Plant Physiol. (1986) 80, 982-9870032-0889/86/80/0982/06/$0 1.00/0

Identification of a Highly Conserved Domain on Phytochromefrom Angiosperms to Algae'

Received for publication August 8, 1985 and in revised form November 27, 1985

MARIE-MICHELE CORDONNIER2, HUBERT GREPPIN, AND LEE H. PRATr*Laboratoire de Physiologie Vegetale, Pavillon des Isotopes, 20 Boulevard d'Yvoy, CH-1211 Geneve 4(M.-M.C., H.G.), and Botany Department, University ofGeorgia, Athens, Georgia 30602 (L.H.P.)

ABSTRACT

A monoclonal antibody (Pea-25) directed to phytochrome from etiol-ated peas (Pisum sativam L., cv Alaska) binds to an antigenic domainthat has been highly conserved throughout evolution. Antigenic cross-reactivity was evaluated by immunoblotting sodium dodecyl sulfate sam-ple buffer extracts prepared from lyophilized tissue samples or freshlyharvested algae. Pea-25 immunostained an approximately 120-kilodaltonpolypeptide from a variety of etiolated and green plant tissues, includingboth monocotyledons and dicotyledons. Moreover, Pea-25 immuno-stained a similarly sized polypeptide from the moss Physcomitrella, andfrom the algae Mougeotia, Mesotaeniwm, and Chlamydomonas. BecausePea-25 is directed to phytochrome, and because it stains a polypeptideabout the size of oat phytochrome, it is likely that Pea-25 is detectingphytochrome in each case. The conserved domain that is recognized byPea-25 is on the nonchromophore bearing, carboxyl half of phytochromefrom etiolated oats. Identification of this highly conserved antigenicdomain creates the potential to expand investigations of phytochrome ata cellular and molecular level to organisms, such as Chlamydomonas,that offer unique experimental advantages.

Even though much is known about phytochrome physiology(21) and its biochemical properties (15, 22), little is known aboutits primary mode of action. One approach to elucidating itsmode of action has involved a search for conserved domains onphytochrome. This search is based upon the assumption that ifthe primary mode ofaction ofphytochrome is similar in differentplants, then the related functional sites on phytochrome shouldhave been conserved throughout evolution (3). Moreover, anti-bodies should be a useful tool to search for such evolutionarilyconserved regions. Examples of successful applications of thisapproach to other systems include detection of a domain onhuman and guinea pig Ia antigens that may have a regulatoryfunction in T-cell activation (28), and identification ofa domainon human transferrin that appears to be involved in receptorbinding (1).

Initial efforts to identify common antigenic determinants onphytochrome were made with polyclonal rabbit antisera andpartially or highly purified phytochrome preparations (3, 14, 17).This approach proved to be limited in scope for a number ofreasons: (a) given that it is often difficult to extract and partiallypurify the undegraded phytochrome required for rigorous analy-

' Supported by Swiss National Funds grant 3:292-0:82 and NationalScience Foundation grants PCM-83 15840 and PCM-83 15882.

2Present address: Biotechnology Research, CIBA-GEIGY Corpora-tion, Research Triangle Park, NC 27709-2257.

sis (2, 11, 26), the range of plants that can be used is restricted;(b) rabbit antisera contain so few cross-reacting immunoglobu-lins (3, 14, 17) that it would be difficult to isolate only those ofinterest; and (c) moreover, this subset ofcross-reacting antibodiespresumably consists of a family of immunoglobulins directed tomultiple antigenic sites on phytochrome, making it difficult toidentify individual sites with precision. However, the availabilityof monoclonal antibodies, each of which is specific for a singleantigenic domain or epitope (9), coupled with immunoblotanalysis of SDS sample buffer extracts, overcomes these earlierlimitations.Most monoclonal antibodies so far tested have exhibited only

limited cross-reactivity among phytochromes from monocotyle-donous and dicotyledonous plants (5, 6, 8, 13, 18). Two mono-clonal antibodies (Oat-12 and Oat-20) that bind to phytochromefrom three monocotyledons and three dicotyledons, however,were identified by enzyme-linked immunosorbent assay (5),while a third antibody (1.3G7F) was observed to bind to bothoat and zucchini phytochrome on immunoblots of SDS poly-acrylamide gels (8). Unfortunately, neither Oat-12 nor Oat-20detects phytochrome well by immunoblotting ofSDS polyacryl-amide gels (M-M Cordonnier, LH Pratt, unpublished observa-tions).No monoclonal antibody has as yet been shown to bind to

phytochrome obtained from sources outside the angiosperms.Here we identify a monoclonal antibody that is directed tophytochrome from etiolated peas and that recognizes an anti-genic domain on a similarly sized polypeptide obtained from avariety of etiolated and green angiosperms, as well as from amoss and three algae.

