Mode of phytochrome B action in the photoregulation of seed ...

The Plant Journal (1998) 13(5), 583–590
Mode of phytochrome B action in the photoregulation of
seed germination in Arabidopsis thaliana
Tomoko Shinomura1 , Hiroko Hanzawa1 ,
Eberhard Schäfer2 and Masaki Furuya1,* 1Hitachi Advanced Research Laboratory, Hatoyama,
Saitama 350–0395, Japan, and
2Institut für Biologie II, Albert Ludwigs Universität,
Schänzlestrasse 1, D-7800 Freiburg, Germany
Summary
Arabidopsis thaliana seeds imbibed for a short duration
show phytochrome B (PhyB)-specific photo-induction of
germination. Using this system, the relationship was
determined between the amount of PhyB in seeds and
photon energy required for PhyB-specific germination in
two transgenic Arabidopsis lines transformed with either
the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA
(RBO). Immunochemical detection of PhyB apoprotein
(PHYB) showed that the expression level of PHYB in ABO
seeds was at least two times higher than that in the
wild-type seeds, but in RBO seeds the PHYB level was
indistinguishable from that in wild-type seeds. The photon
fluence required for induction and photoreversible inhibition
of germination was examined using the Okazaki large
spectrograph. At the wavelengths of 400–710 nm, the ABO
seeds required significantly less photon fluence than wildtype
seeds for induction of germination, whereas the RBO
seeds required similar fluence to wild-type seeds. A critical
threshold wavelength for either induction or inhibition of
germination of ABO seeds shifted towards the longer
wavelengths relative to wild-type seeds. By assuming
that PhyA and PhyB are similar in their photochemical
parameters, amounts of P fr at each wavelength were
calculated. The photon fluence required for 50% germination
was equivalent to the fluence generating a P fr/P tot
ratio of 0.21–0.43 in wild-type seeds, and of 0.035–0.056
in ABO seeds. These results indicate that PhyB-specific
seed germination is not strictly a function of the P fr/
P tot ratio, but is probably a function of the absolute P fr
concentration.
Introduction
Light plays many crucial roles in plant development
(Hendricks and Borthwick, 1967; Mohr and Shropshire,
1983). Phytochrome regulates many of these responses to
Received 14 July 1997; revised 20 October 1997; accepted 30 October 1997.
*For correspondence (fax �81 492 96 7511;
e-mail mfuruya@harl.hitachi.co.jp).
light. It undergoes a reversible photoconversion between
the red light (R)-absorbing form (P r) and the far-red light
(FR)-absorbing form (P fr) upon light irradiation (Butler et al.,
1959). P r and P fr are commonly considered to be the
physiologically inactive form and the active form, respectively
(Borthwick et al., 1952; Butler et al., 1959).
Early papers showed that physiological responses such
as seed germination (Borthwick et al., 1954), hook opening
(Klein et al., 1967) and inhibition of mosocotyl elongation
(Weintraub and Price, 1947) were proportional to the logarithm
of the incident energy, except for the extreme values
near threshold and saturation. The photochemical transformations
of phytochrome in both directions (from P r to
P fr and from P r to P fr) are first-order with respect to
the incident energy (Butler et al., 1964). Therefore, it was
expected that the magnitude of a physiological response
to R and FR would be related in some simple way to the
amount of P fr, which can be measured spectrophotometrically
in vivo. However, many attempts to determine
such a correlation have yielded inconclusive results
(Furuya, 1989, 1993; Hillman, 1967). Explanations for these
results invoke the idea that the bulk of phytochrome
detected spectrophotometrically is inactive, and the active
fraction has different properties from the inactive fraction
(sensitivity for photoconversion, FR reversibility, rate of
dark reversion, etc.) (Hillman, 1967).
Since the discovery of the phytochrome multi-gene
family (Clack et al., 1994; Sharrock and Quail, 1989),
research has been directed to determining which member
of the phytochrome family is responsible for which phytochrome-mediated
response(s). Among them, phytochrome
A (PhyA) and phytochrome B (PhyB) have been the best
analysed so far (Furuya and Schäfer, 1996). The absorption
spectrum of PhyA (see, for example, Butler et al., 1964) is
similar to that of PhyB (Abe et al., 1989; Kunkel et al., 1993).
