Nuclear localization of the Arabidopsis blue light receptor ...

The Plant Journal (1999) 19(3), 289±296 

Nuclear localization of the Arabidopsis blue light receptor 

cryptochrome 2 

Oliver Kleiner 1 , Stefan Kircher 2 , Klaus Harter 2 and 

Alfred Batschauer 1, * 

1 

FB Biologie/Botanik, Philipps-UniversitaÈt, Karl-von-Frisch 

Str., 35032 Marburg, Germany, and 

2 

Biologisches Institut II, Albert-Ludwigs-UniversitaÈt, 

SchaÈnzlestr. 1, 79104 Freiburg, Germany 

Summary 

The cryptochrome blue light photoreceptor family of 

Arabidopsis thaliana consists of two members, CRY1 

and CRY2 (PHH1). CRY2 contains a putative nuclear 

localization signal (NLS) within its C-terminal region. We 

examined whether CRY2 is localized in the nucleus and 

whether the C-terminal region of CRY2 is involved in 

nuclear targeting. Total cellular and nuclear protein 

extracts from Arabidopsis were subjected to 

immunoblot analysis with CRY2-speci®c antibodies. 

Strong CRY2 signals were obtained in the nuclear 

fraction. Fusion proteins consisting of the green 

¯uorescent protein (GFP) and different fragments of 

CRY2 were expressed in parsley protoplasts and the 

localization of the fusion proteins was determined by 

¯uorescence and confocal laser scanning microscopy. 

GFP-fusions containing the entire CRY2 protein or its Cterminal 

region were found exclusively in the nucleus. 

We conclude from these results that CRY2 is localized in 

the nucleus and that nuclear localization is mediated by 

the C-terminal region of CRY2. 

Introduction 

Plants have at least three different photoreceptor systems 

which detect different light qualities. The phytochromes, 

which sense mainly the red and far-red region of the 

spectrum, have already been analyzed in detail (reviewed 

in Batschauer, 1998; Furuya and SchaÈ fer, 1996). The 

molecular characterization of photoreceptors sensing the 

blue and UV-A spectrum started recently. The photomorphogenic 

Arabidopsis mutant hy4 is affected in blue lightregulated 

extension growth of the hypocotyl and some 

other blue light responses (Ahmad and Cashmore, 1993; 

Ahmad et al., 1995; Bagnall et al., 1996; Koornneef et al., 

1980). Ahmad and Cashmore (1993) isolated the HY4 gene, 

which encodes a blue light receptor named CRY1 (crypto- 

Received 23 April 1999; revised 23 June 1999; accepted 23 June 1999. 

*For correspondence (fax +49 6421 281545; 

e-mail batschau@mailer.uni-marburg.de). 

chrome 1) (Lin et al., 1995). The predicted polypeptide of 

CRY1 consists of 681 amino acids with 30% sequence 

identity to class I DNA photolyases (Ahmad and Cashmore, 

1993). Class I DNA-photolyases repair cyclobutane pyrimidine 

dimers by using light energy in the UV-A/blue region 

(for review see Sancar, 1994). CRY1 and the SA-PHR1 

protein from white mustard (Batschauer, 1993), which is 

89% identical with Arabidopsis CRY2, bind the chromophores 

typically present in photolyases, but have no 

photolyase activity (Lin et al., 1995; Malhotra et al., 1995). 

The hy4 mutant is only affected in some blue light 

responses (Ahmad and Cashmore, 1993; Ahmad et al., 

1995; Bagnall et al., 1996; Koornneef et al., 1980), indicating 

the presence of further blue light receptors in Arabidopsis. 

For example, there is strong evidence that a plasma 

membrane associated protein encoded by the NPH1 gene 

is the photoreceptor for phototropism (Christie et al., 1998; 

Huala et al., 1997). 

A second gene related to class I photolyases was 

isolated from Arabidopsis, which was named AT-PHH1 

(Hoffman et al., 1996), or CRY2 (Lin et al., 1996a). This gene 

encodes a protein of 612 amino acids which is 54% 

identical with CRY1 within the N-terminal 500 amino acids. 

CRY2 does not code for a photolyase (Hoffman et al., 1996). 

