Planta

Planta (1988)173:61-72

9 Springer-Verlag 1988

Cross-reactivity of monoclonal antibodies against phytochrome from Zea and Avena Localization of epitopes, and an epitope common to monocotyledons, dicotyledons, ferns, mosses, and a liverwort

Hansj6rg A.W. Schneider-Poetsch1 *, Heribert Schwarz ~, Rudolf Grimm 2 and Wolfhart Rtidiger 2 1 Botanisches Institut der Universitfit zu K61n, Gyrhofstrasse 15, D-5000 K61n 41, and 2 Botanisches Institut der Universit/it Miinchen, Menzinger Strasse 67, D-8000 Mfinchen 19, Federal Republic of Germany

Abstract. The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Arena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg- mol- ~ phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120 125 kg.mo1-1 from several ferns, a liverwort and mosses. This anti-

* To whom correspondence should be addressed Abbreviations: ELISA=enzyme-linked immunosorbent assay; McAb = monoclonal antibody; PBS : phosphate-buffered saline; Pfr (Pr) = far-red-absorbing (red-absorbing) form of phytochrome; S D S - P A G E - s o d i u m dodecyl sulfate-polyacrylanaide gel electrophoresis

body (Z-3BI), which was localized within a 23.5-kg.mo1-1 section of Arena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical. Key words: Arena (phytochrome) - Bryophyta (phytochrome) - Monoclonal antibody (phytochrome) - Phytochrome in different plant phyla - Pteridophyta (phytochrome) - Zea (phytochrome).

Introduction

In 1981 monoclonal antibodies (McAbs) raised against 5-aminolevulinate dehydratase from spinach (Liedgens et al. 1980) were shown to be a very efficient means of finding structural relationships ("conserved" domains) among nondenatured 5aminolevulinate dehydratases from various other plant species (Schneider and Liedgens 1981). Since then the potential offered by McAbs has been exploited by an increasing number of investigators in the field of plant science. The protein most intensively studied has been phytochrome, a regulative protein specific to plant morphogenesis. Raising McAbs against phytochrome was in great part governed by the hope of finding a means of probing the molecular properties of this unique protein. Antibodies discriminating between the red-absorbing (Pr) and the far-red-absorbing (Pfr)

62

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

form have been found (e.g. Thomas et al. 1984; Cordonnier et al. 1985; Shimazaki et al. 1986), but to date their power of discrimination is low. It does not allow the quantification of Pr in the presence of Pfr and vice versa. Some antibodies were selected which interact with conformational changes of phytochrome in vitro (Nagatani et al. 1984; Lumsden et al. 1985), and differences and similarities between phytochromes from etiolated and green plants were detected by others (Abe et al. 1985; Shimazaki and Pratt 1985; Tokuhisha et al. 1985; Schwarz and Schneider 1987). Eventually, binding of McAbs to proteolytic peptides of known location on the total sequence of phytochrome (Hershey et al. 1985) allowed the exact localization of domains for McAbs on phytochrome (Grimm et al. 1986). In order to understand the phytochrome-antibody relations in some detail, we have extended these localization studies in the present investigations. Studies on the cross-reactivity of McAbs against phytochrome were initiated in order to find "conserved" domains, i.e. conformational structures indispensable for the biological function and therefore passed on with a minimum of alteration. However, the degree of cross-reactivity varied greatly with the set of antibodies prepared. For example, of six antibodies raised against phytochrome from rye, two reacted with phytochrome from oats, and none with phytochrome from pea, and moderate cross-reactivity of six McAbs against phytochrome from pea was only detected within some dicotyledons (Saji et al. 1984). An anti-Arena phytochrome antibody that was attributed to a domain on phytochrome near the amino terminus of this protein showed cross-reactivity with zucchini phytochrome (Daniels and Quail 1984). Considerable intersubclass cross-reactivity, however, was demonstrated in experiments with 9 McAbs to phytochrome from pea and 16 to phytochrome from oats and phytochrome from zucchini, lettuce, pea, oats, rye and barley (Cordonnier et al. 1984). In a recent paper a McAb raised against pea (and tentatively localized on the carboxyl-terminus half of phytochrome not containing the chromophore) was presented which crossreacted with phytochrome from monocotyledons, a moss and algae, and with phytochrome from green plants (Cordonnier et al. 1986a). We have prepared about 40 McAbs against phytochrome from Zea and Arena, and in screening these antibodies, we found several antibodies which cross-reacted with 16 other species of the Poaceae, a few cross-reacting with phytochrome of dicotyledons and one, localized within a

23.5-kg-mol - ~ section of phytochrome containing the chromophore, which cross-reacted with phytochrome from illuminated and green plants, dicotyledons, ferns, mosses and a liverwort. Material and methods Phytochromes used for immunization. Phytochromes were isolated from 5- to 6-d-old etiolated Avena sativa (Pirol) or Zea mays seedlings (Badischer Landmais) grown at 23 ~ C. " S m a l l " phytochrome (approx. 60 kg.mol-1) from Arena was prepared according to Thiimmler et al. (1983). " L a r g e " phytochromes from Zea and Arena were enriched by hydroxylapatite chromatography and ammonium-sulfate precipitation (see Vierstra and Quail 1983), redissolved without boiling in sample buffer containing only 0.025% sodium dodecyl sulfate (SDS), and run on gels according to Neville and Glossman (1974). The resulting faintly bluish band in the region of molecular weights 116 to 124 kg-mol-* was eluted with phosphate-buffered saline (PBS) and used for immunization.

