Planta 9 by Springer-Verlag 1978
Planta 14l, 273-277 (1978)
Destruction and Possible de Novo Synthesis of Phytochrome in Subcellular Fractions of Laminae from Arena sativa L. S. Grombein 1, W. Rfidiger 1., and R. H a m p p 2 1 Botanisches Institut, Universitfit Mtinchen, Menzinger StraBe 67, D-8000 Miinchen 19, and 2 Institut ftir Botanik, Technische Universitfit M/inchen, Arcisstrage 21, D-8000 Mtinchen 2, Federal Republic of Germany
Abstract. Phytochrome was determined in etiolated laminae o f A r e n a s a t i v a L , either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0-24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reached a minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids. Key words: A r e n a Phytochrome.
-
Etioplasts - Mitochondria -
Introduction
Whereas the bulk of phytochrome in dark-grown plants is found in the cytoplasm (soluble phytochrome) and is found to be bound to as yet unidentified particulate fractions only after irradiation (Coleman and Pratt, 1974; Mackenzie etal., 1975; Marm~, *
To whom reprint requests should be addressed
1977), small but measurable amounts of phytochrome are found in mitochondria (Manabe and Furuya, 1975) and plastids (Evans and Smith, 1976; Cooke and Kendrick, 1976). The specific activity of phytochrome ( A A A per mg of protein) is smaller in these organelles than in the cytoplasm. An unspecific adsorption of phytochrome onto these organelles could therefore not entirely be ruled out. Evidence against such an unspecific adsorption is provided by the phytochrome-mediated functional changes found in plastids (Cooke and Kendrick, 1976; H a m p p and Schmidt, 1977) and in mitochondria (Manabe and Furuya, 1974; H a m p p and Schmidt, 1977), as well as by binding experiments with 125I-labeled phytochrome to mitochondria (Georgevich et al., 1977). Phytochrome destruction (operationally defined as the disappearance of assayable photoreversibility) has been observed only in intact plants. In grass seedlings, a lag phase of destruction is often observed (Kidd and Pratt, 1973; Schfifer et al., 1975; Guthoff, 1978). Dicotyledons evidence an apparent resynthesis of phytochrome when returned to the dark (Clarkson and Hillman, 1967; Marm~, 1969; Quail et al., 1973a, b). No such reaccumulation of phytochrome in the dark could be observed in grass seedlings (Guthoff, 1978). We describe here a reinvestigation of phytochrome destruction in A r e n a laminae under conditions of apparent resynthesis. The determination of phytochrome in isolated mitochondria and plastids revealed differences between these organelles which shed new light upon the problem of the subcellular localization of phytochrome.
Materials and Methods
Abbreviations: Ptot=total phytochrome; Pr=red absorbing form
of phytochrome; Pfr=far-red absorbing form of phytochrome; ER =endoplasmic reticulum
Oat seedlings (Arena sativa L., var. Arnold) were grown in moist vermiculite at 27~ and high humidity for 6 days in the dark.
0032-0935/78/0141/02;73/$01.00
274 About 50 g of the upper 4 crn of the laminae were harvested in the dark either without pretreatment or after 5 rnin of red irradiation of the intact laminae (Mohr et al., 1964) followed by a dark period of 0, 2, 4, 8 or 24 h. This procedure is usually employed for the investigation of apparent phytochrome resynthesis, and it also prevents the accumulation of large amount of chlorophyll that would interfere with the measurement of phytochrome. The complete phototransformation of phytochrome to Pfr was monitored spectrophotometrically. The procedure for the isolation of mitochondria and etioplasts in a medium containing 0,35 mol 1 ~ sorbitol; 0.001 mol 1 1 MgC12; 0.00l moll -5 KH2PO4; 0.00l moll ~ NaNO~; 0.2% (mass/vol.) bovine serum albumin, Sigma type V, in 0.05 m o l l -5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES), finally adjusted to pH 7.3, has been described previously (Hampp and Wellburn, 1976a, b). Contamination of the etioplast- and mitochondria-enriched fractions was routinely checked by measuring the rates of activity of several marker enzymes: cytochrome-c-oxidase (EC 1.9.3.1, Tolbert et al., 1968), cytochrome-c-oxidase (antimycin A insensitive; Bonnet, 1974), ribulose-l,5-bisphosphate carboxylase (EC 4.1.1.39, Lorimer et al., 1977), and catalase (EC 1.11.1.6, Miflin and Beevers, 1974). With respect to the total homogenate, contamination of the etioplast fraction was considerably below 1% for both cytochrome-c-oxidase and catalase, and that of the mitochondriaenriched fraction about 2% for cataIase and below 1% for RuDPcarboxylase. The level of activity of antimycin A insensitive cytochrome-c-oxidase, an ER marker, was about 1% for b o t h fractions. The amount of phytochrome was determined with a custombuilt dual wavelength spectrophotometer in 3-mm path length cuvettes, using samples prepared with CaCO~ as a scattering agent to eliminate effects of inherent scattering differences within the samples. The method is described in detail by Pratt and Marm6 (1976) and Guthoff (1978). Photoreversibility was measured between 658.6 and 730.5 nm (interference filters, half width 4.9 and 5.0 nm, resp.). Actinic light was obtained from a slide projector unit equipped with interference filters 661.1 nm (half width 12.8 nm) and 729.0 nm (14.0 nm). All filters are of type MDEDDO-VIS, Seavom/France. One unit of phytochrome per ml measured according to this method equals a A (A A) of 5 • 10-5 (clear solution, [0 mm light path). Total homogenate phytochrome and phytochrome associated with the organelles is expressed in terms of these units. Units of phytochrome are related to the total content of phytochrome of the homogenate, yielding the relative content of phytochrome associated with the organelle fractions. Specific activity is expressed as phytochrome concentration (units/ml) per protein concentration (mg/ml). Protein was determined according to Lowry et al. (1951).
