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Plant Physiol. (1998) 117: 797-807
The Expression of Light-Regulated Genes in the
High-Pigment-1
Mutant of Tomato1
Janny L. Peters2, *,
Márta Széll2, and
Richard E. Kendrick
Laboratory for Photoperception and Signal Transduction, Frontier
Research Program, Institute of Physical and Chemical Research (RIKEN),
Hirosawa 2-1, Wako-shi, Saitama, 351-0198 Japan
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ABSTRACT |
Three
light-regulated genes, chlorophyll a/b-binding protein
(CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase
small subunit, and chalcone synthase (CHS), are
demonstrated to be up-regulated in the high-pigment-1
(hp-1) mutant of tomato (Lycopersicon
esculentum Mill.) compared with wild type (WT). However, the
pattern of up-regulation of the three genes depends on the light
conditions, stage of development, and tissue studied. Compared with WT,
the hp-1 mutant showed higher CAB gene
expression in the dark after a single red-light pulse and in the
pericarp of immature fruits. However, in vegetative tissues of
light-grown seedlings and adult plants, CAB mRNA
accumulation did not differ between WT and the hp-1
mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small
subunit mRNA accumulated to a higher level in the
hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was
up-regulated in de-etiolated vegetative hp-1-mutant
tissues only. The CAB and CHS genes were
shown to be phytochrome regulated and both phytochrome A and B1 play a
role in CAB gene expression. These observations support
the hypothesis that the HP-1 protein plays a general repressive role in
phytochrome signal transduction.
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INTRODUCTION |
Light controls many aspects of plant morphogenesis and provides
energy for photosynthesis. Different regions of the spectrum are
perceived by different photoreceptor molecules: the B photoreceptors, the UV photoreceptors, and the R-/FR-sensitive phytochromes.
Phytochromes were physiologically identified 50 years ago, and in the
last two decades different phytochrome types have been purified and cloned from several plant species. Mutants deficient in specific phytochrome family members have been isolated from several species: e.g. Arabidopsis (for review, see Smith, 1995 ), tomato
(Lycopersicon esculentum Mill.) (van Tuinen et al., 1995a,
1995b), and pea (Weller et al., 1995). These mutants are excellent
tools for studying the functions of the different members of the
phytochrome family.
Although there is information about photoperception by phytochromes,
little is known about the signal transduction pathways linking these
receptors with gene expression. Several approaches have been used to
study phytochrome signal transduction pathways. First, using
microinjection, Neuhaus et al. (1993) identified several molecules that
participate in phytochrome-signal transduction. The existence of two
separate pathways was proposed: the cGMP-mediated pathway that leads to
the regulation of CHS genes, and the
Ca2+-calmodulin mediated pathway that regulates
the expression of CAB and RBCS genes. In both
pathways the signal is transduced from phytochrome via a heterotrimeric
G protein, and subsequently the existence of a reciprocal control
mechanism between the pathways has been demonstrated (Bowler et al.,
1994a, 1994b).
A second approach to identify and characterize components and
regulators of phytochrome-signal transduction pathways is the isolation
and characterization of mutants with altered light responses. Constitutive-response mutants such as constitutive photomorphogenesis (cop), de-etiolated (det), and fusca
(fus) in Arabidopsis (for review, see Wei and Deng, 1996),
light-independent (lip) in pea (Frances et al., 1992), and
hyp2 in tobacco (Traas et al., 1995) are excellent tools for
these studies. These mutants fail to exhibit the characteristics of
dark-grown seedlings and show reduced elongation and expanded leaves.
Some also accumulate anthocyanin in the dark. The cloning of the
corresponding genes from Arabidopsis allowed the biochemical
characterization of the affected gene products and provided information
about the possible role and function of these
components in phytochrome-signal transduction (Wei and Deng, 1996). In
addition, mutants in genes affecting phyA and B signaling have been
reported (Whitelam et al., 1993; Ahmad and Cashmore, 1996; Wagner et
al., 1997; Hoecker et al., 1998).
A third approach to identify the components of phytochrome-signal
transduction is to screen directly for mutants altered in the
regulation of particular light-regulated genes. Li et al. (1994, 1995)
isolated Arabidopsis mutants altered in the regulation of
CAB gene expression. These mutants were named doc
(for dark overexpression of CAB) and cue1 (for
CAB underexpressed). The doc mutation affects the
expression of CAB genes and the cue1 mutation
affects the expression of both CAB and RBCS
genes. The expression of CHS genes was neither modified in
doc nor in cue1 mutant plants. In contrast, the
increased CHS expression (icx1) mutant of
Arabidopsis shows enhanced induction of CHS gene expression by light, but no alteration in the level of CAB transcript
accumulation (Jackson et al., 1995).
