Molecular Plant Advance Access originally published online on June 7, 2007
Molecular Plant 2008 1(1):4-14; doi:10.1093/mp/ssm002
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© The Author 2007. Published by Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.
Chemically Induced and Light-Independent Cryptochrome Photoreceptor Activation
a FB Biologie-Pflanzenphysiologie, Philipps-Universität, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany
b Universität Dortmund, FB Chemie-Chemische Biologie, Otto-Hahn-Str. 6, 44227 Dortmund, Germany
c University of Tennessee, Department of Biochemistry, Cellular and Molecular Biology, Walters Life Sciences M407, Knoxville, TN 37996-0840, USA
1 To whom correspondence should be addressed. E-mail batschau{at}staff.uni-marburg.de, fax 49-(0)6421-282-1545.
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Abstract |
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The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP–rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.
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INTRODUCTION |
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Many receptor proteins fulfill their biological function as dimers by physical interaction with proteins of identical or related amino acid sequence. Dimerization can be constitutive or signal induced. One example of receptors that form dimers in the majority of cases are histidine protein kinases, where ligand binding normally activates the kinase activity resulting in cross-phosphorylation of the two subunits (for reviews, see Stock et al., 2000; Grefen and Harter, 2004). In contrast, many animal cell surface receptors shift the equilibrium to the dimeric form upon growth factor binding and initiate the signal transduction cascade by this mechanism (for a review, see Li and Hristova, 2006).
Higher plants possess three classes of unrelated photoreceptor proteins, the phytochromes (phy), the phototropins (phot), and the cryptochromes (cry) (for reviews, see Briggs and Spudich, 2005; Schäfer and Nagy, 2006). In the model plant Arabidopsis thaliana, these three photoreceptor families consist of five, two, and three members, respectively, and all of them form dimers. Phytochromes form constitutive dimers in vivo and in vitro (Brockmann et al., 1987; Tokutomi et al., 1989). The region mediating dimerization of phytochromes resides in their C-terminal domain (Edgerton and Jones, 1994) most probably via their histidine kinase-related domain that is conserved in this region (Stock et al., 2000). The N-terminal region of phytochromes binds the tetrapyrrole chromophore (for a review, see Montgomery and Lagarias, 2002). Recently it has been shown that the C-terminal domain of phytochrome B can be replaced by another protein that is able to dimerize, such as β-glucuronidase (GUS), leading to a hyperactive receptor protein, whereas fusion of the N-terminal domain to a non-dimerizing protein such as green fluorescent protein (GFP) results in a less active receptor (Matsushita et al., 2003). These data demonstrate that dimerization is required for the full biological activity of phytochrome and dispute an essential role for its C-terminal domain in signal transduction. However, besides its role in dimerization, the C-terminal domain in the authentic phytochrome is required for nuclear import.
Phototropins are a class of FMN-binding UV-A/blue light receptors that control phototropism and other movement responses in plants such as chloroplast relocation and stomatal opening (for reviews, see Celaya and Liscum, 2005; Christie and Briggs, 2005). FMN binding is mediated by so-called LOV (light, oxygen, or voltage) domains in phototropins that resemble a subclass of the PAS domain (Christie et al., 1999; Salomon et al., 2000). Interestingly, phototropins possess two LOV domains (LOV1 and LOV2) in their N-terminal region both of which bind FMN (for a review, see Briggs et al., 2001) and perform a photocycle that includes light-induced covalent linkage of the chromophore to a conserved cysteine residue in the LOV domain followed by bond splitting in darkness (Salomon et al., 2000). The quantum efficiency of Arabidopsis phot1 LOV2 is about 10-fold higher than that of LOV1 (Salomon et al., 2001), and analysis of phot mutants unable to perform a photocycle of either LOV1 or LOV2 demonstrated that photochemically active LOV2 but not LOV1 is required for phototropin function (Christie et al., 2002). In vitro studies on LOV1 and LOV2 from oat phot1 have shown that LOV1 dimerizes and that dimerization does not depend on light excitation of LOV1 (Salomon et al., 2004). Whether phototropins form dimers in vivo has not been analyzed in detail yet.
