JXB Advance Access originally published online on June 1, 2007
Journal of Experimental Botany 2007 58(10):2595-2607; doi:10.1093/jxb/erm087
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RESEARCH PAPER |
Functional cross-talk between two-component and phytochrome B signal transduction in Arabidopsis
1Zentrum für Molekularbiologie der Pflanzen/Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany
2Institut für Biologie II/Botanik, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany
3Plant Biology Institute, Biological Research Center, PO Box 521, H-6726 Szeged, Hungary
* To whom correspondence should be addressed. E-mail: klaus.harter{at}uni-tuebingen.de
Received 12 February 2007; Revised 20 March 2007 Accepted 26 March 2007
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Abstract |
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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 functions as a modulator of photomorphogenesis. In concert 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 aspartate to asparagine within the receiver domain creates a version of ARR4 that negatively affects photomorphogenesis. Additional evidence suggests that ARR4 activity 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
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Introduction |
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To monitor the intensity, wavelength, direction, and timing of light, plants have evolved diverse photoreceptor systems. These photoreceptors include the blue light-sensitive cryptochromes and phototropins as well as the well-characterized phytochromes (Schäfer and Nagy, 2006).
Phytochromes (phy) are red (R)/far-red (FR) photoreversible chromoproteins that form dimers with a monomeric molecular mass of 
120 kDa, to which an open tetrapyrrole chromophore is covalently attached (Tu and Lagarias, 2005). They are synthesized in the inactive Pr form in the dark and photoconverted by red light into the photomorphogenically active far-red light-absorbing Pfr form. In addition to these light reactions, Pfr is converted back to Pr by a thermally driven process called dark reversion. Dark reversion has a strong effect on phytochrome function and attenuates phytochrome activity by reducing the amount of active Pfr (Elich and Chory, 1997; Sweere et al., 2001; Fankhauser, 2002; Hennig, 2006). In Arabidopsis thaliana, the phytochrome family comprises five members termed phyA to phyE, with distinct but overlapping modes of photomorphogenic action (Tu and Lagarias, 2005). Whereas phyA controls the very low fluence and far-red high irradiance responses, phyB to phyE mediate responses to continuous red (cR) light and are responsible for the R/FR reversible induction reactions (Schäfer and Nagy, 2006).
In general, the phytochrome molecule is separated into two major domains: an N-terminal chromophore carrying a photosensory domain of 70 kDa and a C-terminal domain of 55 kDa (Fig. 1A). The latter is necessary for dimerization and the import of phy into the nucleus, and comprises two PAS motifs as well as a His kinase-like domain (Sakamoto and Nagatani, 1996; Kircher et al., 1999, 2002). The C-terminal domain has long been considered to transduce the light signal to downstream signalling components. However, recent studies by Nagatani and colleagues have shown that the N-terminal photosensory domain of phyB alone is sufficient for signalling and that the C-terminal domain probably has a function in the modulation of signal transduction (Matsushita et al., 2003; Oka et al., 2004).
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The N-terminal photosensory domain is highly conserved throughout the phytochromes and displays spectral properties indistinguishable from those of the full-length photoreceptors. Within this domain three different regions can be recognized (Fig. 1A): a short N-terminal extension of 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 PHY region is required for the spectral integrity and stabilization of the active Pfr form by reducing the rate of dark reversion, but is not essential for signal transduction (Oka et al., 2004). The chromophore-bearing central region, including the N-terminal extension of phyB, upholds its biological activity in planta and is able to transduce the light signal almost normally to downstream elements in response to irradiation (Oka et al., 2004). The short N-terminal extension has also been shown to play an important role in the function of phytochromes. Truncation of the N-terminal extension results in an increase in the rate of dark reversion, indicating that this domain is not critical for signalling but plays a prominent role in Pfr stabilization (Quail, 1997). In summary, whereas the central region of the photosensory domain mediates light signal transduction, the interplay of both ends—the N-terminal extension and the PHY region—appears to be involved in the stabilization of the active Pfr form, thereby influencing the light sensitivity of phytochromes (Oka et al., 2004). Phytochrome-dependent signal transduction involves the translocation of the active Pfr form from the cytoplasm into the nucleus and the physical interaction with and post-translational modification of downstream signalling elements (Nagatani, 2004; Khanna et al., 2004; Ryu et al., 2005; Al-Sady et al., 2006). In recent years, several elements have been identified, which interact with phytochromes in the cytoplasm and the nucleus and are assumed to participate in downstream light signalling (Ryu et al., 2005; Al-Sady et al., 2006; Schäfer and Nagy, 2006). Among them, only the Arabidopsis response regulator 4 (ARR4) recognizes the N-terminal extension of phyB (Sweere et al., 2001; Fankhauser, 2002). In yeast and plants, the interaction of ARR4 with phyB stabilizes the active Pfr form of the photoreceptor under inductive red light conditions by reducing the rate of dark reversion. Therefore, overexpression of ARR4 resulted in increased red light sensitivity in hypocotyl growth and other phyB-regulated processes (Sweere et al., 2001).
