detection and quantitation of three phytochromes in unimbibed seeds of: avena sativa l

Photochemistry and Photobiology Vol. 56, No. 5, pp. 709-716, 1992 Printed in Great Britain. All rights reserved

0031-8655/92 $05.00+0.00 Copyright @ 1992 Pergamon Press Ltd

DETECTION AND QUANTITATION OF THREE PHYTOCHROMES IN UNIMBIBED SEEDS OF

Avena sativa L.

Yu-CHIE WANG, MARIE-MICHBLE CORDONNIER-PRATT and LEE H. PRATT* Botany Department, University of Georgia, Athens, GA 30602, USA

(Received 5 February 1992; accepted 30 April 1992)

Abstract-Three phytochrome apoproteins in unimbibed seeds of Avena saliva L. were identified with monoclonal antibodies directed to, and specific for, three oat phytochromes with monomeric molecular masses of 125, 124 and 123 kDa [Wang et al.,1991, Planta 184, 96-1041. All three phytochromes were readily detected in embryo-containing portions. Only trace amounts were found in endosperm tissue. Phytochrome photoreversibility was detected after concentration and partial purification of embryo extracts by fractionation with ammonium sulfate, indicating that at least one of these seed phytochromes had its chromophore prosthetic group bound to it. Immunoblot analyses were performed to quantitate each of the three phytochromes in unimbibed seeds. Quantitation of phytochromes in detergent-free extracts led to serious underestimates of phytochrome contents in the unimbibed seeds. In contrast, more than 93% of each of the three phytochromes in the unimbibed seeds was extracted when a modified sodium dodecyl sulfate sample buffer was used as the extraction medium. In such extracts, we measured per embryo 1.40 * 0.12. 1.60 & 0.05 and 6.13 * 0.31 ng of 125-, 124- and 123-kDa phytochrome, respectively.

INTRODUCTION

The discovery of phytochrome resulted in large part from the study of photoreversibly regulated germination of lettuce seeds (Borthwick et a l . , 1952). Since then, a number of studies have reported the presence of phytochrome in seeds of a variety of plant species (see Frankland and Taylorson, 1983; Cone and Kendrick, 1986, for reviews). This photoreceptor is known to play a role in regulating the germination of not only light-sensitive seeds, but also some seeds that germinate in darkness. In the latter cases, far-red light was found to inhibit the germination of otherwise light-insensitive seeds and this inhibitory effect could be at least partially reversed by red light (Kendrick and Frankland, 1969; Spruit and Mancinelli, 1969; Taylorson, 1975; Hou and Simpson, 1990).

During imbibition, seed phytochrome appears in two phases. The first is correlated with hydration of seed proteins, while the second begins several hours after the onset of imbibition (Kendrick et al . , 1969; Zouaghi et a l . , 1972; Hilton and Thomas, 1985). It was suggested that the first phase results from rehydration of pre-existing seed phytochrome, while the second results from the de novo synthesis of phytochrome during imbibition. The pre-existing seed phytochrome is of particular interest because it is probably the phytochrome that regulates seed

*To whom correspondence should be addressed. tAbbreviations: MAb, monoclonal antibody; PAGE, poly-

acrylamide gel electrophoresis; Pfr Pr, far-red- and red- absorbing-form of phytochrome, respectively; SDS, sodium dodecyl sulfate; Tris, 2-amino-2-(hydroxymethyl)-l,3-propanediol.

germination (Adamse et al . , 1988). Moreover, this pre-existing seed phytochrome has been reported to regulate the subsequent de novo synthesis of phytochrome itself during seed germination (Hilton and Thomas, 1987; Thomas et al . , 1989). However, the biochemical status of pre-existing seed phytochrome is still controversial, even from studies of the same plant species. In oats, Hilton and Thomas (1985, 1987) reported that the well-characterized 124-kDa phytochrome is absent from seeds prior to 16 h after onset of imbibition. In contrast, Tokuhisa and Quail (1987) reported that two types of phytochrome, including the 124-kDa type, are detectable in both quiescent and germinating oat seeds. In peas, Konomi et al. (1985) initially reported that there is neither spectrophotometrically nor immunochemically detectable phytochrome in unimbibed embryonic axes. However, in a later study (Konomi et al . , 1987), they found both the so-called type I and type I1 phytochromes in unimbibed embryonic axes.