Cryptochromes, the second class of UV-A/blue light receptors of plants, are related in their sequence to DNA repair enzymes, DNA photolyase, but lack repair activity. In addition, most cryptochromes are distinguished from photolyase by carrying unique C-terminal extensions (for reviews, see Sancar, 2003; Batschauer, 2005). Evidence that these extensions are required for the biological activity of plant cryptochromes came from studies of inactive cry1 alleles that carry mutations in this region (Ahmad et al., 1995). Furthermore, overexpression of the C-terminal domain of either cry1 or cry2 (CCT1, CCT2) resulted in plants that showed a constitutive photomorphogenic (cop) phenotype (Yang et al., 2000) similar to mutants of cop1 or members of the cop9 complex (with typical de-etiolation responses already in darkness) (for a review, see Yi and Deng, 2005). This result strongly suggested that CCT mediates cry signaling which is in line with yeast two-hybrid studies that showed physical interaction of CCT1 and CCT2 with COP1 (Wang et al., 2001; Yang et al., 2001), a general component in light signal transduction. Recently it has been shown (Sang et al., 2005) that cry1 is constitutively dimeric in vivo and in vitro, and that the N-terminal domain, which binds the FAD and MTHF cofactors, mediates dimerization. Moreover, transgenic Arabidopsis lines expressing fusions of CCT1 and CCT2 with GUS, that forms dimers and/or multimers, and which have been used for the previously mentioned studies (Yang et al., 2000), have a cop phenotype, in contrast to lines that express a fusion of CCTs with monomeric proteins [GFP, Arabidopsis (6–4) photolyase] (Sang et al., 2005). Taken together, these data strongly suggest that dimerization of cryptochromes is required for their biological activity and/or that the fused GUS forces the CCT into a conformation that resembles its signaling state.
These results prompted us to investigate whether cry activity could be completely uncoupled from the light signal and switched on by a substitute external signal using chemically induced protein dimerization. The system we used for this purpose consists of the human protein FKBP12 (FK506-binding protein, FKBP) that binds to the natural immunosuppressive drugs rapamycin and FK506 with high affinity (Choi et al., 1996). The FKBP–rapamycin complex binds tightly to another human protein called FRAP or a 93 amino acid domain of FRAP called FRB (Chen et al., 1995). When FKBP and FRB are fused to other proteins, rapamycin-induced heterodimerization of the fusion partners can be achieved. Researchers at ARIAD Pharmaceuticals Inc. have designed FRAP mutants that, in addition to rapamycin, bind chemically modified analogs (rapalogs), such as AP21967, which do not have immunosuppressive activity (Pollock et al., 2002). Using the vectors of the ARIAD ARGENTTM Regulated Heterodimerization Kit, we constructed chimeric genes of FKBP and FRB with Renilla luciferase (RLUC) and yellow fluorescent protein (YFP) for bioluminescence resonance energy transfer (BRET) studies (Xu et al., 1999, 2003; Subramanian et al., 2004b). Results from the studies presented here show that rapalog-induced protein dimerization is applicable to plant cells. In addition, we show that cryptochrome 2 signal transduction can be switched on in darkness by chemical-induced dimerization of its C-terminal domain. Hence, our results also provide the basis for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.
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Results |
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Arabidopsis Cryptochrome 2 Forms Dimers In Vivo
As outlined above, Arabidopsis cry1 homodimerizes constitutively in vitro and in vivo by its N-terminal domain as shown by coimmunoprecipitation analysis, gel filtration, yeast two-hybrid analysis, and in vivo chemical cross-linking studies (Sang et al., 2005). In addition, the same authors showed by yeast two-hybrid assays and in vitro coimmunoprecipitation analysis that cry2 forms homodimers. However, dimerization of cry2 in plant extracts has yet to be demonstrated. We therefore analyzed cry2 dimerization using a transgenic Arabidopsis line expressing full-length cry2 fused with GFP. Western blot analysis of protein extracts isolated from the cry2–GFP line probed with anti-cry2 antibody showed, besides endogenous cry2, an additional band that fits in terms of size with the cry2–GFP fusion protein (Figure 1A). This line also showed nuclear GFP signals (data not shown) as expected from the localization of cry2 in this compartment (Guo et al., 1999; Kleiner et al., 1999). Addition of anti-GFP antibody followed by protein G–agarose incubation depleted the protein extract of the cry2–GFP fusion (Figure 1C). In the pellet fraction, the cry2–GFP protein was detected together with endogenous cry2 (Figure 1C). No cry2 signal was detected in the pellet fraction of wild-type extracts treated with anti-GFP antibody and protein G–agarose (Figure 1B) and in the pellet fraction of protein extracts isolated from the transgenic cry2–GFP line that was not incubated with anti-GFP antibody (Figure 1C), demonstrating that the anti-GFP antibody used is specific for GFP and does not cross-react with cry2. Together, these data indicate physical interaction of cry2 with cry2 in vivo, most probably by formation of dimers. Alternatively, the endogenous cry2 could be located in a complex together with the cry2–GFP fusion protein.