ARR4 is a canonical response regulator (Fig. 1A) of the Arabidopsis type-A subfamily, whose activity is believed to be regulated by a two-component His
Asp phosphorelay—a phosphotransfer mechanism that is initiated by a sensor histidine kinase (Grefen and Harter, 2004; Ferreira and Kieber, 2005). However, although the C-terminal domain of phytochromes displays homology to the transmitter region of histidine kinases, corresponding histidine kinase activity has not been reported for either phyB or for any other higher plant phytochrome (Tu and Lagarias, 2006). This suggests that phyB is probably not the cognate histidine kinase for the aspartate phosphorylation of ARR4. It is conceivable that ARR4 represents the output element of an independent two-component signalling 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 been shown to be up-regulated in response to cytokinin treatment and to function as negative regulators in cytokinin signalling (Kakimoto, 2003; Ferreira and Kieber, 2005). To accomplish this negative function in cytokinin signalling, type-A ARRs are believed to be phosphorelay targets of the cytokinin sensor histidine kinases 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 acting on an as yet unknown clock protein. Thereby, ARR3 and ARR4 signal via phyB into the clock and modulate the circadian phase (Salome et al., 2006). Furthermore, ARR4 and phyB are elements in the input of the cytokinin signal to the circadian phase of the clock (Hanano et al., 2006). From these results, the intriguing idea arises that a cytokinin-triggered two-component system may regulate ARR4 activity on phyB by aspartate phosphorylation (Fankhauser, 2002; Grefen and Harter, 2004).
In this study, the influence of ARR4 on phyB-Pfr stability in plants was examined under extended red light irradiation. In addition, the effects of the expression of a non-phosphorylatable form of ARR4 (ARR4D95N) on photomorphogenesis in Arabidopsis have been analysed and, by biochemical and genetic approaches, upstream regulatory elements have been identified which are likely to participate in the regulation of ARR4 activity. Furthermore, the in planta findings suggest that the phytohormone cytokinin directly modifies the sensitivity status of phyB via phosphorelay. This molecular cross-talk mechanism enables the plant to adjust light responsiveness to endogenous requirements in growth and development.
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Materials and methods |
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Light sources, plant material and growth, and in planta spectroscopic phytochrome measurement
The R, FR, and B light sources and handling of dark-grown seedlings under dim green safety light have been described previously (Kircher et al., 1999; Sweere et al., 2001). To avoid any influence of carbohydrates and growth media components on photomorphogenesis (Fankhauser and Casal, 2004; see Supplementary Fig. S6 at JXB online), Arabidopsis seeds were sown on four layers of filter paper and allowed to imbibe in sterile water in the dark for 24 h at 4 °C. During the following irradiation or dark incubation, seedlings were kept at 23 °C. Hypocotyl length measurements, growth of Arabidopsis plants on soil, determination of flowering time (number of leaves developed at flowering) under long-day conditions (16 h white light/8 h dark), and spectroscopic in planta measurement of total phytochrome and Pfr amounts using a dual-wavelength ratio photometer were performed according to Sweere et al. (2001). The following mutants or transgenic plants have been described previously: ARR4-OX-I, ARR4-OX-II [ARR4-overexpressors in A. thaliana wild-type background (Col.); Sweere et al., 2001], ABO/A– [transgenic A. thaliana overexpressing Arabidopsis phyB (ABO; Wagner et al., 1991) in the phyA mutant (A–) background (fre1-1; Nagatani et al., 1993)], ARR4-OX in the ABO/A– background (Sweere et al., 2001), cre1-1 (Inoue et al., 2001), arr4 (To et al., 2004), and cre1-12/ahk2-2/ahk3-3 (Higuchi et 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 the ARR4 wild-type sequence cloned in the binary, Escherichia coli expression and yeast two-hybrid vectors (for constructs used as templates see Sweere et al., 2001) was performed using the QuickChangeTM XL Mutagenesis System (Stratagene) and appropriate primers according to the manufacturer. The cDNA encoding Arabidopsis AHP1 was cloned into the yeast two-hybrid Gal4 activation domain vector (AD) pGAD424 via BamHI/SalI. The cDNA 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 as template and appropriate primers, and cloned in the yeast two-hybrid Gal4 DNA-binding vector (BD) pGBT9 via BamHI/SalI and EcoRI/SalI, respectively. For reverse transcription–PCR (RT–PCR), total RNA was isolated from green leaves using RNAWizTM (Ambion). Reverse transcription of 1.5 µg of RNA per sample was performed using SuperScript III according to the manufacturer's protocol (Invitrogen). For amplification of cDNA, the PCR was carried out in 25 cycles using Actin2- and ARR4-specific primers.