Phytochrome extraction for enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Coleoptiles with primary leaves were cut and ground in liquid nitrogen in a mortar. Before thawing, a buffer containing 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pH 7.9 and 56 m M mercaptoethanol was added (routinely 5 ml per g of tissue) and phytochrome extracted by homogenization. Phenylmethylsulfonylftuoride (PMSF) was added to a final concentration of 4 m M (see Kerscher and Nowitzki 1982). The extract was centrifuged for 15 rain at 40000-g (4 ~ C) and the liquid between the pellet and the surface layer used in the assays. Phytochromes of other plant species were extracted in the same manner. The amount of buffer was reduced if the phytochrome content was low. Phytochromes of dicotyledons were extracted from etiolated seedlings 2 d after germination, phytochromes of ferns from young, still-growing leaves darkened for 2 d and phytochromes of mosses from the whole plants transferred to darkness for 2 d.

Immunization and production of antibodies. Immunization procedures, antibody production and antibody purification were performed as described by Schwarz and Schneider (1987).

Antibody screening. Phytochrome-positive colonies were detected by a double sandwich EL1SA on polyvinylchloride immunoassay plates (see Schwarz and Schneider 1987). In brief: mouse antibodies from culture supernatants were bound by goat anti-mouse immunoglobulin G (IgG)-coated wells. Added phytochrome was bound if anti-phytochrome antibodies were present in the culture supernatants. Bound phytochrome was detected by rabbit anti-phytochrome polyclonal antibodies which were visualized by goat anti-rabbit IgG antibodies conjugated to peroxidase. Substrates for peroxidase were 2,2'-azinobis-(3-ethylbenz-thiazoline)-sulfonic acid and H202. Immunoblotting. The proteins of phytochrome extracts were diluted with sample buffer (1:1, v/v), processed and separated according to Neville and Glossman (1974) and blotted onto nitrocellulose filters (see Gershoni and Palade 1983). The filters were processed according to Blake et al. (1984) and Johnson et al. (1984)_ After residual binding sites of the filters were blocked by inert proteins, the filters were incubated with culture supernatants diluted 5- to 20-fold or with purified antibodies in concentrations of 1-6 ~tg-ml- 1 of PBS - bovine serum albu-

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

100

"'*"%

\

tO ..Q r

50, (.3 Q.

I

I

'

was preincubated with different concentrations of a McAb and then applied to wells of assay plates coated with the same antibody. Phytochrome still bound to the wells was detected by anti-phytochrome serum. Increasing antibody concentrations in the preincubation mixture resulted in decreasing phytochrome binding to the wells (Fig. 1). If the wells are coated with a different antibody which occupies the same or an adjacent epitope, inhibition of phytochrome binding will also occur. Preincubation was always done at antibody concentrations which resulted in an inhibition of at least 70% if the antibody used for preincubation was also used to coat the wells. A reduction in binding of at least 30% was assessed as positive.

/ I

63

DetetwTination of chain classes. Antibodies bound to immunoas-

'

I

9

2 1 0.25 0.06 concentration of antibody during preincubation (~g.m[ -1) Fig. I. Inhibition of antibody (Z-4B5) binding to phytochrome by preincubation of phytochrome (500 ng. ml-1) with the same antibody. Different concentrations of antibody were incubated with phytochrome (500 ng-ml-~). The incubation mixture was then applied to immunoassay plates coated with the same antibody and the amount of phytochrome bound by this antibody was monitored min (BSA) Tween for 3 h. Thereafter, and after washing, the filters were incubated for 3 h in anti-mouse Ig conjugated to phosphatase (A 5153; Sigma, Miinchen, FRG) diluted 1 to 1000 with PBS-BSA-Tween. Washings with detergents (0.1% Triton X-100, 0.1% SDS) in PBS were followed by incubation in the substrate nitro blue tetrazolium and 5-bromo-4-chloro-3indolyl-phosphate in diethanolamine buffer, pH 9.6. Bands became visible within 5-30 rain.

Phytochrome for Western blot analysis of proteolytic fragments. Undegraded phytochrome (124 kg-mol-1) was isolated from 3.5-d-old etiolated oat seedlings according to Grimm and Riidiger (1986). The purity index for the preparation used for proteolytic fragmentation was A667/A2s o = 0.90 to 0.99. Partial proteoIysis was achieved either with endogenous proteases from oat or with trypsin as described by Grimm et al. (1986). The 80-kg'mo1-1 fragment which had not been described in this latter paper was obtained by digestion with 0.2% trypsin for 30 rain at 4 ~ C. It was only obtained from Pfr but not from Pr. Sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) eleetroblotting onto activated glass-fiber sheets and microsequencing were performed as previously described (Grimm et al. 1986).

Testing of cross-reactivity by ELISA. To test cross-reactivity of the selected antibodies, the test system was similar to that used for antibody screening, except that purified McAbs (3 gg. ml- i PBS) were directly bound to the wells of the test plates, and Arena and Zea phytochromes were replaced by extracts prepared from other plant species. Mutual inhibition of antibodies. Mutual inhibition of antibodies was likewise tested by an assay derived from that used for antibody screening. A phytochrome solution of 500 ng.ml-1

say plates were incubated with chain-class-specific anti-mouse antibodies conjugated to biotin (generous gift from the laboratory of Professor Rajewski, K61n, FRG). Incubation with avidin-conjugated alkaline phosphatase, washings and incubation with p-nitrophenylphosphate in diethanol buffer pH 9.6 followed. All antibodies specified in the present study had a kappa light chain. The heavy chain was predominately gamma 1 and gamma 2A (Z-2D2, Z-3BJ, Z-4A5, Z-6D5). No specific reaction was found for the heavy chain of antibodies A-2A3 and Z-4B5. The set of chain-specific antibodies did not contain anti-gamma 3 antibodies.

