and Berkaloff, C. (!984) in:
[15] [16] [17l [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
February 1988
Advances in Photosynthesis Research, I (Sybesma, C. ed.) pp.69-72, Nijhoff/Junk, The Hague. Berkaloff, C. and Duval, J.C. (1980) Photosynth. Res. l, 127-135. Mortain-Bcrtrand, A., Descolas-Gros, C. and Jupin, H. (1987) Phycologia 26, 262-269. Laemmli, U.K. (1970) Nature 227, 680-685. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Burnette, W. (1981)Anal. Biochem. 112, 195-203. Rechenmann, C. (1987) Thesis, Univ. Paris-Sud, Centre d'Orsay, CNRS Registration no.3363. Kyle, D.J., Haworth, P. and Arntzen, C.J. (1982) Biochim. Biophys. Acta 680, 336-342. Lichfle, C., Berkaloff, C. and Lemoine, Y. (1986) VIIth International Congress of Photosynthesis, Brown Univ., USA, abstr.305-120. Friedman, A.L. and Alberte, R.S. (1987) J. Phycol. 23, 427-433. Dunsmuir, P. and Bedbrook, J. (1983) in: Structure and Function of Plant Genome (Ciferri, O. and Dure, L. eds) pp.221-230, Plenum, New York. Allen, J.F., Sanders, C.E. and Holmes, N.G. (1985) FEBS Lett. 193, 271-275. Holmes, N.G., Sanders, C,E. and Allen, J.F. (1986) Biochem. Soc. Trans. 14, 67-68. Holmes, N.G. and Allen, J.F. (1986) FEBS Lett. 200, 144-148.
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FEB 05604
February 1988
Polypeptide composition of light-harvesting complexes from some brown algae and diatoms L. C a r o n , R. R e m y * a n d C. B e r k a l o f f + Laboratoire Arago, UA C N R S 117, F-66 650 Banyuls sur mer, *Equipe de Gbn~tique Molbculaire des Plantes, UA C N R S 115, Bat. 360, Univ. Paris XI, F-91405 Orsay Cbdex and +Laboratoire des Biomembranes et Surfaces Cellulaires V~g~tales, UA C N R S 311, Ecole Normale Sup~rieure, 46, rue d'Ulm, F- 75230 Paris Cbdex 05, France
Received 28 December 1987 The polypeptide composition of some Chromophyte light-harvestingcomplexeswas investigated by SDS-PAGE and compared to LHCP of higher plants. Accordingto the species, one or two major polypeptideswere found in the range 17-25 kDa, which is noticeablylower than for higher plant LHCP polypeptides.Evidenceis provided for three species LH polypeptidespossessing differentmolecular masses that they all possess some immunologicalanalogy with a maize LHCP. In addition, Fucus serratus LH was proved to be able to phosphorylate. Immunodetection;Light-harvestingcomplex;Phosphorylation;Polypeptide;(Brownalga; Diatom)
1. I N T R O D U C T I O N The Chromophytes (or chlorophyll c-containing algae) play a major role in ocean productivity. Their photosynthetic apparatus differs from that o f green plants, in both ultrastructure and pigment composition. Besides chlorophyll a and c, their chloroplasts contain carotenoids in greater abundance than in green plants. The light-harvesting (LH) function o f these carotenoids has been proved for at least two o f them, peridinin in Dinophyceae [1] and fucoxanthin in brown algae [2,3] and diatoms [4]. The major L H pigment protein complex o f Dinophyceae, extrinsic to thylakoid membranes, has been rather extensively studied [1,5]. In contrast, although L H fractions containing fucoxanthin, Chl a and Chl c have been isolated from different materials by means o f density gradient or electrophoresis (reviews [6-8]) Correspondence address: C. Berkaloff, Laboratoire des Biomembranes et Surfaces Cellulaires V6g6tales, Ecole Normale Sup6rieure, 46 rue d'Ulm, F-75230 Paris Cedex, France Abbreviations: LH, light-harvesting complex; LHCP, light-
harvesting complexof higherplants; PAGE, polyacrylamidegel electrophoresis; PS, photosystem
some confusion remains about their number and pigment composition, probably owing to their high sensitivity to detergents. Their polypeptide composition has been studied in two species, the diatom P h a e o d a c t y l u m t r i c o r n u t u m and the Prymnesiophyte P a v l o v a g y r a n s [9]. For t}. tricorn u t u m , several reports exist [9-12] that do not describe the same number or exactly the same molecular mass o f the constituent polypeptides. In addition, polyclonal antibodies against this last L H complex have been raised [11,12] and tested against total membrane polypeptides of a large variety o f diatoms [11]. In green plants, the L H C P complex has been proven to be implicated in the regulation o f the energy distribution between the two photosystems. The overreduction of the plastoquinone pool triggers the phosphorylation o f L H C P , which modifies the Charge o f L H C P molecules and leads to disconnection o f a part of them from the PS II centers (review [13]). This kind of state transition has not been demonstrated in brown algae or diatoms and, to date, it appears rather that L H of these algae normally transfers energy to both photosystems [4,14]. Furthermore, their chloroplasts do not possess the granal structure needed