S: Grombein et at. : Destruction and Synthesis of Phytochrome
20-rc =
16~ 12-
, o
81
~_ ~" ,.6 5 rain R
=
o--IF
o
.
o
;.
8
li
:;o
dQrk p~riod [h] Fig. 1. Destruction and apparent resynthesis of phytochrome in various fractions; x - - • total homogenate, e - - e - - e plastid fraction, 9 1 6 9 mitochondrial fraction. The values given are representative for 3-4 independent experiments; during the different times of dark treatments, following a red light pulse, there was no significant change in the protein/plastid ratio (about 106 plastids per mg protein)
25
~ 20
.l::
'~ 15 ,, ~ 10
~- 5 5 rain R
D "~0
4
8
12 16 20 24 do.rk period [hi Fig. 2. Phytochrome content of the plastid fraction; ( e - - e - - e ) and the mitochondrial fraction; ( o - - o - - o ) in per cent of the total homogenate
Results
Figure 1 gives the specific activity of phytochrome associated with the total homogenate and with the plastid and the mitochondrial fractions in relation to the different treatments of intact tissue. The amounts within the fractions obtained from etiolated tissue are compared to those measured after 5 min of red irradiation, followed by a dark period of up to 24 h. After 5 min of irradiation with red light, an enhanced specific activity was detected in all three samples when compared with the etiolated stage. This increase in
specific activity was most pronounced for the mitochondrial fraction ( + 140%), and amounted to about 60% for the etioplasts. Additionally, a slight increase in the specific activity of the total homogenate (about 10%) was also determined. In the dark period following red irradiation, phytochrome destruction occurred in all samples, leading to a decrease in the specific activity of the phytochrome. In contrast to the homogenate and the mitochondrial fraction, which showed a continuous decrease in phytochrome activity to levels far below the etio-
S. Grombein et al. : Destruction and Synthesis of Phytochrome
lated level with increasing time of darkness, the plastid fraction phytochrome decreased to a minimum, which was followed by an increase in the amount of associated phytochrome ("apparent resynthesis"). This increase was most pronounced between 4 and 8 h after red irradiation. After 24 h, a level even higher than the dark level was observed. In Figure 2 the amounts of phytochrome identified within the particulate fractions are presented as per cent values of the total homogenate phytochrome. A comparison of the values obtained with organelles from etiolated tissue with those from tissue treated with a pulse of red light shows an enrichment of phytochrome in both particulate fractions. This increase was nearly threefold for the plastids and about twofold for the mitochondria. The different behavior of the two fractions during the subsequent dark period is clearly demonstrated with the per cent distribution data of the phytochrome. The amount of phytochrome associated with mitochondria showed a slightly more intense decrease than that associated with the homogenate; in c o n t r a s t - a f t e r a lag-phase of about 2 h - t h e plastids exhibited a significant increase of associated phytochrome, This is reflected by a dramatic relative increase of the plastid phytochrome from 2.5% to about 23% of the total homogenate phytochrome (Fig. 2). However, there was also an absolute increase of plastid phytochrome with respect to the quantity of seedlings extracted (g fresh weight), which was in contrast to the case with the total homogenate and the mitochondrial fractions (values not shown here).
