Planta
Plauta 133, 169- 177 (1977)
9 by Springer-Verlag 1977
Phytochrome and Phosphotungstate-chromate-positive Vesicles from Cucurbita pepo L. P.H. Quail and J.E. Hughes Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia
Abstract. The phosphotungstic
acid-chromic acid (PTA-CrO3) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from C u c u r b i t a hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTACrO3-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.
Key words: Cell fractionation - C u c u r b i t a - Histochemical staining - Phosphotungstate-chromate staining - Phytochrome - Plasma membrane.
Introduction
Two phytochrome-containing fractions are observed when 20,000 • g pellets from red-irradiated Cucurbita hypocotyl hooks are centrifuged on sucrose gradients. One, which sediments at 31 S, results from the electrostatic adsorption of the pigment to ribonucleoprotein (RNP) from degraded ribosomes and is almost certainly a preparative artifact [7, 18, 19, 24-26]. Phytochrome in the second ( = " h e a v y " ) fraction is PTA-CrO3=phosphotungstate-chromate, ribonucleoprotein Abbreviations."
RNP-
in some way associated with membranous material which has a mean buoyant density of 1.15 g cm-3 [7, 27]. Whether this association might also be artifactual remains undetermined but, in contrast to the 31 S fraction, it does not appear to involve RNP [7]. Polar head groups of phospholipids likewise appear unlikely to be directly involved in the association. Phospholipase C solubilises more than 80% of the [t4C]choline from the "heavy" fraction of prelabelled tissue without releasing the phytochrome [7]. By elimination, this leaves protein as the component most likely to be responsible for the binding. This notion is consistent with the observation that very low levels of deoxycholate release the pigment from the particulate fraction [7]. Attempts to identify the membrane species with which the pigment is associated in the "heavy" fraction thus far indicate that the bulk of the phytochrome is not associated with mitochondria, plastids or ribosome-free endoplasmic reticulum (ER) [7, 18, 27]. Of the remaining cellular membranes, the plasma membrane is, on the basis of accumulated physiological evidence, considered to be a likely site for direct phytochrome-membrane interaction [9]. Accordingly, a determination of the distribution of plasma-membrane vesicles relative to the phytochrome in particulate fractions is of considerable interest [18, 20, 40]. In this paper we report an attempt to use the putatively plasma-membrane-specific phosphotungstate-chromate (PTA-CrO3) staining procedure [12, 29] to identify and quantitate this membrane in sucrose gradient fractions by electron microscopy. During the course of this study it became apparent that, in addition to the plasma membrane, other components, including membranes of the prolamellar body, lipid droplets, and ribosomes, are also densely stained by the procedure. This has led us to question seriously the validity of the method for estimating plasma-membrane contents in isolated particulate
170
fractions, as has been the practice of many authors [10, 12, 13, 31, 34, 36]. Vesicles of plasma-membrane origin cannot be unequivocally identified as such in isolated fractions as long as there exist other membranes or membrane-associated elements that also stain with PTA-CrO3. For this reason the correlation we observe between phytochrome and PTA-CrO3positive material in sucrose-gradient fractions cannot be unambiguously interpreted as evidence of a phytochrome-plasma membrane association in this tissue.
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
discontinuous gradient consisting of steps of 4ml 50% (w/w) and 7.5ml each of 38.5%, 30%, 21% and 10% (w/w) sucrose in the same buffer, EDTA and mercaptoethanol concentrations (pH 7.0) as the resuspension medium. Centrifugation was for 4 h at 27,000 rpm in a Beckman SW 27 rotor. Fractions of 1 ml were collected and assayed for phytochrome, cytochrome-c oxidase, NADPH cytochrome-c reductase, protein and RNA as described below. Percent sucrose (w/w) in the gradient fractions was determined refractometrically.
Materials and Methods
Assays Plant Material Zucchini seedlings (Cucurbita pepo L. cv. Greyzini; Rumseys Seeds, Parramatta, N.S.W., Australia) were grown in the dark on moist paper towels, at 30 ~ C. Harvesting and all subsequent manipulations were performed under green safelight [22] until phytochrome measurements were complete. Hypocotylhook segments, 5 mm long, cut beneath the cotyledonary node of 3-day-old seedlings, were used as starting material.
Irradiation Procedures The excised hooks were irradiated at 25~ with a saturating (5 rain) dose of red light (660 nm, 3800 ergs cm-2 s-1) immediately after excision, using a Zeutschel M3 monochromator (Heinz Zeutschel, Ttibingen, Germany) and a Schott & Gen. (Mainz, Germany) D.I.L. (Doppel-Linienfilter) interference filter.
Phytochrome was measured at 0~ C with a modified [17] Ratiospect (Agricultural Speciality Co., Hyattsville, Md., USA) using CaCO3 as a scattering agent [3]. The measuring beams were 728 and 802 nm and the actinic beams 656 and 737 nm (Schott D.I.L. filters). All values were corrected for "quenching" of the A(AA) signal by sucrose [2l]. The mitochondrial marker enzyme, cytochrome-c oxidase, was measured by recording the rate of oxidation of reduced cytochrome c at 550nm according to Smith [32]. NADPH-dependent cytochrome-c reductase, an endoplasmic reticulum marker, was assayed according to Lord et al. [15] by following the rate of reduction of cytochrome c at 550nm in the presence of NADPH. The latter enzyme activity was not affected by 1 gM antimycin A. RNA was estimated from the UV absorbance of the acid-precipitable, KOH-soluble material in the fractions according to the procedure of Fleck and Munro [6]. Rat-liver RNA was used as a standard. Protein was determined by the method of Lowry et al. [16] using bovine serum albumin as a standard.
