Planta
Planta (1983) 159: 545-553
9 Springer-Verlag 1983
Immunofluorescence visualization of phytochrome in Pisum sativum L. epicotyls using monoclonal antibodies Mary Jane Saunders 1'*, Marie-Mich+le Cordonnier 2, Barry A. Palevitz 1 and Lee H. Pratt 1 1 Department of Botany, University of Georgia, Athens, GA 30602, USA, and 2 Laboratoire de Physiologic v6g+tale, Universit6 de Gen+ve, Pavillon des Isotopes, 20 Bd. d'Yvoy, CH-1211 Gen6ve 4, Switzerland
We have investigated the cellular distribution of phytochrome in epicotyls of dark-grown pea (Pisum sativum L.) seedlings using monoclonal antibodies to pea phytochrome. Screening of the eight available antibodies both by an enzymelinked immunosorbent assay (ELISA) and by their ability to visualize phytochrome in situ by immunocytochemical fluorescence demonstrated that: (1) three antibodies work well for immunofluorescence; (2) none of the eight antibodies discriminates between the red- and the far-red-absorbing forms of phytochrome (Pr, Pfr) as assayed by ELISA; (3) the antigenicity of phytochrome is reduced by fixation with formaldehyde with respect to all eight antibodies; and (4) two antibodies that bind well to formaldehyde-fixed phytochrome as assayed by ELISA do not bind well to phytochrome in situ. Phytochrome is observed in both cortical and stomatal guard cells of the epicotyl and exhibits a homogeneous cytoplasmic distribution in non-irradiated tissue. After red-light (R) treatment phytochrome becomes transiently inaccessible to antibodies. If maintained in the Pfr form for J0 rain at room temperature before fixation, at least a portion of the phytochrome pool becomes accessible to antibodies and assumes a "sequestered" distribution. Both of these effects are almost entirely either prevented or reversed by subsequent far-red light treatment. We believe that the transient inaccessibility of phytochrome to antibodies after R irradiation is not a function of its conformational state. We suggest instead that R treatment rapidly induces an association of phyAbstract.
* Present address: Botany Department, Louisiana State Uni-
versity, Baton Rouge, LA 70803, USA Abbreviations: ELISA=enzyme-linked immunosorbent assay;
FR = far-red light; IgG = immunoglobulin G; Pfr = fa>red-absorbing form of phytochrome; Pr = red-absorbing form of phytochrome; R=red fight
tochrome with a subcellular component that interferes with antibody binding and that the "sequestered" areas represent a phytochrome pool that is distinct from both the diffusely distributed phytochrome in non-irradiated cells and from that phytochrome which is inaccessible to antibodies immediately after R irradiation. Key words: Immunosorbent assay (phymchrome) - Monoclonal antibody - Phytochrome (immuno-
fluorescence) - Pisum (phytochrome).
In~oducfion
Phytochrome is a photosensitive chromoprotein that mediates many developmental responses of plants to light (for recent reviews, see Smith 1975; Pratt 1979, 1982). The most promising hypothesis for its mode of action, albeit not definitively tested, is that phytochrome affects membrane activity after photoconversion from the physiologically inactive red-absorbing form (Pr) to the physiologically active far-red-absorbing form (Pfr) (Hendricks and Borthwick 1967; Marm6 1977). Attempts to obtain membrane-associated phytochrome in vitro and determine its subcellular distribution in vivo have resulted in the following observations. After centrifugation of a crude extract, 5-10% of the pigment is inherently associated with particulate subcellular fractions (Rubinstein et al. 1969). As much as 80%, however, can be induced to pellet with particulate fractions following photoconversion of Pr to Pfr (Quail et al. 1973). Analyses of these fractions, coupled with spectrophotometric assays, have led various researchers to describe phytochrome associations with ~he plasma membrane (Marm6 et al. 1976), endoplasmic reticulum (Williamson et al. 1975; Marm6 et al. 1976),
546
M.J. Saunders et al.: Immunofluorescence visualization of phytochrome in Pisum epicotyls
etioplast envelope (Evans and Smith 1976) and mitochondria (Manabe and Furuya 1975). Immunocytochemical analyses of phytochrome distribution in monocotyledonous plants using polyclonal rabbit antibodies to oat phytochrome have indicated a diffuse cytosolic distribution in dark-grown tissue (Coleman and Pratt 1974a, b), which changes after photoconversion of Pr to Pfr, the pigment becoming "sequestered" in numerous discrete areas about 1 Ixm in size (Mackenzie et al. 1975). The relationship of this sequestering phenomenon to the induction by red light (R) of pelletable phytochrome is unknown. It is possible that both are manifestations of an initial intracellular event in phytochrome-mediated development, namely, association of physiologically active Pfr with a receptor or reaction partner that is itself membrane bound. To learn more about the distribution and intracellular localization of phytochrome and thereby obtain information to test current hypotheses concerning its mode of action, as well as to extend immunocytochemical investigations of phytochrome localization to a dicotyledonous plant and thereby determine whether the previously described sequestering phenomenon might be of general interest, we have used recently developed monoclonal antibodies (Cordonnier et al. 1983) to visualize immunocytochemically phytochrome in epicotyls of etiolated pea seedlings. Monoclonal antibodies provide a greater degree of specificity than can be obtained with polyclonal antisera, leading to increased resolution of immunocytochemical observations. Reactivity of the eight monoclonal antibodies to both Pr and Pfr presently available, either fixed with formaldehyde or unfixed, is also determined by an enzyme-linked immunosorbent assay (ELISA).
Material and methods Plant tissue. Peas (Pisum sativum L., cv. Alaska; Leatherman's Seed Co., Canton, O., USA) were grown in darkness on cellulose packing material (Kimpak 62360; Kimberly-Clark Corp., Neenah, Wis., USA) at 25 ~ C in saturating humidity for 4-6 d. Until fixation was complete, tissue was handled only under green safe lights (Pratt 1973).
Light treatments. Whole seedlings were irradiated with monochromatic light obtained from a 45-W microscope lamp (Unitron Instruments, Plainview, N.Y., USA) equipped with B-40 interference filters (Balzers, Liechtenstein; 666 nm, 20-nm halfband width; 737 nm, 20-nm half-band width). A 20-s irradiation with either R or far-red light (FR) saturated photoconversion as determined by dual wavelength spectrophotometry. After irradiation, either 1-2-ram sections were excised from the hook region and fixed immediately, or the seedlings were
incubated in darkness at room temperature for 10 rain before excision and fixation.