The other constructs used in this study have been described previously: 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 were verified by sequencing. Primer sequences are avaiable upon request.
Expression of Strep-tagged and (His)6-tagged proteins in E. coli and affinity purification on StrepTactin (IBA) or nickel-nitrilotriacetic 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 selected for glufosinate resistance according to Lohrmann et al. (2001) and Sweere 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 and lacZ reporter gene activities were carried out according to Lohrmann et al. (2001). The yeast two-hybrid screen resulting in two million primary transformants was performed as described by Kim et al. (1997). Co-immunopurification of phyB with ARR4 or ARR4D95N from plant extracts using an ARR4-specific antiserum from mouse has been described previously (Sweere et al., 2001).
For co-affinity purification assays, 5 µg of AHP1-Strep, AHP2-Strep, or green fluorescent protein (GFP)-Strep were co-incubated on ice for 2 h with 10 µl of 35S-labelled ARR4 in a final volume of 250 µl of incubation buffer (100 mM TRIS-MOPS pH 8.3, 150 mM NaCl, 1 mM EDTA, 28 mM β-mercaptoethanol). The 35S-labelled ARR4 was generated by in vitro transcription/translation in reticulocyte lysate using the TNT Quick Coupled Transcription/Translation kit (Promega). The co-incubated samples were washed three times with incubation buffer, and bound protein complexes eluted in 50 µl of elution buffer (100 mM TRIS-HCl pH 7.4, 1 mM EDTA, 10 mM desthiobiotin). A 25 µl aliquot of the eluate was applied for immunodetection of ARR4 and 25 µl for detection of Strep-tagged proteins using StepTactin–alkaline phosphatase (AP) conjugate as described by the manufacturer (IBA).
SDS-PAGE, western blot transfer, and immunodetection was performed as described previously (Harter et al., 1994; Sweere et 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 of protoplasts, protein extraction, subcellular fractionation, and generation of microsomal preparations (membrane) were carried out as described (Harter et al., 1994). For in vitro phosphorylation, 5 µg of AHP1-(His)6 or ARR4-Strep were co-incubated for the indicated time period at 25 °C with 2 µg of total PcEP extract or membrane fraction, respectively, and 10 µCi of [
-32P]ATP in a total volume of 30 µl of phosphorylation buffer [1x TEDG buffer: 50 mM TRIS-HCl pH 8.0, 0.5 mM EDTA, 2 mM dithiothreitol (DTT), 50 mM KCl, 5 mM MgCl2, 10% glycerol] containing 1x proteinase inhibitors (Boehringer). The proteins were recovered from the reaction mixture by affinity purification on StrepTactin or Ni-NTA beads as described above, and then subjected to SDS-PAGE and autoradiography. AHP1-to-ARR4 phosphotransfer was initiated by co-incubating [32P]AHP1-(His)6 with 2 µg of unlabelled ARR4-Strep in a final volume of 50 µl of 1x TEDG buffer. After the indicated times, the reaction was stopped by the addition of 5 µl of boiling 5x SDS sample buffer. A 25 µl aliquot of the sample was subjected to autoradiography and 25 µl applied for detection of ARR4-Strep using StepTactin–AP conjugate and of AHP1-(His)6 using Ni-NTA–AP conjugate (Qiagen).
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Results |
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ARR4 stabilizes the active Pfr form of phyB under extended red light irradiation
It was recently reported that ARR4 interacts with the photoreceptor phyB and stabilizes the active Pfr form of the photoreceptor under inductive red light conditions in yeast and in plants by inhibiting dark reversion (Sweere et al., 2001). However, many (red) light-regulated responses, including hypocotyl growth inhibition, rely on continuous irradiation and are light intensity dependent (Schäfer and Nagy, 2006). We therefore tested whether ARR4 also modulates the Pfr/Ptotal ratio of phyB under extended irradiation with red light of different intensity in transgenic Arabidopsis seedlings overexpressing Arabidopsis phyB in a phyA minus background (ABO/A–). As shown in Fig. 1B, the amount of active phyB-Pfr strongly depends on the intensity of red light during an extended irradiation period of 20 min. The effect of Pfr-to-Pr dark reversion on the entire phyB-Pfr pool becomes most noticeable at lower fluence rates. In the presence of ARR4, however, the dark reversion of Pfr back to Pr is inhibited, resulting in higher levels of active phyB-Pfr, especially at lower light intensities (Fig. 1B). To determine whether the higher phyB-Pfr levels could correspond to altered phyB-mediated responses, hypocotyl growth studies were performed in 3-d-old ABO/A– seedlings. Confirming the data published in Sweere et al. (2001), overexpression of ARR4 resulted in seedlings which showed an enhanced hypocotyl growth inhibition response over a wide range of red light intensities (Fig. 1C). These data suggest that ARR4 also causes the stabilization of active phyB-Pfr under extended red light (cR) conditions. Furthermore, this stabilization appears to be, at least to a major extent, responsible for the hypersensitive phenotype observed in cR light-grown ARR4-overexpressing Arabidopsis seedlings.