In contrast, PhyA and PhyB display a marked diversity in
amino acid sequences (Sharrock and Quail, 1989) and
expression patterns (Somers and Quail, 1995). Analysis of
PhyA-null mutants (phyA) and PhyB-null mutants (phyB)
of Arabidopsis has led to the proposal that PhyA and PhyB
have overlapping physiological functions but perceive distinct
features of the light environment, such as wavelength
and intensity. For example, photo-inhibition of hypocotyl
elongation in Arabidopsis is regulated by both PhyA and
PhyB, but they are active under continuous irradiation with
FR and R, respectively (Quail et al., 1995). The photoregulation
of seed germination in Arabidopsis is also mediated
by both PhyA and PhyB, but the action spectra for the
response mediated by these two phytochromes are quite
© 1998 Blackwell Science Ltd 583

584 Tomoko Shinomura et al.
different in their photon fluence and wavelength requirements
(Shinomura et al., 1996).
These data suggest that one of the explanations for the
previous inability to correlate physiological responses with
the amount of P fr was due to the simultaneous spectrophotometric
measurement of multiple phytochromes with
differential responses. In order to address this question,
we concentrated on PhyB-specific seed germination using
PhyB over-expressing transgenic plants. The PhyB role in
seed germination is distinguishable from that of the other
phytochromes when seeds are allowed to imbibe water
for a short period before the light treatment (Shinomura
et al., 1994, 1996). The over-expression level of phytochrome(s)
in seed has not been demonstrated in transgenics
of phytochrome genes, although the expression
level in seedlings were measured both immunochemically
and spectrophotometrically (Wagner et al., 1991).
We describe here light fluence–response relationships
in PhyB over-expressing transgenics that showed different
PhyB levels in seeds. Quantitative correlation between
the amount of PhyB in P fr form in seeds and the light
energy required for PhyB-induced germination will be
discussed.
Results
Immunochemical determination of PHYB amount in
seeds of RBO and ABO
The amount of PhyB apoprotein (PHYB) in seeds was
quantified by immunoblot analysis of crude seed extracts
from two homozygous transgenic lines, transformed with
either the cDNA of the Arabidopsis PhyB gene (ABO) or
the cDNA of the rice PhyB gene (RBO). Using a monoclonal
antibody which reacts with Arabidopsis PHYB, it was demonstrated
that the total amount of PHYB in ABO seeds was
greater than that in the wild-type (WT) seeds (Figure 1b).
The magnitude of the difference in PhyB expression levels
between ABO and WT seeds was smaller than that in
seedlings (Figure 1b). Based on a serial dilution comparison
of the extracts derived from ABO and WT, we estimated
that ABO seeds showed more than twice the level of PHYB
than WT seeds (Figure 1c). However, using the monoclonal
antibody which reacts with both rice PHYB and Arabidopsis
PHYB, the amount of PHYB in RBO seeds was indistinguishable
from the levels observed in WT seeds (Figure 1a).
In contrast, the amount of PHYB in RBO seedlings was
substantially higher than that in WT seedlings (Figure 1a).
In conclusion, both ABO and RBO seeds showed lower
expression levels of PHYB than seedlings, and only ABO
seeds exhibited substantial over-expression of PHYB in
seeds.
Figure 1. Immunochemical detection of Arabidopsis PHYB and rice PHYB
apoproteins extracted from seeds and seedlings of RBO and ABO.
(a) Crude protein extracts from seeds and seedlings of WT and RBO were
loaded (50 μg protein in each lane) and probed with mBT4 which reacts
with both rice PHYB and Arabidopsis PHYB.
(b) Crude protein extracts of WT and ABO were loaded (40 μg protein in
each lane) and probed with mBA2 which reacts with Arabidopsis PHYB.
(c) Crude extract from ABO seeds containing 40 μg protein was serially
diluted to half of the concentration with extraction buffer and probed
with mBA2.
Figure 2. Effect of photon fluence of 667 nm light on induction of PhyBdependent
seed germination.
The experiment was performed on seeds of WT (s), the phyB mutant (r),
RBO (n) and ABO (u).