Fixation procedure. All solutions were made in 0.1 M sodium phosphate, pH 7.4. The fixation schedule was as follows: fix overnight on ice in 4% formaldehyde, freshly prepared from p-formaldehyde; infiltrate 8 h at 4 ~ C with 1 M sucrose in 0.4% formaldehyde; rinse with 1.3 M sucrose; incubate 4 h at room temperature in 1.3 M sucrose; keep at 4 ~ C for not more than 4 d until secfior~ed. Sectioning. Sucrose-infiltrated tissue was trimmed, mounted on a copper block, and frozen rapidly by immersion into liquid nitrogen. Sections, 1 ~m thick, were cut at - 50 ~ C on a Sorval MT-2 ultramicrotome equipped with an FTS cryo-sectioning unit (Dupont, Wilmington, Del., USA). The sections were transferred to glass slides treated with Slipicone Release Spray (Dow Corning, Midland, Mich., USA) using the sucrose dropwire loop method of Tokuyasu (1980). At least six sections were placed on each slide and processed uniformly. Control sections were cut from adjacent areas of the same tissue block. Each experiment was repeated at least three times. Antibodies. All antibodies were prepared in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 140 m M NaCI, pH 7.4) containing 10% lamb serum (No. 200-6070; GIBCO Laboratories, Grand Island, N.Y., USA). Immunostaining involved the sequential application of three different antibodies. The first antibody was either an immunopurified monoclonal antibody to pea phytochrome, designated I-3b2, I-9a2, II-10a6, 1-11a12, 1-13b6, 1-15a3, 1-18al or II-18a4 (see Cordonnier et al. 1983 for details) or, as a control, non-immune mouse immunoglobulin G (IgG; No. 1-5381 ; Sigma Chemical Co., St. Louis, Mo., USA). The antibodies were used at concentrations of 150, 30 and 10 gg/ml for initial screening. Antibody II-18a4, as well as non-immune mouse IgG, was used at 10 gg/ml for all subsequent localization work presented here. The second antibody (used to amplify the signal) was immunopurified rabbit antibody to mouse IgG (see Cordonnier et al. 1983) and was used at 10 ~g/ml. The third antibody was rhodamine-conjugated goat antibody to rabbit IgG (No. 61-266; Miles-Yeda, Rehovot, Israel) and was used at 1 : 400 dilution. The concentrations of second and third antibody used were determined to be optimal by testing several dilutions.
Staining protocol. Labeling of the tissue sections was performed at room temperature by the following protocol: (1) 2% gelatin, 15rain; (2) PBS, 15rain; (3) lamb serum, 15rain; (4) PBS+ 10% lamb serum, rinse; (5) first antibody, 2 h; (6) PBS+ 10% lamb serum, 15 rain; (7) lamb serum, 15 min; (8) PBS+10% lamb serum, rinse; (9) second antibody, 45 rain; (10) PBS+ 10% lamb serum, rinse; (11) lamb serum, 15 rain; (12) PBS+ 10% lamb serum, rinse; (13) third antibody, 45 rain; (14) PBS, three changes of 15 rain each; (15) mount in 90% glycerol, 10% PBS. Lamb serum was used to block non-specific binding sites.
Microscopy. The sections were viewed on a Zetopan incident light fluorescence-phase contrast microscope (C. Reichert Optische Werke, Vienna, Austria). For fluorescence microscopy an HBO 200-W mercury lamp (Osram, West Berlin, Germany) was used in conjunction with a 4 2 546 exciter filter (broad band excitation peak at 546 nm) and a 3 0 G 590 barrier filter (C. Reichert). In addition, a 640-nm short pass filter (Ditric Optics, Hudson, Mass., USA) was placed at the top exit port of the microscope in front of the video camera. The fluorescence signal was amplified by a triple image intensifier vidicon camera
M.J. Saunders et al. : Immunofiuorescence visualization of phytochrome in Pisum epicotyls (Model 9004; Venus Scientific, Farmingdale, N.Y., USA) and viewed and photographed on a high-resolution video monitor (see Palevitz et al. 1981 for a complete description of the video equipment). Beam, signal gain, and black (pedestal) levels were kept constant for all experiments. Photographs were taken on Ilford FP-4 film and developed at ASA 1000 using K o d a k HC110 developer and Factor 8 (MinMax, N o r t h Hollywood, Calif., U S A ; company now defunct). Identical printing conditions were used for all micrographs taken with fluorescence optics. Magnifications of the video images were calibrated with a slide micrometer.
Enzyme-linked immunosorbent assay. The ELISA was carried out under green safe lights (see above) at room temperature. The procedure was as described by Cordonnier et al. (1983), with the following modification. The wells were coated with either 50 [al/well of 6 gg/ml purified pea phytochrome or, as a control for non-specific binding, an equivalent amount of bovine serum albumin (No. A9647; Sigma). Both were diluted either into borate-saline buffer or borate-saline buffer containing 4% formaldehyde. Formaldehyde was washed away upon completion of the initial coating step. Before dilution, the phytochrome was photoconverted to either Pr or Pfr using the light source and filters described above. Monoclonal antibodies were used at 10 gg/ml.
Results and discussion
Antibody screening. Of the eight monoclonal antibodies available, only three (II-10a6, 1-15a3 and II-18a4) are useful for immunocytochemistry at any of the concentrations tested, although they are all strongly positive when assayed against pea phytochrome by ELISA (Table 1). The inability of the remaining five antibodies to bind to phytochrome
547
in fixed tissue sections may result from different causes. The environment of phytochrome in situ may be such that one or more of the antibodies is sterically hindered from binding to it. Alternatively, fixation of the tissue may cross-link or otherwise change phytochrome in such a manner as to decrease its antigenicity. The ELISA data indicate that chemical fixation does reduce the ability of phytochrome to be recognized by all of the antibodies tested (Table i). The binding of antibodies I-3b2, I-%2 and 1-13b6 was reduced to very low levels, which correlates with the immunofluorescence results. The antibodies that work well for immunofiuorescence (II-10a6, I-15a3 and II-18a4) show a moderate level of' binding to fixed phytochrome. However, both I-I la12 and 1-18al display equal or greater binding to fixed phytochrome in the ELISA than II-18a4 but are immunocytochemically negative (Table 1). The lack of in situ binding of 1-11a12 and [-18al may result from differences in the intracellular environment affecting phytochrome accessibility that are not mimicked in an ELISA. The monoclonal antibody II-18a4 was best for immunocytochemical applications and was used at a concentration of 10 gg/ml for all subsequent localization work presented here.
Distribution of phytochrome in dark-grown non-iradiated tissue. Cortical cells demonstrate the l)ighest degree of phytochrome-associated fluorescence (Fig. I). Although epidermal and vascular ,=ells of
Table 1. Comparison of immunofluorescence and ELISA binding of monoclonal antibodies to pea phytochromea Antibody
Immunofluorescence b
ELISA activity *
Non-irradiated 150 I-3b2 I-9a2 II-10a6 1-11a12 1-13b6 I-t5a3 1-18al II-18a4 Non-immune mouse IgG
. + . . + +
30
20 s R 10
150
10
-
-
-
-
. --
-
-
.
. +
. .
++++ . .
. . + + . . ++++ .
. + . . + . . ++++ .
. . .
--
Pr
Pfr
Pr (fixed)
79 47 39 50 40 83 57 60
74 46 42 53 34 90 65 62
16 9 26 32 9 38 49 29
Pfr (fixed)
~16 ~9 " 31 36 10 29 46 32
BSA d
BSA (fixed)
2
3 2 2 1 2 2 4
.
