Cryptochrome Nucleocytoplasmic Distribution and Gene Expression Are Regulated by Light Quality in the Fern
Adiantum capillus-veneris
Takato Imaizumi,
a,b
Takeshi Kanegae,
a
and Masamitsu Wada
a,b,1
a
Department of Biology, Faculty of Science, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji, Tokyo, 192-0397, Japan
b
Department of Regulation Biology, National Institute for Basic Biology, 38, Nishigonaka, Myodaiji, Okazaki, Aichi,444-8585, Japan
Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the mo-lecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptorgenes, designated cryptochrome genes 4 and 5 (
CRY4
and
CRY5
), from the fern
Adiantum capillus-veneris.
Becausepreviously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largestidentified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with pre-viously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the crypto-chromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reactionanalysis indicate that the expression of the newly identified
CRY4
and
CRY5
genes is regulated by light and is under
phytochrome control. The intracellular distribution of reporter
b
-glucuronidase (GUS)–CRY fusion proteins indicatesthat GUS–CRY3 and GUS–CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS–CRY3 is regulatedin a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, orboth might be the photoreceptor that mediates inhibition of spore germination by blue light.
INTRODUCTION
Blue light responses have been known to occur in variousorganisms, including plants, fungi, and bacteria, for manydecades. In plants, phenomena such as phototropism, theinhibition of hypocotyl growth, flavonoid biosynthesis, andstomatal opening all are mediated by blue light photorecep-tors. At least some of these photoreceptors are thought tocontain a flavin chromophore (reviewed in Horwitz, 1994;Senger and Schmidt, 1994). One of the flavin chromophoreclass of photoreceptors, encoded by
CRY1
, was isolatedfrom the Arabidopsis long hypocotyl mutant line
hy4
, whichis defective in blue light inhibition of hypocotyl elongation(Ahmad and Cashmore, 1993). The deduced amino acid se-quence of
CRY1
shows substantial sequence similarity withclass I photolyases, the repair enzymes that split cyclobu-tane pyrimidine dimers by using electrons obtained fromblue light.
CRY1
encodes a 75-kD protein that binds two co-factors, 5,10-methenyltetrahydrofolate and flavin adenine
dinucleotide (FAD), as do the class I photolyases, but theCRY1 protein lacks DNA photorepair activity (Lin et al.,1995; Malhotra et al., 1995). To date, cryptochrome ho-mologs have been identified from four different plant spe-cies: Arabidopsis
CRY2
(
AT-PHH1
; Hoffman et al., 1996; Linet al., 1996a),
Sinapis alba SA-PHR1
(Batschauer, 1993),Chlamydomonas
CPH1
(Small et al., 1995), and
Adiantumcapillus-veneris CRY1
,
CRY2
, and
CRY3
(Kanegae andWada, 1998). The amino acid sequences deduced fromthese genes show remarkable similarity to CRY1 in theirN-terminal domains but little similarity in their C-terminaldomains.
Cryptochromes regulate many blue light responses in Ar-abidopsis. The physiological functions of CRY1 and CRY2appear to overlap to some degree; for example, both CRY1and CRY2 mediate inhibition of hypocotyl elongation and in-duction of anthocyanin synthesis (Lin et al., 1996b, 1998).Furthermore, functional analysis of plants overexpressingchimeric proteins comprising the N-terminal domain ofCRY1 and the C-terminal domain of CRY2, or the N-terminaldomain of CRY2 and the C-terminal domain of CRY1, indi-cates that the N-terminal domains and the C-terminal do-mains of CRY1 and CRY2 are interchangeable (Ahmad et
1
To whom correspondence should be addressed. E-mail [email protected]; fax 81-426-77-2559.
82 The Plant Cell
al., 1998a). In addition to their common functions, both Ara-bidopsis CRY proteins have distinct functions. For example,CRY2 mediates cotyledon expansion and controls timing offlowering (Guo et al., 1998; Lin et al., 1998), whereas en-trainment of the circadian clock by blue light is mediated byCRY1 (Somers et al., 1998).
Very recently, cryptochromes isolated from fruit flies andmice have been reported to play important roles in entrain-ing and maintaining circadian rhythms in these organisms(Stanewsky et al., 1998; van der Horst et al., 1999). On thebasis of amino acid sequence comparisons, cryptochromesare known to be ubiquitous photoreceptors in the plant andanimal kingdoms, despite their distinct evolutionary histories(Cashmore et al., 1999). These findings raise the fascinatingquestion of how individual cryptochromes evolved to per-form diverse functions. To begin to answer this question, it isnecessary to identify the functions of cryptochromes from awide range of organisms. As discussed above, the onlyfunctions of plant cryptochromes known in any detail arethose from Arabidopsis. However, numerous blue light re-sponses have been characterized by focusing on the singlecells and even on the single organelles in lower plants, par-ticularly in mosses and ferns, because of the simple organi-zation of their gametophytes. Thus, identifying the functions ofindividual lower plant cryptochromes is of particular interest.
Many physiological responses are induced by blue light ingametophytes of the fern
A. capillus-veneris
(reviewed inWada and Sugai, 1994). Spore germination is inhibited bybrief irradiation with blue light (Sugai and Furuya, 1985).Phototropism (Hayami et al., 1986), inhibition of tip growth(Kadota et al., 1979), apical swelling (Wada et al., 1978), andsubsequent cell division (Wada and Furuya, 1972, 1978;Miyata et al., 1979) also are regulated by blue light in pro-tonemata. In addition, blue light regulates organelle move-ments, including, for example, the orientational movementsof chloroplasts (Yatsuhashi et al., 1985; Kagawa and Wada,1994). Partial cell irradiation studies have further indicatedthat there are specific intracellular localizations for the bluelight photoreceptors involved in each response (Kadota etal., 1986). For example, blue light photoreceptors involved inthe inhibition of spore germination and cell cycle inductionare shown to be localized in or close to the nuclear compart-ment (Wada and Furuya, 1978; Furuya et al., 1997). As a firststep toward understanding the molecular mechanisms un-derlying these various blue light responses, we sought toclone and characterize the
CRY
gene family from
A. capillus-veneris.
Using the Arabidopsis
CRY1
cDNA as a probe, weobtained nine clones from an
A. capillus-veneris
genomic li-brary and have further classified these clones into fivegroups. Three of the five groups were characterized previ-ously and designated
A. capillus-veneris CRY1
,
CRY2
, and
CRY3
, in accordance with their remarkable similarities withcryptochromes of higher plants (Kanegae and Wada, 1998).
