Proc. Natl. Acad. Sci. USAVol. 84, pp. 359-363, January 1987Biochemistry

Red light-induced formation of ubiquitin-phytochrome conjugates:Identification of possible intermediates of phytochrome degradation

(protein degradation/regulatory photoreceptor/posttransladonal modification)

JOHN SHANKLIN, MERTEN JABBEN, AND RICHARD D. VIERSTRADepartment of Horticulture, University of Wisconsin-Madison, Madison, WI 53706

Communicated by Winslow R. Briggs, September 23, 1986 (received for review June 19, 1986)

ABSTRACT Phytochrome is the photoreceptor that con-trols red light-mediated morphogenesis in higher plants. Itexists in two photointerconvertible forms, a red light-absorbingform, Pr, and a far-red light-absorbing form, Pfr. Becausephotoconversion of Pr to Pfr by a brief light pulse decreases thein vivo half-life of this chromoprotein by a factor of =100, thissystem offers a unique way to modulate the turnover rate of aspecific protein and hence study the mechanisms responsiblefor selective protein degradation. In etiolated oat [Avena sativa(L.)] seedlings, degradation of phytochrome as Pfr followszero-order kinetics as measured both spectrally and by ELISA,with 50% of Pfr lost in 130 min at 270C. Immunoblot analysisof the destruction process with anti-oat phytochrome immu-noglobulins reveals that degradation involves the loss of the124-kDa phytochrome monomer and that proteolytic interme-diates of apparent molecular mass lower than 124 kDa do notaccumulate to detectable levels in vivo (

360 Biochemistry: Shanklin et al.

irradiated by use of a slide projector in conjunction witheither a red (660 nm, 10-nm half-bandwidth) interference filteror a far-red (>720 nm, Coming type CS7-69) cutofffilter. Redlight irradiations were for 5 min and converted =75% ofPr toPfr [assuming 86% Pfr at saturation (12)]. Following irradi-ation, seedlings were maintained in darkness at 270C, and atvarious times, the apical 3-4 cm of the seedling was harvest-ed and rapidly frozen in liquid nitrogen. Frozen tissue washomogenized at 40C for 30 sec in 25% ethylene glycol/50mMTris HCl/70 mM (NH4)2SO4/5 mM Na4EDTA/20 mM sodi-um metabisulfite, pH 8.0, at 40C (2.5 ml/g fresh weight), withthe addition of 4 mM phenylmethylsulfonyl fluoride justbefore use. The extract was made 0.1% (wt/vol) poly(ethyl-enimine) by addition of a 10% (wt/vol) solution (pH 7.8),stirred for 5 min, and clarified at 50,000 x g for 20 min. Thiscrude supernatant was used for most subsequent analyses.Phytochrome was partially purified (80- to 100-fold) byammonium sulfate precipitation followed by hydroxyapatitechromatography (13).Antibody Preparation. Polyclonal immunoglobulins direct-

ed against highly purified oat phytochrome (AW/A280 ratios2 1.00) were raised in both rabbits and chickens. Totalimmunoglobulins were purified from yolks ofchicken eggs bythe method of Polson and von Wechmar (14). Phytochrome-specific immunoglobulins were purified from either rabbitserum or total chicken immunoglobulins by affinity chroma-tography using a column containing oat phytochrome immo-bilized on Affi-Gel 10 (13). Purified mouse monoclonal IgGsdirected against the 6-kDa amino-terminal region of oatphytochrome (designated type 1) were those described byDaniels and Quail (15). Polyclonal rabbit immunoglobulinsdirected against either purified oat or human ubiquitin wereprepared according to Hershko et al. (16). In both cases,ubiquitin was conjugated to bovine gamma-globulin withglutaraldehyde and then boiled in the presence of 0.1%(wt/vol) NaDodSO4 prior to injection. Anti-ubiquitin immu-noglobulins were purified from rabbit serum by affinitychromatography using a column containing the correspond-ing ubiquitin immobilized on Affi-Gel 10.