MATERIALS AND METHODSPlant Materials. Etiolated plants were grown in complete

darkness at 25°C and near saturating humidity as before (2). Theplants tested, their age when harvested, and the tissue harvestedwere: peas (Pisum sativum L., cv Alaska), 7 d, whole shoots;zucchini (Cucurbita pepo L., cv Black Beauty) 5 d, cotyledons;soybeans (Glycine max L., cv unknown), 5 d, cotyledons; spinach(Spinacia oleracea L., cv Nobel), 7 d, whole plant; lettuce (Lac-tuca sativa L., cv Grand Rapids), 5 d, whole plant; rye (Secalecereale L., cv unknown), 6 d, whole shoots; barley (Hordeumvulgare L., cv unknown), 6 d, whole shoots; maize (Zea maysL., cv unknown), 5 d, whole shoots; and oats (Avena sativa, L.,cv Garry), 5 d, whole shoots.

Leaves were harvested from green plants that were obtainedin a variety ofways. Wheat (Triticum aestivum L., cv unknown)was grown for 10 d, in a greenhouse near Geneva under naturalillumination. Maize, spinach, and soybean were grown at 22°C,under Sylvania Grolux lamps with a 12:12-h light:dark cycle for1, 2, and 3 weeks, respectively. Tobacco plants (Nicotiana plum-baginifolia LD) were regenerated from protoplasts that wereoriginally isolated from wild type plants (courtesy ofDr. P. King,

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CONSERVED PHYTOCHROME DOMAIN

Friedrich-Miescher Institute, Basel, Switzerland). Daisy (Bellisperennis L.), dandelion (Taraxacum officinale Weber), birch(Betula pendula Roth), and ivy (Hedra helix L.) were harvestedfrom nature in Geneva, Switzerland.Moss (Physcomitrella patens) was grown on agar prepared

with mineral medium under a natural light cycle at room tem-perature (courtesy of Professor J.-P. Zryd, University of Lau-sanne, Switzerland). Mougeotia sp. was grown autotrophically inliquid culture under continuous illumination. The alga was keptin darkness for 48 h prior to harvest. Mesotaenium caldariorumwas grown autotrophically on an 18:6-h light:dark cycle. It washarvested at the end of a photoperiod and provided to us inlyophilized form by Mr. Daniel G. Kidd and Prof. J. C. Lagarias,University of California, Davis. Chlamydomonas reinhardtiistrain Y l (wild type, provided by Professor J.-D. Rochaix, Uni-versity of Geneva, Switzerland) was grown heterotrophically onacetate-containing medium in darkness. One culture was trans-ferred to continuous illumination for 24 h prior to harvest. Cellswere harvested during exponential growth at a concentration of2 x 106/ml.

Preparation of Plants for Analysis. With the exception ofChlamydomonas, plants were frozen in liquid N2, ground to apowder under liquid N2 in a mortar and pestle, and lyophilized.Special care was taken to ensure that, once frozen, the tissue wasdried without being permitted to thaw at any time. Chlamydo-monas was collected from 200 ml of culture, pelleted by centrif-ugation, and resuspended in 100 ml of water. After collectingagain by centrifugation, the cells were resuspended in 5 to 10 mlof 0.4 M sucrose, 10 mM MgCl2, 140 mM 2-mercaptoethanol, 100mM Tris, pH adjusted to 8.0 at 2°C with HCl. Phenylmethylsul-fonyl fluoride was added to a final concentration of 4 mmimmediately prior to use. The suspended cells were then passedtwice through a precooled French press. The resultant brei wasused without further preparation. Chlamydomonas was providedto us in this prepared state by Dr. Steve Mayfield, University ofGeneva, Switzerland.Phytochrome Preparations. Immunopurified phytochrome

from etiolated oats (A667/A280 = 0.77, greater than 95% pure)was prepared as before (2).

