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The detection of phytochrome in the red alga Acrochaetium DaviesII
Plant Science Letters, 11 (1978) 145--149 © Elsevier/North-Holland Scientific Publishers Ltd.
145
THE DETECTION OF PHYTOCHROME IN THE RED ALGA A CR O C H A E T I U M D A VIESII H.H. VAN DER VELDE and A.M. HEMRIKA-WAGNER
Department of Botany, Biologisch Laboratorium der Vrije Universiteit, De Boelelaan 108 7, Amsterdam-Buitenveldert (The Netherlands) (Received June 17th, 1977) (Accepted October 19th, 1977)
SUMMARY
The occurrence of phytochrome in the red alga Acrochaetium daviesii is demonstrated and the possible importance is discussed of the phytochrome system for red algae submerged deeply in the sea.
INTRODUCTION
In many algae phytochrome-mediated photomorphogenetic responses have been demonstrated [1--12]. The only report of an isolation of phytochrome from an alga is that concerning Mesotaenium caldariorum [13]. Besides the red, far-red reversible phytochrome system other photoreversible, photoregulatory systems have been demonstrated in algae [14--18] and the pigments concerned have been isolated (19--22). Further many blue light effects have been described for algae; a detailed list of these effects is given by Van der Velde [23]. In this paper experiments are described with the aim to demonstrate the occurrence of phytochrome in the red alga Acrochaetium daviesii, in view of laboratory experiments in which Acrochaetium was cultivated under different light colours [ 11,23,24]. MATERIALS AND METHODS
Plant material Axenic cultures were used of the red alga Aerochaetium daviesii (Dillw.) Ntig.(Nemaliales). The Acrochaetium plants were grown under red, white or blue fluorescent light with equal intensity (5 J.m-2.s -1). The cultivation method has been described previously [23,24]. Abbreviations: Pr and Pfr are the red and far-red absorbing forms of phytochrome, respectively.
146
Extraction and partial purification of phytochrome The plant material was homogenized in an ice-cold mortar with some carborundum. Per gram fresh weight 50 ml of the homogenizing buffer (0.05 M Tris--HC1, containing, 1.5 mM EDTA and 2 mM cysteine. HC1 pH 7.4) was used. The homogenate was pressed through a double layer of perlon gauze and the remainder in the perlon gauze was homogenized for a second time. The t w o resulting filtrates were combined and sonified with a Sonifier B-12 (Branson Sonic P o w e r Co) under cooling with a mixture of alcohol and ice, and centrifuged (15 min; 25 000 × g). The resulting s u p e m a t a n t was either used as crude extract or further purified. It was necessary to carry o u t some purification because with crude extracts no good results could be obtained. F o r purification an equal volume of 66% saturated a m m o n i u m sulphate was added to the crude extract. The mixture was centrifuged (15 min; 30 000 × g) and the precipitate was redissolved in a small volume o f 15 mM potassium phosphate buffer (pH 7.5). The solution was dialyzed for 1 h against a large volume of the same buffer to remove the remaining a m m o n i u m sulphate. After dialysis the solution was filtered and used in experiments as the partially purified p h y t o c h r o m e solution. All steps were performed at 4 ° C and as much as possible in the dark. Also all steps o f this partial purification procedure have been tested on etiolated barley seedlings (Hordeum vulgare, var. Zephyr) which are known to contain such amounts o f p h y t o c h r o m e that these can be demonstrated very well with the assay m e t h o d used in this investigation. This purification procedure was based on the extensive purification m e t h o d s of Gardner et al. [ 2 5 ] . Following the complete purification procedure described b y Gardner et al. [25] such a great part of the p h y t o c h r o m e is lost that the partial purification procedure given has been preferred in view of the small amounts of Acrochaetium that were available.