Immunoadsorbents. Antibodies were bound to Sepharose CL-4B (Pharmacia, Freiburg, FRG) according to the method of March et al. (1974).

Results Localization o f epitopes The method applied for localization of epitopes for McAbs on the peptide chain of oat phytoc h r o m e w a s e s s e n t i a l l y t h e s a m e as t h e m e t h o d u s e d b y G r i m m et al. (1986). P a r t i a l p r o t e o l y s i s of purified phytochrome with either trypsin or endogenous proteases under defined conditions yielded fragments which were separated by SDSPAGE. Western blot analysis with diverse McAbs revealed whether the epitope for a particular antib o d y w a s p r e s e n t in t h e r e s p e c t i v e f r a g m e n t (=positive reaction) or not (= no reaction). Since t h e c l e a v a g e sites h a d b e e n l o c a l i z e d e x a c t l y b y sequence analysis of a number of these fragments (see G r i m m et al. J986), w e w e r e a b l e t o l o c a l i z e t h e e p i t o p e s f o r t h e M c A b s ( F i g . 2). Besides those antibodies which had been invest i g a t e d e a r l i e r ( G r i m m et al. 1986), e p i t o p e s f o r s o m e a d d i t o n a l M c A b s w e r e d e t e r m i n e d as f o l lows. Antibodies Z-2B3, Z-4B5 and Z-6D5 reacted with the 118-kg'mol- 1 fragment (which extends f r o m a m i n o - a c i d r e s i d u e 34 t o r e s i d u e 1106) b u t not with the ll3-kg.mol-1 f r a g m e n t ( w h i c h extends from residues 54/63/66 to approx, residue 1093). S i n c e t h e s e a n t i b o d i e s r e a c t e d a l s o 'with a n 80-kg. tool- 1 fragment which contains the blocked amino terminus of phytochrome (data not shown), t h e e p i t o p e m u s t b e l o c a t e d in t h e a m i n o - t e r m i n a l

64 63 54166 1 3&!!i

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies 1088 10"/0 11106 i i i ]129 ,' ;'L ' , , ,

322 143 210 I

"&-I :'= ll--Z-2B3 Z-1C4

I 4

T

426

537 595 625 I I I ,, ,,

,,

Ill

:--IV-"

~v-.-:---vI ~"

Z-3B1

A-1B3 A-CC5 A - FD/.,

7-2A3 A-2A3 Z-3A6 Z-2B4 Z - 2D2 Z-4A5

Z-4B5 Z-BD5

Fig. 2. Schematic drawing of the peptide chain of oat phytochrome. Vertical bars and numbers above the line representing phytochrome indicate analyzed sites of proteolytic cleavage by either trypsin or endogenous proteases from oat. Six regions (I to VI) were found for binding of monoclonal antibodies (McAbs). The McAbs which bind to a particular region are indicated below. The chromophore is located at position 322

Table 1. Mutual inhibition of anti-phytochrome McAbs and comparison with the results of antibody localization (see Fig. 2.). Phytochrome (Zea and Arena, respectively) was preincubated with a McAb (concentration sufficient to give at least 70% inhibition when tested against the same antibody) and then applied to the wells of immunoassay plates coated with the same or another anti-phytochrome McAb. Inhibitions exceeding 30% were taken as positive. Double arrow=mutual inhibition; single arrow= unidirectional inhibition Inhibition of antibodies by other antibodies

Convergences and differences to epitope localization

A) A-FD4*--~A-CC5 --*A-CC3 antibodies raised against 60-kg.mol i phytochrome

A-FD4/A-CC5 localized on the same region (IV)

B) Z-3BI *--~A-1B3 inhibition only observed with Arena phytochrome A-IB3 binds very weakly to Zea phytochrome

Z-3B1/A-I B3 localized on regions closely adjacent (m/IV)

C) A-2A3*--~Z-2A3

A-2A3/Z-2A3 localized on regions closely adjacent

(v/vi) D) Z-4A5 ~ Z-2D2 Inhibition only observed with Zea phytochrome Z-2D2 binds weakly to Arena phytochrome

Z-4A5/Z-2D2 localized on the same region (VI)

E) Z-2B4 binds very weakly to Arena phytochrome

no inhibition, although member of group VI

F) Z-2B34--~Z-3A6*--~Z-4B54--~Z-6D5

Z-2B3/Z-4B5/Z-6D5 localized on the same region (I) Z-3A6 localized on region V

part of the peptide sequence which is present in the 118- and in the 80-kg.mo1-1 fragments but not in the 113-kg-mol-1 fragment. This is the region between residues 54/63/66 (region I on Fig. 2). Likewise, the epitope for the antibodies A-1B3, ACC5 and A - F D 4 was localized between residues 537 and 595 (region IV) because these antibodies reacted with the 118-, J 13- and 59-kg. mol - 1 fragments (residues 66 to 595 in the case of the 59-kgtool -x fragment) but not with the 53 (residues 66 to 537) or 39-kg'mol-1 (residues 66 to 426) fragments.

the antibodies were performed. It may be assumed that mutual inhibition indicates binding of antibodies to the same or a closely adjacent epitope either in the sequence or the tertiary structure. Unidirectional inhibition may be explained by great differences in the binding constants of two different antibodies. Phytochrome was preincubated with a M c A b and then applied to wells of immunoassay plates coated with the same or another antibody. No or reduced binding of phytochrome to the plates indicated that two antibodies were competing for the same domain. If the assignment to a certain group (A to F in Table 1) coincides with the subdivision into a certain region of the peptide chain of phytochrome (see Fig. 2), we can assume that the antibodies occupy the same or an adjacent epitope within the sequence. This was the case for McAbs A-FD4/A-