Crude, phytochrome-containing extracts of etiolated oatshoots were prepared as follows. Tips of freshly harvested shootswere extracted into 50 mm Tris, 0.2 M 2-mercaptoethanol, pHadjusted to 8.5 at room temperature. Immediately prior to ex-traction, phenylmethylsulfonyl fluoride was added to 4 mm,benzamidine to 2 mM, and E-aminocaproic acid to 10 mm.Samples were clarified for 15 min at 1 7,000g prior to immuno-blotting as described below.Monoclonal Antibodies. The new monoclonal antibody to pea

phytochrome (Pea-25) was obtained from a mouse immunizedwith phytochrome that was purified from etiolated pea shootsand that had an A"7/A280 ratio of 0.23 (about 20-40% pure), asdescribed elsewhere (4). The hybridoma producing this antibodywas identified and cloned by limiting dilution as described inCordonnier et al. (6). Oat-22 and Oat-25, both of which aredirected to phytochrome from etiolated oats, have been charac-terized previously (4-6). All three antibodies are of the immu-noglobulin Gl isotype (6). They were all immunopurified fromspent hybridoma medium as before, using a column of immo-bilized rabbit antibodies to mouse immunoglobulins (4).

Immunoblotting. Lyophilized samples were prepared for SDS-PAGE by extraction in a modified SDS sample buffer (6, 27), ata ratio of 45 mg powder to 1 ml sample buffer. Samples wereheated to 100°C for 5 min, cooled, and clarified by centrifugation.Supernatants were used immediately or stored frozen for lateruse. Chlamydomonas was prepared by mixing an aliquot ofextracted brei with an equal volume of triple-strength samplebuffer (12) at 100C and incubating at this temperature for 5

min. Immunopurified oat phytochrome and crude extracts ofetiolated oats were mixed with SDS sample buffer (12) andincubated at 100°C for 5 min.

Prepared samples were electrophoresed on 5 to 10% lineargradient SDS polyacrylamide gels (12), using the electrophoresisbuffer of Studier (23). Electrotransfer of polypeptides to nitro-cellulose (25), transient staining of the nitrocellulose with Pon-ceau S, and immunostaining of the nitrocellulose with Pea-25,rabbit antibodies to mouse immunoglobulins, and alkaline phos-phatase-conjugated goat antibodies to rabbit immunoglobulinswere done as described elsewhere (6, 16).Mol wt standards were obtained from Sigma (mixture SDS-

6H).Epitope Identification. Crude, aqueous extracts of etiolated

oats were assayed by immunoblotting of SDS polyacrylamidegels exactly as described previously (6). Extracts were assayedboth immediately after preparation and following 7 h incubationas Pfr at 22°C. Samples analyzed are the same as those charac-terized elsewhere (6). Phytochrome from etiolated oats was usedfor this purpose because prior characterization of this proteinsimplified data analysis (6, 8, 11, 15, 22, 26).

RESULTS

Since oat phytochrome and the monoclonal antibodies to oatphytochrome that were used here have been well characterizedpreviously (4-6, 15, 19, 20, 22), the specificity of Pea-25 isestablished here with reference to oat phytochrome. Comparativeimmunoblotting ofa dilution series ofa crude extract ofetiolatedoat shoots indicates that Pea-25 stains the same polypeptide asdoes Oat-22 (Fig. la), which previously has been shown to bedirected to phytochrome from etiolated oats (4, 5). In addition,Pea-25 does not appreciably stain other polypeptides, eventhough they are present in much greater amounts than phyto-chrome (Fig. lb). When comparing immunostaining of highlypurified phytochrome (Fig. lc) to that in a crude extract (Fig.la), it is evident that the bands are stained about equally well bythe two antibodies and in approximate proportion to the amountof phytochrome applied to the gel.When tested against whole tissue extracts of a variety of

etiolated plant tissues, both monocotyledons and dicotyledons,Pea-25 immunostains specifically (Fig. 2, center, lanes M, 0, S)or preferentially (Fig. 2, center, lanes B, R, P, Z, S', L) apolypeptide about the size of native phytochrome from etiolatedoats, which is 124 kD (1 1, 26). This polypeptide is, in each case,an exceedingly minor component of the total protein load (Fig.2, right) and is not stained by non-immune mouse immunoglob-ulins (Fig. 2, left).When tested against crude extracts of a variety ofgreen leaves,

Pea-25 again immunostains preferentially a polypeptide the sizeof oat phytochrome (Fig. 3, center; Fig. 4, lane I). The replicablot immunostained with nonimmune mouse immunoglobulinsindicates that much of the stain not associated with the polypep-tide near 120 kD is nonspecific (Fig. 3, bottom).

