The Plant Cell, Vol. 12, 81–95, January 2000, www.plantcell.org © 2000 American Society of Plant Physiologists
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 Wadaa,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 molecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptor
genes, designated cryptochrome genes 4 and 5 ( CRY4 and CRY5), from the fern Adiantum capillus-veneris. Because
previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest
identified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reaction
analysis 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 indicates
that GUS–CRY3 and GUS–CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS–CRY3 is regulated
in a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, or
both might be the photoreceptor that mediates inhibition of spore germination by blue light.
INTRODUCTION
Blue light responses have been known to occur in various
organisms, including plants, fungi, and bacteria, for many
decades. In plants, phenomena such as phototropism, the
inhibition of hypocotyl growth, flavonoid biosynthesis, and
stomatal opening all are mediated by blue light photoreceptors. At least some of these photoreceptors are thought to
contain a flavin chromophore (reviewed in Horwitz, 1994;
Senger and Schmidt, 1994). One of the flavin chromophore
class of photoreceptors, encoded by CRY1, was isolated
from the Arabidopsis long hypocotyl mutant line hy4, which
is defective in blue light inhibition of hypocotyl elongation
(Ahmad and Cashmore, 1993). The deduced amino acid sequence of CRY1 shows substantial sequence similarity with
class I photolyases, the repair enzymes that split cyclobutane pyrimidine dimers by using electrons obtained from
blue light. CRY1 encodes a 75-kD protein that binds two cofactors, 5,10-methenyltetrahydrofolate and flavin adenine
1 To
whom correspondence should be addressed. E-mail [email protected]; fax 81-426-77-2559.
dinucleotide (FAD), as do the class I photolyases, but the
CRY1 protein lacks DNA photorepair activity (Lin et al.,
1995; Malhotra et al., 1995). To date, cryptochrome homologs have been identified from four different plant species: Arabidopsis CRY2 (AT-PHH1; Hoffman et al., 1996; Lin
et al., 1996a), Sinapis alba SA-PHR1 (Batschauer, 1993),
Chlamydomonas CPH1 (Small et al., 1995), and Adiantum
capillus-veneris CRY1, CRY2, and CRY3 (Kanegae and
Wada, 1998). The amino acid sequences deduced from
these genes show remarkable similarity to CRY1 in their
N-terminal domains but little similarity in their C-terminal
domains.
Cryptochromes regulate many blue light responses in Arabidopsis. The physiological functions of CRY1 and CRY2
appear to overlap to some degree; for example, both CRY1
and CRY2 mediate inhibition of hypocotyl elongation and induction of anthocyanin synthesis (Lin et al., 1996b, 1998).
Furthermore, functional analysis of plants overexpressing
chimeric proteins comprising the N-terminal domain of
CRY1 and the C-terminal domain of CRY2, or the N-terminal
domain of CRY2 and the C-terminal domain of CRY1, indicates that the N-terminal domains and the C-terminal domains of CRY1 and CRY2 are interchangeable (Ahmad et
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al., 1998a). In addition to their common functions, both Arabidopsis CRY proteins have distinct functions. For example,
CRY2 mediates cotyledon expansion and controls timing of
flowering (Guo et al., 1998; Lin et al., 1998), whereas entrainment of the circadian clock by blue light is mediated by
CRY1 (Somers et al., 1998).
Very recently, cryptochromes isolated from fruit flies and
mice have been reported to play important roles in entraining and maintaining circadian rhythms in these organisms
(Stanewsky et al., 1998; van der Horst et al., 1999). On the
basis of amino acid sequence comparisons, cryptochromes
are known to be ubiquitous photoreceptors in the plant and
animal kingdoms, despite their distinct evolutionary histories
(Cashmore et al., 1999). These findings raise the fascinating
question of how individual cryptochromes evolved to perform diverse functions. To begin to answer this question, it is
necessary to identify the functions of cryptochromes from a
wide range of organisms. As discussed above, the only
functions of plant cryptochromes known in any detail are
those from Arabidopsis. However, numerous blue light responses have been characterized by focusing on the single
cells and even on the single organelles in lower plants, particularly in mosses and ferns, because of the simple organization of their gametophytes. Thus, identifying the functions of
individual lower plant cryptochromes is of particular interest.
Many physiological responses are induced by blue light in
gametophytes of the fern A. capillus-veneris (reviewed in
Wada and Sugai, 1994). Spore germination is inhibited by
brief 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), and
subsequent cell division (Wada and Furuya, 1972, 1978;
Miyata et al., 1979) also are regulated by blue light in protonemata. In addition, blue light regulates organelle movements, including, for example, the orientational movements
of chloroplasts (Yatsuhashi et al., 1985; Kagawa and Wada,
1994). Partial cell irradiation studies have further indicated
that there are specific intracellular localizations for the blue
light photoreceptors involved in each response (Kadota et
al., 1986). For example, blue light photoreceptors involved in
the inhibition of spore germination and cell cycle induction
are shown to be localized in or close to the nuclear compartment (Wada and Furuya, 1978; Furuya et al., 1997). As a first
step toward understanding the molecular mechanisms underlying these various blue light responses, we sought to
clone and characterize the CRY gene family from A. capillusveneris. Using the Arabidopsis CRY1 cDNA as a probe, we
obtained nine clones from an A. capillus-veneris genomic library and have further classified these clones into five
groups. Three of the five groups were characterized previously and designated A. capillus-veneris CRY1, CRY2, and
CRY3, in accordance with their remarkable similarities with
cryptochromes of higher plants (Kanegae and Wada, 1998).
Here, we report the molecular cloning of the two formerly
unidentified genes, CRY4 and CRY5, and demonstrate both
that cryptochromes in A. capillus-veneris are encoded by a
small gene family comprising at least five members and that
none of the CRY proteins has photoreactivating activity. In
addition, we present the temporal and spatial expression
patterns of individual A. capillus-veneris cryptochromes under various light conditions. This information provides important insights into cryptochrome function in A. capillusveneris.
