Abstract. Phytochrome of oat (Arena sativa L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conver- sion to the ...
Planta 9 by Springer-Verlag 1978
Planta 141, 129- 134 (1978)
Phytochrome Photoreversibility: Empirical Test of the Hypothesis that it Varies as a Consequence of Pigment Compartmentation ~ John M. Mackenzie, jr.t**, Winslow R. Briggs 1, and Lee H. Pratt 2 i Department of Plant Biology,Carnegie Institution of Washington, Stanford, CA 94305, and 2 Department of Biology,Vanderbilt University,Nashville, TN 37235, USA
Abstract. Phytochrome of oat ( A r e n a s a t i v a L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conversion to the far-red-light-absorbing form, Pfr, it is observed only in very small areas within the cell. Comparison of phytochrome photoreversibility measurements to the distribution of the pigment within the cell indicates that the spectral assay is not influenced by the observed compartmentalization of the chromoprotein. However, the observed compartmentalization of phytochrome is correlated with a loss in spectrophotometrically detectable Pr. Key words: tochrome.
Arena
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Immunocytochemistry - Phy-
Introduction Spruit (1972) has suggested that the loss of phytochrome photoreversibility during the destruction reaction (i.e., the loss of spectrally detectable phytochrome in etiolated tissue when present as the FRabsorbing Pfr form) could be accounted for by a compartmentalization of phytochrome rather than by an actual degradation of the chromoprotein. Spruit's argument also can be used to account for any observed changes in photoreversibility. His argument is based upon the so-called "sieve effect," whereby for a given quantity of pigment in a given sample, a uniform distribution of the pigment would yield a relatively high absorbance value as compared to that obtained with the pigment in a compartmentalized distribution. *
C.I.W.-D.P.B. Publication No. 622 Department of Pharmacology, Stanford University Medical Center, Stanford, CA 94305, USA Abbreviations." Pr=red-absorbing form of phytochrome; Pfr=farred-absorbing form of phytochrome; R=red light; FR=far-red light ** Present address."
With regard to phytochrome destruction, immunochemical evidence has already led to the conclusion that destruction probably results from proteolysis of phytochrome rather than from compartmentalization since antigenically detectable phytochrome is lost at the same rate as spectrally detectable phytochrome (Coleman and Pratt, 1974; Pratt et al., 1974). With regard to the more general application of Spruit's argument, Britz et al. (1977) have recently criticized some of the assumptions made by Spruit (1972) and concluded from theoretical arguments that given phytochrome concentrations found in etiolated tissues and the typical sample geometry used for invivo spectral assays, one should not expect any correlation between phytochrome distribution and photoreversibility measurements. Immunocytochemical assay of dark-grown plants shows phytochrome, as the R-absorbing Pr form, to be diffusely distributed throughout the cytoplasm (Coleman and Pratt, 1974a, b; Mackenzie etal., 1975). Similar assay of R-treated tissue indicates that phytochrome becomes associated with discrete areas about 1 gm in size which we will call the sequestered condition (Mackenzie etal., 1975, 1977). If phytochrome is subsequently reconverted to Pr with F R and the tissue incubated in darkness, phytochrome, over a 2-h period, resumes a diffuse distribution comparable to that observed in the dark controls by a process we will refer to as relaxation. The purpose of this paper is to take advantage of these immunocytochemically observed changes in intracellular phytochrome distribution in order to determine empirically whether or not phytochrome distribution affects in vivo photoreversibility measurements. Material and Methods 1. Plant Material and Light Treatments
Oat seeds (Arena sativa L., cv. Garry; Southern States Cooperative, Baltimore, Md., USA), were germinated and grown at 25~ in
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J.M. Mackenzie, Jr. et al. : Phytochrome: Photoreversibility and Compartmentation
complete darkness (Pratt, 1973). Four-d-old shoots were treated with light if appropriate, excised, assayed for photoreversibility when indicated, and then fixed in darkness at 0 ~C in 0.1 M sodium phosphate, pH 7.6, containing 4% formaldehyde prepared from p-formaldehyde (Pratt and Coleman, 1974). All manipulations except for the specified light treatments were carried out under dim green safelights as described in Pratt (1973). R-treatments were for 8 rain at 2 0 ~ 4 ~ with three closely spaced 40-W Sylvania Gro-lux fluorescent lamps at a distance of 20 cm. F R treatments were for 2 rain, also at 20-24~ with light from incandescent lamps at a distance of 15 cm filtered through Plexiglas FRF-700 (Rohm and Haas). Both irradiations were saturating and found by dual-wavelength assay to produce photostationary states equivalent to those produced by 665-nm and 725-nm interference filters, respectively.