As for CRY1, mutant studies enabled identi®cation of the 

function of CRY2. The late ¯owering Arabidopsis mutant 

fha-1 (Koornneef et al., 1991) is defective in the CRY2 gene 

(Guo et al., 1998), indicating that CRY2 is a blue light 

photoreceptor which is involved in measuring the daylength. 

Recently, genes were identi®ed in humans and 

mice which are related to Arabidopsis CRY1 and CRY2. The 

genes were named hCry1, hCry2 (human) and mCry1, 

mCry2 (mouse), respectively (Hsu et al., 1996; Kobayashi 

et al., 1998). Mice strains lacking the Cry2 gene had a 

reduced photic sensitivity and increased phase shifts in 

response to light, indicating that mCry2 has a role in 

circadian photoreception (Thresher et al., 1998). In addition, 

mCry2 deletion led to a lengthening of the freerunning 

period in constant darkness, whereas mice Cry1 

mutants had an accelerated free-running periodicity of 

locomoter activity. In the double mutant, the free-running 

rhythmicity was completely lost, indicating that the 

cryptochromes in mice may directly interact with components 

of the circadian clock (Van der Horst et al., 1999). In 

Drosophila, a cryptochrome-related gene was identi®ed 

which is also involved in resetting the circadian rhythm 

(Emery et al., 1998; Stanewsky et al., 1998). 

The two cryptochromes from mice and humans are 

localized in different cellular compartments. mCry1 was 

ã 1999 Blackwell Science Ltd 289

290 Oliver Kleiner et al. 

found in the mitochondria, whereas most of the mCry2 

was located in the nucleus (Kobayashi et al., 1998). In line 

with the immuno-localization of mCry2, an hCry2±GFP 

fusion protein was targeted to the nucleus (Thresher et al., 

1998). Arabidopsis CRY1 was found to be a soluble protein 

(Lin et al., 1996b), but was also found to be enriched in the 

membrane fraction (Ahmad et al., 1998a). Very recently it 

was shown that a fusion protein consisting of Arabidopsis 

CRY1 and GFP localizes to the nucleus on transient 

expression in onion epidermal cells, indicating that 

Arabidopsis CRY1 is a nuclear protein (Cashmore et al., 

1999). 

Here we demonstrate, by the use of antibodies speci®c 

for CRY2, that Arabidopsis CRY2 is localized in the nucleus, 

as is mCry2 and hCry2. Thus, nuclear localization of CRY2 

is evolutionarily conserved in plants and animals. In order 

to identify the domain responsible for nuclear targeting, 

GFP was fused with de®ned regions of CRY2. The Cterminal 

extension of CRY2, which contains one bipartite 

NLS, is suf®cient for nuclear targeting. 

Results 

Antibodies speci®c for CRY2 

Arabidopsis CRY1 and CRY2 are well conserved within 

the N-terminal region (amino acid positions 1±500), 

sharing 54% identity. In contrast, the C-terminal regions 

of CRY1 and CRY2 differ in length and in sequence, 

except for a short serine-rich stretch (Hoffman et al., 

1996). In Escherichia coli cells we expressed the Cterminal 

region (amino acid position 501±612) of CRY2 

as a His±Taq fusion protein and puri®ed the protein by 

Ni-NTA chromatography. The puri®ed protein was used 

to raise antibodies in rabbits (see Experimental procedures). 

The antiserum was tested for speci®c recognition 

of CRY2 in protein gel blot analysis. Total protein 

extracts were isolated from dark-adapted Arabidopsis 

wild-type (Ler), the cry2 mutant fha-1, and blue lighttreated 

wild-type (Ler) seedlings. fha-1 has a stop codon 

in the CRY2 gene and does not accumulate CRY2 

protein (Guo et al., 1998). Blue light treatment leads to 

a fast and strong decrease in the CRY2 protein level 

(Ahmad et al., 1998b; Guo et al., 1998; Lin et al., 1998), 

whereas CRY1 is light-stable (Lin et al., 1996b). Thus, 

one would expect that the CRY2-speci®c antiserum 

should only give a signal with extracts of dark-adapted 

wild-type seedlings and not with blue light-treated 

seedlings and the fha-1 mutant. Exactly this result was 

observed (Figure 1), demonstrating that the antiserum is 

speci®c for CRY2. The protein detected by the anti-CRY2 

antiserum has an apparent molecular mass in the range 

of 75±80 kDa, which is somewhat higher than the 

calculated molecular mass of CRY2 (69.9 kDa). 