Spectral Measurements. Spectral quantitation of phyto-chrome was by dual-wavelength (A730/A6m) spectroscopyfollowing saturating red or far-red irradiations, using either aShimadzu UV3000 spectrophotometer or a Ratiospect (Ag-ricultural Specialty, Beltsville, MD). The extinction coeffi-cient of 1.2 x 105 liter mol-l'cm-1 for Pr (17) and a photo-equilibrium value of86% Pfr in red light (12) were used for allcalculations of phytochrome content.ELISA. Immunological quantitation of phytochrome was

accomplished by "sandwich" ELISA using chicken immu-noglobulins adsorbed to the wells of microtitration plates(Costar, Cambridge, MA) and rabbit immunoglobulins as theprimary detector. Wells were incubated overnight with 50 ,ulof chicken anti-phytochrome immunoglobulins (12 ,ug/ml) in25mM potassium phosphate (pH 7.5). Remaining nonspecificprotein-binding sites were blocked with 3% (wt/vol) gelatinin 20 mM Tris HCl/180 mM NaCl, pH 9.0, for 60 min at 37°C,and the wells were washed with 10 mM potassium phos-phate/150 mM NaCl/0.02% (wt/vol) NaN3/0.05% (vol/vol)Triton X-100, pH 7.5 (wash solution). Crude extracts orpurified phytochrome (50-,ul samples), diluted to the appro-priate concentrations with 10 mM potassium phosphate/150mM NaCl/0.02% NaN3/0.05% Triton X-100/1% (wt/vol)bovine serum albumin, pH 7.2 (ELISA diluent), were addedto the wells with phytochrome as Pfr and incubated for 2.5 hrat 4°C. Wells were then rinsed three times with wash solutionand incubated for 90 min with 50 Al of rabbit anti-phyto-chrome immunoglobulins (8 ,ug/ml of ELISA diluent). Afterthree additional washes, wells were incubated for 90 min at250C with 50 tkl of alkaline phosphatase-conjugated goat IgGsdirected against rabbit IgGs (5 Ag/ml of ELISA diluent).

Then the wells were washed three times and 100 ,.l ofp-nitrophenyl phosphate (2 mg/ml in 5 mM TrisHCl, pH 9.0)was added to each well. The reactions were terminated by theaddition of 50 ttl of 1 M NaOH, and the extent of enzymeactivity was determined from the absorbance at 405 nm.

Immunoprecipitations. Immunoprecipitations were per-formed as described (18). Phytochrome-containing samples(1 ml) were clarified by centrifugation at 16,000 x g for 5 minand incubated for 60 min at 40C with 26 ,ug of anti-phyto-chrome immunoglobulins. Staphylococcus aureus cells [100,ul of20% (vol/vol) suspension] were added and incubated for20 min followed by centrifugation through a 400-1.l sucrosecushion at 16,000 x g for S min. The pellet was resuspendedand washed twice in 50 mM Tris HCl/150 mM NaCl/Na4-EDTA/0.02% NaN3, pH 7.5, and the final pellet was sus-pended in NaDodSO4/PAGE sample buffer (3) and boiled for3 min.Immunoblot Analysis. Discontinuous NaDodSO4/PAGE

(19) was accomplished using 7% (wt/vol) acrylamide gels(acrylamide:methylene bisacrylamide ratio 30:0.8) and pro-teins were transferred to nitrocellulose (HAHY 304 FO,Millipore) as described (3). Immunoreactive phytochromebands were visualized colorimetrically by using rabbit anti-phytochrome immunoglobulins in conjunction with alkalinephosphatase-conjugated goat IgGs directed against rabbitimmunoglobulins and the phosphatase substrates nitro bluetetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (3).

Ubiquitin conjugates were visualized by a modification ofthe immunoblot method of Haas and Bright (20). Followingtransfer, the nitrocellulose membrane was incubated for 1 hrwith anti-oat- or anti-human ubiquitin immunoglobulins dis-solved at 0.5 ,ug/ml in 25 mM Tris'HCl/150 mM NaCl/2.5%bovine serum albumin/0.02% NaN3, pH 7.5 (immunoblotdiluent). The membrane then was washed with 25 mMTrisHCl/150 mM NaCl, pH 7.5 (Tris/NaCl) for 20 min,followed by a 20-min wash with Tris/NaCl containing 0.05%Triton X-100, and by another 20-min wash with Tris/NaCl.The membrane was incubated for 1 hr with immunoblotdiluent containing 251I-labeled protein A (20 ng/ml; Amer-sham). Initial specific radioactivity ofthe solution was 4 x 10,dpm/ml. The membrane was washed three times and dried.Immunoreactive bands were visualized by autoradiographywith Kodak XAR-5 x-ray film in conjunction with CronexLightning Plus intensifying screens.