Recently, we employed newly produced monoclonal antibodies (MAb)t to characterize phytochromes in oat seedlings and demonstrated that they contain at least three distinct phytochromes (Wang et al., 1991). Here we use these MAb to investigate phytochromes in unimbibed oat seeds and to help resolve the apparent discrepancies in the literature.

MATERIALS AND METHODS

Seed phytochrome exfraction. Oat seeds (Avena sativa L., cv. Garry; Agriculver, Trumansburg, NY, USA) were dehusked and dissected into embryo-containing and embryo-free portions. The embryo-containing portion contains both the embryonic axis and the scutellum. while the embryo-free portion contains primarily endosperm.

709

710 Yu-CHIE WANG et al.

To extract phytochromes from the embryo-containing portions of unimbibed oat seeds, two different protocols were used. The first utilized a detergent-free buffer. Fifty freshly prepared embryo-containing portions were placed in a mortar, to which was added 2.5 mL of 50 mM 2- amino-2-(hydroxymethyl)-l,3-propanediol(Tris)-CI, pH 8.5 at 4°C. 125 pL of 0.2 M iodoacetamide (No. 1-6125; Sigma Chemical Co., St Louis, MO, USA) in 50 mM Tris-CI, pH 7.8 at 4"C, and 25 pL of 0.2 M phenylmethylsulfonyl fluoride (No. P-7626; Sigma) in isopropanol. The tissue was ground thoroughly with a pestle. The homogenate was centrifuged for 30 min at 39000 g and 4°C. The resultant supernatant was filtered through one layer of Miracloth (No. 475855; Calbiochem, San Diego,CA, USA) to remove overlaid lipoidal material. The second protocol utilized sodium dodecyl sulfate (SDS) sample buffer extraction, in which case 50 lyophilized embryo- containing portions (ca 0.14 g) were mixed in a mortar with 3.45 mL. of modified SDS sample buffer [125 mM Tris-CI, pH 6.8 at room temperature, 4% (wtlvol) SDS, 10% (vollvol) 2-mercaptoethanol, 20% (vollvol) glycerol; see Vierstra et al., 19841 at 100°C. The mixture was ground thoroughly with a pestle, after which the homogenate was heated in a boiling water bath for 5 min and then centrifuged at 16000 g for 10 min. The resultant supernatant was recovered and clarified again at 16000 g for 10 min. In order to examine the recovery of phytochromes by this extraction, the pellet from the first centrifugation was extracted and prepared a second time as described above, this time with 1.15 mL of modified SDS sample buffer.

To extract phytochromes from the embryo-free portions of unimbibed seeds, 50 freshly prepared embryo-free cary- opses were mixed in a mortar with 3.5 mL of 50 mM Tris-CI, pH 8.5 at 4"C, 175 pL of 0.2 M iodoacetamide in 50 mM Tris-CI, pH 7.8 at 4"C, and 35 pL of 0.2 M phenylmethylsulfonyl fluoride in isopropanol. The tissue was then extracted and prepared as in the first protocol described above.

All detergent-free extractions were done under dim green light and samples were kept on ice except where otherwise stated.

Protein quantitation. For all detergent-free extracts, protein contents were determined by the method of Brad- ford (1976) using bovine serum albumin as a standard. To quantitate protein content in the extracts prepared with modified SDS sample buffer, acetone was first added to samples to a final concentration of 80% (vol/vol). After 1 h incubation at -20°C samples were centrifuged at 16000 g for 10 min at room temperature. The precipitated proteins were resuspended in 2% (wt/vol) Na,CO,, 0.4% (wtlvol) NaOH, 0.16% (wtlvol) NaK-tartrate, 1% (wtlvol) SDS and quantitated by the method of Lowry et al. (1951), as modified by Markwell et al. (1978).