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/88/$3.50 © 1988 Federation of European BiochemicalSocieties
11
Volume 229, number 1
FEBS LETTERS
f o r this r e g u l a t i o n process. H o w e v e r , the v a r i a b i l i t y o f light i n t e n s i t y a n d q u a l i t y in the n a t u r a l h a b i t a t o f these o r g a n i s m s suggests t h a t s o m e k i n d of equilibration of energy distribution must o p e r a t e in their p h o t o s y n t h e t i c a p p a r a t u s . I n C y a n o b a c t e r i a , in w h i c h t h e t h y l a k o i d a r r a n g e m e n t is also a g r a n a l a n o t h e r k i n d o f r e g u l a t i o n occurs, which seems to implicate protein p h o s p h o r y l a t i o n processes [131. H e r e , we s h o w t h a t the m o l e c u l a r m a s s o f m a j o r polypeptides of LH from different brown algae a n d d i a t o m s m a y v a r y b e t w e e n 17 a n d 22 k D a , t h a t t h e y exhibit i m m u n o l o g i c a l c r o s s - r e a c t i o n s w i t h a n t i b o d i e s o f green p l a n t L H C P a n d m o r e o v e r t h a t b r o w n algae L H a r e c a p a b l e o f light-driven phosphorylation.
2. M A T E R I A L S A N D M E T H O D S Thalli of different species of brown algae were collected at the seashore near the marine laboratories of Roscoff (for Fucus serratus L. Dictyota dichotoma (Hudson) Lamouroux, Laminaria saccharina (L.), Lamouroux, or Banyuls (for Cystoseira mediterranea (Sauvageau) (France). Chloroplasts were prepared according to [15]. Diatom species (P. tricornutum Bohlin, Skeletonema costatum (Greville) Cleve) were cultivated as in [16] and harvested by centrifugation. Chloroplasts or diatom cells, resuspended in a 10 mM HepesNa buffer (pH 7.4), 2 mM MgC12, 2 mM MnC12, 10 mM KCI, 1 M sorbitol, were fragmented with a French pressure cell (136 MPa) in the presence of digitonin at a detergent/Chl ratio of 100 and incubated for 1 h in the same medium. The homogenate was loaded on the top of a sucrose gradient and centrifuged at 140000 x g for 15 h. The LH complexes obtained in the upper part of the gradient were diluted with 0.5 M Tris-HCl buffer, pH 8.0, with protease inhibitors (1 mM benzamidine and phenylmethanesulfonyl fluoride), then concentrated by centrifugation overnight at 200000 x g and 4°C. The pellets were incubated in dissolution buffer, containing 0.06 M Tris, 5% SDS, 1 M mercaptoethanol, for 1 h at room temperature. Samples were loaded on electrophoresis slabs to determine polypeptide composition. The stacking gel was 5°/o acrylamide with 0.1o70 SDS, pH 6.8, the running gel comprising a 10-22% acrylamide gel gradient with 0.1 o70SDS, pH 8.8. The buffer system of Laemmli [17] was used and 0.1 SDS was added to the upper reservoir. Proteins were stained with Coomassie brilliant blue R250. Immunological detection of electroblotted LH apoproteins was based on techniques described by Towbin et al. [18] and Burnette [19]. A polyclonal antibody raised in rabbits against maize LHCP apoproteins was used [20]. A horseradish peroxidase-conjugated IgG (Biosys) was used as antibody directed against the immunoglobulins of the first antiserum. Revelation of the peroxidase activity was performed using 0.015°/0 H202, 0.05°/o 4-chloronaphthol, in 0.02 M Tris, pH 8.2, and 0.9o70 NaCI. 12
February 1988
For thylakoid phosphorylation, the method in [21] was followed except that chlorophyll concentration in the incubation medium was 150/~g/ml and ATP was 100/~M. Illumination was provided by a white light (500/~E. m -2. s-t). 10/~Ci/ml of [y-32P]ATP (Amersham, spec. act. 3000 Ci/mM) was added to the reaction mixture. Dark phosphorylation was performed by adding 20 mM Ha dithionite. To inhibit phosphatases, 20 mM NaF was added to the reaction mixture. Following phosphorylation (15 min), the membranes were immediately pelleted at 20000 x g for 5 min and solubilized for SDS-PAGE [17]. Phosphoproteins were located after 15 days of autoradiography of the stained and dried gel, using X-ray film (Agfa-Gevaert Curix).
