Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis

Journal of Experimental Botany, Vol. 58, No. 10, pp. 2595–2607, 2007

doi:10.1093/jxb/erm087 Advance Access publication 1 June, 2007

RESEARCH PAPER

Functional cross-talk between two-component andphytochrome B signal transduction in Arabidopsis

Virtudes Mira-Rodado1, Uta Sweere2, Christopher Grefen1, Tim Kunkel2, Erzsebet Fejes3, Ferenc Nagy3,

Eberhard Schafer2 and Klaus Harter1,*

1 Zentrum fur Molekularbiologie der Pflanzen/Pflanzenphysiologie, Universitat Tubingen, Auf der Morgenstelle 1,D-72076 Tubingen, Germany2 Institut fur Biologie II/Botanik, Universitat Freiburg, Schanzlestrasse 1, D-79104 Freiburg, Germany3 Plant Biology Institute, Biological Research Center, PO Box 521, H-6726 Szeged, Hungary

Received 12 February 2007; Revised 20 March 2007; Accepted 26 March 2007

Abstract

The A-type response regulator ARR4 is an element in

the two-component signalling network of Arabidopsis.

ARR4 interacts with the N-terminus of the red/far-red

light photoreceptor phytochrome B (phyB) and func-

tions as a modulator of photomorphogenesis. In con-

cert with other A-type response regulators, ARR4 also

participates in the modulation of the cytokinin response

pathway. Here evidence is presented that ARR4 directly

modulates the activity state of phyB in planta, not only

under inductive but also under extended irradiation

with red light. Mutation of the phosphorylatable aspar-

tate to asparagine within the receiver domain creates

a version of ARR4 that negatively affects photomorpho-

genesis. Additional evidence suggests that ARR4 activ-

ity is regulated by a phosphorelay mechanism that

depends on the AHK family of cytokinin receptors.

Accordingly, the ability of ARR4 to function on phyB is

modified by exogenous application of cytokinin. These

results implicate a cross-talk between cytokinin and

light signalling mediated by ARR4. This cross-talk

enables the plant to adjust light reponsiveness to

endogenous requirements in growth and development.

Key words: ARR4, cytokinin, light, phytochrome B, signal

integration, two-component signalling.

Introduction

To monitor the intensity, wavelength, direction, andtiming of light, plants have evolved diverse photoreceptor

systems. These photoreceptors include the blue light-sensitive cryptochromes and phototropins as well as thewell-characterized phytochromes (Schafer and Nagy, 2006).

Phytochromes (phy) are red (R)/far-red (FR) photo-reversible chromoproteins that form dimers with a mono-meric molecular mass of ;120 kDa, to which an opentetrapyrrole chromophore is covalently attached (Tu andLagarias, 2005). They are synthesized in the inactive Prform in the dark and photoconverted by red light into thephotomorphogenically active far-red light-absorbing Pfrform. In addition to these light reactions, Pfr is convertedback to Pr by a thermally driven process called darkreversion. Dark reversion has a strong effect on phyto-chrome function and attenuates phytochrome activity byreducing the amount of active Pfr (Elich and Chory, 1997;Sweere et al., 2001; Fankhauser, 2002; Hennig, 2006). InArabidopsis thaliana, the phytochrome family comprisesfive members termed phyA to phyE, with distinct butoverlapping modes of photomorphogenic action (Tu andLagarias, 2005). Whereas phyA controls the very lowfluence and far-red high irradiance responses, phyB tophyE mediate responses to continuous red (cR) light andare responsible for the R/FR reversible induction reactions(Schafer and Nagy, 2006).

In general, the phytochrome molecule is separated intotwo major domains: an N-terminal chromophore carryinga photosensory domain of ;70 kDa and a C-terminaldomain of ;55 kDa (Fig. 1A). The latter is necessary fordimerization and the import of phy into the nucleus, andcomprises two PAS motifs as well as a His kinase-likedomain (Sakamoto and Nagatani, 1996; Kircher et al.,1999, 2002). The C-terminal domain has long been

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

considered to transduce the light signal to downstreamsignalling components. However, recent studies byNagatani and colleagues have shown that the N-terminalphotosensory domain of phyB alone is sufficient forsignalling and that the C-terminal domain probably hasa function in the modulation of signal transduction(Matsushita et al., 2003; Oka et al., 2004).

The N-terminal photosensory domain is highly con-served throughout the phytochromes and displays spectralproperties indistinguishable from those of the full-lengthphotoreceptors. Within this domain three different regionscan be recognized (Fig. 1A): a short N-terminal extensionof 6–10 kDa, a central chromophore-carrying region of;40 kDa, and the C-terminal PHY region of ;20 kDa(Tu and Lagarias, 2005). In the case of phyB, the PHYregion is required for the spectral integrity and stabiliza-tion of the active Pfr form by reducing the rate of darkreversion, but is not essential for signal transduction (Okaet al., 2004). The chromophore-bearing central region,including the N-terminal extension of phyB, upholds itsbiological activity in planta and is able to transduce thelight signal almost normally to downstream elements inresponse to irradiation (Oka et al., 2004). The shortN-terminal extension has also been shown to play animportant role in the function of phytochromes. Truncationof the N-terminal extension results in an increase in the rateof dark reversion, indicating that this domain is not criticalfor signalling but plays a prominent role in Pfr stabilization(Quail, 1997). In summary, whereas the central region ofthe photosensory domain mediates light signal transduc-tion, the interplay of both ends—the N-terminal extensionand the PHY region—appears to be involved in thestabilization of the active Pfr form, thereby influencing thelight sensitivity of phytochromes (Oka et al., 2004).

Phytochrome-dependent signal transduction involvesthe translocation of the active Pfr form from the cytoplasminto the nucleus and the physical interaction with andpost-translational modification of downstream signallingelements (Nagatani, 2004; Khanna et al., 2004; Ryu et al.,2005; Al-Sady et al., 2006). In recent years, severalelements have been identified, which interact withphytochromes in the cytoplasm and the nucleus and areassumed to participate in downstream light signalling(Ryu et al., 2005; Al-Sady et al., 2006; Schafer and Nagy,2006). Among them, only the Arabidopsis responseregulator 4 (ARR4) recognizes the N-terminal extensionof phyB (Sweere et al., 2001; Fankhauser, 2002). In yeastand plants, the interaction of ARR4 with phyB stabilizesthe active Pfr form of the photoreceptor under inductivered light conditions by reducing the rate of dark reversion.Therefore, overexpression of ARR4 resulted in increasedred light sensitivity in hypocotyl growth and other phyB-regulated processes (Sweere et al., 2001).