RESULTS
Isolation of Additional Cryptochrome Homologs:
A. capillus-veneris CRY4 and CRY5 Genes
When three cryptochrome homologs ( A. capillus-veneris
CRY1, CRY2, and CRY3) were identified from the genomic
library, two additional groups of unidentified clones were
isolated. The partial sequences of these clones showed a
similarity to the photolyase/blue light photoreceptor family
(Kanegae and Wada, 1998). To determine whether these
clones also encode cryptochrome homologs, we isolated
the corresponding cDNAs and determined their nucleotide
sequences by performing 5 9 and 39 rapid amplification of
cDNA ends (RACE) experiments using total RNA isolated
from 1-day dark-imbibed spores as template. The resulting
sequences confirm that these cDNAs encode cryptochrome
homologs because their deduced amino acid sequences
have a high degree of similarity to cryptochromes and share
the features described below that are common to all cryptochromes. Thus, we designated these genes CRY4 and
CRY5 (Figures 1A and 1B).
The longest CRY4 cDNA was 2892 bp in length and contained an open reading frame encoding a predicted protein
of 699 amino acids. The longest CRY5 cDNA was 2828 bp
long and contained an open reading frame of 487 amino acids. By comparing the cDNA and genomic sequences, we
deduced 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 previously
characterized A. capillus-veneris and Arabidopsis CRY genes
(Figure 1B). DNA gel blot analysis showed that both genes
are present in a single copy in the haploid genome (data not
shown). The N-terminal regions of A. capillus-veneris CRY4
and CRY5, which share similarities with photolyase (28.6
and 26.9% identities to Escherichia coli photolyase, respectively), are also well conserved with respect to those of plant
cryptochromes (e.g., Figure 1A). Using BLAST (Altschul et
al., 1997) and FASTA (Lipman and Pearson, 1985; Pearson
and Lipman, 1988) searches, we could not find any sequences that showed notable similarity with any of the C termini. However, A. capillus-veneris CRY3 and CRY4 showed
substantial similarity to each other at their C termini. The
C-terminal conserved amino acid motifs DQMVP and
STAESSSS 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 AB028930
for the gene) and CRY5 (AcCRY5; DDBJ accession number AB028929 for the cDNA and AB028931 for the gene) are compared with Arabidopsis
CRY1 and CRY2 (AtCRY1; Ahmad and Cashmore, 1993; and AtCRY2; Lin et al., 1996a), and the three previously identified A. capillus-veneris
cryptochromes, CRY1, CRY2, and CRY3 (AcCRY1, AcCRY2, and AcCRY3; Kanegae and Wada, 1998). Sequences were aligned by using the
Clustal W program, version 1.74 (Thompson et al., 1994). The numbers at the ends of amino acid sequences indicate the positions of amino acids. 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 positions
of 14 amino acids found in E. coli photolyase to interact with FAD by either direct H bonds (circles) or indirect H bonds (squares) are indicated
below the alignment. The amino acids that are conserved between E. coli photolyase and cryptochromes are marked with closed circles and a
closed square, and those that are not conserved are marked with open circles and an open square. The TGYP motif (asterisks) and the position
of Trp-277 of E. coli photolyase (plus) are indicated below the alignment. STAESSSS motifs found in the C termini of cryptochromes are enclosed with boxes. The putative nuclear localization signals found in C termini of CRY3 and CRY4 are underlined. The N-terminal regions within
the 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.
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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 chromophores (Lin et al., 1995; Malhotra et al., 1995). The amino
acids binding to FAD have been deduced from the crystal
structure of the E. coli photolyase (Park et al., 1995). In total,
10 of the 14 amino acids forming direct or indirect H bonds
to FAD were found to be conserved in CRY4 and CRY5 (Figure 1A), which suggests that CRY4 and CRY5 might contain
FAD as one of the two chromophores. In contrast, the amino
acids binding to 5,10-methenyltetrahydrofolate in the E. coli
photolyase were poorly conserved in CRY4 and CRY5.
These chromophore binding site characteristics are similar
in the other plant cryptochromes. Amino acids presumed to
form a DNA binding site in the E. coli photolyase have been
noted; some of these are also present in CRY4 and CRY5.
However, Trp-277 in the E. coli protein, which is important
for specific binding to pyrimidine dimers (Li and Sancar,
1990), is replaced by Phe in CRY4 and Leu in CRY5, as has
been 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).
Expression Patterns of A. capillus-veneris CRY Genes in
Different Developmental Stages
Because blue light responses have been observed in various
developmental stages of A. capillus-veneris, we examined
the expression patterns of the five CRY genes at different
developmental stages by RNA gel blot analysis. All of the
CRY genes are expressed in low amounts, so we prepared
poly(A)1 RNA to detect the transcripts. As shown in Figure 3,
all of the CRY mRNAs were expressed in both the sporophyte and gametophyte stages. The CRY1 and CRY2 mRNAs showed almost identical accumulation patterns. The
amounts of these mRNAs rose a little after spore germination and stayed at the same level through the haploid and
diploid phases. The amounts of CRY3 mRNA were relatively
higher in protonemata and sporophyte tissues than in either
spores or prothallia. The expression patterns of CRY4 and
CRY5 genes were unique. The CRY4 mRNA was highly concentrated in the spores and in young leaves that had been
grown 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 depend
on the nuclear phase. Both the protonemal and prothallial
tissues examined were cultured under light, whereas the
spores were prepared in the dark. In case the expression of
the CRY4 gene might be downregulated by light, we further
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 can
catalyze DNA repair, we cloned separately the full-length
cDNAs encoding the five A. capillus-veneris CRY proteins
and the E. coli phr gene (positive control) into an E. coli
expression vector and transformed each plasmid into the
photolyase-deficient strain SY2(DE3) of E. coli. These recombinant proteins also contain a calmodulin binding protein (CBP) tag at their N termini, and the expression vector
that contained only the tag protein also was transformed as
a negative control. The expression of the CBP-tagged recombinant CRY proteins was confirmed by protein gel blotting with biotinylated calmodulin and streptavidin–alkaline
phosphatase (data not shown). After inducing synthesis of
recombinant proteins, SY2(DE3) cells harboring each plasmid were exposed to UV light for specified times. These
cells were plated in duplicate, and half the plates were irradiated with photoreactivating light (blue light) for 1 hr before
dark incubation. As shown in Figure 2, blue light exposure
did not change the survival rates of cells carrying either A.
capillus-veneris CRY cDNAs or the negative control but did
increase the survival rate of the cells carrying the E. coli phr
gene. This result indicates that A. capillus-veneris cryptochromes do not function as photolyases in E. coli cells. Together with the characteristics of the primary structures of A.
capillus-veneris CRY proteins as shown in Figure 1, this
finding suggests that A. capillus-veneris CRYs are likely to
be the cryptochromes of this fern.
