Planta (Berl.) 123, 207--215 (1975) 9 by Springer-Verlag 1975
Experimentally Induced Binding of Phytochrome to Mitochondrial and Microsomal Fractions in Etiolated Pea Shoots Katsushi Manabe Biological Institute, Faculty of Science, Nagoya University, Chikusa, Nagoya 464,Japan Masaki Furuya Botany Department, Faculty of Science, University of Tokyo, IIongo, Tokyo 113, Japan Received 10 December 1974; accepted 6 February 1975 Summary. A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0 ~ resulted in very rapid binding of b o t h P r and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The a m o u n t of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of P r a n d Pfr in the bound form was the same as t h a t in 12,000 • g supernatant. After a brief exposure of the segments to red light a t 0 ~ a n d a subsequent dark incubation a t 30 ~ in TristiC1 buffer containing dithiothreitol or EDTA, which b o t h inhibit Pfr decay, the contents of phytochrome in the mitoehondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed b y far-red light. The increase of the phytoehrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of A (AA) per nag protein.
Introduction The importance of phytochrome-induced changes in membrane properties has been emphasized b y many workers (see review by Briggs and Rice, 1972). However, little is known about relationship between photoreversible effects and membrane-bound phytochrome. Recently, Manabe and Furuya (1974) observed that, when isolated pea mitochondria were briefly exposed to 1% light, their ability to reduce exogenous 5~ADP was enhanced within a few minutes and this 1%-light effect was reversed by F1% light, and that photoreversible absorbance changes between 730 and 800 nm were spectrophotometrically detected in the purified mitochondria and their membrane fraction. Quail et al. (i973) demonstrated a light-dependent binding of phytochrome to particulate cell fractions of maize coleoptiles and pumpkin hypocotyl hooks and a reversible difference in the degree of binding of Pr and Pfr. Further, Marm6 et al. (1973) found that brief red irradiation of a buffer extract of squash (Curcurbita) seedlings resulted in a magnesium-dependent binding of Pfr to a particulate fraction. In a preliminary study with pea segments, we noticed that phytochrome binding to mitoehondrial and microsomal fractions occurred very rapidly after R and F R exposures when the segments were kept on ice, and that the phytochrome content in these particulate fractions gradually increased even at a physiological temperature provided the Pfl" decay in the tissue was prevented. Abbreviations: R = red, FI~ ~ far-red; Pr = red-absorbing form of phytochrome, Pfr = far-red-absorbing form of phytochrome. 15 Planta (Berl.), "Col. 123
208
Katsushi Manabe and Mas~ki Furuya
I n this p a p e r we describe some of the characteristics of this b i n d i n g of p h y t o chrome to m i t o c h o n d r i a l a n d microsomal fractions i n pea shoots, a n d consider the n a t u r e of p a r t i c l e - b o u n d p h y t o c h r o m e so far studied. Material and Methods Plant Material, Light 8ource8 and Irradiation. Seeds of Pisum sativum L. cv. Alaska, purchased from Watanabe Seed Co., Kogota, Miyagi, Japan, were germinated in a darkroom on vermiculite saturated with water at 26~ The apical 2-cm-long portions of the shoots were cut with scissors from 5-day-old etiolated seedlings under dim green safelight and were kept in the dark at ca. 0~ on crushed ice until used in the experiments. R light was provided by three 20-W fluorescent tubes (Toshiba FL 20 SW) behind a 3.2 mm thickness of red "Plexiglas" (Rohm and Haas, No. 2444). FR light was supplied by one 400-W incandescent lamp (Matsushita, Reflector lamp) behind ca. 10 cm of water and a 3.2-ram-thick black Plexiglas (l~ohm and Haas, V-58015). The prechilled segments were irradiated with 0.75 W m-2 R light for 3 rain and/or 15 W m-2 Ft~ light for 2 rain with crushed ice. I n some experiments, the segments were incubated immediately after irradiation in 10 mM Tris-ttC1 buffer, pH 7.2, with or without inhibitor for Pfr decay, for periods from 0 to 120 rain at 30~ with reciprocal shaking in darkness. Extraction and Fractionation Procedures. For preparation of particulate fractions, 10 g of shoot segments were placed on ice for 15 rain or longer, and were homogenized by grinding in a chilled mortar with pestle with 20 ml of 10 mM Tris.HC1 buffer (pH 7.2) containing 0.4 M sucrose and I mM dithiothreitol. After filtration through two layers of cheesecloth, the extract was adjusted to pH 7.2 by 0.5 IV[ Tris solution, centrifuged for 10 rain at 1,000• and the supernatant further centrifuged for 10 rain at 12,000 • g. The particle fraction was washed with 20 ml of the extraction buffer described above, and it was then centrifuged at 400 • g for 5 rain. The supernatant further centrifuged for 10 rain at 7,000• and the precipitate was resnspended in 1.75 ml of the same extraction buffer. This suspension was used as "mitochondrial fraction". These entire procedures were carried out in a cold room at 3~ under a dim green safelight. After preparation of the 12,000 • g precipitate the supernatant was further centrifuged at 105,000 • g for 30 rain using the Hitachi RP 65 rotor, and the 12,000 to 105,000 • g sedimentary particle fraction was suspended in 1.5 ml of the extraction buffer. This suspension is called the "microsomal fraction ". The amounts of protein in the samples were determined by the method of Lowry et al. (1951). Phytochrome Determination. Phytochrome was determined with a dual-wavelength difference spectrophotometer (Hitachi Ltd., Japan, model 261), as previously described (Pjon and Furuya, 1968). For the assay, the suspension of particle fractions was pipetted into a cylindrical aluminium cell with glass windows (10 mm in diameter, 16 mm path length) and the cell was m~intained at ca. 0~ with crushed ice before and during measurement. Each value in tables and figures of this paper represents an average of 2-6 samples from, at least, two separate experiments.