ARR4 is a canonical response regulator (Fig. 1A) of theArabidopsis type-A subfamily, whose activity is believed

to be regulated by a two-component His/Asp phospho-relay—a phosphotransfer mechanism that is initiated bya sensor histidine kinase (Grefen and Harter, 2004;Ferreira and Kieber, 2005). However, although the

Fig. 1. ARR4 modulates phytochrome B-Pfr stability and phytochromeB (phyB) function under continuous red light. (A) Schematic represen-tation of Arabidopsis phyB (AtphyB) and the Arabidopsis responseregulator 4 (ARR4). N, N-terminal extension; CENTRAL, centralchromophore-bearing domain; PHY, PHY domain; receiver, receiverdomain with the conserved phosphorylatable Asp95 (D95); out, outputdomain. (B) phyB-Pfr dark reversion in Arabidopsis seedlings. Four-day-old ABO/A– (triangles) or ARR4-overexpressing ABO/A– (circles)seedlings were irradiated for 20 min with red light of the indicatedintensity. The amount of phyB-Pfr (filled symbols) and total phyB(open symbols) was measured in planta at the end of the irradiationperiod using a dual-wavelength ratio photometer. The error barsrepresent the SD (n >3). (C) Hypocotyl elongation response inseedlings. Fluence rate dependence of red light inhibition of hypocotylelongation in 3-d-old ABO/A– (triangles) or ARR4-overexpressingABO/A– (circles) seedlings. D, hypocotyl length in darkness. Error barsrepresent the SD (n >30).

2596 Mira-Rodado et al.

C-terminal domain of phytochromes displays homology tothe transmitter region of histidine kinases, correspondinghistidine kinase activity has not been reported for eitherphyB or for any other higher plant phytochrome (Tu andLagarias, 2006). This suggests that phyB is probably notthe cognate histidine kinase for the aspartate phosphoryla-tion of ARR4. It is conceivable that ARR4 represents theoutput element of an independent two-component signal-ling system which acts on phyB activity and, therefore,red light-regulated photomorphogenesis (Sweere et al.,2001; Fankhauser, 2002; Grefen and Harter, 2004). Type-A response regulators including ARR4 have also beenshown to be up-regulated in response to cytokinintreatment and to function as negative regulators incytokinin signalling (Kakimoto, 2003; Ferreira andKieber, 2005). To accomplish this negative function incytokinin signalling, type-A ARRs are believed to bephosphorelay targets of the cytokinin sensor histidinekinases AHK2, AHK3, and AHK4/CRE1/WOL (Kakimoto,2003; Grefen and Harter, 2004; Ferreira and Kieber, 2005;Riefler et al., 2006). Furthermore, in concert with ARR3,ARR4 maintains the pace of the circadian clock by actingon an as yet unknown clock protein. Thereby, ARR3 andARR4 signal via phyB into the clock and modulate thecircadian phase (Salome et al., 2006). Furthermore, ARR4and phyB are elements in the input of the cytokinin signalto the circadian phase of the clock (Hanano et al., 2006).From these results, the intriguing idea arises that a cytoki-nin-triggered two-component system may regulate ARR4activity on phyB by aspartate phosphorylation (Fankhauser,2002; Grefen and Harter, 2004).

In this study, the influence of ARR4 on phyB-Pfrstability in plants was examined under extended red lightirradiation. In addition, the effects of the expression ofa non-phosphorylatable form of ARR4 (ARR4D95N) onphotomorphogenesis in Arabidopsis have been analysedand, by biochemical and genetic approaches, upstreamregulatory elements have been identified which are likelyto participate in the regulation of ARR4 activity. Further-more, the in planta findings suggest that the phytohor-mone cytokinin directly modifies the sensitivity status ofphyB via phosphorelay. This molecular cross-talk mech-anism enables the plant to adjust light responsiveness toendogenous requirements in growth and development.

Materials and methods

Light sources, plant material and growth, and in planta

spectroscopic phytochrome measurement

The R, FR, and B light sources and handling of dark-grownseedlings under dim green safety light have been describedpreviously (Kircher et al., 1999; Sweere et al., 2001). To avoid anyinfluence of carbohydrates and growth media components onphotomorphogenesis (Fankhauser and Casal, 2004; see Supplemen-tary Fig. S6 at JXB online), Arabidopsis seeds were sown on four

layers of filter paper and allowed to imbibe in sterile water in thedark for 24 h at 4 �C. During the following irradiation or darkincubation, seedlings were kept at 23 �C. Hypocotyl length mea-surements, growth of Arabidopsis plants on soil, determination offlowering time (number of leaves developed at flowering) underlong-day conditions (16 h white light/8 h dark), and spectroscopicin planta measurement of total phytochrome and Pfr amounts usinga dual-wavelength ratio photometer were performed according toSweere et al. (2001). The following mutants or transgenic plantshave been described previously: ARR4-OX-I, ARR4-OX-II [ARR4-overexpressors in A. thaliana wild-type background (Col.); Sweereet al., 2001], ABO/A– [transgenic A. thaliana overexpressingArabidopsis phyB (ABO; Wagner et al., 1991) in the phyA mutant(A–) background (fre1-1; Nagatani et al., 1993)], ARR4-OX in theABO/A– background (Sweere et al., 2001), cre1-1 (Inoue et al.,2001), arr4 (To et al., 2004), and cre1-12/ahk2-2/ahk3-3 (Higuchiet al., 2004).

Plasmid construction, RT-PCR, expression of recombinant

proteins, and generation of transgenic Arabidopsis lines

Site-directed mutagenesis of Asp95 to asparagine (D95N) within theARR4 wild-type sequence cloned in the binary, Escherichia coliexpression and yeast two-hybrid vectors (for constructs used astemplates see Sweere et al., 2001) was performed using theQuickChange� XL Mutagenesis System (Stratagene) and appropri-ate primers according to the manufacturer. The cDNA encodingArabidopsis AHP1 was cloned into the yeast two-hybrid Gal4activation domain vector (AD) pGAD424 via BamHI/SalI. ThecDNA fragments encoding the receiver and output domain of ARR4(ARR4rec, amino acids 1–175; ARR4out, amino acids 176–259)were amplified by PCR using the wild-type ARR4 cDNA astemplate and appropriate primers, and cloned in the yeast two-hybrid Gal4 DNA-binding vector (BD) pGBT9 via BamHI/SalI andEcoRI/SalI, respectively. For reverse transcription–PCR (RT–PCR),total RNA was isolated from green leaves using RNAWiz�(Ambion). Reverse transcription of 1.5 lg of RNA per sample wasperformed using SuperScript III according to the manufacturer’sprotocol (Invitrogen). For amplification of cDNA, the PCR wascarried out in 25 cycles using Actin2- and ARR4-specific primers.