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Bioluminescence Resonance Energy Transfer Studies Confirm Induced Dimerization of FKBP and FRB in Plant Cells
In vivo dimerization of FKBP and FRB by rapamycin and rapalogs has been shown for mammalian and yeast cells (Ho et al., 1996; Rivera et al., 1996; De and Gambhir, 2005). To our knowledge, however, the applicability of this system in plant cells has not been demonstrated. We therefore constructed chimeric genes consisting of either FKBP or FRB fused in-frame to the N-terminus of either YFP or RLUC. The dimerizing proteins were separated from the BRET partners by an insertion of nine alanine residues to allow higher flexibility for physical interaction of the BRET partners (Subramanian et al., 2004b). These gene constructs were introduced into onion epidermal cells by particle gun bombardment, and BRET signals were monitored 40 h after transfection in the absence or presence of 2 µM AP21967. As shown in Figure 2, the combination of RLUC with YFP gave BRET signals with yellow/blue ratios of 0.8, which is in the range for proteins that do not physically interact (Subramanian et al., 2004b). In contrast, the positive control (combination RLUC–COP1 with YFP–COP1) showed higher yellow/blue ratios than the negative control, with values in the range of 1.1 (Figure 2), as expected, because COP1 forms dimers (Torii et al., 1998; Subramanian et al., 2004a). The BRET signals of the cells expressing both combinations of RLUC and YFP fused to FKBP or FRB increased strongly after induction (with yellow/blue ratios up to 1.6) in contrast to the negative and the positive controls (Figure 2). These data demonstrate that dimerization of FKBP and FRB is induced by AP21967 in intact plant cells. We further analyzed the applicability of this system in chemically transfected protoplasts of Arabidopsis cell cultures, and investigated how stable the BRET signal was over time and how it depends on the concentration of the inducer. The induction of dimerization of the FKBP–YFP and FRB–RLUC fusion proteins by AP21967 was analyzed in a concentration range between 0 and 1000 nM, and BRET ratios were measured up to 240 min after inducer application. As shown in Figure 3, even 10 nM inducer resulted in an increase in the BRET signal. The BRET signals reached a plateau within 20 min and then remained relatively constant over the analyzed time period of 240 min, indicating that the dimers were stable. Concentrations of inducer above 10 nM yielded higher BRET signals, and the maximum level was reached with the highest concentration tested (1000 nM). These data show that the rapalog-inducible system works in a dose-dependent fashion in plant cells surrounded by a cell wall (onion epidermal cells) as well as in plant protoplasts that lack a cell wall.
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Chemically Induced Dimerization of CCT2 Induces the Expression of a Blue Light-Controlled Reporter Gene in Darkness
Having shown the productive FKBP–FRB dimerization by BRET analysis, dimerization of CCT2 was investigated. Fusion constructs of CCT2 with FKBP and FRB were introduced into Arabidopsis protoplasts and their expression studied by immununoblot analysis using antibodies specific for CCT2. As shown in Figure 4, this antibody specifically detects endogenous cry2, as confirmed by the immunoblot signal at 69.5 kDa and the disappearance of the cry2 signal in blue light-treated protoplasts (compare lanes 2 and 3 in Figure 4). Cry2 is a light-labile photoreceptor that is rapidly degraded under high fluence rate UV-A/blue light (Lin et al., 1998). In addition, the fusion proteins of FKBP–CCT2 and FRB–CCT2 were specifically detected by the cry2 antibody when individually expressed in protoplast (Figure 4, lanes 4 and 5, respectively), since these bands were missing in the untransformed control cells (Figure 4, lane 6). The mobility of the detected proteins is in accordance with the calculated molecular masses of the CCT2 fusions (FKBP–CCT2, 26 kDa; FRB–CCT2, 25.4 kDa). Double transformants expressed the fusion proteins of FKBP–CCT2 and FRB–CCT2 to similar levels, and the level of the CCT2 fusion proteins was similar in AP21967-treated and untreated cells (Figure 4, compare lanes 1 and 2). In conclusion, the immunoblot data show that FKBP–CCT2 and FRB–CCT2 fusion proteins are efficiently expressed in Arabidopsis cells and yield in higher levels than the endogenous cry2, as expected, since CCT2 fusions were expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter.