Hydroxyapatite-purijied phytochromes. Phytochromes in the shoots of 4-day-old, etiolated and of 10- to ll-day- old, greenhouse-grown oats were extracted, fractionated with poly(ethy1enimine) and ammonium sulfate, and chro- matographed through hydroxyapatite as before (Pratt et al., 1991b). The absolute amount of phytochrome was estimated spectrophotometrically as before (Pratt et al., 1991 b) .

Photoreversibility of seed phytochromes. To measure the photoreversibility of seed phytochromes, 500 or 1000 embryo-containing portions of unimbibed seeds were extracted into detergent-free buffer as described above using proportionately greater volumes of 50 mM Tris buffer and protease inhibitors. Saturated ammonium sulfate solution (pH 7.8 and 4°C) was added to these crude extracts to a final concentration of 48% of saturation. After stirring for 3 min and centrifugation at 39000 g and 4°C for 30 min, the resultant pellets were resuspended in 5 mL of 50 mM Tris-CI, pH 7.8 at PC, and clarified

by centrifugation at 39000 g and 4°C for 10 min. These clarified samples were used to measure photoreversible absorbance changes (AM) with a custom-built dual-wavelength spectrophotometer similar to that described before (Pratt et al., 1985). Measuring wavelengths were either 653 and 728 nm, or 666 and 728 nm.

Antibodies. MAb GO-4 and GO-7, directed to 125- and 123-kDa phytochromes in oat seedlings, have been recently characterized (Pratt et al . , 1991c; Wang et al., 1991). MAb Oat-8, Oat-13, Oat-22, Oat-25 and Oat-28 are directed to 124-kDa phytochrome from etiolated oat shoots and detect five spatially separated epitopes on this protein (Pratt et al., 1988; Thompson et al., 1989).

Immunoprecipitation. To each 500-pL microcentrifuge tube was added 2CO p L of detergent-free embryo extract (equivalent to the extract from 5.7 embryos), 10 pg of MAb (GO-4 or Oat-22) in 10 mM sodium phosphate, 140 mM NaCI, pH 7.4 (PBS), or non-immune mouse IgGl (No. M-9261; Sigma), and 25 pL of blocking solution [PBS supplemented with 1% (wtlvol) bovine serum albumin and 0.02% (wtlvol) sodium azide]. The final volume was brought to 275 pL with 50 mM Tris-CI, pH 7.8 at 4°C. These mixtures were incubated overnight at 4°C with constant, gentle agitation in darkness or occasionally under dim green light. The next morning, 150 pL of a 10% (wt/vol) suspension of prewashed, formalin-treated Staphylococcus aureus (No. P-7155; Sigma) in 0.1 M boric acid, 25 mM sodium borate, 75 mM NaCI, pH 8.5, was added to each tube and incubated for 1 h at 4°C in darkness. The incubated mixtures were centrifuged at 16000 g for 10 min and then separated into supernatant and precipitate fractions. Proteins in the supernatant fractions, which were first precipitated by acetone as described above, and in the precipitate fractions were each mixed with 100 pL of 62.5 mMTris-CI, pH 6.8 at room temperature, 5% (vollvol) 2-mercaptoethanol, 2% (wtlvol) SDS, 10% (vollvol) glycerol, 0.001% (wtlvol) bromophenol blue (Laemmli, 1970). After heating in a boiling water bath for 3 min, followed by centrifugation at 16,000 g for 10 min at room temperature, the clarified samples were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Electrophoresis, electroblotting and immunostaining. SDS-PAGE, electroblotting and immunostaining were performed as before (Wang et al., 1991). All MAb, as well as non-immune mouse IgG, were applied to blots at 3 pg mL-', except where noted otherwise in the figure legends.