Mutual inhibition of anti-phytochrome McAbs In order to obtain more information about the number of recognized epitopes and their spatial distance, assays testing the mutual inhibition of

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

65

Table 2A, B. Cross-reactivity of McAbs raised against Z e a and A r e n a phytochrome with proteins extracted from other species as tested by enzyme immunoassay. Phytochromes were fixed by a McAb and detected by rabbit anti-phytochrome ( A ~ e n a ) serum. Numbers from 0 to 10 indicate the percentage (10 = 100%) of reaction [see absolute absorbance values (A405; valid for A and B) measured after 1 h] as compared to the reaction with a saturating concentration of the homologous phytochromes. Reactions of 5% or less were neglected. A Reactions with extracts from grasses; B Reactions with extracts from dicotyledons + and indicate samples with spectrophotometrically detectable and undetectable amounts of phytochrome, respectively A

Z-6D5 Z-4B5

Z-3A6 Z-2B3

Z-2D2 Z-2B4

Z-4A5 Z-3B1

Z-2A3

Zea mays

10

10

10

10

10

10

10

10

10

8

1

9

8

8

+

Arena

sativa

10

11

10

10

4

1

10

34

10

10

10

10

10

10

+

Arena

elatior

10

10

11

9

9

-

11

33

9

9

10

10

9

-

-

1

11

7

8 8

5 5

9 9

9 9

8 6

+ +

9 9

10 10

10 10

10 10

-

-

23 5

8 9

8

2

8

8

7

+

10

8

2

9

8

5

+

9

9

8

8

-

-

6

11

8

4

6 9

6 10

5 11

6 10

-

2

17

10 9

11 9

10 10

10 8

-

2

10 1

7

1

8

4

5

1

-

10

10

9

10

8 6 11 10 10 1 9

8 5 10 9 8 1 8

6 1 11 8 8 3 8

+ / + + + / /

Hordeum vulgare Secale cereale Triticum Triticum

aestivum tricocoides

Oryza sativa Dactylis glomerata Loliumperenne S e t a r i a viridis Setaria macrostachya

8

Sorghum

cafforum

Panicum Panieum

esculentum crus-galli

3

6

3

3

-

9

10

11

10

1

Panicum

miliaceum

1

8

6

6

A4o5

1.14

1.12

1.11

1.12

B

Z-6D5 Z-4B5

Brassica nappus Brassica oleracea Raphanus sativus

. . .

Pisum sativum Phaseolus mungo

-

Vicia faba Glycine soja

. 1

Solanum Cucurbita

tuberosum pepo

.

. .

-

-

. 1

-

. 1

.

10

9 8

9 3 1 1

3

1

2

1

1

1

2

/

1

2

9

2

2

8 3

4 1

9 2

8 2

8 5

+ +

0.19

1.10

1.06

0.56

1.09

1.06

1.07

Z-2A3

A-2A3 A-1B3 A-FD4 A-CC5 A-CC3

1.16

Z-4A5 Z-3B1

1

5

4

/

3

3 2 4

1 2 3

/ + +

u

1

-

-

4 3

9

-

2 2

2

-

-

-

. -

.

2

4

10

.

.

.

.

1.02

Z-2D2 Z-2B4 .

. 1

1

0.91

6

7 6 9 8 8 3 8

-

-

Z-3A6 Z-2B3

.

2

1

A-2A3 A-1B3 A-FD4 A-CC5 A-CC3

.

. 2

.

.

-

_

1

-

CC5/A-CC3, for Z-4A5/Z-2D2 and for Z-2B3/Z4B5/6D5. If inhibition is found in the ELISA with undenatured phytochrome by McAbs which bind to different epitopes according to Western blot analysis, we have to assume that the epitopes are close to each other in the tertiary structure but not in sequence. This was the case for M c A b pairs Z-3B1/A-1B3, A-2A3/Z-2A3 and Z-3A6/Z-2B3, Z3A6/Z-4B5, Z-3A6/Z-6D5. Assignment to different groups in Table I but to the same subdivision in Fig. 2 means that the epitopes are localized on the same proteolytic fragment but are located distantly enough not to inhibit each other. This was the case for M c A b pairs A-1B3/A-CC5, A-1B3/AFD4, for Z-2A3/Z-3A6 and for A-2A3/Z-2D2, A2A3/Z-4A5. In summary, the investigated antibodies probably bind to nine different epitopes within the phytochrome amino-acid sequence.

1

-

2

_

-1-

1

2

-

4

3

-

Cross-reactivity of McAbs with heterologous phytochrornesfrom grasses and dicotyledons under ELISA conditions The double-sandwich assay, which was originally used to screen and select anti-phytochrome McAbs (fixation o f p h y t o c h r o m e by a M c A b and detection of bound phytochrome by polyclonal anti-phytochrome rabbit sera), was adopted here to test the cross-reactivity of these selected McAbs with native phytochrome from heterologous origins. If the anti-phytochrome serum contains any antibody cross-reacting with phytochrome of the species under investigation, this assay will reliably, albeit with changing intensities, indicate McAbs which have bound this phytochrome. The magnitude of the reaction was recorded in relation to a saturating concentration (about

