Planta

Planta (1993)189:391-396

9 Springer-Verlag1993

Spatial distribution of three phytochromes in dark- and light-grown Avena sativa L. Yu-Chie Wang*, Marie-Mich~le Cordonnier-Pratt, and Lee H. Pratt** Botany Department, University of Georgia, Athens, GA 30602, USA Received 10 August; accepted 5 October 1992 Abstract. We have addressed two issues regarding the spatial distribution of three phytochromes in 3-d-old oat ( A v e n a s a t i v a L.) seedlings. Three monoclonal antibodies, GO-4, GO-7 and Oat-22, were used as probes. Each antibody detects only one of the phytochromes. The first issue is whether any of the phytochromes might be membrane-bound. To address this issue the abundance of each phytochrome in extracts prepared with either a detergent-free or a detergent-containing buffer was compared by immunoblot assay. The detergent-free buffer was formulated to extract only soluble protein, while the detergent-containing buffer was intended to extract both soluble and membrane proteins. None of the data indicate that any of these three phytochromes is membrane-bound in either a dark- or a light-grown seedling. The second issue is whether these three phytochromes are distributed differentially in 3-d-old dark- and lightgrown seedlings. When seedlings were dissected into shoots, scutellums, and roots, all three phytochromes were detected in all three fractions from both dark- and light-grown seedlings. Each of the three phytochromes was most abundant in the shoot and least abundant in the root, except that in light-grown seedlings type I, etiolated-tissue phytochrome was more abundant in the root than in either the shoot or the scutellum. When the equivalent fractions dissected from different seedlings were compared, those dissected from dark-grown seedlings contained a higher quantity of each of the three phytochromes than did those dissected from light-grown seedlings, except that green-tissue, type II phytochromes did not differ significantly in the roots. At this level of resolution, no evidence was obtained to indicate a sub-

* Present address: Instituteof MolecularBiology,AcademiaSinica, Nankang, Taipei, Taiwan 11529, ROC ** To whom correspondence should be addressed; FAX: 1 (706) 542 1805 Abbreviations: Da = Dalton; MAb = monoelonal antibody; NIM = non-immune mouse immunoglobulin G (IgG); SDS = sodium dodecylsulfate

stantive difference among the three phytochromes in their spatial distribution. Key words: A v e n a - Phytochrome (spatial distribution) - Spatial distribution (phytochrome)

Introduction The recent discovery of multiple phytochromes (Quail 1991 for review) renews interest in two questions pertaining to the spatial distribution of phytochrome. The first question derives from the hypothesis that the primary mechanism of phytochrome action is at the membrane level (Hendricks and Borthwick 1967): are any phytochromes membrane-bound? At present, a major difficulty with this hypothesis is that while physiological studies support it (Marm6 1977; Roux 1986 for reviews), direct immunovisualization of phytochrome does not (Pratt 1986 for review). In its red-absorbing form, phytochrome is distributed diffusely throughout the cytosol, while in its far-red-absorbing form, phytochrome is sequestered into numerous discrete areas (Mackenzie et al. 1975; Saunders et al. 1983; Cope and Pratt 1992). In neither case has a unique association of phytochrome with an organelle or membrane been observed (McCurdy and Pratt 1986; Speth et al. 1986; Warmbrodt et al. 1989). Nevertheless, these immunolocalization experiments have been performed only with antibodies produced by immunizing animals with phytochrome purified from etiolated seedlings. The presence of multiple, immunochemically distinct phytochromes in a single plant raises the possibility that one of these newly discovered phytochromes might be membrane-bound. The second question concerns the distribution of phytochrome within a plant. Phytochrome distribution has been investigated both spectrophotometrically (e.g., Briggs and Siegelman 1965; Pjon and Furuya 1968 ; Kondo et al. 1973) and immunochemically (e.g., Pratt and Coleman 1971, 1974; Schwarz and Schneider 1987).