Pea-25 also immunostains a polypeptide, which is about thesize of phytochrome from etiolated oats, on immunoblots ofcrude extracts of the moss Physcomitrella (Fig. 3, lane M') andthe algae Mougeotia (Fig. 3, lane M"), Chlamydomonas (Fig. 4,lanes C and C'), and Mesotaenium (Fig. 5, lane Me). In the caseof Chlamydomonas, the outcome is essentially the same regard-less of whether the cells were harvested from a dark-grownculture, or from a culture kept in the light for 24 h immediatelyprior to harvest (Fig. 4, lanes C' and C, respectively).Immunoblotting of crude extracts of etiolated oat shoots in-

dicates that while all three antibodies bind undegraded phyto-chrome as expected (Fig. 6, lanes 1-3), they stain differentproteolytically derived fragments following incubation for 7 h at22°C. Oat-25 and Oat-22 both immunostain prominently apolypeptide at 72 kD, while Oat-22 also stains one at 66 kD (Fig.

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CORDONNIER ET AL.

0-22 P-25 -

aPonceau 0-22 P-25---

b

a, sq - -

C

-m

FIG. 1. Immunoblot of crude extracts of etiolated oat shoots (panels a and b) and of immunopurified phytochrome from etiolated oats (panel c).Panels a and b, the central two lanes contained the greatest quantity of extract. The amount of extract added decreased symmetrically as indicatedby the arrows. Phytochrome quantities, which were estimated by photoreversibility assay, were 300, 100, 30, 10, and 3 ng. The nitrocellulose wasfirst stained with Ponceau S and photographed (panel b) before being cut in two and immunostained with Oat-22 or Pea-25 as indicated. Panel c,nitrocellulose was immunostained with Oat-22 or Pea-25 as indicated. Phytochrome quantities added to each lane were as described for panels aand b. Positions of mol wt standards and their sizes in kD are indicated on the left.

R B M 0 P Z S SF L R B M 0 P Z S S L R B M 0 P Z S Sam. mp& om

IFs

200-

.. -"

44-

31-

FIG. 2. Immunoblot of crude SDS-sample buffer extracts of lyophilized, etiolated plant tissues. Two replica blots were prepared: one was stainedtransiently with Ponceau S (right panel) prior to being immunostained with Pea-25 at I ,ug/ml (center panel); the other was immunostained withnonimmune mouse immunoglobulins at 1 ug/ml (left panel). Sample loads were: rye (R), 10 Ml; barley (B), 10 M; maize (M), 5 l; oat (0), 1 Ml; pea(P), 20 ul; zucchini (Z), 20 Ml; soybean (S), 20 1A; spinach (S'), 20 Ml; lettuce (L), 20 M1. Positions of mol wt standards and their sizes in kD areindicated.

6, lanes 4 and 5). Pea-25 immunostains uniquely a polypeptideat 52 kD (Fig. 6, lane 6).

DISCUSSIONPea-25 binds to phytochrome from etiolated oats as indicated

by its ability to immunostain this protein when highly purified

(Fig. lc). Moreoever, with respect to polypeptides extracted frometiolated oat shoots (Fig. lb), Pea-25 is as specific for phyto-chrome as is Oat-22 (Fig. la). Thus, since Pea-25, which wasdirected against phytochrome from etiolated peas, recognizeswell phytochrome from etiolated oats, since it is highly specificto phytochrome with respect to other proteins, at least from

200-

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44-

116-92.5-66-

984 Plant Physiol. Vol. 80, 1986

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CONSERVED PHYTOCHROME DOMAIN

W M DD' TS" SB' M'M" '~~~I

200-

116- *1*t ,,7...... 92.5-

66-.4 . . - -_ ...** , _

44-31-

P-25- NIMFIG. 4. Immunoblot of crude extracts of green ivy and Chlamydo-

monas. Two replica blots were prepared: one was immunostained withPea-25 at 1 jg/ml (left), the other with nonimmune mouse immuno-globulins at I Ag/ml (right). Sample loads were: ivy (I), 35 Ml; Chiamy-domonas harvested after 24 h in light (C) and from a dark-grown culture(C'), 5 Ml. An extract of lyophilized, etiolated oats (0, I Ml) was loadedinto an intervening lane as a size reference. Positions of mol wt standardsand their sizes in kD are indicated.