In this paper we examine a putative phytochrome signal transduction
mutant of tomato, the hp-1 (high-pigment-1) mutant. This monogenic recessive hp-1 mutant was first identified in 1917 (Reynard, 1956) and exhibits higher anthocyanin content, shorter
hypocotyl (Kerr, 1965; Mochizuki and Kamimura, 1985; Peters et al.,
1989), and darker green foliage (Jarret et al., 1984) and fruits
(Thompson, 1962) when compared with WT. The HP-1 gene has
been recently mapped to chromosome 2 (Yen et al., 1997). Soressi (1975)
identified a recessive hp-2 mutant, which is phenotypically
similar but nonallelic to hp-1 and maps to chromosome 1 (van
Tuinen et al., 1997). Attempts to isolate Arabidopsis counterparts of
the tomato hp mutants have been reported (Ichikawa et al.,
1996), but await detailed analysis.
Although the nature of the hp mutations is still unclear,
detailed physiological characterization of the hp-1 mutant
provided a valuable insight into phytochrome signal transduction
processes. The hp-1 mutant has high levels of anthocyanin
and reduced height of light-grown seedlings (Peters et al., 1992a;
Kerckhoffs et al., 1997a). Furthermore, the photoinduction of several
enzymes in biochemical pathways: Phe ammonia lyase (Goud et al., 1991), nitrate reductase, nitrite reductase, and amylase (Goud and Sharma, 1994), are amplified in the hp-1 mutant compared with WT.
All of these features have been shown previously to be phytochrome regulated, and therefore, it was concluded that the hp-1
mutant shows exaggerated phytochrome responses (Kerckhoffs and
Kendrick, 1997). The apparent phenocopying of the hp-1
mutant's phenotype and immature fruit color as a result of phyA
overexpression in tomato (Boylan and Quail, 1989) is consistent with
this idea. However, in vivo spectrophotometric and immunochemical
analysis failed to provide evidence that the hp-1 mutant is
a photoreceptor mutant (Peters et al., 1992b; Kerckhoffs et al.,
1997a). Therefore, it was proposed that the hp-1 mutation is
associated with an amplification step in the phytochrome-transduction
chain (Peters et al., 1992b; Kerckhoffs et al., 1997a; Kerckhoffs
and Kendrick, 1997). This conclusion is supported by the recent
observation using specific phytochrome family-member-deficient mutants,
that it is phyA and phyB1 that play a dominant role in the seedling
anthocyanin response (Kerckhoffs et al., 1997b). In the
phytochrome-amplification model, phytochrome responses are envisaged to
be under the constraint of the HP-1 gene product. Both B and
the hp-1 mutation appear to be able to relieve this
constraint (Peters et al., 1989, 1992b).
The dark-green immature fruit color of the hp-1 mutant
compared with WT is due to higher chlorophyll levels (Sanders et al., 1975; Kerckhoffs, 1996) and the mature hp-1-mutant fruits
have a higher lycopene and carotene content and increased levels of ascorbic acid than those of WT (Thompson, 1962). Recently, the plastid
copy number in the hypocotyls and the Suc and flavonoid contents of
ripe fruits have been reported to be elevated in the hp-1
mutant compared with WT (Yen et al., 1997).
In this paper we characterize the effect of the hp-1
mutation on CAB, RBCS, and CHS gene
expression at different developmental stages using the most extreme
allele available (hp-1w).
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
The tomato (Lycopersicon esculentum Mill.) genotypes
used in the experiments were hp-1w (Peters
et al., 1989);
hp-1w,fri1
(far-red light insensitive),
deficient in phyA (Kerckhoffs et al., 1997b);
hp-1w,tri1
(temporarily red-light
insensitive), deficient in phyB1 (Kerckhoffs et al., 1997b)
in the genetic backgrounds MoneyMaker (MM) or breeding line GT.
For the experiments with seedlings, seeds were surface sterilized for 3 min in a 1% (v/v) dilution of commercial bleach and rinsed for 5 min
in Milli-Q water (Milli-RO 8 water purification system, Millipore).
Seeds were sown at noon on 0.6% (w/v) agar medium containing 0.46 g L 1 Murashige-Skoog basal salts
(Murashige-Skoog, 1962) in plastic tissue culture containers (Plantcon,
Flow Laboratories Inc., McLean, VA) and germinated in a FR-6113A growth
chamber (Koito, Tokyo, Japan) at 25°C. To germinate seedlings in
absolute darkness, tissue culture containers were wrapped in aluminum
foil, put in a black velvet sack, and grown in a dark room at 25°C.
In the light-pulse experiments R (27 µmol m2
s1) was obtained from FL20SRF fluorescent tubes
(National, Osaka, Japan) filtered through a red, plastic filter
(Shinkolite A no. 102, Mitsubishi Rayon Corp., Tokyo, Japan)
and FR (33 µmol m2
s1) from FL20S-FR74 fluorescent tubes
(Toshiba) wrapped with one layer of Polycolor no. 22 and one layer of
Polycolor no. 72 film (Tokyo Butai Shomei Co., Tokyo, Japan). B (11 µmol m2 s1) was
obtained from FL20S.B fluorescent tubes (Toshiba). WL-grown seedlings
were germinated in 16-h WL (120 µmol m2
s1 PAR) 8-h dark cycles at 25°C. WL was
obtained from FL20SD SDL fluorescent tubes (National).