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We next studied whether CCT2 dimerization could activate the expression of a cotransfected reporter gene construct that is under cryptochrome control. This reporter gene construct (LRU4:GUS) consists of a minimal 35S promoter fused with four copies of the light regulatory unit (LRU) of Arabidopsis chalcone synthase (CHS) in front of the GUS reporter (Hartmann et al., 1998). It has been shown previously that the expression of Arabidopsis chalcone synthase is induced by blue light via cryptochrome (Ahmad et al., 1995; Jackson and Jenkins, 1995) as the specific role of the LRU in this process (Hartmann et al., 1998). In order to normalize the measured GUS activity between the various experiments, a control construct was cotransformed expressing RLUC under control of the CaMV 35S promoter, and the GUS activities that were measured by a fluorometric assay were normalized to the RLUC control. When transfected protoplasts were kept in complete darkness, AP21967 induced the LRU4-driven GUS expression by a factor of five (Figure 5, compare column 1 with column 2), demonstrating that the induced dimerization of CCT2 results in the activation of cry signaling. In the absence of inducer the likewise transformed protoplasts kept in blue light showed GUS levels that were about eight times higher than in darkness (Figure 5, compare column 1 with column 3), confirming that the expression of the LRU4:GUS construct is controlled by blue light most probably through the endogenous cryptochromes. When AP21967 was added to protoplasts under blue light, GUS expression was even enhanced (Figure 5, compare columns 3 and 4). This additional stimulating effect of the inducer on LRU4:GUS expression under blue light is discussed below. Controls that were transformed with FKBP–YFP and FRB–YFP fusion constructs instead of the CCT2 constructs did not show any AP21967 induction of GUS expression in darkness (Figure 5, compare column 1 with 5) and, in the absence of chemical inducer, the blue light response of FKBP–YFP/FRB–YFP-transformed cells was nearly the same as the blue light response in protoplasts expressing the CCT2 fusions that were not treated with inducer (Figure 5, compare column 3 with 6). Together these data show that the enhanced expression of LRU4:GUS is indeed specific for CCT2 when present as a dimer.
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CCT2 Dimerization Induces the Expression of the Endogenous CHS and CHI Genes
To investigate whether the chemically induced dimerization of CCT2 has no unspecific effects and also allows induction of the expression of endogenous genes, we cotransfected FKBP–CCT2 and FRB–CCT2 into Arabidopsis protoplasts and analyzed the expression of the constitutively expressed ubiquitin (UBQ), the blue light-induced CHS, and another blue light-induced flavonoid biosynthetic gene, chalcone isomerase (CHI), in the absence and presence of AP21967 by real-time PCR. CHS transcript levels showed strong induction by blue light treatment, as expected (Figure 6B). When the protoplasts that were cotransfected with FKBP–CCT2 and FRB–CCT2 were incubated with AP21967 in darkness, an 11-fold increase in the CHS transcript level was detected (Figure 6A). This induction is caused by CCT2 dimerization since non-transfected protoplasts did not respond to the inducer (Figure 6A). The smaller induction rates by AP21967 compared with blue light (compare Figure 6A and B) can be explained at least in part by the fact that only those cells which have been successfully transfected with the FKBP–CCT2 and the FRB–CCT2 construct can respond to the inducer, whereas the light signal is perceived by all cells (see Discussion). Like CHS, CHI has been shown to be induced by blue light, and the kinetics of their induction in etiolated Arabidopsis seedlings are similar, with a strong increase in their transcript levels after 2.5 h of blue light irradiation (Kubasek et al., 1992). Similar to CHS, we found up-regulation of CHI transcripts by the AP21967 inducer in dark-cultivated protoplasts expressing the CCT2 constructs (Figure 6C), and a strong increase of CHI transcript levels after blue light treatment (Figure 6D). Together these data demonstrate that CCT2 dimerization activates the expression not only of the cotransfected LRU4:GUS construct but also of the endogenous CHS and CHI genes that are under cryptochrome control.
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DISCUSSION |
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Plant cryptochromes form dimers via their N-terminal photosensory domain (Sang et al., 2005) and possess a well-defined output domain, the unique C-terminal extension (CCT). The observation that the photosensory domain, when replaced by GUS, leads to a constitutively active photoreceptor (Yang et al., 2000) whereas fusions of CCT with a monomeric protein result in an inactive photoreceptor (Sang et al., 2005) led to the conclusion that cryptochromes require dimerization for their biological activity (Sang et al., 2005). Unfortunately, the putative biological activity of CCTs that were not fused to other proteins could not be analyzed due to the fact that these peptides are unstable in plant cells (Yang et al., 2000).
These results motivated us to investigate whether an inducible dimerization system could be used to trigger a chemical but light-independent activation of cryptochrome signaling. For this purpose we have chosen the rapalog-inducible dimerization of two human proteins, FKBP and FRB, that has been used before in mammalian and yeast cells (Ho et al., 1996; Rivera et al., 1996; De and Gambhir, 2005) but not in plant cells. As proof-of-principle for the applicability of this system to plants we tested induced FKBP–FRB dimerization by fusing the dimerization partners RLUC and YFP and analyzed BRET between RLUC and YFP in ballistically transformed onion epidermal cells. An increase in BRET signals of the two FKBP–FRB combinations was only observed after adding the inducer AP21967 (Figure 2), whereas the BRET signals of the negative control (RLUC with YFP) and the positive control (RLUC–COP1 with YFP–COP1) showed no difference in the BRET signals before and after application of the inducer. These data demonstrate that AP21967 has no unspecific effects on protein dimerization and provides, to our knowledge for the first time, evidence that rapalog-induced dimerization is also feasible in plant cells. Since bombarded cells with intact cell walls responded to the inducer, the plant cell wall does not seem to be a serious barrier for the uptake of the inducer. Accordingly, we consider this system to be applicable also for whole plants.