The function of ARR4 on phyB is phosphorylation dependent
Because ARR4 is a response regulator (Fig. 1A), it was considered whether ARR4 activity on phyB may be regulated by phosphorylation. To investigate whether the conserved phosphor-accepting aspartate residue (Asp95) within the receiver domain of ARR4 may become phosphorylated, a cell-free phosphorelay system was used based on evacuolated parsley (P. crispum) cell culture protoplasts (PcEP; Harter et al., 1994) and recombinant Strep-tagged ARR4 proteins, either in the wild-type form or in a form in which the Asp95 has been mutated to asparagine (ARR4D95N). Mutation of the conserved aspartate to asparagine within receiver domains usually abolishes phosphorylation and creates loss-of-function variants of response regulators (West and Stock, 2001). For phosphorylation experiments, wild-type ARR4 or ARR4D95N were incubated in the presence of [-32P]ATP in total extracts of PcEP. The proteins were then recovered by affinity purification and analysed for incorporation of radioactive phosphate. Whereas wild-type ARR4 was efficiently phosphorylated, modification of ARR4D95N was not observed (Fig. 2A). This result shows that ARR4 is phosphorylated by a plant extract in vitro. Furthermore, these data indicate that Asp95 is required for ARR4 modification and represents the target amino acid residue for a plant aspartate phosphorylation activity (Hwang et al., 2002).
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-ARR4). (B) ARR4 and ARR4D95N interact with the N-terminal domain of phyB (amino acids 1–173) in the yeast two-hybrid system. The interaction of the indicated fusion proteins was monitored by the activity of the LacZ reporter gene (β-galactosidase). Error bars represent the SD (n >3). (C) ARR4 and ARR4D95N interact with phyB in planta. PhyB was co-purified from crude extracts of plants overexpressing either ARR4 (ARR4-OX) or ARR4D95N (ARR4D95N-OX) using an ARR4-specific antiserum (Whether the Asp95 to asparagine mutation influences the capacity of ARR4 to interact with phyB was determined by interaction 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 with phyB in yeast is independent of whether the photoreceptor is present in the active Pfr or inactive Pr form (A Viczian, E Schäfer, and K Harter, unpublished data). Co-purification experiments using red light-irradiated total extracts from plants overexpressing ARR4 or ARR4D95N revealed a very similar result (Fig. 2C; Sweere et al., 2001). In summary, the mutation of Asp95 to asparagine does not seem to interfere with the interaction of ARR4 with phyB.
To determine whether the mutation of Asp95 to asparagine interferes with the potential of the response regulator to act on phyB in vivo, transgenic Arabidopsis plants expressing ARR4D95N under the control of the 35S promoter (see Supplementary Fig. S1A at JXB online) were generated and the corresponding seedlings were analysed for altered photomorphogenic response in light of different wavelengths. In contrast to wild-type ARR4 overexpressors, which showed a hypersensitive hypocotyl growth response specifically to red light (Sweere et al., 2001), ARR4D95N-expressing seedlings exhibited a reduced reaction not only to red but also to blue and far-red light (Fig. 3A). Detailed fluence rate/response analyses for one selected ARR4D95N-expressing line indicate that the altered photomorphogensis is due to the reduced sensitivity of the seedlings to light (see Supplementary Fig. S2 at JXB online). The inhibitory effect of ARR4D95N on phyB-mediated processes was also observed for flowering. In contrast to ARR4-overexpressing plants, which flower later than wild type under long-day conditions (16 h light/8 h dark), ARR4D95N-expressing plants set fewer leaves prior to bolting and flowered earlier than the wild type (Fig. 3B). This hyposensitive phenotype of the ARR4D95N-expressing plants is similar to that of an arr4 loss-of-function mutant published by To et al. (2004). Under the present growth conditions, the arr4 mutant displayed a significantly reduced response to red light but, in contrast to the ARR4D95N overexpressors, a wild-type reaction to blue and far-red light (Fig. 3C; see Supplementary Fig. S3 at JXB online). If ARR4D95N is ectopically expressed in ABO/A– plants (see Supplementary Fig. S1B at JXB online), the strong effect on photomorphogenesis is even more dramatic. ABO/A– plants display the typical phyB overexpressor phenotype, showing a very compact rosette with short internodes and dark-green leaves with short petioles (Fig. 3D). This phenotype can be further enhanced by the overexpression of wild-type ARR4 (Fig. 3D). In contrast, the expression of ARR4D95N caused ABO/A– plants (F1 generation) to develop leaves with extremely long petioles and stems with long internodes—a phenotype which is very similar to that of phyB mutants (Fig. 3D). However, due to strong silencing effects in the F2 and following generations, the phyB-Pfr dark reversion of ABO/A– seedlings expressing ARR4D95N could not be determined. The effect of ARR4D95N on photoreversible phyB in yeast was therefore studied (Sweere et al., 2001). In contrast to wild-type ARR4, ARR4D95N was not able to stabilize the active Pfr form of phyB and the photoreceptor showed dark reversion kinetics similar to those observed in cells not expressing a response regulator (Fig. 4).