Figure 1. Characterization of antibodies speci®c for Arabidopsis CRY2. 

Protein gel blot analysis performed with total protein extracts from 6days-old 

Arabidopsis seedlings which have either been dark adapted for 

72 h (dark) or kept in blue light for 24 h (blue) before harvest. Dark 

adapted Arabidopsis Landsberg erecta wild-type (Ler dark), blue lighttreated 

Landsberg erecta wild-type (Ler blue), dark adapted fha-1 mutant 

(fha-1 dark). Per lane 50 mg of protein were loaded and probed with the 

antiserum raised against the C-terminal region of CRY2 (a). The blot was 

stripped and reprobed with antibodies against DnaK (b) as a control for 

equal loading and transfer. The arrow indicates the CRY2 signal. 

Molecular masses of standard proteins are indicated. 

Nuclear localization of CRY2 

As outlined above, Arabidopsis CRY1 and CRY2 share high 

similarity in the N-terminal region between amino acids 1± 

500. In contrast, the C-terminal regions of CRY1 and CRY2 

are not well conserved. By database searching we 

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 289±296

Figure 2. Putative nuclear localization signal of CRY2. 

The amino acid sequence of Arabidopsis thaliana CRY2 (positions 541± 

558) is shown in an alignment with the bipartite NLS of nuclear localized 

proteins. Basic amino acids are shown in bold. The bipartite motif is 

boxed in. 

identi®ed in the C-terminal region of CRY2 a nuclear 

localization sequence (NLS; amino acid positions 541±558), 

which belongs to the group of bipartite NLS (Figure 2). The 

CRY2 NLS is very similar to NLS of proteins, for which 

nuclear localization has already been demonstrated 

(GoÈ rlich and Mattaj, 1996; Kobayashi et al., 1998; 

Thresher et al., 1998). 

Due to the presence of an NLS in CRY2 we reasoned that 

CRY2 could be located in the nucleus. Therefore, we tested 

the cellular localization of CRY2 by immunological methods. 

Total cellular and nuclear protein extracts were 

isolated from Arabidopsis seedlings which were darkadapted 

for 72 h (see Experimental procedures). Fifty 

micrograms of total cellular proteins and 5 mg of nuclear 

proteins were separated by SDS-PAGE and probed in 

protein gel blot analysis. The purity of the nuclear fraction 

was tested with antibodies against nuclear (histone), 

cytosolic (HSC70) and mitochondrial (DnaK) proteins. The 

nuclear fraction gave signals with the anti-histone antibody 

but no signals with the anti-HSC70 and anti-DnaK 

antibodies (Figure 3), demonstrating that the nuclear 

fraction was not contaminated by proteins from other 

cellular compartments. Protein gel blot analysis using the 

CRY2-speci®c antibody gave a single signal with the total 

cellular protein extract (Figure 3). In the nuclear fraction we 

also detected a single band with the CRY2-speci®c antibody. 

This band was more diffuse than in the total cellular 

extract and of a slightly higher mobility. We assume that 

this was caused by some degradation of CRY2 during the 

isolation of nuclei, although protease inhibitors were 

present during the whole procedure. The intensity of the 

signal in the nuclear fraction was at least the same as in 

the total cellular protein fraction, though only one-tenth of 

the amount of protein was loaded (Figure 3). We conclude 

from these data that CRY2 is located in the nucleus. 

ã Blackwell Science Ltd, The Plant Journal, (1999), 19, 289±296 

Nuclear localization of CRY2 291 

Figure 3. Immunoblot detection of CRY2 in nuclear extracts. 