Since the BRET signals remained high and constant even for several hours after application of the inducer (Figure 3), the formed FKBP–FRB dimers and the AP21967 inducer seem to be essentially stable in the plant, allowing its use also for applications that require the presence of dimers over long time periods. Such a strong association of FKBP with FRB could, however, fail to mimic protein–protein interactions that are weak or transient.
As outlined above, expression of CCT1 and CCT2 causes a cop phenotype only when fused with the dimerizing GUS protein (Yang et al., 2000; Sang et al., 2005). Moreover, plant cryptochromes exist as dimers (Sang et al., 2005 and Figure 1), suggesting that dimerization of these receptors is required for their biological activity. However, one cannot exclude that the dimerizing protein (GUS) used in these studies forced the CCT into a conformation that mimics the signaling state. The approach we have taken allows a distinction to be made between the two possibilities since the same CCT2 fusions are tested either as monomers or as dimers.
To analyze whether fusions of CCT2 with FKBP and FRB become biologically active when they are induced to dimerize, we first checked the expression levels of these proteins in transfected protoplasts. The immunoblot shown in Figure 4 demonstrates clearly that protein fusions of CCT2 with FKBP and FRB can be overexpressed in protoplasts, singly or together, without obvious deleterious effects. Moreover, their protein level was not affected by addition of the AP21967 inducer or by exposure to 80 µmol m–2 s–1 of blue light, while endogenous cry2 (
70 kDa) was unstable under blue light (Figure 4), as expected from studies on cry2 stability in Arabidopsis seedlings (Lin et al., 1998).
As a readout for CCT2 activity in transient assays, we used a chimeric gene construct encoding GUS under control of four copies of the LRU of the Arabidopsis CHS gene (Hartmann et al., 1998). CHS expression is under control of phyA and cryptochromes (Ahmad et al., 1995; Jackson and Jenkins, 1995; Batschauer et al., 1996), as well as the regulators COP1 and HY5 (Ang et al., 1998; Osterlund et al., 2000; Holm et al., 2002; Seo et al., 2003; Saijo et al., 2003). Arabidopsis protoplasts were cotransfected with the LRU4:GUS reporter gene as well as with 35S:RLUC as a reference gene to normalize for transformation efficiency. In addition, FKBP–CCT2 and FRB–CCT2 were cotransfected. Addition of AP21967 clearly resulted in a 5-fold activation of LRU4:GUS expression in darkness (Figure 5). This effect was evidently mediated by CCT2 dimerization because it was not observed when YFP fusions to FKBP and FRB were used instead of CCT2. Moreover, the activation of gene expression by CCT2 dimerization could also be detected in blue light, although blue light alone caused activation of LRU4:GUS, as expected. The additional stimulating effect of the inducer on LRU4:GUS expression under blue light compared with the blue light control (Figure 5) is probably caused by the fact that the CCT2 fusion proteins are light stable, in contrast to endogenous cry2 (Figure 4). Consequently, the level of active cryptochrome (cry1 and cry2) decreases during the blue light treatment, but this decrease is compensated by the chemical activation of CCT2.
The applicability of the FKBP–FRB dimerizing system for gene activation in whole transgenic plants requires uptake of the inducer through either leaves or roots, and the activation of endogenous genes. The observed AP21967 induction of reporter gene activity in ballistically transformed onion epidermis cells (Figure 2) indicates that the plant cell wall does not cause relevant difficulties in the uptake of this inducer. However, other barriers such as the leaf cuticle could hinder or reduce uptake. Accordingly, a systematic investigation of rapalog-induced dimerization in whole plants is required. These experiments are currently in progress in our laboratory.