" The pea phytochrome used for the ELISA was partially purified by brushite chromatography, a m m o n i u m sulfate fractionation, and diethylaminoethyl-agarose chromatography (see Pratt 1973; Cordonnier et al. 1983). The ratio of absorbance at 667 n m to 280 nm with phytochrome as Pr was 0.1 b Immunofluorescence activity was evaluated visually after immunostaining either non-irradiated tissue o r tissue irradiated with R for 20 s immediately prior to fixation. Antibodies were used at 150, 30 or 10 i~g/ml as indicated c ELISA activity is presented as absorbance at 400 nm for a 5-ram light path, x 100. Monoclonal antibodies were tested at 10 gg/ml d B S A = b o v i n e serum albumin
548
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
Figs. 1-3 (a, b). Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown non-irradiated pea cpicotyl tissue, demonstrating the distribution of phytochrome-associated fluorescence at the tissue level. Bar= 10 vLm; x 1000. Fig. 1, Pea tissue section treated with II-18a4 IgG (10 gg/ml). The epidermal cells (e) appear dark compared with the brightly fluorescent cortical ceils (e). Fig. 2. Pea tissue section treated with II-18a4 IgG (10 gg/ml). Cortical cells (c) are fluorescent while vascular tissue (v) is less bright. Fig. 3. Control pea tissue section treated with non-immune mouse IgG (10 gg/ml). Only slight fluorescence is discernable from cortical (c) and epidermal (e) cells. Print exposure and development conditions for b were the same as those for Figs. 1 b and 2b
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
549
Fig. 4a, b, Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown non-irradiated pea epicotyl epidermal cells treated with II-18a4 IgG (10 gg/ml). Note the bright phytochrome-associated fluorescence in guard cells (g). Bar = 10 gm; x 1500
tissue treated with antibody to pea phytochrome are somewhat brighter than those in control tissue treated with non-immune mouse IgG as the primary antibody, they are markedly less bright than cortical cells (Figs. 1-3). Most of the phytochrome associated with the hook region of pea epicotyls is in the cortical cells. Although most epidermal cells do not bind antibody well, stomatal guard cells treated with II18a4 IgG are brightly fluorescent (Fig. 4). Guard cells in control tissue are not fluorescent (data not shown). This demonstration of high phytochrome levels in guard cells is consistent with a possible role for phytochrome in stomatal action (Habermann 1973; Holmes and Klein 1983). Additionally, since pea guard cells contain chloroplasts (Singh and Srivastava 1973), the presence ofphytochrome may be related to its role in mediating chloroplast development (Mohr 1977). Phytochrome in non-irradiated pea cortical cells is restricted in its distribution to the cytosol (Fig. 5). There is no staining of plastids or vacuoles. Furthermore, the level of nuclear fluorescence is so low that it is difficult to determine whether it is indicative of the presence of phytochrome. The cytoplasmic distribution of phytochrome in non-irradiated pea tissue is similar to the pattern described previously for monocotyledonous tissues (Coleman and Pratt 1974a, b). While these observations correlate well with the fact that phytochrome acts as a soluble protein when extracted from dark-grown tissue (see Pratt 1979), they are also consistent with a possible association of phytochrome with a widely distributed
cytoplasmic matrix membrane system.
or
endoplasmic-reticuIum
Distribution of phytochrome in light-treated tissue. None of the monoclonal antibodies tested binds significantly to phytochrome in pea epicotyl tissue that was exposed to 20 s R and fixed immediately (Table 1). Similar results with both non-irradiated and R-irradiated tissues were obtained using polyclonal rabbit antiphytochrome antibodies that were immunopurified with a column of immobilized pea phytochrome (data not shown; see Cordonnier and Pratt 1982). The lack of phytochromerelated fluorescence in R-irradiated cells is therefore not a consequence of some specific property or properties of the monoclonal antibodies. The degree of fluorescence observed in R-treated cortical cells using antibody II-18a4 (Fig. 6) is approximately the same as that observed with epidermal ceils in non-irradiated tissue. Non-irradiated tissue processed in parallel as a control stains consistently with II-18a4 as described above (Fig. 5) and control R-irradiated tissue treated with non-immune mouse IgG does not stain (Table 1). If the R treatment was followed immediately by a 20-s F R treatment, most cells stained with II-18a4 as if they h a d never been irradiated (Fig. 7). Inl some instances all the cortical cells were brightly fluorescent but at other times a few cortical cells did not stain and resembled R-irradiated cells. A possible interpretation of these results is that these monoclonal antibodies are specific!for phytochrome in the Pr conformation and therefore do not bind Pfr in red-irradiated tissue. To test this
Figs. 5--7a, b. Paired phase-contrast (a) and fluorescence (b) micrographs of dark-grown pea epicotyl tissue treated with II-18a4 IgG (10 gg/ml). B a r = 10 gin; x 1000. Fig. 5. Intracellular distribution of phytochrome in cortical cells of non-irradiated tissue. Note the bright cytoplasm (cy), markedly reduced fluorescence in nuclei (nu) and plastids (arrows) and lack of fluorescence in vacuoles (v) and nucleoli (n). Fig. 6. Cortical cells of pea tissue exposed to 20 s R and fixed immediately at 0 ~ C. Note reduced fluorescence in cytoplasm (ey) (compare with Fig. 5). Nuclei (nu) and vacuoles (v) are still discernable as darker regions. Print exposure and development conditions were the same as in Fig. 5. Fig. 7. Cortical and epidermal cells of pea tissue exposed sequentially to 20 s R, 20 s F R and then fixed immediately at 0 ~ C. Note that bright cortical cells (e) and dark epidermal cells (e) exhibit a fluorescence pattern similar to that of non-irradiated tissue (Figs. 1, 5)
M.J. Saunderset al. : Immunofluorescencevisualizationof phytochromein Pisumepicotyls interpretation, we compared by ELISA phytochrome treated in four ways: (1) Pr; (2) Pfr; (3) fixed Pr; (4) fixed Pfr. None of the antibodies to pea phytochrome discriminate between Pr and Pfr, whether fixed or unfixed (Table 1). These data are consistent with the previously noted observation that polyclonal rabbit antibodies to pea phytochrome, which would not be expected to discriminate between Pr and Pfr (based upon previous work with comparable antisera to oat phytochrome; Pratt 1973), do not detect the pigment when tissue is fixed immediately after R irradiation. We therefore conclude that a conformational change of phytochrome is not solely responsible for the loss of the ability of the monoclonal antibodies to recognize this pigment in R-irradiated tissue. An alternative explanation is that there has been a change in the molecular environment of phytochrome within the cortical cells after R treatment that either blocks or obscures the antigenic sites on the chromoprotein and causes it to become "cryptic." These results cannot be considered as definitive evidence for binding of phytochrome to a receptor after R treatment; however, that is one possible interpretation. If a 10-rain dark incubation before fixation follows the R treatment, small but discrete fluorescent areas become visible within the cytoplasm of cortical cells (Fig. 8). The epidermal and vascular tissues look similar to R-treated tissue that was immediately fixed as well as to non-irradiated tissue. The bright regions in cortical cells do not correspond to any organelle recognizable by phase contrast microscopy. These fluorescent areas are similar in size and distribution to the "sequestered phytochrome" regions described for oat tissue (Mackenzie et al. 1975). Whether or not "cryptic" phytochrome remains cannot be determined. The physiological role of these sequestered regions in oats is not known but the appearance of similar areas in pea tissue after R treatment indicates that it may well be a general phenomenon. In addition, these latter observations (Fig. 8) indicate that II-18a4 IgG can bind to phytochrome in the Pfr form in situ. The ability of the antibody to bind phytochrome in these "sequestered" regions may also indicate that the presumed association between phytochrome and another subcellular component produced immediately after R treatment does not exist in the sequestered regions. These regions may represent a new phytochrome pool that appears only over time after photoconversion to Pfr. If tissue is irradiated for 20 s with R, incubated
551
in darkness for 10 rain at room temperature, irradiated for 20 s with FR and then fixed immediately, most or all of the cortical cells again exhibit fluorescence similar to that found in non-irradiated tissue (in contrast to previous observations with oats; Mackenzie et al. 1975). A few cells retain the sequestered distribution that is seen with R-treated tissue that is incubated in darkness before fixation (Fig. 9). It is difficult to determine if the cells that have very bright cytoplasmic fluorescence have retained sequestered regions within the cytoplasm. Therefore, one cannot be certain whether the diffusely distributed phytochrome visible after FRtreatment arises from the cryptic pool or from the sequestered pool. This result may be indicative, however, of multiple pools of phytochrome after light treatment. It is possible that in the R-treated cells that display the sequestering phenomenon we are not visualizing all the phytochrome, but instead some remains both cytoplasmic and cryptic. FR treatment would then make this cryptic pool available again for binding by antibodies. While there are, as already noted, some obvious similarities between the present observations and those obtained previously with monocotyledonous plants (Coleman and Pratt 1974a, b; Mackenzie et al. 1975), there are also some apparent differences. Phytochrome is found in nonirradiate,d cells to be diffusely distributed throughout the cytosol both in the dicotyledonous plant studied here (Fig. 5) and in the grass seedlings previously investigated (Coleman and Pratt 1974a, b). The inaccessibility of phytochrome to antibodies described here for cells fixed immediately after R (Fig. 6), however, has not been reported for grass seedlings. To determine whether the difference is real or apparent will require that the experimental protocol used here also be used with monocotyledonous plants. The sequestering of Pfr described here (Fig. 8) is also similar to that described previously for grass seedlings (Mackenzie et al. 1975). A potential difference with the earlier observations, however, is the apparent rapid F R reversibility (Fig. 9). As just discussed, however, it is not possible to discriminate between an effect of FR on reversal of sequestering as opposed to an effect on reversal of "cryptic" phytochrome that may remain even in the presence of the sequestered phytochrome.