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3).In summary, the data indicate that ARR4 interferes with light and phyB signalling and that Asp95 modification has a major impact on the ability of ARR4 to modulate the activity of phyB in vivo.
The ARR4 activity appears to be regulated by a two-component signalling pathway
Although higher plant phytochromes are ancestral two-component histidine kinases, HisAsp phosphorelay activity could not yet be demonstrated for these photoreceptors (Tu and Lagarias, 2006). Therefore, it is conceivable to assume that Asp95 phosphorylation of ARR4 occurs through an independent two-component system. To identify two-component proteins potentially acting 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 Arabidopsis histidine phosphotransfer domain-containing (HPt) protein 1 (AHP1; Hwang et al., 2002). The DBP1 gene encodes a DNA-binding protein of unknown function whose expression is up-regulated in response to auxin treatment (Alliotte et al., 1989). Because AHP proteins link via phosphorelay response regulators to hybrid histidine kinases (West and Stock, 2001; Grefen and Harter, 2004), further analysis was concentrated on the interaction of ARR4 with AHP1. Detailed yeast two-hybrid studies on the basis of growth on interaction selective media and enzymatic β-galactosidase reporter gene assays revealed that AHP1 associates with full-length ARR4 and the receiver domain, but not with the output domain (Fig. 5A, B).
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A phosphorelay approach was initiated to corroborate the yeast two-hybrid results and to investigate whether AHP1 is able to transfer phosphoryl residues from plant membranes to ARR4. (His)6-tagged AHP1 was incubated in a reaction mixture containing microsomal fractions of PcEP and [
-32P]ATP. Recovery by affinity chromatography followed by autoradiography showed that AHP1 was phosphorylated in a plant membrane-dependent manner (Fig. 5C). A kinetic analysis revealed that the phosphorylation of AHP1 occurred very rapidly and reached a steady-state equilibrium within 60 s (Fig. 5D). The recovered radioactively phosphorylated AHP1 was then used to perform phosphorelay experiments on ARR4. Strep-tagged ARR4 and (His)6-tagged AHP1 were co-incubated, and the transfer of phosphoryl residues on ARR4 was monitored by autoradiography. As shown in Fig. 5E, radioactive phosphoryl residues appeared on ARR4 only in the presence of AHP1. Furthermore, the transfer of phosphate from AHP1 to ARR4 was rapid and started immediately after the onset of co-incubation (Fig. 5E). In conclusion, the yeast two-hybrid interaction results and the biochemical data suggest that AHP1 interacts with ARR4 and is able to transfer phosphoryl residues from plant membranes to the response regulator. With one exception (AHK5), all plant histidine kinases are thought to be membrane-bound receptors (Hwang et al., 2002). It is therefore conceivable that the plant membrane-associated activity which phosphorylates AHP1 in vitro originates from a hybrid histidine kinase. Studies in yeast have shown that AHP1 interacts with several plant hybrid histidine kinases, including the cytokinin receptors and the ethylene receptor ETR1 (see Supplementary Fig. S4 at JXB online; Urao et al., 2000; Dortay et al., 2006). It is therefore possible that ARR4 phosphorylation and activity are regulated by cytokinin receptor-dependent two-component signalling mechanisms. Using a physiological approach it was therefore determined whether Arabidopsis photomorphogenesis is changed in a cytokinin receptor mutant background. The cre1-12/ahk2-2/ahk3-3 triple loss-of-function mutant was chosen (Higuchi et al., 2004). The mutant seedlings were grown for 3 d under continuous red, far-red, or blue light of different intensities, or kept in darkness. (Fig. 6A; see Supplementary Fig. S5 at JXB online). The growth response displayed that the triple mutant seedlings were less sensitive to red light, especially at lower intensities, and to blue light (Fig. 6A; see Supplementary Fig. S5 at JXB online). No difference was observed in far-red light (Fig. 6A). To determine the role of histidine kinase activity, the cre1-1 mutant which carries a point mutation in one of the catalytic domains crucial for histidine kinase activity was also analysed (Inoue et al., 2001). Compared with wild type, cre1-1 seedlings showed a longer hypocotyl under all light conditions (Fig. 6B) and flowered earlier under long-day conditions (see Supplementary Fig. S6 at JXB online). Notably, the altered photomorphogenic phenotype of the the cre1-1 single mutant is similar to that of plants overexpressing non-phosphorylatable ARR4D95N, whereas the hypocotyl growth response of the cre1-12/ahk2-2/ahk3-3 triple mutant is similar to that of the arr4 mutant. In general, this suggests that the capacity of plants to react to light seems to be reduced in the absence of cytokinin receptor- and ARR4-dependent signalling pathways.