3. R E S U L T S A N D D I S C U S S I O N
3.1. Isolation o f the L H fractions A f t e r d i g i t o n i n t r e a t m e n t , similar g r a d i e n t p a t terns were o b t a i n e d f r o m all t h e screened species. A s a l r e a d y d e s c r i b e d for Fucus [3], t h r e e m a j o r t y p e s o f b a n d s c o u l d be o b s e r v e d : (i) o n e o r t w o h e a v y green, P S I - e n r i c h e d f r a c t i o n s ; (ii) in s o m e cases a few a b u n d a n t m e d i a n f r a c t i o n s w i t h P S I I characteristics; (iii) in the u p p e r p a r t o f the grad i e n t a large z o n e with a n intense o r a n g e - b r o w n c o l o r : in s o m e e x p e r i m e n t s t w o d i f f e r e n t c o l o r e d layers c o u l d b e d i s t i n g u i s h e d in this zone. I n this case, o n l y the l o w e r o f t h e t w o layers was used for t h e p r e s e n t s t u d y , since e x c i t a t i o n fluorescence s p e c t r a h a d a l r e a d y [3] d e m o n s t r a t e d t h e a c t u a l l i g h t - h a r v e s t i n g role o f this f r a c t i o n . Its p i g m e n t c o m p o s i t i o n h a d been a n a l y s e d b y m e a n s o f H P L C . It c o n t a i n e d all t h e m a i n p i g m e n t s p r e s e n t in the w h o l e c h l o r o p l a s t b u t with a n e n r i c h m e n t in c h l o r o p h y l l c a n d f u c o x a n t h i n , a n d conversely a d e p l e t i o n in c h l o r o p h y l l a a n d ~ - c a r o t e n e [22].
3.2. Polypeptide composition o f L H fractions T h e p o l y p e p t i d e c o m p o s i t i o n s o b s e r v e d in L H f r a c t i o n s f r o m t h e 6 s t u d i e d species a r e listed in t a b l e 1. T h e L H f r a c t i o n s o f all the tested species exhibited one or two major polypeptide comp o n e n t s . E l e c t r o p h o r e s i s o f the d i f f e r e n t s a m p l e s ( f i g . l ) clearly s h o w e d t h a t the m o l e c u l a r m a s s o f these p o l y p e p t i d e s v a r i e d f r o m 17 t o 21 k D a acc o r d i n g t o t h e species. T h e n u m b e r o f b a n d s in this molecular mass range varied from one to three and t h e i r relative a b u n d a n c e d i f f e r e d . S o m e o t h e r p o l y p e p t i d e s were p r e s e n t b e y o n d this r a n g e , b u t in m u c h l o w e r quantities; in b r o w n a l g a e t h e y were o n l y seen o n o v e r l o a d e d gels. T h e y were relatively m o r e a b u n d a n t a n d also m o r e
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Table 1 Presently reported polypeptide compositions of LH complexes from brown algae and diatoms Species
Major LH polypeptides (kDa) This work Other reports
Brown algae Fucus serratus Cystoseira mediterranea Dictyota dichotoma Laminaria saccharina
21.5, 23 21, 22 20, 19 20.5, 21.5
Diatoms Phaeodactylum tricornutum
18.5, 18
16, 16.4, 17,
17.5 [9] 16.4, 16.9 [10]
17.5, 18 [11] 19, 19.5 [12] Skeletonema costatum
22, 19.5
Fig.2. (A) Coomassie blue-stained gel. (B) Western blot and immunodetection using LHCP antiserum. Lanes: (1) protein markers, (2) Fucus serratus LH, O) Dictyota dichotoma LH, (4) Phaedactylum tricornutum LH, (5) polypeptides from pea thylakoids. In order to obtain clear immunodetection, lane 5 in B was loaded with 5-times less protein than the corresponding lane in A.