The other constructs used in this study have been describedpreviously: AHP1/pET24b [AHP1-(His)6] in Lohrmann et al.(2001) and ARR4/pASK-IBA2 (ARR4-Strep), phyB1–173/pGBT9(BD-phyB1–173), ARR4/pGBT9 (BD-ARR4), and ARR4/pGAD424(AD-ARR4) in Sweere et al. (2001). PCR-generated cDNAs wereverified by sequencing. Primer sequences are avaiable upon request.

Expression of Strep-tagged and (His)6-tagged proteins in E. coliand affinity purification on StrepTactin (IBA) or nickel-nitrilotri-acetic acid resin (Ni-NTA, Qiagen) have been reported previously(Lohrmann et al., 2001).Arabidopsis plants were transformed by in planta infiltration

using the Agrobacterium tumefaciens strain GV3101 and selectedfor glufosinate resistance according to Lohrmann et al. (2001) andSweere et al. (2001).

Yeast two-hybrid interaction assay and screen, co-immuno

and co-affinity purification, SDS-PAGE, western blot, and

immunodetection

Yeast transformation and determination of the ADE2 growth andlacZ reporter gene activities were carried out according toLohrmann et al. (2001). The yeast two-hybrid screen resulting intwo million primary transformants was performed as described byKim et al. (1997). Co-immunopurification of phyB with ARR4 orARR4D95N from plant extracts using an ARR4-specific antiserumfrom mouse has been described previously (Sweere et al., 2001).

Two-component system/light signal integration 2597

For co-affinity purification assays, 5 lg of AHP1-Strep, AHP2-Strep, or green fluorescent protein (GFP)-Strep were co-incubatedon ice for 2 h with 10 ll of 35S-labelled ARR4 in a final volume of250 ll of incubation buffer (100 mM TRIS-MOPS pH 8.3,150 mM NaCl, 1 mM EDTA, 28 mM b-mercaptoethanol). The35S-labelled ARR4 was generated by in vitro transcription/trans-lation in reticulocyte lysate using the TNT Quick CoupledTranscription/Translation kit (Promega). The co-incubated sampleswere washed three times with incubation buffer, and bound proteincomplexes eluted in 50 ll of elution buffer (100 mM TRIS-HCl pH7.4, 1 mM EDTA, 10 mM desthiobiotin). A 25 ll aliquot of theeluate was applied for immunodetection of ARR4 and 25 ll fordetection of Strep-tagged proteins using StepTactin–alkaline phos-phatase (AP) conjugate as described by the manufacturer (IBA).

SDS-PAGE, western blot transfer, and immunodetection wasperformed as described previously (Harter et al., 1994; Sweereet al., 2001).

Generation of evacuolated protoplasts, cell fractionation, and

in vitro phosphotransfer assay

Preparation of protoplasts from a heterotrophic cell culture of parsley(Petroselinum crispum; Dangl et al., 1987), evacuolization ofprotoplasts, protein extraction, subcellular fractionation, and gener-ation of microsomal preparations (membrane) were carried out asdescribed (Harter et al., 1994). For in vitro phosphorylation, 5 lg ofAHP1-(His)6 or ARR4-Strep were co-incubated for the indicatedtime period at 25 �C with 2 lg of total PcEP extract or membranefraction, respectively, and 10 lCi of [c-32P]ATP in a total volumeof 30 ll of phosphorylation buffer [13 TEDG buffer: 50 mMTRIS-HCl pH 8.0, 0.5 mM EDTA, 2 mM dithiothreitol (DTT),50 mM KCl, 5 mM MgCl2, 10% glycerol] containing 13 pro-teinase inhibitors (Boehringer). The proteins were recovered fromthe reaction mixture by affinity purification on StrepTactin or Ni-NTA beads as described above, and then subjected to SDS-PAGEand autoradiography. AHP1-to-ARR4 phosphotransfer was initiatedby co-incubating [32P]AHP1-(His)6 with 2 lg of unlabelled ARR4-Strep in a final volume of 50 ll of 13 TEDG buffer. After theindicated times, the reaction was stopped by the addition of 5 ll ofboiling 53 SDS sample buffer. A 25 ll aliquot of the sample wassubjected to autoradiography and 25 ll applied for detection ofARR4-Strep using StepTactin–AP conjugate and of AHP1-(His)6

using Ni-NTA–AP conjugate (Qiagen).

Results

ARR4 stabilizes the active Pfr form of phyB underextended red light irradiation

It was recently reported that ARR4 interacts with thephotoreceptor phyB and stabilizes the active Pfr form ofthe photoreceptor under inductive red light conditions inyeast and in plants by inhibiting dark reversion (Sweereet al., 2001). However, many (red) light-regulatedresponses, including hypocotyl growth inhibition, rely oncontinuous irradiation and are light intensity dependent(Schafer and Nagy, 2006). We therefore tested whetherARR4 also modulates the Pfr/Ptotal ratio of phyB underextended irradiation with red light of different intensity intransgenic Arabidopsis seedlings overexpressing Arabi-dopsis phyB in a phyA minus background (ABO/A–). Asshown in Fig. 1B, the amount of active phyB-Pfr strongly

depends on the intensity of red light during an extendedirradiation period of 20 min. The effect of Pfr-to-Pr darkreversion on the entire phyB-Pfr pool becomes mostnoticeable at lower fluence rates. In the presence ofARR4, however, the dark reversion of Pfr back to Pr isinhibited, resulting in higher levels of active phyB-Pfr,especially at lower light intensities (Fig. 1B). To de-termine whether the higher phyB-Pfr levels could corre-spond to altered phyB-mediated responses, hypocotylgrowth studies were performed in 3-d-old ABO/A– seed-lings. Confirming the data published in Sweere et al.(2001), overexpression of ARR4 resulted in seedlingswhich showed an enhanced hypocotyl growth inhibitionresponse over a wide range of red light intensities(Fig. 1C). These data suggest that ARR4 also causes thestabilization of active phyB-Pfr under extended red light(cR) conditions. Furthermore, this stabilization appears tobe, at least to a major extent, responsible for thehypersensitive phenotype observed in cR light-grownARR4-overexpressing Arabidopsis seedlings.

