An Arabidopsis protein closely related to ... - Wiley Online Library

The Plant Journal (2003) 35, 93±103

doi: 10.1046/j.1365-313X.2003.01787.x

An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles Tatjana Kleine1, Peter Lockhart2 and Alfred Batschauer1, 1 FB Biologie/P¯anzenphysiologie, Philipps-UniversitaÈt, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany, and 2 Allan Wilson Centre for Molecular Ecology and Evolution, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand Received 21 March 2003; accepted 31 March 2003. For correspondence (fax 49 6421 28 21545; e-mail [email protected]).

Summary Cryptochromes (CRYs) are blue/UV-A photoreceptors related to the DNA repair enzyme DNA photolyase. They have been found in plants, animals and most recently in the cyanobacterium Synechocystis. Closely related to the Synechocystis cryptochrome is the Arabidopsis gene At5g24850. Here, we show that the encoded protein of At5g24850 binds ¯avin adenine dinucleotide (FAD). It has no photolyase activity, and is likely to function as a photoreceptor. We have named it At-cry3 to distinguish it from the other Arbabidopsis cryptochrome homologues At-cry1 and At-cry2. At-cry3 carries an N-terminal sequence, which mediates import into chloroplasts and mitochondria. Furthermore, we show that At-cry3 binds DNA. DNA binding was also demonstrated for the Synechocystis cryptochrome, indicating that both photoreceptors could have similar modes of action. Based on the ®nding of a new cryptochrome class in bacteria and plants, it has been suggested that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. However, our phylogenetic analyses are also consistent with an alternative explanation that the presence of cryptochromes in the plant nuclear genome is the result of dual horizontal gene transfer. That is, CRY1 and CRY2 genes may originate from an endosymbiotic ancestor of modern-day a-proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern-day cyanobacteria. Keywords: cryptochrome, Arabidopsis, Synechocystis, photoreceptor evolution, organelle targeting, DNA binding.

Introduction Cryptochromes (CRYs) were ®rst identi®ed in Arabidopsis thaliana. Largely through studies of mutants and plants overexpressing cryptochromes, they have been shown to encode blue/UV-A photoreceptors (for review, see Lin, 2002). They are related to DNA photolyases, enzymes, which catalyse the repair of UV-B photoproducts (cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts) by using the energy of photons in the blue/UV-A region (for review, see Sancar, 2000). Cryptochromes and DNA photolyases not only have homology in their amino acid sequences but also carry the same chromophores (Lin et al., 1995; Malhotra et al., 1995). In contrast to DNA photolyase, most of the cryptochromes contain extensions at their C-terminus, which are considered to be signalling domains from protein±protein interaction studies (Ahmad et al., 1998; Jarillo et al., 2001; Wang et al., 2001), and the constitutive photomorphogenic phenotype of Arabidopsis ß 2003 Blackwell Publishing Ltd

seedlings overexpressing such C-terminal domains (Yang et al., 2000). However, CRYs were identi®ed in Sinapis alba and the fern Adiantum capillus-veneris that have no Cterminal extension but still seem to have photoreceptor function (Batschauer, 1993; Imaizumi et al., 2000). Therefore, it is yet dif®cult to draw a general picture of the function of the C-terminal domains in CRY signalling. In Arabidopsis, two cryptochromes (cry1, cry2) have been described so far. In blue light, cry1 plays a major role in the de-etiolation response, and in the induction of anthocyanin formation and chalcone synthase gene expression (for review, see Lin, 2002). Cry2 seems to play only a minor role in the de-etiolation response of Arabidopsis, but without it, a strong retardation in ¯owering under long-day conditions is observed, indicating that cry2 is involved in day-length perception (Guo et al., 1998). Besides seed plants, CRYs have also been found in algae, ferns and 93