Discussion
The present results show an association of phytochrome with the purified fractions of etioplasts and mitochondria (for purity see Materials and Methods). Both fractions together yield about 4.5% of Ptot in the homogenate from etiolated laminae (Fig. 2). This value corresponds to values reported earlier, e.g. 0.6-1.5% of P~o~in barley etioplasts (Evans and Smith, 1976) and 2-3% of Ptot in pea mitochondria (Manabe and Furuya, 1975). Comparable values have been obtained for pelletable phytochrome from etiolated seedlings, i.e. 5-10% of Ptot in the 20,000-45,000 g pellet in the dark and in the absence of Mg 2+ (Grombein et al., 1975; Pratt and Marm~, 1976; Fuad and Yu, 1977; Jose, 1977). An additional amount of about 5% of Pto~ is found in the microsomal fraction (Manabe and Furuya, 1975). However, contamination with this fraction (ER
275
and microbody marker enzymes tested) has been excluded in our preparations. The specific activity per mg protein in purified barley etioplasts was about 25 35% of that of the crude homogenate (Evans and Smith, 1976). In terms of specific activity, comparable results were obtained with oat etioplasts isolated according to a similar technique ( ~ 4 0 % ; Fig. 1). An increase of Pto~ after red irradiation (Fig. 1) was reported earlier (Schfifer etal., 1975; Guthoff, 1978) but is not yet understood. A possible explanation could be a light-induced transformation of spectrophotometrically undetectable precursors into photoreversible phytochrome, The change in distribution after red irradiation (Fig. 2) signifies an enhancement of phytochrome association with particulate fractions as a result of red light treatment. This enhancement should not be confused with the enhanced pelletability of the bulk phytochrome observed by extraction with magnesiumcontaining buffers (Quail et al., 1973a; Boisard et al., 1974; Quail, 1975; Grombein et al., 1975; Pratt and Marm6, 1976), but it is comparable to the small increase in phytochrome pelletability after red irradiation in the absence of magnesium (Grombein et al., 1975; Pratt and Marm6, 1976). Few attempts have been made to identify the phytochrome-associating components, or to quantify the amount of phytochrome bound to defined subcellular fractions after irradiation with red light. The results presented here show that the relative increase in particle association of the etioplast fraction is twice that of the mitochondrial fraction (Fig. 2). When the values are given in terms of A (AA) x mg -1 protein, the increase is more pronounced with mitochondria. In any event, both fractions exhibit a significant red light-induced association of phytochrome, For mitochondria a similar increase in pigment was observed by Manabe and Furuya (1975), but only during a dark period of incubation in a medium inhibiting the degradation of Pfr following a brief exposure of segments to red light. To the best of our knowledge, no comparable results have been obtained with plastids. Georgevich et al. (1977) demonstrated that specific binding of ~25I-labeled Pfr tO mitochondria is saturated at 0.1 ng phytochrome/~tg mitochondrial protein. This value is in the same order of magnitude as our value after red irradiation. The specificity was demonstrated by competitive inhibition of 12SI-Pfr binding by unlabeled Pfr (and not by other proteins). During the first two hours of darkness after R-irradiation phytochrome destruction occurred in the homogenate as well as in the plastid and mitochondrial fractions. One cannot conclude from these results that
276
destruction occurs at the cell organelles themselves because a slow release of phytochrome from the organelles is also possible. Although a slow release of the bulk pelletable phytochrome from the particulate fraction has only been observed after re-transformation to Pr (Quail et al., 1973c; Yamamoto and Furuya, 1975; Pratt and Marm6, 1976; Marm6, 1977) (which was not the case in our experiments), such a possibility cannot be excluded at present for phytochrome in the plastid and mitochondrial fractions. Whereas the total homogenate and the mitochondrial fraction showed a further net destruction of phytochrome after 2 h in the dark, the plastid phytochrome slowly increased and reached the level of the dark control after an 8 h dark period. If this increase is to be explained as a redistribution of the cytoplasmic phytochrome, drastic changes must be assumed respective of the plastid affinity for phytochrome: from 0 to 2 h a decrease, from 2 to 24 h a manyfold increase of affinity (see Fig. 2). Another, more plausible explanation is considered here: the newly appearing phytochrome could signify de novo synthesis of phytochrome within plastids. Although the amount of phytochrome detectable in this fraction is small, it is far from being insignificant in relation to the possible reactions it can control (Evans and Smith, 1976). Therefore, the increase of plastid-associated phytochrome could indicate a unique function of plastids in light-mediated responses-especially in the green p l a n t - a n d will be subject to further investigation. The absence of correlation in the behavior of etioplast and mitochondrial fractions gives additional support to our assumption that we are indeed dealing. with phytochrome specifically bound to these organelles and confirms the suggestion of several authors that this proposed association of phytochrome with mitochondria and etioplasts is biologically meaningful (Manabe and Furuya, 1974; Cooke et al., 1975; Hampp and Schmidt, 1977; Schmidt and Hampp, 1977). The skillful technical assistance of Mrs. Schoy-Tribbensee and Mr. Harzer is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.