syringe needle was inserted into one of the slots and used to inject cold 0.1 M sodium phosphate, pH 7.6, into the sample to improve its thermal conductivity. Air bubbles were removed by gentle tapping, and a thermocouple was inserted into the sample to monitor temperature continuously throughout the measurements. The temperature was lowered by adding NaC1 to the ice bath of the cuvette holder. If at any time the sample reached - 2~C the sample froze and was immediately abandoned. If the sample temperature exceeded - 0 . 5 ~ at any time during measurement, except where specifically required, the sample was also abandoned. The samples were nraintained at - I ~ for at least 5 min before measurements were begun; they were measured through 5 consecutive R, F R actinic irradiations of 45 s each.
2. Immunocytochemistry
Results
Fixed tissue was heated to 50~ for 5 min and embedded in paraffin (Pratt and Coleman, 1974). Phytochrome was visualized by sequential treatment of 8-gin sections mounted on microscope slides with specific rabbit anti-phytochrome serum (Pratt and Coleman, 1971 ; Pratt, 1973), sheep anti-rabbit immunoglobulin serum, and soluble, purified rabbit antiperoxidase-peroxidase complex (Sternberger et al., 1970). Peroxidase was then cytochemically stained with 3,3'diaminobenzidine and HzO 2. As controls for every experiment, sections adjacent to those presented were utilized; they were treated identically to the experimental sections except that non-immune rabbit serum was substituted for the specific anti-phytochrome serum. In no case did visible stain appear in the controls, demonstrating that no endogenous peroxidase activity or non-specific staining was present. Thus, visible contrast in the bright-field micrographs represents diaminobenzidine reaction product specifically associated with antigenically active phytochrome. Details of the procedure have been presented elsewhere (Pratt and Coleman, 1974). Each immunocytochemical experiment was performed on two separate lots of tissue. Photography was performed with a Reichert Zetopan microscope using a 63 x dry planachromat objective (N.A. 0.8I) with a Reichert photoautomat system (American Optical Corp., Reichert Division, San Francisco, Cal., USA) for 35-ram film. Highspeed Ektachrome transparencies were printed in color and then photographed and reprinted in black and white. All of the pictures from any one experiment were taken on a single film, and the photographs processed in a single batch to avoid any source of variation between pictures that could be attributed to differences in photographic processing. Cells presented are parenchyma cells in the coleoptile about 0.3 mm from the tip (Pratt and Coleman, 1971).
Phytochrome in samples measured without special precautions to control temperature as described above (temperature =ca. 4 ~ as measured with a thermocouple inserted into the sample) was found by subsequent immunocytochemical assay to have become sequestered during the spectral assay (Fig. 1). Thus, in all remaining measurements the precautions for temperature control described above were utilized. Immunocytochemical assay of samples measured through 5 R, FR irradiation cycles at - I~ and fixed at 0 ~C after either a terminal FR (Fig. 2 a) or R irradiation (Fig. 2b) indicated that neither spectral assay at - 1 ~ nor fixation at 0~ permitted the phytochrome redistribution seen at 4~ (Fig. 1) or higher temperatures (Mackenzie et al., 1975, 1977). If plants were pretreated with 8 min R at 20 ~C prior to spectral assay at - I ~ and then fixed at 0~ phytochrome
3. Spectral Measurements For in-vivo spectral measurements, 15 shoots (total sample weight 0.6 g) were used; these were cut into 3-5-mm sections and packed gently into a chilled aluminum cuvette with a 1-cm 2 cross-sectional area. Photoreversibility at 665 and 727 nm was assayed in a custombuilt, dual-wavelength spectrophotometer (Kidd and Pratt, 1973). For measurements at - I~ the cuvette was modified to allow precise control of sample temperature (Mackenzie, 1976). A lucite plug was inserted into the cuvette to isolate the sample from any warming effects at the bottom of the cuvette. A standard sample was then prepared on top of the lower plug as above. A second lucite plug, which contained four 1-mm 2 slots along the sides, was placed over the sample to prevent the sections from floating and to isolate them thermally fl'om the top of the cuvette. A
Fig. l a and b. Bright-field photomicrographs of Arena coleoptile cells after immunocytochemical staining of tissue fixed at 0 ~ C. a Dark control tissue handled as a sample for normal spectral measurement but without any actinic irradiation, b Tissue assayed for photoreversibility through 5 red, far-red actinic irradiation cycles at 4~ (with terminal far-red irradiation) and then fixed immediately. Note loss of stain throughout cytosol and appearance of intensely stained discrete areas (arrows). 590 x ; b a r = 2 5 ~ITI