Densirometry. Immunostained blots were scanned and analyzed with a commercial image analysis system (Analytical Imaging Concepts, Irvine, CA. USA), which consists of a video camera and monitor, a camera control unit, an IBM-compatible 80386 personal computer and operating software (IM4000). This system is designed for use with a microscope. To adapt it to our purpose, a Zuiko 50-mm auto-macro lens (Olympus Optical Co., Tokyo, Japan) was mounted on the video camera. When scanning a blot, the average densities of phytochrome bands of known and unknown quantities were measured using a "window" of constant size. The same "window" size was also used to measure the average density of the nitrocellulose immediately below each phytochrome band. These background densities were then subtracted from the measured densities of corresponding phytochrome bands. For each sample, three independent blots with 3 replicates on each blot (9 replicates total) were measured. Data are presented as means t standard errors.

RESULTS

Three phytochromes in unimbibed seeds Detergent-free extracts of embryo-containing a n d

embryo-free portions of unimbibed seeds were ana-

Three phytochromes in unimbibed seeds 711

Em En Em En Em En Em En GO-4 0-22 NIM S E P S E P

GO-4 0-22 GO-7 NIM

Figure 1. Immunoblot analysis of phytochromes in embryo (Em) and endosperm (En) portions of unimbibed oat seeds. Protein extracted without detergent from 0.39 embryos (Em) and 0.39 endosperms (En) were subjected to 10% SDS PAGE, electroblotted and immunostained by GO-4, Oat-22 (0-22), GO-7 and non-immune mouse

IgG (NIM).

lyzed on the basis of equal organism number (0.39), which serendipitously contained comparable amounts of proteins (46.6 pg per embryo-containing portion vs 49.4 pg per embryo-free portion). The immunoblot (Fig. 1) shows that each of GO-4, Oat- 22 and GO-7 readily detects a phytochrome apoprotein in the extract of embryo-containing portions. At the sample load used here, GO-4 and possibly GO-7 stain phytochrome at best only very weakly in the extract of embryo-free portions (Fig. 1).

While we have demonstrated previously that these three MAb detect three distinct phytochromes in oat seedlings (Wang et al., 1991), it is possible that unimbibed seeds have a unique phytochrome that contains epitopes recognized by any two or all three MAb used here. To determine whether this is the case, phytochromes in a detergent-free extract of embryo-containing portions were immunoprecipitated with GO-4 or Oat-22. GO-7 is not suitable for this application (Pratt et al., 1991c) and therefore was not included. The resultant supernatants and precipitates were then separated and analyzed by immunoblotting with three MAb (GO-4, Oat-22 and GO-7) and non-immune mouse IgGl (Fig. 2). GO-4 and Oat-22 each detect the phytochrome immunoprecipitated by itself, but not that immunoprecipitated by the other MAb. GO-7 does not detect phytochrome in either immunoprecipitate. On the inverse, GO-4 stains phytochrome remaining in the supernatant after immunoprecipitation by Oat-22, while Oat-22 stains what remains in the supernatant after immunoprecipitation by GO-4. As anticipated (Wang et al . , 1991), GO-7 stains phytochrome in both supernatants. None of the three phytochromes appears in the pellet when non- immune mouse IgGl is used as a control.

Is one of the three phytochromes in unimbibed seeds the same as that which is abundant in etiolated oat shoots?

As already noted there are conflicting data con- cerning the presence in unimbibed oat seed of the

Figure 2. Immunoblot analysis followine immunoDreciDitation

S E P GO-4

GO-?

0-2 2

NIM

of embryo phytochromes bv GO-4. Oat-22 (0-22)

and non'limmune mous; IgGl (NiM), as indicated'at the top. After addition of and incubation with Sruphylococcus uureus, each sample was separated by centrifugation into supernatant (S) and precipitate (P) fractions. Following 10% SDS PAGE, proteins equivalent to those from 0.38 embryos were electrotransferred onto nitiocellulose and, as indicated at the right, were immunostained by GO-4, GO-7, Oat-22 and non-immune mouse IgG1. As a positive control, unfractionated embryo extract (E) was included.