Assay of phytochrome content The presence of p h y t o c h r o m e was demonstrated with the aid of an Aminco DW-2 s p e c t r o p h o t o m e t e r set in the dual-wavelength position. The m o n o c h r o m a t o r s of the s p e c t r o p h o t o m e t e r were set at 730 and 800 nm, according to Smith [27] in order to avoid interference with the phytochrome assay caused b y changes in the absorbance of the solutions due to irreversible changes in the protochlorophyll. The procedure of the phytochrome assay was: (a) preparation o f extract; (b) illumination o f extract with far-red light (Pfr ~ Pr); (c) transfer o f the cuvette with the extract to the s p e c t r o p h o t o m e t e r in the dark and determination of the difference in absorbance at 800 and at 730 nm. This difference is called AAfr; (d) illumination o f extract with red light (Pr ~ Pfr); (e) transfer o f the cuvette with the extract to the s p e c t r o p h o t o m e t e r in the dark and determination o f the difference in absorbance at 800 and 730 nm. This difference is called/x Ar.
147
Red and far-red illuminations were performed in a dark room using a waterbath o f + 10 cm as a heat filter between the light source and the cuvette with the extract. Between the heat filter and the cuvette was a holder in which two metal-interference filters of the type Ffltraflex B-40 (Balzers) could be placed: one with kmax = 732 nm for far-red illumination and one with ~'max = 652 nm for red illumination. Illuminations were performed for 5 min; the light intensity of the white light directed to the coloured filters was + 2000 lux. By red light not all Pr is converted to Pfr" In fact an equilibrium is reached with 81% o f the phytochrome in the Pfr form and 19% in the Pr form. The relation of the total phytochrome content with the difference between hAft a n d A A r is:
P t o t a l = 1.25 X k X ~ ( ~ A ) = 1.25 X k X ( ~ A f r - - ~ A r } , in w h i c h k is an u n k n o w n c o n s t a n t . T h e r e l a t i v e a m o u n t s o f p h y t o c h r o m e g i v e n in A ( A A ) / g f r e s h w e i g h t .
are
RESULTS
Red and far-red illuminations caused distinct differences in absorbance in the extracts, indicating that phytochrome is present (Fig. 1). The relative phyto0.010
0.008
0.006
I 0.004
C~
0.002
L.
£.
t.
0
//
//-4/
/J
// t
//---/~ 50S
t
Time
>
Fig. 1. Demonstration of phytochrome in a partially purified extract from Acrochaetium daviesii grown under blue light with the aid of a dual-wavelength spectrophotometer. A(Ao~o--AT~o) was recorded after red or far-red illumination of the extract.
148 TABLE I RELATIVE PHYTOCHROME CONTENT (IN zx (A A)/G FRESH WEIGHT) IN PARTIALLY PURIFIED EXTRACTS FROM A C R O C H A E T I U M DA VIESII GROWN UNDER RED, WHITE OR BLUE LIGHT Cultivation light
Phytochrome content ( • 10 -3)
Red White Blue
10.7 13.4 10.2
chrome contents of Acrochaetium plants grown under red, white or blue light are given in Table I. Plants grown under red or blue light contained the same amount of phytochrome while plants grown under white light seemed to have a somewhat higher pbytochrome content. It should be considered that as a consequence of the purification procedure -+ 30% of the protein present in the crude extract has been removed. As it is unknown whether the removed part of the protein contained some phytochrome or not, no corrections can be made in the calculation of the relative phytochrome contents for such a loss. DISCUSSION