Here, we report the molecular cloning of the two formerlyunidentified genes,
CRY4
and
CRY5
, and demonstrate boththat cryptochromes in
A. capillus-veneris
are encoded by a
small gene family comprising at least five members and thatnone of the CRY proteins has photoreactivating activity. Inaddition, we present the temporal and spatial expressionpatterns of individual
A. capillus-veneris
cryptochromes un-der various light conditions. This information provides im-portant insights into cryptochrome function in
A. capillus-veneris.
RESULTS
Isolation of Additional Cryptochrome Homologs:
A. capillus-veneris CRY4
and
CRY5
Genes
When three cryptochrome homologs (
A. capillus-venerisCRY1
,
CRY2
, and
CRY3
) were identified from the genomiclibrary, two additional groups of unidentified clones wereisolated. The partial sequences of these clones showed asimilarity to the photolyase/blue light photoreceptor family(Kanegae and Wada, 1998). To determine whether theseclones also encode cryptochrome homologs, we isolatedthe corresponding cDNAs and determined their nucleotidesequences by performing 5
9
and 3
9
rapid amplification ofcDNA ends (RACE) experiments using total RNA isolatedfrom 1-day dark-imbibed spores as template. The resultingsequences confirm that these cDNAs encode cryptochromehomologs because their deduced amino acid sequenceshave a high degree of similarity to cryptochromes and sharethe features described below that are common to all crypto-chromes. Thus, we designated these genes
CRY4
and
CRY5
(Figures 1A and 1B).The longest
CRY4
cDNA was 2892 bp in length and con-tained an open reading frame encoding a predicted proteinof 699 amino acids. The longest
CRY5
cDNA was 2828 bplong and contained an open reading frame of 487 amino ac-ids. By comparing the cDNA and genomic sequences, wededuced that the
CRY4
gene comprises five exons and the
CRY5
gene comprises four exons. The intron positions in
CRY4
and
CRY5
are very similar to those in the previouslycharacterized
A. capillus-veneris
and Arabidopsis
CRY
genes(Figure 1B). DNA gel blot analysis showed that both genesare present in a single copy in the haploid genome (data notshown). The N-terminal regions of
A. capillus-veneris
CRY4and CRY5, which share similarities with photolyase (28.6and 26.9% identities to
Escherichia coli
photolyase, respec-tively), are also well conserved with respect to those of plantcryptochromes (e.g., Figure 1A). Using BLAST (Altschul etal., 1997) and FASTA (Lipman and Pearson, 1985; Pearsonand Lipman, 1988) searches, we could not find any se-quences that showed notable similarity with any of the C ter-mini. However,
A. capillus-veneris
CRY3 and CRY4 showedsubstantial similarity to each other at their C termini. TheC-terminal conserved amino acid motifs DQMVP andSTAESSSS found in other plant cryptochromes (Kanegae
Cryptochrome Localization and Expression 83
Figure 1. Alignment of Amino Acid Sequences of Arabidopsis and A. capillus-veneris Cryptochromes.
The deduced amino acid sequences of A. capillus-veneris (Ac) CRY4 (AcCRY4; DDBJ accession number AB028928 for the cDNA and AB028930for the gene) and CRY5 (AcCRY5; DDBJ accession number AB028929 for the cDNA and AB028931 for the gene) are compared with ArabidopsisCRY1 and CRY2 (AtCRY1; Ahmad and Cashmore, 1993; and AtCRY2; Lin et al., 1996a), and the three previously identified A. capillus-veneriscryptochromes, CRY1, CRY2, and CRY3 (AcCRY1, AcCRY2, and AcCRY3; Kanegae and Wada, 1998). Sequences were aligned by using theClustal W program, version 1.74 (Thompson et al., 1994). The numbers at the ends of amino acid sequences indicate the positions of amino ac-ids. The gaps are shown by dots.(A) Residues present in four or more of the sequences are highlighted (BOXSHADE; http://ulrec3.unil.ch/software/BOX_form.html). The positionsof 14 amino acids found in E. coli photolyase to interact with FAD by either direct H bonds (circles) or indirect H bonds (squares) are indicatedbelow the alignment. The amino acids that are conserved between E. coli photolyase and cryptochromes are marked with closed circles and aclosed square, and those that are not conserved are marked with open circles and an open square. The TGYP motif (asterisks) and the positionof Trp-277 of E. coli photolyase (plus) are indicated below the alignment. STAESSSS motifs found in the C termini of cryptochromes are en-closed with boxes. The putative nuclear localization signals found in C termini of CRY3 and CRY4 are underlined. The N-terminal regions withinthe lines were used to construct the phylogenetic tree shown in Figure 8.(B) The intron insertion positions of cryptochrome genes are indicated by the arrowheads on the partial amino acid alignment.
84 The Plant Cell
and Wada, 1998; Lin et al., 1998) were found in CRY4(DQRVP and STAESSSS, respectively) but not in CRY5.
The cryptochrome apoproteins (Arabidopsis CRY1 and
S.alba
SA-PHR1) interact noncovalently with FAD chro-mophores (Lin et al., 1995; Malhotra et al., 1995). The aminoacids binding to FAD have been deduced from the crystalstructure of the
E. coli
photolyase (Park et al., 1995). In total,10 of the 14 amino acids forming direct or indirect H bondsto FAD were found to be conserved in CRY4 and CRY5 (Fig-ure 1A), which suggests that CRY4 and CRY5 might containFAD as one of the two chromophores. In contrast, the aminoacids binding to 5,10-methenyltetrahydrofolate in the
E. coli
photolyase were poorly conserved in CRY4 and CRY5.These chromophore binding site characteristics are similarin the other plant cryptochromes. Amino acids presumed toform a DNA binding site in the
E. coli
photolyase have beennoted; some of these are also present in CRY4 and CRY5.However, Trp-277 in the
E. coli
protein, which is importantfor specific binding to pyrimidine dimers (Li and Sancar,1990), is replaced by Phe in CRY4 and Leu in CRY5, as hasbeen observed in the previously identified cryptochromes(Figure 1A). Moreover, CRY proteins from Arabidopsis and
S. alba
possess no photolyase activity (Lin et al., 1995;Malhotra et al., 1995; Hoffman et al., 1996).
A. capillus-veneris
CRY Proteins Do Not Catalyze DNA Photorepair in a Photolyase-Deficient
E. coli
Strain
To investigate whether
A. capillus-veneris
CRY proteins cancatalyze DNA repair, we cloned separately the full-lengthcDNAs encoding the five
A. capillus-veneris
CRY proteinsand the
E. coli phr
gene (positive control) into an
E. coli
expression vector and transformed each plasmid into thephotolyase-deficient strain SY2(DE3) of
E. coli.
These re-combinant proteins also contain a calmodulin binding pro-tein (CBP) tag at their N termini, and the expression vectorthat contained only the tag protein also was transformed asa negative control. The expression of the CBP-tagged re-combinant CRY proteins was confirmed by protein gel blot-ting with biotinylated calmodulin and streptavidin–alkalinephosphatase (data not shown). After inducing synthesis ofrecombinant proteins, SY2(DE3) cells harboring each plas-mid were exposed to UV light for specified times. Thesecells were plated in duplicate, and half the plates were irra-diated with photoreactivating light (blue light) for 1 hr beforedark incubation. As shown in Figure 2, blue light exposuredid not change the survival rates of cells carrying either
A.capillus-veneris CRY
cDNAs or the negative control but didincrease the survival rate of the cells carrying the
E. coli phr
gene. This result indicates that
A. capillus-veneris
crypto-chromes do not function as photolyases in
E. coli
cells. To-gether with the characteristics of the primary structures of
A.capillus-veneris
CRY proteins as shown in Figure 1, thisfinding suggests that
A. capillus-veneris
CRYs are likely tobe the cryptochromes of this fern.