For the experiments with adult plants and fruits, seeds were sown in
the greenhouse in a 4:1 vermiculite/granular-clay-based compost
mixture. After 1 month plants were transplanted to pots (19 cm
[diameter] × 15 cm [height] for vegetative tissues of adult plants
and 27.1 cm × 28.6 cm for fruits) containing 2:1
vermiculite/granular-clay based compost mixture and transferred to a
phytotron KG-206HL-D (Koito) with 16-h WL (250 µmol
m2 s1 PAR) 8-h dark
cycles at 25°C. Vegetative plant material was harvested 2 months
after sowing and frozen in liquid nitrogen. The frozen material was
stored in a  135°C freezer until use. After the first fruit(s) on a
plant became red, all fruits of that particular plant were harvested.
Harvest was always at noon because of diurnal mRNA fluctuations of
CAB genes in tomato fruits (Piechulla and Gruissem,
1987). Directly after harvest a picture was taken of the fruits from
one plant. The age, diameter, length, and weight of each fruit were
measured and samples were taken for the chlorophyll assay. The
remaining material was separated into pericarp (the outer wall of
the pericarp including the epidermis) and the inner section (radial
and inner wall of the pericarp, placental tissue, and locular
cavity with seeds), and frozen in liquid nitrogen. The frozen material
was stored in a 135°C freezer until use.
Chlorophyll Assay
To determine the chlorophyll content in the fruits, samples were
taken from the equator of the fruits using an 11-mm cork borer. From
this sample the pericarp and about 5 mm of the inner section (for
definition, see above) directly bordering the pericarp were cut. For
each fruit the chlorophyll was extracted from one pericarp and one
inner-section disc. The fruit discs were placed in 15-mL tubes (Falcon)
and incubated in darkness for at least 48 h at 65°C in DMSO
(after the work of Hiscox and Israelstam, 1979). Samples were
re-extracted with DMSO until no extra chlorophyll could be extracted,
and the samples were always kept in the dark. When the samples were
cooled to room temperature, A649 and
A665 were determined
spectrophotometrically. Chlorophyll a and b were calculated on a gram fresh weight basis, using the equations for ethanol published by Lichtenthaler and Wellburn (1983).
Anthocyanin Assay
Anthocyanin was extracted from cotyledons and hypocotyls of
seedlings with 0.6 mL of acidified (0.3% HCl, v/v) methanol for 48 h. The extraction was carried out by shaking the samples in darkness at room temperature for 48 h. At the end of the
extraction 0.45 mL of water and 1.2 mL of chloroform were added.
Samples were vortexed and centrifuged for 20 min at 4500g.
The A535 of the upper
anthocyanin-containing phase was determined spectrophotometrically (DU650, Beckman).
RNA Gel-Blot Analysis
Total RNA was isolated by a modification of the method of Loening
(1969) and was previously described by Peters and Silverthorne (1995).
RNA was electrophoresed in 1% (w/v) agarose gels containing Mops
buffer (20 mM Mops, 1 mM EDTA, and 5 mM sodium acetate, pH 7.0) and 6.7% (v/v) formaldehyde.
Gels were soaked in distilled water to remove the formaldehyde (three
changes of 20 min each) and visualized by staining with ethidium
bromide (1 µg/mL) prior to blotting onto Hybond N+
membranes (Amersham). Protocol no. 4 of the VacuGene XL blotting system
(Pharmacia LKB Biotechnology, Bromma, Sweden) was used for vacuum
transfer of RNA.
The blots were prehybridized overnight at 42°C in 50% (v/v)
formamide, 5× Denhardt's reagent (1× Denhardt's reagent is 0.02% [w/v] Ficoll Type 400 Sigma, 0.02% [w/v] PVP, and 0.02% [w/v] bovine albumin Fraction V, Sigma), 0.1% (w/v) SDS, 5× SSC (1× SSC is
150 mM NaCl and 15 mM sodium citrate, pH 7.0),
and 50 µg/mL salmon sperm DNA (0.5 mL per cm2
blot).
The coding region of the tomato CAB-1 (Pichersky et al.,
1985) and RBCS-2 (Pichersky et al., 1986) and
CHS1 (O'Neill et al., 1990) genes were used to synthesize
DNA probes by random priming using the Rediprime DNA labeling
system (Amersham). To remove unincorporated nucleotides 1 µL of 10%
SDS and 2 µL of denatured salmon sperm (10 mg/mL) were added to the
50-µL reaction mix in an ultra-free microcentrifuge tube
(Ultrafree-C3 TGC, Nihon Millipore Ltd., Tokyo, Japan) and spun at room
temperature (5 min, 5000g). After washing the labeled DNA
with 100 µL of sterile water, the probe was recovered from the upper
part of the Millipore tube, denatured (by boiling for 5 min), and put
on ice until use. For hybridization, the appropriate probe (specific
activity 0.5 dpm/µg) was added to the hybridization buffer (0.1 mL of
prehybridization buffer per cm2 blot).
Hybridizations were carried out overnight at 42°C. As a loading
control each blot was rehybridized with a 17-base oligonucleotide complementary to the 18S rRNA (Gallo-Meager et al., 1992). The oligonucleotide was labeled by phosphorylation with T4 polynucleotide kinase and the hybridization was carried out as described by
Gallo-Meager et al. (1992).