392

Y.-C. Wang et al. : Spatial distribution of three phytochromes in Arena

T h e s e studies, h o w e v e r , focused p r i m a r i l y o n e t i o l a t e d seedlings. Little is k n o w n a b o u t p h y t o c h r o m e d i s t r i b u t i o n in l i g h t - g r o w n plants. M o r e o v e r , since p r e v i o u s s p e c t r o p h o t o m e t r i c studies q u a n t i t a t e d p h y t o c h r o m e in i n t a c t o r g a n s a n d o r g a n systems, these d a t a n o t o n l y r e p r e s e n t the collective a b u n d a n c e o f different p h y t o c h r o m e s , b u t also suffer f r o m the p o s s i b i l i t y t h a t different organs and organ systems have dissimilar optical properties, as p o i n t e d o u t b y Briggs a n d S i e g e l m a n (1965). A n d , the i m m u n o c h e m i c a l studies o n l y used a n t i b o d i e s dir e c t e d to p h y t o c h r o m e f r o m e t i o l a t e d seedlings a n d t h e r e f o r e p r i m a r i l y p r o v i d e i n f o r m a t i o n for o n l y one m e m b e r o f the p h y t o c h r o m e family. We have previously demonstrated that oats contain three p h y t o c h r o m e s , w h i c h h a v e m o n o m e r i c m a s s e s o f 125-, 124-, a n d 1 2 3 - k i l o d a l t o n ( k D a ) ( W a n g et al. 1991). T h e 1 2 4 - k D a p h y t o c h r o m e is m o s t a b u n d a n t in e t i o l a t e d seedlings a n d is a n e t i o l a t e d - t i s s u e , t y p e I p h y t o c h r o m e d e r i v i n g f r o m a p h y A gene ( W a n g et a]. 1993). In contrast, the 125- a n d 1 2 3 - k D a p h y t o c h r o r n e s p r e d o m i n a t e in l i g h t - g r o w n seedlings ( W a n g et al. 1993) a n d are greentissue, t y p e II p h y t o c h r o m e s ( Q u a i l 1991). H e r e we a d dress in o a t s the a b o v e t w o q u e s t i o n s c o n c e r n i n g the s p a t i a l d i s t r i b u t i o n o f these three p h y t o c h r o m e s . T h r e e m o n o c l o n a ] a n t i b o d i e s ( M A b s ) , e a c h specific to o n l y one o f the three p h y t o c h r o m e s , were used as p r o b e s .

Material and methods Oat seedlinys. Oat seeds (Arena sativa L., cv. Garry; Agriculver, Trumansburg, N.Y., USA) were germinated in sand in plant culture containers (No. 26-720-02; Flow Laboratories, Inc., McLean, Va., USA) in the presence or absence of light as previously described (Wang et al. 1993). At the end of the third day after the onset of imbibition, entire seedlings were harvested as described in Wang et al. (1993). Some harvested seedlings were dissected with a razor blade into shoots, scutellums, and roots. Harvested tissue was frozen rapidly in liquid N2, after which it was lyophilized. Darkgrown seedlings were harvested and dissected under dim green light, while light-grown seedlings were harvested and dissected under white laboratory illumination from fluorescent tubes. Phytochrome extraction and protein quantitation. Phytochrome was extracted with either a detergent-flee buffer [50 mM 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris)-C1, pH 8.5 at 4~ C, 10 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride] or a hot ( ~ 100~ C), modified sodium-dodeeyl-sulfate (SDS) sample buffer [125 mM Tris-C1, pH 6.8 at room temperature, 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% glycerol; see Vierstra et al. 1984] at a ratio of 1 ml to 40 mg of lyophilized tissue, as described in Wang et al. (1992). Initial detergent-free extracts were clarified by centrifugation (model J2-21 centrifuge; Beckman Instruments Co., Fullerton, Calif., USA) for 30 min at 36000 9g at 4~ C (Et , Gr-, see Fig. 1), while SDS-sample-buffer extracts were clarified in a microcentrifuge (model 5415; Eppendorf, Westbury, N.Y., USA) for 10min at 16000. 9 at room temperature (Et+, G r + , see Fig. 1). In some