Expression Patterns of
A. capillus-veneris CRY
Genes in Different Developmental Stages
Because blue light responses have been observed in variousdevelopmental stages of
A. capillus-veneris
, we examinedthe expression patterns of the five
CRY
genes at differentdevelopmental stages by RNA gel blot analysis. All of the
CRY
genes are expressed in low amounts, so we preparedpoly(A)
1
RNA to detect the transcripts. As shown in Figure 3,all of the
CRY
mRNAs were expressed in both the sporo-phyte and gametophyte stages. The
CRY1
and
CRY2
mR-NAs showed almost identical accumulation patterns. Theamounts of these mRNAs rose a little after spore germina-tion and stayed at the same level through the haploid anddiploid phases. The amounts of
CRY3
mRNA were relativelyhigher in protonemata and sporophyte tissues than in eitherspores or prothallia. The expression patterns of
CRY4
and
CRY5
genes were unique. The
CRY4
mRNA was highly con-centrated in the spores and in young leaves that had beengrown in the dark but not in the other tissues examined. The
CRY5
mRNA was observed mainly in sporophyte tissues,suggesting that expression of the
CRY5
gene might dependon the nuclear phase. Both the protonemal and prothallialtissues examined were cultured under light, whereas thespores were prepared in the dark. In case the expression ofthe
CRY4
gene might be downregulated by light, we further
Figure 2. Comparison of Photoreactivation (PR) Effects of A. capil-lus-veneris CRY Genes with E. coli Photolyase on Survival Rates ofthe Photolyase-Deficient E. coli Strain.
The photolyase-deficient strain SY2(DE3) carrying each CRY cDNAin the pCAL-n-EK expression vector was exposed by UV light andplated in duplicate. Before overnight incubation in the dark, oneplate of duplicates subsequently was irradiated with blue light (1B)for PR, but the others were not (2B). Each symbol represents theaverage of three independent survival rates of the cells carrying thefollowing plasmids: pCALCRY1, closed circles; pCALCRY2, opencircles; pCALCRY3, closed triangles; pCALCRY4, open triangles;pCALCRY5, closed squares; pCALPHR, open squares with positivecontrol; and pCALSTOP, pluses with negative control.
Cryptochrome Localization and Expression 85
investigated the amount of CRY mRNAs that accumulatedduring light-dependent spore germination.
Both Red and Blue Light Altered the Expression of CRY Genes during Light-Dependent Spore Germination
As was evident from the previous RNA gel blot analysis, theamounts of CRY transcripts were low in all the tissues inves-tigated. Therefore, we used competitive polymerase chainreaction (PCR) to measure the changes in amount of individ-ual CRY transcripts. The expression patterns of the CRY1,CRY2, and CRY3 genes were very similar (Figure 4B). Theaccumulated amounts of these mRNAs showed a 10-fold(CRY1 and CRY2) or sixfold (CRY3) increase in the first dayafter spore imbibition, but the amounts did not vary muchduring the following 6 days in the dark. When the sporeswere transferred to red light, which induces spore germina-tion, the amounts of CRY1, CRY2, and CRY3 mRNAs in-
creased two- to threefold within 12 hr and stayed at thislevel for as long as 72 hr. Blue light, which inhibits sporegermination, had almost no effect on the amounts of eitherCRY1 or CRY2 mRNA expressed but did affect the amountof CRY3 mRNA, causing a fivefold decrease. Unexpectedly,it was found that not only the amount of CRY4 mRNA butalso that of CRY5 mRNA was apparently regulated by lightduring spore germination. However, the ways in whichmRNA accumulation was regulated by light included bothinhibition and induction (Figures 4A and 4B). For CRY4, theamount of mRNA was reduced by z50-fold during the first24 hr after the onset of red light irradiation; blue light re-duced the level fivefold. Accumulation of CRY5 mRNA, how-ever, was induced rapidly after the onset of exposure toboth red and blue light, and it kept increasing to 300- to400-fold the initial dark level during the first 12 hr. ThemRNA amount then decreased for the next 12 hr, afterwhich it remained at a level 20- to 40-fold higher than that ofthe dark control. All of the major changes in expression ofthe five CRY genes occurred before spore germination (Fig-ure 4B).
Phytochrome Involvement in the Regulation of CRY4 and CRY5 mRNA Accumulation
To determine which photoreceptor is involved in regulatingCRY4 and CRY5 gene expression, we irradiated sporesbriefly in combinations of red, blue, or far-red light, and theexpression patterns of both genes were measured by com-petitive PCR. Our results indicate that the amounts of bothCRY4 and CRY5 mRNAs are regulated, at least in part, byphytochrome (Figures 5A and 5B). The accumulatedamounts of CRY4 and CRY5 mRNAs were examined at 24and 12 hr, respectively, after the light treatments, becausethe differences in the extents of their expression with orwithout light are clearly discernable at those times (see Fig-ure 4A). The effect of red light on the accumulation of bothmRNAs was cancelled by subsequent irradiation with far-red light and vice versa (Figures 5A and 5B). This red/far-redreversibility is a diagnostic characteristic of phytochromeregulation.
A blue light pulse reduced the amount of CRY4 mRNA ac-cumulation to that observed under continuous irradiationwith blue light; subsequent exposure to far-red light did notalter the amount expressed. This indicates that CRY4 ex-pression also was regulated by a blue light photoreceptor. Incontrast, a blue light pulse was not sufficient to induce alarge increase in CRY5 transcript abundance, and subse-quent irradiation with far-red light decreased the amounteven further. Moreover, the amount of mRNA present aftersimultaneous irradiation with continuous blue and far-redlight was 20 times less than that measured after continuousblue light irradiation. Together, these results suggest thatcontinuous blue light induction of CRY5 mRNA occurredmainly by way of a phytochrome.
Figure 3. Expression Patterns of Five CRY mRNAs in Different De-velopmental Stages.
Poly(A)1 RNA (4 mg) from spores, protonemata, prothallia, light-grown sporophytic leaves (Leaves [L]), and dark-grown leaves(Leaves [D]) was analyzed by RNA gel blotting by using specificprobes corresponding to the C-terminal region of each CRY cDNA.The gametophyte tissues were obtained under the following cultureconditions: imbibition for 1 day in darkness (Spores); imbibition for 4days in darkness and incubation for an additional 3 days under redlight (Protonemata); and incubation for 1 month under white light(Prothallia).
86 The Plant Cell
Intracellular Localization of CRYs under DifferentLight Conditions
Physiological observations suggest that the blue light pho-toreceptor that mediates inhibition of spore germination and
induction of the cell cycle is localized in or close to the nu-cleus (Kadota et al., 1986; Furuya et al., 1997). To determinewhether any cryptochromes are located in the nucleus, wetransiently expressed in fern gametophytes fusion genes con-taining each CRY cDNA inserted as a translational fusion to
Figure 4. Competitive PCR Analysis of CRY mRNA Accumulation during Light-Dependent Spore Germination.
The amounts of each CRY transcript were determined by using competitive PCR. Spores imbibed in the dark were transferred to either red lightor blue light. The amounts of CRY transcripts were measured at various times after transfer. This experiment was repeated twice in its entiretywith very similar results in each case.(A) Representative gel images of competitive PCR for CRY4 and CRY5. The PCR products derived from cDNA (closed arrowheads) and compet-itor (open arrowheads) are indicated on the gel image at each 1-hr red light irradiation sample after 4 days of incubation in the dark. The copynumbers of the competitors (from 101 to 108 copies) in each reaction tube are shown at the top of the sequential gel images. DS, dry spores; 1D,1-day dark-imbibed spores; 4D1hB, 1-hr blue light irradiation samples after 4 days of incubation in the dark. The remaining samples are labeledsimilarly, with R indicating red light.(B) Relative amounts of expression of five CRY genes and the germination rates in the same samples. Each point represents the average ofthe amounts of accumulation from two experiments. The symbols indicate the light conditions: circles, dark; squares, red light; triangles, bluelight. The amounts of cDNA were determined from densitometric analysis of the gel images by using National Institutes of Health Image soft-ware.