Figure 2. Comparison of Photoreactivation (PR) Effects of A. capillus-veneris CRY Genes with E. coli Photolyase on Survival Rates of
the Photolyase-Deficient E. coli Strain.
The photolyase-deficient strain SY2(DE3) carrying each CRY cDNA
in the pCAL-n-EK expression vector was exposed by UV light and
plated in duplicate. Before overnight incubation in the dark, one
plate of duplicates subsequently was irradiated with blue light (1B)
for PR, but the others were not (2B). Each symbol represents the
average of three independent survival rates of the cells carrying the
following plasmids: pCALCRY1, closed circles; pCALCRY2, open
circles; pCALCRY3, closed triangles; pCALCRY4, open triangles;
pCALCRY5, closed squares; pCALPHR, open squares with positive
control; and pCALSTOP, pluses with negative control.
Cryptochrome Localization and Expression
85
creased two- to threefold within 12 hr and stayed at this
level for as long as 72 hr. Blue light, which inhibits spore
germination, had almost no effect on the amounts of either
CRY1 or CRY2 mRNA expressed but did affect the amount
of CRY3 mRNA, causing a fivefold decrease. Unexpectedly,
it was found that not only the amount of CRY4 mRNA but
also that of CRY5 mRNA was apparently regulated by light
during spore germination. However, the ways in which
mRNA accumulation was regulated by light included both
inhibition and induction (Figures 4A and 4B). For CRY4, the
amount of mRNA was reduced by z50-fold during the first
24 hr after the onset of red light irradiation; blue light reduced the level fivefold. Accumulation of CRY5 mRNA, however, was induced rapidly after the onset of exposure to
both red and blue light, and it kept increasing to 300- to
400-fold the initial dark level during the first 12 hr. The
mRNA amount then decreased for the next 12 hr, after
which it remained at a level 20- to 40-fold higher than that of
the dark control. All of the major changes in expression of
the five CRY genes occurred before spore germination (Figure 4B).
Figure 3. Expression Patterns of Five CRY mRNAs in Different Developmental Stages.
Poly(A)1 RNA (4 mg) from spores, protonemata, prothallia, lightgrown sporophytic leaves (Leaves [L]), and dark-grown leaves
(Leaves [D]) was analyzed by RNA gel blotting by using specific
probes corresponding to the C-terminal region of each CRY cDNA.
The gametophyte tissues were obtained under the following culture
conditions: imbibition for 1 day in darkness (Spores); imbibition for 4
days in darkness and incubation for an additional 3 days under red
light (Protonemata); and incubation for 1 month under white light
(Prothallia).
investigated the amount of CRY mRNAs that accumulated
during 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, the
amounts of CRY transcripts were low in all the tissues investigated. Therefore, we used competitive polymerase chain
reaction (PCR) to measure the changes in amount of individual CRY transcripts. The expression patterns of the CRY1,
CRY2, and CRY3 genes were very similar (Figure 4B). The
accumulated amounts of these mRNAs showed a 10-fold
(CRY1 and CRY2) or sixfold (CRY3) increase in the first day
after spore imbibition, but the amounts did not vary much
during the following 6 days in the dark. When the spores
were transferred to red light, which induces spore germination, the amounts of CRY1, CRY2, and CRY3 mRNAs in-
Phytochrome Involvement in the Regulation of CRY4 and
CRY5 mRNA Accumulation
To determine which photoreceptor is involved in regulating
CRY4 and CRY5 gene expression, we irradiated spores
briefly in combinations of red, blue, or far-red light, and the
expression patterns of both genes were measured by competitive PCR. Our results indicate that the amounts of both
CRY4 and CRY5 mRNAs are regulated, at least in part, by
phytochrome (Figures 5A and 5B). The accumulated
amounts of CRY4 and CRY5 mRNAs were examined at 24
and 12 hr, respectively, after the light treatments, because
the differences in the extents of their expression with or
without light are clearly discernable at those times (see Figure 4A). The effect of red light on the accumulation of both
mRNAs was cancelled by subsequent irradiation with farred light and vice versa (Figures 5A and 5B). This red/far-red
reversibility is a diagnostic characteristic of phytochrome
regulation.
A blue light pulse reduced the amount of CRY4 mRNA accumulation to that observed under continuous irradiation
with blue light; subsequent exposure to far-red light did not
alter the amount expressed. This indicates that CRY4 expression also was regulated by a blue light photoreceptor. In
contrast, a blue light pulse was not sufficient to induce a
large increase in CRY5 transcript abundance, and subsequent irradiation with far-red light decreased the amount
even further. Moreover, the amount of mRNA present after
simultaneous irradiation with continuous blue and far-red
light was 20 times less than that measured after continuous
blue light irradiation. Together, these results suggest that
continuous blue light induction of CRY5 mRNA occurred
mainly by way of a phytochrome.
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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 light
or blue light. The amounts of CRY transcripts were measured at various times after transfer. This experiment was repeated twice in its entirety
with 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 competitor (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 copy
numbers 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 labeled
similarly, 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 of
the amounts of accumulation from two experiments. The symbols indicate the light conditions: circles, dark; squares, red light; triangles, blue
light. The amounts of cDNA were determined from densitometric analysis of the gel images by using National Institutes of Health Image software.
Intracellular Localization of CRYs under Different
Light Conditions
Physiological observations suggest that the blue light photoreceptor that mediates inhibition of spore germination and
induction of the cell cycle is localized in or close to the nucleus (Kadota et al., 1986; Furuya et al., 1997). To determine
whether any cryptochromes are located in the nucleus, we
transiently expressed in fern gametophytes fusion genes containing each CRY cDNA inserted as a translational fusion to
Cryptochrome Localization and Expression
b-glucuronidase (GUS). Because both red and blue light are
known to cause various responses during the early stages of
gametophyte development, the transfected cells were incubated under either red light or blue light or in the dark to examine the effects of light on the intracellular distribution of
CRY proteins. Representative intracellular staining patterns of
GUS–CRY fusion proteins are shown in Figure 6. We observed the same distribution patterns of each GUS fusion
protein in both the basal cells and the tip cells. The intracellular localization of GUS activity varied slightly among individual
cells, even those expressing a single fusion construct, probably because of damage from particle bombardment. Therefore, we show the tendency of the intracellular distribution
pattern of each GUS–CRY fusion protein in Figure 7.