References Boisard, J., Marm+, D., Briggs, W.R. : In vivo properties of membrane-bound phytochrome. Plant Physiol. 54, 272-276 (1974) Bonner, W.D.Jr.: Plant mitochondria. In: Experimental plant physiol., San Pietro, A. ed. pp. 125 133. Saint Louis: Mosby 1974 Clarkson, D.T., Hillman, W.S.: Apparent phytochrome synthesis in Pisum tissue. Nature 213, 468 470 (1967)
S. Grombein et al. : Destruction and Synthesis of Phytochrome Coleman, R.A., Pratt, L.H.: Subcellular localization of the redabsorbing form of phytochrome by immunocytochemistry. Planta 121, 119-131 (1974) Cooke, R.J., Saunders, P.F., Kendrick, R.E.: Red-light induced production of gibberellin-like substances in homogenates of etiolated wheat leaves and in suspensions of intact etioplasts. Planta 124, 319 328 (1975) Cooke, R.J., Kendrick, R.E. : Phytochrome controlled gibberellin metabolism in etioplast envelopes. Planta 131, 303-307 (1976) Evans, A., Smith, H. : Spectrophotometric evidence for the presence ofphytochrome in the envelope membranes of barley etioplasts. Nature 259, 323 325 (1976) Fuad, N., Yu, R. : Far red and blue light-induced binding of phytochrome to a subcellular fraction of maize coleoptiles. Z. Pflanzenphysiol. 81, 304 307 (1977) Georgevich, G., Cedel T.E., Roux S.J.: Use of 125I-labeled phytochrome to quantitate phytochrome binding to membranes of Arena sativa. Proc. Natl. Acad. Sci. USA 74, 4439-4443 (1977) Grombein, S., Rgdiger, W., Pratt, L., Marmb, D.: Phytochrome pelletability in extracts of Arena shoots. Plant Sci. Lett. 5, 275 280 (1975) Guthoff, Ch. : Untersuchungen zur Destruktion von Phytochrom. Doctoral Dissertation, UniversitS~t Mtinchen (1978) Hampp, R., Wellburn, A.R.: Changes in the permeability of the inner mitochondrial membrane associated with plastid development. Planta 131, 21 26 (1976a) Hampp, R., Wellburn, A.R.: Early changes in the envelope permeability of developing chloroplasts. J. exp. Bot. 27, 778-784 (1976b) Hampp, R., Schmidt, H.-W. : Regulation of membrane properties of mitochondria and plastids during chloroplast development. I. The action of phytochrome in situ. Z. Pflanzenphysiol. 82, 68 77 (1977) Jose, A.M. : Gel Filtration of Particle-bound Phytochrome. Planta 134, 287-293 (1977) Kidd, G.H., Pratt, L.H.: Phytochrome destruction. An apparent requirement for protein synthesis in the induction of destruction mechanism. Plant Physiol. 52, 309 311 (1973) Lorimer, G.H., Badger, M.R., Andrews, T.J.: D-Ribulose-l.5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. Anal. Biochem. 78, 66 75 (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Mackenzie, J.M.Jr., Coleman, R.A., Briggs, W.R., Pratt, L.H.: Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form. Proc. Natl. Acad. Sci. USA 72, 799-803 (1975) Manabe, K., Furuya, M.: Phytochrome-dependent reduction of nicotinamide nucleotides in the mitochondrial fraction isolated from etiolated pea epicotyls. Plant Physiol. 53, 343-347 (1974) Manabe, K., Furuya, M. : Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots. Planta 123, 207-215 (1975) Marm+, D.: Photometrische Messungen am Phytochromsystem von Senfkeimlingeu (Sinapis alba L.). Planta 88, 43 57 (1969) Marm~, D. : Phytochrome : Membranes as possible sites of primary action. Ann. Rev. Plant Physiol. 28, 173-198 (1977) Miflin, B.J., Beevers, H. : Isolation of intact plastids from a range of plant tissues. Plant Physiol. 53, 870-874 (1974) Mohr, H., Meyer, U., Hartmann, K. : Die Beeinflussung der Farnsporen-Keimung (Osmunda cinnamomea L. und O. claytoniana L.) fiber das Phytochromsystem und die Photosynthese. Planta 61), 483496 (1964)
S. Grombein et al. : Destruction and Synthesis of Phytochrome Pratt, L.H., Marm~, D.: Red light-enhanced phytochrome pelletability: re-examination and further characterization. Plant Physiol. 58, 686-692 (1976) Quail, P.H. : Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L. Planta 123, 223-234 (1975) Quail, P.H., Schfifer, E., Marm6, D. : De Novo Synthesis of Phytochrome in Pumpkin Hooks. Plant Physiol. 52, 124-127 (1973a) Quail, P.H., SchS.fer, E., Marm~, D.: Turnover of Phytochrome in Pumpkin Cotyledons. Plant Physiol. g2, 128-131 (1973b) Quail, P.H., Marm~, D., Schfifer, E. : Particle bound phytochrome from maize and pumpkin. Nature New Biol. 245, 189 191 (1973c) Sch/ifer, E., Lassig, T.-U., Schopfer, P.: Photocontrol of phytochrome destruction in grass seedlings. The influence of wave-
277 length and irradiance. Photochem. Photobiol. 22, 193-202 (1975) Schmidt, H.-W., Hampp, R.: Regulation of membrane properties of mitochondria and plastids during chloroplast development. II. The action ofphytochrome in a cell-free system. Z. Pflanzenphysiol. 82, 428 434 (1977) Tolbert, N.E., Oeser, A., Kisaki, T., Hagemann, R.H., Yamazaki, R.K.: Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. Biol. Chem. 243, 5179-5184 (1968) Yamamoto, K.T., Furuya, M.: Photoreversible binding in vitro of cytosolic phytochrome to particulate fraction isolated from pea epicotyles. Planta 127~ 177-186 (1975) Received 8 March; accepted 9 May 1978
Planta 14l, 273-277 (1978)
Destruction and Possible de Novo Synthesis of Phytochrome in Subcellular Fractions of Laminae from Arena sativa L. S. Grombein 1, W. Rfidiger 1., and R. H a m p p 2 1 Botanisches Institut, Universitfit Mtinchen, Menzinger StraBe 67, D-8000 Miinchen 19, and 2 Institut ftir Botanik, Technische Universitfit M/inchen, Arcisstrage 21, D-8000 Mtinchen 2, Federal Republic of Germany
Abstract. Phytochrome was determined in etiolated laminae o f A r e n a s a t i v a L , either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0-24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reached a minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids. Key words: A r e n a Phytochrome.