Cryptochrome Localization and Expression 87
b-glucuronidase (GUS). Because both red and blue light areknown to cause various responses during the early stages ofgametophyte development, the transfected cells were incu-bated under either red light or blue light or in the dark to ex-amine the effects of light on the intracellular distribution ofCRY proteins. Representative intracellular staining patterns ofGUS–CRY fusion proteins are shown in Figure 6. We ob-served the same distribution patterns of each GUS fusionprotein in both the basal cells and the tip cells. The intracellu-lar localization of GUS activity varied slightly among individualcells, even those expressing a single fusion construct, proba-bly because of damage from particle bombardment. There-fore, we show the tendency of the intracellular distributionpattern of each GUS–CRY fusion protein in Figure 7.
The GUS–CRY3 and GUS–CRY4 proteins showed clearnuclear localization, but GUS–CRY1, GUS–CRY2, andGUS–CRY5 did not. The GUS–CRY3 protein tended to ac-cumulate in the nucleus in the dark and in red light but not inblue light. The GUS–CRY4 protein was predominantly nu-clear under all the light conditions examined. The degree ofnuclear enrichment of GUS staining in GUS–CRY3—expressing cells under red light seemed to be less than thatin dark-incubated cells (see Figure 6). In the dark, a few ofthe cells showed nuclear enrichment in GUS–CRY1 andGUS–CRY2; in those cells, GUS activity also was observedin the cytoplasm. In onion epidermal cells, we also observednuclear localization of GUS–CRY3 and GUS–CRY4 but notof GUS–CRY1, GUS–CRY2, and GUS–CRY5 (data notshown). The expression rates of all GUS–CRY fusion pro-teins in A. capillus-veneris—especially those of GUS–CRY1and GUS–CRY2—under red or blue light were much lowerthan their expression rates in the dark. This observation sug-gests that a light-dependent protein degradation mecha-nism similar to that seen for Arabidopsis CRY2 (Lin et al.,1998) and Drosophila CRY (Emery et al., 1998) might alsooperate in A. capillus-veneris.
The N-terminal regions of cryptochromes, especially ofthe A. capillus-veneris CRYs, are highly conserved with re-spect to each other (see Figure 1A). Nonetheless, our resultsindicate that the subcellular distributions of these crypto-chromes differ, with only GUS–CRY3 and GUS–CRY4showing clear nuclear localization. This implies that the di-vergent C-terminal regions of these cryptochromes mightplay important roles in the determination of subcellular dis-tribution. In addition, small basic amino acid clusters (e.g.,KRKAK), which might function as monopartite nuclear local-ization signals, are present in the conserved regions foundat the CRY3 and CRY4 C termini (Figure 1A). To test thispossibility, we generated fusion genes encoding GUS at theN-terminal half and CRY3 or CRY4 C-terminal regions at theC-terminal half (GUS–CRY3C and GUS–CRY4C) and trans-formed these into A. capillus-veneris cells. The results showthat the light-dependent intracellular localization patterns ofGUS–CRY3C and the nuclear localization pattern of GUS–CRY4C are similar to those of GUS–CRY3 and GUS–CRY4(Figures 6 and 7). This finding suggests that the C-terminal
regions of both CRY3 and CRY4 are required to importthese proteins into the nucleus.
DISCUSSION
Including the two additional cryptochrome genes reportedhere, the A. capillus-veneris cryptochrome gene family com-prises at least five members, all of which are expressed dur-ing the haploid and diploid phases of the life cycle (see
Figure 5. Effects of Pulse Irradiation with Red, Far-Red, and BlueLight on the Regulation of CRY4 and CRY5 Gene Expression.
The effects of subsequent pulse irradiation (10 min each) with eitherred light (R) and far-red light (FR) or blue light (B) and far-red light(FR) on CRY4 and CRY5 transcript accumulation were measured us-ing competitive PCR. Four-day dark-imbibed spores were irradiatedin turn with red light and far-red light pulses or with blue light andfar-red light. After 24- and 12-hr (h) dark incubation (for CRY4 andCRY5, respectively), the expression rates shown were observed.CRY5 was also simultaneously irradiated with continuous blue light(Bc) and far-red light (FRc).(A) Representative gel images of competitive PCR for CRY4 andCRY5.(B) Relative amounts of CRY4 and CRY5 mRNAs. Each bar showsthe average from two experiments. The gray, dotted, and black hori-zontal lines in each panel indicate the amounts of CRY4 and CRY5cDNAs after the same lengths (24 and 12 hr) of continuous red (Rc)and blue (Bc) light exposure and of darkness (Dc), respectively (seeFigure 4B).
88 The Plant Cell
Figure 6. Representative Images of the Intracellular Distribution of GUS–CRY Fusion Proteins in Germinating Cells.
Intracellular distribution of GUS–CRY fusion proteins under different light conditions is shown.(A) to (C) GUS–CRY1.(D) to (F) GUS–CRY2.(G) to (I) GUS–CRY3.(J) to (L) GUS–CRY4.(M) to (O) GUS–CRY5.(P) to (R) GUS.(S) to (U) GUS–CRY3C (CRY3 C terminus).(V) to (X) GUS–CRY4C.Protonemal cells expressing various GUS–CRY fusion proteins were incubated under red light ([A], [D], [G], [J], [M], [P], [S], and [V]), blue light([B], [E], [H], [K], [N], [Q], [T], and [W]), or in the dark ([C], [F], [I], [L], [O], [R], [U], and [X]) for 16 hr and stained under the same light condi-tions. The cells showing GUS activity were photographed by using Nomarski optics (left panels); the fluorescence micrographs show the posi-tion of the nuclei in the same cells after staining with 49,6-diamidino-2-phenylindole (right panels). Note that under fluorescence, chlorophyllautofluoresces red and spore coats appear bluish white. Bar in (X) 5 20 mm for (A) to (X).
Cryptochrome Localization and Expression 89
Figure 3). Until now, the two Arabidopsis CRY genes repre-sented the only completely characterized family (Ahmad andCashmore, 1993; Lin et al., 1998). The five-member genefamily that we report here for the fern A. capillus-veneris isthe largest cryptochrome gene family characterized to date.Considering the complexity of blue light–induced phenom-ena found in this fern, it is not surprising that this fern has alarge number of blue light photoreceptors.