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Cytokinin modulates the activity of ARR4 to function on phyB
The data presented here indicate that cytokinin may exert a direct influence on plant photomorphogenesis, thereby utilizing the AHK–AHP1–ARR4 phosphorelay signalling system. To gain further support for this conclusion, wild-type seedlings and seedlings overexpressing ARR4 were treated with either red light or cytokinin alone or coincidently with red light and cytokinin, and the hypocotyl growth response was determined. Compared with wild type, ARR4-overexpressing seedlings revealed a reduced response to cytokinin but an enhanced reaction to red light (Fig. 7A, B). Coincident treatment of seedlings with red light and cytokinin reveals an additive interaction between both signals on a physiological level. In comparison with non-treated seedlings, cytokinin renders ARR4-overexpressing seedlings less responsive to red light, whereas the hyposensitive cytokinin response observed in darkness disappeared in the presence of red light (Fig. 7B).
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The reduced light response might be a direct result of the cytokinin-induced interference of ARR4 with phyB dynamics. To test this hypothesis, 3-d-old dark-grown ABO/A– seedlings and ABO/A– seedlings overexpressing ARR4 were mock treated or pre-treated for 2 h with cytokinin and then irradiated for 5 min with red light. The seedlings were transferred back to darkness and total phyB and phyB-Pfr amounts were measured in planta (Fig. 7C). As reported previously (Sweere et al., 2001), in the absence of cytokinin, ARR4 stabilizes the active Pfr form of phyB rendering the plants hypersensitive to red light (Fig. 7C). In contrast, when the seedlings were pre-treated with cytokinin, an enhanced dark reversion was observed in ABO/A– and the stabilizing effect of ARR4 on phyB-Pfr was no longer detectable (Fig. 7C). This suggests that the responsiveness of seedlings to red light may be altered by cytokinin.
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Discussion |
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Stability of phyB-Pfr is modulated by ARR4 under red light
In accordance with a previous report (Sweere et al., 2001), it is shown that ARR4 also reduces the rate of phyB-Pfr dark reversion under extended irradiation especially at low R light intensity. However, the possibility that ARR4 might (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 in a fluence rate-dependent increase of the phyB PfrPfr isoform and, thus, in plants that are more sensitive to cR light. Although discussed controversially (Chen et al., 2003), the data suggest that PfrPfr homodimers are predominantly responsible for phyB function. Besides the enhanced accumulation, cytokinin-activated ARR4 could also interfere with the localization of the PfrPfr isoform into nuclear speckles (NS). Independently of whether the NS are the location for phyB-Pfr sequestration or initiation sites for signalling, one would expect that ARR4 overexpression or loss of ARR4 function changes the equilibrium between dispersed and NS-localized phyB Pfr inside the nucleus (Chen et al., 2003). Several approaches including the ectopic expression of ARR4, ARR4D95N, and ARR4D95E in phyB–GFP-expressing plants are presently in progress to address these questions. In summary, by regulation of its intracellular level and/or activity, ARR4 is able to adjust the red light sensory phyB system within a wide range of photosensitivity.
Overexpression of ARR4 renders Arabidopsis seedlings hypersensitive to red light in various phyB-dependent responses (Fig. 1C; Sweere et al., 2001). In addition, arr4 loss-of-function mutant seedlings show a hyposensitive response to red light. This appears to be in contrast to the data published by To and colleagues, in which loss-of-function mutations in ARR3, ARR4, ARR5, and ARR6 independently or together resulted in an increased sensitivity of hypocotyl growth to red light in Arabidopsis seedlings. However, this hypersensitivity of the type-A ARR mutants is caused by sucrose and MS in the growth media—conditions which strongly interfere with photomorphogenic response pathways (see Supplementary Fig. S7 at JXB online; Sheen, 1990; Fankhauser and Casal, 2004). Notably, compared with wild type, soil-grown arr4, arr3,4, and arr3,4,5,6 mutant plants increasingly show an altered shade avoidance response similar 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 was detected in cytokinin receptor triple mutant seedlings (ahk2-5/ahk3-7/cre1-2). Besides the different growth conditions and mutant alleles, the length of the seedlings under red and far-red light suggests that the applied light intensities reached or were very close to saturation. However, as shown in Supplementary Fig. S4 at JXB online, significant differences in light sensitivity can hardly be detected under saturating light conditions.