FIG. 3. Immunoblot of crude SDS-sample buffer extracts of greenleaves, a moss, and an alga. Two replica blots were prepared: one wasstained transiently with Ponceau S (top panel) prior to being immuno-stained with Pea-25 at I jug/ml (center panel); the other was immuno-stained with nonimmune mouse immunoglobulins at I Mig/ml (bottompanel). Sample loads were: wheat (W), 20 gl; maize (M), 20 du; daisy (D),10 IA; dandelion (D'), 10 jul; tobacco (T), 10 Ml; spinach (S'), 10 jsl;soybean (S), 20 gl; birch (B'), 20 gl; moss (M'), 20 Ml; Mougeotia (Me),10 Ml. Unlabeled intervening lanes and the lane at the extreme right, allofwhich contained too little protein to be seen with the Ponceau S stain,were loaded with I Ml of lyophilized, etiolated oat extract, which servesas a size reference. Positions of molecular weight standards and theirsizes in kD are indicated.

etiolated oat shoots, and since in immunoblots it stains fromeach plant or alga a polypeptide of approximately the same sizeas phytochrome from etiolated oats (Figs. 2-5), it is most likelythat in each case Pea-25 is immunostaining phytochrome.

It is possible, of course, that Pea-25 is immunostaining adifferent polypeptide that, although it is not phytochrome, issimilar in size. Even in this instance, however, it is evident thatthis polypeptide carries an epitope that is essentially identical tothat found on phytochrome. It is not a polypeptide that interactswith immunoglobulins nonspecifically, since it is not detectedwith nonimmune mouse immunoglobulins (Figs. 2-5), nor withseveral other monoclonal antibodies to phytochrome that havebeen tested (data not shown). Moreover, the polypeptide beingstained is a trace component of the total sample load as revealedwith Ponceau S (Figs. 1-3). Thus, it is evident that the affinityof Pea-25 for this polypeptide must be high in every case. It ispossible to estimate that if the polypeptide being stained fromgreen plants is phytochrome, then it represents from about 1part in 20,000 to 1 part in 100,000 of the total protein appliedto the gel (15). The ability ofPea-25 to immunostain this putative

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CORDONNIER ET AL.

MeO MeO I 2345 6

200-

92.5-

66-

44-

31 2ss ;N

4__l

P-25 ---NIM ---FIG. 5. Immunoblot of a crude extract of Mesotaenium. Two replica

blots were prepared, each loaded with 20 zA of Mesotaenium (Me): onewas immunostained with Pea-25 at 1 ,ug/ml (left), the other with non-immune mouse immunoglobulins at 1 jig/ml (right). As a size reference,1 ul of an extract of lyophilized, etiolated oats (0) was loaded. Positionsof mol wt standards and their sizes in kD are indicated.

phytochrome polypeptide, even under these conditions, is notsurprising given that with this antibody as little as 1 ng ofphytochrome applied to a polyacrylamide gel can be detected inthe derived immunoblot (unpublished observations). The rela-tively high level ofbackground stain is also not unexpected underthese conditions, especially since it is for the most part associatedwith polypeptides of relatively high abundance (Fig. 3).

It is not surprising that Pea-25 immunostains a putative phy-tochrome polypeptide in extracts of angiosperms, since theseplants are well known to contain this chromoprotein (21). Sim-ilarly, since Physcomitrella and Mougeotia both exhibit phyto-chrome-mediated responses (7, 10), and since Mesotaenium hasbeen shown by spectrophotometric assay to contain phyto-chrome (24), immunodetection of a phytochrome-like polypep-tide from these organisms is not too unexpected. Immunodetec-tion of a phytochrome-like polypeptide from Chlamydomonas,however, is novel since there is no a priori expectation that itshould be present in this alga.