We also confirmed, using real-time PCR, that the induction of CCT2 activity by AP21967 causes up-regulation of the endogenous CHS and CHI transcript levels (Figure 6). The seemingly low induction rates for CHS and CHI expression by CCT2 dimerization in dark samples compared with light-treated samples that were not incubated with inducer are most probably caused by the fact that only a small fraction of the protoplasts was transformed. With the applied transformation protocol, we routinely observe transformation rates in the range of 20% or less. Accordingly, only cells of this fraction can respond to the inducer, whereas light treatment induces CHS and CHI expression in all cells. In addition, blue light activates not only cryptochromes but also phytochromes, and both induce CHS expression as mentioned above. Furthermore, when overexpressed as a GUS fusion, CCT2 is less active than CCT1 in the hypocotyl inhibition response and anthocyanin induction (Yang et al., 2000), suggesting that even stronger effects could be achieved by inducing CCT1 dimerization.
Our studies specifically addressed the question of whether a plant photoreceptor can be designed to act independently of light but can be induced chemically via protein dimerization. The presented data show that this approach is feasible, thereby allowing the signal transduction of a single photoreceptor to be analyzed specifically in darkness. In addition, this approach makes it possible to study the specific role of a single photoreceptor under light conditions that activate the whole spectrum of plant photoreceptors. In contrast to loss-of-function studies that compare responses in wild type and mutants, the chemically induced dimerization allows gain-of-function studies where one line is being studied before and after induction. In contrast to other systems such as Cre/loxP that uses induced gene activation or suppression based on site-specific recombination via transcriptional activation of the CRE recombinase (Austin et al., 1981; Sternberg and Hamilton, 1981; Odell et al., 1990), or chemically induced synthetic promoters (Gatz, 1997), the FKBP–FRB system has the advantage of being reversible, either by washing out the inducer or by competitive inhibition of the inducer (Mootz et al., 2003). Finally, it allows faster induction since it does not depend on transcriptional activation.
As outlined above, not only cryptochromes but also phytochromes and phototropins form dimers. In the case of phytochromes it has been shown that the dimeric form is biologically active (Matsushita et al., 2003). Their dimerizing domain resides in the C-terminal domain that can be replaced by GUS, still yielding an active photoreceptor when nuclear localization signals were added to allow nuclear entry of the fusion protein (Matsushita et al., 2003). However, the N-terminal domain that binds the bilin chromophore does not activate phytochrome signaling in darkness. Therefore, both dimerization and light are required for the activation of phy, thus preventing study of phytochrome signaling in darkness. Nevertheless, we consider it feasible to study the effects of temporal activation of single phytochrome members during light development by induced dimerization. Similarly, the dimerization of phototropins through their LOV1 domain (Salomon et al., 2004) could be considered as a target for induced dimerization, although the role of dimerization for the biological activity of these photoreceptors is still elusive.
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Gene Constructs
Constructs for BRET experiments were based on pBS-35S-Ala-RLUC (GenBank accession no. AY189982) and pBS-35S-Ala-YFP (GenBank accession no. AY189984). As positive controls, RLUC–COP1 and YFP–COP1 were used (Subramanian et al., 2004a). Fragments of FKBP and FRB with restriction sites for NcoI at the 5' end and NotI at the 3' end were generated by PCR. FKBP spanning amino acid residues 15–234 was amplified from pC4M-F2E (ARGENTTM Regulated Heterodimerization Kit, ARIAD Pharmaceuticals, Inc., Cambridge, MA, USA) by using the primers 5'-CCATGGCGTCTAGAGGAGTGCAGGTGGAA-3' (NcoI site underlined) and 5'-TGCGGCCGCACTAGTTTCCAGTTTTAGAAGCTC-3' (NotI site underlined). FRB spanning amino acid residues 3–99 was amplified from pC4-RHE (ARGENTTM Regulated Heterodimerization Kit, ARIAD Pharmaceuticals, Inc.) using the primers 5'-CCATGGCTTCTAGAATCCTCTGGCAT-3' (NcoI site underlined) and 5'-TGCGGCCGCACTAGTCTTTGAGATTCGTCGGAA-3' (NotI site underlined). The PCR products were subcloned into pGEM®-T (Promega, Madison, WI, USA), sequenced to verify their identity, and then ligated into NcoI and NotI sites of pBS-Ala-RLUC and pBS-Ala-YFP, resulting in plasmids pBS-FKBP-Ala-RLUC, pBS-FRB-Ala-RLUC, pBS-FKBP-Ala-YFP, and pBS-FRB-Ala-YFP.
The plasmids for regulated dimerization of CCT2 (pBS-FKBP-CCT2 and pBS-FRB-CCT2) were generated by excising the RLUC fragment from pBS-FKBP-Ala-RLUC and pBS-FRB-Ala-RLUC, respectively, with NotI and BglII, and replaced by a 384 bp fragment coding for CCT2 (amino acid positions 489–612). The CCT2 fragment was PCR amplified by primers 5'-GCGGCCGCACGTGAAGCACAGATCATG-3' (NotI site underlined) and 5'-AGATCTTCATTTGCAACATTTTTTCC-3'(BglII site underlined), subcloned into pGEM®-T (Promega), and sequenced to verify its identity. The expression of the BRET and CCT2 constructs in plant cells was driven by the CaMV 35S promoter.