Results R a p i d Binding o / P h y t o c h r o m e to Mitochondrial and Mierosomal Fractions at L o w Temperature Apical 2-cm-long shoot segments k e p t on ice for at least 15 rain were exposed to a l t e r n a t i n g R a n d F R , a n d t h e p h y t o c h r o m e contents i n the three subcellular fractions isolated from the samples were d e t e r m i n e d to find a n y rapid changes of p h y t o c h r o m e d i s t r i b u t i o n a t ca. 0 ~ The results in T a b l e 1 show t h a t t h e 12,000 • s u p e r n a t a n t was n o t signifi c a n t l y affected b y the light t r e a t m e n t s while R i r r a d i a t i o n of the tissue caused a significant increase of t h e p h y t o c h r o m e c o n t e n t i n the m i t o c h o n d r i a l a n d
Particle-bound Phytochrome in Peas
209
Table 1. Effects of R and FR light on intraeellular distribution of phytochrome in pea shoot segments kept at 0 ~ R, 0.75 W m -2 red light for 3 rain; FR, 15 W m-2 far-red light for 2 min. Irradiation
Distribution
Phytochrome content (A(AA) • 106 mg-1 protein)
Phytochrome Protein (A (AA) • (mg/gfw a) 10a/gfwa)
Pr
Mitoehondrial 12000 x g sprit, b Microsomal
0.48 15.1 0.57
0.34 7.99 0.83
142 188 68
0
FR
Mitoehondrial 12000 X g spnt. b Microsomal
0.49 18.9 0.72
0.33 8.87 0.96
144 212 72
2 1 3
R
Mitochondrial 12000 x g spnt. b Microsomal
0.71 16.4 1.89
0.33 9.18 0.97
43 35 27
174 144 169
R-FR
Mitochondrial 12000 x 9 spnt. b Mierosomal
0.72 15.2 1.92
0.30 9.43 0.99
228 159 190
8 2 3
R-FR-R
Mitochondrial 12000 X g spnt. b Microsomal
0.87 16.9 2.0
0.35 9.04 0.99
45 36 28
205 151 173
Before exposure
Fraction
P~
1
0
To~l
142 189 68 146 213 75 217 179 196 236 161 193 250 187 201
a gfw = 1 g flesh weight tissue equivalent. b s p n t . = supernatant.
microsomal fractions. I r r a d i a t i o n with F R did n o t significantly affect t h e p h y t o chrome contents in the particle fractions. The R - i n d u c e d effect in t h e mitochondrial a n d microsomal fractions was n e i t h e r F R reversible, n o r c u m u l a t i v e i n successive irradiations with R light. The v e r y rapid increase of p h y t o c h r o m e cont e n t s in the particulate fractions a t 0 ~ is t e r m e d " r a p i d p h y t o c h r o m e b i n d i n g " . The degree of r a p i d b i n d i n g did n o t change for, a t least, 1 h after R i r r a d i a t i o n if t h e tissues were k e p t on ice.