-
Etioplasts - Mitochondria -
Introduction
Whereas the bulk of phytochrome in dark-grown plants is found in the cytoplasm (soluble phytochrome) and is found to be bound to as yet unidentified particulate fractions only after irradiation (Coleman and Pratt, 1974; Mackenzie etal., 1975; Marm~, *
To whom reprint requests should be addressed
1977), small but measurable amounts of phytochrome are found in mitochondria (Manabe and Furuya, 1975) and plastids (Evans and Smith, 1976; Cooke and Kendrick, 1976). The specific activity of phytochrome ( A A A per mg of protein) is smaller in these organelles than in the cytoplasm. An unspecific adsorption of phytochrome onto these organelles could therefore not entirely be ruled out. Evidence against such an unspecific adsorption is provided by the phytochrome-mediated functional changes found in plastids (Cooke and Kendrick, 1976; H a m p p and Schmidt, 1977) and in mitochondria (Manabe and Furuya, 1974; H a m p p and Schmidt, 1977), as well as by binding experiments with 125I-labeled phytochrome to mitochondria (Georgevich et al., 1977). Phytochrome destruction (operationally defined as the disappearance of assayable photoreversibility) has been observed only in intact plants. In grass seedlings, a lag phase of destruction is often observed (Kidd and Pratt, 1973; Schfifer et al., 1975; Guthoff, 1978). Dicotyledons evidence an apparent resynthesis of phytochrome when returned to the dark (Clarkson and Hillman, 1967; Marm~, 1969; Quail et al., 1973a, b). No such reaccumulation of phytochrome in the dark could be observed in grass seedlings (Guthoff, 1978). We describe here a reinvestigation of phytochrome destruction in A r e n a laminae under conditions of apparent resynthesis. The determination of phytochrome in isolated mitochondria and plastids revealed differences between these organelles which shed new light upon the problem of the subcellular localization of phytochrome.
Materials and Methods
Abbreviations: Ptot=total phytochrome; Pr=red absorbing form
of phytochrome; Pfr=far-red absorbing form of phytochrome; ER =endoplasmic reticulum
Oat seedlings (Arena sativa L., var. Arnold) were grown in moist vermiculite at 27~ and high humidity for 6 days in the dark.
0032-0935/78/0141/02;73/$01.00
274 About 50 g of the upper 4 crn of the laminae were harvested in the dark either without pretreatment or after 5 rnin of red irradiation of the intact laminae (Mohr et al., 1964) followed by a dark period of 0, 2, 4, 8 or 24 h. This procedure is usually employed for the investigation of apparent phytochrome resynthesis, and it also prevents the accumulation of large amount of chlorophyll that would interfere with the measurement of phytochrome. The complete phototransformation of phytochrome to Pfr was monitored spectrophotometrically. The procedure for the isolation of mitochondria and etioplasts in a medium containing 0,35 mol 1 ~ sorbitol; 0.001 mol 1 1 MgC12; 0.00l moll -5 KH2PO4; 0.00l moll ~ NaNO~; 0.2% (mass/vol.) bovine serum albumin, Sigma type V, in 0.05 m o l l -5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES), finally adjusted to pH 7.3, has been described previously (Hampp and Wellburn, 1976a, b). Contamination of the etioplast- and mitochondria-enriched fractions was routinely checked by measuring the rates of activity of several marker enzymes: cytochrome-c-oxidase (EC 1.9.3.1, Tolbert et al., 1968), cytochrome-c-oxidase (antimycin A insensitive; Bonnet, 1974), ribulose-l,5-bisphosphate carboxylase (EC 4.1.1.39, Lorimer et al., 1977), and catalase (EC 1.11.1.6, Miflin and Beevers, 1974). With respect to the total homogenate, contamination of the etioplast fraction was considerably below 1% for both cytochrome-c-oxidase and catalase, and that of the mitochondriaenriched fraction about 2% for cataIase and below 1% for RuDPcarboxylase. The level of activity of antimycin A insensitive cytochrome-c-oxidase, an ER marker, was about 1% for b o t h fractions. The amount of phytochrome was determined with a custombuilt dual wavelength spectrophotometer in 3-mm path length cuvettes, using samples prepared with CaCO~ as a scattering agent to eliminate effects of inherent scattering differences within the samples. The method is described in detail by Pratt and Marm6 (1976) and Guthoff (1978). Photoreversibility was measured between 658.6 and 730.5 nm (interference filters, half width 4.9 and 5.0 nm, resp.). Actinic light was obtained from a slide projector unit equipped with interference filters 661.1 nm (half width 12.8 nm) and 729.0 nm (14.0 nm). All filters are of type MDEDDO-VIS, Seavom/France. One unit of phytochrome per ml measured according to this method equals a A (A A) of 5 • 10-5 (clear solution, [0 mm light path). Total homogenate phytochrome and phytochrome associated with the organelles is expressed in terms of these units. Units of phytochrome are related to the total content of phytochrome of the homogenate, yielding the relative content of phytochrome associated with the organelle fractions. Specific activity is expressed as phytochrome concentration (units/ml) per protein concentration (mg/ml). Protein was determined according to Lowry et al. (1951).