Fifty micrograms of total cellular (total) and 5 mg of nuclear (nuclear) 

protein extract were analyzed by immunoblotting with antibodies against 

CRY2 (Anti-CRY2). The blot was stripped and reprobed with antibodies 

against histone H2A/H2B (Anti-H2A/2B), HSC70 (Anti-HSC70) and DnaK 

(Anti-DnaK) as controls for nuclear, cytosolic and mitochondrial proteins, 

respectively. 

The C-terminal domain of CRY2 mediates nuclear 

localization 

The data obtained with CRY2-speci®c antibodies demonstrated 

that CRY2 is localized in the nucleus. In order to 

de®ne the region of CRY2 which is responsible for nuclear 

targeting, we constructed fusion proteins of the green 

¯uorescent protein (GFP) and three different regions of 

CRY2 (Figure 4). The gene constructs were introduced into 

parsley protoplasts, expressed, and the localization of the 

chimeric GFP-proteins was studied by ¯uorescence and 

confocal laser scanning microscopy. The full-length CRY2± 

GFP fusion protein was found exclusively in the nucleus 

(Figure 4), con®rming the results obtained by protein gel 

blot analysis. In the case where the putative NLS located in 

CRY2 between amino acid positions 541 and 558 (Figure 2) 

is functional in nuclear targeting, one would expect that 

the fusion protein consisting of GFP and the C-terminal 

region of CRY2 would be located in the nucleus, and this 

was indeed observed (Figure 4). Since the fusion protein of 

GFP with the N-terminal region of CRY2 was detected in 

the cytoplasm (Figure 4), we conclude that the C-terminal 

region is necessary and suf®cient for nuclear targeting of 

CRY2.


Discussion 

We have analyzed the cellular localization of CRY2 by two 

different approaches. First, CRY2-speci®c antibodies gave 

signals only with one protein of the expected size in the 

nuclear fraction. Second, the CRY2-GFP fusion protein was 

found exclusively in the nucleus. Therefore, Arabidopsis 

CRY2 is obviously a nuclear protein. 

Figure 4. Localization of CRY2-GFP fusion 

proteins in parsley protoplasts. 

Shown are epi¯uorescence (left), light 

microscopical images (middle) and confocal 

sections (right) of parsley protoplasts 

transiently expressing fusion proteins of 

mGFP4 with full-length CRY2 (CRY2(1±612)/ 

GFP), the N-terminal part of CRY2 (CRY2(1± 

500)/GFP) or the C-terminal part of CRY2 

(CRY2(501±612)/GFP). After transformation 

by electroporation the protoplasts were kept 

for 24 h in darkness. The left and middle 

panels show identical protoplasts. The GFP 

¯uorescence of confocal sections is 

presented in red. Arrows indicate the 

position of nuclei. 

The GFP approach also enabled us to test which part of 

CRY2 mediates nuclear targeting. Since the fusion protein 

of GFP with the C-terminal region of CRY2 was found 

exclusively in the nucleus, we assume that the bipartite 

NLS in the C-terminal region is responsible for the nuclear 

targeting of CRY2. The chromophore-binding domain is 

lacking in the C-terminal CRY2-GFP fusion protein. We 


conclude therefore that there is no need for the presence of 

chromophores for the nuclear import of CRY2. 

Furthermore, import occurs in darkness and seems not 

to be controlled by light. 

Genes were identi®ed in humans and mice, which are 

related in sequence to Arabidopsis CRY1 and CRY2 (Hsu 

et al., 1996; Kobayashi et al., 1998; Todo et al., 1996; Van 

der Spek et al., 1996). None of these genes encode 

photolyase and it was suggested that they could have a 

blue light receptor function (Hsu et al., 1996). Mice mutated 

in Cry2 showed an increased phase shift in circadian 

rhythm in response to light pulses. In addition, the freerunning 

period was extended, indicating that mCry2 is a 

photoreceptor which entrains the endogenous oscillator 

and interacts with components of the circadian clock 

(Thresher et al., 1998). The Arabidopsis cry2 mutant is 

delayed in ¯owering under long-day conditions compared 

to the wild type (Guo et al., 1998). Therefore, Arabidopsis 

CRY2 seems to have a similar function in the entrainment 

of the circadian clock as the animal cryptochromes. The 

fact that related proteins, which have blue light photoreceptor 

function, synchronize circadian rhythms in organisms 

as distantly related as plants and mammals is very 

interesting from an evolutionary point of view. 