Fig. 1. Coomassiebine-stained gel loaded with 2/~g chlorophyll. LH from 0anes): (1) Cystoseira mediterranea, (2) Skeletonema costatum, (3) Laminaria saccharina, (4) Fucus serratus, (5) Phaedactylum tricornutum, (6) Dictyota dichotoma.
numerous in the two diatoms' L H (figs 1,2). Probably, these minor bands were not constituents o f L H itself, but originated f r o m other membranes components still present in the gradient fraction. It must be noted that, whereas in P. t r i c o r n u t u m two successive detergent incubations seem to be needed to purify the 20 k D a range polypeptides [11,12], we obtained the same result after only digitonln action in the brown algal fractions. Concerning P. t r i c o r n u t u m , if we compare our results with published analyses (table 1), we note t h a t the molecular mass estimations o f the m a j o r polypeptide were closer to the value given by Friedm a n and Alberte [11] than to those reported in [9,10]. It is noteworthy that the polypeptide composition o f S. c o s t a t u m L H was very different f r o m that observed in P. t r i c o r n u t u m . Concerning brown algae L H , to our knowledge no molecular mass estimation has yet been published. All species tested here showed L H polypeptide with lower molecular mass than the current value observed for L H C P o f green plants. 13
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3.3. Immunochemical analogy with higher plant LHCP In order to determine whether there was a molecular analogy in the polypeptide composition of LH complexes from Chlorophytes and Chromophytes, an immunological study was performed using a maize LHCP polyclonal antibody. The resulting immunoblot is presented in fig.2. As a control the cross-reaction with pea LHCP polypeptides is given in lane 5. Although LH apoproteins of brown algae (lanes 2,3) and diatoms (lane 4) have noticeably lower molecular masses than higher plants, it is clear that the maize LHCP antibody recognizes the different Chromophyte LH. This indicates that at least some peptide sequences are common to Chromophyte and Chlorophyte LH proteins. However, as a polyclonal antibody was used, it cannot be ascertained whether it was the same peptide sequence that was recognized in the different LH investigated. Recently, very interesting data were published showing that a monospecific polyclonal antibody raised against the two apoproteins (17.5 and 18kDa) of the major light-harvesting pigment-protein from the diatom P. tricornutum [23] cross-reacted with neither LH polypeptides of three Chlorophyte algal divisions (Chrysophyta, Cryptophyta, Pyrrophyta) nor two classes of Chlorophyta algae. This probably indicated that the antibodies used in [23] recognize by chance a very sharp sequence. The maize LHCP antibody used in our work showed monospecific reactions with three constituent polypeptides of 29, 27 and 25 kDa of the maize LHCP [20] and with similar polypeptides of pea LHCP; therefore, larger sequences were probably recognized, which could explain why a cross-reaction was found here with P. tricornutum LH. As the different polypeptides of green plant LHCP are known to be coded by a multiple family of genes [24], it might be suggested that an analogous family is present in brown algae and diatoms, and that different genes of this family are expressed in the different genera or species, explaining the variability in molecular mass from one to another. 3.4. Phosphorylation o f brown alga L H Owing to this newly demonstrated homology between LH of brown algae and green plants, it 14
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A
B
Fig.3. Evidenceshowingthat Fucus serratus LH ca~ be phosphorylated. IsolatedFucusmembraneswerephosphorylatedin vitro with [-r-32p]ATP. (A) Coomassieblue-staine~dgel loaded with 5/Lg chlorophyll; lanes: 1, light-phosphorylatedmembranes; 2, dark control; 3, dark plus dithionite. (B) Autoradiogram of gel in A. seemed interesting to investigate whether brown alga LH was able to be phosphorylated and perhaps to play a role in light adaptation. Fig.3 demonstrates that Fucus LH was able to phosphorylate under light, or in darkness in the presence of dithionite, as higher plant LHCP. If photosynthetic membrane phosphorylation has now been demonstrated in organisms other than higher plants such as Cyanobacteria [25] and some purple photosynthetic bacteria [26,27], it is to our knowledge+the first time that such a possibility has been reported with regard to brown algae. At present, it is not possible to ascertain if this phosphorylation is related with modification of the light energy distribution between the two photosystems.