In partially purified extracts from cultures of Acrochaetium daviesii grown under red, white or blue light the presence of phytochrome could be demonstrated. In crude extracts the assay was disturbed, probably by the action of proteolytic enzymes (see ref. 25). Cultures grown under red, white or blue light all contained approximately the same amount of phytochrome. Total phytochrome contents as determined in extracts do not necessarily give exact information on the in vivo situation. Firstly, phytochrome can exist in diverse more or less aggregated forms which might be liable to protein denaturation to a different extent. Secondly, in the extraction of phytochrome no discrimination is made between the in vivo active and non.active portions of the phytochrome. The relative phytochrome content determined for Acrochaetium daviesii is very well in agreement with that assayed for the green alga Mesotaenium caldariorum [13] and that for barley (15 • 10 -3 A (AA)/g fresh Weight) assayed in this investigation. Phytochrome-mediated photomorphogenetic responses in algae are known from laboratory experiments. Recently a possible control on growth (rhythm of mitosis) by phytochrome has been reported for an unidentified Acrochaetium species [11]. Therefore it is important that phytoehrome could now be demonstrated in Acrochaetium daviesii. Some authors question the usefulness of the phytochrome system for
149
red algae submerged deeply in the sea because fax-red light does not penetrate very deeply into the seawater [26]. Phytochrome m a y yet be very useful for these algae because Pr and Pfr have an absorption peak in the blue region of the spectrum and these peaks differ in height [27]. Blue light is k n o w n to penetrate deeply into the seawater [28,29]. It is obvious that the involvement of the phytochrome system should be taken into account in the interpretation both of the effects of different light c o l o u r s o n c u l t u r e s o f red algae (forAcrochaetium see refs. 11 a n d 23), a n d o f t h e d i f f e r e n c e s f o u n d b e t w e e n algae h a r v e s t e d f r o m d i f f e r e n t h a b i t a t s ( d e p t h s ) in t h e sea. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
R. Rethy, Z. Pflanzenphysiol.,59 (1968) 100. S. Takatori and K. Imahori, Phycologia, 10 (1971) 221. W. Haupt, Planta,53 (1959) 484. H. Neuschler-Wirth,Z. Pflanzenphysiol.,63 (1970) 283. W. Haupt and R. Thiele,Planta, 56 (1961) 388. K.L. Giles,Can. J. Bot.,48 (1970) 1343. M.J. Lipps, ~ Phycol., 9 (1973) 237. R. Forward and D. Davenport, Science, 161 (1968) 1028. H.-G. Rentschler,Planta,76 (1967) 65. M.J. Dring,Nature, 215 (1967) 1411. J.P.Larpent and M. Ducher, C.R. Soc. Biol.,169 (1975) 1501. N. Richardson, J. Phycol.,6 (1970) 215. A.O. Taylor and B.A. Bonner, Plant Physiol.,42 (1967) 762. Y. Fujitaand A. Hattori,Plant CellPhysiol.,1 (1960) 293. N. Lazaroff, Photomorphogenesis and nostocacean development, in N.G. Cart and B.A. Whitton (Eds.),The Biology of Blue-green Algae, Blackwell,Oxford, 1973, pp. 279--319. S. Diakoff and J. Scheibe, Plant Physiol.,51 (1973) 382. S. Diakoff and J. Scheibe, Physiol.Plant.,34 (1975) 125. J.C.O'Kelly,J.P. Durant and D.L. Stockdale,Photochem. Photobiol.,20 (1974) 47. J. Scheibe, Science, 176 (1972) 1037. G.S. BjSrn and L.O. BjSrn, Physiol.Plant.,36 (1976) 297. J.P.Thomas and J.C. OTLelly, Photochem. Photobiol.,17 (1973) 469. J.P.Thomas and J.C. O'Kelly,J.K. Hardman and E.F. Aldridge,Photochem. Photobiol., 22 (1975) 135. H.H. van der Velde, The growth of Acrochaetium under red, white and blue light, Thesis Vrije Universiteit, Amsterdam, 1977, pp. 1--160. H.H. van der Velde, P. Guiking and D. van der Wulp, Z. Pflanzenphysiol., 76 (1975) 95. G. Gardner, C.S. Pike, H.V. Rice and W.R. Briggs, Plant Physiol., 48 (1971) 686. J.A. West, J. Phycol., 4 (1968) 89. H. Smith, Phytochrome and Photomorphogenesis, McGraw-Hill, London, 1975, pp. 54--85. T. Lewing, The vegetation in the sea, in T. Lewing, H.A. Hoppe and O.J. Schmid (eds.), Marine Algae. A Survey of Research and Utilization, Cram, De Gruyter & Co, Hamburg, 1969, pp. 1--46. M.J. Dring, Light quality and the photomorphogenesis of algae in marine environments, in D.J. Crisp (Ed.), Fourth European Marine Biology symposium, Cambridge University Press, Cambridge, 1971, pp. 375--392.