Specificity of the immunocytochernical staining procedure. Demonstration of immunocytochemical specificity for the antigen of interest is a major concern (see Petrusz t983 for recent discussion). Several arguments indicate that we can have a high
552
M.J. Sauttders et al. : Immunoftuorescencevisualization of phytochrome in Pisum epicotyls
Figs. 8, 9a, b. Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown pea epicotyl tissue treated with II-18a4 IgG (10 gg/ml). Bar=10 gin; x 1000- Fig. 8. Cortical cells of pea tissue treated with 20 s R, 10 rain dark incubation and then fixed at 0~ C. Note the bright "sequestered" regions (arrows) visible in the cytoplasm but absent in the nuclei (nu) and plastids (p). Fig. 9. Cortical cells of pea tissue treated with 20 s R, 10 rain dark incubation, 20 s FR and then fixed at 0~ C. Note the bright cytoplasmic (cy) fluorescence in many cells, similar to that seen in non-irradiated cells (Figs. 1, 5), or bright "sequestered" regions (arrows) in other cells, similar to those seen in R-irradiated, dark incubated cells (Fig. 8) degree o f confidence in the immunospecificity o f the observations presented here. (1) While not emphasized above, it is i m p o r t a n t to note that the three m o n o c l o n a l antibodies, as well as the immunopurified polyclonal antibodies, all yield the same results. It is unlikely that all would give rise to the same i m m u n o s t a i n i n g artifact. (2) Since the immunostaining results are strongly and rapidly influenced in photoreversible fashion by R and F R , it is again most likely that p h y t o c h r o m e itself is the only antigen being visualized. (3) Since the m o n o c l o n a l antibodies have all been shown to be directed to p h y t o c h r o m e (Cordonnier et al. 1983), the question of antigenic nonspecificity does not
arise as it does with a polyclonal preparation, even though the possibility of staining an immunologically cross-reacting antigen c a n n o t be excluded by this consideration. (4) Controls, which were run in parallel with all experimental sections, indicated invariably that there was no nonspecific binding of second or third antibodies to the tissue sections. Thus, the entire available evidence indicates strongly that all immunofluorescence is associated with phytochrome. While adsorption of antibodies with highly purified antigen is often used as yet a n o t h e r control, this control is inappropriate here since its purpose is to determine whether after adsorption any antibodies remain that bind to the
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
tissue sections. Since adsorption by phytochrome of the immunopurified monoclonal antibodies used here would remove all antibody (Cordonnier et al. 1983), this control would be in fact less rigorous than that used here, which was the substitution of non-immune IgG for the monoclonal antibody. Conclusions
Firstly, we have demonstrated the use of sucroseembedded, cryosectioned tissue for indirect immunofluorescence staining with monoclonal antibodies to pea phytochrome and have determined that of those avaialble, 1I-18a4 is the most suitable for immunocytochemistry. Secondly, we have determined that an ELISA as a method for pre-selecting monoclonal antibodies is not always predictive but can give useful preliminary results. Thirdly, we have extended information about phytochrome localization to dicotyledonous tissue and described high concentrations of phytochrome in both cortical and guard cells. We have determined, as described previously for grass seedlings (Coleman and Pratt 1974a, b), that the subcellular localization of phytochrome in dark-grown pea tissue is diffuse rather than organelle-associated. Finally, we have described two phenomena that occur after R treatment of a dicotyledonous seddling. Phytochrome (as Pfr) initially becomes cryptic and, subsequently, a portion of the phytochrome pool becomes sequestered. These events are either prevented or reversed by F R and are indicative of changes associated with phytochrome phototransformation that are not solely a result of its form. The photoreversible change in the ability ofphytochrome to be bound by antibodies may indicate a rapid association of Pfr with a subcellular reaction partner after R irradiation. These observations, while not conclusive, are nevertheless consistent with the hypothesis that phytochrome binds to an intracellular receptor as an initial step in its mode of action. This work was supported by grants from the National Science Foundation (PCM80-22159 to L.H.P. and PCM81-04470 to B.A.P.). We thank Sally Kroehnke for darkroom assistance.
References Coleman, R.A., Pratt, L.H. (1974 a) Electron microscopic localization of phytochrome in plants using an indirect antibodylabeling method. J. Histochem. Cytochem. 22, 1039-1047 Coleman, R.A., Pratt, L.H. (1974b) Subcellular localization of the red-absorbing form of phytochrome by immunocytochemistry. Planta 121, 119-131 Cordonnier, M.-M., Pratt, L.H. (1982) Immunopurification and initial characterization of dicotyledonous phytochrome. Plant Physiol. 69, 360-365 Cordonnier, M.-M., Smith, C. Greppin, H., Pratt, L.H. (1983)
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Production and purification of monoclonal antibodies to Pisum and Arena phytochrome. Planta 158, 369-376 Epel, B.L., Butler, W.L., Pratt, L.H., Tokuyasu, K.T. (1980) Immunofluorescence localization studies of the Pr and Pfr forms of phytochrome in the coleoptile tips of oats, corn and wheat. In: Photoreceptors and plant development, pp. 121-133, De Greef, J., ed. Antwerpen University Press, Antwerpen Evans, A., Smith, H. (1976) Spectrophotometric evidence for the presence of phytochrome in the envelope membranes of barley etioplasts. Nature (London) 259, 323-325 Habermann, H.M. (i973) Evidence for two photoreactions and possible involvement of phytochrome in light dependent stomatal opening. Plant Physiol. 51, 543-548 Hendricks, S.B., Borthwick, H.A. (1967) The functiQn ofphytochrome in regulation of plant growth. Proc. Natl. Acad. Sci. USA 58, 2125-2130 Holmes, M.G., Klein, W.H. (1983) Photocontrol of stomatal movement. In: Photomorphogenesis in plants; (Book of Abstr.), p. 22, Sundqvist, C., ed. University of Lund Mackenzie, J.M., Jr., Coleman, R.A., Briggs, W.R.,iPratt , L.H. (1975) Reversible redistribution of phytochrom~ within the cell upon conversion to its physiologically activelform. Proc. Natl. Acad. Sci. USA 72, 799-803 Manabe, K., Furuya, M. (1975) Experimentally induced binding of phytochrome to mitochondrial and micrdsomal fractions in etiolated pea shoots. Planta 123, 207-2i5 Marm6, D. (1977) Phytochrome: membranes as possible sites of primary action. Annu. Rev. Plant Physiol. 28, 173-198 Marm~, D., Bianco, J., Gross, J. (]976) Evidence for phytochrome binding to plasma membrane and endopIasmic reticulum. In: Light and plant development, pp. 95-110, Smith, H., ed. Butterworths, London Mohr, H. (1977) Phytochrome and chloroplast development. Endeavor 1, 107-114 Palevitz B.A., O'Kane, D.J., Kobres, R.E., Raikhel, N.V. (1981) The vacuole system in stomatal cells of Allium. Vacuole movements and changes in morphologyi in differentiating cells as revealed by epifluorescence, video and electron microscopy. Protoplasma 109, 23-55 Petrusz, P. (1983) Essential requirements for the validity of immunocytochemical staining procedures. J.i Histochem. Cytochem. 