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On the localization and orientation of phytochrome molecules in corn coleoptiles (Zea mays L.)
Institute fur Biologie II und III der Universitat Freiburg
On the Localization and Orientation of Phytochrome Molecules in Corn Coleoptiles (Zea mays L.) DIETER MARME and EBERHARD SCHAFER Received January 7, 1972
Summary Corn coleoptiles (Zea mays L.) were irradiated using linearly polarized red and far red light vibrating parallel and normal to the longitudinal axis. The amount of converted phytochrome was then measured spectrophotometric ally in vivo. Both red and far red light, when vibrating normal to the longitudinal axis, convert 15-20 ~/o more phytochrome than when vibrating parallel to this axis. The most probable explanation is: The phytochrome is located in or associated with the plasmalemma and orientated along a screw line with an angle of inclination of about 35°.
To know how phytochrome acts means in part to know where phytochrome is located in the cell. During the last 10 years considerable data have been accumulated suggesting that the pigment may be a part of or associated with large molecular structures. GORDON (1961), SIEGELMAN and FIRER (1964), RUBINSTEIN et al. (1969) using biochemical methods obtained pelletable phytochrome. Detailed analysis of the polarotropic response of Dryopteris by ETZOLD (1965) and HAUPT'S (1969) experiments with polarized microbeams on the chloroplast movement of Mougeotia demonstrate that phytochrome in these organisms is located and orientated in the outermost portion of the cytoplasm and is probably associated with the plasmalemma. PRATT (1971) obtained evidence using immunological techniques that some phytochrome in Avena sativa L. is associated with membranes. HENDRICKS and BORTHWICK (1967) have summarized a number of plant responses which indicate that phytochrome acts on membrane permeability as a first or early step in its control of plant growth and development. In this paper data will be presented which were obtained by spectrophotometric measurements in vivo of the phytochrome in maize coleoptiles after irradiation with linearly polarized red and far red light. 10 mm segments of six-day old dark grown coleoptiles were used. The tip, about 5 mm, was discarded. A single cell from these segments has an average length of about 100 fl, and an average width of about 30 fl,. The segments were cut with a razor blade parallel to the longitudinal axis into two halves to avoid light scattering, and thus depolarization, as much as possible. The coleoptile-halves were put between two glass plates and placed behind the polarizer. The irradiation was performed with a Leitz Prado 500 through Schott DIL interference filters with}. = 658 and 756 nm. The Prado light source emits partially polarized light; therefore a scatter plate was placed Z. Pjlanzenphysiol. Bd. 67. S. 192-194. 1972.
Orientation of Phytochrome
193
into the light beam to be sure that only completely depolarized light falls on the polarizer. The various directions of polarization could be obtained by rotating the polarizer. An analyser was used as control. To be sure that the same light dose with the same degree of polarization passes through the coleoptiles, the depolarization and the surface reflection of the incoming light were measured for the two directions of polarization. To test the dependence of the light reflection on the position of the coleoptile with respect to the electrical light vector, the total transmission at 658 and 756 nm was measured for these two normal directions of polarization. The monochromatic light of a Cary 14 was completely depolarized by introducing a scatter plate into the light beam. The polarizer was placed in front of the exit slit to which a coleoptile half was attached. The intensity of the transmitted light was the same for both directions of polarization, no selective reflection of the polarized light could be detected. To test the depolarization of the linearly polarized light by the tissue, a coleoptile half was put between polarizer and analyser. When the longitudinal axis of the coleoptile was parallel or normal to the electrical vector of the incoming light, the analyser remained dark but when the coleoptile was rotated by 45 0 the analyser became illuminated. These results show that depolarization for both directions of the incoming light used in the experiments could be neglected. From these data one can assume that the same light doses with the same degree of polarization for both directions of the electrical vector reach the phytochrome molecules in the coleoptile. The results are shown in the table. Table 1 The mean values of P fr and P, represented in this table, are arbitrary units for 18 coleoptiles with 12-18 parallel measurements. The standard deviation is about ± 3 % of each value. The red light dose of 2 X 104 erg/cm 2 and the far red light dose of 2 X 105 erg/cm 2 were given at 25° C. The measurements of P fr and P were performed at 0 0 C. light-dark red treatment normal
Pfr P
40 70
red red unparallel polarized
35 70
40 70
red normal +4 hdark
red parallel +4hdark
red normal + far red normal + 4 hdark
red normal + far red parallel +4 h dark
29
35
48
40
Spectrophotometric measurements of [Ph'] (see a, c below) immediately after irradiation or of [P] (b) after irradiation with polarized light plus 4 hours dark (allowing time for the destruction of Pfr) yield the following results. a) Polarized red light vibrating normal to the longitudinal axis of the coleoptile converts about 15 % more P r to P l r than the same light dose vibrating parallel to the longitudinal axis. b) Polarized red light vibrating parallel to the coleoptile followed by 4 hours dark yields about 20 (I/ o more total phytochrome than the corresponding experiment with normal vibrating light. Z. Pjlanzenphysiol. Bd. 67. S. 192-194. 1972.