Photon fluence of R required for seed germination in
ABO
Experiments were designed to determine the photon
fluence requirement of 667 nm light for PhyB-specific
germination. WT (ecotype Nossen) seeds effectively
germinated when irradiated with a fluence higher than
20 μmol m –2 of 667 nm light and reached a maximum at a
fluence of 200 μmol m –2 (Figure 2). This range of photon
fluence is equivalent to the PhyB-specific germination
previously reported in A. thaliana ecotype Landsberg erecta
(Shinomura et al., 1996). Moreover, seeds of the phyB
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 583–590

mutant (established by crossing with Nossen) did not show
photo-induction of germination when they were allowed
to imbibe water for 3 h (Figure 2), again similar to the phyB
mutant of Landsberg erecta (Shinomura et al., 1994).
To determine whether an increase of over-expressed
PhyB causes an increase or decrease in the photon fluence
required for induction of PhyB-specific germination, we
examined the effect of various ranges of photon fluence
of R (667 nm) on the photo-induction of RBO and ABO seed
germination. The RBO seeds showed similar sensitivity to
that of WT seeds (Figure 2). In contrast, ABO seeds showed
more sensitive induction of germination than that of WT;
photo-induction occurred by irradiation with a fluence
higher than 7 μmol m –2 of 667 nm light and reached a
maximum at a fluence of 70 μmol m –2 (Figure 2).
The present results (Figure 2) together with data on
immunochemical detection of PHYB in seeds of RBO and
ABO (Figure 1) indicate that the higher the amount of PHYB
in seeds, the less photon fluence of R required for the
PhyB-specific photo-induction of seed germination.
Shift of effective range of wavelength in ABO
In order to assess whether the over-expression of PHYB
leads to a shift in the effective range of wavelength for
induction of germination, we examined the effect of various
photon fluences of monochromatic light in the spectral
range from 300–720 nm on germination using the Okazaki
large spectrograph. RBO showed similar sensitivity to WT
at all wavelengths examined (data not shown). Representative
fluence–response curves for induction of germination
in WT and ABO are presented in Figure 3.
Based on the similarity and differences between the
fluence–response curves of WT and of ABO, the range of
wavelength was divided into three groups. In the wavelength
range 300–350 nm, both WT and ABO showed
similar fluence–response curves (Figure 3). In the range
400–690 nm, both reached a maximum of 100% germination,
but the ABO seeds germinated with a lower photon
fluence than WT seeds (Figure 3). Wavelengths longer than
690 nm showed complicated fluence–response curves.
From 694 nm, the final germination percentages of WT
seeds decreased, and irradiation with monochromatic light
longer than 700 nm did not induce germination within
the fluence range examined (less than 100 mmol m –2 )
(Figure 3). In contrast, ABO seeds germinated to 100% by
irradiation with monochromatic light from 694–700 nm
(Figure 3). From 705–715 nm, the final germination percentages
of ABO seeds decreased (Figure 3), and irradiation
with monochromatic light longer than 720 nm did not
induce germination within the fluence range examined
(less than 100 mmol m –2 , data not shown). Thus, in WT
seeds, a critical threshold of response to light wavelength
occurred between 700 and 705 nm. In contrast, the corres-
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 583–590
Mode of phytochrome B action in seed germination 585
Figure 3. Effect of photon fluence of 300–715 nm light on induction of PhyBdependent
seed germination.
Representative fluence-response relationships for WT (s) and ABO (u)
seeds are shown. Wavelengths are shown in each section.
ponding critical wavelength for ABO shifted in the direction
of longer wavelength. An explanation for these phenomena
may relate to the photoconversion of phytochrome at
photostationary state (see Discussion).
Photoreversible inhibition of germination in ABO
To determine whether the over-expressed PHYB has any
physiological effects on PhyB-specific photoreversible
regulation in seeds, we examined R/FR reversibility of seed
germination in WT, ABO and RBO. All of the lines clearly
exhibited R/FR reversibility by irradiation with R
(5.8 mmol m –2 ) and then FR (18 mmol m –2 ) (data not
shown). This result establishes that over-expressed PHYB
does not interfere with the R/FR reversible regulation of
seed germination within the photon fluence range
examined.