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mosses, as well as in animals and human beings (for review, see Deisenhofer, 2000; Lin, 2002). The plant CRYs are more closely related to the class I CPD photolyases, whereas animal CRYs are more closely related to the (6-4) photolyases, indicating that CRYs may have evolved independently in plants and animals (Cashmore et al., 1999; Kobayashi et al., 2000). In the cyanobacterium Synechocystis sp. PCC 6803, there exist two open-reading frames (sll1629 and slr0854) with strong sequence similarity and inferred homology to photolyase and cryptochrome. It was shown that slr0854 encodes a photolyase whereas sll1629 has no photolyase activity, indicating that sll1629 encodes a cryptochrome-type photoreceptor (Hitomi et al., 2000; Ng and Pakrasi, 2001). Very recently, further support for the photoreceptor function of sll1629 came from solving its crystallographic structure (Brudler et al., 2003). Although the overall structure of Synechocystis cryptochrome is very similar to the structure of class I CPD photolyase, there are clear differences at the FAD-binding site and the thymine dimer pocket, which abolish photolyase function. Interestingly, this cryptochrome retained the ability of photolyases for sequence-independent DNA binding (Brudler et al., 2003), which was also shown for mouse cry1 (Kobayashi et al., 1998). Most closely related to the Synechocystis cryptochrome is the Arabidopsis protein At5g24850, which has 50% identity with sll1629 over a stretch of 400 amino acids. We demonstrate here that At5g24850 carries a dual targeting signal that mediates import into chloroplasts and mitochondria. Furthermore, we show that At5g24850 binds FAD non-covalently, has no photolyase activity and binds non-sequence speci®c to DNA. Although the photoreceptor function of At5g24850 has not de®nitely been proven yet, we assume such a function and therefore name it At-cry3. In the evolution of chloroplasts, it is well accepted that following endosymbiotic uptake of a cyanobacteria-like organism, horizontal gene transfer has moved many genes from the endosymbiont to the host nucleus (CavalierSmith, 2002; Martin et al., 1998; Rujan and Martin, 2001; The Arabidopsis Genome Initiative, 2000). In this respect, it seems likely that plant nuclear genomes may have received their phytochrome sequences from the endosymbiont because similar photoreceptor genes were identi®ed in cyanobacteria and in even more distantly related eubacteria (Davis et al., 1999; Hughes et al., 1997; Kehoe and Grossman, 1996; Yeh et al., 1997). Phylogenetic analysis of the cryptochrome/photolyase family led to the de®nition of a new cryptochrome class that includes not only the Synechocystis cryptochrome and Arabidopsis At-CRY3, but also cryptochromes identi®ed in Drosophila and Homo sapiens. The cryptochromes of this class were named cryptochrome DASH (for Drosophila, Arabidopsis, Synechocystis, Homo sapiens) (Brudler et al., 2003). The same authors concluded from the existence of closely related cryptochromes in

bacteria and plants that cryptochromes evolved before the divergence of prokaryotes and eukaryotes. Findings from the analyses that we present here also support the close phylogenetic relationship between the Synechocystis cryptochrome and Arabidopsis CRY3. However, Arabidopsis CRY1 and CRY2 are more distantly related to Synechocystis cryptochrome than Arabidopsis CRY3. CRY1 and CRY2 sequences are more closely related to those from a-proteobacteria, which are considered to be progenitors of mitochondria (for review, see Gray et al., 2001). This ®nding raises the possibility that plants may have received their cryptochrome photoreceptors by dual horizontal gene transfer, one homologue from an endosymbiotic ancestor of chloroplasts and one from an endosymbiotic ancestor of mitochondria.

Results Arabidopsis cry3 is closely related to Synechocystis cryptochrome and targeted to organelles In Arabidopsis, two cryptochromes (cry1 and cry2) and two photolyases were discovered and analysed in detail (for review, see Lin, 2002). There are also other members of the cryptochrome/photolyase family present in Arabidopsis with unknown function. One of these genes is At-CRY3 (At5g24850) also named Arabidopsis cryptochrome DASH (Brudler et al., 2003). The expression of this gene is veri®ed by the presence of corresponding ESTs in the databases (ETG NP424316) and our own studies (data not shown). The identity between At-CRY3 and Synechocystis cryptochrome (sll1629) is 50% over a stretch of 400 amino acids, much higher than between sll1629 and Arabidopsis CRY1 and CRY2 (less than 20% identity, see Figure 1). Database searches (http://www.inra.fr/servlets/WebPredotator; http:// www.cbs.dtu.dk/services/TargetP) predicted with high probability the presence of a targeting sequence for import into chloroplast and mitochondria within the N-terminal 40 amino acids (MAASSLSLSSPLSNPLRRFTLHHLHLSKKPLSS SSLFLCS). To test whether these predictions held true, we performed in vitro import studies with 35S-Met-labelled full-length At-cry3 protein and pea chloroplasts. Indeed, we observed a protease-resistant band of lower mobility than the TNT-produced protein after the incubation with chloroplasts (Figure 2). In samples where the chloroplasts were disrupted, after importing the protein, with Triton X100 and treated with thermolysin, the signal was completely gone. These ®ndings indicate that At-cry3 is imported into chloroplasts. Further proof for chloroplast targeting comes from confocal studies of GFP fusions expressed in protoplasts prepared from a green Arabidopsis cell culture. The GFP signal from the full-length At-cry3 fusion protein merged perfectly well with chlorophyll ¯uorescence signals ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103