320 mg powder

160 mg powder

detergent-free buffer extraction supernatant (Et- or Gr-)

I SDS sample buffer

extraction

pellet [

supernatant (Et+ or Or+)

1/2 pellet

1

pellet

supernatant (Et-/+ or Gr-/+)

pellet

Et-

Et-/-

Et-/+

Et+

Et+/+

56.9

8.3

20.0

79.3

5.6

Fig. 1. Schematic presentation of protein extraction using detergentfree and-or SDS sample buffer. Extracts were prepared beginning with either dark-grown, etiolated oat seedlings (Et) or light-grown, green seedlings (Gr), using buffer either without ( - ) or with ( + )

pellet

supernatant (Et+/+ or Or+/+)

SDS sample J buffer extraction

3-d-old dark-grown seedlings Protein content (mg per gram of lyophilized powder)

SDS sample buffer extraction

1/2 pellet

detergent-free buffer extraction supernatant ( E t - / - or Gr-,/-)

pellet

3-d-old light-grown seedlings Or- G r - / 51.4

5.6

Gr-/+

8r+

20.5

76.8

Or+/+ 4.1

detergent. If a fraction derives from two sequential extractions, it is designated - / - , - / + or + / +, where the two symbols indicate whether detergent was present in the first and second extract buffers, respectively

Y.-C. Wang et al. : Spatial distribution of three phytochromes in Arena

393

cases (Fig. 1), subsequent pellets were extracted a second time in the same way, except that only one-third the original volume of extraction buffer was used. This second extract, prepared with either detergent-free or SDS sample buffer, was separated into supernatant and pellet fractions by centrifugation in an Eppendorf microcentrifuge for I0 rain at 16000 9g and either 4 ~ C (Et-/-, Gr-/-, see Fig. 1) or room temperature ( E t a + , G r - / + , E t + / + , G r + / + , see Fig. 1). Extracted protein was quantitated by a modified Lowry method (Markwell et al. 1978) after precipitation of protein with acetone (Wang et al. 1992).

Antibodies. Three MAbs, GO-4, Oat-22, and GO-7, were used. They have been shown to be specific to 125-, 124-, and 123-kDa oat phytochrome, respectively (Pratt et al. 1991; Wang et al. 1991, 1992). Non-immune mouse IgGs (NIM) were purchased (No. 1-5381; Sigma Chemical Co., St. Louis, Mo., USA). Electrophoresis, electroblotting, immunostaining and immunoquantitation. Protein was electrophoresed in 7.5% SDS-polyacrylamide gels, electrotransferred onto nitrocellulose, and immunostained as before (Wang et al. 1991). The MAbs GO-4 and GO-7, and NIM were applied to blots at 3 lag 9ml-1, while Oat-22 was applied at 0.3 lag " ml- 1 Phytochrome was quantitated as described in Wang et al. (1992). Immunoblots containing both unknown samples and a dilution series of known quantities of one of the three phytochromes were scanned with a commercial image analysis system (Analytical Imaging Concept, Irvine, Calif., USA). Phytochrome quantities in unknown samples were extrapolated from standard curves consisting of relative band density as a function of phytochrome amount. Each unknown sample was quantitated on at least three independent blots. Each value is presented as the mean• standard error.

Results

Extraction with different buffers. L y o p h i l i z e d , 3 - d - o l d d a r k - a n d l i g h t - g r o w n seedlings were e x t r a c t e d w i t h detergent-free buffer a n d - o r w i t h h o t S D S s a m p l e buffer. E x t r a c t s were f r a c t i o n a t e d as d e p i c t e d in Fig. 1. T h e a m o u n t o f p r o t e i n r e c o v e r e d in each f r a c t i o n is given at the b o t t o m o f Fig. 1. O n a n e q u a l - p r o t e i n basis, supern a t a n t s f r o m initial e x t r a c t i o n s w i t h d e t e r g e n t - f r e e buffer