Identification of an antibody that recognizes phytochrome, or

24-". * ^

72-66-52-

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FIG. 6. Epitope location for Pea-25. Aliquots of a crude extract ofetiolated oat shoots, either immediately after preparation (lanes 1-3) orfollowing 7 h incubation as Pfr in darkness at 22C (lanes 4-6), wereelectrophoresed on a 5 to 10% gradient SDS polyacrylamide gel. Afterelectrotransfer to nitrocellulose, the polypeptides were immunostainedwith I jig/ml of Oat-25 (lanes 1 and 4), Oat-22 (lanes 2 and 5), or Pea-25 (lanes 3 and 6), following which the individual strips were reconsti-tuted into their original geometry. Indicated sizes of immunostainedpolypeptides in kD were derived from a standard curve.

at least a polypeptide about the size of native oat phytochrome,from such a diverse range of organisms creates new possibilitiesfor investigating phytochrome function. In particular, if thephytochrome-like polypeptide from Chlamydomonas is phyto-chrome, then it becomes possible to initiate genetic investigationsrelated to the mode of action of this morphogenically activechromoprotein that would be difficult to perform with higherplants. Attempts to identify phytochrome-mediated responses inChlamydomonas appear to be justified based upon the datapresented here.The highly conserved nature of the epitope recognized by Pea-

25 indicates that it may play a critical role in the molecularfunction of phytochrome, and that detailed characterization ofthis epitope is justified. Screening ofa large panel ofmonoclonalantibodies to phytochrome indicated that, while a few discrimi-nate between Pr and Pfr (6; Y Shimazaki, M-M Cordonnier, LHPratt, unpublished observations), Pea-25 does not, at least by theassay method that was used (6).

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CONSERVED PHYTOCHROME DOMAIN

Initial epitope mapping data (Fig. 6) indicate that Pea-25 bindsto the nonchromophore bearing, carboxyl half of phytochrome.As discussed in detail elsewhere (6), Oat-25 detects an epitopethat includes primary structure within 6 kD of the amino ter-minus of undegraded oat phytochrome, while Oat-22 recognizesan epitope on the same half of the phytochrome polypeptide,but further removed from the amino terminus. Thus, the 72-kD,proteolytically derived peptide that is detected by Oat-25 (Fig. 6,lane 4) must derive from the amino terminus end of phyto-chrome. It is this half of the phytochrome polypeptide that isknown to contain the chromophore (8). Since Pea-25 does notstain this 72-kD peptide (Fig. 6, lane 6), it presumably mustrecognize an epitope at the other end of phytochrome. Theability of Pea-25 to immunostain a 52-kD peptide (Fig. 6, lane6), which is the difference between undegraded phytochrome of124 kD and the 72-kD chromophore-bearing peptide, is consist-ent with this presumption. Thus, even though the half of phyto-chrome that includes the epitope for Pea-25 does not contain thechromophore, and is not required for phytochrome photorever-sibility (15, 22), it does contain the most highly conserveddomain yet identified on this chromoprotein.

Acknowledgments-We thank Marie-Claire Pfeiffer, Caroline Gabus, StephanPost, and Donna Tucker for their excellent technical assistance. We are grateful toDr. Pat King, Professor Jean-Pierre Zryd, Dr. Steve Mayfield, Professor Jean-DavidRochaix, Mr. Daniel G. Kidd, and Professor J. C. Lagarias for providing us withsamples.

LITERATURE CITlED

1. BARTEK J, V VIKLICKY~, A STRATIL 1985 Phylogenetically more conservativeepitopes among monoclonal antibody-defined antigenic sites of humantransferrin are involved in receptor binding. Br J Haematol 59: 435-441

2. CORDONNIER M-M, LH PRATT 1982 Immunopurification and initial charac-terization of dicotyledonous phytochrome. Plant Physiol 69: 360-365

3. CORDONNIER M-M, LH PRATT 1982 Comparative phytochrome immuno-chemistry as assayed by antisera against both monocotyledonous and dico-tyledonous phytochrome. Plant Physiol 70: 912-916

4. CORDONNIER M-M, C SMITH, H GREPPIN, LH PRATT 1983 Production andpurification of monoclonal antibodies to Pisum and Avena phytochrome.Planta 158: 369-376

5. CORDONNIER M-M, H GREPPIN, LH PRATT 1984 Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to Pisum and Avenaphytochrome. Plant Physiol 74: 123-127