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REFERENCES [1] Prezelin, B.B. and Haxo, T. (1976) Planta 128, 132-141. [2] Barrett, J.A. and Anderson, J.M. (1980) Biochim. Biophys. Acta 590, 309-323. [3] Caron, L., Dubacq, J.P., Berkaloff, C. and Jupin, H. (1985) Plant Cell Physiol. 26, 131-139. [4] Owens, T.G. and Wold, E.R. (1986) Plant Physiol. 80, 732-738. [5] Song, P.S., Koka, P., Pr~zelin, B.B. and Haxo, T. (1976) Biochemistry 15, 4422-4427. [6] Dural, J.C., Jupin, H. and Berkaloff, C. (1983) Physiol. V6g. 6, 1145-1157. [7] Anderson, J.M. and Barrett, J.A. (1986)in: Encyclopedia of Plant Physiology, Photosynthesis III (Staehelin, L.A. and Arntzen, C.J. eds) pp.269-284, Springer, Berlin. [8] Caron, L. and Brown, J. (1987) Plant Cell Physiol. 28, 775-785. [9] Fawley, M.W., Morton, J.S., Stewart, K.D. and Mattox, K.R. (1987) J. Phycol. 23, 377-381. [10] Gugllelmeili, L.A. (1984) Biochim. Biophys. Acta 766, 45-50. [11] Friedman, A.L. and Alberte, R.S. (1984) Plant Physiol. 76, 483-489. [12] Fawley, M.W. and Grossman, A.R. (1986) Plant Physiol. 81, 149-155. [13] Williams, W.P. and Allen, J.F. (1987) Photosynth. Res. 13, 19-45. [14] Caron, L., Jupin, H. and Berkaloff, C. (!984) in:
[15] [16] [17l [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
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Advances in Photosynthesis Research, I (Sybesma, C. ed.) pp.69-72, Nijhoff/Junk, The Hague. Berkaloff, C. and Duval, J.C. (1980) Photosynth. Res. l, 127-135. Mortain-Bcrtrand, A., Descolas-Gros, C. and Jupin, H. (1987) Phycologia 26, 262-269. Laemmli, U.K. (1970) Nature 227, 680-685. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Burnette, W. (1981)Anal. Biochem. 112, 195-203. Rechenmann, C. (1987) Thesis, Univ. Paris-Sud, Centre d'Orsay, CNRS Registration no.3363. Kyle, D.J., Haworth, P. and Arntzen, C.J. (1982) Biochim. Biophys. Acta 680, 336-342. Lichfle, C., Berkaloff, C. and Lemoine, Y. (1986) VIIth International Congress of Photosynthesis, Brown Univ., USA, abstr.305-120. Friedman, A.L. and Alberte, R.S. (1987) J. Phycol. 23, 427-433. Dunsmuir, P. and Bedbrook, J. (1983) in: Structure and Function of Plant Genome (Ciferri, O. and Dure, L. eds) pp.221-230, Plenum, New York. Allen, J.F., Sanders, C.E. and Holmes, N.G. (1985) FEBS Lett. 193, 271-275. Holmes, N.G., Sanders, C,E. and Allen, J.F. (1986) Biochem. Soc. Trans. 14, 67-68. Holmes, N.G. and Allen, J.F. (1986) FEBS Lett. 200, 144-148.
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