Extraction and Sucrose Gradient Centrifugation Electron Microscopy Prechilled tissue was homogenized for 15 s in an Ultra Turrax blender (Janke and Kunkel, Staufen/Br., Germany) at 6700 rpm in ice-cold extraction medium (pH 7.5) containing 35 mM N-morpholino-3-propansulfonic acid (MOPS), 250 mM sucrose, 3 mM EDTA and 14 mM 2-mercaptoethanol. A solution: tissue ratio of 3:1 (v/w) was used, giving a final homogenate pH of 7.0-7.1. The brei was squeezed through nylon cloth and pre-centrifuged at 500 x g for 10 rain. The resultant 500xg supernatant was centrifuged at 20,000 x g for 30 min. The 20,000 x g pellet was resuspended in 25 mM MOPS, 3 mM EDTA, 250 mM sucrose and 14 mM 2-mercaptoethanol pH 7.0 (standard resuspension buffer =SRB). A 3-ml aliquot (35 mg protein from 6 g tissue) was layered onto a
Visible bands were collected from the four lower-most gradient interfaces designated "mitochondria" (50%/38.5%), "heavy" phytochrome (38.5%/30%), "smooth E R " (30%/21%) and " 3 1 S " (21%/10%) for convenience. The bands were diluted 1:1 with SRB and pelleted at 100,000 x g for 1 h in a Beckman Ti 50 rotor. The pellets were fixed for 8 h in 2.5% glutaraldehyde in 25raM phosphate buffer, pH 7.0, and postfixed for 2h at 0~ in 2% OsO4 in the same buffer. For ease of further handling, the pellets were then stabilised by addition of a solution of 2.5% (w/v) bovine serum albumin followed by fixation with 2.5% glutaraldehyde for 30 min [2]. Excess glutaraldehyde
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO 3 Stain
was removed by washing and the pellets were then dehydrated through a graded acetone series and embedded in Spurr's resin [33]. Excised segments of hypocotyl-hook tissue for microscopy were fixed, post-fixed, dehydrated and embedded as described for the pellets (except for the bovine-serum-albumin stabilisation step). Sections were cut on a Reichert OMU3 ultramicrotome (Reichert Optische Werke, Vienna, Austria) and transferred to grids, Phosphotungstic acidchromic acid staining of the sections on the grids was according to the procedure of Roland et al. [29]. Briefly, the grids were placed in 1% (v/v) periodic acid for 30 rain; this was followed by thorough washing in 3-4 changes of distilled water. The grids were then transferred to 1% (v/v) phosphotungstic acid in 10% (v/v) chromic acid for 10 rain before further thorough washing in 3-4 changes of distilled water. All operations were performed at room temperature. The unstained control in Figure 4a was treated identically except that the grids remained in distilled water instead of being transferred to the PTA-CrO3 solution after the periodic-acid treatment. The sections were examined using a JEOL (Japanese Electronic Optics [Ltd.], Tokyo, Japan) 100B electron microscope. The proportion of membranes staining positively with PTA-CrO3 in each gradient fraction was determined morphometrically [37]. Interrupted line-grid transparent overlays were placed on electron micrographs of stained gradient fractions. Both the total number of line-membrane intersections and the number of linePTA-CrO3-positive-membrane intersections were scored. The latter was expressed as a percentage of the former, and considered to represent the proportion of the total membrane in each fraction reacting positively to the stain. Between 8 and 13 micrographs from each gradient fraction were scored and averaged to obtain the values reported. Some sections were stained with uranyl acetate and lead citrate [28] for comparison.
171
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~50
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" 40
Fig. l a-c. Distribution profiles of various components in a 20,000 x g pellet from red-irradiated Cucurbita hypocotyl hooks following centrifugation on a discontinuous sucrose gradient, (o--o Phytochrome; 9 9 cytochrome-e oxidase; , - - j , N A D P H cytocbrome-c rednctase; o~ 9 protein; A - - A R N A ; . . . % (w/w) sucrose
Results
Previous studies using linear sucrose gradients have shown that the bulk of the phytochrome in the socalled " h e a v y " fraction occupies a density intermediate between the mitochondrial and smooth ER markers [7, 27]. The discontinuous gradient shown in Figure 1 was designed, on this basis, to provide a fraction enriched in phytochrome-bearing components and minimally contaminated with mitochondria and ER. The interface fractions thus obtained w e r e - as already mentioned-nominally designated "mitochondria", "heavy" phytochrome, "smooth
E R " and "31 S". Each has been assessed :for PTACrO3-positive material in an attempt to determine the quantitative distribution of putative plasma membrane vesicles across the gradient. Electron micrographs of these fractions stained with PTA-CrO3 are shown in Figure 2, Mitochondria predominate in the densest band (Fig. 2a). The "heavy" phytochrome fraction contains smooth membrane vesicles with some mitochondria (Fig. 2b). The "smooth E R " band also consists of smooth-surfaced vesicles with no evidence of the presence of membrane-bound ribo-
172
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO 3 Stain
Table l. Distribution of various components on a discontinuous sucrose gradient following centrifugation Of a 20,000 x g pellet from red-irradiated Cucurbita hypocotyl hooks" Fraction
"Mitochondria" " H e a v y " phytochrome "Smooth E R " "31 S"
Amount of component under each peak u Phytochrome Cyt. c oxid.
Cyt. c red. c
RNA
Protein
(%)
(%)
(%)
(%)
(%)
18 52 18 12
75 25 0 0
4 20 70 6
14 18 23 45
23 28 29 20
PTA-CrO3-positive material as % of total membrane in each fraction d
16 26 13 6
" See Figure 1 for experimental details b Data computed from Figure 1 by integrating the amount of each component under a given peak as follows: "mitochondria", fractions 1-8; " h e a v y " phytochrome, fractions 9 15; " s m o o t h ER", fractions 16-22; "31S", fractions 23 36 c The cytochrome c reductase values here have been corrected for a blank value not subtracted from the values in Figure 1 a See Material and Methods for morphometric quantitation of membranes in gradient fractions
somal particles (Fig. 2c). This 1s consistent with the density of the ER marker (1.12 g cm -3) which indicates that the membranes are ribosome-free [15, 30, 38]. Barely visible amounts of material can be pelleted from the "31 S" interface. That material which can be obtained includes some small, smoothsurfaced membrane vesicles (Fig. 2d). Previous studies with [14C]choline indicate that only about 3% of the total membrane phospholipid on the gradient locates in the 31 S band [7]. Figure 2b illustrates the differential staining of some of the vesicles with PTA-CrO3. Quantitative estimates of the proportion of such vesicles in each gradient fraction, together with the relative distributions of other components, are presented in Table 1. An enrichment of both phytochrome and PTA-CrO3positive material is observed in the ~ heavy" fraction. This fraction contains 52% of the total pigment on the gradient but only 25 and 20%, respectively, of the cytochrome-c oxidase and reductase present. Furthermore, there is an apparently good correlation in the distribution of phytochrome and PTA-CrO3-positive vesicles across the separate gradient fractions (data not shown). Thus on the basis of the procedures and criteria employed in other reports, the present data might be interpreted to indicate a correlated distribution of phytochrome and "plasma membrane". We feel, however, that a closer examination of the reputed specificity of PTA-CrO3 for the plasma membrane casts doubt upon its reliability for unequivocally identifying and quantitating this membrane following tissue homogenisation. Figures 3 and 4 illustrate the argument. It is clear that in addition to the plasma membrane, prolamellar-body membranes (Fig. 3a), the contents of presumptive lipid droplets (Fig. 3b), and especially ribosomes (Fig. 3a, b) are densely
stained as a result of this treatment. Figure 4 indicates, by means of successive steps in the staining procedure carried out on serial sections, that the ribonucleoprotein (RNP) of the ribosomes is selectively stained over and above the intrinsic electron-density of the particles. It is clear, therefore, that non-plasmamembrane components also have the capacity to appear PTA-CrO3-positive in sub-cellular fractions from this tissue.