The GUS–CRY3 and GUS–CRY4 proteins showed clear
nuclear localization, but GUS–CRY1, GUS–CRY2, and
GUS–CRY5 did not. The GUS–CRY3 protein tended to accumulate in the nucleus in the dark and in red light but not in
blue light. The GUS–CRY4 protein was predominantly nuclear under all the light conditions examined. The degree of
nuclear enrichment of GUS staining in GUS–CRY3—
expressing cells under red light seemed to be less than that
in dark-incubated cells (see Figure 6). In the dark, a few of
the cells showed nuclear enrichment in GUS–CRY1 and
GUS–CRY2; in those cells, GUS activity also was observed
in the cytoplasm. In onion epidermal cells, we also observed
nuclear localization of GUS–CRY3 and GUS–CRY4 but not
of GUS–CRY1, GUS–CRY2, and GUS–CRY5 (data not
shown). The expression rates of all GUS–CRY fusion proteins in A. capillus-veneris—especially those of GUS–CRY1
and GUS–CRY2—under red or blue light were much lower
than their expression rates in the dark. This observation suggests that a light-dependent protein degradation mechanism similar to that seen for Arabidopsis CRY2 (Lin et al.,
1998) and Drosophila CRY (Emery et al., 1998) might also
operate in A. capillus-veneris.
The N-terminal regions of cryptochromes, especially of
the A. capillus-veneris CRYs, are highly conserved with respect to each other (see Figure 1A). Nonetheless, our results
indicate that the subcellular distributions of these cryptochromes differ, with only GUS–CRY3 and GUS–CRY4
showing clear nuclear localization. This implies that the divergent C-terminal regions of these cryptochromes might
play important roles in the determination of subcellular distribution. In addition, small basic amino acid clusters (e.g.,
KRKAK), which might function as monopartite nuclear localization signals, are present in the conserved regions found
at the CRY3 and CRY4 C termini (Figure 1A). To test this
possibility, we generated fusion genes encoding GUS at the
N-terminal half and CRY3 or CRY4 C-terminal regions at the
C-terminal half (GUS–CRY3C and GUS–CRY4C) and transformed these into A. capillus-veneris cells. The results show
that the light-dependent intracellular localization patterns of
GUS–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
87
regions of both CRY3 and CRY4 are required to import
these proteins into the nucleus.
DISCUSSION
Including the two additional cryptochrome genes reported
here, the A. capillus-veneris cryptochrome gene family comprises at least five members, all of which are expressed during the haploid and diploid phases of the life cycle (see
Figure 5. Effects of Pulse Irradiation with Red, Far-Red, and Blue
Light on the Regulation of CRY4 and CRY5 Gene Expression.
The effects of subsequent pulse irradiation (10 min each) with either
red light (R) and far-red light (FR) or blue light (B) and far-red light
(FR) on CRY4 and CRY5 transcript accumulation were measured using competitive PCR. Four-day dark-imbibed spores were irradiated
in turn with red light and far-red light pulses or with blue light and
far-red light. After 24- and 12-hr (h) dark incubation (for CRY4 and
CRY5, 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 and
CRY5.
(B) Relative amounts of CRY4 and CRY5 mRNAs. Each bar shows
the average from two experiments. The gray, dotted, and black horizontal lines in each panel indicate the amounts of CRY4 and CRY5
cDNAs after the same lengths (24 and 12 hr) of continuous red (Rc)
and blue (Bc) light exposure and of darkness (Dc), respectively (see
Figure 4B).
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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 conditions. The cells showing GUS activity were photographed by using Nomarski optics (left panels); the fluorescence micrographs show the position of the nuclei in the same cells after staining with 49,6-diamidino-2-phenylindole (right panels). Note that under fluorescence, chlorophyll
autofluoresces red and spore coats appear bluish white. Bar in (X) 5 20 mm for (A) to (X).
Cryptochrome Localization and Expression
Figure 3). Until now, the two Arabidopsis CRY genes represented the only completely characterized family (Ahmad and
Cashmore, 1993; Lin et al., 1998). The five-member gene
family that we report here for the fern A. capillus-veneris is
the largest cryptochrome gene family characterized to date.
Considering the complexity of blue light–induced phenomena found in this fern, it is not surprising that this fern has a
large number of blue light photoreceptors.
The N termini of these five cryptochromes are highly conserved, and the amino acid residues necessary for interaction with FAD and presumptive DNA binding are found
mostly in the same positions as in the E. coli photolyase.
However, as we have shown, none of these cryptochromes
has photolyase activity. Their characteristics, therefore, are
the same as in previously identified cryptochromes, suggesting that the same redox electron transfer mechanism
from blue light photons by way of FAD also functions in A.
capillus-veneris CRY proteins. The STAESSSS-related amino
acid motif also is found at the C termini of CRY2, CRY3, and
CRY4 (STAESTS, PMTESSSS, and STAESSSS, respectively). Although the function of this motif is unknown, it has
been reported to be important for the phosphorylation of
cryptochrome 1 by phytochrome A in vitro (Ahmad et al.,
1998b). Physiological responses in A. capillus-veneris that are
regulated antagonistically or cooperatively by phytochrome
and blue light photoreceptors include spore germination,
phototropism, the cell cycle, and chloroplast movement
(Wada and Sugai, 1994). We have identified three phytochrome 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 expressed in yeast have serine/threonine kinase activity. Thus,
in the signal transduction cascades upstream of some photoresponses in A. capillus-veneris, direct interaction of phytochromes and cryptochromes might happen by way of the
STAESSSS motif by phosphorylation. However, the A. capillus-veneris CRY1, CRY5, Chlamydomonas CPH1, and S. alba
SA-PHR1 sequences do not possess the STAESSSS motif,
which suggests other blue light signal transduction cascades that do not involve phosphorylation of this motif are
present.