586 Tomoko Shinomura et al.
Figure 4. Effect of photon fluence of 690–820 nm on photoreversible
inhibition of PhyB-dependent germination.
WT (s) and ABO (u) seeds were exposed to saturating R and subsequently
irradiated with monochromatic light. Representative fluence–response
relationships are shown.
To assess whether PHYB over-expressing seeds are different
from WT seeds in terms of their requirements for
light quantity or wavelength for photoreversible inhibition
of germination, fluence–response curves at 690–820 nm
were determined (Figure 4). In the spectral range of 715–
820 nm, ABO required a higher photon fluence level than
WT to reverse the promotion of seed germination induced
by previous irradiation with R (1.5 mmol m –2 ); for example,
ABO seeds required more than 12 times greater fluence of
750 nm than WT seeds (Figure 4). The photoreversible
inhibition of germination by FR therefore required higher
fluences in the case of ABO than WT, which is opposite to
the case for the induction of germination by R.
The effective range of light wavelengths for photoreversible
inhibition of germination was examined. Reversibility
was observed in the range of 690–820 nm for WT seeds
(Figure 4). In contrast, in ABO seeds, reversibility was not
observed at wavelengths shorter than 710 nm, but was
observed at wavelengths longer than 715 nm (Figure 4).
Thus, a critical threshold of light wavelength required for
photoreversible inhibition of WT seed germination was
postulated between 690 and 698 nm. In contrast, the corresponding
wavelength for ABO seeds shifted to the direction
of longer wavelengths between 710 nm and 715 nm. These
results correlated well with the shift of the critical wavelength
observed in the induction of germination (Figure 3).
Discussion
The present study demonstrated that the expression level
of PHYB in both ABO and RBO was substantially lower in
seeds than in seedlings (Figure 1). In previous papers, the
PHYB expression levels in phytochrome over-expressing
transgenic plants were determined in the seedling stage
of development (Boylan and Quail, 1989, 1991; Cherry
et al., 1991; Kay et al., 1989; Wagner et al., 1991). In contrast,
relatively little evidence has been provided for the amount
of phytochrome in seeds, either using spectrophotometric
techniques (see reviews, Frankland and Taylorson, 1983;
Shinomura, 1997) or immunochemical techniques (Konomi
et al., 1985, 1987). These results showed that the total
amount of phytochrome in seeds is very low. Because
PHYB levels in Arabidopsis are spectrophotometrically
undetectable in WT seedlings (Wagner et al., 1991) and in
WT seeds (Hanzawa et al., unpublished data), only immunochemical
techniques have been successful in determining
the level of PHYB (Shinomura et al., 1994, 1996). The
differences in the PHYB expression level observed between
seeds and seedlings (Figure 1) may be due to the differential
activation of the cauliflower mosaic virus 35S promoter
or the differential stability of PHYB between these two
developmental stages.
Transgenic Arabidopsis lines over-expressing PhyB are
powerful tools to investigate the role of PhyB as they allow
the effects of changes to be studied in the absolute amount
of PhyB in vivo (Wagner et al., 1991). The present study
showed that over-expressed PhyB in ABO seeds functions
in a similar way to the endogenous PhyB in terms of the R/
FR-reversible regulation of germination by brief irradiation
(Figure 4). This observation is consistent with the results
demonstrating R/FR reversibility obtained from the test of
inhibition of hypocotyl elongation growth in ABO and RBO
by intermittent irradiation with R and/or FR (McCormac
et al., 1993b).