/

+

66

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

Fig. 3A, B. Detection of Arena and Zea phytochrome by various purified McAbs by immunoblotting. The antibodies were applied at a concentration of 1 ~tg.ml 1. The blot filter was cut into strips which were incubated with different antibodies. SDS-PAGE 7.5-10%. A Detection of phytochrome from etiolated plants. Total amount of phytochromes applied per gel was 1 gg. Incubation in substrate: I, 15 rain; II, 60 rain. B Detection of phytochrome from green Zea shoots (primary leaves just before penetrating the coleoptile) continuously illuminated (photon fluence rate 100 gmol. m - 2. s- 1, cool-white fluorescent tubes; Sylvania, Danvers, Mass., USA). Phytochrome was isolated by antibody Z-4B5 bound to Sepharose (0.5 mg.ml 1). The Sepharose-phytochrome complex was heated with sample buffer and the supernatant after dilution with water (1 : 1, v/v) was applied to the gel. Incubation in substrate: III, 30 rain; IV, 120 rain. H = h e a v y chain, L=light chain of Z-4B5

1 t.tg'm1-1, see Schwarz and Schneider 1987) of the homologous phytochrome. In experiments with the supernatants of crude phytochrome extracts from 16 different etiolated grass seedlings, only a few McAbs failed to give a positive reaction (Table 2A). Even when phytochrome concentrations evaded spectrophotometric detection (PerkinElmer 555, 10-mm light path; Perkin-Elmer, Oberlingen, FRG) the ELISA gave a positive result. In a series of cases the reaction was as strong as with the homologous phytochrome, in a few cases even stronger (see antibody Z-3B1). The greatest losses in cross-reactivity were found with three an-

tibodies binding to a domain between 116- and 118-kg'mol-1 phytochrome (see Grimm et al. 1986). That this region of phytochrome was not lost by proteolysis during the assay procedures was shown by an anti-Arena phytochrome antibody (A-2A3) binding to this same region. With dicotyledons, cross-reactivity was much lower (Table 2 B). Some antibodies, however, were still reactive with some extracts from etiolated dicotyledon seedlings despite the limitation imposed by the use of a serum raised against the phytochrome of a monocotyledon. Further experiments were therefore done by immunoblotting, although

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

67

Fig. 4A, B. Examples for the width of the spectrum of plants which possess proteins detectable by anti-phytochrome antibodies. A Detection of phytochrome from various grasses with antibody Z-4B5 (1 gg.ml 1) on immunoblots; SDS-PAGE 6%. All samples were prepared as described in Materials and methods, and 50 gl were put into each slot. B Detection of phytochrome-like proteins from ferns and mosses with antibody Z-3BJ (4 g g . m l - 1 ) ; SDS-PAGE 7.5-15%. The species were extracted with buffer 1 : 1 (w/v), except Psilotum shoots (1 : 10), and 3 ng of Zea phytochrome were applied. Species extracted : Adiantum fulvum, Asp[enium

spec., Cyclosorus dentatus, Funaria hygrometrica, Marchantia polymc,rpha, Platycerium bifurcatum, Psilotim triquetrum, Selaginella martensii, Sphagnum auriculatum

antibodies recognizing the undenatured antigen in an ELISA need not necessarily recognize the SDSdenatured one. The species which reacted most positively in the ELISA belonged to the Chenopodiacea, Cucurbitaceae, Brassicaceae and Fabaceae.

Cross-reactivity assayed by immunoblot analysis Monocotyledons. As described, the present set of McAbs against phytochrome was selected by an assay using native phytochrome. After experiments with McAbs against native 5-aminolevulinate dehydratase and Helix pomatia lectin (data not

shown) it was astonishing to find that all of these antibodies recognized Arena and Zea phytochrome denatured by SDS-PAGE and immunoblotting. On the whole it seems that the pattern of binding differences of anti-Zea and anti-Arena phytochrome antibodies with respect to binding to the homologous and heterologous antigen in ELISA experiments is to a certain extent reflected by the results of immunoblotting. Antibodies raised against Arena phytochrome bound more weakly to the Zea antigen than to Arena phytochrome, i.e., the band intensity appeared to be minor when comparable amounts of the phytochromes were applied to the gels. Vice versa, a similar tendency was observed when McAbs raised against Zea phy-

68

H.A.W. Schneider-Poetschet al. : Epitopes of monoclonalanti-phytochromeantibodies

tochrome were applied to the two antigens, even though the differences in some cases appeared to be less well expressed. All antibodies recognized the heterologous antigen (Fig. 3 A). However, it is clear that epitope differences exist between phytochromes from Zea and Arena. Antibody Z-4B5 was used to demonstrate that the results of the ELISA experiments with respect to the detection of heterologous phytochromes were reliable. Immunoblots of extracts of 14 different etiolated grass seedlings revealed a protein in the molecular-weight range of Arena phytochrome (Fig. 4A). Young shoots which were darkened for 2 d on fully grown Tradescantia and Dendrobium plants and which were treated likewise did not give a positive reaction.

They possibly indicate fragments of phytochrome which are favourably bound.

Dicotyledons. Antibodies A-FD4, Z-3B1 and Z-4B5 had reacted in ELISA experiments with dicotyledons. A mixture of them applied in immunoblot experiments also detected dicotyledonous phytochromes, or at least proteins in the molecularweight range of 120 to 125 kg.mo1-1 (Fig. 5A, 5 C). The main bands are considered to be undegraded phytochrome. Substituting antibody Z-4A5 for the antibody mixture resulted in a similar pattern of positive-reacting species; however, background reactions not observed with extracts of grasses were also present (Fig. 5B). Members of the same families found to give a positive reaction in ELISA experiments reacted under immunoblot conditions. No reaction was given by Lactuca, Vicia and Lupinus. The farthest-reaching cross-reactivity must be ascribed to antibody Z-3BI. This is the antibody which binds to a phytochrome fragment of 23.5 kg. mo1-1 (residues 210 to 426) containing the chromophore (see Fig. 2). Conspicuously, this antibody gave the lowest relative reactions when tested in ELISA experiments (see Table 2). In experiments comparing antibody binding to proteins of spinach, mustard, cucumber and pea (Fig. 5 D), other antibodies only partially revealed the proteins in the 120- to 125-kg-mo1-1 region. Antibody A-FD4 raised against " s m a l l " ('60 kg. m o l - 1) Arena phytochrome, however, still showed g o o d labelling with spinach, mustard and pea. One may speculate that after Z-3B1 this antibody marks another conserved region. The antibodies showing weaker or missing cross-reactivity bound to a domain in the vicinity of the carboxyl terminus of phytochrome. Although phenylmethylsulfonylfluoride was added to the homogenization mixture, additional bands, probably the result of phytochrome degradation, could not always be avoided.