Fig. 2. Comparison of phytochrome amounts in initial detergentfree buffer extracts ( E t - and G r - for dark- and light-grown samples, respectively) and initial SDS-sample-buffer extracts (Et + and G r + for dark- and light-grown samples, respectively). Proteins were separated in 7.5% SDS polyacrylamide gels, electroblotted onto nitrocellulose, and immunostained with MAbs GO-4, Oat-22 (0-22), and GO-7, and NIM. Each lane was loaded with 20 lag of protein except that the two lanes stained with Oat-22 in the left panel were loaded with 2 lag of protein

Fig. 3. Comparison of phytochrome amounts in different fractions derived from the pellets obtained from clarification of initial detergent-free buffer extracts. Lyophilized, 3-d-old oat seedlings were extracted with detergent-flee buffer. After clarification by centrifugation, supernatants (Et-- and G r - for dark- and light-grown samples, respectively) were removed and pellets were divided into two equal fractions by weight. Each of these two fractions was extracted again either with detergent-flee buffer (Et--/-- and G r - - / - - for dark- and light-grown samples, respectively) or hot SDS sample buffer ( E t - - / + and G r - / + for dark- and light-grown samples, respectively). Proteins were separated in 7.5% SDS polyacrylamide gels, electroblotted onto nitrocellulose, and immunostained with MAbs GO-4, Oat-22 (0-22), and GO-7, and NIM. The amount of protein loaded in each lane is indicated at the bottom of each column, except that in the left panel lanes stained with Oat-22 were loaded with only one-tenth as much protein as indicated ( E t - a n d G r - ) c o n t a i n m o r e o f e a c h o f the t h r e e p h y t o c h r o m e s t h a n d o the s u p e r n a t a n t s f r o m initial e x t r a c tions with S D S s a m p l e buffer (Et + a n d G r + ) (Fig. 2). Pellets d e r i v e d f r o m c l a r i f i c a t i o n o f the initial e x t r a c t s p r e p a r e d w i t h d e t e r g e n t - f r e e buffer were d i v i d e d b y weight i n t o t w o e q u a l f r a c t i o n s a n d e x t r a c t e d a g a i n w i t h either d e t e r g e n t - f r e e buffer ( E t - / and Gr-/-) or w i t h S D S s a m p l e buffer ( E t - / + and Gr-/+) (Fig. 1). O n a n e q u a l - p r o t e i n basis, s u p e r n a t a n t s f r o m initial ext r a c t i o n s w i t h d e t e r g e n t - f r e e buffer ( E t - a n d G r - ) c o n tain s u b s t a n t i a l l y m o r e o f each o f the three p h y t o c h r o m e s t h a n d o the c o r r e s p o n d i n g s u p e r n a t a n t s f r o m the s e c o n d e x t r a c t i o n s ( E t - / - , Et-/+ and Gr-/-, Gr-/+ ; see Fig. 3). T h e s u p e r n a t a n t s f r o m the s e c o n d e x t r a c t i o n s p r e p a r e d either w i t h d e t e r g e n t - f r e e buffer (Et-/and Gr-/-) o r w i t h buffer c o n t a i n i n g S D S (Et-/+ and Gr-/+) h a v e a b o u t the s a m e a m o u n t o f each o f the three p h y t o c h r o m e s w h e n 42% as m u c h protein from Et-/was l o a d e d as f r o m E t - / + (Fig. 3, left p a n e l ) o r w h e n 27% as m u c h p r o t e i n f r o m Gr-/was l o a d e d as f r o m G r - / + (Fig. 3, right panel).