Relationship between R-light Dosages and the Degree o / R a p i d Binding o / P r and P/r Prechilled segments of pea shoots were irradiated a t 0 ~ with various dosages of R light j u s t before extraction, a n d p h y t o e h r o m e contents a n d percent of Pfr formed were d e t e r m i n e d in the mitochondrial fraction a n d the 12,000 >
Experimentally Induced Binding of Phytochrome to Mitochondrial and Microsomal Fractions in Etiolated Pea Shoots Katsushi Manabe Biological Institute, Faculty of Science, Nagoya University, Chikusa, Nagoya 464,Japan Masaki Furuya Botany Department, Faculty of Science, University of Tokyo, IIongo, Tokyo 113, Japan Received 10 December 1974; accepted 6 February 1975 Summary. A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0 ~ resulted in very rapid binding of b o t h P r and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The a m o u n t of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of P r a n d Pfr in the bound form was the same as t h a t in 12,000 • g supernatant. After a brief exposure of the segments to red light a t 0 ~ a n d a subsequent dark incubation a t 30 ~ in TristiC1 buffer containing dithiothreitol or EDTA, which b o t h inhibit Pfr decay, the contents of phytochrome in the mitoehondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed b y far-red light. The increase of the phytoehrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of A (AA) per nag protein.
Introduction The importance of phytochrome-induced changes in membrane properties has been emphasized b y many workers (see review by Briggs and Rice, 1972). However, little is known about relationship between photoreversible effects and membrane-bound phytochrome. Recently, Manabe and Furuya (1974) observed that, when isolated pea mitochondria were briefly exposed to 1% light, their ability to reduce exogenous 5~ADP was enhanced within a few minutes and this 1%-light effect was reversed by F1% light, and that photoreversible absorbance changes between 730 and 800 nm were spectrophotometrically detected in the purified mitochondria and their membrane fraction. Quail et al. (i973) demonstrated a light-dependent binding of phytochrome to particulate cell fractions of maize coleoptiles and pumpkin hypocotyl hooks and a reversible difference in the degree of binding of Pr and Pfr. Further, Marm6 et al. (1973) found that brief red irradiation of a buffer extract of squash (Curcurbita) seedlings resulted in a magnesium-dependent binding of Pfr to a particulate fraction. In a preliminary study with pea segments, we noticed that phytochrome binding to mitoehondrial and microsomal fractions occurred very rapidly after R and F R exposures when the segments were kept on ice, and that the phytochrome content in these particulate fractions gradually increased even at a physiological temperature provided the Pfl" decay in the tissue was prevented. Abbreviations: R = red, FI~ ~ far-red; Pr = red-absorbing form of phytochrome, Pfr = far-red-absorbing form of phytochrome. 15 Planta (Berl.), "Col. 123
208
Katsushi Manabe and Mas~ki Furuya
I n this p a p e r we describe some of the characteristics of this b i n d i n g of p h y t o chrome to m i t o c h o n d r i a l a n d microsomal fractions i n pea shoots, a n d consider the n a t u r e of p a r t i c l e - b o u n d p h y t o c h r o m e so far studied. Material and Methods Plant Material, Light 8ource8 and Irradiation. Seeds of Pisum sativum L. cv. Alaska, purchased from Watanabe Seed Co., Kogota, Miyagi, Japan, were germinated in a darkroom on vermiculite saturated with water at 26~ The apical 2-cm-long portions of the shoots were cut with scissors from 5-day-old etiolated seedlings under dim green safelight and were kept in the dark at ca. 0~ on crushed ice until used in the experiments. R light was provided by three 20-W fluorescent tubes (Toshiba FL 20 SW) behind a 3.2 mm thickness of red "Plexiglas" (Rohm and Haas, No. 2444). FR light was supplied by one 400-W incandescent lamp (Matsushita, Reflector lamp) behind ca. 10 cm of water and a 3.2-ram-thick black Plexiglas (l~ohm and Haas, V-58015). The prechilled segments were irradiated with 0.75 W m-2 R light for 3 rain and/or 15 W m-2 Ft~ light for 2 rain with crushed ice. I n some experiments, the segments were incubated immediately after irradiation in 10 mM Tris-ttC1 buffer, pH 7.2, with or without inhibitor for Pfr decay, for periods from 0 to 120 rain at 30~ with reciprocal shaking in darkness. Extraction and Fractionation Procedures. For preparation of particulate fractions, 10 g of shoot segments were placed on ice for 15 rain or longer, and were homogenized by grinding in a chilled mortar with pestle with 20 ml of 10 mM Tris.HC1 buffer (pH 7.2) containing 0.4 M sucrose and I mM dithiothreitol. After filtration through two layers of cheesecloth, the extract was adjusted to pH 7.2 by 0.5 IV[ Tris solution, centrifuged for 10 rain at 1,000• and the supernatant further centrifuged for 10 rain at 12,000 • g. The particle fraction was washed with 20 ml of the extraction buffer described above, and it was then centrifuged at 400 • g for 5 rain. The supernatant further centrifuged for 10 rain at 7,000• and the precipitate was resnspended in 1.75 ml of the same extraction buffer. This suspension was used as "mitochondrial fraction". These entire procedures were carried out in a cold room at 3~ under a dim green safelight. After preparation of the 12,000 • g precipitate the supernatant was further centrifuged at 105,000 • g for 30 rain using the Hitachi RP 65 rotor, and the 12,000 to 105,000 • g sedimentary particle fraction was suspended in 1.5 ml of the extraction buffer. This suspension is called the "microsomal fraction ". The amounts of protein in the samples were determined by the method of Lowry et al. (1951). Phytochrome Determination. Phytochrome was determined with a dual-wavelength difference spectrophotometer (Hitachi Ltd., Japan, model 261), as previously described (Pjon and Furuya, 1968). For the assay, the suspension of particle fractions was pipetted into a cylindrical aluminium cell with glass windows (10 mm in diameter, 16 mm path length) and the cell was m~intained at ca. 0~ with crushed ice before and during measurement. Each value in tables and figures of this paper represents an average of 2-6 samples from, at least, two separate experiments.