Washes of nylon membranes (Hybond N+, Amersham) were
performed in 2× SSC, 0.1% SDS (3 × 10 min at room temperature),
and 0.1× SSC, 0.1% SDS (2 times for 30 min at 65°C). The signals
were visualized and quantitated with a phosphor imager (Fujix BAS 2000, Fuji, Japan).
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RESULTS |
Effect of a Single R Pulse on CAB and
RBCS Gene Expression
In tomato seedlings the expression of CAB genes is
controlled by phytochrome (Sharrock et al., 1988; Wehmeyer et al.,
1990). Wehmeyer et al. (1990) showed that CAB mRNA
accumulation reached a maximum 4 h after a R pulse. In contrast to
CAB mRNA, they could not easily demonstrate phytochrome
regulation of RBCS mRNA. To determine whether the
CAB and RBCS gene expression in the
hp-1w-mutant seedlings is controlled by
phytochrome, we first studied the kinetics of expression of both genes
after a single R pulse. To this end, 4-d-old etiolated seedlings were
irradiated with a 10-min saturating R pulse and returned to the dark.
Samples were collected immediately (0 h) and at 1, 2, 4, 6, and 8 h after the R pulse. Control seedlings were maintained in continuous
darkness and harvested simultaneously with the seedlings harvested
immediately after the R pulse. Very low levels of CAB gene
expression were detectable in dark-grown WT seedlings (Fig.
1). In contrast to WT, the
hp-1w-mutant seedlings exhibited a
substantial level of CAB gene expression in the dark. A R
pulse induced CAB mRNA accumulation in both WT and
hp-1w-mutant seedlings and maximum
expression occurred 4 h after the light-pulse treatment. However,
at this time point the hp-1w mutant
accumulated more transcript than WT. The RBCS mRNA
accumulation in dark-grown hp-1w-mutant
seedlings was about 3-fold higher than the levels observed in WT.
Although a R pulse slightly induced RBCS gene expression in
WT seedlings, no induction was observed in
hp-1w-mutant seedlings.
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| Figure 1.
Effect of a 10-min R pulse on the
CAB and RBCS mRNA abundance in etiolated
4-d-old WT and hp-1w-mutant tomato
seedlings. A, For the RNA blots shown, RNA was extracted directly (0 h), 1, 2, 4, 6, and 8 h after onset of the R pulse. The control
(lanes C) represents the CAB mRNA amount in seedlings
that did not receive a R pulse but were kept in continuous darkness. As
a loading control the blots were probed with an 18S rRNA probe. B, The
CAB and RBCS mRNA abundance was
quantified using a phosphor imager and the mean values
(±SE) are shown for CAB and
RBCS, respectively. A value of 100% on the ordinate
represents the maximum steady-state mRNA detected within the
experiment.
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Phytochrome Regulation of CAB Gene Expression in
Photomorphogenic Mutants
Figure 1 showed that in hp-1w-mutant
seedlings CAB but not RBCS gene expression could
be induced by a single R pulse. Therefore, we limited our experiment to
study the involvement of phytochrome in light-regulated gene expression
to CAB gene expression. Since various photomorphogenic
mutants of tomato are available, we used phyA-deficient,
hp-1w,fri1,
and phyB1-deficient,
hp-1w,tri1,
double mutants in addition to the hp-1w
mutant and WT. All genotypes were grown in continuous darkness for
4 d and exposed to a 10-min R pulse, 15-min FR pulse, or 10-min R
pulse followed by a 15-min FR pulse. Control seedlings were kept in
continuous darkness during the experiment. Whole seedlings were
harvested 4 h after the light pulse(s) when maximum response occurs (Fig. 1).
In WT, CAB mRNA accumulation was induced by a 10-min R pulse
and could be partially reversed by a FR pulse (Fig.
2). This incomplete reversal can be
explained by a partial escape from FR reversibility during the R
pretreatment. The response after FR alone probably reflects the VLFR
component of CAB mRNA accumulation (Sharrock et al., 1988).
Recently, a similar VLFR component of CAB gene expression
was shown for Arabidopsis (Hamazato et al., 1997). In agreement with
the data in Figure 1, a substantial level of CAB gene
expression was detected in dark-grown
hp-1w-mutant seedlings (Fig. 2). Due to the
hp-1w mutation, the
hp-1w,fri1 and
hp-1w,tri1 double
mutants also showed CAB gene expression in the dark,
although at a reduced level compared with the
hp-1w monogenic mutant. To account for the
possible effect of harvest under green safe light on CAB
gene expression, the seedlings were also harvested in total darkness
(in Fig. 2, DD) and compared with samples harvested under green safe
light (Fig. 2, D). Figure 2 shows that when seedlings were harvested in
complete darkness, the hp-1w mutation
resulted in significant CAB mRNA accumulation. Moreover, the
hp-1w mutant exhibited a higher response to
all light-pulse treatments. The phyA-deficient
hp-1w,fri1
double mutant exhibited approximately 30% of the CAB gene
expression induced by a single R pulse in the
hp-1w mutant. A similar reduction in
CAB mRNA accumulation could be seen when the
fri1 mutant was compared with WT (data not
shown). This implies a role for phyA in the low fluence response,
either directly or indirectly. The effect of FR and R/FR treatments are
markedly reduced in the
hp-1w,fri1
mutant compared with the hp-1w mutant,
which verifies the role of phyA in the VLFR proposed previously (Casal
et al., 1994; Botto et al., 1996; Shinomura et al., 1996). In the
phyB1-deficient
hp-1w,tri1
double mutant, the CAB gene expression induced by a R pulse
was also reduced (about 50%) compared with the
hp-1w mutant. This clearly indicates that
phyB1 also plays a role in the regulation of CAB gene
expression.