Organelle-targeted cryptochrome

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Figure 1. Comparison of plant, Synechocystis and a-proteobacteria sequences of the cryptochrome/photolyase family. Shown is an alignment of protein sequences from Synechocystis sp. PCC 6803 cryptochrome (sll1629, accession number S74850), Arabidopsis CRY3 (At5g24850, accession number NP-568461), Arabidopsis CRY1 (accession number Q43125), Arabidopsis CRY2 (accession number Q96524), and the a-proteobacterium Caulobacter crescentus (Cc PHR, accession number NP-420241). Amino acid positions conserved in Synechocystis cryptochrome (sll1629) and Arabidopsis CRY3 are marked by plus signs in the upper line; those conserved in Arabidopsis CRY1, CRY2 and the Caulobacter protein are marked by asterisks in the lower line. Positions conserved in all proteins are dark shaded.

of chloroplasts (Figure 3a). In control cells that expressed only GFP, a homogenous ¯uorescence signal was seen throughout the cytosol and the nucleus, with no signal in the chloroplasts (Figure 3b). A truncated version of At-cry3, lacking the 40-amino-acid N-terminal targeting signal and fused to GFP, showed no ¯uorescence in the chloroplasts. Instead, a homogenous distribution of GFP throughout the cytosol was observed (Figure 3c). A construct containing the N-terminal 63 amino acids of At-CRY3 fused to GFP showed essentially the same ¯uorescence signal as ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103

the full-length protein fusion with strong staining in chloroplasts (Figure 3d). We conclude from these data that the At-cry3 protein is targeted to chloroplasts, and that its N-terminal sequence is required and suf®cient for chloroplast targeting. The GFP fusion proteins containing either full-length or the N-terminal fragment of At-CRY3 showed staining in chloroplasts as well as in other smaller structures with a size of about 0.5±1 mm in diameter (Figure 3a,d). This size ®ts with the size of mitochondria. For an unambiguous

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Tatjana Kleine et al. ¯uorescence emission is typical for FAD (Weber, 1959). Thin layer chromatography of the supernatant resulted in a single ¯uorescent spot with the same Rf-value as authentic FAD (data not shown). The calculated ratio of FAD and At-CRY3 molecules was 0.7, indicating that each At-CRY3 protein binds one FAD. Taken together, we conclude that At-CRY3 binds non-covalently FAD in 1 : 1 stoichiometry. As for Synechocystis cryptochrome (Brudler et al., 2003; Hitomi et al., 2000), we could not identify a second chromophore attached to At-CRY3. At-cry3 lacks photolyase activity

Figure 2. At-cry3 is imported into chloroplasts. In vitro import study with pea chloroplasts and in vitro synthesised 35S-Metlabelled At-cry3. Lane 1: In vitro translation product. Lane 2: Radiolabelled At-cry3 incubated with pea chloroplasts. Lane 3: Thermolysin-treated import reaction. Lane 4: Triton X-100- and thermolysin-treated import assay. The arrows indicate the positions of the precursor (p) and the mature (m) protein. The labelled proteins were visualised by autoradiography after SDS±PAGE.

identi®cation of mitochondria, we stained the cells with mitotracker after expression of the GFP fusion proteins. Fusions of GFP with either full-length At-cry3 or with its N-terminus showed GFP ¯uorescence in the mitochondria (Figure 3e,g), whereas the N-terminal truncated At-cry3 fused with GFP showed the same homogenous distribution in the cytosol (Figure 3f) as the GFP control (Figure 3b). Taken together, we conclude from the in vitro import and GFP localisation data that At-cry3 contains a functional dual targeting signal mediating transport into chloroplasts and mitochondria.

The sequence of At-CRY3 does not allow an unambiguous prediction of its function. We therefore tested whether expression of At-cry3 in the photolyase-de®cient E. coli strain KY1225 can restore photoreactivation. Expression of the At-cry3 protein in KY1225 cells was con®rmed by SDS±PAGE (data not shown). As shown in Figure 5(a), there was no signi®cant difference in the survival rate of KY1225 cells transformed with either the At-CRY3 construct or the empty vector (pQE-60) after treatment with UV-B and subsequently followed by photoreactivating light. Under the same conditions, the corresponding E. coli wild-type strain (KY1056) showed strong photoreactivation (Figure 5a) as KY1225 cells that expressed the Arabidopsis CPD photolyase as a further control (Figure 5b). In accordance with the results from the complementation tests, we could not detect any signi®cant photolyase activity in vitro in extracts from E. coli cells expressing At-cry3 (data not shown). At-cry3 binds to DNA