The function of ARR4 on phyB is phosphorylationdependent

Because ARR4 is a response regulator (Fig. 1A), it wasconsidered whether ARR4 activity on phyB may beregulated by phosphorylation. To investigate whether theconserved phosphor-accepting aspartate residue (Asp95)within the receiver domain of ARR4 may becomephosphorylated, a cell-free phosphorelay system was usedbased on evacuolated parsley (P. crispum) cell cultureprotoplasts (PcEP; Harter et al., 1994) and recombinantStrep-tagged ARR4 proteins, either in the wild-type formor in a form in which the Asp95 has been mutated toasparagine (ARR4D95N). Mutation of the conservedaspartate to asparagine within receiver domains usuallyabolishes phosphorylation and creates loss-of-functionvariants of response regulators (West and Stock, 2001).For phosphorylation experiments, wild-type ARR4 orARR4D95N were incubated in the presence of [c-32P]ATPin total extracts of PcEP. The proteins were thenrecovered by affinity purification and analysed for in-corporation of radioactive phosphate. Whereas wild-typeARR4 was efficiently phosphorylated, modification ofARR4D95N was not observed (Fig. 2A). This result showsthat ARR4 is phosphorylated by a plant extract in vitro.Furthermore, these data indicate that Asp95 is required forARR4 modification and represents the target amino acidresidue for a plant aspartate phosphorylation activity(Hwang et al., 2002).

Whether the Asp95 to asparagine mutation influences thecapacity of ARR4 to interact with phyB was determined byinteraction studies. In yeast two-hybrid assays, ARR4D95N

associated with the N-terminus of phyB similarly to wild-type ARR4 (Fig. 2B). The interaction of ARR4D95N withphyB in yeast is independent of whether the photoreceptor


is present in the active Pfr or inactive Pr form (A Viczian,E Schafer, and K Harter, unpublished data). Co-purificationexperiments using red light-irradiated total extracts from

plants overexpressing ARR4 or ARR4D95N revealed a verysimilar result (Fig. 2C; Sweere et al., 2001). In summary,the mutation of Asp95 to asparagine does not seem tointerfere with the interaction of ARR4 with phyB.

To determine whether the mutation of Asp95 toasparagine interferes with the potential of the responseregulator to act on phyB in vivo, transgenic Arabidopsisplants expressing ARR4D95N under the control of the 35Spromoter (see Supplementary Fig. S1A at JXB online)were generated and the corresponding seedlings wereanalysed for altered photomorphogenic response in lightof different wavelengths. In contrast to wild-type ARR4overexpressors, which showed a hypersensitive hypocotylgrowth response specifically to red light (Sweere et al.,2001), ARR4D95N-expressing seedlings exhibited a re-duced reaction not only to red but also to blue and far-redlight (Fig. 3A). Detailed fluence rate/response analyses forone selected ARR4D95N-expressing line indicate that thealtered photomorphogensis is due to the reduced sensitiv-ity of the seedlings to light (see Supplementary Fig. S2at JXB online). The inhibitory effect of ARR4D95N onphyB-mediated processes was also observed for flowering.In contrast to ARR4-overexpressing plants, which flowerlater than wild type under long-day conditions (16 h light/8 h dark), ARR4D95N-expressing plants set fewer leavesprior to bolting and flowered earlier than the wild type(Fig. 3B). This hyposensitive phenotype of theARR4D95N-expressing plants is similar to that of an arr4loss-of-function mutant published by To et al. (2004).Under the present growth conditions, the arr4 mutantdisplayed a significantly reduced response to red light but,in contrast to the ARR4D95N overexpressors, a wild-typereaction to blue and far-red light (Fig. 3C; see Supplemen-tary Fig. S3 at JXB online). If ARR4D95N is ectopicallyexpressed in ABO/A– plants (see Supplementary Fig. S1Bat JXB online), the strong effect on photomorphogenesis iseven more dramatic. ABO/A– plants display the typicalphyB overexpressor phenotype, showing a very compactrosette with short internodes and dark-green leaves withshort petioles (Fig. 3D). This phenotype can be furtherenhanced by the overexpression of wild-type ARR4(Fig. 3D). In contrast, the expression of ARR4D95N causedABO/A– plants (F1 generation) to develop leaves withextremely long petioles and stems with long internodes—aphenotype which is very similar to that of phyB mutants(Fig. 3D). However, due to strong silencing effects in theF2 and following generations, the phyB-Pfr dark rever-sion of ABO/A– seedlings expressing ARR4D95N couldnot be determined. The effect of ARR4D95N on photo-reversible phyB in yeast was therefore studied (Sweereet al., 2001). In contrast to wild-type ARR4, ARR4D95N

was not able to stabilize the active Pfr form of phyBand the photoreceptor showed dark reversion kineticssimilar to those observed in cells not expressing a responseregulator (Fig. 4).

Fig. 2. Non-phosphorylatable ARR4D95N interacts with phyB. (A)Phosphorylation of ARR4 targets Asp95. Strep-tagged ARR4 orARR4D95N were incubated for 15 min at 4 �C in a reaction mixcontaining [c-32P]ATP and the indicated components. Proteins wererecovered by affinity chromatography and analysed for incubation oflabelled phosphate (I, [32P]ARR4-Strep) or subjected to immunodetec-tion using an ARR4-specific antiserum (II, a-ARR4). (B) ARR4 andARR4D95N interact with the N-terminal domain of phyB (amino acids1–173) in the yeast two-hybrid system. The interaction of the indicatedfusion proteins was monitored by the activity of the LacZ reporter gene(b-galactosidase). Error bars represent the SD (n >3). (C) ARR4 andARR4D95N interact with phyB in planta. PhyB was co-purified fromcrude extracts of plants overexpressing either ARR4 (ARR4-OX) orARR4D95N (ARR4D95N-OX) using an ARR4-specific antiserum (a-ARR4). As a control, the corresponding pre-immunoserum was used(pre-ARR4). PhyB was detected with the monoclonal antibody mAT1(Lopez-Juez et al., 1992).


In summary, the data indicate that ARR4 interferes withlight and phyB signalling and that Asp95 modification hasa major impact on the ability of ARR4 to modulate theactivity of phyB in vivo.