For construction of the binary vector encoding the fusion of GFP with full-length cry2, the CRY2-GFP coding region was excised from plasmid CRY2(1–612)/GFP-pMAV4 (Kleiner et al., 1999) with BamHI and SacI. The binary vector pPCV812 (Koncz et al., 1989) was cut with the same restriction enzymes and the uidA gene replaced by CRY2-GFP under control of the CaMV 35S promoter. The resulting vector was introduced into the Agrobacterium strain GV3101 (Koncz and Schell, 1986) and used for transformation of A. thaliana ecotype Landsberg erecta by the floral dip protocol (Clough and Bent, 1998).
Plant Materials and Transformation
Onion epidermal cells were bombarded with gene constructs coated onto gold particles using the PDS-1000 He particle gun (Bio-Rad, Munich, Germany) and BRET assays performed as described (Subramanian et al., 2004a). After the initial BRET measurements, AP21967 was added to a final concentration of 2 µM and BRET ratios measured again 60 and 120 min later.
For protoplast transfection, A. thaliana cell suspension cultures were used. Cell cultures were heterotroph (ecotype Columbia, derived from root tissue). Cells were protoplasted with 1% (w/v) cellulase ‘Onzuka R-10’ and 0.25% (w/v) macerozyme R-10 (both enzymes from Serva, Heidelberg, Germany) in 0.4 M mannitol, 8 mM CaCl2 for 6 h at 50 rpm and 25°C. Protoplasts were transfected in several batches according to published procedures (Dangl et al., 1987). After transfection, the protoplast batches transformed with the same plasmid were pooled and split again to ensure equal transformation efficiencies. Incubation overnight was either in darkness or under blue light (see below). For GUS assays, immunoblot, and real-time analysis, the inducer AP21967 (dissolved in 100% ethanol) or an equal volume of 100% ethanol (as mock control) was added to the samples to a final concentration of 1 µM inducer directly after transfection. For BRET experiments, AP21967 (10, 100, or 1000 nM final concentration) was added 16 h after transfection. The yellow-to-blue ratio for t0 was determined directly before adding the inducer.
For light treatments, samples were kept in broad-band blue light (
max 436 nm, photon fluence rate 80 µmol m–2 s–1) (Schäfer, 1977) for 16 h. Controls were kept in darkness for the same duration.
Coimmunoprecipitation Studies
Arabidopsis wild-type and T2 seedlings expressing cry2–GFP (both in the Ler background) were grown at 24°C in complete darkness on filter paper after stratification at 4°C for 2 d and induction of germination by white light for 2 h. Harvesting and all subsequent steps were done in darkness or red light at 4°C to avoid cry2 degradation. Proteins were extracted by grinding seedlings in liquid nitrogen in the presence of 1 ml of extraction buffer [400 mM NaCl, 100 mM Tris–HCl, pH 7.5, 10 % (v/v) glycerol, 1 mM dithiothreitol (DTT), 1% (v/v) igepal CA-630 (Sigma, Steinheim, Germany)] per mg of seedlings. After thawing on ice, samples were centrifuged (20 000 g, 10 min), and the supernatants were removed and centrifuged as before. The protein concentration of the extracts was determined by the amido-black method (Popov et al., 1975). For pull-down experiments, aliquots of the protein extracts containing 1 mg of protein were incubated with goat anti-GFP antibody (Rockland, Gilbertsville, PA, USA) in a final dilution of 1:200 for 1 h followed by incubation with 100 µl of protein G–agarose (Roche, Penzberg, Germany; 1:2 diluted with extraction buffer) overnight under continuous shaking. Agarose beads were pelleted (20 000 g, 1 min) and resuspended three times in 1 ml of extraction buffer followed by incubation after each resuspension for 10 min and centrifugation. After washing, the beads were resuspended in 100 µl of SDS sample buffer [62.5 mM Tris–HCl, pH 6.8, 4 M urea, 0.7 M β-mercaptoethanol, 2% (w/v) SDS, 0.002 % (w/v) bromphenol blue, 10% (v/v) glycerol] and boiled for 10 min. After centrifugation (20 000 g, 10 min, 25°C), 20 µg of protein from each sample or 25 µl of the immunoprecipated protein solutions were separated by 10% Laemmli SDS–PAGE (Laemmli, 1970) together with SDS7B marker (Sigma, St Louis, MO, USA), blotted onto nitrocellulose membranes, and probed with cry2-specific antibodies as described (Kleiner et al., 1999).