124-kDa phytochrome that is abundant in etiolated seedlings. Consequently, extracts of embryo-containing portions of unimbibed oat seeds were subjected to immunoblot analysis with five MAb that detect 124-kDa phytochrome but not 123- and 125- kDa phytochromes (data not shown; Shimazaki and Pratt, 1985). Each of these five MAb detected phytochrome in such extracts (Fig. 3). Because of (i) the heavy protein load applied to the gel, (ii) the fact that phytochrome is a very minor component of the protein applied, and (iii) the relatively long

M 8 13 2225 28 Figure 3. Immunoblot analysis of 124-kDa phytochrome in a detergent-free extract of embryo-containing portions of oat seeds. For each lane embryo proteins from 0.36 organisms were separated by 10% SDS PAGE and electrotransferred onto nitrocellulose. Individual strips were immunostained with one of five MAb directed to 124-kDa phytochrome [Oat-8 (8), Oat-13 (13), Oat-22 (22), Oat- 25 (25) and Oat-28 (28)J and with non-immune mouse IgG (M). The arrowhead indicates a line drawn on the nitrocellulose before cutting strips for immunostaining. This line permits precise repositioning of the strips after

staining.

712 Yu-CHIE WANC et al.

incubation in substrate solution, other polypeptides were also stained, especially in the case of Oat-13. Nonetheless, it is evident that each MAb stains well a polypeptide the size of phytochrome.

Phytochrome photoreversibility in extracts of unimbibed seeds

Detergent-free extracts of embryo-containing portions of unimbibed seeds, even after fractionation with ammonium sulfate, were too turbid and contained too little phytochrome to permit accurate spectrophotometric quantitation of phytochrome in them. Moreover, as shown below, phytochrome yields resulting from detergent-free sample preparation were unacceptably low. Nevertheless, a AM of about 0.002 per 1000 embryo-containing portions is obtained when the extract is assayed in a cuvette with a 5-cm path length, indicating that at least one of the seed phytochromes is immediately photoreversible upon hydration.

Phytochrome quantitation in unimbibed seeds

In preliminary experiments phytochrome contents in detergent-free and in SDS sample buffer extracts were compared by immunoblot assay. It is evident that SDS sample buffer extracts contain consider- ably more of each of the three phytochromes than do aqueous extracts (Fig. 4; see also below) indicating that the latter are unsuitable for phytochrome quantitation. To determine how much of each of the three phytochromes was recovered by a single SDS sample buffer extraction, the pellet obtained upon clarification of the initial extract was extracted a second time with SDS sample buffer. The initial and second extracts were found to contain 93 and 770, respectively, of total extractable protein. Since

0.4 0.2 S A S A

GO- 4 - . -

c

c

0- 22

GO-7

Figure 4. Immunoblot analysis of phytochromes in detergent-free and SDS sample buffer extracts of embryo-containing portions of oat seeds. Phytochromes were extracted with either detergent-free buffer, followed by fractionation with 48% saturation ammonium sulfate (A), or with SDS sample buffer (S). Protein from 0.2 and 0.4 embryos was subjected to 7.5% SDS PAGE, transferred onto nitrocellulose, and immunostained with GO-4, GO-7, Oat-22 (0- 22, applied at 0.3 pg mL-’), and non-immune mouse

IgGl (NIM).

1 2

GO -4

0-22

GO-7

N I M 93 7 ( % I

Figure 5 . Comparative evaluation of the contents of the three phytochromes in first and second extracts of ernbryo- containing portions of oat seeds. Lyophilized embryo- containing portions of unimbibed oat seeds were extracted twice with SDS sample buffer. Equal protein amount (50 pg) from the first (1) and the second (2) extracts were separated by 7.5% SDS PAGE and electroblotted onto nitrocellulose. Strips were immunostained with GO-4, GO-7, Oat-22 (0-22, 0.3 p,g mL-’) and non-immune mouse IgG (NIM). The numbers at the bottom indicate the percentage of total extractable protein in the two

extracts.

the initial extract contains more of each of the three phytochromes than does the second on an equal protein basis (Fig. 5), it is concluded that the initial extract recovered more than 93% of each of the three phytochromes. Thus, a single SDS sample buffer extraction was used for phytochrome quantitation.