Phytochrome from green plants. In another experiment, binding of antibody Z-3B1 to phytochromes from etiolated plants continuously illuminated for 24 h or from plants grown under continuous illumination was assayed. If green plants were grown under a natural light-dark regime, the time of harvest would certainly by crucial. Phytochrome is degraded during the light period and regenerated in the dark. Distinct reactions of plants illuminated for a long time were found with extracts from species containing greater amounts of phytochrome in the etiolated state: maize, spinach, cucurbit (Fig. 6A). In a control experiment, extracts of maize shoots (primary leaves just penetrating the coleoptile) which were grown under continuous illumination were shaken with antibody Z-3BI bound to Sepharose in order to accumulate phytochrome. After the sedimented immunoadsorbent was heated with sample buffer, the procedures already described were followed. A clear band in the 125-kg.mol-1 region indicated that this antibody had bound to the native as well as to the denatured antigen of green maize. Similar results were obtained with green Psilotum harvested 8 h after the onset of light in a light-dark cycle (Fig. 6 B). Some additional bands scarcely seen on blots with nonpreadsorbed phytochrome from Psilotum darkened for 2 d may be assumed to be degradation products of this phytochrome. Although phytochrome degradation under illumination proceeds rapidly within a few hours (for degradation kinetics in Zea, see Schwarz and Schneider 1987) the possibility cannot be excluded that the reaction measured in the present experiments is still based on residual "etiolated" and not on " g r e e n " phytochrome. In a parallel experiment, phytochrome from green maize plants was

Mosses, a liverwort and ferns. Finally, antibody Z3B1 was found to detect phytochrome-like proteins in several mosses, a liverwort and ferns which were darkened for 2 d. Immunoblots of extracts of Funaria, Marchantia, Sphagnum, Asplenium, Psilotum, and Selaginella showed labelling in the region of crucial molecular weight. No reaction was found with Adianthum, Blechnum, Cyclosorus, and Platycerium (Fig. 4 B). At present it is not clear whether failed reactions were the result of phytochrome concentrations below the level of detection or of altered epitopes.

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

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Fig. 5A-D. Detection of various phytochrome-like proteins from dicotyledons by immunoblotting. A Detection by an antibody mixture (Z-3BI, Z-4B5, A-FD4). B Detection by antibody Z-4A5. C Detection similar to A. Species giving doubtful :reactions were extracted 1 :l (w/v) with buffer. Each antibody concentration was 4 gg.m1-1. Three ng of reference Zea phytochrome was applied, 50 ~tl of sample preparation; SDS-PAGE 5-10%. Species applied: Brassica napus rapifera, B. oleracea capitata, B. oleracea gongyIodes, Cucurbita pepo, G@cine soja, Lens culinaris, Phaseolus mungo, Pisum sativum, Raphanus sativus, Sinapis alba, Solanum tuberosum (young shoots), Spinacia oleracea. D Spinacia oleracea, Pisum sativum, Brassica nigra and Cucurbita pepo extracts tested by diverse McAbs on immunoblots. Relevant sections of the whole blot are shown. Three ng of Zea phytochrome were applied and 50 gl of sample preparation, respectively. Arrows indicate the 125-kg-mol-1 region

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H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonal anti-phytochrome antibodies

also bound by antibody Z-4B5 (Fig. 3B). Phytochrome from maize plants grown under continuous illumination was isolated by antibody Z-4B5, blotted onto nitrocellulose and then analyzed by the other antibodies. The relative band intensities appeared to be different from those obtained with "etiolated" Zea (or Arena) phytochrome (cf. Fig. 3). However, most of the antibodies against Zea phytochrome reacted with phytochrome from the green plants. These findings may indicate that different populations of phytochrome exist in extracts of the green Zea plants, and that these are to a certain degree discriminated by the antibodies. The reaction of anti-Arena phytochrome antibodies with Zea phytochromes from green plants was doubtful, but still visible with antibody ACC3. To date, no information is available as to whether phytochrome from green plants, which is reported to be immunologically and spectrophotometrically distinct from phytochrome from etiolated ones (Cordonnier et al. 1986b), is a (or the) morphogenetically active form, the less so if both phytochromes are present at the same time. In any case, it is clear that antibody Z-3B1 exhibits properties which surpass the potential of other single antibodies. Screening of about 25 more of our antibodies did not reveal a second antibody with comparable properties. It will be of great interest to localize the epitope for this antibody further. Discussion Fig. 6A, B. Detection of phytochrome extracted from illuminated seedlings. A Detection of phytochrome without concentration prior to blotting. The standard 3 ng of Z e a phytochrome was applied as reference. Seedlings were extracted 1 : 5 (w/v), and 60 gl of extract diluted 1 : 1 (v/v) with sample buffer were applied to the gels in twofold serial dilutions; SDS-PAGE 7.5-15%. The figure shows the relevant section of the whole gel. B Detection of phytochrome immunoprecipitated from extracts of green plants by antibody Z-3BI bound to Sepharose (0.1 mg.ml *) prior to immunoblotting with antibody Z-3B1. Five ml of extract (1:5, w/v) were shaken with 50 gl of the immunoadsorbent for 1.5 h. The immunosorbent was heated with 50 gl of sample buffer and after a 1 : 1 (v/v) dilution with distilled water an aliquot of 40 Ill was applied to the gel (7.5-15%). Standard: 200, 116.25, 92.5, 66.2, 45kg'mo1-1 (BioRad, Mfinchen, FRG). Lane I, 1 : phytochrome from plants darkened for 2 d, not adsorbed prior to immunoblotting; lane L 2: immunoadsorbent alone; lane I, 3: phytochrome from green plants 8 h after the onset of light in a light-dark cycle; lane L 4 : phytochrome from plants darkened for six weeks (fourfold dilution of 14); lane II, 1: immunoadsorbent alone; lane II, 2: phytochrome from plants permanently illuminated; lane II, 3: phytochrome from plants illuminated for 24 h; lane H, 4: trace amounts of phytochrome from etiolated plants; lane H, 5: twice the amount o f / / , 4. H = heavy chain, L = l i g h t chain of Z-3B1