The N termini of these five cryptochromes are highly con-served, and the amino acid residues necessary for interac-tion with FAD and presumptive DNA binding are foundmostly in the same positions as in the E. coli photolyase.However, as we have shown, none of these cryptochromeshas photolyase activity. Their characteristics, therefore, arethe same as in previously identified cryptochromes, sug-gesting that the same redox electron transfer mechanismfrom blue light photons by way of FAD also functions in A.capillus-veneris CRY proteins. The STAESSSS-related aminoacid motif also is found at the C termini of CRY2, CRY3, andCRY4 (STAESTS, PMTESSSS, and STAESSSS, respec-tively). Although the function of this motif is unknown, it hasbeen reported to be important for the phosphorylation ofcryptochrome 1 by phytochrome A in vitro (Ahmad et al.,1998b). Physiological responses in A. capillus-veneris that areregulated antagonistically or cooperatively by phytochromeand blue light photoreceptors include spore germination,phototropism, the cell cycle, and chloroplast movement(Wada and Sugai, 1994). We have identified three phyto-chrome genes from A. capillus-veneris (Okamoto et al., 1993;Nozue et al., 1998a, 1998b). Recently, Yeh and Lagarias(1998) showed that phytochromes from oat and alga ex-pressed in yeast have serine/threonine kinase activity. Thus,in the signal transduction cascades upstream of some pho-toresponses in A. capillus-veneris, direct interaction of phy-tochromes and cryptochromes might happen by way of theSTAESSSS motif by phosphorylation. However, the A. capil-lus-veneris CRY1, CRY5, Chlamydomonas CPH1, and S. albaSA-PHR1 sequences do not possess the STAESSSS motif,which suggests other blue light signal transduction cas-cades that do not involve phosphorylation of this motif arepresent.
Cryptochromes have been found in divergent speciesacross the biological kingdom (Cashmore et al., 1999). Ofparticular interest is how cryptochromes evolved in plants.To address this question, we performed a phylogenetic treeanalysis using 10 full-length cryptochrome cDNA sequences(alga, Chlamydomonas [Small et al., 1995]; moss, Phy-scomitrella patens [Imaizumi et al., 1999]; fern, A. capillus-veneris [Kanegae and Wada, 1998; this article]; dicots, Ara-bidopsis [Ahmad and Cashmore, 1993; Lin et al., 1996a] andS. alba [Batschauer, 1993]) and related partial sequencesfound in the database. The regions of each N terminus indi-cated in Figure 1 were used for construction of the tree, andthe bootstrap values from 1000 replicates were calculatedby using the neighbor-joining method. The tree was rootedby using Chlamydomonas CPH1 as the outgroup (Figure 8).
Although some branching points are not reliable because ofthe low bootstrap values, the branching order of crypto-chromes and the related sequences closely correspond tothe organismal phylogeny. As in the phylogenetic analysis ofanother plant photoreceptor, phytochrome (Mathews andSharrock, 1997), multiple cryptochrome lineages in crypto-gams are clearly separable from those in seed plant clusters.
Consistent with the amino acid sequence alignment, A.capillus-veneris CRY1 and CRY2, and CRY3 and CRY4 arein each cluster. Three recent independent gene duplicationsmight have occurred to yield CRY3 and CRY4, CRY1/CRY2and CRY5, and CRY1 and CRY2. This information, together
Figure 7. Intracellular Distribution of GUS–CRY Fusion Proteins un-der Different Light Conditions.
Distribution patterns of GUS activities in individual transformed cellswere classified into two groups: (1) nuclear enrichment of GUS activ-ity was seen (nucleus [Nuc.] . cytoplasm [Cyto.]) or (2) nuclear en-richment of GUS activity was not seen (Nuc. < Cyto.). These cellsexpressed each GUS–CRY fusion protein under red light (stripedbars), blue light (open bars), or in the dark (black bars). From pro-tonemal cells prepared under the same experimental conditions asin Figure 6, we randomly selected cells that showed GUS activities.The first group (Nuc. . Cyto.) contained cells that showed clear nu-clear enrichment with various degrees of cytoplasmic GUS staining;and the second group (Nuc. < Cyto.) contained cells that showednuclear staining indistinguishable from or less than cytoplasmicstaining. The data were obtained from between three and seven in-dependent experiments. In total, we examined 45, 119, and 104cells (red light, blue light, and dark treatment, respectively) for GUS–CRY1; 30, 76, and 117 cells for GUS–CRY2; 117, 129, and 114 cellsfor GUS–CRY3; 120, 120, and 124 cells for GUS–CRY4; 105, 139,and 139 cells for GUS–CRY5; 144, 113, and 130 cells for GUS; 120,136, and 111 cells for GUS–CRY3C; and 141, 128, and 119 cells forGUS–CRY4C. For each experiment, we calculated the percentage ofpopulation of each group, dividing the number of cells belonging toeach group by the total number of cells examined in that experi-ment. The bars show the average percentages of populationsderived from all experiments. Standard errors of the mean are indi-cated.
90 The Plant Cell
with the mRNA accumulation pattern of CRY1 and CRY2(Figure 3) and the intracellular distribution of CRY1/CRY2and CRY3/CRY4 (Figures 6 and 7), indicates that CRY1/CRY2 and CRY3/CRY4 might comprise subfamilies of A.capillus-veneris cryptochromes. Because the diversity ofcryptogam phytochrome genes is thought to result fromtaxon-specific diversification, it is of great interest to learnwhether the diversification of A. capillus-veneris CRY genesalso occurred taxon specifically, as in the case of the crypto-gam phytochromes, or whether cryptogams have more thanone cryptochrome subfamily, as has been suggested for thephytochromes of seed plants. In seed plants, cryptochrome-related partial sequences from monocots are closely relatedto Arabidopsis CRY1, whereas sequences from dicots are inthe same cluster as Arabidopsis CRY2. It is also of interestto know whether seed plant cryptochromes diverged beforeor after the diversification of dicots and monocots. To ad-dress these questions requires identifying additional crypto-chrome genes from other plant species.
In A. capillus-veneris, the previous physiological obser-vations indicated that blue light photoreceptors were in-volved in phototropism (Hayami et al., 1992), apicalswelling (Wada et al., 1978), and chloroplast orientationalmovement (Yatsuhashi et al., 1987) localized on or close to
the plasma membrane in protonemata. Figures 6 and 7show that CRY1, CRY2, and CRY5 proteins are localizedin the cytoplasm. We could not find the putative mem-brane-spanning domains in CRY1, CRY2, or CRY5 proteins.However, some data showed that Arabidopsis CRY1 protein,which was not noted to contain any membrane-spanning do-mains, was enriched in the membrane fractions (Ahmad etal., 1998c). Possibly, therefore, some portions of CRY1,CRY2, or CRY5 proteins may attach to the plasma mem-brane where they might be involved in blue light–inducedphototropism, apical swelling, chloroplast orientational move-ment in protonemata, or some combination of these re-sponses.
Blue light suppresses the entry into the S phase of the firstmitosis leading to spore germination (Uchida and Furuya,1997). In protonemata, blue light photoreceptors also con-trol shortening of the G1 phase (Miyata et al., 1979). The bluelight photoreceptors involved in these responses are local-ized in or close to the nucleus (Wada and Furuya, 1978;Furuya et al., 1997). Such physiological observations indi-cate that the blue light photoreceptor mediating inhibition ofspore germination must be present in or close to the nu-cleus before the onset of blue light irradiation. Althoughwhether these blue light photoreceptors are derived fromthe maternal cytoplasm or are newly translated during imbi-bition in the dark is unknown, CRY1, CRY2, CRY3, andCRY4 genes would be the candidates for such receptors,given our results showing that these mRNAs accumulate indark-imbibed spores (Figure 4). In addition, we demon-strated that GUS–CRY3 and GUS–CRY4 apparently accu-mulate in the nucleus in dark-incubated protonemal cells(Figures 6 and 7).