Phytochrome standards were prepared from detergent-free extracts of 4-day-old etiolated or 10- to 11-day-old greenhouse-grown oat seedlings purified through hydroxyapatite chromatography. The overwhelming majority of phytochrome in etiolated oats is the 124-kDa type (Tokuhisa and Quail, 1987; Pratt et al., 1991a). For the preparation of the 124- kDa phytochrome standard it was therefore assumed that all of the phytochrome is of this type. Conversely, 124-kDa phytochrome is virtually absent from greenhouse-grown oats (Pratt et al., 1991a). Thus, we assumed that all phytochrome in the partially purified extract from such oats was either 123 or 125 kDa in size. The proportion that is 125-kDa phytochrome was determined by immunoprecipitation with a saturating quantity of GO-4 (data not shown; see also Fig. 1 in Pratt et al., 1991~). It was assumed that all phytochrome precipitated by this MAb was of the 125-kDa type. It is not known whether there are additional phytochromes in an oat plant. We therefore assumed that the phytochrome not precipitated by GO-4 was the 123-kDa type. If either of the previous assumptions should prove later to be wrong, it would then only be necessary to recognize that what is called here 123- andor 125-kDa phytochrome is, in fact, the sum of two (or more) phytochromes, or that an as yet unidentified phytochrome has simply gone undetected.

For immunoblot quantitation of seed phyto-


5.0 S 2.5 A 1.25 S .63 A .31 S .16 A .08 (W) GO-4

0-22

GO-7

Figure 6. Immunoblot quantitation of three phytochromes in detergent-free and SDS sample buffer extracts of embryo-containing portions of oat seeds. Phytochromes were extracted with either detergent-free buffer, followed by fractionation with 48% saturation ammonium sulfate (A), or with SDS sample buffer (S). A 2-fold dilution series of known quantities (from 5 to 0.08 ng) of 125-, 124-, and 123-kDa phytochromes were prepared from hydroxyapatite-purified phytochromes from either 4-day- old etiolated or 10- to 11-day-old greenhouse-grown oats. Protein equivalent to that from either 0.15 (SDS sample buffer extract) or 0.4 (detergent-free extract) organisms and the indicated quantities of each of the three standards ( 5 , 2.5, 1.25, 0.63, 0.31, 0.16 and 0.08 ng) were separated by 7.5% SDS PAGE and electrotransferred onto nitrocellulose. Blots were immunostained with GO-4, Oat-22 (0- 22, applied at 0.3 pg mL-’) or GO-7, which detect 125-, 124- and 123-kDa phytochrome, respectively. An additional blot containing 5 ng of 12.5, 124- and 123-kDa phytochrome in different lanes was immunostained at the same time with non-immune mouse IgG; no bands were

observed (blot not shown).

chromes, embryo extracts were analyzed on the same blot as serial dilutions of known quantities of the three phytochrome standards (Fig. 6). It was evident in these blots that the partially purified phytochromes used as standards reveal multiple bands, while the phytochromes in the seed extracts appear as single bands corresponding to the band

40 t

a V d (d n

I I I I I 0.1 0.33 1 3.3 10

Phytochrome amount (ng) Figure 7. Representative standard curves for immunoblot quantitation of three phytochromes. Immunostained phytochrome bands such as those in Fig. 6 were scanned with an image analysis system. Relative band densities were determined as a function of the amount of 125- kDa phytochrome (125-kDa), 124-kqa phytochrome (124- kDa), or 123-kDa phytochrome (123-kDa) electrophor- esed. Data are averages from three independent blots, presented as means 2 standard errors. Relative densities of bands derived from embryo extracts were all within the

ranges of these standard curves.