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| Figure 2.
Effect of no light pulse (lanes D), a 10-min R,
15-min FR, and 10-min R followed by 15-min FR pulse (R/FR) on the
CAB mRNA abundance in etiolated 4-d-old tomato
seedlings. The genotypes used were WT, hp-1w
mutant,
hp-1w,fri1, and
hp-1w,tri1 double
mutants. A, For the RNA gel blots shown, RNA was isolated 4 h
after the light pulse(s) and probed with a CAB cDNA
probe. As a loading control the blots were probed with an 18S rRNA
probe. B, The CAB mRNA abundance was quantified using a
phosphor imager and the mean values (±SE) are shown. A
value of 100% on the ordinate represents the maximum steady-state mRNA
detected within the experiment.
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Phytochrome Regulation of CHS Gene Expression
Although a single R pulse could induce CAB gene
expression (Figs. 1 and 2), the same light treatment was ineffective
for the induction of CHS gene expression (data not shown).
Therefore, an irradiation schedule used by Peters et al. (1992b), which
is efficient in inducing phytochrome regulation of anthocyanin
biosynthesis, was applied to determine if phytochrome regulates
CHS gene expression. The WT and
hp-1w-mutant seedlings were grown in
darkness for 4 d and exposed to a 12-h R or B pretreatment
followed by no pulse, a 10-min R pulse, 15-min FR pulse, or a 10-min R
pulse followed by a 15-min FR pulse. Control seedlings were kept in
darkness during the experiment. Figure 3
shows that neither WT nor hp-1w-mutant
seedlings accumulated detectable levels of CHS mRNA when grown in complete darkness. In seedlings that were given R and B
pretreatments, a R pulse induced high levels of CHS
expression in the WT. This effect could be reversed by FR. In the
hp-1w mutant R and B pretreatments followed
by a R pulse resulted in a significant amplification of CHS
gene expression compared with WT, and a FR pulse could not reverse the
level of expression to the same extent as in the WT. In summary, the
total level of response is enhanced in the
hp-1w mutant compared with WT for all
treatments. Thus, by using light pretreatments, CHS gene
expression was shown to be regulated by phytochrome in tomato.
Moreover, the phytochrome-induced, R/FR reversible, CHS mRNA
accumulation response is significantly enhanced in the
hp-1w mutant compared with WT after R
pretreatment, but not significantly affected after B pretreatment. This
indicates that B alone can enhance the phytochrome response in WT to an
amount similar to that in the hp-1w mutant.
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| Figure 3.
Effect of a 12-h R or B pretreatment terminated
with no light pulse (lanes D), a 10-min R, 15-min FR, and 10-min R
followed by 15-min FR pulse (R/FR) on the CHS mRNA
abundance in etiolated 4-d-old WT and
hp-1w-mutant seedlings of tomato. A, For the
RNA gel blots shown, RNA was isolated 4 h after the light pulse(s)
and probed with a CHS1 cDNA probe. As a loading control
the blots were probed with an 18S rRNA probe. B, The CHS
mRNA abundance was quantified using a phosphor imager and the mean
values (±SE) are shown. A value of 100% on the ordinate
represents the maximum steady-state mRNA detected within the
experiment.
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Circadian Rhythm of CAB Gene Expression
Accumulation of CAB mRNA shows a circadian rhythm in
tomato with a maximum level of expression at noon (Kellman et al.,
1993). A difference in the circadian rhythm of CAB mRNA
accumulation of WT and hp-1w-mutant plants
could result in misleading conclusions when studying tomato plants
grown in light/dark cycles. Therefore, we studied the effect of the
hp-1w mutation on the circadian rhythm.
Seedlings were grown in 16-h WL, 8-h dark cycles (lights on at 6 AM; light off at 10 PM) and transferred to
continuous darkness at 8 AM of d 5 (Fig.
4, WL/D  D/D). Control seedlings were
kept in WL/D cycles and showed diurnal oscillations (Fig. 4, WL/D).
Whole seedlings were harvested every 4 h for 3 d, starting at
8 AM on the 4th d after sowing. No differences in the
diurnal oscillations of CAB gene expression of WT and
hp-1w-mutant seedlings were observed (Fig.
4, WL/D). The brief, 2-h exposure to WL before the transfer to
continuous darkness resulted in a phase shift, and the subsequent peak
of CAB gene expression occurred at 8 AM instead
of at noon in both WT and hp-1w mutant
(Fig. 4, WL/D D/D). Although a slight difference in dampening of
the CAB gene expression between WT and
hp-1w-mutant seedlings may exist, the
pattern and quantitative level of CAB mRNA accumulation in
the two genotypes is very similar.