31, 17%179 Pratt, L.H. (1973) Comparative immunochemistry of phytochrome, Plant Physiol. 51,203-209 Pratt, L.H. (1979) Phytochrome: function and pro.perties. Photochem. Photobiol. Rev. 4, 59-~24 Pratt, L.H. (1982) Phytochrome: the protein moiety. Annu. Rev. Plant Physiol. 33, 55%582 Quail, P.H., Marmfi, D., Sch/ifer, E. (1973) Particle-bound phytochrome from maize and pumpkin. Nature (Eondon) New Biol. 245, 189-191 Rubinstein, B., Drury, K.S., Park, R.B. (1969)Evidence for bound phytochrome in oat seedlings. Plant, Physiol. 44, 105-109 Singh, A.P., Srivastava, L.M. (1973) The fine structure of pea stomata. Protoplasma 76, 61-82 Smith, H. (1975) Phytochrome and photom0rphogenesis. McGraw-Hill, London Tokuyasu, K.T. (1980) Immunochemistry of ultrathin frozen sections. Histochem. J. 12, 181 203 Williamson, F.A., Morr6, D.J., Jaffe, M.J. (19751) Association of phytochrome with rough surfaced endoplasNic reticulum fractions from soybean hypocotyls. Plant Physiol. 56, 738-743 Received 20 April; accepted 3 October 1983
Planta (1983) 159: 545-553
9 Springer-Verlag 1983
Immunofluorescence visualization of phytochrome in Pisum sativum L. epicotyls using monoclonal antibodies Mary Jane Saunders 1'*, Marie-Mich+le Cordonnier 2, Barry A. Palevitz 1 and Lee H. Pratt 1 1 Department of Botany, University of Georgia, Athens, GA 30602, USA, and 2 Laboratoire de Physiologic v6g+tale, Universit6 de Gen+ve, Pavillon des Isotopes, 20 Bd. d'Yvoy, CH-1211 Gen6ve 4, Switzerland
We have investigated the cellular distribution of phytochrome in epicotyls of dark-grown pea (Pisum sativum L.) seedlings using monoclonal antibodies to pea phytochrome. Screening of the eight available antibodies both by an enzymelinked immunosorbent assay (ELISA) and by their ability to visualize phytochrome in situ by immunocytochemical fluorescence demonstrated that: (1) three antibodies work well for immunofluorescence; (2) none of the eight antibodies discriminates between the red- and the far-red-absorbing forms of phytochrome (Pr, Pfr) as assayed by ELISA; (3) the antigenicity of phytochrome is reduced by fixation with formaldehyde with respect to all eight antibodies; and (4) two antibodies that bind well to formaldehyde-fixed phytochrome as assayed by ELISA do not bind well to phytochrome in situ. Phytochrome is observed in both cortical and stomatal guard cells of the epicotyl and exhibits a homogeneous cytoplasmic distribution in non-irradiated tissue. After red-light (R) treatment phytochrome becomes transiently inaccessible to antibodies. If maintained in the Pfr form for J0 rain at room temperature before fixation, at least a portion of the phytochrome pool becomes accessible to antibodies and assumes a "sequestered" distribution. Both of these effects are almost entirely either prevented or reversed by subsequent far-red light treatment. We believe that the transient inaccessibility of phytochrome to antibodies after R irradiation is not a function of its conformational state. We suggest instead that R treatment rapidly induces an association of phyAbstract.
* Present address: Botany Department, Louisiana State Uni-
versity, Baton Rouge, LA 70803, USA Abbreviations: ELISA=enzyme-linked immunosorbent assay;
FR = far-red light; IgG = immunoglobulin G; Pfr = fa>red-absorbing form of phytochrome; Pr = red-absorbing form of phytochrome; R=red fight
tochrome with a subcellular component that interferes with antibody binding and that the "sequestered" areas represent a phytochrome pool that is distinct from both the diffusely distributed phytochrome in non-irradiated cells and from that phytochrome which is inaccessible to antibodies immediately after R irradiation. Key words: Immunosorbent assay (phymchrome) - Monoclonal antibody - Phytochrome (immuno-
fluorescence) - Pisum (phytochrome).
In~oducfion
Phytochrome is a photosensitive chromoprotein that mediates many developmental responses of plants to light (for recent reviews, see Smith 1975; Pratt 1979, 1982). The most promising hypothesis for its mode of action, albeit not definitively tested, is that phytochrome affects membrane activity after photoconversion from the physiologically inactive red-absorbing form (Pr) to the physiologically active far-red-absorbing form (Pfr) (Hendricks and Borthwick 1967; Marm6 1977). Attempts to obtain membrane-associated phytochrome in vitro and determine its subcellular distribution in vivo have resulted in the following observations. After centrifugation of a crude extract, 5-10% of the pigment is inherently associated with particulate subcellular fractions (Rubinstein et al. 1969). As much as 80%, however, can be induced to pellet with particulate fractions following photoconversion of Pr to Pfr (Quail et al. 1973). Analyses of these fractions, coupled with spectrophotometric assays, have led various researchers to describe phytochrome associations with ~he plasma membrane (Marm6 et al. 1976), endoplasmic reticulum (Williamson et al. 1975; Marm6 et al. 1976),
546
M.J. Saunders et al.: Immunofluorescence visualization of phytochrome in Pisum epicotyls
etioplast envelope (Evans and Smith 1976) and mitochondria (Manabe and Furuya 1975). Immunocytochemical analyses of phytochrome distribution in monocotyledonous plants using polyclonal rabbit antibodies to oat phytochrome have indicated a diffuse cytosolic distribution in dark-grown tissue (Coleman and Pratt 1974a, b), which changes after photoconversion of Pr to Pfr, the pigment becoming "sequestered" in numerous discrete areas about 1 Ixm in size (Mackenzie et al. 1975). The relationship of this sequestering phenomenon to the induction by red light (R) of pelletable phytochrome is unknown. It is possible that both are manifestations of an initial intracellular event in phytochrome-mediated development, namely, association of physiologically active Pfr with a receptor or reaction partner that is itself membrane bound. To learn more about the distribution and intracellular localization of phytochrome and thereby obtain information to test current hypotheses concerning its mode of action, as well as to extend immunocytochemical investigations of phytochrome localization to a dicotyledonous plant and thereby determine whether the previously described sequestering phenomenon might be of general interest, we have used recently developed monoclonal antibodies (Cordonnier et al. 1983) to visualize immunocytochemically phytochrome in epicotyls of etiolated pea seedlings. Monoclonal antibodies provide a greater degree of specificity than can be obtained with polyclonal antisera, leading to increased resolution of immunocytochemical observations. Reactivity of the eight monoclonal antibodies to both Pr and Pfr presently available, either fixed with formaldehyde or unfixed, is also determined by an enzyme-linked immunosorbent assay (ELISA).