S: Grombein et at. : Destruction and Synthesis of Phytochrome
20-rc =
16~ 12-
, o
81
~_ ~" ,.6 5 rain R
=
o--IF
o
.
o
;.
8
li
:;o
dQrk p~riod [h] Fig. 1. Destruction and apparent resynthesis of phytochrome in various fractions; x - - • total homogenate, e - - e - - e plastid fraction, 9 1 6 9 mitochondrial fraction. The values given are representative for 3-4 independent experiments; during the different times of dark treatments, following a red light pulse, there was no significant change in the protein/plastid ratio (about 106 plastids per mg protein)
25
~ 20
.l::
'~ 15 ,, ~ 10
~- 5 5 rain R
D "~0
4
8
12 16 20 24 do.rk period [hi Fig. 2. Phytochrome content of the plastid fraction; ( e - - e - - e ) and the mitochondrial fraction; ( o - - o - - o ) in per cent of the total homogenate
Results
Figure 1 gives the specific activity of phytochrome associated with the total homogenate and with the plastid and the mitochondrial fractions in relation to the different treatments of intact tissue. The amounts within the fractions obtained from etiolated tissue are compared to those measured after 5 min of red irradiation, followed by a dark period of up to 24 h. After 5 min of irradiation with red light, an enhanced specific activity was detected in all three samples when compared with the etiolated stage. This increase in
specific activity was most pronounced for the mitochondrial fraction ( + 140%), and amounted to about 60% for the etioplasts. Additionally, a slight increase in the specific activity of the total homogenate (about 10%) was also determined. In the dark period following red irradiation, phytochrome destruction occurred in all samples, leading to a decrease in the specific activity of the phytochrome. In contrast to the homogenate and the mitochondrial fraction, which showed a continuous decrease in phytochrome activity to levels far below the etio-
S. Grombein et al. : Destruction and Synthesis of Phytochrome
lated level with increasing time of darkness, the plastid fraction phytochrome decreased to a minimum, which was followed by an increase in the amount of associated phytochrome ("apparent resynthesis"). This increase was most pronounced between 4 and 8 h after red irradiation. After 24 h, a level even higher than the dark level was observed. In Figure 2 the amounts of phytochrome identified within the particulate fractions are presented as per cent values of the total homogenate phytochrome. A comparison of the values obtained with organelles from etiolated tissue with those from tissue treated with a pulse of red light shows an enrichment of phytochrome in both particulate fractions. This increase was nearly threefold for the plastids and about twofold for the mitochondria. The different behavior of the two fractions during the subsequent dark period is clearly demonstrated with the per cent distribution data of the phytochrome. The amount of phytochrome associated with mitochondria showed a slightly more intense decrease than that associated with the homogenate; in c o n t r a s t - a f t e r a lag-phase of about 2 h - t h e plastids exhibited a significant increase of associated phytochrome, This is reflected by a dramatic relative increase of the plastid phytochrome from 2.5% to about 23% of the total homogenate phytochrome (Fig. 2). However, there was also an absolute increase of plastid phytochrome with respect to the quantity of seedlings extracted (g fresh weight), which was in contrast to the case with the total homogenate and the mitochondrial fractions (values not shown here).