The function of ARR4 on phyB relies on Asp95 phosphorylation
The phosphorylation mutant version of ARR4 (ARR4D95N) is still able to interact with phyB in yeast and in plants, suggesting that the action of ARR4 on phyB is not regulated by phosphorylation-dependent association. On the other hand, expression of ARR4D95N causes Arabidopsis to be hyposensitive to light and converts ABO/A– plants, which display a strong phyB-overexpressing phenotype, into a phyB-like mutant. Furthermore, ARR4D95N is not able to stabilize phyB-Pfr in 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 plants again supports the idea that ARR4 has a function in modulating phyB sensitivity by directly altering the dynamics of the photoreceptor. Work is presently in progress to inactivate the ARR4 gene or ectopically express ARR4, ARR4D95N, and ARR4D95E in the ABO/A– background under the control of alternative promoters for additional phyB dark reversion measurements substantiating the data derived from the ARR4 and ARR4D95N overexpressors in wild-type background. The present study also indicates that phosphorylation of Asp95 appears to be crucial for the action of ARR4 on phyB in planta.
ARR4D95N-expressing plants show reduced sensitivity not only to red light but also to far-red and blue light, indicating interference with phyA and blue light receptor signalling. Because a physical interaction could not be detected, a direct influence of ARR4 on the phyA photoreceptor dynamics is not likely (Sweere et al., 2001; A Viczian, E Schäfer, and K Harter, unpublished data). However, the existence of an intense cross-talk between the different photoreceptor systems in higher plants has been well documented (Casal, 2000). The altered photomorphogenic responses of ARR4D95N-expressing and arr4 loss-of-function seedlings could therefore be a result of modified signalling cross-talk induced by the altered activity of phyB. On the other hand, the possibility cannot be excluded that, by titrating out phosphorylated AHP proteins or by inducing another imbalance in the two-component signalling network, ARR4D95N overexpression or loss of ARR4 function interfere with the activity of other type-A ARRs functioning directly or indirectly on phyA and blue light photoreceptor signal transduction.
The photomorphogenic function of phyB depends on cytokinin signalling
The availability of protein fractions from plant cells (PcEP) enabled a phosphorylation cascade, which originates from a plant histidine kinase activity and comprises AHP1 and ARR4, to be established for the first time. The ability of ARR4 to function as a phosphohistidine phosphatase on AHPs (AHP2) was suggested earlier by Imamura et al. (1999). However, by performing the phosphorylation reaction on ice, it could be shown that the phosphoryl residue is covalently attached to ARR4. Furthermore, the mutation of Asp95 to asparagine abolishes the modification of ARR4, suggesting that the observed phosphotransfers could reflect a canonical phosphorelay. Furthermore, yeast two-hybrid studies and co-affinity purification experiments suggested that the interaction and phosphotransfer between AHP1 and ARR4 are relatively specific (data shown here; Urao et al., 2000; Dortay et al., 2006). From these data, it can be concluded that AHP1 and ARR4 may form a default pathway for specific signalling purposes.
Yeast two-hybrid studies and indirect HisAsp phosphorelay experiments in E. coli indicate that AHP1 interacts with the cytoplasmic domain of all Arabidopsis cytokinin receptors and of ETR1, but not with those of ERS1, ATHK1, and CKI1 (see Supplementary Fig. S3 at JXB online; Urao et al., 2000; Suzuki et al., 2001b; Dortay et al., 2006). On the basis of these data, it is reasonable to assume that a cytokinin receptor-dependent phosphorelay may regulate the activity of ARR4 to modify phyB dynamics. The present physiological approach, using the cytokinin receptor triple mutant and cre1-1 which expresses a non-functional histidine kinase (Inoue et al., 2001), demonstrated that these plants are photomorphogenic mutants with reduced sensitivity to light of different wavelengths. The phenotype of the triple mutant and the arr4 loss-of-function line indicate a direct functional relationship of these two-component signalling elements in phyB-Pfr stabilization. In contrast, the phenotype of the cre1-1 mutant and the ARR4D95N overexpressors suggests a modification of other light signal response pathways by dominant negative effects which depend on two-component signalling. In summary, these data suggest that the capacity of plants to react to light seems to be reduced in the absence of a functional cytokinin receptor–ARR4 signalling mechanism and depends 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 overexpression renders ABO/A– seedlings hypersensitive to red light (Sweere et al., 2001). In contrast, seedlings overexpressing ARR4 display a hyposensitive phenotype in the presence of cytokinin in darkness (Osakabe et al., 2002). Coincident treatment with both stimuli reveals an interaction between the light and cytokinin signal response pathways in seedlings overexpressing ARR4. In the presence of light, the response of the seedlings to cytokinin is enhanced. On the other hand, the hypersensitivity of seedlings to cR light is lost in the presence of cytokinin. The cytokinin effect on the red light response pathway could be directly linked to altered phyB dark reversion. In ABO/A– seedlings pretreated with cytokinin an enhanced dark reversion is observed, and the stabilizing effect of ARR4 on the amount of phyB-Pfr is lost. The data suggest that, under these experimental conditions, cytokinin has an immediate effect on phyB photoreceptor dynamics and red light-mediated photomorphogenesis. The signalling pathway which gives rise to this effect might be represented by a two-component phosphorelay system consisting of at least one cytokinin hormone receptor, AHP1, or other phosphotransfer proteins and the response regulator ARR4.