Material and methods Plant tissue. Peas (Pisum sativum L., cv. Alaska; Leatherman's Seed Co., Canton, O., USA) were grown in darkness on cellulose packing material (Kimpak 62360; Kimberly-Clark Corp., Neenah, Wis., USA) at 25 ~ C in saturating humidity for 4-6 d. Until fixation was complete, tissue was handled only under green safe lights (Pratt 1973).
Light treatments. Whole seedlings were irradiated with monochromatic light obtained from a 45-W microscope lamp (Unitron Instruments, Plainview, N.Y., USA) equipped with B-40 interference filters (Balzers, Liechtenstein; 666 nm, 20-nm halfband width; 737 nm, 20-nm half-band width). A 20-s irradiation with either R or far-red light (FR) saturated photoconversion as determined by dual wavelength spectrophotometry. After irradiation, either 1-2-ram sections were excised from the hook region and fixed immediately, or the seedlings were
incubated in darkness at room temperature for 10 rain before excision and fixation.
Fixation procedure. All solutions were made in 0.1 M sodium phosphate, pH 7.4. The fixation schedule was as follows: fix overnight on ice in 4% formaldehyde, freshly prepared from p-formaldehyde; infiltrate 8 h at 4 ~ C with 1 M sucrose in 0.4% formaldehyde; rinse with 1.3 M sucrose; incubate 4 h at room temperature in 1.3 M sucrose; keep at 4 ~ C for not more than 4 d until secfior~ed. Sectioning. Sucrose-infiltrated tissue was trimmed, mounted on a copper block, and frozen rapidly by immersion into liquid nitrogen. Sections, 1 ~m thick, were cut at - 50 ~ C on a Sorval MT-2 ultramicrotome equipped with an FTS cryo-sectioning unit (Dupont, Wilmington, Del., USA). The sections were transferred to glass slides treated with Slipicone Release Spray (Dow Corning, Midland, Mich., USA) using the sucrose dropwire loop method of Tokuyasu (1980). At least six sections were placed on each slide and processed uniformly. Control sections were cut from adjacent areas of the same tissue block. Each experiment was repeated at least three times. Antibodies. All antibodies were prepared in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 140 m M NaCI, pH 7.4) containing 10% lamb serum (No. 200-6070; GIBCO Laboratories, Grand Island, N.Y., USA). Immunostaining involved the sequential application of three different antibodies. The first antibody was either an immunopurified monoclonal antibody to pea phytochrome, designated I-3b2, I-9a2, II-10a6, 1-11a12, 1-13b6, 1-15a3, 1-18al or II-18a4 (see Cordonnier et al. 1983 for details) or, as a control, non-immune mouse immunoglobulin G (IgG; No. 1-5381 ; Sigma Chemical Co., St. Louis, Mo., USA). The antibodies were used at concentrations of 150, 30 and 10 gg/ml for initial screening. Antibody II-18a4, as well as non-immune mouse IgG, was used at 10 gg/ml for all subsequent localization work presented here. The second antibody (used to amplify the signal) was immunopurified rabbit antibody to mouse IgG (see Cordonnier et al. 1983) and was used at 10 ~g/ml. The third antibody was rhodamine-conjugated goat antibody to rabbit IgG (No. 61-266; Miles-Yeda, Rehovot, Israel) and was used at 1 : 400 dilution. The concentrations of second and third antibody used were determined to be optimal by testing several dilutions.
Staining protocol. Labeling of the tissue sections was performed at room temperature by the following protocol: (1) 2% gelatin, 15rain; (2) PBS, 15rain; (3) lamb serum, 15rain; (4) PBS+ 10% lamb serum, rinse; (5) first antibody, 2 h; (6) PBS+ 10% lamb serum, 15 rain; (7) lamb serum, 15 min; (8) PBS+10% lamb serum, rinse; (9) second antibody, 45 rain; (10) PBS+ 10% lamb serum, rinse; (11) lamb serum, 15 rain; (12) PBS+ 10% lamb serum, rinse; (13) third antibody, 45 rain; (14) PBS, three changes of 15 rain each; (15) mount in 90% glycerol, 10% PBS. Lamb serum was used to block non-specific binding sites.
Microscopy. The sections were viewed on a Zetopan incident light fluorescence-phase contrast microscope (C. Reichert Optische Werke, Vienna, Austria). For fluorescence microscopy an HBO 200-W mercury lamp (Osram, West Berlin, Germany) was used in conjunction with a 4 2 546 exciter filter (broad band excitation peak at 546 nm) and a 3 0 G 590 barrier filter (C. Reichert). In addition, a 640-nm short pass filter (Ditric Optics, Hudson, Mass., USA) was placed at the top exit port of the microscope in front of the video camera. The fluorescence signal was amplified by a triple image intensifier vidicon camera
M.J. Saunders et al. : Immunofiuorescence visualization of phytochrome in Pisum epicotyls (Model 9004; Venus Scientific, Farmingdale, N.Y., USA) and viewed and photographed on a high-resolution video monitor (see Palevitz et al. 1981 for a complete description of the video equipment). Beam, signal gain, and black (pedestal) levels were kept constant for all experiments. Photographs were taken on Ilford FP-4 film and developed at ASA 1000 using K o d a k HC110 developer and Factor 8 (MinMax, N o r t h Hollywood, Calif., U S A ; company now defunct). Identical printing conditions were used for all micrographs taken with fluorescence optics. Magnifications of the video images were calibrated with a slide micrometer.
Enzyme-linked immunosorbent assay. The ELISA was carried out under green safe lights (see above) at room temperature. The procedure was as described by Cordonnier et al. (1983), with the following modification. The wells were coated with either 50 [al/well of 6 gg/ml purified pea phytochrome or, as a control for non-specific binding, an equivalent amount of bovine serum albumin (No. A9647; Sigma). Both were diluted either into borate-saline buffer or borate-saline buffer containing 4% formaldehyde. Formaldehyde was washed away upon completion of the initial coating step. Before dilution, the phytochrome was photoconverted to either Pr or Pfr using the light source and filters described above. Monoclonal antibodies were used at 10 gg/ml.
Results and discussion
Antibody screening. Of the eight monoclonal antibodies available, only three (II-10a6, 1-15a3 and II-18a4) are useful for immunocytochemistry at any of the concentrations tested, although they are all strongly positive when assayed against pea phytochrome by ELISA (Table 1). The inability of the remaining five antibodies to bind to phytochrome
547
in fixed tissue sections may result from different causes. The environment of phytochrome in situ may be such that one or more of the antibodies is sterically hindered from binding to it. Alternatively, fixation of the tissue may cross-link or otherwise change phytochrome in such a manner as to decrease its antigenicity. The ELISA data indicate that chemical fixation does reduce the ability of phytochrome to be recognized by all of the antibodies tested (Table i). The binding of antibodies I-3b2, I-%2 and 1-13b6 was reduced to very low levels, which correlates with the immunofluorescence results. The antibodies that work well for immunofiuorescence (II-10a6, I-15a3 and II-18a4) show a moderate level of' binding to fixed phytochrome. However, both I-I la12 and 1-18al display equal or greater binding to fixed phytochrome in the ELISA than II-18a4 but are immunocytochemically negative (Table 1). The lack of in situ binding of 1-11a12 and [-18al may result from differences in the intracellular environment affecting phytochrome accessibility that are not mimicked in an ELISA. The monoclonal antibody II-18a4 was best for immunocytochemical applications and was used at a concentration of 10 gg/ml for all subsequent localization work presented here.