Furthermore, results presented here which demonstrate 

that Arabidopsis CRY2 is localized within the nucleus as is 

Cry2 in mice and humans (Kobayashi et al., 1998; Thresher 

et al., 1998) may well be very important for the further 

understanding of the molecular mechanism by which the 

light signal is transduced to the clock. Studies on the 

circadian rhythm of cab gene transcription in photoreceptor 

mutants demonstrate that besides cryptochromes, 

phytochromes also entrain the circadian clock (Somers 

et al., 1998). At least one member of the phytochrome 

family (phytochrome B) is also nuclear localized 

(Sakamoto and Nagatani, 1996) and the accumulation of 

phyB in the nucleus is induced by red light (Yamaguchi 

et al., 1999). Phytochrome B seems to have an antagonistic 

role to CRY2 in ¯ower induction. Whereas the cry2 mutant 

is delayed in ¯owering, the phyB mutant ¯owers earlier 

(Guo et al., 1998). 

How the different photoreceptors act together is still 

poorly understood. Recently, direct molecular interaction 

between PhyA and CRY1 in the yeast two-hybrid system 

has been demonstrated (Ahmad et al., 1998c). If CRY2 

would physically interact with another photoreceptor, 

PhyB would be a good candidate since both photoreceptors 

are located in the nucleus. However, phyB is lightstable 

and operates under low and high intensity (red) 

light conditions (for review see Batschauer, 1998; Furuya 

and SchaÈ fer, 1996), whereas CRY2 disappears under high 

intensity (blue) light conditions (Ahmad et al., 1998b; Lin 

et al., 1998). CRY1 is light-stable (Ahmad et al., 1998b; Lin 

et al., 1996b), whereas PhyA rapidly disappears under low 


and high intensity (red) light conditions (for review see 

Batschauer, 1998; Furuya and SchaÈ fer, 1996). Models for 

the molecular interaction of photoreceptors must therefore 

consider the effects of light on photoreceptor stability as 

well as on the cellular localization of the photoreceptors. 

The nuclear localization of CRY2 which we presented here 

may facilitate the identi®cation of interacting partners by 

focussing on those which are located in the nucleus. 

Experimental procedures 

Plant material, growth conditions and light treatment 

Arabidopsis thaliana ecotype Landsberg erecta (Ler) and the fha-1 

mutant (Koornneef et al., 1991), which has Ler background, were 

used. Plants were grown on four layers of Whatman paper soaked 

with distilled water in plastic containers. Germination was 

induced by a 24 h incubation at 4°C followed by white light 

treatment for 1 h. Plants were kept at 25°C for 72 h in a 16 h/8 h 

light/dark cycle (light intensity 250 mmol m ±1 s ±1 ) in a growth 

chamber and dark-adapted afterwards for 72 h. In some experiments 

plant were treated with blue light (¯uence rate 4.1 W m ±2 ) 

for 24 h before harvest, as described previously (Kaiser et al., 

1995). All manipulations were carried out in orange-safe light 

(Osram L36 W/62). 

Isolation of total cellular proteins 

Seedlings were quick-frozen and ground in liquid nitrogen. One 

hundred microlitres of SDS sample buffer (62.5 mM Tris±HCl 

pH 6.8, 4 M urea, 0.7 M b-mercaptoethanol, 2% (w/v) SDS, 0.002% 

(w/v) bromphenol blue, 10% glycerol) were added per mg of plant 

material. Samples were boiled for 10 min and clari®ed by 

centrifugation (20 000 g for 10 min). 

Isolation of nuclear proteins 


Twenty grams of the aerial part from Arabidopsis seedlings, 

which were dark-adapted for 72 h, were used for the preparation 

of nuclei according to a published procedure (Willmitzer and 

Wagner, 1981). During the whole procedure, the following 

protease inhibitors were present: AEBSF (1 mM), bestatin 

(10 mM), pepstatin A (1 mM), E-64 (10 mM), leupetin (100 mM), 1,10phenanthroline 

(100 mM). Nuclei were resuspended in SDSsample 

buffer, boiled for 10 min and clari®ed by centrifugation 

(20 000 g for 10 min). 