These results are consistent with the notion that CRY3and/or CRY4 genes could encode the blue light photorecep-tors regulating spore germination. Under natural light condi-tions, the ratio between blue light and red light may differ,depending on the surrounding environment, but both arealways present. Even after the steps leading to sporegermination are activated by light signals by way of aphytochrome, any blue light photoreceptors working antag-onistically would interfere with germination. Although wecould not find any amino acid sequences that fit the consen-sus sequence of the nuclear export signal in the C-terminalregion of CRY3 (Bogerd et al., 1996; Kim et al., 1996), ourdata showed the apparent blue light–dependent export ofGUS–CRY3 fusion protein from the nuclei of dark-growncells to the cytoplasm (Figures 6 and 7). If CRY3 is the bluelight photoreceptor of the inhibition of spore germination,the nuclear export of CRY3 might be one of the importantmechanisms to cause the inhibition of spore germination.CRY4 seems to localize in the nucleus under all light condi-tions examined, but the accumulation of CRY4 mRNA isregulated by light quality. If CRY4 is the blue light photore-ceptor that inhibits spore germination, the suppression ofCRY4 mRNA accumulation observed under red light (Figures4 and 5) might reduce the interruption of the germination
Figure 8. Phylogenetic Analysis of Plant Cryptochromes.
The regions of partial amino acid sequences used to generate thephylogenetic tree are shown in Figure 1. The neighbor-joiningmethod was used to calculate bootstrap values from 1000 replicates(http://www.genome.ad.jp/) and to draw the tree by using TreeViewsoftware (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Thenumbers located at branch points are the bootstrap values. Gen-Bank/DDBJ/EMBL accession numbers for each cryptochrome DNAsequence, except for those given in the legend of Figure 1, are asfollows: Angiopteris for Angiopteris evecta, X99261; Avena for Avenasativa, X99262; CPH1 for Chlamydomonas CPH1, L07561; Equise-tum for Equisetum arvense, X99263; Maize1 and Maize2 for Zeamays, X99265 and X99266; Mougeotia for Mougeotia scalaris,AJ000694; Physcomitrella for P. patens, AB027528; SA-PHR1 for S.alba SA-PHR1, X72019; and TomatoCRY2 for Lycopersicon escu-lentum, AJ000695.
Cryptochrome Localization and Expression 91
processes by blue light, allowing uninterrupted completionof germination. Currently, the answers to these questionsremain obscure, but abundant physiological data suggestthat cryptochromes might work as negative photoswitchesfor cell cycle promotion in germination of A. capillus-venerisspores.
METHODS
Plant Materials and Light Sources
Spores of Adiantum capillus-veneris were harvested in either 1994 or1995 in a greenhouse at Tokyo Metropolitan University. Spores weresterilized with 10% (v/v) antiformin (NaClO) containing 0.1% (v/v) Tri-ton X-100. After three rinses with sterile distilled water, the sporeswere suspended in 1:10 strength of modified Murashige and Skoogculture medium (Wada and Furuya, 1970) and sown on a cellophanesheet that covered the same Murashige and Skoog medium solidi-fied with 0.5% (w/v) agar. The spores were incubated under differentlight conditions at 25 6 18C. The gametophytes were collected on fil-ter paper by using vacuum filtration; they were then frozen immedi-ately in liquid nitrogen and stored at 2808C before use. Allmanipulations after sowing were performed under a dim green safe-light (Kadota et al., 1984). Sporophytes were cultivated in the green-house under natural light conditions. After all leaves were cut, younglight-grown leaf tissue was collected as emerging croziers. Somesporophytes were transferred into complete darkness after removalof all the leaves. Young dark-grown leaves (croziers) were collectedin the dark for 3 to 4 weeks under a safelight. This tissue, too, wasfrozen immediately and stored at 2808C.
White light (25.7 mmol m22 sec 21) was obtained from broad range–emitting fluorescent tubes (model FL40SD; Toshiba Ltd., Tokyo, Ja-pan). Red light (9.9 mmol m22 sec 21) was obtained from either red-emitting fluorescent tubes (model FL20S-Re-66; Toshiba Ltd.) or flu-orescent tubes (model FL40SD; Toshiba Ltd.) filtered through a redplastic sheet (model Acrylite 102; Mitsubishi Rayon, Tokyo, Japan).Far-red light (9.2 mmol m22 sec 21) was provided by far-red-emittingfluorescent tubes (model FL20S-FR-74; Toshiba Ltd.) and filteredthrough the same red plastic sheet (model Acylite 102; MitsubishiRayon) and a far-red filter (IR-1; Koto Electronic Company Ltd.,Urawa, Japan). Blue light (4.6 mmol m22 sec 21) was obtained fromblue-emitting fluorescent tubes (models FL20S-B and FL20S-BW;Toshiba Ltd.) filtered through a blue plastic sheet (model Acrylite302; Mitsubishi Rayon). The light conditions described above wereused throughout all experiments.
DNA and RNA Isolation
Genomic DNA and total RNA derived from either gametophytes orsporophytes were isolated by using the cetyltrimethylammoniumbromide (CTAB) method (Kanegae and Wada, 1998), with somemodifications. When gametophyte tissue was ground with a mortarand pestle, an amount of sterilized quartz sand equal to 10 times thevolume (w/w) was added to each sample to help break the cell wallsin liquid nitrogen. Frozen tissue was incubated with an amount of 2 3CTAB solution (1 3 CTAB solution is 2% [w/v] CTAB, 0.1 M Tris-HCl,pH 8.0, 20 mM EDTA, and 1.4 M NaCl) equal to 10 times the volume
(w/v) containing 5% (v/v) 2-mercaptoethanol at 608C. The chloroformtreatments were repeated three times. DNA was precipitated with2-propanol. RNA was precipitated twice with LiCl. The precipitatewas dissolved in Tris-EDTA or diethyl pyrocarbonate–treated water.To purify poly(A)1 RNA, we used mRNA purification kits (AmershamPharmacia Biotech, Buckinghamshire, UK) according to the manu-facturer’s protocol.
Cloning of A. capillus-veneris Cryptochrome 4 and 5 Genes
Three clones (l-1, l-3, and l-5) contained the same gene (CRY4).The other clone (l-2), corresponding to CRY5, was isolated from theA. capillus-veneris genomic l phage library by using the full-lengthArabidopsis CRY1 cDNA as a probe (Kanegae and Wada, 1998).These clones were subcloned into the plasmid vector pBluescript IISK1 (Stratagene, La Jolla, CA). Both genomic clones and cDNAclones were sequenced by using either fluorescent-labeled primersor dye terminators (BigDye terminator cycle sequencing ready reac-tion; Applied Biosystems, Foster City, CA). For isolation of CRY4 andCRY5 cDNAs, a 39 rapid amplification of cDNA ends (RACE) kit (LifeTechnologies, Rockville, MD) and a 59 RACE kit (Life Technologies)were used according to the system protocols. In 39 RACE, cDNAsynthesized from 1 mg of total RNA derived from 1-day dark-imbibedspores was amplified by gene-specific primers: 59-CCAAGCTTGGGG-AGGAGAG-39 for CRY4 and 59-AGGGAAGCCAGATGAAGAGC-39
for CRY5. Both primary polymerase chain reaction (PCR) productswere amplified with the same nested primer (59-CCYYTDGTKGAT-GCHGGVATG-39). In 59 RACE, first-strand cDNAs were synthesizedfrom the same total RNA used in 39 RACE by using gene-specificprimers: 59-AAGCCTCAGCATCAGT-39 for CRY4 and 59-AACTAA-TGGTCGTGGA-39 for CRY5. The cDNAs were then amplified by PCRwith nested gene-specific primers: 59-CCCAAGCACATCAGACTC-CAAA-39 for CRY4 and 59-TCATCCATCCGTTCGAGTTC-39 for CRY5.Primary PCR products that were between 1 and 2.5 kb long were re-covered and reamplified by using another nested gene-specificprimer: 59-AGACTATGACTCGCACTCGGTT-39 for CRY4 and 59-CCC-ATGTCCAAGGTAGCTGC-39 for CRY5. Afterward, 39- and 59 RACEproducts were cloned into the plasmid vector pGEM-T Easy (Pro-mega, Madison, WI) and sequenced.