Discussion
The present results show an association of phytochrome with the purified fractions of etioplasts and mitochondria (for purity see Materials and Methods). Both fractions together yield about 4.5% of Ptot in the homogenate from etiolated laminae (Fig. 2). This value corresponds to values reported earlier, e.g. 0.6-1.5% of P~o~in barley etioplasts (Evans and Smith, 1976) and 2-3% of Ptot in pea mitochondria (Manabe and Furuya, 1975). Comparable values have been obtained for pelletable phytochrome from etiolated seedlings, i.e. 5-10% of Ptot in the 20,000-45,000 g pellet in the dark and in the absence of Mg 2+ (Grombein et al., 1975; Pratt and Marm~, 1976; Fuad and Yu, 1977; Jose, 1977). An additional amount of about 5% of Pto~ is found in the microsomal fraction (Manabe and Furuya, 1975). However, contamination with this fraction (ER
275
and microbody marker enzymes tested) has been excluded in our preparations. The specific activity per mg protein in purified barley etioplasts was about 25 35% of that of the crude homogenate (Evans and Smith, 1976). In terms of specific activity, comparable results were obtained with oat etioplasts isolated according to a similar technique ( ~ 4 0 % ; Fig. 1). An increase of Pto~ after red irradiation (Fig. 1) was reported earlier (Schfifer etal., 1975; Guthoff, 1978) but is not yet understood. A possible explanation could be a light-induced transformation of spectrophotometrically undetectable precursors into photoreversible phytochrome, The change in distribution after red irradiation (Fig. 2) signifies an enhancement of phytochrome association with particulate fractions as a result of red light treatment. This enhancement should not be confused with the enhanced pelletability of the bulk phytochrome observed by extraction with magnesiumcontaining buffers (Quail et al., 1973a; Boisard et al., 1974; Quail, 1975; Grombein et al., 1975; Pratt and Marm6, 1976), but it is comparable to the small increase in phytochrome pelletability after red irradiation in the absence of magnesium (Grombein et al., 1975; Pratt and Marm6, 1976). Few attempts have been made to identify the phytochrome-associating components, or to quantify the amount of phytochrome bound to defined subcellular fractions after irradiation with red light. The results presented here show that the relative increase in particle association of the etioplast fraction is twice that of the mitochondrial fraction (Fig. 2). When the values are given in terms of A (AA) x mg -1 protein, the increase is more pronounced with mitochondria. In any event, both fractions exhibit a significant red light-induced association of phytochrome, For mitochondria a similar increase in pigment was observed by Manabe and Furuya (1975), but only during a dark period of incubation in a medium inhibiting the degradation of Pfr following a brief exposure of segments to red light. To the best of our knowledge, no comparable results have been obtained with plastids. Georgevich et al. (1977) demonstrated that specific binding of ~25I-labeled Pfr tO mitochondria is saturated at 0.1 ng phytochrome/~tg mitochondrial protein. This value is in the same order of magnitude as our value after red irradiation. The specificity was demonstrated by competitive inhibition of 12SI-Pfr binding by unlabeled Pfr (and not by other proteins). During the first two hours of darkness after R-irradiation phytochrome destruction occurred in the homogenate as well as in the plastid and mitochondrial fractions. One cannot conclude from these results that
276
destruction occurs at the cell organelles themselves because a slow release of phytochrome from the organelles is also possible. Although a slow release of the bulk pelletable phytochrome from the particulate fraction has only been observed after re-transformation to Pr (Quail et al., 1973c; Yamamoto and Furuya, 1975; Pratt and Marm6, 1976; Marm6, 1977) (which was not the case in our experiments), such a possibility cannot be excluded at present for phytochrome in the plastid and mitochondrial fractions. Whereas the total homogenate and the mitochondrial fraction showed a further net destruction of phytochrome after 2 h in the dark, the plastid phytochrome slowly increased and reached the level of the dark control after an 8 h dark period. If this increase is to be explained as a redistribution of the cytoplasmic phytochrome, drastic changes must be assumed respective of the plastid affinity for phytochrome: from 0 to 2 h a decrease, from 2 to 24 h a manyfold increase of affinity (see Fig. 2). Another, more plausible explanation is considered here: the newly appearing phytochrome could signify de novo synthesis of phytochrome within plastids. Although the amount of phytochrome detectable in this fraction is small, it is far from being insignificant in relation to the possible reactions it can control (Evans and Smith, 1976). Therefore, the increase of plastid-associated phytochrome could indicate a unique function of plastids in light-mediated responses-especially in the green p l a n t - a n d will be subject to further investigation. The absence of correlation in the behavior of etioplast and mitochondrial fractions gives additional support to our assumption that we are indeed dealing. with phytochrome specifically bound to these organelles and confirms the suggestion of several authors that this proposed association of phytochrome with mitochondria and etioplasts is biologically meaningful (Manabe and Furuya, 1974; Cooke et al., 1975; Hampp and Schmidt, 1977; Schmidt and Hampp, 1977). The skillful technical assistance of Mrs. Schoy-Tribbensee and Mr. Harzer is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft.