6. CORDONNIER M-M, H GREPPIN, LH PRATT 1985 Monoclonal antibodies withdiffering affinities to the red-absorbing and far-red-absorbing forms of phy-tochrome. Biochemistry 24: 3246-3253

7. COVE DJ, A SCHILD, NW ASHTON, E HARTMANN 1978 Genetic and physiolog-ical studies of the effect of light on the development of the moss, Physcomi-trella patens. Photochem Photobiol 27: 249-254

8. DANIELS SM, PH QUAIL 1984 Monoclonal antibodies to three separate do-mains on 124 kilodalton phytochrome from Avena. Plant Physiol 76: 622-626

9. GODING JW 1983 Monoclonal Antibodies: Principles and Practice. AcademicPress, London

10. HAUPT W 1970 Localization of phytochrome in the cell. Physiol Veg 8: 551-563

11. KERSCHER L, S NowITZKI 1982 Western blot analysis ofa lytic process in vitrospecific for the red light absorbing form of phytochrome. FEBS Lett 146:173-176

12. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

13. NAGATANI A, KT YAMAMOTO, M FURUYA, T FUKUMOTO, A YAMASHITA 1983Production and characterization of monoclonal antibodies to rye (Secalecereale) phytochrome. Plant Cell Physiol 24: 1143-1149

14. PRATT LH 1973 Comparative immunochemistry of phytochrome. Plant Phys-iol 51: 203-209

15. PRATT LH 1982 Phytochrome: the protein moiety. Annu Rev Plant Physiol33: 557-582

16. PRATT LH 1984 Phytochrome immunochemistry. In H Smith, MG Holmes,eds, Techniques in Photomorphogenesis. Academic Press, London, pp 201-226

17. RICE HV, WR BRIGGS 1973 Immunochemistry of phytochrome. Plant Physiol51: 939-945

18. SAJi H, A NAGATANI, KT YAMAMOTO, M FURUYA, T FUKUMOTO, A YAMASH-ITA 1984 Cross-reactivity of monoclonal antibodies against rye and peaphytochrome with phytochromes extracted from eight different plant species.Plant Sci Lett 37: 57-61

19. SHIMAZAKI Y, LH PRATT 1985 Immunochemical detection with rabbit poly-clonal and mouse monoclonal antibodies of different pools of phytochromefrom etiolated and green Avena shoots. Planta 164: 333-344

20. SHIMAZAKI Y, M-M CORDONNIER, LH PRATT 1983 Phytochrome quantitationin crude extracts of Avena by enzyme-linked immunosorbent assay withmonoclonal antibodies. Planta 159: 534-544

21. SHROPSHIRE W JR, H MOHR, eds 1983 Photomorphogenesis. In Encyclopediaof Plant Physiology, New Series, Vol 16. Springer-Verlag, Heidelberg

22. SMITH WO 1983 Phytochrome as a molecule. In W Shropshire Jr, H Mohr,eds, Encyclopedia ofPlant Physiology, New Series, Vol 16A. Springer-Verlag,Heidelberg, pp 96-118

23. STUDIER FW 1973 Analysis of bacteriophage T7 early RNAs and proteins onslab gels. J Mol Biol 79: 237-248

24. TAYLOR AO, BA BONNER 1967 Isolation of phytochrome from the algaMesotaenium and liverwort Sphaerocarpos. Plant Physiol 42: 762-766

25. TOWBIN H, T STAEHELIN, J GORDON 1979 Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: procedure and some ap-plications. Proc Natl Acad Sci USA 76: 4350-4354

26. VIERSTRA RD, PH QUAIL 1982 Native phytochrome: inhibition of proteolysisyields a homogeneous monomer of 124 kilodaltons from Avena. Proc NatlAcad Sci USA 79: 5272-5276

27. VIERSTRA RD, M-M CORDONNIER, LH PRATT, PH QUAIL 1984 Native phy-tochrome: immunoblot analysis of relative molecular mass and in-vitroproteolytic degradation for several plant species. Planta 160: 521-528

28. ZWEIG SE, S FERRONE, EM SHEVACH 1984 Monoclonal antibodies directedagainst human Ia antigens detect an evolutionarily conserved epitope onguinea pig Ia antigens with unique functional properties. J Leukocyte Biol35: 101-113

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