Discussion
We have attempted here by means of the widely used and putatively plasma-membrane-specific PTA-CrO3 cytochemical staining procedure [10, 12, 13, 29, 31, 34-36] to compare the distributions of phytochrome and plasma membrane on sucrose density gradients. Our data indicate a positive correlation in the distributions of the pigment and PTA-CrO3-positive vesicles (Table 1). Other observations, however, call into question the fundamental assumption upon which the quantitative validity of this procedure rests: namely, that only plasma membrane vesicles are preferentially stained in particulate fractions. Prolamellar-body membranes, presumptive lipid droplets, and ribosomes all stain positively with PTACrO 3 in situ in addition to the plasma membrane (Figs. 3, 4). The potential for erroneously scoring prolamellar body fragments as plasma-membrane vesicles in gradient fractions is obvious. The possibility also exists that the PTA-CrO3-positive material from lipid droplets might become associated with membranes other than the plasma membrane during or after homogenisation. Cross-exchange of lipids between membranes in vitro is well documented [14, 39]. The staining of ribosomal RNP by the PTA-CrO3
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
173
Fig. 2a-d. Electron micrographs of fractions recovered from the interfaces of a discontinuous sucrose gradient analogous to that shown in Figure 1, The fractions correspond to those nominally designated in Table 1 as "mflochondria" (a), "heavy" phytochrome (b), "smooth ER" (e) and "31 S" (d). Sections have been stained according to the PTA-CrO3 procedure, p=PTA-CrO3-positive vesicle. Bar=l gm; •
procedure has been m e n t i o n e d recently in passing [13] but without discussion o f the potential problems posed by this observation. The presence o f significant levels o f R N P in all the fractions u n d e r consideration here is well d o c u m e n t e d (Fig. 1; [7, 24, 26]. This
R N P is o f ribosomal origin but is partially degraded [2@ The absence f r o m the m e m b r a n e vesicles in the gradient fractions o f readily identifiable intact ribosomal particles (Fig. 2) is consistent with this observation and is evidence that the R N P present is in other than
174
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
Fig. 3a and b. Electron micrographs of intact Cucurbita hypocotyl-hook tissue stained according to the PTA-CrOa procedure, a illustrates staining of plasma membrane (pro), ribosomes (r) and prolamellar body membranes (pb) by this procedure. Bar: 0.5 lain; x 54,600. b depicts the staining of the contents of presumptive lipid droplets (s) (note absence of bounding membrane and appearance when stained with uranyl acetate-lead citrate; inset x 19,900) as well as of ribosomes (r) and plasmamembrane (pro). Bar: 0.5 ~tm; x 31,300
its native globular configuration. One pertinent possibility is that the R N P is adsorbed in an extended configuration to the surfaces of vesicle bilayers. Staining of this R N P might then generate PTA-CrO3-positive vesicles indistinguishable from true plasma m e m b r a n e vesicles (cf. Fig. 5 in [1]), This would lead to false positive scoring of PTA-CrO3-stained vesicles as plasm a - m e m b r a n e fragments. The ready adsorption of
ribosomal R N P to other proteinaceous cytoplasmic components is well documented [4, 5, 23, 25]. Affinity is higher for basic than for acidic proteins so that differential adsorption to vesicles of varied origin may be expected and could lead to selective staining of some profiles over others. The granularity and discontinuity of the stain around the profiles of individual vesicles c o m m o n l y observed (Fig. 2; [12, 29, 31, 36])
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
175
Fig. 4a and b. Electron micrographs of serial sections from intact Cucurbita hypocotyl-hook tissue, a Sections treated with periodic acid but not with PTA-CrO3; b Sections treated with periodic acid followed by staining with PTA-CrO3 (= normal PTA-CrO3 procedure). r ribosomes, pm plasma membrane. Bar: 0.5 ~tm, • 38,200 might also be consistent with the staining of membrane-bound RNP. Another report of the failure of the PTA-CrO3 procedure to give totally selective staining of the plasma m e m b r a n e has recently appeared [35]. Tonoplast and ER membranes were also found to be PTA-CrO3positive in the tissue used. The opportunities for falsely scoring PTA-CrO3-positive profiles as plasma m e m b r a n e are thus apparently abundant, This might explain the questionably high contents of " p l a s m a
m e m b r a n e " in particulate fractions reported by some authors using this technique. As much as 29% of the total homogenate m e m b r a n e complement has been claimed to be " p l a s m a m e m b r a n e " on this basis [361. We conclude that despite an apparent correlation in the distribution of phytochrome and PTA-CrO3positive m e m b r a n e vesicles from Cucurbita, the data do not necessarily constitute evidence of a phytochrome-plasma m e m b r a n e association. This is because
176
the PTA-CrO3 procedure cannot be considered to unequivocally identify plasma-membrane fragments once the architecture of the cell is disrupted by homogenisation. Moreover, we concur with Thorn et al. [35] in suggesting that this reservation might apply equally well to other studies purporting to provide evidence of plasma-membrane-associated components from plant tissue. The literature contains numerous reports that salt-stimulated ATPase [10], /~glucan synthetase [36], indoleacetic acid-[ll], N-1naphthylphthalamic acid-J12] and helminthosporoside-[34] binding activities, and phytochrome [20, 40] are all associated with plasma-membrane-rich fractions. A high sterol-to-phospholipid ratio has likewise been attributed to these fractions [8, 10]. Without exception, however, these claims are based either directly or indirectly on the use of the PTA-CrO3 stain for quantitating putative plasma membrane vesicles in the particulate fractions in question. It is suggested that, in the absence of confirmatory evidence from an independent means of monitoring plasma membrane in tissue homogenates, such conclusions should be considered tentative. Acknowledgements. The authors wish to thank Drs. B.E.S. Gunning and Th. Hendriks for helpful discussions, and Ms. E.A. Gallagher for competent technical assistance. Professor D.J. Cart provided research facilities.