Photoreactivation Analysis
Full-length coding regions of five CRYs and an Escherichia coli pho-tolyase (phr) gene (pRT2; kindly provided by T. Todo, Kyoto Univer-sity, Kyoto, Japan) were cloned into the expression vector pCAL-n-EK (affinity LIC cloning and protein purification kit; Stratagene) ac-cording to the manufacturer’s procedure. The primers used for PCRcloning were the following: 59-GACGACGACAAGGCCTGCACAATT-GTGTGGTT-39 and 59-GGAACAAGACCCGTGAGTTTCTGAGAC-AATCT-39 for pCALCRY1; 5 9-GACGACGACAAGGCGGCACAC-ACAATTGTGGC-39 and 59-GGAACAAGACCCGTCTGACCCTTAACT-TCAAC-39 for pCALCRY2; 59-GACGACGACAAGGCAAAATCATGT-ACCGTTGT-39 and 59-GGAACAAGACCCGTCACGTCAGTTTAAACA-AC-39 for pCALCRY3; 59-GACGACGACAAGGCAAAACCTTGTACA-ATAGT-39 and 59-GGAACAAGACCCGTCACCGATGCATTTTTTCG-39
for pCALCRY4; 59-GACGACGACAAGACCACCTCTACAACCATT-GT-39 and 59-GGAACAAGACCCGTAGCTGCAGACTAATCAAC-39 forpCALCRY5; and 59-GACGACGACAAGACTACCCATCTGGTCTGG-TT-39 and 59-GGAACAAGACCCGTATTGCCTGACGCGTCTGT-39
92 The Plant Cell
for pCALPHR. These recombinant proteins possess a calmodulinbinding protein (CBP) tag in the N terminus, so a vector expressingonly the CBP was generated to insert synthetic oligonucleotides con-sisting of three stop codons (TGATAATAG) into the cloning site as anegative control vector (pCALSTOP). The resulting clones were se-quenced to confirm that they contained the correct inserts. A photo-lyase-deficient E. coli strain SY2 (JM107; phr::Cmr uvrA::Kmr
recA::Tetr; Yasuhira and Yasui, 1992; kindly provided by T. Todo)was lysogenized with lDE3 prophage (Novagen, Madison, WI) toenable the expression system to use the T7 RNA polymerase pro-moter. Plasmids pCALCRY1, pCALCRY2, pCALCRY3, pCALCRY4,pCALCRY5, pCALPHR, and pCALSTOP were transformed into theresulting SY2(DE3) cells. The SY2(DE3) cells harboring each plasmidwere cultured until the OD600 reached 0.6; then isopropylthiogalacto-pyranoside was added to a final concentration of 1 mM. After an ad-ditional 3.5-hr incubation at 378C in the dark, the cells were collectedand washed with phosphate buffer (pH 6.8; Yamamoto, 1985). Thecells were resuspended in phosphate buffer at a concentration of 1to 2 3 107 cells per mL. The cell suspensions, in 30-mm-diameterplastic dishes, were exposed to UV light (0.2 mmol m22 sec 21) ob-tained from a germicidal light tube (model GL-20; Toshiba Ltd.) andthen plated in duplicate in two to three dilution series. For photoreac-tivation, half the group of Luria-Bertani plates were irradiated withblue light for 1 hr before overnight incubation in the dark. The numberof surviving colonies was independently counted three times to cal-culate the survival rates. After resuspension of the cells, all manipu-lations were performed under red light (model National FL20S.R-F;Matsusita Electric Co., Osaka, Japan).
RNA Gel Blot Analysis
Poly(A)1 RNA (4 mg per lane) was electrophoresed on 1.2% formal-dehyde-agarose gels and transferred to nylon membranes (HybondN1; Amersham Pharmacia Biotech) over a period of 14 to 16 hr with20 3 SSC (1 3 SSC is 0.15 M NaCl and 0.015 M sodium citrate). Forhybridization and detection, a chemifluorescence-labeled gene de-tection kit (Gene Images; Amersham Pharmacia Biotech) was usedaccording to the manufacturer’s instruction. The 39 regions of eachCRY cDNA (nucleotide sequence positions 1451 to 2457 for theCRY1 cDNA, 1691 to 2754 for the CRY2 cDNA, 1448 to 2440 for theCRY3 cDNA, 1634 to 2857 for the CRY4 cDNA, and 1328 to 2444 forthe CRY5 cDNA) were amplified by PCR to be used as probes. Theblots were hybridized with the probes at 658C. The membranes werewashed in 1 3 SSC, 0.1% (w/v) SDS, and then in 0.1 3 SSC, 0.1%(w/v) SDS, for 15 min each time at 658C. Cross-hybridization did notoccur when we performed DNA gel blot analyses with the sameprobes at the lower stringency condition (0.5 3 SSC, 0.1% [w/v] SDSat 608C for the second wash; data not shown).
Competitive Reverse Transcription–PCR Analysis
DNA competitors were designed to have different sequences onboth 59 and 39 ends in which each set of CRY-specific primers couldbe annealed but with the same sequences derived from l phageDNA in between (Competitive DNA construction kit; Takara ShuzoCo., Kyoto, Japan). Five primer sets were used for PCR synthesis ofeach competitor: 59-CAGAATTTATGCCCGAGTCCGCGTGAGTATTAC-GAAGGTG-39 and 5 9-TATGGAAACAGGAAGACCAGTGAAGAC-GACGCGAAATTCA-39 for CRY1 competitor; 59-GCACATTCACAC-
AACTCTCCGCGTGAGTATTACGAAGGTG-39 and 59-CTAATGTTG-GTGTAAGGAGGTGAAGACGACGCGAAATTCA-39 for CRY2 com-petitor; 59-GCCCATTGAGAGCCATTAAGGCGTGAGTATTACGAA-GGTG-39 and 59-AGCTCACGACGGGATCAAATTGAAGACGACGCG-AAATTCA-39 for CRY3 competitor; 59-CTCTGACAATGGTGACTT-CTGCGTGAGTATTACGAAGGTG-39 and 59-AGACAGCAGTGGCAG-GAACATGAAGACGACGCGAAATTCA-39 for CRY4 competitor; and59-ACAATCCACCAATGAGCCCAGCGTGAGTATTACGAAGGTG-39
and 59-GAACTCGAACGGATGGATGATGAAGACGACGCGAAATTCA-39 for CRY5 competitor.