Cryptochromes have been found in divergent species
across the biological kingdom (Cashmore et al., 1999). Of
particular interest is how cryptochromes evolved in plants.
To address this question, we performed a phylogenetic tree
analysis using 10 full-length cryptochrome cDNA sequences
(alga, Chlamydomonas [Small et al., 1995]; moss, Physcomitrella patens [Imaizumi et al., 1999]; fern, A. capillusveneris [Kanegae and Wada, 1998; this article]; dicots, Arabidopsis [Ahmad and Cashmore, 1993; Lin et al., 1996a] and
S. alba [Batschauer, 1993]) and related partial sequences
found in the database. The regions of each N terminus indicated in Figure 1 were used for construction of the tree, and
the bootstrap values from 1000 replicates were calculated
by using the neighbor-joining method. The tree was rooted
by using Chlamydomonas CPH1 as the outgroup (Figure 8).
89
Figure 7. Intracellular Distribution of GUS–CRY Fusion Proteins under Different Light Conditions.
Distribution patterns of GUS activities in individual transformed cells
were classified into two groups: (1) nuclear enrichment of GUS activity was seen (nucleus [Nuc.] . cytoplasm [Cyto.]) or (2) nuclear enrichment of GUS activity was not seen (Nuc. < Cyto.). These cells
expressed each GUS–CRY fusion protein under red light (striped
bars), blue light (open bars), or in the dark (black bars). From protonemal cells prepared under the same experimental conditions as
in Figure 6, we randomly selected cells that showed GUS activities.
The first group (Nuc. . Cyto.) contained cells that showed clear nuclear enrichment with various degrees of cytoplasmic GUS staining;
and the second group (Nuc. < Cyto.) contained cells that showed
nuclear staining indistinguishable from or less than cytoplasmic
staining. The data were obtained from between three and seven independent experiments. In total, we examined 45, 119, and 104
cells (red light, blue light, and dark treatment, respectively) for GUS–
CRY1; 30, 76, and 117 cells for GUS–CRY2; 117, 129, and 114 cells
for 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 for
GUS–CRY4C. For each experiment, we calculated the percentage of
population of each group, dividing the number of cells belonging to
each group by the total number of cells examined in that experiment. The bars show the average percentages of populations
derived from all experiments. Standard errors of the mean are indicated.
Although some branching points are not reliable because of
the low bootstrap values, the branching order of cryptochromes and the related sequences closely correspond to
the organismal phylogeny. As in the phylogenetic analysis of
another plant photoreceptor, phytochrome (Mathews and
Sharrock, 1997), multiple cryptochrome lineages in cryptogams 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 are
in each cluster. Three recent independent gene duplications
might have occurred to yield CRY3 and CRY4, CRY1/CRY2
and CRY5, and CRY1 and CRY2. This information, together
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Figure 8. Phylogenetic Analysis of Plant Cryptochromes.
The regions of partial amino acid sequences used to generate the
phylogenetic tree are shown in Figure 1. The neighbor-joining
method was used to calculate bootstrap values from 1000 replicates
(http://www.genome.ad.jp/) and to draw the tree by using TreeView
software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). The
numbers located at branch points are the bootstrap values. GenBank/DDBJ/EMBL accession numbers for each cryptochrome DNA
sequence, except for those given in the legend of Figure 1, are as
follows: Angiopteris for Angiopteris evecta, X99261; Avena for Avena
sativa, X99262; CPH1 for Chlamydomonas CPH1, L07561; Equisetum for Equisetum arvense, X99263; Maize1 and Maize2 for Zea
mays, 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 esculentum, AJ000695.
with the mRNA accumulation pattern of CRY1 and CRY2
(Figure 3) and the intracellular distribution of CRY1/CRY2
and CRY3/CRY4 (Figures 6 and 7), indicates that CRY1/
CRY2 and CRY3/CRY4 might comprise subfamilies of A.
capillus-veneris cryptochromes. Because the diversity of
cryptogam phytochrome genes is thought to result from
taxon-specific diversification, it is of great interest to learn
whether the diversification of A. capillus-veneris CRY genes
also occurred taxon specifically, as in the case of the cryptogam phytochromes, or whether cryptogams have more than
one cryptochrome subfamily, as has been suggested for the
phytochromes of seed plants. In seed plants, cryptochromerelated partial sequences from monocots are closely related
to Arabidopsis CRY1, whereas sequences from dicots are in
the same cluster as Arabidopsis CRY2. It is also of interest
to know whether seed plant cryptochromes diverged before
or after the diversification of dicots and monocots. To address these questions requires identifying additional cryptochrome genes from other plant species.
In A. capillus-veneris, the previous physiological observations indicated that blue light photoreceptors were involved in phototropism (Hayami et al., 1992), apical
swelling (Wada et al., 1978), and chloroplast orientational
movement (Yatsuhashi et al., 1987) localized on or close to
the plasma membrane in protonemata. Figures 6 and 7
show that CRY1, CRY2, and CRY5 proteins are localized
in the cytoplasm. We could not find the putative membrane-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 domains, was enriched in the membrane fractions (Ahmad et
al., 1998c). Possibly, therefore, some portions of CRY1,
CRY2, or CRY5 proteins may attach to the plasma membrane where they might be involved in blue light–induced
phototropism, apical swelling, chloroplast orientational movement in protonemata, or some combination of these responses.
Blue light suppresses the entry into the S phase of the first
mitosis leading to spore germination (Uchida and Furuya,
1997). In protonemata, blue light photoreceptors also control shortening of the G1 phase (Miyata et al., 1979). The blue
light photoreceptors involved in these responses are localized in or close to the nucleus (Wada and Furuya, 1978;
Furuya et al., 1997). Such physiological observations indicate that the blue light photoreceptor mediating inhibition of
spore germination must be present in or close to the nucleus before the onset of blue light irradiation. Although
whether these blue light photoreceptors are derived from
the maternal cytoplasm or are newly translated during imbibition in the dark is unknown, CRY1, CRY2, CRY3, and
CRY4 genes would be the candidates for such receptors,
given our results showing that these mRNAs accumulate in
dark-imbibed spores (Figure 4). In addition, we demonstrated that GUS–CRY3 and GUS–CRY4 apparently accumulate in the nucleus in dark-incubated protonemal cells
(Figures 6 and 7).