Distribution of phytochrome in dark-grown non-iradiated tissue. Cortical cells demonstrate the l)ighest degree of phytochrome-associated fluorescence (Fig. I). Although epidermal and vascular ,=ells of
Table 1. Comparison of immunofluorescence and ELISA binding of monoclonal antibodies to pea phytochromea Antibody
Immunofluorescence b
ELISA activity *
Non-irradiated 150 I-3b2 I-9a2 II-10a6 1-11a12 1-13b6 I-t5a3 1-18al II-18a4 Non-immune mouse IgG
. + . . + +
30
20 s R 10
150
10
-
-
-
-
. --
-
-
.
. +
. .
++++ . .
. . + + . . ++++ .
. + . . + . . ++++ .
. . .
--
Pr
Pfr
Pr (fixed)
79 47 39 50 40 83 57 60
74 46 42 53 34 90 65 62
16 9 26 32 9 38 49 29
Pfr (fixed)
~16 ~9 " 31 36 10 29 46 32
BSA d
BSA (fixed)
2
3 2 2 1 2 2 4
.
" The pea phytochrome used for the ELISA was partially purified by brushite chromatography, a m m o n i u m sulfate fractionation, and diethylaminoethyl-agarose chromatography (see Pratt 1973; Cordonnier et al. 1983). The ratio of absorbance at 667 n m to 280 nm with phytochrome as Pr was 0.1 b Immunofluorescence activity was evaluated visually after immunostaining either non-irradiated tissue o r tissue irradiated with R for 20 s immediately prior to fixation. Antibodies were used at 150, 30 or 10 i~g/ml as indicated c ELISA activity is presented as absorbance at 400 nm for a 5-ram light path, x 100. Monoclonal antibodies were tested at 10 gg/ml d B S A = b o v i n e serum albumin
548
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
Figs. 1-3 (a, b). Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown non-irradiated pea cpicotyl tissue, demonstrating the distribution of phytochrome-associated fluorescence at the tissue level. Bar= 10 vLm; x 1000. Fig. 1, Pea tissue section treated with II-18a4 IgG (10 gg/ml). The epidermal cells (e) appear dark compared with the brightly fluorescent cortical ceils (e). Fig. 2. Pea tissue section treated with II-18a4 IgG (10 gg/ml). Cortical cells (c) are fluorescent while vascular tissue (v) is less bright. Fig. 3. Control pea tissue section treated with non-immune mouse IgG (10 gg/ml). Only slight fluorescence is discernable from cortical (c) and epidermal (e) cells. Print exposure and development conditions for b were the same as those for Figs. 1 b and 2b
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
549
Fig. 4a, b, Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown non-irradiated pea epicotyl epidermal cells treated with II-18a4 IgG (10 gg/ml). Note the bright phytochrome-associated fluorescence in guard cells (g). Bar = 10 gm; x 1500
tissue treated with antibody to pea phytochrome are somewhat brighter than those in control tissue treated with non-immune mouse IgG as the primary antibody, they are markedly less bright than cortical cells (Figs. 1-3). Most of the phytochrome associated with the hook region of pea epicotyls is in the cortical cells. Although most epidermal cells do not bind antibody well, stomatal guard cells treated with II18a4 IgG are brightly fluorescent (Fig. 4). Guard cells in control tissue are not fluorescent (data not shown). This demonstration of high phytochrome levels in guard cells is consistent with a possible role for phytochrome in stomatal action (Habermann 1973; Holmes and Klein 1983). Additionally, since pea guard cells contain chloroplasts (Singh and Srivastava 1973), the presence ofphytochrome may be related to its role in mediating chloroplast development (Mohr 1977). Phytochrome in non-irradiated pea cortical cells is restricted in its distribution to the cytosol (Fig. 5). There is no staining of plastids or vacuoles. Furthermore, the level of nuclear fluorescence is so low that it is difficult to determine whether it is indicative of the presence of phytochrome. The cytoplasmic distribution of phytochrome in non-irradiated pea tissue is similar to the pattern described previously for monocotyledonous tissues (Coleman and Pratt 1974a, b). While these observations correlate well with the fact that phytochrome acts as a soluble protein when extracted from dark-grown tissue (see Pratt 1979), they are also consistent with a possible association of phytochrome with a widely distributed
cytoplasmic matrix membrane system.
or
endoplasmic-reticuIum
Distribution of phytochrome in light-treated tissue. None of the monoclonal antibodies tested binds significantly to phytochrome in pea epicotyl tissue that was exposed to 20 s R and fixed immediately (Table 1). Similar results with both non-irradiated and R-irradiated tissues were obtained using polyclonal rabbit antiphytochrome antibodies that were immunopurified with a column of immobilized pea phytochrome (data not shown; see Cordonnier and Pratt 1982). The lack of phytochromerelated fluorescence in R-irradiated cells is therefore not a consequence of some specific property or properties of the monoclonal antibodies. The degree of fluorescence observed in R-treated cortical cells using antibody II-18a4 (Fig. 6) is approximately the same as that observed with epidermal ceils in non-irradiated tissue. Non-irradiated tissue processed in parallel as a control stains consistently with II-18a4 as described above (Fig. 5) and control R-irradiated tissue treated with non-immune mouse IgG does not stain (Table 1). If the R treatment was followed immediately by a 20-s F R treatment, most cells stained with II-18a4 as if they h a d never been irradiated (Fig. 7). Inl some instances all the cortical cells were brightly fluorescent but at other times a few cortical cells did not stain and resembled R-irradiated cells. A possible interpretation of these results is that these monoclonal antibodies are specific!for phytochrome in the Pr conformation and therefore do not bind Pfr in red-irradiated tissue. To test this
Figs. 5--7a, b. Paired phase-contrast (a) and fluorescence (b) micrographs of dark-grown pea epicotyl tissue treated with II-18a4 IgG (10 gg/ml). B a r = 10 gin; x 1000. Fig. 5. Intracellular distribution of phytochrome in cortical cells of non-irradiated tissue. Note the bright cytoplasm (cy), markedly reduced fluorescence in nuclei (nu) and plastids (arrows) and lack of fluorescence in vacuoles (v) and nucleoli (n). Fig. 6. Cortical cells of pea tissue exposed to 20 s R and fixed immediately at 0 ~ C. Note reduced fluorescence in cytoplasm (ey) (compare with Fig. 5). Nuclei (nu) and vacuoles (v) are still discernable as darker regions. Print exposure and development conditions were the same as in Fig. 5. Fig. 7. Cortical and epidermal cells of pea tissue exposed sequentially to 20 s R, 20 s F R and then fixed immediately at 0 ~ C. Note that bright cortical cells (e) and dark epidermal cells (e) exhibit a fluorescence pattern similar to that of non-irradiated tissue (Figs. 1, 5)
M.J. Saunderset al. : Immunofluorescencevisualizationof phytochromein Pisumepicotyls interpretation, we compared by ELISA phytochrome treated in four ways: (1) Pr; (2) Pfr; (3) fixed Pr; (4) fixed Pfr. None of the antibodies to pea phytochrome discriminate between Pr and Pfr, whether fixed or unfixed (Table 1). These data are consistent with the previously noted observation that polyclonal rabbit antibodies to pea phytochrome, which would not be expected to discriminate between Pr and Pfr (based upon previous work with comparable antisera to oat phytochrome; Pratt 1973), do not detect the pigment when tissue is fixed immediately after R irradiation. We therefore conclude that a conformational change of phytochrome is not solely responsible for the loss of the ability of the monoclonal antibodies to recognize this pigment in R-irradiated tissue. An alternative explanation is that there has been a change in the molecular environment of phytochrome within the cortical cells after R treatment that either blocks or obscures the antigenic sites on the chromoprotein and causes it to become "cryptic." These results cannot be considered as definitive evidence for binding of phytochrome to a receptor after R treatment; however, that is one possible interpretation. If a 10-rain dark incubation before fixation follows the R treatment, small but discrete fluorescent areas become visible within the cytoplasm of cortical cells (Fig. 8). The epidermal and vascular tissues look similar to R-treated tissue that was immediately fixed as well as to non-irradiated tissue. The bright regions in cortical cells do not correspond to any organelle recognizable by phase contrast microscopy. These fluorescent areas are similar in size and distribution to the "sequestered phytochrome" regions described for oat tissue (Mackenzie et al. 1975). Whether or not "cryptic" phytochrome remains cannot be determined. The physiological role of these sequestered regions in oats is not known but the appearance of similar areas in pea tissue after R treatment indicates that it may well be a general phenomenon. In addition, these latter observations (Fig. 8) indicate that II-18a4 IgG can bind to phytochrome in the Pfr form in situ. The ability of the antibody to bind phytochrome in these "sequestered" regions may also indicate that the presumed association between phytochrome and another subcellular component produced immediately after R treatment does not exist in the sequestered regions. These regions may represent a new phytochrome pool that appears only over time after photoconversion to Pfr. If tissue is irradiated for 20 s with R, incubated