References 1. Adelman, M.R., Blobel, G., Sabatini, D.D. : An improved fractionation procedure for the preparation of rat liver membranebound ribosomes. J. Cell Biol. 56, 191-205 (1973) 2. Atkinson, A.W., Gunning, B.E.S., John, P.C.L. : Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry, and incorporation of 14C-acetate studied in synchronous cultures. Planta (Bed.) 107, 1-32 (1972) 3. Butler, W.L., Norris, K.H.: The spectrophotometry of dense light-scattering material. Arch. Biochem. Biophys. 87, 31-40 (1960) 4. Dallner, G. : Isolation of rough and smooth microsomes- general. In: Methods in Enzymology, vol. XIII, Biomembranes, pt. A, p. 191-215, Fleischer, S., Packer, L., eds. New York: Acad. Press 1974 5.Das, N.K., Alfert, M.: Binding of labeled ribonucleic acid to basic proteins, a major difficulty in ribonucleic acid-deoxyribonucleic acid hybridisation in fixed cells in situ. J. Histochem. Cytochem. 17, 418-425 (1969) 6. Fleck, A., Munro, H.N. : The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta (Amst.) 55, 571 583 (1962) 7. Gressel, J., Quail, P.H.: Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C. Plant Cell Physiol. 17, 925-940 (1976) 8. Hartmann, M.A., Normand, G., Benveniste, P. : Sterol composition of plasma membrane enriched fraction from maize coleoptiles. Plant Sci. Lett. 5, 287-292 (1975)
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain 9. Haupt, W.: Localisation of phytochrome within the cell. In: Phytochrome, p. 553-569, Mitrakos, K., Shropshire, W., Jr., eds. New York: Acad. Press 1972 10. Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W.: Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membrane. Proc. nat. Acad. Sci. (Wash.)69, 3307-3311 (1972) l 1. Kasamo, K., Yamaki, T.: In vitro binding of IAA to plasma membrane-rich fractions containing Mg2+-activated ATPase from mung bean hypocytyls. Plant Cell Physiol. 17, 149-164 (1976) 12. Lembi, C.A., Morr6, D.J., Thomson, K.S., Hertel, R.: N-1Naphthylphthalamic-acid-binding activity of plasma membrane-rich fraction from maize coleoptiles. Planta (Berl.) 99, 37-45 (1971) 13. Leonard, R.T., Van der Woude, W.J.: Isolation of plasma membranes from corn roots by sucrose gradient centrifugation. Plant Physiol. 57, 105-114 (1976) 14. Lord, J.M.: Phospholipid synthesis and exchange in castor bean endosperm homogenates. Plant Physiol. 57, 218-223 (1976) 15. Lord, J.M., Kagawa, T., Moore, T.S,, Beevers, H.: Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J. Cell Biol. 57, 659--667 (1973) 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 17. Marm6, D. : An automatic recording device for measuring phytochrome with a dual wavelength ratiospect. Planta 88, 58-60 (1969) 18. Marm6, D. : Binding properties of the plant photoreceptor phytochrome to membranes. J. Supramol. Struct. 2, 751 768 (t974) 19. Marm6, D., Boisard, J., Briggs, W.R.: In vitro binding properties of phytochrome to a membrane fraction. Proc. nat. Acad. Sci. (Wash.) 70, 3861-3865 (1973) 20. Matin+, D., Bianco, J., Gross, J.: Evidence for phytochrome binding to plasma membrane and endoplasmic reticulum. In: Light and plant development, Smith, H., ed., in press. London: Butterworth (in press) 21. Marm6, D., Gross, J. : Remarks on photoreversibility as a measure for phytochrome. Book of Abstr., Ann. Eur. Photomorph. Syrup., Smith, H., ed., p. 88, 1975 22. Mohr, H., Appuhn, U. : Die Keimung yon Lactuca-Ach/inen unter dem Einflul3 des Phytochromsystems und der Hochenergiereaktion der Photomorphogenese. Planta (Bed.) 60, 274-288 (1963) 23. Petermann, M.L. : The physical and chemical properties of ribosomes. Amsterdam: Elsevier 1964 24. Quail, P.H.: Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L. Planta (Bed.), 123, 223-234 (1975) 25. Quail, P.H.: Particle-bound phytochrome: The nature of the interaction between pigment and particulate fractions. Planta (Berl.) 123, 235-246 (1975) 26. Quail, P.H., Gressel, J. : Particle-bound phytochrome: Interaction of the pigment with ribonucleoprotein material from Cueurbita pepo L. In: Light and Plant Development, Smith, H., ed., London Butterworth (in press) 27. Quail, P.H., Gallagher, E.A., Wellburn, A.R. : Membrane-associated phytochrome: Non-coincidence with plastid marker profiles on sucrose gradients. Photochem. Photobiol., in press 28. Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212 (1963) 29. Roland, J.C., Lembi, C.A., Morr6, D.J.: Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Tech. 47, 195-200 (1972)
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain 30. Sabatini, D.D., Tashiro, Y., Palade, G.E. : On the attachment of ribosomes to microsomal membranes. J. molec. Biol. 19, 503~-524 (1966) 31. Shore, G., Maclachlan, G.A.: The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkaliinsoluble fi-l,4-glucan (cellulose) synthetase activities. J. Cell. Biol. 64, 557-571 (1975) 32. Smith, L. : Cytochrome c oxidase. In: Methods in biochemical analysis, p. 427-434, Glick, D., ed. New York: Interscience 1955 33. Spurr, A.R. : A iow viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastr. Res. 26, 31-43 (1969) 34. Strobel, G.A., Hess, W.M. : Evidence for the presence of toxinbinding protein on the plasma membrane of sugar cane cells. Proc. nat. Acad. Sci. (Wash.) 7I, 1413 1417 (1974) 35. Thorn, M., Laetsch, W.M., Maretzki, A.: Isolation of membranes from sugar cane cell suspensions: Evidence for a plasma membrane enriched fraction. Plant Sci. Lett. 5, 245-253 (1975)
177 36. Van der Woude, W.J., Lembi, C.A., Morr6, D.J., Kindinger, J.I., Ordin, L. : fi-glucan synthetases of plasma membrane and golgi apparatus from onion stem. Plant Physiol. 54, 333 340 (1974) 37. Weibel, E.R. : Stereological principles for morphometry in electron microscope cytology. Internat. Rev. Cytol. 26, 235 302 (1969) 38. Wibo, M., Amar-Costesac, A., Berthet, J., Beaufay, H.: Electron microscope examination of subcellular fractions. J. Cell Biol. 51, 52-71 (1971) 39. Wirtz, K.W.A. : Transfer of phospholipids between membranes. Biochim. Biophys. Acta 344, 95-117 (1974) 40. Yu, R. : Characterisation of phytochrome-containing particles obtained by glutaraldehyde pre-fixation of maize coleoptiles. J. Exp. Bot. 26, 808 821 (1975)
Received 21 July; accepted 17 August 1976
Plauta 133, 169- 177 (1977)
9 by Springer-Verlag 1977
Phytochrome and Phosphotungstate-chromate-positive Vesicles from Cucurbita pepo L. P.H. Quail and J.E. Hughes Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2601, Australia
Abstract. The phosphotungstic
acid-chromic acid (PTA-CrO3) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from C u c u r b i t a hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTACrO3-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.