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| Figure 4.
Circadian rhythmic CAB mRNA
accumulation in WT and hp-1w-mutant
seedlings of tomato. A, For the RNA gel blots shown, seedlings were
grown in 16-h WL (WL, 6 AM-10 PM), 8-h dark
(lanes D, 10 PM-6 AM) cycles. To describe the
diurnal CAB transcript oscillations (WL/D), samples were
collected every 4 h starting with 4-d-old seedlings at 8 AM. To study the circadian rhythm of CAB
genes (WL/D D/D), seedlings were transferred to darkness on d 5 at 8 AM. As a loading control the blots were
probed with an 18S rRNA probe. B, The CAB mRNA abundance
was quantified using a phosphor imager and the mean values
(±SE) are shown. A value of 100% on the ordinate
represents the mRNA abundance detected on d 4 at noon.
{/ANNT;;;left;top}
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Organ-Specific Expression of CAB, RBCS, and
CHS Genes
At least one of the light-regulated genes studied, the
RBCS gene family, exhibits organ-specific expression in
tomato (Sugita and Gruissem, 1987; Wanner and Gruissem, 1991). Since
the hp-1w-mutant seedlings showed elevated
responses for the genes studied, it is important to compare the
organ-specific expression patterns of these genes in seedlings and
adult plants of the WT and hp-1w mutant.
Such a comparison can answer the question of whether more mRNA
accumulates in the same organs or whether the distribution patterns
also change due to the hp-1w mutation.
Seedlings were grown in either continuous darkness or in 16-h WL, 8-h
dark cycles for 4 d. The CAB, RBCS, and
CHS mRNA accumulation in cotyledons and hypocotyls is shown
in Figure 5. None of the genes were
expressed in the roots (data not shown). The patterns of mRNA
accumulation in hp-1w-mutant seedlings
differ from that of WT in several aspects. CAB mRNA in
dark-grown hypocotyls and cotyledons and RBCS mRNA in dark-grown hypocotyls accumulated to a much higher level in the hp-1w mutant than WT (Fig. 5). Moreover, a
higher CHS mRNA accumulation was observed in WL/D-grown
hp-1w-mutant seedlings compared with WT.
The CHS mRNA accumulation was significantly increased in
cotyledons of the hp-1w mutant compared
with WT (Fig. 5), which correlates well with the anthocyanin
accumulation data for the WT and hp-1w
mutant (A535 per three cotyledon pairs was
0.065 ± 0.004 and 0.449 ± 0.015 for WT and
hp-1w mutant, respectively;
A535 per three hypocotyls was 0.323 ± 0.022 and 0.600 ± 0.038 for WT and
hp-1w mutant, respectively). The
RBCS gene expression was always higher in the
hp-1w mutant, irrespective of the light
conditions under which the plants were grown (Fig. 5). As in the R and
B pretreatment experiment (Fig. 3), no CHS mRNA accumulation
was observed in the dark. Accumulation of CHS mRNA reached a
higher level in WL/D-grown hp-1w-mutant
seedlings than in WT. In contrast to RBCS and
CHS, CAB gene expression was only higher in
seedlings grown in darkness.
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| Figure 5.
CAB, RBCS, and
CHS mRNA abundance in cotyledons (lanes Cot) and
hypocotyls (lanes Hyp) of 4-d-old WT and
hp-1w-mutant seedlings of tomato. A, For the
RNA gel blots shown, RNA was isolated from seedlings grown in dark (D)
or 16-h WL, 8-h dark cycles (WL/D). As a loading control the blots were
probed with a 18S rRNA probe. B, The CAB,
RBCS, and CHS mRNA abundance was
quantified using a phosphor imager and the mean values
(±SE) are shown. A value of 100% on the ordinate
represents the maximum steady-state mRNA detected within the
experiment.
|
|
To investigate the CAB, RBCS, and CHS
mRNA levels in the leaves, stems, and roots of adult plants, plants
were grown under 16-h WL, 8-h dark cycles for 8 weeks. None of the
genes were expressed in the roots of adult WT and
hp-1w-mutant plants (Fig.
6). As in 4-d-old, WL/D-grown seedlings
(Fig. 5), the CAB mRNA accumulation in adult plant parts was
not higher in the hp-1w mutant than WT.
However, both RBCS and CHS gene expression were significantly higher in stems of the hp-1w
mutant compared with WT. These data on the organ-specific expression of
light-regulated genes in seedling and adult plants show that the three
genes studied are differentially affected by the
hp-1w mutation.
View larger version (22K):
[in this window]
[in a new window]
| Figure 6.