These results are consistent with the notion that CRY3
and/or CRY4 genes could encode the blue light photoreceptors regulating spore germination. Under natural light conditions, the ratio between blue light and red light may differ,
depending on the surrounding environment, but both are
always present. Even after the steps leading to spore
germination are activated by light signals by way of a
phytochrome, any blue light photoreceptors working antagonistically would interfere with germination. Although we
could not find any amino acid sequences that fit the consensus sequence of the nuclear export signal in the C-terminal
region of CRY3 (Bogerd et al., 1996; Kim et al., 1996), our
data showed the apparent blue light–dependent export of
GUS–CRY3 fusion protein from the nuclei of dark-grown
cells to the cytoplasm (Figures 6 and 7). If CRY3 is the blue
light photoreceptor of the inhibition of spore germination,
the nuclear export of CRY3 might be one of the important
mechanisms to cause the inhibition of spore germination.
CRY4 seems to localize in the nucleus under all light conditions examined, but the accumulation of CRY4 mRNA is
regulated by light quality. If CRY4 is the blue light photoreceptor that inhibits spore germination, the suppression of
CRY4 mRNA accumulation observed under red light (Figures
4 and 5) might reduce the interruption of the germination
Cryptochrome Localization and Expression
processes by blue light, allowing uninterrupted completion
of germination. Currently, the answers to these questions
remain obscure, but abundant physiological data suggest
that cryptochromes might work as negative photoswitches
for cell cycle promotion in germination of A. capillus-veneris
spores.
METHODS
Plant Materials and Light Sources
Spores of Adiantum capillus-veneris were harvested in either 1994 or
1995 in a greenhouse at Tokyo Metropolitan University. Spores were
sterilized with 10% (v/v) antiformin (NaClO) containing 0.1% (v/v) Triton X-100. After three rinses with sterile distilled water, the spores
were suspended in 1:10 strength of modified Murashige and Skoog
culture medium (Wada and Furuya, 1970) and sown on a cellophane
sheet that covered the same Murashige and Skoog medium solidified with 0.5% (w/v) agar. The spores were incubated under different
light conditions at 25 6 18C. The gametophytes were collected on filter paper by using vacuum filtration; they were then frozen immediately in liquid nitrogen and stored at 2808C before use. All
manipulations after sowing were performed under a dim green safelight (Kadota et al., 1984). Sporophytes were cultivated in the greenhouse under natural light conditions. After all leaves were cut, young
light-grown leaf tissue was collected as emerging croziers. Some
sporophytes were transferred into complete darkness after removal
of all the leaves. Young dark-grown leaves (croziers) were collected
in the dark for 3 to 4 weeks under a safelight. This tissue, too, was
frozen 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, Japan). Red light (9.9 mmol m22 sec 21) was obtained from either redemitting fluorescent tubes (model FL20S-Re-66; Toshiba Ltd.) or fluorescent tubes (model FL40SD; Toshiba Ltd.) filtered through a red
plastic sheet (model Acrylite 102; Mitsubishi Rayon, Tokyo, Japan).
Far-red light (9.2 mmol m22 sec 21) was provided by far-red-emitting
fluorescent tubes (model FL20S-FR-74; Toshiba Ltd.) and filtered
through the same red plastic sheet (model Acylite 102; Mitsubishi
Rayon) and a far-red filter (IR-1; Koto Electronic Company Ltd.,
Urawa, Japan). Blue light (4.6 mmol m22 sec 21) was obtained from
blue-emitting fluorescent tubes (models FL20S-B and FL20S-BW;
Toshiba Ltd.) filtered through a blue plastic sheet (model Acrylite
302; Mitsubishi Rayon). The light conditions described above were
used throughout all experiments.
DNA and RNA Isolation
Genomic DNA and total RNA derived from either gametophytes or
sporophytes were isolated by using the cetyltrimethylammonium
bromide (CTAB) method (Kanegae and Wada, 1998), with some
modifications. When gametophyte tissue was ground with a mortar
and pestle, an amount of sterilized quartz sand equal to 10 times the
volume (w/w) was added to each sample to help break the cell walls
in liquid nitrogen. Frozen tissue was incubated with an amount of 2 3
CTAB 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
91
(w/v) containing 5% (v/v) 2-mercaptoethanol at 608C. The chloroform
treatments were repeated three times. DNA was precipitated with
2-propanol. RNA was precipitated twice with LiCl. The precipitate
was dissolved in Tris-EDTA or diethyl pyrocarbonate–treated water.
To purify poly(A)1 RNA, we used mRNA purification kits (Amersham
Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer’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 the
A. capillus-veneris genomic l phage library by using the full-length
Arabidopsis CRY1 cDNA as a probe (Kanegae and Wada, 1998).
These clones were subcloned into the plasmid vector pBluescript II
SK1 (Stratagene, La Jolla, CA). Both genomic clones and cDNA
clones were sequenced by using either fluorescent-labeled primers
or dye terminators (BigDye terminator cycle sequencing ready reaction; Applied Biosystems, Foster City, CA). For isolation of CRY4 and
CRY5 cDNAs, a 39 rapid amplification of cDNA ends (RACE) kit (Life
Technologies, Rockville, MD) and a 59 RACE kit (Life Technologies)
were used according to the system protocols. In 39 RACE, cDNA
synthesized from 1 mg of total RNA derived from 1-day dark-imbibed
spores was amplified by gene-specific primers: 59-CCAAGCTTGGGGAGGAGAG-39 for CRY4 and 59-AGGGAAGCCAGATGAAGAGC-39
for CRY5. Both primary polymerase chain reaction (PCR) products
were amplified with the same nested primer (59-CCYYTDGTKGATGCHGGVATG-39). In 59 RACE, first-strand cDNAs were synthesized
from the same total RNA used in 39 RACE by using gene-specific
primers: 59-AAGCCTCAGCATCAGT-39 for CRY4 and 59-AACTAATGGTCGTGGA-39 for CRY5. The cDNAs were then amplified by PCR
with nested gene-specific primers: 59-CCCAAGCACATCAGACTCCAAA-39 for CRY4 and 59-TCATCCATCCGTTCGAGTTC-39 for CRY5.