RESULTSIn accord with previous observations on many plant species(9-11), phytochrome in etiolated oat seedlings was rapidlydegraded in vivo (as measured spectrally or by ELISA) afterphotoconversion of Pr to Pfr (Fig. 1). We conclude thatdegradation is specific for Pfr, because the loss of totalphytochrome in red light-irradiated seedlings can be account-ed for entirely by the loss of Pfr (Fig. 1) and because nomeasurable loss of phytochrome was observed in controlseedlings containing almost exclusively Pr [far-red or unir-radiated (Fig. 1)]. Pfr degradation began immediately afterphotoconversion and followed zero-order kinetics, with 130min required to degrade 50% of Pfr. Immunoblot analysiswith anti-oat-phytochrome immunoglobulins (Fig. 1 Upper)revealed that the 124-kDa oat phytochrome monomer isspecifically lost after Pfr formation (Fig. 1). Degradationkinetics obtained spectrally deviated from those obtained byELISA; this has been observed previously and is thought toresult from a spectrally active but immunologically distinctpool of stable phytochrome (10).

In an attempt to detect in vivo intermediates of Pfrdegradation, crude oat extracts were rapidly prepared atvarious times after Pfr formation. These extracts weresubjected to immunoblot analysis with anti-oat-phytochrome

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FIG. 1. Kinetics of phytochrome degradation in etiolated oatseedlings after photoconversion to Pfr. Oat seedlings were eitherkept in darkness (c, *) or irradiated with red (o, e, A) or far-red (o,*) light at zero time and then incubated at 27TC in darkness. Atvarious times, tissue was rapidly frozen and homogenized, andphytochrome content in the crude extract was assayed spectrally(open symbols), by sandwich ELISA (filled symbols), or by im-munoblot analysis (gel lanes). Pfr content in red light-irradiatedseedlings (A) was measured spectrally and is expressed as a percent-age of the total phytochrome content. Immunoblot analysis withanti-oat phytochrome immunoglobulins was done after NaDod-SO4/PAGE of equal volumes of crude extract prepared at each timepoint. Only the region of the blot surrounding the 124-kDa oatphytochrome monomer is shown. R, FR, and D indicate samplesprepared from red light-irradiated tissue, far-red light-irradiatedtissue, and unirradiated tissue, respectively.

immunoglobulins, either directly or following immunoprecip-itation of phytochrome with anti-oat phytochrome immuno-globulins. The extraction conditions used have been shown(13) to minimize posthomogenization proteolysis of thechromoprotein. From such analyses, we were unable todetect any intermediate(s) with apparent molecular masses20 kDa that appeared to be specific to the invivo catabolism of Pfr (unpublished data). The immunoblotmethods used can detect as little as 10 pg of the undegradedoat phytochrome monomer. Because the samples subjectedto immunoblotting contained .150 ng of phytochrome, thisfailure suggests that the large intermediates (=60 kDa)expected to be generated during Pfr breakdown in vivorepresent 124 kDa was similar, within experimental variability, to thatobserved with anti-phytochrome antibody (Fig. 2). Highermolecular mass polypeptides were detected within S min afterred light irradiation, reached maximal levels (as a percentageof the total phytochrome pool) at -'90 min, and declinedthereafter. (Note that the samples in Fig. 3 were adjusted togive equal phytochrome content and as a result do notrepresent the relative content of the proteins in vivo.) Thesepolypeptides were not observed in samples prepared fromeither (i) unirradiated tissue; (ii) unirradiated tissue that washomogenized and then irradiated with red light; or (iii) fromtissue chilled to ice temperature, irradiated with red light, andimmediately frozen (Figs. 3 and 4; unpublished data). This

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362 Biochemistry: Shanklin et al.