Abundance of CAB,
RBCS, and CHS mRNA in young leaves (lanes
Le), stems (lanes St), and roots (lanes Ro) of 8-month-old WT and
hp-1w-mutant tomato plants. A, For the RNA
gel blots shown, RNA was isolated from adult plants grown in 16-h WL,
8-h dark cycles. As a loading control the blots were probed with a 18S
rRNA probe. B, The CAB, RBCS, and
CHS mRNA abundance was quantified using a phosphor
imager and the mean values (±SE) are shown. A value of
100% on the ordinate represents the maximum steady-state mRNA detected
within the experiment.
|
|
To investigate CAB and RBCS gene expression and
chlorophyll content during fruit development, we analyzed fruits at
seven different physiological ages. The levels of CAB and
RBCS expression in the pericarp were found to be highest in
young fruits and they both gradually declined during fruit development
(Fig. 7A). The expression level of both
CAB and RBCS was amplified in the
hp-1w-mutant fruits compared with those of
WT. The increased level of CAB and RBCS
expression in the pericarp correlates well with its approximate 5-fold
higher chlorophyll content (25-d-old fruits contain 100 and 25 µg
chlorophyll g1 fresh weight in the
hp-1w mutant and WT, respectively; Fig.
7B).
View larger version (37K):
[in this window]
[in a new window]
| Figure 7.
A, The CAB and RBCS
mRNA accumulation in the pericarp of the
hp-1w-mutant and WT tomato fruit during
fruit ripening. For both CAB and RBCS
mRNA accumulation, the amount of mRNA in the pericarp of the youngest
hp-1w-mutant fruit was set at 100%. As a
loading control the blots were probed with a 18S rRNA probe. B, A truss
of WT (left) and hp-1w-mutant (right)
immature tomato fruits. The fruits of the
hp-1w mutant are darker green and have an
elongated shape when compared with WT.
|
|
| |
DISCUSSION |
The results presented indicate that CAB,
RBCS, and CHS gene expression is up-regulated in
the hp-1w mutant compared with WT. Two of
these genes, CAB and CHS, were shown to be
phytochrome regulated in the hp-1w mutant
(Figs. 2 and 3). However, their pattern of up-regulation is different
and dependent on the stage of development and tissue studied. For
instance, if we had only investigated gene expression in WL-grown
seedlings, we would have only seen up-regulation of RBCS and
CHS, but not CAB, compared with WT in the
hp-1w mutant (Figs. 4-6). In other words,
in WL-grown seedlings, CAB gene expression appears to be
saturated and is comparable in WL-grown WT and
hp-1w-mutant seedlings (Figs. 4-6). In
contrast, dark-grown hp-1w-mutant seedlings
accumulate higher levels of CAB (and RBCS)
transcripts than WT. In dark-grown seedlings of hp-1,
up-regulation of enzyme activity for Phe ammonia lyase (Goud et al.,
1991), nitrate reductase, nitrite reductase, and amylase (Goud and
Sharma, 1994) have been previously reported. All of these enzymes have
been shown to be phytochrome regulated (Goud et al., 1991; Goud and
Sharma, 1994). Taken together, these findings indicate that the
hp-1 mutation causes changes in the dark at the molecular
level. Unlike the dark "de-etiolated" Arabidopsis mutants such as
cop, det, and fus, there are no
visible differences between dark-grown WT and hp-1-mutant seedlings. Therefore, the hp-1
mutation appears to affect only a subset of responses regulated by
cop, det, and fus genes. The
CAB mRNA measured in dark-grown
hp-1w-mutant seedlings could be due to an
amplification of the response to the residual Pfr available in the
seeds, and probably reflects a phyA-mediated VLFR, which is very
difficult to test experimentally. The results with the phyA-deficient
hp-1w,fri1 double
mutant suggest that this is the case.
R induction of CAB was similar in the
hp-1w-mutant and WT seedlings (Fig. 1) and
confirms the observation of Wehmeyer et al. (1990). However, the
difference between hp-1w and WT observed in
darkness was retained. The RBCS gene expression level was
always higher in hp-1w seedlings than those
of WT, regardless of the conditions under which they were grown. In
fact, the high RBCS gene expression in the dark could not be
further enhanced by a single pulse of R, whereas in the WT a gradual
elevation of expression was observed (Fig. 1), which never attained the
level in hp-1w. We can therefore conclude
that the hp-1w mutation affects both
CAB and RBCS expression (Figs. 1, 2, 5, and 6).
Studies involving doc and cue1 mutants suggested
that different biochemical pathways downstream of phytochrome regulate CAB and RBCS gene expression (Li et al., 1994,
1995). Therefore, the differential effect of the hp-1
mutation on the expression of CAB and RBCS genes
may be related to the differences in the role of HP-1 in these
pathways.
The hp-1-mutant has high anthocyanin levels in both
seedlings and adult plants (Kerckhoffs et al., 1997a) and increased
flavonoid accumulation in ripe fruits (Yen et al., 1997). The
CHS transcript accumulation of the enzyme that is the first
committed step of flavonoid biosynthesis also shows a higher level in
the hp-1 mutant than WT (Figs. 3, 5, and 6). Since no
CHS gene expression could be observed in dark-grown or
single pulse-treated seedlings, a 12-h R or B pretreatment irradiation
schedule known to be effective in anthocyanin production (Peters et
al., 1989) was used to study CHS gene expression (Fig. 3).