A gradient series from 101 to 108 copies per mL of competitors wasused for the analysis. Complementary DNA was synthesized from 2mg of each total RNA pretreated with DNase I (Stratagene) by usingan oligo(dT) primer according to the supplied procedure (SUPER-SCRIPT preamplification system for first-strand cDNA synthesis; LifeTechnologies). One-tenth volume of the reaction solution of cDNAand 1 mL of each concentration of competitors were put into thesame reaction tube. The locations of the primers were designed tocontain the last intron of each CRY gene so that we could distinguishwhether the amplified fragments were derived from cDNA or fromcontaminated genomic DNA. PCR was performed with the followingprimer sets: 59-CAGAATTTATGCCCGAGTCC-39 and 59-TATGGAAAC-AGGAAGACCAG-39 for CRY1; 59-GCACATTCACACAACTCTCC-39 and59-CTAATGTTGGTGTAAGGAGG-39 for CRY2; 59-GCCCATTGAGAG-CCATTAAG-39 and 59-AGCTCACGACGGGATCAAAT-39 for CRY3;59-CTCTGACAATGGTGACTTCT-39 and 59-AGACAGCAGTGGCAG-GAACA-39 for CRY4; and 59-GAACTCGAACGGATGGATGA-39 and59-ACAATCCACCAATGAGCCCA-39 for CRY5. The PCR schedulewas 1 min at 948C; 40 cycles of 10 sec at 988C, 30 sec at 558C, and30 sec at 728C; and then 3 min at 728C. Equal volumes of PCR prod-ucts were separated in 3% (w/v) agarose gels. The expected lengthsof amplified fragments derived either from each CRY cDNA or fromeach competitor were 0.5 and 0.4 kb, respectively. The gel imageswere captured and analyzed by using the public domain National In-stitutes of Health Image program (http://rsb.info.nih.gov/nih-image/).
b-Glucuronidase–CRY Plasmid Construction
A PCR-based cloning method (seamless cloning kit; Stratagene) wasused to construct vectors containing b-glucuronidase (GUS)–CRYfusion genes. pBI221 plasmid (Clontech, Palo Alto, CA) that con-tained a cauliflower mosaic virus 35S promoter–driven GUS genewas used as a template. The primers used for making GUS–CRYconstructs were as follows (forward primer and reverse primer, re-spectively): 59-AGTTACTCTTCAGCTACCGAGCTCGAATTTCCCCGA-39 and 59-AGTTACTCTTCATTGTTTGCCTCCCTGCTGCGGTTT-39
for amplifying the pBI221 vector that has a specific in-frame cloningsite; 59-AGTTACTCTTCACAAGCCTGCACAATTGTGTGGTTTCGG-39
and 59-AGTTACTCTTCAAGCGAGAACCAACCAACCGCCACATTC-39
for CRY1 insert; 59-AGTTACTCTTCACAAGCGGCACACACAATT-GTGGCACAC-39 and 59-AGTTACTCTTCAAGCCCAGGCTTTGATCCT-AGTGGGTGT-39 for CRY2 insert; 59-AGTTACTCTTCACAAGCAAAA-TCATGTACCGTTGTGTGG-39 and 59-AGTTACTCTTCAAGCCAGTGC-AACTGCACGTCAGTTTAAAC-39 for CRY3 insert; 59-AGTTACTCT-TCACAAGCAAAACCTTGTACAATAGTGTGG-39 and 59-AGTTACTCT-TCAAGCCGTCACCAGCCCCACTCATGAAGA-39 for CRY4 insert;59-AGTTACTCTTCACAAACCACCTCTACAACCATTGTCTGGC-39 and59-AGTTACTCTTCAAGCGCTGGGGCTCTGAGACATCCTTTC-39 forCRY5 insert; 59-AGTTACTCTTCACAAGTGGGCGATTCAATTGCG-AAGGTC-39 for CRY3C insert; and 59-AGTTACTCTTCACAACCA-
Cryptochrome Localization and Expression 93
CCACTGGTTTCAACTCACTCC-39 for CRY4C insert. To amplifyCRY3C and CRY4C inserts, we used the same reverse primers as forthe CRY3 and CRY4 inserts. Both junctions between vector and am-plified CRY inserts and CRY inserts themselves were sequenced toconfirm whether or not these fragments were inserted in the sameframe correctly.
Analysis of GUS Intracellular Localization in Protonemal Cells
Gold particles (1.6 mm in diameter) coated with GUS–CRY or pBI221plasmids were introduced into the two-celled protonemata, whichwere incubated for 4 days in the dark and for an additional 3 days un-der red light, by using a biolistic gun transformation system (DuPont,Wilmington, DE). To bombard gold particles, we used 1350-psi rup-ture discs according to the manufacturer’s procedure. Transfectedcells were incubated in red light, blue light, or in the dark for 16 hr at258C and then stained with 100 mM sodium phosphate, pH 7.0, 1mM EDTA, 0.3% (v/v) Triton X-100, 0.5 mM potassium ferricyanide,0.5 mM potassium ferrocyanide, 0.1 mg/mL 49,6-diamidino-2-phe-nylindole, and 1 mM X-gluc. Staining was performed under the samelight conditions at 378C overnight. Cells showing GUS staining wereselected under low magnifying power (310 to 25) through a stere-omicroscope, and the intracellular localization of GUS products wasobserved under a microscope (3400). The optic images of GUS ac-tivity and 49,6-diamidino-2-phenylindole staining were obtained byusing a microscope equipped with Nomarski optics. A dim greensafelight was used in every step of the transfections and staining.Transfections using each recombinant plasmid were repeated atleast five times.
ACKNOWLEDGMENTS
We thank Dr. Takeshi Todo for providing the SY2 strain and the pRT2plasmid. We are grateful to Dr. Jane Silverthorne (University of Califor-nia, Santa Cruz) for critical reading of the manuscript. We thank Hyun-Sook Park for 39 RACE analysis and Kayoko Hara, Eitetsu Sugiyama,and Takako Yasuki for their technical assistance. This work was sup-ported in part by the Grant-in-Aid for International Scientific Research(Joint Research) (Grant Nos. 07044206 and 09044232) and for Scien-tific Research (B) (Grant Nos. 07458196 and 09440270) from theMinistry of Education, Science, Sports, and Culture of Japan; bygrants from the Mitsubishi Foundation, PROBRAIN (Program for Pro-motion of Basic Research Activities for Innovative Biosciences), andthe NOVARTIS Foundation to M.W.; by the Grant-in-Aid for Encour-agement of Young Scientists (Grant No. 10740373) from the Ministryof Education, Science, Sports, and Culture of Japan to T.K.; and alsoby a grant from Research Fellowships of the Japan Society for the Pro-motion of Science for Young Scientists (Grant No. 10639301) to T.I.
Received July 19, 1999; accepted November 1, 1999.
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DOI 10.1105/tpc.12.1.81 2000;12;81-95Plant Cell
Takato Imaizumi, Takeshi Kanegae and Masamitsu WadaAdiantum capillus-venerisQuality in the Fern
Cryptochrome Nucleocytoplasmic Distribution and Gene Expression Are Regulated by Light
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