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FIG. 3. Time course of appearance of ubiquitin-phytochromeconjugates in etiolated oat seedlings following photoconversion toPfr. Oat seedlings were irradiated with red light and then incubatedat 270C in darkness. At the times indicated, tissue was rapidly frozenand homogenized and phytochrome was partially purified by ammo-nium sulfate precipitation and hydroxyapatite chromatography.Phytochrome was immunoprecipitated from the pooled hydroxyap-atite fractions with anti-oat phytochrome immunoglobulins andsubjected to NaDodSO4/PAGE and immunoblot analysis with anti-oat ubiquitin immunoglobulins. An equal amount of immunoprecip-itated phytochrome was applied to each gel lane (determined spec-trally). Detection ofthe unmodified, 124-kDa phytochrome monomer(arrowheads) in each lane is the result ofnonspecific immunoglobulinbinding. The heavily stained band of lower molecular mass in eachlane represents the heavy chain of rabbit anti-phytochrome IgG usedfor the immunoprecipitations.

demonstrated that their formation required red light andoccurred in vivo. These polypeptides had apparent molecularmasses similar to those observed with anti-phytochromeantibody (Fig. 2) but, in contrast, exhibited greater im-munorecognition with the anti-ubiquitin antibody with in-creasing size. In fact, the 129-kDa species observed promi-nently with anti-phytochrome antibody gave only a faint bandwith the anti-ubiquitin antibody.Based on their copurification with phytochrome and im-

munoreaction with both antibody types, we concluded thatthese higher molecular mass polypeptides represent ubiqui-tinated forms of phytochrome. Further evidence was provid-ed by the ability of anti-human ubiquitin antibody to alsorecognize this ladder of phytochrome polypeptides formedafter red light irradiation (Fig. 4 A and B). Since theanti-human ubiquitin immunoglobulins were elicited againsta highly purified protein from erythrocytes (20), it is unlikelythat they would recognize plant proteins other than thosecontaining ubiquitin sequence. In addition, the failure ofnonspecific immunoglobulins to recognize this ladder whenused either to immunoprecipitate or to immunoblot phyto-chrome-containing samples eliminated the possibility thatnonspecific binding was responsible for the observed signals(Fig. 4 C and D).From phytochrome-containing samples purified by hy-

droxyapatite chromatography followed by immunoprecipita-tion, several discrete ubiquitin-phytochrome conjugateswere detected by immunoblotting (Figs. 3 and 4). However,immunoblot analysis of immunoprecipitates obtained direct-ly from crude extracts revealed substantial heterogeneity inapparent molecular mass of these conjugates. In addition tothe discrete size classes detected previously (Fig. 4A), asmear of phytochrome-ubiquitin conjugates up to 200 kDa

FIG. 4. Immunoblot analysis of ubiquitin-phytochrome conju-gates with various immunoglobulin preparations. Oat seedlings wereirradiated with red light and frozen immediately (t = 0) or incubatedfor 90 min at 270C in darkness (t = 90 min) before freezing. Frozentissue was homogenized and phytochrome was immunoprecipitatedeither directly from the crude extract (E) or after partial purificationof phytochrome by ammonium sulfate precipitation and hydroxyap-atite chromatography (A-D). Immunoprecipitations were performedwith either anti-oat phytochrome immunoglobulins (A, B, C, and E)or an equivalent amount of nonimmune immunoglobulins (D).Immunoprecipitates were then subjected to NaDodSO4/PAGE andimmunoblot analyses with either anti-oat ubiquitin immunoglobulins(A, D, and E), anti-human ubiquitin immunoglobulins (B), or anequivalent amount of nonimmune immunoglobulins (C). In A, B, C,and E, equal amounts of phytochrome were applied in the 0- and90-min lanes (determined spectrally). Lanes in D contained equalvolumes of immunoprecipitates. Detection of the unmodified, 124-kDa phytochrome monomer (arrowheads) in each lane is the resultof nonspecific immunoglobulin binding. The heavily stained band atlower molecular mass in each lane represents the heavy chain ofrabbit anti-phytochrome IgG used for the immunoprecipitations.

could be observed after prolonged autoradiographic expo-sure (Fig. 4E). The appearance and disappearance of thisconjugate smear paralleled that observed for the discretebands purified by hydroxyapatite chromatography.