Irrespective of whether a R or B pretreatment was given, significantly
higher levels of CHS transcripts accumulated in the
hp-1w-mutant seedlings compared with WT
(Fig. 3). These CHS mRNA accumulation data correlate
reasonably well with the anthocyanin accumulation data under the same
irradiation conditions (Peters et al., 1989). When WL-grown seedlings
were examined, we also found a strong correlation between
CHS abundance and anthocyanin content (results given in text
and Fig. 5).
The expression of CAB and RBCS genes decreased
with increasing fruit age in both WT and
hp-1w-mutant fruits (Fig. 7). In the
pericarp these data show a positive correlation with the chlorophyll
data (data not shown). The pericarp of green fruits is known to be
photosynthetically active, and this activity decreases during
chloroplast/chromoplast differentiation (Piechulla and Gruissem,
1987). Earlier work of Piechulla et al. (1985) showed that mRNA for
photosynthetic polypeptides disappear during fruit ripening. These
changes of mRNA levels correlated with alterations that occur at the
photosynthesis and polypeptide level (Piechulla and Gruissem, 1987).
Work of Meehan et al. (1996) using Arabidopsis transgenics that
expressed a CAB promotor fused to a GUS reporter gene shows
that GUS activities were positively correlated with chlorophyll content
and cell size. Therefore, the transcription of nuclear genes for
chloroplast components could be modulated by chloroplast numbers, which
increase with cell size.
Our results support the proposal that the hp-1 mutation
amplifies phytochrome responses (for review, see Kerckhoffs and
Kendrick, 1997): the R induction of both CAB and
CHS gene expression was higher in the hp-1 mutant
compared with WT and was shown to be mediated by phytochrome (Figs. 2
and 3). Since the R induction of CAB gene expression is
considerably lower in the phyA-deficient, hp-1w,fri1 mutant
than the hp-1w mutant (Fig. 2), phyA
appears to modulate the magnitude of the low fluence response.
Phytochrome-mediated CAB gene expression in tomato has a
VLFR component (Sharrock et al., 1988) and this response, as indicated
by the level induced by FR alone, is much reduced in the
hp-1w,fri1 mutant
compared with the hp-1w mutant (Fig. 2).
These data support the conclusion that phyA mediates the VLFR (Casal et
al., 1994; Botto et al., 1996; Shinomura et al., 1996). In addition,
phyB1 plays a role in CAB gene expression. The induction of
CAB transcript accumulation is reduced in the phyB1-deficient,
hp-1w,tri1 mutant
compared with the hp-1w mutant (Fig. 2).
Thus, both phyA and phyB1 play a role in CAB gene expression
in tomato. Reed et al. (1994) and Hamazato et al. (1997) came to a
similar conclusion when they studied phyA and
phyB mutants in Arabidopsis.
The data presented support the hypothesis that the HP-1 protein has a
repressive role in phytochrome-signal transduction. The pattern of
up-regulation observed for CAB, RBCS, and
CHS gene expression depends on the light conditions, stage
of development, and tissue studied. To date, hp-like
mutations have not been described in other plant species. The dark
phenotype of the hp-1 mutant is more subtle compared with
de-etiolated mutants such as cop, det, and
fus. Considering the higher levels of anthocyanin responses and CHS mRNA accumulation, the tomato hp-1 mutant
is somewhat similar to the icx1 mutant of Arabidopsis
(Jackson et al., 1995). The major difference between the two mutations
is that, whereas the icx1 mutation affects only the signal
transduction processes leading to the regulation of CHS
expression, the tomato hp-1 mutation also affects the
expression of genes (CAB and RBCS) encoding
proteins for the photosynthetic apparatus. This suggests that the
hp-1 mutation acts on an upstream signal transduction
event(s) that leads to the altered pattern of gene expression.
Therefore, HP-1 is proposed to be a fundamental phytochrome signal
transduction regulator, and the cloning of its gene and molecular
characterization is eagerly awaited.
| |
FOOTNOTES |
1
This work was supported by a Science and
Technology Agency fellowship of the Japan International Science and
Technology Exchange Center to J.L.P.
2
These two authors contributed
equally to this paper.
*
Corresponding author; e-mail peters{at}postman.riken.go.jp; fax
81-48-462-9405.
Received December 15, 1997;
accepted April 6, 1998.
| |
ABBREVIATIONS |
Abbreviations:
B, blue light.
CAB, chlorophyll
a/b-binding protein.
CHS, chalcone
synthase.
FR, far-red light.
R, red light.
RBCS, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit.
VLFR, very low fluence response.
WL, white light.
WT, wild type.
| |
ACKNOWLEDGMENTS |
We thank Ferenc Nagy for the CAB-1 cDNA clone,
William Gruissem for the RBCS-2 cDNA clone, John Yoder for
the CHS1 clone, and Shannon Frances for reading the manuscript and
making suggestions. The WT and hp-1w-mutant
seeds were supplied by Maarten Koornneef and colleagues of the
Department of Genetics, Wageningen Agricultural University, The
Netherlands.
| |
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A. Srinivas, R. K. Behera, T. Kagawa, M. Wada, and R. Sharma
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Top
Abstract
Introduction