At-CRY3 binds FAD as chromophore All plant blue-light photoreceptors identi®ed so far are ¯avoproteins (Lin, 2002) carrying either FAD (cryptochromes) or ¯avin mononucleotide (FMN) (phototropins). To determine whether At-CRY3 also binds ¯avin, we expressed it as a 6xHis-tagged fusion in Escherichia coli cells to obtain suf®cient material for spectroscopic analyses. Most of the expressed protein was soluble and bright yellow indicative of the presence of ¯avin. After boiling the puri®ed protein in acidic SDS buffer, the absorption spectrum of the supernatant was taken (Figure 4a). The absorption spectrum of the released chromophore (Figure 4a, line A) was essentially the same as that of authentic FAD (Figure 4a, line B) with maxima at around 375 and 450 nm. To distinguish between FAD and the other two non-dinucleotide forms, FMN and ribo¯avin, ¯uorescence emission spectra of the released chromophore were taken at different pH values. Excitation at 450 nm resulted in ¯uorescence emission with a maximum at 530 nm (Figure 4b). The ¯uorescence emission was pH dependent with eightfold higher ¯uorescence at pH 4.0 than at pH 8.0 (Figure 4b). This pH dependency of

For mouse cry1 (Kobayashi et al., 1998) and Synechocystis cryptochrome (Brudler et al., 2003), DNA binding has been demonstrated. We therefore analysed whether the organelle-targeted At-cry3 is also able to bind DNA and performed mobility shift assays with in vitro translated At-cry3. As shown in Figure 6, At-cry3 bound to the randomly chosen double-stranded DNA probe resulting in a shifted band. As the shifted band could be out competed to the same extent with either the unlabelled probe or with competitor DNA of unrelated sequence, we conclude that binding of At-cry3 to DNA is not sequence speci®c. Besides double-stranded DNA, we also used single-stranded DNAs as competitors. As shown in Figure 6, single-stranded DNAs were as effective as double-stranded DNAs in competition experiments. Therefore, binding of At-cry3 to DNA is neither double-strand nor sequence speci®c. Discussion In Arabidopsis, four members of the cryptochrome/ photolyase family have been described and functionally ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103


Figure 3. At-cry3 is imported into chloroplasts and mitochondria. Confocal images of Arabidopsis protoplasts expressing GFP constructs. (a, e) Protoplasts transformed with full-length At-cry3-GFP fusion construct. (b) Protoplasts transformed with unfused GFP-construct as control. (c, f) Protoplast transformed with fusion construct of GFP with N-terminal truncated (D1±40) At-cry3. (d, g) Protoplast transformed with construct of GFP fused to the N-terminus (amino acids 1±63) of At-cry3. (e±g) Protoplasts were incubated with mitotracker to visualise the mitochondria. The scale bars represent 20 mm.

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103

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Figure 4. At-CRY3 binds FAD. Escherichia coli-expressed At-cry3 was denatured by incubating at 658C for 15 min in the presence of 0.1 M HCl and 0.8% SDS. The supernatant with the released chromophore was used for spectroscopy. (a) Absorption spectrum. Line A, released chromophore; line B, absorption spectrum of standard FAD. (b) Fluorescence emission spectra. Excitation was at 450 nm. The emission spectra were taken at pH 4.0 and pH 8.0.

characterised so far: CRY1, CRY2, a class II CPD photolyase, and a (6-4) photolyase (for review, see Lin, 2002). However, in the Arabidopsis database (http://mips.gsf.de/proj/thal/ db/index.html), we found further members of this protein family. Among those, At5g24850 shows surprisingly high identity (50% over a stretch of 400 amino acids) with the Synechocystis cryptochrome sll1629 (Figure 1). This Arabidopsis protein was recently named Arabidopsis CRY DASH (Brudler et al., 2003) and is named At-cry3 in this paper. Database searches (http://www.inra.fr/servlets/WebPredotator; http://www.cbs.dtu.dk/services/TargetP) predicted a targeting signal at the N-terminus of At-CRY3 for import either in chloroplasts or in mitochondria. There are several examples of proteins in the literature, which are targeted to both organelles (for review, see Peeters and Small, 2001). We have functionally tested these predictions and found by in vitro import studies that At-cry3 is indeed transported

into chloroplasts (Figure 2). The imported protein was synthesised in vitro in the presence of FAD. However, for technical reasons, the incorporation of the chromophore could not be analysed in these experiments, and consequently, it remains an open question whether a chromophore is required for import. The import of At-cry3 into chloroplasts was con®rmed by confocal laser scanning analysis of GFP fusions expressed transiently in protoplasts prepared from green Arabidopsis cell cultures (Figure 3a±d). Using the same approach and staining of mitochondria with mitotracker, transport of the GFP fusion protein into mitochondria was also observed (Figure 3e). As deletion of the predicted N-terminal targeting signal abolished transport into both organelles, and on the contrary, the fusion of this signal peptide to GFP led to targeting into mitochondria and chloroplasts, we believe that the observed transport into both organelles is speci®c. Earlier studies on the