Key words: Cell fractionation - C u c u r b i t a - Histochemical staining - Phosphotungstate-chromate staining - Phytochrome - Plasma membrane.
Introduction
Two phytochrome-containing fractions are observed when 20,000 • g pellets from red-irradiated Cucurbita hypocotyl hooks are centrifuged on sucrose gradients. One, which sediments at 31 S, results from the electrostatic adsorption of the pigment to ribonucleoprotein (RNP) from degraded ribosomes and is almost certainly a preparative artifact [7, 18, 19, 24-26]. Phytochrome in the second ( = " h e a v y " ) fraction is PTA-CrO3=phosphotungstate-chromate, ribonucleoprotein Abbreviations."
RNP-
in some way associated with membranous material which has a mean buoyant density of 1.15 g cm-3 [7, 27]. Whether this association might also be artifactual remains undetermined but, in contrast to the 31 S fraction, it does not appear to involve RNP [7]. Polar head groups of phospholipids likewise appear unlikely to be directly involved in the association. Phospholipase C solubilises more than 80% of the [t4C]choline from the "heavy" fraction of prelabelled tissue without releasing the phytochrome [7]. By elimination, this leaves protein as the component most likely to be responsible for the binding. This notion is consistent with the observation that very low levels of deoxycholate release the pigment from the particulate fraction [7]. Attempts to identify the membrane species with which the pigment is associated in the "heavy" fraction thus far indicate that the bulk of the phytochrome is not associated with mitochondria, plastids or ribosome-free endoplasmic reticulum (ER) [7, 18, 27]. Of the remaining cellular membranes, the plasma membrane is, on the basis of accumulated physiological evidence, considered to be a likely site for direct phytochrome-membrane interaction [9]. Accordingly, a determination of the distribution of plasma-membrane vesicles relative to the phytochrome in particulate fractions is of considerable interest [18, 20, 40]. In this paper we report an attempt to use the putatively plasma-membrane-specific phosphotungstate-chromate (PTA-CrO3) staining procedure [12, 29] to identify and quantitate this membrane in sucrose gradient fractions by electron microscopy. During the course of this study it became apparent that, in addition to the plasma membrane, other components, including membranes of the prolamellar body, lipid droplets, and ribosomes, are also densely stained by the procedure. This has led us to question seriously the validity of the method for estimating plasma-membrane contents in isolated particulate
170
fractions, as has been the practice of many authors [10, 12, 13, 31, 34, 36]. Vesicles of plasma-membrane origin cannot be unequivocally identified as such in isolated fractions as long as there exist other membranes or membrane-associated elements that also stain with PTA-CrO3. For this reason the correlation we observe between phytochrome and PTA-CrO3positive material in sucrose-gradient fractions cannot be unambiguously interpreted as evidence of a phytochrome-plasma membrane association in this tissue.
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
discontinuous gradient consisting of steps of 4ml 50% (w/w) and 7.5ml each of 38.5%, 30%, 21% and 10% (w/w) sucrose in the same buffer, EDTA and mercaptoethanol concentrations (pH 7.0) as the resuspension medium. Centrifugation was for 4 h at 27,000 rpm in a Beckman SW 27 rotor. Fractions of 1 ml were collected and assayed for phytochrome, cytochrome-c oxidase, NADPH cytochrome-c reductase, protein and RNA as described below. Percent sucrose (w/w) in the gradient fractions was determined refractometrically.
Materials and Methods
Assays Plant Material Zucchini seedlings (Cucurbita pepo L. cv. Greyzini; Rumseys Seeds, Parramatta, N.S.W., Australia) were grown in the dark on moist paper towels, at 30 ~ C. Harvesting and all subsequent manipulations were performed under green safelight [22] until phytochrome measurements were complete. Hypocotylhook segments, 5 mm long, cut beneath the cotyledonary node of 3-day-old seedlings, were used as starting material.
Irradiation Procedures The excised hooks were irradiated at 25~ with a saturating (5 rain) dose of red light (660 nm, 3800 ergs cm-2 s-1) immediately after excision, using a Zeutschel M3 monochromator (Heinz Zeutschel, Ttibingen, Germany) and a Schott & Gen. (Mainz, Germany) D.I.L. (Doppel-Linienfilter) interference filter.
Phytochrome was measured at 0~ C with a modified [17] Ratiospect (Agricultural Speciality Co., Hyattsville, Md., USA) using CaCO3 as a scattering agent [3]. The measuring beams were 728 and 802 nm and the actinic beams 656 and 737 nm (Schott D.I.L. filters). All values were corrected for "quenching" of the A(AA) signal by sucrose [2l]. The mitochondrial marker enzyme, cytochrome-c oxidase, was measured by recording the rate of oxidation of reduced cytochrome c at 550nm according to Smith [32]. NADPH-dependent cytochrome-c reductase, an endoplasmic reticulum marker, was assayed according to Lord et al. [15] by following the rate of reduction of cytochrome c at 550nm in the presence of NADPH. The latter enzyme activity was not affected by 1 gM antimycin A. RNA was estimated from the UV absorbance of the acid-precipitable, KOH-soluble material in the fractions according to the procedure of Fleck and Munro [6]. Rat-liver RNA was used as a standard. Protein was determined by the method of Lowry et al. [16] using bovine serum albumin as a standard.