The present study demonstrated, for the first time, the
quantitative relationship between PhyB and its effect on
seed germination. Previous work has investigated the
photon fluence of R and FR required for seed germination of
transgenic tobacco over-expressing oat PHYA (McCormac
et al., 1993a), but that work was carried out without the
knowledge of the differential roles of endogenous PhyA
and PhyB for seed germination. In contrast, the present
study demonstrated that the seeds expressing twice the
total amount of PHYB relative to WT (Figure 1) required
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 583–590

Mode of phytochrome B action in seed germination 587
Table 1. Photon fluence required for induction of 50% germination in the wild-type and ABO seeds, and calculated P fr/P tot ratio
Wavelength T a P fr/P r ratio at photo- WT (No-0) ABO
(nm) equilibrium
RF a Calculated P fr/P r RF a Calculated P fr/P r
(μmol m –2 ) ratio (μmol m –2 ) ratio
300 0.11 0.66 c 1100 0.60 660 0.50
350 0.063 0.75 c 1600 0.66 460 0.34
400 0.14 0.57 c 4200 0.57 130 0.10
480 0.37 0.45 c 18 000 0.43 670 0.048
505 0.42 0.51 c 11 000 0.40 840 0.056
530 0.46 0.68 c 2300 0.25 320 0.042
550 0.49 0.80 c 1800 0.37 180 0.049
610 0.55 0.89 c 170 0.26 23 0.042
667 0.58 0.87 c 56 0.25 8.7 0.045
680 0.58 0.80 c 64 0.21 10 0.038
690 0.59 0.61 c 240 0.29 25 0.040
694 0.58 0.49 c 670 0.39 37 0.039
696 0.58 0.42 c 1900 0.42 41 0.035
698 0.58 0.36 c 12 000 0.36 61 0.040
700 0.57 0.30 c,d N b – 68 0.034
705 0.62 0.18 d N b – 200 0.051
710 0.62 0.102 c,d N b – 430 0.050
715 0.61 0.041 d N b – N b –
720 0.61 0.020 c,d N b – N b –
a The required photon fluence (RF) was calculated as follows: RF � F � T, where F is the measured fluence required for induction of 50%
germination based on the fluence response curves (Figures 2 and 3), and T is the transmittance of the seed coat at each wavelength.
b N means that 50% germination was not induced by the maximum fluence examined (1 mol m –2 ).
c Mancinelli (1994).
d Schäfer et al. (1975).
less than one-sixth of the photon fluence for induction of
PhyB-dependent germination (Figures 2, 3 and Table 1).
Likewise, for photoreversible inhibition of germination, the
PhyB over-expressing seeds required more than 12 times
the fluence of FR than WT (Figure 4). In addition, we
demonstrated that the PhyB over-expressing seeds show
a shift of the critical wavelength for PhyB-dependent
germination (Figures 3 and 4).
These data are consistent with the hypothesis that a
certain absolute amount of PhyB photoconverted to P fr is
required for the induction of seed germination. By definition,
PHYB over-expressing seeds contain more P r than
WT seeds; therefore, they should require a smaller R
photon fluence than WT seeds to achieve similar levels of
P fr for induction of germination. This is exactly what was
observed in WT and ABO seeds. Likewise, in the case of
photoreversible inhibition of germination, the PHYB overexpressing
seeds should require a larger photon fluence
of FR for reversing P fr to P r, because PHYB over-expressing
seeds should contain a larger amount of P fr than WT seeds
by previous irradiation with R. Again, ABO seeds showed
such an increased requirement of FR photon fluence to
reverse seed germination.
It has long been discussed whether the phytochrome
response is determined by the amount of P fr or by the ratio
of P fr to total phytochrome (P fr/P tot ratio). The P fr/P tot ratio
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 583–590
is estimated as a function of light irradiance (photon
fluence and wavelength) and photoconversion parameters
irrespective to the absolute amount of phytochrome
(Schäfer et al., 1983). Relatively good correlation between
response and P fr/P tot ratio have been reported for stem
elongation under continuous irradiation of light (Morgan
and Smith, 1976; Smith, 1982). Similarly, photo-induction
of seed germination after brief irradiation with light has
been explained in relation to the P fr/P tot ratio. For example,
a good correlation was reported for the germination of
light-requiring weed seeds (Taylorson and Borthwick, 1969)
and for the germination of partially light-requiring lettuce
seeds (Mancinelli, 1994). In these previous experiments,
only P fr/P tot ratios were calculated, but the absolute concentration
of P fr was not measured because this measurement
is difficult in seeds (Frankland and Taylorson, 1983).