DISCUSSIONThe immunological evidence presented here demonstratesthat ubiquitin-phytochrorne conjugates are produced in etio-lated oat seedlings following red light irradiation. Copurifica-tion of these conjugates with phytochrome and their immu-noprecipitation with anti-phytochrome immunoglobulins pre-pared against highly purified phytochrome preclude thepossibility that they represent non-phytochrome conjugates.Likewise, detection by both anti-oat- and anti-human ubiq-uitin antibodies discounts the possibility that these observa-tions result from contaminating immunoglobulins in either ofour anti-ubiquitin preparations. The increased antigenicity ofthe individual conjugates with anti-ubiquitin immunoglob-ulins as a function of higher molecular mass would beexpected based on an increased availability of antigenicdeterminants on such conjugates as additional ubiquitins areattached. The poor recognition of the 129-kDa polypeptide byanti-ubiquitin immunoglobulins may indicate that the ubiq-uitin moiety is not readily accessible in this species.

Although the effect of bifurcation (isopeptide bond forma-tion) on the apparent size of large proteins such as phyto-chrome is unknown, it is possible that the predominantspecies observed here represent phytochrome conjugatedwith one to seven ubiquitin molecules. The exact linkagesite(s) within the chromoprotein have not been identified, butthe facts that ubiquitin can be linked to either a- or s-amino

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Proc. Natl. Acad. Sci. USA 84 (1987) 363

groups on the target protein or to itself once conjugated(22-24) and that oat phytochrome contains 64 lysines (25)suggest that many potential linkage sites are available. Thepossibility that these sites are accessible only when phyto-chrome is in the Pfr form may help explain the preferentialconjugation of ubiquitin to this spectral form.The physiological significance of ubiquitin-phytochrome

conjugation is as yet unresolved. To our knowledge, thismodification represents the first posttranslational modifica-tion reported for phytochrome that occurs in vivo and isselective for one spectral form (Pfr). The involvement ofubiquitin conjugation in the degradation of many short-livedcytoplasmic proteins (4) and the fact that phytochrome israpidly degraded as these conjugates are formed suggest thatthey represent intermediates in the degradation of Pfr. Theapparent energy dependence of Pfr destruction (26) could bepartially explained by the known ATP requirement forubiquitin conjugation (1, 2). It is also possible that Pr isdegraded in the same manner but that the in vivo concentra-tion of Pr-ubiquitin conjugates are below detectable levels asa result of the slow turnover rate of Pr. The levels of ubiquitinconjugates do not coincide with the levels of Pfr. This mayindicate that reactions other than those directly involved inconjugation are rate-limiting for phytochrome destruction.This possibility is consistent with earlier observations (11)that the rate of Pfr disappearance is not regulated by theamount of Pfr but by a highly efficient but rate-limiting darkreaction. However, when considering the various roles ofubiquitination in cell physiology (2, 5, 6), alternative func-tion(s) for ubiquitin-phytochrome conjugates, including thepossibility that they represent the active form of the photo-receptor, cannot be dismissed.Because ubiquitin conjugation appears to serve as a com-

mitted step for protein catabolism (1, 2), it is possible that thespecificity of phytochrome degradation resides in the selec-tive ubiquitination of Pfr. Further investigation of the mo-lecular mechanism(s) involved in preferential ligation ofubiquitin to Pfr may provide insights into how cells recognizeand selectively degrade intracellular proteins.

We thank A. L. Haas for providing anti-human ubiquitin IgGs andS. M. Daniels and P. H. Quail for supplying the monoclonal antibodyto oat phytochrome. This work was supported by grants from theNational Science Foundation (DMB-8409210), the United StatesDepartment of Agriculture Competitive Grants Research Office(85-CRCR-1-1547), and the Research Division of the College of

Agriculture and Life Science (Hatch 2858) of the University ofWisconsin-Madison.

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