Extraction and Sucrose Gradient Centrifugation Electron Microscopy Prechilled tissue was homogenized for 15 s in an Ultra Turrax blender (Janke and Kunkel, Staufen/Br., Germany) at 6700 rpm in ice-cold extraction medium (pH 7.5) containing 35 mM N-morpholino-3-propansulfonic acid (MOPS), 250 mM sucrose, 3 mM EDTA and 14 mM 2-mercaptoethanol. A solution: tissue ratio of 3:1 (v/w) was used, giving a final homogenate pH of 7.0-7.1. The brei was squeezed through nylon cloth and pre-centrifuged at 500 x g for 10 rain. The resultant 500xg supernatant was centrifuged at 20,000 x g for 30 min. The 20,000 x g pellet was resuspended in 25 mM MOPS, 3 mM EDTA, 250 mM sucrose and 14 mM 2-mercaptoethanol pH 7.0 (standard resuspension buffer =SRB). A 3-ml aliquot (35 mg protein from 6 g tissue) was layered onto a
Visible bands were collected from the four lower-most gradient interfaces designated "mitochondria" (50%/38.5%), "heavy" phytochrome (38.5%/30%), "smooth E R " (30%/21%) and " 3 1 S " (21%/10%) for convenience. The bands were diluted 1:1 with SRB and pelleted at 100,000 x g for 1 h in a Beckman Ti 50 rotor. The pellets were fixed for 8 h in 2.5% glutaraldehyde in 25raM phosphate buffer, pH 7.0, and postfixed for 2h at 0~ in 2% OsO4 in the same buffer. For ease of further handling, the pellets were then stabilised by addition of a solution of 2.5% (w/v) bovine serum albumin followed by fixation with 2.5% glutaraldehyde for 30 min [2]. Excess glutaraldehyde
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO 3 Stain
was removed by washing and the pellets were then dehydrated through a graded acetone series and embedded in Spurr's resin [33]. Excised segments of hypocotyl-hook tissue for microscopy were fixed, post-fixed, dehydrated and embedded as described for the pellets (except for the bovine-serum-albumin stabilisation step). Sections were cut on a Reichert OMU3 ultramicrotome (Reichert Optische Werke, Vienna, Austria) and transferred to grids, Phosphotungstic acidchromic acid staining of the sections on the grids was according to the procedure of Roland et al. [29]. Briefly, the grids were placed in 1% (v/v) periodic acid for 30 rain; this was followed by thorough washing in 3-4 changes of distilled water. The grids were then transferred to 1% (v/v) phosphotungstic acid in 10% (v/v) chromic acid for 10 rain before further thorough washing in 3-4 changes of distilled water. All operations were performed at room temperature. The unstained control in Figure 4a was treated identically except that the grids remained in distilled water instead of being transferred to the PTA-CrO3 solution after the periodic-acid treatment. The sections were examined using a JEOL (Japanese Electronic Optics [Ltd.], Tokyo, Japan) 100B electron microscope. The proportion of membranes staining positively with PTA-CrO3 in each gradient fraction was determined morphometrically [37]. Interrupted line-grid transparent overlays were placed on electron micrographs of stained gradient fractions. Both the total number of line-membrane intersections and the number of linePTA-CrO3-positive-membrane intersections were scored. The latter was expressed as a percentage of the former, and considered to represent the proportion of the total membrane in each fraction reacting positively to the stain. Between 8 and 13 micrographs from each gradient fraction were scored and averaged to obtain the values reported. Some sections were stained with uranyl acetate and lead citrate [28] for comparison.
171
100.
'~,
"'"
~50
/l phytochrome
/
"-;-
"~
I". oo
Iol
401f/ ,
l,oo
g
... 9
2o
lo
I00~-
"~
,,
| ii cyt.c / L ~ 80t ii
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9
(b)
//
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protein & RNA
.
)
/
l/ cyt. c ]/ red"
]
~,o,o , ~ H\ 10
20
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fraction number
" 40
Fig. l a-c. Distribution profiles of various components in a 20,000 x g pellet from red-irradiated Cucurbita hypocotyl hooks following centrifugation on a discontinuous sucrose gradient, (o--o Phytochrome; 9 9 cytochrome-e oxidase; , - - j , N A D P H cytocbrome-c rednctase; o~ 9 protein; A - - A R N A ; . . . % (w/w) sucrose
Results
Previous studies using linear sucrose gradients have shown that the bulk of the phytochrome in the socalled " h e a v y " fraction occupies a density intermediate between the mitochondrial and smooth ER markers [7, 27]. The discontinuous gradient shown in Figure 1 was designed, on this basis, to provide a fraction enriched in phytochrome-bearing components and minimally contaminated with mitochondria and ER. The interface fractions thus obtained w e r e - as already mentioned-nominally designated "mitochondria", "heavy" phytochrome, "smooth
E R " and "31 S". Each has been assessed :for PTACrO3-positive material in an attempt to determine the quantitative distribution of putative plasma membrane vesicles across the gradient. Electron micrographs of these fractions stained with PTA-CrO3 are shown in Figure 2, Mitochondria predominate in the densest band (Fig. 2a). The "heavy" phytochrome fraction contains smooth membrane vesicles with some mitochondria (Fig. 2b). The "smooth E R " band also consists of smooth-surfaced vesicles with no evidence of the presence of membrane-bound ribo-
172
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO 3 Stain
Table l. Distribution of various components on a discontinuous sucrose gradient following centrifugation Of a 20,000 x g pellet from red-irradiated Cucurbita hypocotyl hooks" Fraction
"Mitochondria" " H e a v y " phytochrome "Smooth E R " "31 S"
Amount of component under each peak u Phytochrome Cyt. c oxid.
Cyt. c red. c
RNA
Protein
(%)
(%)
(%)
(%)
(%)
18 52 18 12
75 25 0 0
4 20 70 6
14 18 23 45
23 28 29 20
PTA-CrO3-positive material as % of total membrane in each fraction d
16 26 13 6
" See Figure 1 for experimental details b Data computed from Figure 1 by integrating the amount of each component under a given peak as follows: "mitochondria", fractions 1-8; " h e a v y " phytochrome, fractions 9 15; " s m o o t h ER", fractions 16-22; "31S", fractions 23 36 c The cytochrome c reductase values here have been corrected for a blank value not subtracted from the values in Figure 1 a See Material and Methods for morphometric quantitation of membranes in gradient fractions
somal particles (Fig. 2c). This 1s consistent with the density of the ER marker (1.12 g cm -3) which indicates that the membranes are ribosome-free [15, 30, 38]. Barely visible amounts of material can be pelleted from the "31 S" interface. That material which can be obtained includes some small, smoothsurfaced membrane vesicles (Fig. 2d). Previous studies with [14C]choline indicate that only about 3% of the total membrane phospholipid on the gradient locates in the 31 S band [7]. Figure 2b illustrates the differential staining of some of the vesicles with PTA-CrO3. Quantitative estimates of the proportion of such vesicles in each gradient fraction, together with the relative distributions of other components, are presented in Table 1. An enrichment of both phytochrome and PTA-CrO3positive material is observed in the ~ heavy" fraction. This fraction contains 52% of the total pigment on the gradient but only 25 and 20%, respectively, of the cytochrome-c oxidase and reductase present. Furthermore, there is an apparently good correlation in the distribution of phytochrome and PTA-CrO3-positive vesicles across the separate gradient fractions (data not shown). Thus on the basis of the procedures and criteria employed in other reports, the present data might be interpreted to indicate a correlated distribution of phytochrome and "plasma membrane". We feel, however, that a closer examination of the reputed specificity of PTA-CrO3 for the plasma membrane casts doubt upon its reliability for unequivocally identifying and quantitating this membrane following tissue homogenisation. Figures 3 and 4 illustrate the argument. It is clear that in addition to the plasma membrane, prolamellar-body membranes (Fig. 3a), the contents of presumptive lipid droplets (Fig. 3b), and especially ribosomes (Fig. 3a, b) are densely
stained as a result of this treatment. Figure 4 indicates, by means of successive steps in the staining procedure carried out on serial sections, that the ribonucleoprotein (RNP) of the ribosomes is selectively stained over and above the intrinsic electron-density of the particles. It is clear, therefore, that non-plasmamembrane components also have the capacity to appear PTA-CrO3-positive in sub-cellular fractions from this tissue.