Table 1. Video densitometric quantitation of three phytochromes in immunoblots of SDS sample buffer and detergent-free extracts of embryo-containing portions of oat seeds. Detergent-free extracts were fractionated with

ammonium &fate at 48% of saturation

Phytochrome amount (ng embryo-’ * SE)

Phytochrome size SDS Detergent-free ( k W

125 1.40 i 0.12 0.38 5 0.03 124 1.60 & 0.05 0.83 2 0.04

6.13 2 0.31 1.75 2 0.07 123

of highest molecular mass for the corresponding protein standard. The reason for this observation is that the seed extracts were prepared by a protocol intended to prevent post-homogenbation modification, while the partial purification of the standards leads to degradation by endogenous proteases (Vierstra et af., 1984; Wang et al., 1991). To accom- modate this partial proteolysis of the standards, the area included within the “window” of the video densitometer was adjusted for each phytochrome type to be large enough to encompass not only the area of undegraded phytochrome but also the band(s) of degraded phytochrome immediately below. Unknown samples were loaded such that the amounts of the three phytochromes were within the ranges of the standard curves as determined by video densitometry (Fig. 7). Averaged results from a total of nine independent blots document that 123-kDa phytochrome is most abundant, while 124- and 125-kDa phytochromes are present in roughly equivalent amounts (Table 1).

DISCUSSION

Most previous immunochemical studies of phytochrome in light-grown oats have relied upon antibodies directed to phytochrome from etiolated shoots (Tokuhisa et al., 1985; Shimazaki and Pratt, 1985, 1986; Cordonnier et al., 1986; Tokuhisa and Quail, 1987, 1989). Because ‘these antibodies react well with phytochrome from dark-grown tissues, but poorly or not at all with phytochrome from light- grown tissues, it was concluded that the phytochromes predominating in the two types of tissue are different (see Cordonnier, 1989, for review). When such antibodies were used to investigate phytochromes in seeds, two phytochromes were also reported, again because the antibodies recognized two immunochemically distinct pools (Hilton and Thomas, 1985, 1987; Tokuhisa and Quail, 1987).

Even though antibodies to phytochrome from etiolated oats detect two pools, only one of these (the original immunogen) is recognized efficiently by the antibodies (e.g. Tokuhisa et al., 1985; Shima- zaki and Pratt, 1985, 1986). The second pool is defined by the inability of such antibodies to bind

714 Yu-CniE WANG et al.

efficiently to the phytochrome that is present. While this immunochemically distinct phytochrome was initially referred to as a second type (e.g. Tokuhisa et al., 1985; Shimazaki and Pratt, 1985), such nega- tive evidence could not exclude the possibility that there are more than two phytochromes. Molecular genetic data for phytochrome in Arabidopsis has subsequently demonstrated that one should expect to find at least three phytochromes in a single plant (Sharrock and Quail, 1989). Generation of MAb using phytochrome from light-grown oats as immunogen (Pratt et al., 1991c) has verified this expec- tation, at least for oats (Wang et al., 1991). These new MAb permit, moreover, independent detection and quantitation of all three phytochromes by positive assay, rather than by inference from the inability of antibodies directed to one type of phytochrome to bind to another type.

Three phytochromes in unimbibed oat seeds

We have found previously (Wang et al., 1991) that each of the three MAb used here (GO-4, Oat- 22 and GO-7) binds specifically to only one of three phytochromes from oat seedlings. Immunoblot analysis of phytochrome immunoprecipitated by GO-4 and Oat-22 (Fig. 2) confirms that this speci- ficity applies equally well to the three phytochromes identified in unimbibed oat seeds (Fig. 1). Only the MAb used to precipitate a phytochrome also immunostained it on a blot, while the other MAb immunostained only the phytochrome remaining in the supernatant (Fig. 2). That is, no one phytochrome is being detected by more than one MAb. These three MAb are therefore specific probes for the quantitation of the three phytochromes, at least one of which is likely responsible for photoregulation of seed germination (e.g. Adamse et al., 1988) and for regulation of the de novo synthesis of phytochrome during germination (Hilton and Thomas, 1987; Thomas et al., 1989). MAb GO-7, Oat-22 and GO-4 serve as probes for 123-, 124- and 125-kDa phytochromes, respectively. The 124-kDa phytochrome is abundant in etiolated shoots, while 123- and 125-kDa phytochromes predominate in light-grown tissues (Wang et al., 1991).