Fig. 2a-f. Bright-field photomicrographs of Avena coleoptile cells following phytochrome localization of a dark control tissue spectrally assayed with 5 red, far-red actinic irradiation cycles at - I ~ with a terminal far-red light irradiation; b dark control tissue as in a except with a terminal red irradiation; e tissue irradiated with 8 rain red light at 20~ spectrally assayed at - I ~ as in a with terminal far-red irradiation; d tissue treated as in e except with terminal red irradiation; e tissue irradiated with 8 rain red light followed immediately with 2 rain far-red light and kept 2 h in darkness at 24~ before spectral assay at - I ~ as in a with terminal far-red irradiation; f tissue treated as in e except with terminal red irradiation in assay. 590 x ; b a r = 2 5 ~m
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J.M. Mackenzie, Jr. et al. : Phytochrome: Pbotoreversibility and C o m p a r t m e n t a t i o n
was sequestered whether the terminal irradiation in the spectrophotometer was FR (Fig. 2c) or R (Fig. 2d). Whole plants given R followed by FR light with subsequent incubation for 2 h in darkness were also assayed as above. In this case, whether the termi,
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Time, min Fig. 3. Photoreversibility of p h y t o c h r o m e in Arena coleoptile tissue as a function of time in darkness at 20~ after red, far-red irradiation of intact tissue (o). The initial value of t00% was between 0.040 and 0.050 depending upon the specific lot of tissue. Individual values at 2 h are after dark incubation (u). 2 min actinic far-red alone followed by 2 h incubation (A). 8 min actinic red followed by 2 h dark incubation (v). Each point is the average of 3 independent experiments
nal irradiation was FR (Fig. 2e) or R (Fig. 2t), phytochrome has a diffuse distribution comparable to that seen in the dark controls (Fig. 2a, b). The samples preirradiated with R (Fig. 2c, d) has 88% of the photoreversibility observed in the dark controls while the samples pretreated with a R, FR cycle followed by a dark incubation (Fig. 2e, f) had only 70% of the photoreversibility of the dark controls. The kinetics of this apparent destruction of Pr were measured separately, using normal sample-handling procedures (Fig. 3). The 30% loss of photoreversibility after a R, FR cycle indicated above occurs over a 90-min period after which there is no significant change. FR-irradiated and dark controls show a small apparent synthesis of phytochrome. R led to the expected decrease of about 50% in photoreversibility over a 2-h interval. Because it was suspected that destruction during irradiation and sample preparation at 20 ~ rather than compartmentalization, was the reason for the 12% loss in the R treated sample (Fig. 2c, d) an experiment was designed to eliminate this possible contribution of destruction. A control was spectratly assayed for phytochrome at - 1~C, warmed to 10 ~ C, given a R irradiation, kept for 5 min at 10~ and then spectrally assayed a second time but at 10~
Fig. 4a and h. Bright-field photomicrographs of Arena coleoptlle cells tollowmg lmmunocytocnemlcal s m m m g a alter spectral mcasu~cm~m through 5 red, far-red cycles at - I ~ and b after similar measurement at - 1 ~ followed by warming to 10~ irradiation for 45 s with red light using the source built into the spectrophotometer in order not to disturb the sample, and spectral measurement at 10~ with 5 red, far-red cycles. Tissue was transferred directly from the cuvette for fixation at 0~ and immunocytochemical assay. 7 3 0 x ; b a r = 2 5 lam
J.M. Mackenzie,Jr. et aI. : Phytochrome:Photoreversibilityand Compartmentation Fixation at 0~ and subsequent localization indicated that phytochrome distribution had changed from diffuse (Fig. 4a) to sequestered (Fig. 4b). The A(AA) for the sample in Figure 4b was 0.041 at both - l ~ when phytochrome was presumed to have a diffuse distribution (Fig. 2a, b, 4a), and at 10~ when it was found to have a sequestered distribution (Fig. 4b). Phytochrome distribution in the same sample used for Figure 4b could not be determined prior to warming to 10~ without disturbing sample geometry and thus negating the value of this experiment. Five additional samples were assayed spectrally first at - I ~ then at 4~ and finally at 10~ and in only one measurement did the observed photoreversibility deviate from the initial value observed at - I ~ by as much as 0.001A.