Antibodies which exhibit properties such as binding to phytochromes from species that belong to other classes and subclasses or binding to domains functionally involved in conformational changes of phytochrome are rare. The present investigations, which started with the selection of about 40 clones producing antibodies against phytochrome (see also Schwarz and Schneider 1987) revealed only a single antibody with the potential to bind a great many diverse phytochromes. Including the findings of other authors (see literature cited in the Introduction and Cordonnier and coworkers), only two of an estimated number of 200 McAbs have been shown to exhibit cross-reactivity surpassing the borders of a class. Two different domains have been characterized by these antibodies. The antibody of the present study has been localized on a section of phytochrome of 23.5 kg. mo1-1 (amino-acid residues 210 to 426) encompassing the chromophore (see Grimm et al. 1986 and this paper); the other one has tentatively been

H.A.W. Schneider-Poetsch et al. : Epitopes of monoclonaI anti-phytochrome antibodies

ascribed to the nonchromophore-bearing carboxyl half of phytochrome (Cordonnier et al. 1986a). The former antibody was raised against phytochrome from a monocotyledon (Zea), the latter against phytochrome from a dicotyledon (Pisum). The low percentage of antibodies with a potential greater than that of the bulk of the raised antibodies may be due to the low frequency of respective domains on the antigen and- or to a greater immunogenicity of the other domains. In either case, in polyclonal anti-sera these antibodies would be likely to be overlooked. Idiotypes tolerating a certain degree of alteration of their epitope may also be considered. It is tempting to assume that domains recognized by an antibody in a great variety of species are highly conserved. However, such a generalization needs reconfirmation by a thorough screening of more phytochromes, especially within the genera or families which show cross-reacting members, by studies comparing quantitative spectrophotometric measurements with the immunological behaviour. To date it is not always clear as to whether a missing immunoreaction is the result of missing phytochrome or of a changed epitope. In the not very probable case that antibody Z-3BI binds to the chromophore (which may be buried in a cleft of the apoprotein) itself, all phytochromes should be bound. Findings indicating a loss in the ability to recognize an epitope within a genus or a family would mean that changes or conservations are not necessarily bound to greater taxonomic categories, although these categories would certainly be revealed by the sum of possible antibody reactions. In the present experiments, for example, most of the antibodies detect phytochromes from grasses, but only a few detected phytochromes from dicotyledons. Differences in the relative band intensities of phytochromes assayed on immunoblots by different antibodies, however, indicate that even the recognized epitopes are not necessarily identical. If they are identical, differences in antibody binding may be interpreted as the result of influences of amino-acid changes in the environment of the epitope. The degree of conservation of these epitopes would be revealed if the means were worked out for comparing the amino-acid sequences of different phytochromes. The antibodies used in the present investigation, as well as about another 25 antibodies, were selected by a double sandwich ELISA (Schwarz and Schneider 1987) which used nondenatured phytochrome. All of these antibodies also proved to recognize SDS-denatured phytochrome on im-

71

munoblots. Working with other proteins (5-aminolevulinate dehydratase, Helix pomatia lectin, data not shown) showed that the overall similar behaviour of McAbs against denatured phytochrome was the exception. In contrast to phytochrome these other proteins consist of six more or less similar subunits (see Liedgens et al. 1983; Gc, ldstein and Hayes 1978). One may speculate as to whether this structure gives rise to a greater number of discontinuous epitopes. However, we do not know whether all epitopes recognized by our McAbs against phytochrome are continuous. One may assume that even in random coils on immunoblots, distinct conformations may be preserved. Studies with smaller peptides than those hitherto used to localize epitopes (Grimm etal. ]986 and this paper) may give an answer to this question~ Small peptides, however, do not necessarily preserve the epitope conformation of the greater fragment. Polyclonal antibodies raised against a synthetic undecapeptide and binding to the homologous dodecapeptide encompassing the chromophore did not bind to native or denatured phytochrome (Mercurio et al. 1986). In any case, the exact localization of the epitope of antibody Z-3B1 will give more insight into function-structure relations of phytochrome. In addition, antibody Z-3BI will be a means of isolating and of characterizing phytochromes less well known than Arena phytochrome, or of analyzing the state and localization of phytochrome in these plants. The investigations were supported by grants from the Stiftung Volkswagenwerk, Hannover; the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt. We thank Ms. Birgit Braun for carefully assisting in ELISA and immunoblotting experiments.

References Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 26, 1387-1399 Blake, M.S., Johnston, K.H., Russel-Jones, G.J., Gotschlich, E.C. (1984) A rapid sensitive method for detection of alkaline phosphatase conjugated anti-antibody on Western blots. Anal. Biochem. 136, 175 179 Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1984) Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to Pisum and Arena phytochrome. Plant Physiol. 74, 982 987 Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1985) Monoclonal antibodies with different affinities to the red-absorbing and far-red-absorbing forms of phytochrome. Biochemistry 24, 3246-3253 Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1986a) Identifi-

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cation of a highly conserved domain on phytochrome from angiosperms to algae. Plant Physiol. 80, 982-987 Cordonnier, M.-M., Greppin, H., Pratt, L.H. (1986b) Phytochrome from green Arena shoots characterized with monoclonal antibody to phytochrome from etiolated Pisum shoots. Biochemistry 25, 76527666 Daniels, S.M., Quail, P.H. (1984) Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Arena. Plant Physiol. 76, 622-626 Gershoni, J., Palade, G. (1983) Protein blotting. Principles and applications. Anal. Biochem. 131, 1 15 Goldstein, I.J., Hayes, C. (1978) The lectins: Carbohydratebinding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem. 35, 127-340 Grimm, R., Lottspeich, F., Schneider, H.A.W., Rfidiger, W. ( 1986) Investigations of the peptide chain of 124 kD a phytochrome. Localization of proteolytic fragments and epitopes for monoclonal antibodies. Z. Naturforsch. 41e, 993-1000 Grimm, R., Rfidiger, W. (1986) A simple and rapid method for isolation of 124 kDa oat phytochrome. Z. Naturforsch. 41e, 988-992 Hershey, H.P., Barker, R.F., Idler, K.B., Lissemore, J.L., Quail, P.H. (1985) Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Arena. Nucl. Acid. Res. 13, 8543-8559 Johnson, D.A., Gautsch, J.W., Sportsman, J.R., Elder, J.H. (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Technol. 1, 3 8 Kerscher, L., Nowitzki, S. (1982) Western blot analysis of a lyric process in vitro specific for the red light absorbing form of phytochrome. FEBS Lett. 146, 173 176 Liedgens, W., Grfitzmann, R., Schneider, H.A.W. (1980) Highly efficient purification of the labile plant enzyme 5aminolevulinate dehydratase (EC4.2.1.24) by means of monoclonal antibodies. Z. Naturforsch. 35e, 958-962 Liedgens, W., Liitz, C., Schneider, H.A.W. (1983) Molecular properties of 5-aminolevulinic acid dehydratase from Spinacia oleracea. Eur. J. Biochem. 135, 75-79 Lumsden, P.J., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Effect of monoclonal antibodies on the in vitro Pfr dark reversion of pea phytochrome. Plant Cell Physiol. 26, 1313-1322 March, S.C., Parkish, I., Cuatrecasas, P. (1974) A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60, 149-152 Mercurio, F.M., Houghten, R.A., Lagarias, J.C. (1986) Sitedirected antisera to the chromophore binding site of phyto-

chrome. Characterization and cross-reactivity. Arch. Biochem. Biophys. 248, 3542 Nagatani, A., Yamamoto, K.T., Furuya, M., Fukumoto, T., Yamashita, A. (1984) Production and characterization of monoclonal antibodies which distinguish different surface structures of pea (Pisum sativum cv. Alaska) phytochrome. Plant Cell Physiol. 25, 1059-1068 Neville, D.M., Glossmann, M. (1974) Molecular weight determin'ation of membrane protein and glycoprotein subunits by discontinuous gel electrophoresis. Methods Enzymol. 32, 923-1002 Saji, H., Nagatani, A., Yamamoto, K.T., Furuya, M., Fukumoto, T., Yamashita, A. (1984) Cross-reactivity of monoclonal antibodies against rye and pea phytochrome with phytochromes extracted from eight different plant species. Plant Sci. Lett. 37, 57-61 Schneider, H.A.W., Liedgens, W. (1981) An evolutionary tree based on monoclonal antibody-recognized surface features of a plastid enzyme (5-aminolevulinate dehydratase). Z. Naturforsch. 36e, 44-50 Schwarz, H., Schneider, H.A.W. (1987) Immunological assay of phytochrome in small sections of roots and other organs of maize (Zea mays L.) seedlings. Planta 170, 152-160 Shimazaki, Y., Cordonnier, M.-M., Pratt, L.H. (1986) Identification with monoclonal antibodies of a second antigenic domain of Arena phytochrome that changes upon its photoconversion. Plant Physiol. 82, 109-113 Shimazaki, Y., Pratt, L.H. (1985) Immunological detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Arena shoots. Planta 164, 333-344 Thomas, B., Penn, S.E., Butcher, G.W., Galfre, G. (1984) Discrimination between the red- and far-red-absorbing forms of phytochrome from Arena sativa L. by monoclonal antibodies. Planta 160, 382-384 Thiimmler, F., Rfidiger, W., Cmiel, E., Schneider, S. (1983) Chromopeptides from phytochrome and phycocyanine, NMR studies of the Pfr and Pr chromophore of phytochrome and E, Z isomeric chromophores of phycocyanin. Z. Naturforsch. 38e, 359 368 Tokuhisa, J.G., Daniels, S.M., Quail, P.H. (1985) Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in lightgrown Arena sativa L. Planta 164, 321-332 Vierstra, R.D., Quail, P.H. (1983) Purification and initial characterization of 124 kilodalton phytochrome from Arena. Biochemistry 22, 2498 2505 Received 25 March; accepted 12 August 1987