The ARR4 activity appears to be regulated by atwo-component signalling pathway

Although higher plant phytochromes are ancestral two-component histidine kinases, His/Asp phosphorelayactivity could not yet be demonstrated for these photo-receptors (Tu and Lagarias, 2006). Therefore, it isconceivable to assume that Asp95 phosphorylation of

ARR4 occurs through an independent two-componentsystem. To identify two-component proteins potentiallyacting on ARR4, a yeast two-hybrid screen was performed(see Materials and methods for details). From five ARR4-interacting clones, two were identified to encode DBP1,two a DBP1-like protein, and one the Arabidopsishistidine phosphotransfer domain-containing (HPt) protein1 (AHP1; Hwang et al., 2002). The DBP1 gene encodesa DNA-binding protein of unknown function whoseexpression is up-regulated in response to auxin treatment(Alliotte et al., 1989). Because AHP proteins link viaphosphorelay response regulators to hybrid histidine

Fig. 3. ARR4D95N-overexpressing and arr4 loss-of-function Arabidopsis plants display a hyposensitive photomorphogenic phenotype. (A)Hypocotyl elongation response of wild-type (WT) compared with ARR4D95N-OX-I and ARR4D95N-OX-II seedlings. Seedlings were grown for 3 dunder continuous red (cR, 3.2 lmol m�2 s�1), far-red (cFR, 0.4nlmol m�2 s�1), or blue (cB, 119 lmol m�2 s�1) light, or kept in darkness (D). Errorbars represent the SE (n >30). (B) Number of leaves at flowering of wild-type (WT), ARR4D95N-OX-I, and ARR4D95N-OX-II plants grown underlong-day conditions (16 h white light/8 h dark). Error bars represent the SD (n >30). (C) Relative hypocotyl elongation response of wild-type (WT)compared with arr4 loss-of-function mutant (arr4) seedlings. Seedlings were grown and irradiated as described in A (cB, 0.4 lmol m�2 s�1; cR,0.7 lmol m�2 s�1; FR, 0.3 lmol m�2 s�1). Error bars represent the SE (n >30). (D) Phenotype of ABO/A–, ARR4-OX/ABO/A–, ARR4D95N-OX/ABO/A– (F1 generation), and phyB mutant plants grown for 24 d (panel I) or 32 d (panel II) in white light under long-day conditions (16 h light/8 hdark). Representatives from several independent transgenic lines are shown. The scale bars represent 2.5 cm.


kinases (West and Stock, 2001; Grefen and Harter, 2004),further analysis was concentrated on the interaction ofARR4 with AHP1. Detailed yeast two-hybrid studies on thebasis of growth on interaction selective media and enzy-matic b-galactosidase reporter gene assays revealed thatAHP1 associates with full-length ARR4 and the receiverdomain, but not with the output domain (Fig. 5A, B).

A phosphorelay approach was initiated to corroboratethe yeast two-hybrid results and to investigate whetherAHP1 is able to transfer phosphoryl residues from plantmembranes to ARR4. (His)6-tagged AHP1 was incubatedin a reaction mixture containing microsomal fractions ofPcEP and [c-32P]ATP. Recovery by affinity chromatog-raphy followed by autoradiography showed that AHP1was phosphorylated in a plant membrane-dependent manner(Fig. 5C). A kinetic analysis revealed that the phosphory-lation of AHP1 occurred very rapidly and reacheda steady-state equilibrium within 60 s (Fig. 5D). Therecovered radioactively phosphorylated AHP1 was thenused to perform phosphorelay experiments on ARR4.Strep-tagged ARR4 and (His)6-tagged AHP1 were co-incubated, and the transfer of phosphoryl residues onARR4 was monitored by autoradiography. As shown inFig. 5E, radioactive phosphoryl residues appeared onARR4 only in the presence of AHP1. Furthermore, thetransfer of phosphate from AHP1 to ARR4 was rapid andstarted immediately after the onset of co-incubation(Fig. 5E). In conclusion, the yeast two-hybrid interactionresults and the biochemical data suggest that AHP1interacts with ARR4 and is able to transfer phosphorylresidues from plant membranes to the response regulator.

With one exception (AHK5), all plant histidine kinasesare thought to be membrane-bound receptors (Hwanget al., 2002). It is therefore conceivable that the plant

membrane-associated activity which phosphorylatesAHP1 in vitro originates from a hybrid histidine kinase.Studies in yeast have shown that AHP1 interacts withseveral plant hybrid histidine kinases, including thecytokinin receptors and the ethylene receptor ETR1 (seeSupplementary Fig. S4 at JXB online; Urao et al., 2000;Dortay et al., 2006). It is therefore possible that ARR4phosphorylation and activity are regulated by cytokininreceptor-dependent two-component signalling mech-anisms. Using a physiological approach it was thereforedetermined whether Arabidopsis photomorphogenesis ischanged in a cytokinin receptor mutant background. Thecre1-12/ahk2-2/ahk3-3 triple loss-of-function mutant waschosen (Higuchi et al., 2004). The mutant seedlings weregrown for 3 d under continuous red, far-red, or blue lightof different intensities, or kept in darkness. (Fig. 6A; seeSupplementary Fig. S5 at JXB online). The growthresponse displayed that the triple mutant seedlings wereless sensitive to red light, especially at lower intensities,and to blue light (Fig. 6A; see Supplementary Fig. S5 atJXB online). No difference was observed in far-red light(Fig. 6A). To determine the role of histidine kinaseactivity, the cre1-1 mutant which carries a point mutationin one of the catalytic domains crucial for histidine kinaseactivity was also analysed (Inoue et al., 2001). Comparedwith wild type, cre1-1 seedlings showed a longer hypo-cotyl under all light conditions (Fig. 6B) and floweredearlier under long-day conditions (see SupplementaryFig. S6 at JXB online). Notably, the altered photomorpho-genic phenotype of the the cre1-1 single mutant is similarto that of plants overexpressing non-phosphorylatableARR4D95N, whereas the hypocotyl growth response ofthe cre1-12/ahk2-2/ahk3-3 triple mutant is similar to thatof the arr4 mutant. In general, this suggests that thecapacity of plants to react to light seems to be reduced inthe absence of cytokinin receptor- and ARR4-dependentsignalling pathways.