145
THE DETECTION OF PHYTOCHROME IN THE RED ALGA A CR O C H A E T I U M D A VIESII H.H. VAN DER VELDE and A.M. HEMRIKA-WAGNER
Department of Botany, Biologisch Laboratorium der Vrije Universiteit, De Boelelaan 108 7, Amsterdam-Buitenveldert (The Netherlands) (Received June 17th, 1977) (Accepted October 19th, 1977)
SUMMARY
The occurrence of phytochrome in the red alga Acrochaetium daviesii is demonstrated and the possible importance is discussed of the phytochrome system for red algae submerged deeply in the sea.
INTRODUCTION
In many algae phytochrome-mediated photomorphogenetic responses have been demonstrated [1--12]. The only report of an isolation of phytochrome from an alga is that concerning Mesotaenium caldariorum [13]. Besides the red, far-red reversible phytochrome system other photoreversible, photoregulatory systems have been demonstrated in algae [14--18] and the pigments concerned have been isolated (19--22). Further many blue light effects have been described for algae; a detailed list of these effects is given by Van der Velde [23]. In this paper experiments are described with the aim to demonstrate the occurrence of phytochrome in the red alga Acrochaetium daviesii, in view of laboratory experiments in which Acrochaetium was cultivated under different light colours [ 11,23,24]. MATERIALS AND METHODS
Plant material Axenic cultures were used of the red alga Aerochaetium daviesii (Dillw.) Ntig.(Nemaliales). The Acrochaetium plants were grown under red, white or blue fluorescent light with equal intensity (5 J.m-2.s -1). The cultivation method has been described previously [23,24]. Abbreviations: Pr and Pfr are the red and far-red absorbing forms of phytochrome, respectively.
146
Extraction and partial purification of phytochrome The plant material was homogenized in an ice-cold mortar with some carborundum. Per gram fresh weight 50 ml of the homogenizing buffer (0.05 M Tris--HC1, containing, 1.5 mM EDTA and 2 mM cysteine. HC1 pH 7.4) was used. The homogenate was pressed through a double layer of perlon gauze and the remainder in the perlon gauze was homogenized for a second time. The t w o resulting filtrates were combined and sonified with a Sonifier B-12 (Branson Sonic P o w e r Co) under cooling with a mixture of alcohol and ice, and centrifuged (15 min; 25 000 × g). The resulting s u p e m a t a n t was either used as crude extract or further purified. It was necessary to carry o u t some purification because with crude extracts no good results could be obtained. F o r purification an equal volume of 66% saturated a m m o n i u m sulphate was added to the crude extract. The mixture was centrifuged (15 min; 30 000 × g) and the precipitate was redissolved in a small volume o f 15 mM potassium phosphate buffer (pH 7.5). The solution was dialyzed for 1 h against a large volume of the same buffer to remove the remaining a m m o n i u m sulphate. After dialysis the solution was filtered and used in experiments as the partially purified p h y t o c h r o m e solution. All steps were performed at 4 ° C and as much as possible in the dark. Also all steps o f this partial purification procedure have been tested on etiolated barley seedlings (Hordeum vulgare, var. Zephyr) which are known to contain such amounts o f p h y t o c h r o m e that these can be demonstrated very well with the assay m e t h o d used in this investigation. This purification procedure was based on the extensive purification m e t h o d s of Gardner et al. [ 2 5 ] . Following the complete purification procedure described b y Gardner et al. [25] such a great part of the p h y t o c h r o m e is lost that the partial purification procedure given has been preferred in view of the small amounts of Acrochaetium that were available.