References Boisard, J., Marm+, D., Briggs, W.R. : In vivo properties of membrane-bound phytochrome. Plant Physiol. 54, 272-276 (1974) Bonner, W.D.Jr.: Plant mitochondria. In: Experimental plant physiol., San Pietro, A. ed. pp. 125 133. Saint Louis: Mosby 1974 Clarkson, D.T., Hillman, W.S.: Apparent phytochrome synthesis in Pisum tissue. Nature 213, 468 470 (1967)
S. Grombein et al. : Destruction and Synthesis of Phytochrome Coleman, R.A., Pratt, L.H.: Subcellular localization of the redabsorbing form of phytochrome by immunocytochemistry. Planta 121, 119-131 (1974) Cooke, R.J., Saunders, P.F., Kendrick, R.E.: Red-light induced production of gibberellin-like substances in homogenates of etiolated wheat leaves and in suspensions of intact etioplasts. Planta 124, 319 328 (1975) Cooke, R.J., Kendrick, R.E. : Phytochrome controlled gibberellin metabolism in etioplast envelopes. Planta 131, 303-307 (1976) Evans, A., Smith, H. : Spectrophotometric evidence for the presence ofphytochrome in the envelope membranes of barley etioplasts. Nature 259, 323 325 (1976) Fuad, N., Yu, R. : Far red and blue light-induced binding of phytochrome to a subcellular fraction of maize coleoptiles. Z. Pflanzenphysiol. 81, 304 307 (1977) Georgevich, G., Cedel T.E., Roux S.J.: Use of 125I-labeled phytochrome to quantitate phytochrome binding to membranes of Arena sativa. Proc. Natl. Acad. Sci. USA 74, 4439-4443 (1977) Grombein, S., Rgdiger, W., Pratt, L., Marmb, D.: Phytochrome pelletability in extracts of Arena shoots. Plant Sci. Lett. 5, 275 280 (1975) Guthoff, Ch. : Untersuchungen zur Destruktion von Phytochrom. Doctoral Dissertation, UniversitS~t Mtinchen (1978) Hampp, R., Wellburn, A.R.: Changes in the permeability of the inner mitochondrial membrane associated with plastid development. Planta 131, 21 26 (1976a) Hampp, R., Wellburn, A.R.: Early changes in the envelope permeability of developing chloroplasts. J. exp. Bot. 27, 778-784 (1976b) Hampp, R., Schmidt, H.-W. : Regulation of membrane properties of mitochondria and plastids during chloroplast development. I. The action of phytochrome in situ. Z. Pflanzenphysiol. 82, 68 77 (1977) Jose, A.M. : Gel Filtration of Particle-bound Phytochrome. Planta 134, 287-293 (1977) Kidd, G.H., Pratt, L.H.: Phytochrome destruction. An apparent requirement for protein synthesis in the induction of destruction mechanism. Plant Physiol. 52, 309 311 (1973) Lorimer, G.H., Badger, M.R., Andrews, T.J.: D-Ribulose-l.5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. Anal. Biochem. 78, 66 75 (1977) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Mackenzie, J.M.Jr., Coleman, R.A., Briggs, W.R., Pratt, L.H.: Reversible redistribution of phytochrome within the cell upon conversion to its physiologically active form. Proc. Natl. Acad. Sci. USA 72, 799-803 (1975) Manabe, K., Furuya, M.: Phytochrome-dependent reduction of nicotinamide nucleotides in the mitochondrial fraction isolated from etiolated pea epicotyls. Plant Physiol. 53, 343-347 (1974) Manabe, K., Furuya, M. : Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots. Planta 123, 207-215 (1975) Marm+, D.: Photometrische Messungen am Phytochromsystem von Senfkeimlingeu (Sinapis alba L.). Planta 88, 43 57 (1969) Marm~, D. : Phytochrome : Membranes as possible sites of primary action. Ann. Rev. Plant Physiol. 28, 173-198 (1977) Miflin, B.J., Beevers, H. : Isolation of intact plastids from a range of plant tissues. Plant Physiol. 53, 870-874 (1974) Mohr, H., Meyer, U., Hartmann, K. : Die Beeinflussung der Farnsporen-Keimung (Osmunda cinnamomea L. und O. claytoniana L.) fiber das Phytochromsystem und die Photosynthese. Planta 61), 483496 (1964)
S. Grombein et al. : Destruction and Synthesis of Phytochrome Pratt, L.H., Marm~, D.: Red light-enhanced phytochrome pelletability: re-examination and further characterization. Plant Physiol. 58, 686-692 (1976) Quail, P.H. : Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L. Planta 123, 223-234 (1975) Quail, P.H., Schfifer, E., Marm6, D. : De Novo Synthesis of Phytochrome in Pumpkin Hooks. Plant Physiol. 52, 124-127 (1973a) Quail, P.H., SchS.fer, E., Marm~, D.: Turnover of Phytochrome in Pumpkin Cotyledons. Plant Physiol. g2, 128-131 (1973b) Quail, P.H., Marm~, D., Schfifer, E. : Particle bound phytochrome from maize and pumpkin. Nature New Biol. 245, 189 191 (1973c) Sch/ifer, E., Lassig, T.-U., Schopfer, P.: Photocontrol of phytochrome destruction in grass seedlings. The influence of wave-
277 length and irradiance. Photochem. Photobiol. 22, 193-202 (1975) Schmidt, H.-W., Hampp, R.: Regulation of membrane properties of mitochondria and plastids during chloroplast development. II. The action ofphytochrome in a cell-free system. Z. Pflanzenphysiol. 82, 428 434 (1977) Tolbert, N.E., Oeser, A., Kisaki, T., Hagemann, R.H., Yamazaki, R.K.: Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. Biol. Chem. 243, 5179-5184 (1968) Yamamoto, K.T., Furuya, M.: Photoreversible binding in vitro of cytosolic phytochrome to particulate fraction isolated from pea epicotyles. Planta 127~ 177-186 (1975) Received 8 March; accepted 9 May 1978