To determine whether the seed germination data
obtained from the present study correlate with the P fr/P tot
ratios present in the seed samples used, we calculated
P fr/P tot ratios from photon fluences for each data point
at three different wavelengths (505, 667, 690 nm). These
calculations were based on the photochemical parameters
of PhyA (Kelly and Lagarias, 1985; Lagarias et al., 1987;
Mancinelli, 1994), because PhyB has similar photochemical
characteristics to PhyA (P.-S. Song, personal communication).
Figure 5 shows germination percentages versus

588 Tomoko Shinomura et al.
Figure 5. Relationship between calculated P fr/P tot ratios and germination
percentage for induction of germination in WT and ABO seeds.
Data from Figures 2 and 3 were plotted as germination percentages versus
calculated P fr/P tot ratio. WT seeds irradiated with 505 nm (d), 667 nm (j)
and 690 nm (m) and ABO seeds irradiated with 505 nm (s), 667 nm (u),
690 nm (n) are shown.
calculated values of P fr/P tot ratio. In theory, the same P fr/
P tot ratio given with different light wavelengths should
induce the same level of seed germination, if the P fr/P tot
ratio determines the response. This was demonstrated
when the data for different wavelengths were compared
within either WT seeds or ABO seeds (Figure 5). However,
the germination of ABO seeds was induced at lower P fr/
P tot ratio than that of WT seeds (Figure 5). This result
strongly suggests that the seed germination is not determined
by the P fr/P tot ratio itself.
To analyse the difference in P fr/P tot ratio between WT
and ABO at various wavelengths, the photon fluence
required for 50% induction of germination were measured
from Figures 2 and 3, and the corresponding P fr/P tot ratio
was estimated (Table 1). Within the wavelength range of
480–698 nm, 50% induction of WT seeds was induced by
exposure to a photon fluence which is able to generate a
P fr/P tot ratio of 0.21–0.43. In contrast, ABO seeds require a
lower fluence which is able to generate P fr/P tot ratio of
0.035–0.056. These calculated values are consistent with
the results in Figure 5, and confirm again the assertion that
seed germination in Arabidopsis is not simply regulated by
a mechanism determined exclusively by P fr/P tot ratio.
The shift of the critical threshold of light wavelength for
either induction or photoreversible inhibition of germination
between WT and ABO seeds is also understood in
similar terms. Theoretically, if the P fr/P tot ratio determines
seed germination, the critical threshold wavelength for
induction of germination in PhyB over-expressing seeds
should not change from that of WT seeds. P fr/P tot ratio is
determined by wavelength and photon fluence, irrespective
to the absolute amount of phytochrome (Schäfer et al.,
1983). However, Figures 3 and 4 showed a shift of the
critical wavelength in ABO seeds. WT seeds showed a
critical wavelength between 700 and 705 nm which should
generate a P fr/P tot ratio of approximately 0.2 in the photostationary
state (Schäfer et al., 1975). In contrast, ABO
seeds showed a longer critical wavelength between 715
and 720 nm which generated a P fr/P tot ratio of
approximately 0.04 (Schäfer et al., 1975). This difference of
critical wavelength again demonstrated that the P fr/P tot
ratio did not determine the PhyB-dependent germination
of Arabidopsis seed, but that a certain absolute amount of
P fr was probably the determining factor.
However, the quantitative relationship between the
amount of PhyB and the amount of photon fluence required
for PhyB-dependent germination did not show a simple
proportional relationship. For example, ABO seeds (which
had twice as much PHYB as WT seeds) required less than
one-sixth as much R (667 nm) photon fluence for induction
of germination and 12 times more FR (750 nm) photon
fluence for inhibition of germination relative to WT seeds.
These results suggest that photoperception by the PhyB
monomer molecule is not the only limiting factor for
PhyB signal transduction towards seed germination. In the
future, measurements of the photochemical parameters of
PhyB in vitro, photoconversion and dark reversion kinetics
of PhyB in vivo, behaviour of PhyB dimer molecules, and
detection of PhyB signal transduction components are
expected to provide additional insight into how plants
perceive environmental light and adjust seed germination
accordingly.
Experimental procedures
Plant materials
Arabidopsis thaliana (L.) Heynh., ecotype Nossen, and two derivative
transgenic homozygous lines were used. The cDNA of Arabidopsis
PHYB (for ABO) and rice PHYB (for RBO) were fused to the
cauliflower mosaic virus 35S promoter (Wagner et al., 1991). The
phyB mutant seeds were obtained by crossing the phyB-1 allele
(ecotype Landsberg erecta) into WT (ecotype Nossen) twice.