Transient transformation of protoplasts by 

electroporation 

For transient expression of GFP-constructs, protoplasts were 

prepared from a dark-grown parsley (Petroselinum crispum) cell 

culture 6 days after subculturing according to a published 

procedure (Dangl et al., 1987). Electroporation of protoplasts 

was performed in 0.4 cm cuvettes at 320 V and 125 mF (W®`, 

pulse time: 10 ms) using a Gene Pulser II (Biorad, Munich, 

Germany) at room temperature as described previously (Kircher 

et al., 1999). After electroporation, protoplasts were diluted in 5 ml 

HA-medium (Dangl et al., 1987) containing 0.4 M sucrose, transferred 

to Petri dishes and kept in darkness for 24 h.


Light, epi¯uorescence and confocal scanning microscopy 

For light and epi¯uorescence microscopy, 80 ml of protoplast 

suspensions were transferred to a glass slide and analyzed in an 

Axiovert microscope (Zeiss, Oberkochen, Germany). Excitation of 

GFP was performed with standard FITC ®lters (Kircher et al., 1999). 

Documentation was done by photography with an automatic 

Contax 167 MT camera on Kodak 64T ®lm. Confocal laser 

scanning microscopy (DM RBE, Leica, Bensheim, Germany) was 

performed as described by Kircher et al. (1999). 

Expression of His±taq fusion protein and the production 

of a CRY2-speci®c antibody 

A63His±Taq fusion of the C-terminal region (amino acid 501±612) 

of CRY2 in the vector pQE-30 (Quiagen, Hilden, Germany) was 

expressed in E. coli M15[pREP4] cells (Quiagen, Hilden, Germany) 

at 37°C after induction with 1 mM IPTG. The fusion protein was 

puri®ed with an Ni-NTA column according to the manufacturer's 

protocol under denaturing conditions (Quiagen, Hilden, 

Germany). One milligram of the puri®ed protein was used for 

raising antiserum in a rabbit. The immunization was performed 

by Eurogentec (Searing, Belgium). The speci®city of the antiserum 

was tested using E. coli expressed CRY2. The limit of 

detection in gel blot analysis was below 10 ng of protein (data 

not shown). 

SDS-PAGE and gel blot analysis 

Proteins were fractionated on 6% SDS-PAGE (SchaÈ gger and von 

Jagow, 1987) and blotted on PVDF membranes (Macherey-Nagel, 

DuÈ ren, Germany). The blot was blocked with 7% non-fat dry milk 

in TBST (20 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) and 

incubated with anti-CRY2 antiserum (1 : 1000 dilution). The blot 

was then incubated with the secondary antibody (donkey antirabbit 

IgG; Amersham-Pharmacia, Freiburg, Germany) conjugated 

with horseradish peroxidase (1: 10 000 dilution). After each 

incubation step with antibodies the blot was washed with TBST. 

Immunodetection was undertaken with the ECL-plus system 

(Amersham-Pharmacia, Freiburg, Germany) according to the 

manufacturer's instructions. The same procedure was used for 

antibodies against (cytosolic) HSC70 (Neumann et al., 1989), 

histone H2A/ H2B (Mazzolini et al., 1989) and DnaK (Neumann 

et al., 1989). The anti-HSC70 antiserum was diluted 1 : 5000, the 

anti-histone antiserum 1 : 1000 and the anti-DnaK antiserum 

1 : 1000. The molecular weight marker was SDS-7B (Sigma, 

Deisenhofen, Germany). Protein concentration was determined 

according to Popov et al. (1975). 

Gene construction 

Construct for E. coli expression. The region of CRY2, encoding 

amino acids 501±612 (CRY2(501±612)), was ampli®ed by PCR 

using the cDNA clone PHH1 (Hoffman et al., 1996) as template. 

PCR was performed with a proof-reading polymerase (Vent R q - 

polymerase, New England Biolabs, Schwalbach, Germany). The 

following primer combination was used: 5¢-GAGCTCATTGT- 

AGCAGATAGCTTCGAGGCC-3¢ (upstream primer), 5¢-GTCGAC- 

TTGGCAACCATTTTTTCCCAAACTTGT-3¢ (downstream primer). 