Discussion
We have attempted here by means of the widely used and putatively plasma-membrane-specific PTA-CrO3 cytochemical staining procedure [10, 12, 13, 29, 31, 34-36] to compare the distributions of phytochrome and plasma membrane on sucrose density gradients. Our data indicate a positive correlation in the distributions of the pigment and PTA-CrO3-positive vesicles (Table 1). Other observations, however, call into question the fundamental assumption upon which the quantitative validity of this procedure rests: namely, that only plasma membrane vesicles are preferentially stained in particulate fractions. Prolamellar-body membranes, presumptive lipid droplets, and ribosomes all stain positively with PTACrO 3 in situ in addition to the plasma membrane (Figs. 3, 4). The potential for erroneously scoring prolamellar body fragments as plasma-membrane vesicles in gradient fractions is obvious. The possibility also exists that the PTA-CrO3-positive material from lipid droplets might become associated with membranes other than the plasma membrane during or after homogenisation. Cross-exchange of lipids between membranes in vitro is well documented [14, 39]. The staining of ribosomal RNP by the PTA-CrO3
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
173
Fig. 2a-d. Electron micrographs of fractions recovered from the interfaces of a discontinuous sucrose gradient analogous to that shown in Figure 1, The fractions correspond to those nominally designated in Table 1 as "mflochondria" (a), "heavy" phytochrome (b), "smooth ER" (e) and "31 S" (d). Sections have been stained according to the PTA-CrO3 procedure, p=PTA-CrO3-positive vesicle. Bar=l gm; •
procedure has been m e n t i o n e d recently in passing [13] but without discussion o f the potential problems posed by this observation. The presence o f significant levels o f R N P in all the fractions u n d e r consideration here is well d o c u m e n t e d (Fig. 1; [7, 24, 26]. This
R N P is o f ribosomal origin but is partially degraded [2@ The absence f r o m the m e m b r a n e vesicles in the gradient fractions o f readily identifiable intact ribosomal particles (Fig. 2) is consistent with this observation and is evidence that the R N P present is in other than
174
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
Fig. 3a and b. Electron micrographs of intact Cucurbita hypocotyl-hook tissue stained according to the PTA-CrOa procedure, a illustrates staining of plasma membrane (pro), ribosomes (r) and prolamellar body membranes (pb) by this procedure. Bar: 0.5 lain; x 54,600. b depicts the staining of the contents of presumptive lipid droplets (s) (note absence of bounding membrane and appearance when stained with uranyl acetate-lead citrate; inset x 19,900) as well as of ribosomes (r) and plasmamembrane (pro). Bar: 0.5 ~tm; x 31,300
its native globular configuration. One pertinent possibility is that the R N P is adsorbed in an extended configuration to the surfaces of vesicle bilayers. Staining of this R N P might then generate PTA-CrO3-positive vesicles indistinguishable from true plasma m e m b r a n e vesicles (cf. Fig. 5 in [1]), This would lead to false positive scoring of PTA-CrO3-stained vesicles as plasm a - m e m b r a n e fragments. The ready adsorption of
ribosomal R N P to other proteinaceous cytoplasmic components is well documented [4, 5, 23, 25]. Affinity is higher for basic than for acidic proteins so that differential adsorption to vesicles of varied origin may be expected and could lead to selective staining of some profiles over others. The granularity and discontinuity of the stain around the profiles of individual vesicles c o m m o n l y observed (Fig. 2; [12, 29, 31, 36])
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain
175
Fig. 4a and b. Electron micrographs of serial sections from intact Cucurbita hypocotyl-hook tissue, a Sections treated with periodic acid but not with PTA-CrO3; b Sections treated with periodic acid followed by staining with PTA-CrO3 (= normal PTA-CrO3 procedure). r ribosomes, pm plasma membrane. Bar: 0.5 ~tm, • 38,200 might also be consistent with the staining of membrane-bound RNP. Another report of the failure of the PTA-CrO3 procedure to give totally selective staining of the plasma m e m b r a n e has recently appeared [35]. Tonoplast and ER membranes were also found to be PTA-CrO3positive in the tissue used. The opportunities for falsely scoring PTA-CrO3-positive profiles as plasma m e m b r a n e are thus apparently abundant, This might explain the questionably high contents of " p l a s m a
m e m b r a n e " in particulate fractions reported by some authors using this technique. As much as 29% of the total homogenate m e m b r a n e complement has been claimed to be " p l a s m a m e m b r a n e " on this basis [361. We conclude that despite an apparent correlation in the distribution of phytochrome and PTA-CrO3positive m e m b r a n e vesicles from Cucurbita, the data do not necessarily constitute evidence of a phytochrome-plasma m e m b r a n e association. This is because
176
the PTA-CrO3 procedure cannot be considered to unequivocally identify plasma-membrane fragments once the architecture of the cell is disrupted by homogenisation. Moreover, we concur with Thorn et al. [35] in suggesting that this reservation might apply equally well to other studies purporting to provide evidence of plasma-membrane-associated components from plant tissue. The literature contains numerous reports that salt-stimulated ATPase [10], /~glucan synthetase [36], indoleacetic acid-[ll], N-1naphthylphthalamic acid-J12] and helminthosporoside-[34] binding activities, and phytochrome [20, 40] are all associated with plasma-membrane-rich fractions. A high sterol-to-phospholipid ratio has likewise been attributed to these fractions [8, 10]. Without exception, however, these claims are based either directly or indirectly on the use of the PTA-CrO3 stain for quantitating putative plasma membrane vesicles in the particulate fractions in question. It is suggested that, in the absence of confirmatory evidence from an independent means of monitoring plasma membrane in tissue homogenates, such conclusions should be considered tentative. Acknowledgements. The authors wish to thank Drs. B.E.S. Gunning and Th. Hendriks for helpful discussions, and Ms. E.A. Gallagher for competent technical assistance. Professor D.J. Cart provided research facilities.