Primary PCR products that were between 1 and 2.5 kb long were recovered and reamplified by using another nested gene-specific
primer: 59-AGACTATGACTCGCACTCGGTT-39 for CRY4 and 59-CCCATGTCCAAGGTAGCTGC-39 for CRY5. Afterward, 39- and 59 RACE
products were cloned into the plasmid vector pGEM-T Easy (Promega, Madison, WI) and sequenced.
Photoreactivation Analysis
Full-length coding regions of five CRYs and an Escherichia coli photolyase (phr) gene (pRT2; kindly provided by T. Todo, Kyoto University, Kyoto, Japan) were cloned into the expression vector pCALn-EK (affinity LIC cloning and protein purification kit; Stratagene) according to the manufacturer’s procedure. The primers used for PCR
cloning were the following: 59-GACGACGACAAGGCCTGCACAATTGTGTGGTT-39 and 59-GGAACAAGACCCGTGAGTTTCTGAGACAATCT-39 for pCALCRY1; 5 9-GACGACGACAAGGCGGCACACACAATTGTGGC-39 and 59-GGAACAAGACCCGTCTGACCCTTAACTTCAAC-39 for pCALCRY2; 59-GACGACGACAAGGCAAAATCATGTACCGTTGT-39 and 59-GGAACAAGACCCGTCACGTCAGTTTAAACAAC-39 for pCALCRY3; 59-GACGACGACAAGGCAAAACCTTGTACAATAGT-39 and 59-GGAACAAGACCCGTCACCGATGCATTTTTTCG-39
for pCALCRY4; 59-GACGACGACAAGACCACCTCTACAACCATTGT-39 and 59-GGAACAAGACCCGTAGCTGCAGACTAATCAAC-39 for
pCALCRY5; and 59-GACGACGACAAGACTACCCATCTGGTCTGGTT-39 and 59-GGAACAAGACCCGTATTGCCTGACGCGTCTGT-39
92
The Plant Cell
for pCALPHR. These recombinant proteins possess a calmodulin
binding protein (CBP) tag in the N terminus, so a vector expressing
only the CBP was generated to insert synthetic oligonucleotides consisting of three stop codons (TGATAATAG) into the cloning site as a
negative control vector (pCALSTOP). The resulting clones were sequenced to confirm that they contained the correct inserts. A photolyase-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) to
enable the expression system to use the T7 RNA polymerase promoter. Plasmids pCALCRY1, pCALCRY2, pCALCRY3, pCALCRY4,
pCALCRY5, pCALPHR, and pCALSTOP were transformed into the
resulting SY2(DE3) cells. The SY2(DE3) cells harboring each plasmid
were cultured until the OD600 reached 0.6; then isopropylthiogalactopyranoside was added to a final concentration of 1 mM. After an additional 3.5-hr incubation at 378C in the dark, the cells were collected
and washed with phosphate buffer (pH 6.8; Yamamoto, 1985). The
cells were resuspended in phosphate buffer at a concentration of 1
to 2 3 107 cells per mL. The cell suspensions, in 30-mm-diameter
plastic dishes, were exposed to UV light (0.2 mmol m22 sec 21) obtained from a germicidal light tube (model GL-20; Toshiba Ltd.) and
then plated in duplicate in two to three dilution series. For photoreactivation, half the group of Luria-Bertani plates were irradiated with
blue light for 1 hr before overnight incubation in the dark. The number
of surviving colonies was independently counted three times to calculate the survival rates. After resuspension of the cells, all manipulations 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% formaldehyde-agarose gels and transferred to nylon membranes (Hybond
N1; Amersham Pharmacia Biotech) over a period of 14 to 16 hr with
20 3 SSC (1 3 SSC is 0.15 M NaCl and 0.015 M sodium citrate). For
hybridization and detection, a chemifluorescence-labeled gene detection kit (Gene Images; Amersham Pharmacia Biotech) was used
according to the manufacturer’s instruction. The 39 regions of each
CRY cDNA (nucleotide sequence positions 1451 to 2457 for the
CRY1 cDNA, 1691 to 2754 for the CRY2 cDNA, 1448 to 2440 for the
CRY3 cDNA, 1634 to 2857 for the CRY4 cDNA, and 1328 to 2444 for
the CRY5 cDNA) were amplified by PCR to be used as probes. The
blots were hybridized with the probes at 658C. The membranes were
washed 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 not
occur when we performed DNA gel blot analyses with the same
probes at the lower stringency condition (0.5 3 SSC, 0.1% [w/v] SDS
at 608C for the second wash; data not shown).
Competitive Reverse Transcription–PCR Analysis
DNA competitors were designed to have different sequences on
both 59 and 39 ends in which each set of CRY-specific primers could
be annealed but with the same sequences derived from l phage
DNA in between (Competitive DNA construction kit; Takara Shuzo
Co., Kyoto, Japan). Five primer sets were used for PCR synthesis of
each competitor: 59-CAGAATTTATGCCCGAGTCCGCGTGAGTATTACGAAGGTG-39 and 5 9-TATGGAAACAGGAAGACCAGTGAAGACGACGCGAAATTCA-39 for CRY1 competitor; 59-GCACATTCACAC-
AACTCTCCGCGTGAGTATTACGAAGGTG-39 and 59-CTAATGTTGGTGTAAGGAGGTGAAGACGACGCGAAATTCA-39 for CRY2 competitor; 59-GCCCATTGAGAGCCATTAAGGCGTGAGTATTACGAAGGTG-39 and 59-AGCTCACGACGGGATCAAATTGAAGACGACGCGAAATTCA-39 for CRY3 competitor; 59-CTCTGACAATGGTGACTTCTGCGTGAGTATTACGAAGGTG-39 and 59-AGACAGCAGTGGCAGGAACATGAAGACGACGCGAAATTCA-39 for CRY4 competitor; and
59-ACAATCCACCAATGAGCCCAGCGTGAGTATTACGAAGGTG-39
and 59-GAACTCGAACGGATGGATGATGAAGACGACGCGAAATTCA39 for CRY5 competitor.