GUS Assay
GUS assays were performed according to published procedures (Jefferson, 1987). In brief, the protoplasts were ruptured with a needle and syringe in 300 µl of extraction buffer (0.1 M potassium phosphate, 1 mM DTT, pH 7.8), the lysate cleared by centrifugation (20 000 g, 15 min, 4°C), and an aliquot removed for RLUC activity measurements. The crude extracts were diluted 1:1 with substrate solution [1 mg of 4-methylumbelliferyl-β-D-glucuronide (Duchefa, Haarlem, The Netherlands) in 1 ml of extraction buffer] and incubated at 37°C with shaking at 300 rpm. For each time point, 20 µl of the reaction solution were mixed with 1 ml of 0.2 M Na2CO3. Triplicates were made for each sample and time point. Fluorescence emission at = 445 nm was measured with a Shimadzu RF-5301 PC series spectrofluorophotometer with excitation at = 360 nm.
Luciferase Measurements
Luciferase measurements and the calculations of standardized specific GUS activities were done according to published protocols (Hartmann et al., 1998) except that another luciferase buffer was used. In brief, 10–30 µl of protoplast crude extract was diluted with extraction buffer (0.1 M potassium phosphate, 1 mM DTT, pH 7.8) to a final volume of 150 µl and mixed with 50 µl of RLUC buffer [80 mM glycine, 60 mM ATP, 40 mM MgSO4, 1% (w/v) bovine serum albumin (BSA), pH 7.8] and 1 µl of coelenterazine solution (Biotium, Hayward, CA, USA; final concentration 1 µM) 30 s before measurement. Luminescence intensity was determined in four pairs of 10 s readings. Intensities were detected three times for each sample, and mean values were used for correction of fluorescence increase determined in the GUS assay.
Protein Isolation from Protoplasts and Immunoblot Analysis
Protein extraction from protoplasts was done by rupturing the cells with a needle and syringe in the presence of 7% (v/v) trichloroacetic acid (TCA). For each sample, 10 µg of total protein were separated by 15% SDS–PAGE (Laemmli, 1970). As protein standard, PageRulerTM (Fermentas, St. Leon-Rot, Germany) was used. Immunoblot ECL analysis (GE Healthcare, Solingen, Germany) was done as described (Kleiner et al., 1999) using primary antibodies raised against Arabidopsis CCT2 (Lin et al., 1998).
RNA Preparation and Real-Time PCR
Total RNA was extracted using an RNeasy kit (Qiagen, Hilden, Germany) and treated with DNase I (Invitrogen). cDNA synthesis was performed with SuperScriptTM II RNase H reverse transcriptase and oligo(dT) (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Real-time PCR was performed using iQTM SYBR green supermix I with an icycler machine (both from Bio-Rad, Munich, Germany), following the instructions of Bio-Rad. Fragments of CHS, CHI, and UBQ10 were amplified with the following primers: CHS, 5'-CCAGAGAAGGAGCCATGTAAGC-3' and 5'-CATGACCGACCTCAAGGAGAAG-3'; CHI, 5'-GGCTCTCTTACGGTTGCGTT-3' and 5'-GTTCTTCCCGATGATAGATTCC-3'; UBQ, 5'-GCAAGAGTTCTGCCATCCTCC-3' and 5'-CGGGAAAGACGATTACTCTTGAGG-3'.
PCR efficiencies were calculated with LinRegPCR (Ramakers et al., 2003). Data treatment was as described (Pfaffl, 2001). Gene expression data were represented relative to the maximum value among all data sets after normalization to the UBQ control.
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Acknowledgements |
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We thank ARIAD Pharmaceuticals, Inc. (Cambridge, MA, USA; www.ariad.com/regulationkits) for providing materials of the ARGENTTM Regulated Heterodimerization Kit, Stefan Meier (Universität Marburg, Germany) and Jörg Kämper (Max-Planck Institute of Terrestrial Microbiology, Marburg, Germany) for support in real-time PCR, Bernd Weisshaar (Universität Bielefeld, Germany) for the LRU4:CHS construct, Margaret Ahmad (Universite Paris VI, France) for Arabidopsis anti-cry2 antiserum, Csaba Koncz (Max-Planck-Institute for Plant Breeding, Cologne, Germany) for Arabidopsis cell cultures, the binary vector pPCV812, and the Agrobacterium strain GV3101, Hans-Peter Braun (Universität Hannover, Germany) for the POM 34 antiserum, and Oxana Panajotowa, Agnes Debelius, and Elvira Stumpf for excellent technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (grant BA985/7-3 to A.B) and by NSF (grant MCB-0114653 to A.G.v.A).
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