Results R a p i d Binding o / P h y t o c h r o m e to Mitochondrial and Mierosomal Fractions at L o w Temperature Apical 2-cm-long shoot segments k e p t on ice for at least 15 rain were exposed to a l t e r n a t i n g R a n d F R , a n d t h e p h y t o c h r o m e contents i n the three subcellular fractions isolated from the samples were d e t e r m i n e d to find a n y rapid changes of p h y t o c h r o m e d i s t r i b u t i o n a t ca. 0 ~ The results in T a b l e 1 show t h a t t h e 12,000 • s u p e r n a t a n t was n o t signifi c a n t l y affected b y the light t r e a t m e n t s while R i r r a d i a t i o n of the tissue caused a significant increase of t h e p h y t o c h r o m e c o n t e n t i n the m i t o c h o n d r i a l a n d
Particle-bound Phytochrome in Peas
209
Table 1. Effects of R and FR light on intraeellular distribution of phytochrome in pea shoot segments kept at 0 ~ R, 0.75 W m -2 red light for 3 rain; FR, 15 W m-2 far-red light for 2 min. Irradiation
Distribution
Phytochrome content (A(AA) • 106 mg-1 protein)
Phytochrome Protein (A (AA) • (mg/gfw a) 10a/gfwa)
Pr
Mitoehondrial 12000 x g sprit, b Microsomal
0.48 15.1 0.57
0.34 7.99 0.83
142 188 68
0
FR
Mitoehondrial 12000 X g spnt. b Microsomal
0.49 18.9 0.72
0.33 8.87 0.96
144 212 72
2 1 3
R
Mitochondrial 12000 x g spnt. b Microsomal
0.71 16.4 1.89
0.33 9.18 0.97
43 35 27
174 144 169
R-FR
Mitochondrial 12000 x 9 spnt. b Mierosomal
0.72 15.2 1.92
0.30 9.43 0.99
228 159 190
8 2 3
R-FR-R
Mitochondrial 12000 X g spnt. b Microsomal
0.87 16.9 2.0
0.35 9.04 0.99
45 36 28
205 151 173
Before exposure
Fraction
P~
1
0
To~l
142 189 68 146 213 75 217 179 196 236 161 193 250 187 201
a gfw = 1 g flesh weight tissue equivalent. b s p n t . = supernatant.
microsomal fractions. I r r a d i a t i o n with F R did n o t significantly affect t h e p h y t o chrome contents in the particle fractions. The R - i n d u c e d effect in t h e mitochondrial a n d microsomal fractions was n e i t h e r F R reversible, n o r c u m u l a t i v e i n successive irradiations with R light. The v e r y rapid increase of p h y t o c h r o m e cont e n t s in the particulate fractions a t 0 ~ is t e r m e d " r a p i d p h y t o c h r o m e b i n d i n g " . The degree of r a p i d b i n d i n g did n o t change for, a t least, 1 h after R i r r a d i a t i o n if t h e tissues were k e p t on ice.
Relationship between R-light Dosages and the Degree o / R a p i d Binding o / P r and P/r Prechilled segments of pea shoots were irradiated a t 0 ~ with various dosages of R light j u s t before extraction, a n d p h y t o e h r o m e contents a n d percent of Pfr formed were d e t e r m i n e d in the mitochondrial fraction a n d the 12,000 >