194
D . MAR ME and E. SCHAFER
as light vibratin g C) The same dose of unpolar ized red light converts as much P r -+ Plr normal to the coleoptile. an angle of inFrom these data one can calculate for a simple screw type model, 0 the cell surface to clinatio n of about 35 with the assumption that all P r is parallel and that the dichroic ratio is 10. orientat ion after To test whethe r the phytochrome molecules change their dichroic d far red polarize with ed perform was ent experim an the absorpt ion of red light, to normal g vibratin light, red d polarize with ion irradiat an revertin g light following inal axis reverts the coleoptile axis. Far red light vibratin g normal to the longitud These data are light. red far g vibratin parallel than P to r about 20 Ofo more Plr back to the cell surface. not contrad ictionar y to a change of the dichroic orientat ion normal
Acknowledgement ns and for calculati on The authors are grateful to Prof. W. HAUPT for stimulat ing discussio ipt. This work was manuscr the reading for QUAIL P. Dr. and model, type of the screw 46). (SFB schaft. supporte d by the Deutsche Forschungsgemein
References ETZoLD, H.: Planta (Berl.) 64, 254 (1965). Buchman, Elsevier Publishi ng GORDON, S. A.: Progress in Photobio logy, edit.: Christen sen and Compan y 441 (1961). 88, 183 (1969). HAUPT, W., G. MORTEL and J. WINKELNKEMPER: Planta (Bed.) 58, 2125 (1967). HENDRICKS, S. B., and H. A. BORTHWICK: Proc. Nat!. Acad. Sci. (1971). 2431 68, Sci. Acad. Nat. Proc. H.: L. PRATT, 105 (1969). RUBINSTEIN, B., K. S. DRURY and R. B. PARK: Plant Physio!. 44, SIEGELMAN, H. W., and E. M. FIRER: Biochem istry 3, 418 (1964). III und II der Univers itat, DIETER MARME und EBERHARD SCHAFER, Institut flir Biologie D-78 Freiburg , Germany , Schanzlestra~e 9-11.
z.
Pjlanzen physiol. Bd. 67. S. 192-194 . 1972.
On the Localization and Orientation of Phytochrome Molecules in Corn Coleoptiles (Zea mays L.) DIETER MARME and EBERHARD SCHAFER Received January 7, 1972
Summary Corn coleoptiles (Zea mays L.) were irradiated using linearly polarized red and far red light vibrating parallel and normal to the longitudinal axis. The amount of converted phytochrome was then measured spectrophotometric ally in vivo. Both red and far red light, when vibrating normal to the longitudinal axis, convert 15-20 ~/o more phytochrome than when vibrating parallel to this axis. The most probable explanation is: The phytochrome is located in or associated with the plasmalemma and orientated along a screw line with an angle of inclination of about 35°.
To know how phytochrome acts means in part to know where phytochrome is located in the cell. During the last 10 years considerable data have been accumulated suggesting that the pigment may be a part of or associated with large molecular structures. GORDON (1961), SIEGELMAN and FIRER (1964), RUBINSTEIN et al. (1969) using biochemical methods obtained pelletable phytochrome. Detailed analysis of the polarotropic response of Dryopteris by ETZOLD (1965) and HAUPT'S (1969) experiments with polarized microbeams on the chloroplast movement of Mougeotia demonstrate that phytochrome in these organisms is located and orientated in the outermost portion of the cytoplasm and is probably associated with the plasmalemma. PRATT (1971) obtained evidence using immunological techniques that some phytochrome in Avena sativa L. is associated with membranes. HENDRICKS and BORTHWICK (1967) have summarized a number of plant responses which indicate that phytochrome acts on membrane permeability as a first or early step in its control of plant growth and development. In this paper data will be presented which were obtained by spectrophotometric measurements in vivo of the phytochrome in maize coleoptiles after irradiation with linearly polarized red and far red light. 10 mm segments of six-day old dark grown coleoptiles were used. The tip, about 5 mm, was discarded. A single cell from these segments has an average length of about 100 fl, and an average width of about 30 fl,. The segments were cut with a razor blade parallel to the longitudinal axis into two halves to avoid light scattering, and thus depolarization, as much as possible. The coleoptile-halves were put between two glass plates and placed behind the polarizer. The irradiation was performed with a Leitz Prado 500 through Schott DIL interference filters with}. = 658 and 756 nm. The Prado light source emits partially polarized light; therefore a scatter plate was placed Z. Pjlanzenphysiol. Bd. 67. S. 192-194. 1972.