Phytochrome abundance in different organs. T h r e e - d a y old d a r k - a n d l i g h t - g r o w n seedlings were dissected into shoots, scutellums, a n d roots. T h e s e o r g a n s a n d o r g a n systems were e x t r a c t e d i n d e p e n d e n t l y w i t h h o t S D S s a m ple buffer. P h y t o c h r o m e a m o u n t s in these t h r e e f r a c t i o n s were d e t e r m i n e d b y i m m u n o b l o t a s s a y b o t h o n a per-

394

Y.-C. Wang et al. : Spatial distribution of three phytochromes in Arena

Fig. 6a, b. Comparison of three phytochromes in equivalent fracFig, 4. Comparison of three phytochromes in three fractions of dark-grown oat seedlings. Three-day-old dark-grown seedlings were dissected into shoots (Sh), scutellums (Sc), and roots (Rt). Each fraction was extracted with hot SDS sample buffer. Proteins were separated in 7.5% SDS polyacrylamide gels, electroblotted onto nitrocellulose, and immunostained with MAbs GO-4, Oat-22 (0-22), and GO-7, and NIM. a Each lane was loaded with protein equivalent to that obtained from 0.05 seedlings, b, e Each lane was loaded with 20 lag of protein. In e the gels were stained with Coomassie blue

Fig. $. Comparison of three phytochromes in shoots, scutellums and roots of light-grown seedlings. Details are as in the legend to Fig. 4, except that fractions were derived from light-grown seedlings.

tions from oat seedlings grown in the presence or absence of light. Three-day-old dark-grown (D) and light-grown (L) seedlings were dissected into shoot (Sh), scutellum (Sc), and root (Rt). Each fraction was extracted with hot SDS sample buffer. Proteins were separated in 7.5% SDS polyacrylamide gels, electroblotted onto nitrocellulose, and immunostained with MAbs GO-4, Oat-22 (O22), and GO-7, and NIM. a Proteins from shoots and scutellums were loaded at an amount equivalent to that from 0.05 seedlings while proteins from roots were loaded at an amount equivalent to that from 0.1 seedlings, b Each lane was loaded with 20 lag protein organism basis and on a per-unit-protein basis. In darkg r o w n seedlings, 124-kDa p h y t o c h r o m e is m o s t abundant in the shoot (Fig. 4). Both 125- and 123-kDa phytochromes are m o s t a b u n d a n t in the s h o o t when evaluated on a per-organism basis (Fig. 4a), but are a b o u t equal in a b u n d a n c e in the shoot and the scutellum when evaluated on a per-unit-protein basis (Fig. 4b). The r o o t contains the least o f each o f these three p h y t o c h r o m e s (Fig. 4). In light-grown seedlings, 124-kDa p h y t o c h r o m e is relatively more a b u n d a n t in the r o o t and only barely detectable in the shoot and scutellum, on b o t h a perorganism and per-unit-protein basis (Fig. 5). The 125and 123-kDa p h y t o c h r o m e s are m o s t a b u n d a n t in the shoot on a per-organism basis, but are a b o u t equal in a b u n d a n c e in the shoot and the scutellum on a per-unitprotein basis (Fig. 5). While not evident in these blots, 123-kDa p h y t o c h r o m e is detected in extracts o f roots f r o m seedlings g r o w n in the light as well as in darkness (see Fig. 6 below). P h y t o c h r o m e levels in organs and o r g a n systems f r o m seedlings grown with or w i t h o u t light have also been c o m p a r e d directly. In m o s t cases, roots, shoots and scutellums dissected f r o m d a r k - g r o w n seedlings contain substantially m o r e o f each o f the three p h y t o c h r o m e s than do those fractions dissected f r o m light-grown seedlings, regardless o f whether p h y t o c h r o m e levels are evaluated on a per-organism (Fig. 6a) or a per-unit-protein (Fig. 6b) basis. The only exceptions are that in the r o o t there is little or no light-dark difference in the levels o f 123- and 125-kDa p h y t o c h r o m e s (Fig. 6), and that in the scutellum if there is a difference for 123-kDa phyto-

Y.-C. Wang et al.: Spatial distribution of three phytochromes in Arena Table 1. Quantitation of extractable

protein and phytochromes in different organs from 3-d-old dark- and light-grown oat seedlings

Tissue

395 Protein a (mg' organ-1)

Phytochrome b (rig- (mg protein)-l) 125 kDa

124 kDa

123 kDa

Shoot Etiolated Light-grown

0.33 0.30

16.84- 1.8 7.44- 0.9

10704-90