Cytokinin modulates the activity of ARR4 tofunction on phyB

The data presented here indicate that cytokinin may exerta direct influence on plant photomorphogenesis, therebyutilizing the AHK–AHP1–ARR4 phosphorelay signallingsystem. To gain further support for this conclusion, wild-type seedlings and seedlings overexpressing ARR4 weretreated with either red light or cytokinin alone orcoincidently with red light and cytokinin, and thehypocotyl growth response was determined. Comparedwith wild type, ARR4-overexpressing seedlings revealeda reduced response to cytokinin but an enhanced reactionto red light (Fig. 7A, B). Coincident treatment ofseedlings with red light and cytokinin reveals an additiveinteraction between both signals on a physiological level.In comparison with non-treated seedlings, cytokininrenders ARR4-overexpressing seedlings less responsive

Fig. 4. ARR4D95N is not able to stabilize phyB-Pfr in yeast. Yeast cellsexpressing photoreversible phyB together with wild-type ARR4 (tri-angles), ARR4D95N (squares), or no response regulator (circles) wereirradiated with a red light pulse and transferred to darkness. At theindicated time points, the amount of Pfr (filled symbols) and total phy(open symbols) was measured in vivo (see Materials and methods fordetails). The experiment was repeated three times with at least threemeasurements per time point. Error bars represent the SD (n >3).


to red light, whereas the hyposensitive cytokinin responseobserved in darkness disappeared in the presence of redlight (Fig. 7B).

The reduced light response might be a direct result ofthe cytokinin-induced interference of ARR4 with phyBdynamics. To test this hypothesis, 3-d-old dark-grownABO/A– seedlings and ABO/A– seedlings overexpressing

ARR4 were mock treated or pre-treated for 2 h withcytokinin and then irradiated for 5 min with red light. Theseedlings were transferred back to darkness and totalphyB and phyB-Pfr amounts were measured in planta(Fig. 7C). As reported previously (Sweere et al., 2001), inthe absence of cytokinin, ARR4 stabilizes the active Pfrform of phyB rendering the plants hypersensitive to red

Fig. 5. ARR4 interacts with the HPt protein AHP1 and accepts phosphate residues from plant microsomal phosphorylation activity via AHP1 invitro. (A) Yeast two-hybrid interaction studies. The indicated two-hybrid constructs were transformed in the yeast strain PJ69-4A and the activity ofthe reporter gene was assayed by growth on interaction selective media (CSM-L,W, A). Growth on CSM-L,W served as transformation control. AD,Gal4 activation domain; BD, Gal4 DNA-binding domain; Rec, receiver domain; out, output domain. (B) As in (A) but reporter gene activity (LacZ)was determined by the enzymatic b-galactosidase test. (C) AHP1 is phophorylated in vitro by plant microsomal fractions. (His)6-tagged AHP1 wasincubated for 15 min at 4 �C in a reaction mix containing [c-32P]ATP and the indicated components. AHP1-(His)6 was recovered by Ni-NTA affinitychromatography and analysed for incubation of labelled phosphate {[32P]AHP1-(His)6}. (D) Kinetic analysis of AHP1-(His)6 phosphorylationin vitro. The assay was performed as described under (C), and AHP1-(His)6 was recovered from the reaction mixture at the indicated time points.Radioactive labelling of AHP1-(His)6 was detected by autoradiography and the presence of AHP1 protein by affinity detection using Ni-NTA–APconjugate [a-(His)6]. (E) AHP1 transfers phosphoryl residues on ARR4 in vitro. [32P]AHP1-(His)6 was generated and affinity-purified as describedunder (C), and then co-incubated with Strep-tagged ARR4. At the indicated time points, samples were harvested and analysed for [32P]phosphateincorporation by autoradiography (I). The presence of ARR4-Strep and AHP1-(His)6 was verified by detection with StrepTactin–AP (II) or Ni-NTA–AP (III) conjugate.


light (Fig. 7C). In contrast, when the seedlings were pre-treated with cytokinin, an enhanced dark reversion wasobserved in ABO/A– and the stabilizing effect of ARR4on phyB-Pfr was no longer detectable (Fig. 7C). Thissuggests that the responsiveness of seedlings to red lightmay be altered by cytokinin.

Discussion

Stability of phyB-Pfr is modulated byARR4 under red light

In accordance with a previous report (Sweere et al., 2001),it is shown that ARR4 also reduces the rate of phyB-Pfrdark reversion under extended irradiation especially at lowR light intensity. However, the possibility that ARR4might (also) influence the photoconversion rate of phyB-Pfr under continuous R light cannot be entirely excluded.In any case, the interaction of ARR4 with phyB results ina fluence rate-dependent increase of the phyB PfrPfr

isoform and, thus, in plants that are more sensitive to cRlight. Although discussed controversially (Chen et al.,2003), the data suggest that PfrPfr homodimers arepredominantly responsible for phyB function. Besides theenhanced accumulation, cytokinin-activated ARR4 couldalso interfere with the localization of the PfrPfr isoforminto nuclear speckles (NS). Independently of whether theNS are the location for phyB-Pfr sequestration orinitiation sites for signalling, one would expect thatARR4 overexpression or loss of ARR4 function changesthe equilibrium between dispersed and NS-localized phyBPfr inside the nucleus (Chen et al., 2003). Severalapproaches including the ectopic expression of ARR4,ARR4D95N, and ARR4D95E in phyB–GFP-expressingplants are presently in progress to address these questions.In summary, by regulation of its intracellular level and/oractivity, ARR4 is able to adjust the red light sensory phyBsystem within a wide range of photosensitivity.

Overexpression of ARR4 renders Arabidopsis seedlingshypersensitive to red light in various phyB-dependentresponses (Fig. 1C; Sweere et al., 2001). In addition, arr4loss-of-function mutant seedlings show a hyposensitiveresponse to red light. This appears to be in contrast to thedata published by To and colleagues, in which loss-of-function mutations in ARR3, ARR4, ARR5, and ARR6independently or together resulted in an increased sensi-tivity of hypocotyl growth to red light in Arabidopsisseedlings. However, this hypersensitivity of the type-AARR mutants is caused by sucrose and MS in the growthmedia—conditions which strongly interfere with photo-morphogenic response pathways (see SupplementaryFig. S7 at JXB online; Sheen, 1990; Fankhauser andCasal, 2004). Notably, compared with wild type, soil-grown arr4, arr3,4, and arr3,4,5,6 mutant plants in-creasingly show an altered shade avoidance responsesimilar to that observed for phyB mutants (To et al.,2004). In contrast to the present results, Riefler et al.(2006) reported that no altered red light sensitivity wasdetected in cytokinin receptor triple mutant seedlings(ahk2-5/ahk3-7/cre1-2). Besides the different growth con-ditions and mutant alleles, the length of the seedlingsunder red and far-red light suggests that the applied lightintensities reached or were very close to saturation.However, as shown in Supplementary Fig. S4 at JXBonline, significant differences in light sensitivity canhardly be detected under saturating light conditions.

