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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.
983
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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-
116-92.5-
66-
44-
116-92.5-66-
984 Plant Physiol. Vol. 80, 1986
"'I114 41 4111k-,ddr,ik
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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
200-
116-92.5-66-
44-31-
985
4x
--7 ^
'k
40 $14 40 400At",
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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-
_9-000_go
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.
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