A gradient series from 101 to 108 copies per mL of competitors was
used for the analysis. Complementary DNA was synthesized from 2
mg of each total RNA pretreated with DNase I (Stratagene) by using
an oligo(dT) primer according to the supplied procedure (SUPERSCRIPT preamplification system for first-strand cDNA synthesis; Life
Technologies). One-tenth volume of the reaction solution of cDNA
and 1 mL of each concentration of competitors were put into the
same reaction tube. The locations of the primers were designed to
contain the last intron of each CRY gene so that we could distinguish
whether the amplified fragments were derived from cDNA or from
contaminated genomic DNA. PCR was performed with the following
primer sets: 59-CAGAATTTATGCCCGAGTCC-39 and 59-TATGGAAACAGGAAGACCAG-39 for CRY1; 59-GCACATTCACACAACTCTCC-39 and
59-CTAATGTTGGTGTAAGGAGG-39 for CRY2; 59-GCCCATTGAGAGCCATTAAG-39 and 59-AGCTCACGACGGGATCAAAT-39 for CRY3;
59-CTCTGACAATGGTGACTTCT-39 and 59-AGACAGCAGTGGCAGGAACA-39 for CRY4; and 59-GAACTCGAACGGATGGATGA-39 and
59-ACAATCCACCAATGAGCCCA-39 for CRY5. The PCR schedule
was 1 min at 948C; 40 cycles of 10 sec at 988C, 30 sec at 558C, and
30 sec at 728C; and then 3 min at 728C. Equal volumes of PCR products were separated in 3% (w/v) agarose gels. The expected lengths
of amplified fragments derived either from each CRY cDNA or from
each competitor were 0.5 and 0.4 kb, respectively. The gel images
were captured and analyzed by using the public domain National Institutes 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) was
used to construct vectors containing b-glucuronidase (GUS)–CRY
fusion genes. pBI221 plasmid (Clontech, Palo Alto, CA) that contained a cauliflower mosaic virus 35S promoter–driven GUS gene
was used as a template. The primers used for making GUS–CRY
constructs were as follows (forward primer and reverse primer, respectively): 59-AGTTACTCTTCAGCTACCGAGCTCGAATTTCCCCGA39 and 59-AGTTACTCTTCATTGTTTGCCTCCCTGCTGCGGTTT-39
for amplifying the pBI221 vector that has a specific in-frame cloning
site; 59-AGTTACTCTTCACAAGCCTGCACAATTGTGTGGTTTCGG-39
and 59-AGTTACTCTTCAAGCGAGAACCAACCAACCGCCACATTC-39
for CRY1 insert; 59-AGTTACTCTTCACAAGCGGCACACACAATTGTGGCACAC-39 and 59-AGTTACTCTTCAAGCCCAGGCTTTGATCCTAGTGGGTGT-39 for CRY2 insert; 59-AGTTACTCTTCACAAGCAAAATCATGTACCGTTGTGTGG-39 and 59-AGTTACTCTTCAAGCCAGTGCAACTGCACGTCAGTTTAAAC-39 for CRY3 insert; 59-AGTTACTCTTCACAAGCAAAACCTTGTACAATAGTGTGG-39 and 59-AGTTACTCTTCAAGCCGTCACCAGCCCCACTCATGAAGA-39 for CRY4 insert;
59-AGTTACTCTTCACAAACCACCTCTACAACCATTGTCTGGC-39 and
59-AGTTACTCTTCAAGCGCTGGGGCTCTGAGACATCCTTTC-39 for
CRY5 insert; 59-AGTTACTCTTCACAAGTGGGCGATTCAATTGCGAAGGTC-39 for CRY3C insert; and 59-AGTTACTCTTCACAACCA-
Cryptochrome Localization and Expression
CCACTGGTTTCAACTCACTCC-39 for CRY4C insert. To amplify
CRY3C and CRY4C inserts, we used the same reverse primers as for
the CRY3 and CRY4 inserts. Both junctions between vector and amplified CRY inserts and CRY inserts themselves were sequenced to
confirm whether or not these fragments were inserted in the same
frame correctly.
Analysis of GUS Intracellular Localization in Protonemal Cells
Gold particles (1.6 mm in diameter) coated with GUS–CRY or pBI221
plasmids were introduced into the two-celled protonemata, which
were incubated for 4 days in the dark and for an additional 3 days under red light, by using a biolistic gun transformation system (DuPont,
Wilmington, DE). To bombard gold particles, we used 1350-psi rupture discs according to the manufacturer’s procedure. Transfected
cells were incubated in red light, blue light, or in the dark for 16 hr at
258C and then stained with 100 mM sodium phosphate, pH 7.0, 1
mM 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-phenylindole, and 1 mM X-gluc. Staining was performed under the same
light conditions at 378C overnight. Cells showing GUS staining were
selected under low magnifying power (310 to 25) through a stereomicroscope, and the intracellular localization of GUS products was
observed under a microscope (3400). The optic images of GUS activity and 49,6-diamidino-2-phenylindole staining were obtained by
using a microscope equipped with Nomarski optics. A dim green
safelight was used in every step of the transfections and staining.
Transfections using each recombinant plasmid were repeated at
least five times.
ACKNOWLEDGMENTS
We thank Dr. Takeshi Todo for providing the SY2 strain and the pRT2
plasmid. We are grateful to Dr. Jane Silverthorne (University of California, Santa Cruz) for critical reading of the manuscript. We thank HyunSook Park for 39 RACE analysis and Kayoko Hara, Eitetsu Sugiyama,
and Takako Yasuki for their technical assistance. This work was supported in part by the Grant-in-Aid for International Scientific Research
(Joint Research) (Grant Nos. 07044206 and 09044232) and for Scientific Research (B) (Grant Nos. 07458196 and 09440270) from the
Ministry of Education, Science, Sports, and Culture of Japan; by
grants from the Mitsubishi Foundation, PROBRAIN (Program for Promotion of Basic Research Activities for Innovative Biosciences), and
the NOVARTIS Foundation to M.W.; by the Grant-in-Aid for Encouragement of Young Scientists (Grant No. 10740373) from the Ministry
of Education, Science, Sports, and Culture of Japan to T.K.; and also
by a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Grant No. 10639301) to T.I.
Received July 19, 1999; accepted November 1, 1999.
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Quality in the Fern Adiantum capillus-veneris
Takato Imaizumi, Takeshi Kanegae and Masamitsu Wada
Plant Cell 2000;12;81-95
DOI 10.1105/tpc.12.1.81
This information is current as of July 31, 2017
References
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