Orientation of Phytochrome
193
into the light beam to be sure that only completely depolarized light falls on the polarizer. The various directions of polarization could be obtained by rotating the polarizer. An analyser was used as control. To be sure that the same light dose with the same degree of polarization passes through the coleoptiles, the depolarization and the surface reflection of the incoming light were measured for the two directions of polarization. To test the dependence of the light reflection on the position of the coleoptile with respect to the electrical light vector, the total transmission at 658 and 756 nm was measured for these two normal directions of polarization. The monochromatic light of a Cary 14 was completely depolarized by introducing a scatter plate into the light beam. The polarizer was placed in front of the exit slit to which a coleoptile half was attached. The intensity of the transmitted light was the same for both directions of polarization, no selective reflection of the polarized light could be detected. To test the depolarization of the linearly polarized light by the tissue, a coleoptile half was put between polarizer and analyser. When the longitudinal axis of the coleoptile was parallel or normal to the electrical vector of the incoming light, the analyser remained dark but when the coleoptile was rotated by 45 0 the analyser became illuminated. These results show that depolarization for both directions of the incoming light used in the experiments could be neglected. From these data one can assume that the same light doses with the same degree of polarization for both directions of the electrical vector reach the phytochrome molecules in the coleoptile. The results are shown in the table. Table 1 The mean values of P fr and P, represented in this table, are arbitrary units for 18 coleoptiles with 12-18 parallel measurements. The standard deviation is about ± 3 % of each value. The red light dose of 2 X 104 erg/cm 2 and the far red light dose of 2 X 105 erg/cm 2 were given at 25° C. The measurements of P fr and P were performed at 0 0 C. light-dark red treatment normal
Pfr P
40 70
red red unparallel polarized
35 70
40 70
red normal +4 hdark
red parallel +4hdark
red normal + far red normal + 4 hdark
red normal + far red parallel +4 h dark
29
35
48
40
Spectrophotometric measurements of [Ph'] (see a, c below) immediately after irradiation or of [P] (b) after irradiation with polarized light plus 4 hours dark (allowing time for the destruction of Pfr) yield the following results. a) Polarized red light vibrating normal to the longitudinal axis of the coleoptile converts about 15 % more P r to P l r than the same light dose vibrating parallel to the longitudinal axis. b) Polarized red light vibrating parallel to the coleoptile followed by 4 hours dark yields about 20 (I/ o more total phytochrome than the corresponding experiment with normal vibrating light. Z. Pjlanzenphysiol. Bd. 67. S. 192-194. 1972.
194
D . MAR ME and E. SCHAFER
as light vibratin g C) The same dose of unpolar ized red light converts as much P r -+ Plr normal to the coleoptile. an angle of inFrom these data one can calculate for a simple screw type model, 0 the cell surface to clinatio n of about 35 with the assumption that all P r is parallel and that the dichroic ratio is 10. orientat ion after To test whethe r the phytochrome molecules change their dichroic d far red polarize with ed perform was ent experim an the absorpt ion of red light, to normal g vibratin light, red d polarize with ion irradiat an revertin g light following inal axis reverts the coleoptile axis. Far red light vibratin g normal to the longitud These data are light. red far g vibratin parallel than P to r about 20 Ofo more Plr back to the cell surface. not contrad ictionar y to a change of the dichroic orientat ion normal
Acknowledgement ns and for calculati on The authors are grateful to Prof. W. HAUPT for stimulat ing discussio ipt. This work was manuscr the reading for QUAIL P. Dr. and model, type of the screw 46). (SFB schaft. supporte d by the Deutsche Forschungsgemein
References ETZoLD, H.: Planta (Berl.) 64, 254 (1965). Buchman, Elsevier Publishi ng GORDON, S. A.: Progress in Photobio logy, edit.: Christen sen and Compan y 441 (1961). 88, 183 (1969). HAUPT, W., G. MORTEL and J. WINKELNKEMPER: Planta (Bed.) 58, 2125 (1967). HENDRICKS, S. B., and H. A. BORTHWICK: Proc. Nat!. Acad. Sci. (1971). 2431 68, Sci. Acad. Nat. Proc. H.: L. PRATT, 105 (1969). RUBINSTEIN, B., K. S. DRURY and R. B. PARK: Plant Physio!. 44, SIEGELMAN, H. W., and E. M. FIRER: Biochem istry 3, 418 (1964). III und II der Univers itat, DIETER MARME und EBERHARD SCHAFER, Institut flir Biologie D-78 Freiburg , Germany , Schanzlestra~e 9-11.
z.
Pjlanzen physiol. Bd. 67. S. 192-194 . 1972.