Figure 5. Arabidopsis cry3 has no photolyase activity. (a) Photolyase de®cient Escherichia coli KY1225 cells were transformed with the expression vector pQE-60 (circle) as control or with pQE-60 carrying the fulllength At-CRY3-coding region (triangle). As further control, photolyase-pro®cient wild-type KY1056 E. coli cells (square) were included. Cells were plated on solidi®ed LB medium and UV-B treated for times as indicated. One batch of plates was transferred to darkness immediately after the UV-B treatment (closed symbols); the other batch was given photoreactivating light for 60 min (open symbols). (b) Same as (a), but pQE-30 transformed KY1225 cells served as control (circle) and Arabidopsis photolyase AT-PHR1 cloned in vector pQE-30 (triangle) was expressed in KY1225 cells. Colonies were counted after 16-h incubation at 378C in darkness. Error bars represent SEM.



Figure 6. Arabidopsis cry3 binds DNA. At-cry3 synthesised in vitro in the presence of FAD was incubated with a 32P-labelled double-stranded oligonucleotide probe of random sequence and binding reactions separated on non-denaturing gels. Competitor DNAs were included in the binding reactions (lanes 2±9) in 5- and 50-fold excess either as single (ss) or double (ds) strands as indicated. The sequence of competitor DNAs was either identical to the probe (self) or unrelated. Unprogrammed reticulocyte lysate did not produce a shifted band with the chosen probe (not shown). Free, free probe; cry3, cry3-speci®c shifted band.

cellular localisation of cryptochromes in plants have shown that Arabidopsis cry2 is mostly nuclear (Guo et al., 1999; Kleiner et al., 1999a), whereas cry1 is mostly cytosolic in light-grown plants (Guo et al., 1999). Fern cryptochromes show either nuclear or cytosolic localisation (Imaizumi et al., 2000). However, the localisation of a cryptochromelike protein in chloroplasts and mitochondria has not been described before. This is in contrast to localisation studies on mouse cry1, which have shown that this protein is transported into the nucleus and the mitochondria (Kobayashi et al., 1998). As outlined above, the sequence of At-CRY3 does not allow prediction of either photolyase or cryptochrome function. The lack of a C-terminal extension in At-CRY3 is not a strong argument against cryptochrome function because one of the fern CRYs also lacks such an extension (Imaizumi et al., 2000). Class I CPD photolyases have been studied extensively, and several amino acids have been identi®ed, which are essential for either catalysis or cofactor activation (for review, see Sancar, 2000). The At-CRY3 protein contains several of these residues (e.g. Trp306, Trp359, Trp382; numbering according to positions in Saccharomyces cerevisiae photolyase). However, the presence of these `signature' residues also does not allow the prediction of photolyase function as indicated by sequence alignments of the photolyase/cryptochrome family members (Kobayashi et al., 2000; Yasui and Eker, 1998) and most ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103

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recently by solving the crystal structure of the Synechocystis cryptochrome (Brudler et al., 2003). Our data presented in Figure 5 argue strongly against photolyase function of At-cry3. Further evidence against photolyase function of At-cry3 is the observation that the protein is localised in chloroplasts. Photolyase activity could not be detected in chloroplasts of dicot plants (Hada et al., 2000). So far, we have no direct proof that At-cry3 is a cryptochrome-type photoreceptor. However, as a result of the high similarity with Synechocystis sll1629 for which cryptochrome function was shown (Brudler et al., 2003), the presence of an FAD chromophore (Figure 4) and the lack of photolyase activity (Figure 5), it is tempting to speculate that At-cry3 could well have photoreceptor function. This is currently being investigated further in our laboratory. Besides targeting to organelles, we have also shown that At-cry3 binds DNA (Figure 6). Interestingly, for mouse cry1 (Kobayashi et al., 1998) and Synechocystis cry (Brudler et al., 2003), DNA binding was also shown. This indicates that DNA binding is an important feature conserved in many cryptochromes from all kingdoms and provides insight into the molecular mechanism of cryptochrome signalling. Furthermore, binding of cryptochromes to DNA is not sequence speci®c as shown for At-cry3 (Figure 6), Synechocystis CRY-DASH (Brudler et al., 2003) and mouse cry1 (Kobayashi et al., 1998). Photolyases, together with their high af®nity to UV-B photoproducts, also have lower af®nity to non-damaged DNA that is sequence unspeci®c (Sancar, 2000). It seems that this latter feature of DNA photolyases is conserved in the related cryptochromes. Cryptochromes have been found in plants, animals, human beings (for reviews, see Deisenhofer, 2000; Lin, 2002) and cyanobacteria (Brudler et al., 2003). Although the role of CRYs as photoreceptors in vertebrates is still under debate (for review, see Van Gelder, 2002), cumulative evidence obtained from studies in plants and Drosophila suggest such a role (for reviews, see Devlin and Kay, 1999; Lin, 2002). Sequence alignments have shown that the plant CRYs are more closely related to the class I CPD photolyases, whereas the animal CRYs group closer to the (6-4) photolyases. This has led to the suggestion that plant and animal CRYs may have evolved independently (Cashmore et al., 1999; Kobayashi et al., 2000). In addition, Kanai et al. (1997) concluded from sequence comparisons of photolyases and cryptochromes that the CRYs might have diverged from photolyases before the appearance of eukaryotes. A similar conclusion was made from the discovery of a novel cryptochrome class (CRY DASH) having members in Drosophila, Arabidopsis (At-CRY3), Synechocystis and human beings (Brudler et al., 2003). Alternatively, plants could have received their cryptochromes by horizontal gene transfer from a cyanobacterium-type endosymbiont, which gave rise to the chloroplasts. This was suggested,