Discussion
The previously observed sequestering of phytochrome to a discrete localization within the cell (Mackenzie et al., 1975, 1977) occurs during spectral measurement of samples at 4~ which are irradiated only by the actinic sources built into the spectrophotometer (Fig. 1). The process is therefore very fast even at low temperature (occurring within the ca. 5 rain required for the spectral assay), and as a result one must utilize even lower temperatures to test Spruit's (1972) argument as summarized in the Introduction. If tissue is measured at - I ~ and fixed at 0~ the redistribution can be stopped whether fixation is with phytochrome as Pr (Fig. 2a) or as 75% Pfr:25% Pr (Fig. 2b) (Pratt, 1975). Thus, it becomes possible to test Spruit's argument empirically. The rapid sequestering of phytochrome upon R irradiation (Figs. 1, 2c, d) contradicts the prediction that gradual sequestering of phytochrome into a smaller area would cause the loss of photoreversibility observed during destruction (Spruit, 1972; Kendrick and Smith, 1976). In addition, the observed compartmentalization of phytochrome is associated with a loss of photoreversibility of only 12% (Fig. 2 c, d) and this may result from proteolysis, not sequestering, as will be discussed below. It is also clear that at a time when phytochrome is relaxing to its more diffuse distribution (Fig. 2e, f) there is a loss of spectrally detectable phytochrome when Spruit's argument would predict an increase. It would have been helpful to have some quantitative technique for monitoring the sequestering and relaxing processes spectrally in the sections since such a method would permit direct comparison of the kinetics of phytochrome destruction with the kinetics of change in phytochrome distribution. Indeed, Britz
133
et al. (1977b) used a two-wavelength microspectrophotometric technique to quantify light-induced changes in the intracellular distribution of phytochrome in nodal tissue of vice after immunocytochemical localization carried out precisely as described in the present paper. Attempts to use the same technique with oat tissue failed, however, since unlike the case with rice, the initial distribution of pigment before irradiation was already very inhomogeneous and variability was far too great to obtain useful information. However, although quantitation was not possible, the dramatic changes such as those seen in Figures 1, 2, and 4, together with the spectral measurements, appear sufficient to support the arguments presented here. Two questions are raised by the photoreversibility measurements presented in Figures 2 and 3. First, how does one explain the loss of phytochrome as Pr (Figs. 2e, f, 3)? Second, is the observed 12% loss seen in Figure 2c, d caused by a small sieve effect as predicted by Spruit (1972) or by destruction that occurs during the 8-rain irradiation at 20~ and subsequent sample preparation time (total time before cooling about 15 min)? With respect to the first question, apparent destruction of Pr in oat shoots following R, FR irradiation has been reported previously by Dooskin and Mancinelli (1968) and Chorney and Gordon (1966), although they did not present a detailed time course. This apparent destruction stops abruptly at about 90 rain (Fig, 3). Mackenzie et al. (1975, 1977) have shown that this is about the time at which phytochrome is no longer associated with discrete areas, and thus indicates that the loss may be specific for phytochrome in the sequestered condition. If this loss of photoreversibility arises from the same mechanism as the better-characterized Pfr destruction (Frankland, 1972) it would indicate that destruction is specific with reference to phytochrome location within the cell rather than to the form of phytochrome. A second possibility is that Pr produced by a R, FR cycle is different than that present in non-light-treated tissue. With respect to the second question raised above, the evidence in Figure 4 demonstrates clearly that if sample geometry is undisturbed and the temperature is kept sufficiently low to inhibit destruction during the spectral assay, there is no detectable loss of photoreversibility under conditions where phytochrome goes from a diffuse (Fig. 4a) to a sequestered (Fig. 4b) distribution. Unfortunately, one must presume that the sample was in a diffuse distribution during the initial - I ~ assay. However, in all cases examined, including the three presented in this paper (Fig. 2a, b, 4a), spectral assay at - I ~ and fixation
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at 0 ~ p r e v e n t e d the sequestering of p h y t o c h r o m e seen at w a r m e r temperatures, Hence, the observed 12% loss of photoreversibility r e p o r t e d for the sample illustrated in F i g u r e 2c, d w o u l d a p p e a r to result f r o m p h y t o c h r o m e proteolysis (Pratt et al., 1974) rather t h a n p h y t o c h r o m e c o m p a r t m e n t a l i z a t i o n . U n d e r the c o n d i t i o n s used here, one w o u l d predict this 12% loss to occur d u r i n g R i r r a d i a t i o n a n d sample preparation since n o lag phase precedes the onset of destruct i o n ( K i d d a n d Pratt, 1973). Thus, the o b s e r v a t i o n s r e p o r t e d here are consistent with the theoretical p r e d i c t i o n of Britz et al. (1977) that with small a b s o r b a n c e values a n d a large n u m b e r of cell layers, in-vivo spectroscopy should be unaffected by changes in the d i s t r i b u t i o n of the p i g m e n t in the cell. This research was supported by National Science Foundation Grants GB-17057 and PCM75-19125 to L.H.P.J.M.M. was supported in part by National Institutes of Health Training Grant T01GM0036 and by the Department of Plant Biology, Carnegie Institution of Washington, Stanford.