The function of ARR4 on phyB relies onAsp95 phosphorylation

The phosphorylation mutant version of ARR4(ARR4D95N) is still able to interact with phyB in yeastand in plants, suggesting that the action of ARR4 on phyBis not regulated by phosphorylation-dependent associa-tion. On the other hand, expression of ARR4D95N causesArabidopsis to be hyposensitive to light and converts

Fig. 6. ahk2/ahk3/cre1 triple and cre1-1 single cytokinin receptormutant Arabidopsis seedlings display a hyposensitive photomorpho-genic phenotype. (A) Relative hypocotyl elongation response of 3-d-oldahk2/ahk3/cre1 triple mutant seedlings. Seedlings were grown for 3 dunder continuous red (cR), far-red (cFR), or blue (cB) light, or kept indarkness (D). Error bars represent the SE (n >30). (B) Relativehypocotyl elongation response of 3-d-old cre1-1 mutant seedlings(cre1-1) compared with wild type (WT). Seedlings were grown andirradiated as described above. Error bars represent the SE (n >30).


ABO/A– plants, which display a strong phyB-overexpressing phenotype, into a phyB-like mutant.Furthermore, ARR4D95N is not able to stabilize phyB-Pfrin yeast by inhibition of the Pfr-to-Pr dark reversion.Thus, the hyposensitive response of the ARR4D95N-expressing and arr4 loss-of-function seedlings and plantsagain supports the idea that ARR4 has a function in

modulating phyB sensitivity by directly altering thedynamics of the photoreceptor. Work is presently inprogress to inactivate the ARR4 gene or ectopically expressARR4, ARR4D95N, and ARR4D95E in the ABO/A– back-ground under the control of alternative promoters foradditional phyB dark reversion measurements substantiat-ing the data derived from the ARR4 and ARR4D95N

overexpressors in wild-type background. The present studyalso indicates that phosphorylation of Asp95 appears to becrucial for the action of ARR4 on phyB in planta.

ARR4D95N-expressing plants show reduced sensitivitynot only to red light but also to far-red and blue light,indicating interference with phyA and blue light receptorsignalling. Because a physical interaction could not bedetected, a direct influence of ARR4 on the phyAphotoreceptor dynamics is not likely (Sweere et al., 2001;A Viczian, E Schafer, and K Harter, unpublished data).However, the existence of an intense cross-talk betweenthe different photoreceptor systems in higher plants hasbeen well documented (Casal, 2000). The altered photo-morphogenic responses of ARR4D95N-expressing and arr4loss-of-function seedlings could therefore be a result ofmodified signalling cross-talk induced by the alteredactivity of phyB. On the other hand, the possibility cannotbe excluded that, by titrating out phosphorylated AHPproteins or by inducing another imbalance in the two-component signalling network, ARR4D95N overexpressionor loss of ARR4 function interfere with the activity ofother type-A ARRs functioning directly or indirectly onphyA and blue light photoreceptor signal transduction.

The photomorphogenic function of phyB depends oncytokinin signalling

The availability of protein fractions from plant cells(PcEP) enabled a phosphorylation cascade, which origi-nates from a plant histidine kinase activity and comprisesAHP1 and ARR4, to be established for the first time. Theability of ARR4 to function as a phosphohistidine

Fig. 7. Cytokinin regulates phyB-Pfr stability within a light/hormonesignalling network. Absolute (A) and relative (B) values of thehypocotyl elongation response of two independent ARR4-overexpress-ing lines (ARR4-OX-I, ARR4-OX-II) compared with the wild type. In(B), the wild-type response at each treatment was set to 100%.Seedlings were grown for 3 d under continuous red light (cR) withoutexogenous cytokinin, under continuous red light in the presence ofcytokinin (cR+cyt.), in darkness in the presence of cytokinin (D+cyt.),or in darkness without exogenous cytokinin (D). The cytokinin usedwas kinetin at a concentration of 0.5 lM. Error bars represent the SD (n>30). (C) Cytokinin modulates the ARR4 activity on stabilization ofphyB-Pfr. Four-day-old dark-grown ABO/A– or ARR4-overexpressingABO/A– seedlings were pretreated for 2 h with cytokinin (2 lMkinetin, large filled triangles and squares, respectively) or mock-treated(small filled triangles and squares, respectively), then irradiated for5 min with red light and afterwards transferred back to darkness. Atthe indicated time points the amount of Pfr (filled symbols) or totalphyB (open symbols) was measured in planta. Error bars represent theSD (n >3).


phosphatase on AHPs (AHP2) was suggested earlierby Imamura et al. (1999). However, by performing thephosphorylation reaction on ice, it could be shown thatthe phosphoryl residue is covalently attached to ARR4.Furthermore, the mutation of Asp95 to asparagine abol-ishes the modification of ARR4, suggesting that theobserved phosphotransfers could reflect a canonical phos-phorelay. Furthermore, yeast two-hybrid studies andco-affinity purification experiments suggested that theinteraction and phosphotransfer between AHP1 andARR4 are relatively specific (data shown here; Uraoet al., 2000; Dortay et al., 2006). From these data, it canbe concluded that AHP1 and ARR4 may form a defaultpathway for specific signalling purposes.

Yeast two-hybrid studies and indirect His/Asp phos-phorelay experiments in E. coli indicate that AHP1interacts with the cytoplasmic domain of all Arabidopsiscytokinin receptors and of ETR1, but not with those ofERS1, ATHK1, and CKI1 (see Supplementary Fig. S3at JXB online; Urao et al., 2000; Suzuki et al., 2001b;Dortay et al., 2006). On the basis of these data, it isreasonable to assume that a cytokinin receptor-dependentphosphorelay may regulate the activity of ARR4 tomodify phyB dynamics. The present physiological ap-proach, using the cytokinin receptor triple mutant andcre1-1 which expresses a non-functional histidine kinase(Inoue et al., 2001), demonstrated that these plants arephotomorphogenic mutants with reduced sensitivity tolight of different wavelengths. The phenotype of the triplemutant and the arr4 loss-of-function line indicate a directfunctional relationship of these two-component signallingelements in phyB-Pfr stabilization. In contrast, thephenotype of the cre1-1 mutant and the ARR4D95N

overexpressors suggests a modification of other lightsignal response pathways by dominant negative effectswhich depend on two-component signalling. In summary,these data suggest that the capacity of plants to react tolight seems to be reduced in the absence of a functionalcytokinin receptor–ARR4 signalling mechanism anddepends on a histidine kinase activity of at least AHK4/CRE1/WOL (Inoue et al., 2001; Suzuki et al., 2001a).