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Figure 7. Unrooted weighted phylogenetic tree for sequences from the photolyase/cryptochrome family. Tree building details have been provided in the text. Branches shown with bold lines were supported by >95% bootstrap values. The grouping of plant CRY proteins and a-proteobacteria (Mesorhizobium, Agrobacterium, Caulobacter) received 63% non-parametric bootstrap support. The alternative grouping of plant CRY proteins of the CRY1/CRY2 family with At-CRY3 and sll1629 received 0% support. Arabidopsis CRY1, CRY2 and CRY3 are indicated in bold letters.



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for example, for the phytochrome photoreceptors, as related photoreceptors were discovered in cyanobacteria such as Synechocystis (Hughes et al., 1997; Yeh et al., 1997). As outlined above, Arabidopsis CRY1 and CRY2 have only about 20% identity with Synechocystis cryptochrome. A phylogenetic tree, which includes photolyase and cryptochrome sequences from many species of all kingdoms (Figure 7), clearly shows this distance. Furthermore, our analyses provide no evidence for grouping CRY1 and CRY2 with Synechocystis cryptochrome, in contrast to At-CRY3. It is thus unlikely that CRY1 and CRY2 have evolved from At-CRY3 or from a cyanobacterial progenitor. Our phylogeny (Figure 7) suggests that the closest relatives of the plant CRY1 and CRY2 protein family are sequences from the a-proteobacteria. As a-proteobacteria are considered to be progenitors of mitochondria (Gray et al., 2001), it is tempting to speculate that plant nuclear genomes have received their cryptochromes through horizontal gene transfer by two independent events, one class (CRY1 and CRY2 ) from an endosymbiotic a-proteobacteria-like ancestor and one class (CRY3 ) from an endosymbiotic cyanobacteria-like ancestor.

In vitro import assays were performed in a volume of 400 ml, with chloroplasts (equivalent to 50 mg chlorophyll) re-suspended in buffer containing 2 mM ATP, 20 mM potassium gluconate, 5 mM methionine, 20 mM NaHCO3, 2% bovine serum albumin, 330 mM sorbitol, 50 mM Hepes/KOH, pH 7.6 and 3 mM MgCl2. After addition of labelled in vitro translation product, the import mixture was incubated for 30 min at 258C. The reaction was terminated by centrifugation (1 min, 1000 g, 108C), and re-suspension of the chloroplasts in 300 ml of the washing buffer (330 mM sorbitol, 50 mM Hepes/KOH, pH 7.6, 3 mM MgCl2). The suspension was divided into three 100-ml aliquots. The ®rst one was incubated with 1 mM ®nal concentration CaCl2 for 10 min on ice, the second one was subjected to thermolysin treatment (0.15 mg ml 1 thermolysin, Roche Applied Sciences, Mannheim, Germany) for 10 min on ice, and the third one was incubated in 0.2% (v/v) Triton X-100 for 5 min at 258C prior to thermolysin treatment. Afterwards, EDTA was added to a ®nal concentration of 10 mM. The ®rst two reactions were centrifuged as above, washed in 300 ml washing buffer, and the pellets were re-suspended in SDS sample buffer. The sample treated with Triton was precipitated with 4 volumes of acetone, centrifuged (15 min, 20 000 g, 48C), dried and then re-suspended in SDS sample buffer. The samples were separated on 9% SDS-polyacrylamide gels (Laemmli, 1970). After electrophoresis, the gels were incubated in an ampli®cation ¯uorographic reagent (Amersham Biosciences, Freiburg, Germany), dried and exposed on X-ray ®lms (Amersham Biosciences, Freiburg, Germany) for 2 days at 708C.