References Britz, S.J., Mackenzie, J.M., Jr., Briggs, W.R.: The use of twowavelength microspectrophotometry to quantify light-induced changes in the intracellular distribution of phytochrome determined by immunocytochemical localization. Carnegie Inst. Year Book 76, 274-278 (1977a) Britz, S.J., Mackenzie, J.M., Jr., Briggs, W.R. : Non-homogeneous pigment distribution, multiple cell layers, and the determination of phytochrome by in vivo spectrophotometry. Photochem. Photobiol. 25, 137-140 (1977b) Chorney, W., Gordon, S.A. : Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings. Plant Physiol. 41, 891-896 (1966) Coleman, R.A., Pratt, L.H.: Subcelluiar localization of the redabsorbing form of phytochrome by immunocytochemistry. Planta 121, 119-131 (1974a) Coleman, R.A., Pratt, L.H. : Electron microscopic localization of phytochrome in plants using an indirect antibody-labeling method. J. Histochem. Cytochem. 22, 1039-1047 (1974b)
Coleman, R.A., Pratt, L.H.: Phytochrome: immunocytochemical assay of synthesis and destruction. Planta 119, 221-231 (1974c) Dooskin, R.H., Mancinelli, A.L, : Phytochrome decay and coleoptile elongation in Arena following various light treatments. Bull. Torrey Bot. Club 95, 474-487 (1968) Frankland, B.: Biosynthesis and dark transformations of phytochrome. In: Phytochrome, pp. 195~25, Mitrakos, K., Shropshire, W., eds. London: Academic Press 1972 Kidd, G.H., Pratt, L.H.: Phytochrome destruction: An apparent requirement for protein synthesis in the induction of the destruction mechanism. Plant Physiol. 52, 309 311 (1973) Mackenzie, J.M., Jr. : Phytochrome distribution and redistribution as assayed by immunocytochemistry. Ph. D. thesis, Harvard University, Cambridge, Mass. (1976) Mackenzie, J.M., Jr., Briggs, W.R., Pratt, L.H. : Intracellular phytochrome distribution as a function of its molecular form and of destruction. Amer. J. Bot. (1978) in press 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. Nat. Acad. Sci. USA 72, 799-803 (1975) Pratt, L.H. : Comparative immunochemistry of phytochrome. Plant Physiol. 51, 203-209 (1973) Pratt, L.H. : Photochemistry of high molecular weight phytochrome in vitro. Photochem. Photobiol. 22, 33-36 (1975) Pratt, L.H., Coleman, R.A.: Immunocytochemical localization of phytochrome. Proc. Nat. Acad. Sci. USA 68, 2431-2435 (1971) Pratt, L.H., Coleman, R.A. : Phytochrome distribution in etiolated grass seedlings as assayed by an indirect antibody-labelling method. Amer. J. Bot. 61, 195 202 (1974) Pratt, L.H., Kidd, G.H., Coleman, R.A.: An immunochemica[ characterization of the phytochrome destruction reaction. Biochim. Biophys. Acta 365, 93-107 (1974) Spruit, C.J.P. : Estimation of phytochrome by spectrophotometry in vivo Instrumentation and interpretation. In: Phytochrome, pp. 77-104, Mitrakos, K., Shropshire, W. eds. London: Academic Press 1972 Sternberger, L.A., Hardy, P.H,, Jr., Cuculis, J.J., Meyer, H.G.: The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18, 315-333 (1970)
Received 19 December 1977; accepted 14 March 1978