A very similar mechanism for the functional interaction of ARR4 and phyB was recently proposed for the light-dependent cytokinin input to the circadian clock (Hanano et al., 2006). Here, cytokinin activates ARR4 which in turn alters phyB-Pfr stability. The activated phyB-Pfr changes the gene expression of the core clock genes in a phase-dependent manner.
The negative effect of exogenously applied cytokinin on the red light response pathway in ARR4-overexpressing ABO/A– seedlings and on phyB-Pfr levels via a positively acting phosphorelay is difficult to reconcile with the observation that cytokinin triple mutant and cre1-1 mutant plants and plants expressing non-phosphorylatable ARR4D95N show a light-hyposensitive phenotype. A possible explanation for this phenomenon is that high concentrations of exogenously applied cytokinin induce a strong feedback inhibition resulting in a dramatic down-regulation of the responsiveness of the entire cytokinin two-component signalling network. This in turn would lead to a significant decrease in the function of ARR4 on phyB. How ARR4 activity is repressed is uncertain. Because ARR4 transcript accumulation is at a maximum 2 h after cytokinin treatment (Brandstatter and Kieber, 1998), a down-regulation of ARR4 is unlikely, although hormone-induced ARR4 protein degradation cannot be entirely 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 the responsiveness of seedlings to cytokinin is likewise unknown.
From a formally physiological point of view, cytokinin and light response pathways might act independently or sequentially through common signalling intermediates (Thomas et al., 1997, and references therein). Overexpression of ARR4 in combination with coincident cytokinin and light treatments suggests signalling cross-talk. However, for a better molecular understanding of this cross-talk, information has to be gained about the proportion of ARR4 acting on the cytokinin and the red light pathway, and the relative contribution of these pathways in controlling the hypocotyl elongation response.
A similar cross-talk has been reported for the interaction of light and ethylene which functions through cytokinin in the regulation of hypocotyl growth. In dark-grown seedlings of the ein2 ethylene-insensitive mutant, higher fluence rates were required to obtain a hypocotyl growth inhibition comparable with that of the wild type (Smalle et al., 1997). One explanation for the higher fluence rate requirements in ein2 is that the defect in the ein2 allele may influence a component of the light response pathway very similarly to the mechanism performed by ARR4 in the light–cytokinin interaction. On the other hand, when Arabidopsis plants were grown under light in the presence of ethylene, a marked induction of hypocotyl elongation occurred (Smalle et al., 1997). The elongation response was absent or weakened in several ethylene-insensitive mutants or induced in the absence of ethylene in the ctr1 mutant. Thus, depending on light conditions, ethylene can induce opposite effects in the growth response of Arabidopsis hypocotyls.
In summary, a molecular cross-talk and signal integration mechanism between phytohormone and light response pathways in higher plants was uncovered. This molecular integration machinery enables the plant cells to adjust the light reponsiveness and competence to endogenous requirements in growth and development or to needs imposed by other exogenous signals (e.g. pathogen attack, abiotic stress, photoperiod) very often mediated by hormones. Two-component signal transduction systems appear to play a major role in providing the molecular backbone for this signal integration machinery.
Supplementary material
The following supplementary material is available at JXB online.
Figure S1. ARR4D95N transcript and protein levels in transgenic Arabidopsis plants.
Figure S2. Light fluence-rate dependence of hypocotyl growth in ARR4D95N overexpressing Arabidopsis seedlings.
Figure S3. Light fluence-rate dependence of hypocotyl growth in the arr4 mutant.
Figure S4. Yeast two-hybrid interaction analysis of AHP1 with Arabidopsis histidine kinases.
Figure S5. Red light fluence-rate dependence of hypocotyl growth in Arabidopsis cre1-1/ahk2-2/ahk3-3 cytokinin receptor mutant seedlings.
Figure S6. Flowering phenotype of the Arabidopsis cre1-1 mutant.
Figure S7. Photomorphogenic phenotype of in the Arabidopsis arr4 mutant in red light under different growth conditions.
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Acknowledgements |
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We thank T Kakimoto and J Kieber for providing mutant seeds. We are also grateful to S Hummel and D Wanke for technical support, as well as to F de Courcy and E Fischer for proofreading and help in editing the manuscript. This work was supported by HHMI International Scholar and OTKA grants to FN, and DFG grants to ES (SFB388) and KH (AFGN/HA 2146/5; HA 2146/7).
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