Experimental procedures

In vivo photolyase assays

DNA manipulations Polymerase chain reactions and other routine DNA manipulations were performed as described in Current Protocols in Molecular Biology (John Wiley and Sons, Inc., New York, USA). All PCRampli®ed fragments were veri®ed by sequencing.

Construction of GFP and 6xHis fusions The cDNA sequence of A. thaliana At-CRY3 was obtained from the MIPS A. thaliana database (http://mips.gsf.de/proj/thal/db/index. html). The sequences corresponding to the full length, the ®rst 63 amino acids and an N-terminal deletion construct (amino acids 40±569) of At-CRY3 were PCR-ampli®ed from A. thaliana ecotype Landsberg erecta (Ler). cDNA was produced using pairs of primers as follows: At-CRY3: #1 50 -CCATGGCGGCTTCCTCTCTCTC-30 , #2 50 -CCATGGCAGGACCATTGTGTCTAGAAC-30 ; AtCRY3(40±569): #3 50 -CCATGGGATCCGCCGCAAAAATGAACGA-30 , #2; and At-CRY3(1±63): #1, #4 50 -CCATGGCGGCGACGGAGTCAATCTCTT-30 . The ampli®ed fragments were cut with NcoI and cloned in-frame into the NcoI site of the GFP expression vector pAVA393 (Von Arnim et al., 1998) or into the 6xHis expression vector pQE-60 (Qiagen, Hilden, Germany), respectively.

In vitro import experiments Chloroplasts were isolated from Pisum sativum plants and puri®ed by density gradient centrifugation according to Bonk et al. (1997). In vitro transcription and translation were performed using the coupled reticulocyte transcription/translation system (TNT system, Promega, Mannheim, Germany) in the presence of 0.3 mM FAD and [35S]methionine (1000 Ci mmol 1, Hartmann Analytic, Braunschweig, Germany) according to the manufacturer's protocol. ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 93±103

Escherichia coli strain KY1225 (phr±) cells (Akasaka and Yamamoto, 1991) were transformed with pQE-60, pQE-60/AtCRY3 or pQE-30/At-PHR1, encoding A. thaliana CPD photolyase (Kleiner et al., 1999b). Overnight cultures of E. coli KY1056 (phr) and transformed KY1225 were diluted 1 : 20, grown to an OD600 of 0.6 at 378C and protein expression induced by adding isopropylthio-b-D-galactoside (IPTG) to a ®nal concentration of 1 mM. Cells were grown for another 3.5 h at 378C. The cells were placed on ice, and different dilutions of the cells were spread on LB agar plates. The cells were irradiated with UV-B (1 Philips TL40/W12 lamp; distance 53 cm, Philips Lighting B.V., Eindhoven, The Netherlands) for the indicated times. Half of the plates were immediately transferred to darkness after UV-B treatment; the other half were given photoreactivating white light (photon ¯uence rate 50 mmol m 2 sec 1) for 60 min. Control cells were not UV-B treated and transferred to darkness directly after plating. Colonies were counted after overnight incubation at 378C.

Transformation of Arabidopsis protoplasts Protoplasts were prepared from an Arabidopsis Ler mesophyll cell culture and transformed essentially as described (Altmann et al., 1992). In brief, protoplasts were transformed chemically with 50 mg of the respective plasmid DNAs and incubated in the dark for 24 h before confocal microscope analysis.

GFP, chlorophyll and mitotracker visualisation Arabidopsis protoplasts were examined with a Leica TCS SP2 confocal laser scanning microscope using an HCX PL APO 40x/ 1.25±0.75 oil CS objective. Staining of mitochondria was performed by incubating protoplasts for 30 min in mitotracker solution (5 nM CMTM Ros (Molecular Probes, Leiden, The Netherlands), in 0.5 M mannitol, 15 mM NaCl, 12.5 mM CaCl2, 0.5 mM KCl, 0.5 mM

102 Tatjana Kleine et al. glucose, pH 5.8) directly before microscopic analysis. GFP and chlorophyll were excited at 488 nm, mitotracker at 543 nm, and the resulting ¯uorescence was ®ltered using a beam splitter (TD 488/543/633 for GFP and chlorophyll ¯uorescence; DD 488/543 for GFP and mitotracker ¯uorescence) and detected by two different photomultiplier tubes with a bandwidth of 500±520 and 625± 720 nm for GFP and chlorophyll ¯uorescence, respectively, or 500±520 and 565±585 nm for GFP and mitotracker ¯uorescence, respectively. Images were captured with the Leica Confocal Software and processed with Adobe Photoshop 5.5.