Restriction sites for SacI and SalI were introduced into the 

upstream and downstream primer, respectively (underlined in 

the primer sequences). PCR products were subcloned into the 

pCR q II-TOPO vector (Invitrogen, De Schelp, the Netherlands) after 

the addition of 3¢A-overhangs with Taq-polymerase post-ampli®cation. 

The resulting plasmids were cut with SacI and SalI to 

release the insert which was ligated into the SacI/SalI sites of the 

E. coli expression vector pQE-30 (Quiagen, Hilden, Germany). 

GFP constructs. Three constructs containing the coding region 

of GFP and varying regions of CRY2 were made. Construct 

CRY2(1±612)/GFP contained the complete coding region of CRY2 

(comprising amino acids 1±612), construct CRY2(1±500)/GFP 

contained the N-terminal region of CRY2 (comprising amino 

acids 1±500), construct CRY2(501±612)/GFP contained the Cterminal 

region of CRY2 (comprising amino acids 501±612). The 

different regions of CRY2 were ampli®ed by PCR using VentR q - 

polymerase (New England Biolabs, Schwalbach, Germany) and 

the cDNA clone PHH1 (Hoffman et al., 1996) as template. The 

following primer combinations were used. Construct CRY2(1± 

612)/GFP: 5¢ CCCGGGATGAAGATGGACAAAAAGACTATAGTT-3¢ 

(upstream primer), 5¢-CCCGGGTTGGCAACCATTTTTTCCCAAAC- 

TTGT-3¢ (downstream primer). Construct CRY2(1±500)/GFP: 

5¢ CCCGGGATGAAGATGGACAAAAAGACTATAGTT-3¢ (upstream 

primer), 5¢-CCCGGGATCAGGTGCTGCTCCGATCATGATCTG-3¢ 

(downstream primer). Construct CRY2(501±612)/GFP: 5¢ GGATCC- 

AACAATGGCAGATAGCTTCGAGGCCTTA-3¢ (upstream primer), 

5¢-CCCGGGTTGGCAACCATTTTTTCCCAAACTTGT-3¢ (downstream 

primer). Restriction sites for SmaI present in the 

oligonucleotides except for the upstream primer of construct 

CRY2(501±612)/GFP are underlined. In the upstream primer for 

construct CRY2(501±612)/GFP a BamHI-site (underlined) and an 

optimized translation initiation signal (italics) around the start 

codon ATG (bold) were introduced. PCR products were subcloned 

into the pCR q II-TOPO vector (Invitrogen, De Schelp, the 

Netherlands) after the addition of 3¢A-overhangs with Taqpolymerase 

post-ampli®cation. The resulting plasmids were cut 

with SmaI (constructs CRY2(1±612)/GFP and CRY2(1±500)/GFP) or 

with BamHI/SmaI (construct CRY2(501±612)/GFP) to release the 

inserts which were ligated in frame with the coding region of 

mGFP4 (Haseloff et al., 1997) in the vector pMAV4 (Kircher et al., 

1999). All constructs were checked by sequencing. 

Acknowledgements 

We thank Oxana Panajotow for excellent technical assistance and 

help in making the ®gures and Dr Helen Steele (University of 

Marburg, Germany) for critical reading of the manuscript. We also 

thank Dr L. Nover (University of Frankfurt, Germany) for the gift of 

antisera against HSC70 and DnaK and Dr G. Philipps (University of 

Strasbourg, France) for the gift of antiserum against histone H2A/ 

H2B. This research was funded by a grant from the Deutsche 

Forschungsgemeinschaft to A.B. (BA958/5±2) and from the Human 

Frontier Science Program to K.H. (RG0043/1997-M). 

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Note added in proof 

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The paper The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light-dependent 

post-transcriptional mechanism by Hongwei Guo, Hien Duong, Nha Ma and Chentao Lin, which is based on independent 

research, reached the same conclusion as our work at the same time and is published on pages 279±287 in this issue of 

The Plant Journal.

Nuclear localization of the Arabidopsis blue light receptor ...

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