Light treatment and germination assay
Seeds were surface-sterilized and sown onto 0.7% (w/v) aqueous
agar medium in plastic Petri dishes in lots of 80–100 seeds each,
then immediately exposed to FR (18 mmol m –2 ), inhibiting PhyBdependent
dark germination as described previously (Shinomura
et al., 1994). They were kept in total darkness for 3–9 h at 23°C
before being transferred to the appropriate light treatment. After
the light treatment, seeds were kept in darkness for 7 days at 23°C
to allow germination to proceed. Germination was scored using
a microscope to assess radicle emergence. To normalize experimental
differences in germination percentage, the relative
germination percentage (Rλ i) was calculated as follows: R λi �
(G λi – G D)/(G R – G D), where G λi is the germination percentage at
each wavelength at each photon fluence, G D is the germination
percentage in darkness, and G R is the germination percentage
upon irradiation with the saturating fluence of R (3 mmol m –2 ).
Photon fluence was plotted against observed seed germination at
30 different wavelengths from 300 to 820 nm at intervals of 2–
50 nm. Each curve was fitted by the least-squares method. To test
photoreversible inhibition of germination, seeds were exposed to
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 583–590

saturating R (1.5 mmol m –2 ) and subsequently irradiated with
monochromatic light. Monochromatic light was generated by the
Okazaki large spectrograph (Watanabe et al., 1982). The total
fluence was varied by changing the duration (1–30 min) and/or
fluence rate (5.0 � 10 –4 to 3.5 � 10 1 μmol m –2 s –1 ) of the radiation
with threshold boxes and neutral density filters (ND1–50, Hoya,
Tokyo, Japan). Fluence rate was measured by a photon density
meter (PFDM-200LX, Rayon Industrial, Kawasaki, Japan) or a
optical power meter (Model 1830-C, Newport, CA, USA).
P fr/P tot calculations
Photon fluence irradiated was standardized to incident fluence by
the transmittance of the seed coat (%) at each wavelength measured
with a microspectrophotometer (MPM800, Zeiss,
Oberkochen, Germany). P fr/P tot ratios at each wavelength were
calculated from the value of standardized photon fluence on the
basis of photoconversion kinetics of PhyA previously reported
(Mancinelli, 1994; Schäfer et al., 1975).
Immunochemical detection
For detection of PHYB in seeds, crude extracts were prepared
from about 2 � 10 3 seeds and analysed immunochemically as
described previously (López-Juez et al., 1992). Seeds were homogenized
after incubation for 3 h in the dark on aqueous 0.8%
(w/v) agar medium under illumination with a green safety lamp.
Seedlings grown under continuous irradiation with white light for
7 days were used. To detect Arabidopsis PHYB in extracts from
ABO seeds and seedlings, a monoclonal antibody mBA2 (raised
against recombinant Arabidopsis PHYB) (Shinomura et al., 1996)
was used. To detect both rice PHYB and Arabidopsis PHYB in
extracts from RBO, a monoclonal antibody mBT4 (raised against
recombinant tobacco PHYB) (López-Juez et al., 1992) was used.
To quantify the level of over-expression in comparison with WT,
a dilution series of extracts from WT and ABO seeds was prepared
and reacted with mBA2.
Acknowledgements
We thank Professor P. H. Quail for providing plant material and
for critical suggestions on the present work, Professor N. Murata
and Dr M. Watanabe for hosting us at the National Institute for
Basic Biology (NIBB), Drs J. Reed and J. Jelesko for useful
comments and discussion, M. Kubota for assistance with the
operation of the Okazaki large spectrograph, R. Katayanagi for
assistance with plant cultivation, and F. Tsunekawa for measuring
the transmittance of the seed coat. The work was supported in
part by a grant from the Program for Promotion of Basic Research
Activity for Innovative Biosciences to M.F. The experiments were
partly carried out under HARL projects B2018 and B2023 and NIBB
Cooperative Research Programs for OLS 95–528 and 96–522.
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