Expression of At-cry3 in E. coli and characterisation of the chromophore Escherichia coli M15[pREP4] cells harbouring At-CRY3(40±569) in vector pQE-60 were grown in 50 ml of LB medium containing 100 mg ml 1 ampicillin and 40 mg ml 1 kanamycin at 378C overnight. The overnight culture was used to inoculate 1 l of the same medium. Cells were grown at 378C to A600 0.6, and induced with 1 mM IPTG. After induction, cells were grown for another 4 h, and harvested by centrifugation at 5000 g for 20 min. The pellet was resuspended in 25 ml of the lysis buffer (50 mM NaPi, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM EDTA, 1 mg ml 1 lysozyme), incubated at 48C for 60 min, and sonicated thereafter for 12 30 sec. After removing the cell debris by centrifugation (60 min, 48 000 g, 48C), the supernatant was incubated with 1 ml of Ni2-nitrilotriacetic acid agarose (Qiagen, Hilden, Germany) for 90 min on ice with continuous shaking. The slurry was transferred to a chromatography column and washed with 50 ml of the lysis buffer (without lysozyme). The At-cry3 protein was eluted with 2.5 ml of the elution buffer (20 mM Tris±HCl, pH 7.6, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, 250 mM imidazole). The samples were concentrated with centriprep ®lter devices (Amicon, Beverly, MA, USA) according to the manufacturer's instructions, and stored in the elution buffer without imidazole. All steps were carried out using a red safe light. SDS±PAGE con®rmed that the eluted protein was essentially pure. For the identi®cation of the chromophore, the chromophore was released from the protein by incubation of At-cry3 (in the elution buffer) at 658C for 15 min in the presence of 0.1 M HCl. Absorption spectra were recorded with a UV-3000 spectrophotometer (Shimadzu, Kyoto, Japan), and ¯uorescence emission spectra with a RF-540 spectro¯uorometer (Shimadzu, Kyoto, Japan).

Gel mobility assays Twenty picomoles of a 57-base oligonucleotide with random sequence was radiolabelled with 32P-gATP (Hartmann Analytic, Braunschweig, Germany) and T4 polynucleotide kinase (MBI-Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. After labelling and puri®cation, the oligonucleotide was annealed with its complementary strand and used in gel mobility assays. Gel mobility assays were performed in 25-ml reactions (50 mM Tris±HCl, 50 mM NaCl, 5% glycerol, 0.1 mM EDTA, 1 mM DTT, pH 7.5) containing 4 ml of TNT system either unprogrammed or programmed with the At-CRY3 construct, 0.02 pmol 32P-labelled double-stranded probe (80 000 c.p.m.), and competitor DNAs as indicated. Reactions were incubated at 258C for 30 min, followed by non-denaturing electrophoresis in 5% polyacrylamid gels in Tris base, boric acid, EDTA (TBE) buffer (22 mM Tris, 22 mM boric acid, 0.5 mM EDTA) at 10 V cm 1. After electrophoresis, gels were vacuum-dried and exposed to X-ray ®lms between intensifying screens at 708C.

Phylogenetic analyses Sequences were retrieved from GenBank and aligned using CLUS(the alignment ®le used is available from the authors by request). A data block of 260 homologous positions containing no gaps was used for tree building under minimum evolution as implemented in PAUP (Swofford, 2001). Distances were not corrected under symmetric substitution models, as their assumptions would be inappropriate for these multigene data. Mean character difference values were used. Heuristic search settings allowed negative branch lengths, but were set to zero for tree-score calculation. The starting tree(s) for global optimisation was ®rst obtained via neighbour joining. Tree bisection reconnection (TBR) re-arrangements were used to improve the global tree score. Non-parametric bootstrapping was used with 1000 replicates being made.

TALX

Acknowledgements We thank Oxana Panajotowa and Agnes Debelius for excellent technical assistance, Dr Uwe Maier and Julia Prechtl (University of Marburg, Germany) for help with the in vitro import studies, Dr Csaba Koncz (MPI fuÈr ZuÈchtungsforschung, Cologne, Germany) for the green Arabidopsis cell culture, and Dr Franz Grolig and Markus MuÈller (University of Marburg, Germany) for help in confocal studies. This research was funded in part by a grant from the Deutsche Forschungsgemeinschaft to A.B. (BA985/7±2) and by the Optodynamic Centre, University of Marburg. Peter Lockhart thanks the Alexander von Humboldt Foundation and the New Zealand Marsden Fund for his ®nancial support.

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