References 1. Adelman, M.R., Blobel, G., Sabatini, D.D. : An improved fractionation procedure for the preparation of rat liver membranebound ribosomes. J. Cell Biol. 56, 191-205 (1973) 2. Atkinson, A.W., Gunning, B.E.S., John, P.C.L. : Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry, and incorporation of 14C-acetate studied in synchronous cultures. Planta (Bed.) 107, 1-32 (1972) 3. Butler, W.L., Norris, K.H.: The spectrophotometry of dense light-scattering material. Arch. Biochem. Biophys. 87, 31-40 (1960) 4. Dallner, G. : Isolation of rough and smooth microsomes- general. In: Methods in Enzymology, vol. XIII, Biomembranes, pt. A, p. 191-215, Fleischer, S., Packer, L., eds. New York: Acad. Press 1974 5.Das, N.K., Alfert, M.: Binding of labeled ribonucleic acid to basic proteins, a major difficulty in ribonucleic acid-deoxyribonucleic acid hybridisation in fixed cells in situ. J. Histochem. Cytochem. 17, 418-425 (1969) 6. Fleck, A., Munro, H.N. : The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta (Amst.) 55, 571 583 (1962) 7. Gressel, J., Quail, P.H.: Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C. Plant Cell Physiol. 17, 925-940 (1976) 8. Hartmann, M.A., Normand, G., Benveniste, P. : Sterol composition of plasma membrane enriched fraction from maize coleoptiles. Plant Sci. Lett. 5, 287-292 (1975)
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain 9. Haupt, W.: Localisation of phytochrome within the cell. In: Phytochrome, p. 553-569, Mitrakos, K., Shropshire, W., Jr., eds. New York: Acad. Press 1972 10. Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W.: Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membrane. Proc. nat. Acad. Sci. (Wash.)69, 3307-3311 (1972) l 1. Kasamo, K., Yamaki, T.: In vitro binding of IAA to plasma membrane-rich fractions containing Mg2+-activated ATPase from mung bean hypocytyls. Plant Cell Physiol. 17, 149-164 (1976) 12. Lembi, C.A., Morr6, D.J., Thomson, K.S., Hertel, R.: N-1Naphthylphthalamic-acid-binding activity of plasma membrane-rich fraction from maize coleoptiles. Planta (Berl.) 99, 37-45 (1971) 13. Leonard, R.T., Van der Woude, W.J.: Isolation of plasma membranes from corn roots by sucrose gradient centrifugation. Plant Physiol. 57, 105-114 (1976) 14. Lord, J.M.: Phospholipid synthesis and exchange in castor bean endosperm homogenates. Plant Physiol. 57, 218-223 (1976) 15. Lord, J.M., Kagawa, T., Moore, T.S,, Beevers, H.: Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J. Cell Biol. 57, 659--667 (1973) 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 17. Marm6, D. : An automatic recording device for measuring phytochrome with a dual wavelength ratiospect. Planta 88, 58-60 (1969) 18. Marm6, D. : Binding properties of the plant photoreceptor phytochrome to membranes. J. Supramol. Struct. 2, 751 768 (t974) 19. Marm6, D., Boisard, J., Briggs, W.R.: In vitro binding properties of phytochrome to a membrane fraction. Proc. nat. Acad. Sci. (Wash.) 70, 3861-3865 (1973) 20. Matin+, D., Bianco, J., Gross, J.: Evidence for phytochrome binding to plasma membrane and endoplasmic reticulum. In: Light and plant development, Smith, H., ed., in press. London: Butterworth (in press) 21. Marm6, D., Gross, J. : Remarks on photoreversibility as a measure for phytochrome. Book of Abstr., Ann. Eur. Photomorph. Syrup., Smith, H., ed., p. 88, 1975 22. Mohr, H., Appuhn, U. : Die Keimung yon Lactuca-Ach/inen unter dem Einflul3 des Phytochromsystems und der Hochenergiereaktion der Photomorphogenese. Planta (Bed.) 60, 274-288 (1963) 23. Petermann, M.L. : The physical and chemical properties of ribosomes. Amsterdam: Elsevier 1964 24. Quail, P.H.: Particle-bound phytochrome: Association with a ribonucleoprotein fraction from Cucurbita pepo L. Planta (Bed.), 123, 223-234 (1975) 25. Quail, P.H.: Particle-bound phytochrome: The nature of the interaction between pigment and particulate fractions. Planta (Berl.) 123, 235-246 (1975) 26. Quail, P.H., Gressel, J. : Particle-bound phytochrome: Interaction of the pigment with ribonucleoprotein material from Cueurbita pepo L. In: Light and Plant Development, Smith, H., ed., London Butterworth (in press) 27. Quail, P.H., Gallagher, E.A., Wellburn, A.R. : Membrane-associated phytochrome: Non-coincidence with plastid marker profiles on sucrose gradients. Photochem. Photobiol., in press 28. Reynolds, E.S. : The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208212 (1963) 29. Roland, J.C., Lembi, C.A., Morr6, D.J.: Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Tech. 47, 195-200 (1972)
P.H. Quail and J.E. Hughes: Phytochrome and PTA-CrO3 Stain 30. Sabatini, D.D., Tashiro, Y., Palade, G.E. : On the attachment of ribosomes to microsomal membranes. J. molec. Biol. 19, 503~-524 (1966) 31. Shore, G., Maclachlan, G.A.: The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkaliinsoluble fi-l,4-glucan (cellulose) synthetase activities. J. Cell. Biol. 64, 557-571 (1975) 32. Smith, L. : Cytochrome c oxidase. In: Methods in biochemical analysis, p. 427-434, Glick, D., ed. New York: Interscience 1955 33. Spurr, A.R. : A iow viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastr. Res. 26, 31-43 (1969) 34. Strobel, G.A., Hess, W.M. : Evidence for the presence of toxinbinding protein on the plasma membrane of sugar cane cells. Proc. nat. Acad. Sci. (Wash.) 7I, 1413 1417 (1974) 35. Thorn, M., Laetsch, W.M., Maretzki, A.: Isolation of membranes from sugar cane cell suspensions: Evidence for a plasma membrane enriched fraction. Plant Sci. Lett. 5, 245-253 (1975)
177 36. Van der Woude, W.J., Lembi, C.A., Morr6, D.J., Kindinger, J.I., Ordin, L. : fi-glucan synthetases of plasma membrane and golgi apparatus from onion stem. Plant Physiol. 54, 333 340 (1974) 37. Weibel, E.R. : Stereological principles for morphometry in electron microscope cytology. Internat. Rev. Cytol. 26, 235 302 (1969) 38. Wibo, M., Amar-Costesac, A., Berthet, J., Beaufay, H.: Electron microscope examination of subcellular fractions. J. Cell Biol. 51, 52-71 (1971) 39. Wirtz, K.W.A. : Transfer of phospholipids between membranes. Biochim. Biophys. Acta 344, 95-117 (1974) 40. Yu, R. : Characterisation of phytochrome-containing particles obtained by glutaraldehyde pre-fixation of maize coleoptiles. J. Exp. Bot. 26, 808 821 (1975)
Received 21 July; accepted 17 August 1976