All three seed phytochromes are found almost exclusively in the embryo-containing portion of an unimbibed seed (Fig. 1) consistent with earlier work of Hilton and Thomas (1985). At least one of these phytochromes is readily photoreversible (see Results), indicating that it already has chromophore attached and is potentially able to function in the absence of de novo synthesis of either apoprotein or chromophore.

Hilton and Thomas (1985) have reported that there is no 124-kDa phytochrome in oat seeds prior to 16 h after the onset of imbibition. In contrast, Tokuhisa and Quail (1987) have concluded that it is present, because antibodies to 124-kDa phyto-

chrome immunoprecipitate a polypeptide of this size from extracts of quiescent as well as germinating seeds. Nevertheless, because some antibodies to 124-kDa phytochrome can immunoprecipitate and immunostain 125-kDa phytochrome (Pratt et al., 1991a; Wang et al., 1991), it is not possible to discriminate unambiguously between these two phytochromes using polyclonal antibodies to 124- kDa phytochrome, as Tokuhisa and Quail (1987) had to do. The results presented here (Fig. 3) dem- onstrate, however, that the conclusion of Tokuhisa and Quail (1987) was probably correct. There is a phytochrome in unirnbibed oat seeds that shares five spatially distinct epitopes (Pratt et al., 1988; Thompson et al., 1989) with 124-kDa phytochrome. Since it is unlikely that a different seed phytochrome would by chance contain all five of these epitopes, it appears that these MAb are detecting the same 124-kDa phytochrome in unimbibed seeds that also predominates in etiolated seedlings. While Hilton and Thomas (1985) used a different cultivar of oats than did ourselves and Tokuhisa and Quail (1987), it seems more likely that the discrepancy arises from a lack of assay sensitivity in the case of Hilton and Thomas than from the absence of 124-kDa phytochrome because of cultivar differences as Thomas et al. (1989) have suggested. While it might be the case, it therefore seems premature to con- clude that a phytochrome other than the 124-kDa type controls the expression of 124-kDa phytochrome during seed germination (Hilton and Thomas, 1987; Thomas et al., 1989).

Quantitation of phytochromes in unimbibed seeds

Spectrophotometric quantitation of phytochrome from unimbibed oat seeds, while relatively easy, is impractical because (1) it is difficult to obtain optically clear samples with defined measuring light path, (2) the method is relatively insensitive, (3) and it must be done with concentrated detergent- free extracts, which can lead to recovery problems. Not only did we substantially underestimate phytochrome quantities in detergent-free extracts (Figs. 4 and 6; Table l ) , but the magnitude of these underestimates varied by a factor of two, being 1.9-fold for 124-kDa phytochrome and 3.7-fold for 125-kDa phytochrome. Whether this potential problem exists with other tissues is unknown, but must nevertheless be considered in interpretation of quantitation data. A more critical limitation of spectral assay for the present application, however, is that it cannot discriminate among the different phytochromes.

Given the above considerations, seed phytochromes have been quantitated here by immunoblot analyses of SDS sample buffer extracts of lyophilized tissue. This method recovers at least 93% of each of the three phytochromes (Fig. 5). An inherent limitation of this immunochemical approach is that it does not distinguish between


phytochrome holoprotein and apoprotein. Each embryo-containing portion of a seed con-

tains 9.1 ng of the three phytochromes (Table 1). Since there is negligible phytochrome in the embryo-free portion (Fig. l), it can be concluded that each seed contains 9.1 ng, of which 15, 18 and 67% are 1 2 5 , 124- and 123-kDa phytochrome, respectively. Whether each of these phytochromes is biologically active and, if so, is responsible for a unique set of morphogenic responses remains to be seen. It is, however, evident that each of the three is present in sufficient quantity to play an important role in early events associated with seed germination and seedling development.

Acknowledgements-We thank Drs Elizabeth Williams and Tammy Sage (Botany Department, University of Georgia, USA) for generously permitting us to use their image analysis system. This research was supported by USDA NRICGP Grant 91-37100-6490 and DOE Grant DE-AC-09-81SR10925 to L.H.P.

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