551
in darkness for 10 rain at room temperature, irradiated for 20 s with FR and then fixed immediately, most or all of the cortical cells again exhibit fluorescence similar to that found in non-irradiated tissue (in contrast to previous observations with oats; Mackenzie et al. 1975). A few cells retain the sequestered distribution that is seen with R-treated tissue that is incubated in darkness before fixation (Fig. 9). It is difficult to determine if the cells that have very bright cytoplasmic fluorescence have retained sequestered regions within the cytoplasm. Therefore, one cannot be certain whether the diffusely distributed phytochrome visible after FRtreatment arises from the cryptic pool or from the sequestered pool. This result may be indicative, however, of multiple pools of phytochrome after light treatment. It is possible that in the R-treated cells that display the sequestering phenomenon we are not visualizing all the phytochrome, but instead some remains both cytoplasmic and cryptic. FR treatment would then make this cryptic pool available again for binding by antibodies. While there are, as already noted, some obvious similarities between the present observations and those obtained previously with monocotyledonous plants (Coleman and Pratt 1974a, b; Mackenzie et al. 1975), there are also some apparent differences. Phytochrome is found in nonirradiate,d cells to be diffusely distributed throughout the cytosol both in the dicotyledonous plant studied here (Fig. 5) and in the grass seedlings previously investigated (Coleman and Pratt 1974a, b). The inaccessibility of phytochrome to antibodies described here for cells fixed immediately after R (Fig. 6), however, has not been reported for grass seedlings. To determine whether the difference is real or apparent will require that the experimental protocol used here also be used with monocotyledonous plants. The sequestering of Pfr described here (Fig. 8) is also similar to that described previously for grass seedlings (Mackenzie et al. 1975). A potential difference with the earlier observations, however, is the apparent rapid F R reversibility (Fig. 9). As just discussed, however, it is not possible to discriminate between an effect of FR on reversal of sequestering as opposed to an effect on reversal of "cryptic" phytochrome that may remain even in the presence of the sequestered phytochrome.
Specificity of the immunocytochernical staining procedure. Demonstration of immunocytochemical specificity for the antigen of interest is a major concern (see Petrusz t983 for recent discussion). Several arguments indicate that we can have a high
552
M.J. Sauttders et al. : Immunoftuorescencevisualization of phytochrome in Pisum epicotyls
Figs. 8, 9a, b. Paired phase contrast (a) and fluorescence (b) micrographs of dark-grown pea epicotyl tissue treated with II-18a4 IgG (10 gg/ml). Bar=10 gin; x 1000- Fig. 8. Cortical cells of pea tissue treated with 20 s R, 10 rain dark incubation and then fixed at 0~ C. Note the bright "sequestered" regions (arrows) visible in the cytoplasm but absent in the nuclei (nu) and plastids (p). Fig. 9. Cortical cells of pea tissue treated with 20 s R, 10 rain dark incubation, 20 s FR and then fixed at 0~ C. Note the bright cytoplasmic (cy) fluorescence in many cells, similar to that seen in non-irradiated cells (Figs. 1, 5), or bright "sequestered" regions (arrows) in other cells, similar to those seen in R-irradiated, dark incubated cells (Fig. 8) degree o f confidence in the immunospecificity o f the observations presented here. (1) While not emphasized above, it is i m p o r t a n t to note that the three m o n o c l o n a l antibodies, as well as the immunopurified polyclonal antibodies, all yield the same results. It is unlikely that all would give rise to the same i m m u n o s t a i n i n g artifact. (2) Since the immunostaining results are strongly and rapidly influenced in photoreversible fashion by R and F R , it is again most likely that p h y t o c h r o m e itself is the only antigen being visualized. (3) Since the m o n o c l o n a l antibodies have all been shown to be directed to p h y t o c h r o m e (Cordonnier et al. 1983), the question of antigenic nonspecificity does not
arise as it does with a polyclonal preparation, even though the possibility of staining an immunologically cross-reacting antigen c a n n o t be excluded by this consideration. (4) Controls, which were run in parallel with all experimental sections, indicated invariably that there was no nonspecific binding of second or third antibodies to the tissue sections. Thus, the entire available evidence indicates strongly that all immunofluorescence is associated with phytochrome. While adsorption of antibodies with highly purified antigen is often used as yet a n o t h e r control, this control is inappropriate here since its purpose is to determine whether after adsorption any antibodies remain that bind to the
M.J. Saunders et al. : Immunofluorescence visualization of phytochrome in Pisum epicotyls
tissue sections. Since adsorption by phytochrome of the immunopurified monoclonal antibodies used here would remove all antibody (Cordonnier et al. 1983), this control would be in fact less rigorous than that used here, which was the substitution of non-immune IgG for the monoclonal antibody. Conclusions
Firstly, we have demonstrated the use of sucroseembedded, cryosectioned tissue for indirect immunofluorescence staining with monoclonal antibodies to pea phytochrome and have determined that of those avaialble, 1I-18a4 is the most suitable for immunocytochemistry. Secondly, we have determined that an ELISA as a method for pre-selecting monoclonal antibodies is not always predictive but can give useful preliminary results. Thirdly, we have extended information about phytochrome localization to dicotyledonous tissue and described high concentrations of phytochrome in both cortical and guard cells. We have determined, as described previously for grass seedlings (Coleman and Pratt 1974a, b), that the subcellular localization of phytochrome in dark-grown pea tissue is diffuse rather than organelle-associated. Finally, we have described two phenomena that occur after R treatment of a dicotyledonous seddling. Phytochrome (as Pfr) initially becomes cryptic and, subsequently, a portion of the phytochrome pool becomes sequestered. These events are either prevented or reversed by F R and are indicative of changes associated with phytochrome phototransformation that are not solely a result of its form. The photoreversible change in the ability ofphytochrome to be bound by antibodies may indicate a rapid association of Pfr with a subcellular reaction partner after R irradiation. These observations, while not conclusive, are nevertheless consistent with the hypothesis that phytochrome binds to an intracellular receptor as an initial step in its mode of action. This work was supported by grants from the National Science Foundation (PCM80-22159 to L.H.P. and PCM81-04470 to B.A.P.). We thank Sally Kroehnke for darkroom assistance.
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