Cytokinin directly modulates phyB dynamics

As shown here and reported previously, ARR4 over-expression renders ABO/A– seedlings hypersensitive tored light (Sweere et al., 2001). In contrast, seedlingsoverexpressing ARR4 display a hyposensitive phenotypein the presence of cytokinin in darkness (Osakabe et al.,2002). Coincident treatment with both stimuli reveals aninteraction between the light and cytokinin signal re-sponse pathways in seedlings overexpressing ARR4. Inthe presence of light, the response of the seedlings tocytokinin is enhanced. On the other hand, the hypersensi-tivity of seedlings to cR light is lost in the presence of

cytokinin. The cytokinin effect on the red light responsepathway could be directly linked to altered phyB darkreversion. In ABO/A– seedlings pretreated with cytokininan enhanced dark reversion is observed, and the stabiliz-ing effect of ARR4 on the amount of phyB-Pfr is lost. Thedata suggest that, under these experimental conditions,cytokinin has an immediate effect on phyB photoreceptordynamics and red light-mediated photomorphogenesis.The signalling pathway which gives rise to this effectmight be represented by a two-component phosphorelaysystem consisting of at least one cytokinin hormonereceptor, AHP1, or other phosphotransfer proteins and theresponse regulator ARR4.

A very similar mechanism for the functional interactionof ARR4 and phyB was recently proposed for the light-dependent cytokinin input to the circadian clock (Hananoet al., 2006). Here, cytokinin activates ARR4 which inturn alters phyB-Pfr stability. The activated phyB-Pfrchanges the gene expression of the core clock genes ina phase-dependent manner.

The negative effect of exogenously applied cytokinin onthe red light response pathway in ARR4-overexpressingABO/A– seedlings and on phyB-Pfr levels via a positivelyacting phosphorelay is difficult to reconcile with theobservation that cytokinin triple mutant and cre1-1 mutantplants and plants expressing non-phosphorylatableARR4D95N show a light-hyposensitive phenotype. Apossible explanation for this phenomenon is that highconcentrations of exogenously applied cytokinin inducea strong feedback inhibition resulting in a dramatic down-regulation of the responsiveness of the entire cytokinintwo-component signalling network. This in turn wouldlead to a significant decrease in the function of ARR4 onphyB. How ARR4 activity is repressed is uncertain.Because ARR4 transcript accumulation is at a maximum2 h after cytokinin treatment (Brandstatter and Kieber,1998), a down-regulation of ARR4 is unlikely, althoughhormone-induced ARR4 protein degradation cannot beentirely excluded. Another possibility is that a repressor,which is induced by high concentrations of cytokinin,may inhibit the function of ARR4 on phyB. Conversely,the molecular mechanism with which light regulates theresponsiveness of seedlings to cytokinin is likewiseunknown.

From a formally physiological point of view, cytokininand light response pathways might act independently orsequentially through common signalling intermediates(Thomas et al., 1997, and references therein). Over-expression of ARR4 in combination with coincidentcytokinin and light treatments suggests signalling cross-talk. However, for a better molecular understanding of thiscross-talk, information has to be gained about the pro-portion of ARR4 acting on the cytokinin and the red lightpathway, and the relative contribution of these pathwaysin controlling the hypocotyl elongation response.


A similar cross-talk has been reported for the interaction oflight and ethylene which functions through cytokinin in theregulation of hypocotyl growth. In dark-grown seedlings ofthe ein2 ethylene-insensitive mutant, higher fluence rateswere required to obtain a hypocotyl growth inhibitioncomparable with that of the wild type (Smalle et al., 1997).One explanation for the higher fluence rate requirements inein2 is that the defect in the ein2 allele may influencea component of the light response pathway very similarly tothe mechanism performed by ARR4 in the light–cytokinininteraction. On the other hand, when Arabidopsis plantswere grown under light in the presence of ethylene,a marked induction of hypocotyl elongation occurred(Smalle et al., 1997). The elongation response was absentor weakened in several ethylene-insensitive mutants orinduced in the absence of ethylene in the ctr1 mutant. Thus,depending on light conditions, ethylene can induce oppositeeffects in the growth response of Arabidopsis hypocotyls.

In summary, a molecular cross-talk and signal integra-tion mechanism between phytohormone and light re-sponse pathways in higher plants was uncovered. Thismolecular integration machinery enables the plant cells toadjust the light reponsiveness and competence to endoge-nous requirements in growth and development or to needsimposed by other exogenous signals (e.g. pathogen attack,abiotic stress, photoperiod) very often mediated byhormones. Two-component signal transduction systemsappear to play a major role in providing the molecularbackbone for this signal integration machinery.

Supplementary material

The following supplementary material is available atJXB online.

Figure S1. ARR4D95N transcript and protein levels intransgenic Arabidopsis plants.

Figure S2. Light fluence-rate dependence of hypocotylgrowth in ARR4D95N overexpressing Arabidopsis seedlings.

Figure S3. Light fluence-rate dependence of hypocotylgrowth in the arr4 mutant.

Figure S4. Yeast two-hybrid interaction analysis ofAHP1 with Arabidopsis histidine kinases.

Figure S5. Red light fluence-rate dependence ofhypocotyl growth in Arabidopsis cre1-1/ahk2-2/ahk3-3cytokinin receptor mutant seedlings.

Figure S6. Flowering phenotype of the Arabidopsiscre1-1 mutant.

Figure S7. Photomorphogenic phenotype of in theArabidopsis arr4 mutant in red light under differentgrowth conditions.

Acknowledgements

We thank T Kakimoto and J Kieber for providing mutant seeds. Weare also grateful to S Hummel and D Wanke for technical support,

as well as to F de Courcy and E Fischer for proofreading and helpin editing the manuscript. This work was supported by HHMIInternational Scholar and OTKA grants to FN, and DFG grants toES (SFB388) and KH (AFGN/HA 2146/5; HA 2146/7).

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Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis

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