Assay of phytochrome content The presence of p h y t o c h r o m e was demonstrated with the aid of an Aminco DW-2 s p e c t r o p h o t o m e t e r set in the dual-wavelength position. The m o n o c h r o m a t o r s of the s p e c t r o p h o t o m e t e r were set at 730 and 800 nm, according to Smith [27] in order to avoid interference with the phytochrome assay caused b y changes in the absorbance of the solutions due to irreversible changes in the protochlorophyll. The procedure of the phytochrome assay was: (a) preparation o f extract; (b) illumination o f extract with far-red light (Pfr ~ Pr); (c) transfer o f the cuvette with the extract to the s p e c t r o p h o t o m e t e r in the dark and determination of the difference in absorbance at 800 and at 730 nm. This difference is called AAfr; (d) illumination o f extract with red light (Pr ~ Pfr); (e) transfer o f the cuvette with the extract to the s p e c t r o p h o t o m e t e r in the dark and determination o f the difference in absorbance at 800 and 730 nm. This difference is called/x Ar.
147
Red and far-red illuminations were performed in a dark room using a waterbath o f + 10 cm as a heat filter between the light source and the cuvette with the extract. Between the heat filter and the cuvette was a holder in which two metal-interference filters of the type Ffltraflex B-40 (Balzers) could be placed: one with kmax = 732 nm for far-red illumination and one with ~'max = 652 nm for red illumination. Illuminations were performed for 5 min; the light intensity of the white light directed to the coloured filters was + 2000 lux. By red light not all Pr is converted to Pfr" In fact an equilibrium is reached with 81% o f the phytochrome in the Pfr form and 19% in the Pr form. The relation of the total phytochrome content with the difference between hAft a n d A A r is:
P t o t a l = 1.25 X k X ~ ( ~ A ) = 1.25 X k X ( ~ A f r - - ~ A r } , in w h i c h k is an u n k n o w n c o n s t a n t . T h e r e l a t i v e a m o u n t s o f p h y t o c h r o m e g i v e n in A ( A A ) / g f r e s h w e i g h t .
are
RESULTS
Red and far-red illuminations caused distinct differences in absorbance in the extracts, indicating that phytochrome is present (Fig. 1). The relative phyto0.010
0.008
0.006
I 0.004
C~
0.002
L.
£.
t.
0
//
//-4/
/J
// t
//---/~ 50S
t
Time
>
Fig. 1. Demonstration of phytochrome in a partially purified extract from Acrochaetium daviesii grown under blue light with the aid of a dual-wavelength spectrophotometer. A(Ao~o--AT~o) was recorded after red or far-red illumination of the extract.
148 TABLE I RELATIVE PHYTOCHROME CONTENT (IN zx (A A)/G FRESH WEIGHT) IN PARTIALLY PURIFIED EXTRACTS FROM A C R O C H A E T I U M DA VIESII GROWN UNDER RED, WHITE OR BLUE LIGHT Cultivation light
Phytochrome content ( • 10 -3)
Red White Blue
10.7 13.4 10.2
chrome contents of Acrochaetium plants grown under red, white or blue light are given in Table I. Plants grown under red or blue light contained the same amount of phytochrome while plants grown under white light seemed to have a somewhat higher pbytochrome content. It should be considered that as a consequence of the purification procedure -+ 30% of the protein present in the crude extract has been removed. As it is unknown whether the removed part of the protein contained some phytochrome or not, no corrections can be made in the calculation of the relative phytochrome contents for such a loss. DISCUSSION
In partially purified extracts from cultures of Acrochaetium daviesii grown under red, white or blue light the presence of phytochrome could be demonstrated. In crude extracts the assay was disturbed, probably by the action of proteolytic enzymes (see ref. 25). Cultures grown under red, white or blue light all contained approximately the same amount of phytochrome. Total phytochrome contents as determined in extracts do not necessarily give exact information on the in vivo situation. Firstly, phytochrome can exist in diverse more or less aggregated forms which might be liable to protein denaturation to a different extent. Secondly, in the extraction of phytochrome no discrimination is made between the in vivo active and non.active portions of the phytochrome. The relative phytochrome content determined for Acrochaetium daviesii is very well in agreement with that assayed for the green alga Mesotaenium caldariorum [13] and that for barley (15 • 10 -3 A (AA)/g fresh Weight) assayed in this investigation. Phytochrome-mediated photomorphogenetic responses in algae are known from laboratory experiments. Recently a possible control on growth (rhythm of mitosis) by phytochrome has been reported for an unidentified Acrochaetium species [11]. Therefore it is important that phytoehrome could now be demonstrated in Acrochaetium daviesii. Some authors question the usefulness of the phytochrome system for
149
red algae submerged deeply in the sea because fax-red light does not penetrate very deeply into the seawater [26]. Phytochrome m a y yet be very useful for these algae because Pr and Pfr have an absorption peak in the blue region of the spectrum and these peaks differ in height [27]. Blue light is k n o w n to penetrate deeply into the seawater [28,29]. It is obvious that the involvement of the phytochrome system should be taken into account in the interpretation both of the effects of different light c o l o u r s o n c u l t u r e s o f red algae (forAcrochaetium see refs. 11 a n d 23), a n d o f t h e d i f f e r e n c e s f o u n d b e t w e e n algae h a r v e s t e d f r o m d i f f e r e n t h a b i t a t s ( d e p t h s ) in t h e sea. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
R. Rethy, Z. Pflanzenphysiol.,59 (1968) 100. S. Takatori and K. Imahori, Phycologia, 10 (1971) 221. W. Haupt, Planta,53 (1959) 484. H. Neuschler-Wirth,Z. Pflanzenphysiol.,63 (1970) 283. W. Haupt and R. Thiele,Planta, 56 (1961) 388. K.L. Giles,Can. J. Bot.,48 (1970) 1343. M.J. Lipps, ~ Phycol., 9 (1973) 237. R. Forward and D. Davenport, Science, 161 (1968) 1028. H.-G. Rentschler,Planta,76 (1967) 65. M.J. Dring,Nature, 215 (1967) 1411. J.P.Larpent and M. Ducher, C.R. Soc. Biol.,169 (1975) 1501. N. Richardson, J. Phycol.,6 (1970) 215. A.O. Taylor and B.A. Bonner, Plant Physiol.,42 (1967) 762. Y. Fujitaand A. Hattori,Plant CellPhysiol.,1 (1960) 293. N. Lazaroff, Photomorphogenesis and nostocacean development, in N.G. Cart and B.A. Whitton (Eds.),The Biology of Blue-green Algae, Blackwell,Oxford, 1973, pp. 279--319. S. Diakoff and J. Scheibe, Plant Physiol.,51 (1973) 382. S. Diakoff and J. Scheibe, Physiol.Plant.,34 (1975) 125. J.C.O'Kelly,J.P. Durant and D.L. Stockdale,Photochem. Photobiol.,20 (1974) 47. J. Scheibe, Science, 176 (1972) 1037. G.S. BjSrn and L.O. BjSrn, Physiol.Plant.,36 (1976) 297. J.P.Thomas and J.C. OTLelly, Photochem. Photobiol.,17 (1973) 469. J.P.Thomas and J.C. O'Kelly,J.K. Hardman and E.F. Aldridge,Photochem. Photobiol., 22 (1975) 135. H.H. van der Velde, The growth of Acrochaetium under red, white and blue light, Thesis Vrije Universiteit, Amsterdam, 1977, pp. 1--160. H.H. van der Velde, P. Guiking and D. van der Wulp, Z. Pflanzenphysiol., 76 (1975) 95. G. Gardner, C.S. Pike, H.V. Rice and W.R. Briggs, Plant Physiol., 48 (1971) 686. J.A. West, J. Phycol., 4 (1968) 89. H. Smith, Phytochrome and Photomorphogenesis, McGraw-Hill, London, 1975, pp. 54--85. T. Lewing, The vegetation in the sea, in T. Lewing, H.A. Hoppe and O.J. Schmid (eds.), Marine Algae. A Survey of Research and Utilization, Cram, De Gruyter & Co, Hamburg, 1969, pp. 1--46. M.J. Dring, Light quality and the photomorphogenesis of algae in marine environments, in D.J. Crisp (Ed.), Fourth European Marine Biology symposium, Cambridge University Press, Cambridge, 1971, pp. 375--392.