Expression of nuclear genes that encode the A and B subunits of
chloroplast glyceraldehyde-3-phosphate dehydrogenase
(GAPA and GAPB) of Arabidopsis is
known to be regulated by light. We used a negative selection approach
to isolate mutants that were defective in light-regulated expression of
the GAPA gene. Two dominant mutants belonging to the
same complementation group, uga1-1 and
uga1-2, were then characterized. These two mutants showed a dramatic reduction in GAPA mRNA level in both
mature plants and seedlings. Surprisingly, mutations in
uga1-1 and uga1-2 had no effect on the
expression of GAPB and several other light-regulated genes. In addition, we found that the chloroplast
glyceraldehyde-3-phosphate dehydrogenase enzyme activity of the mutants
was only slightly lower than that of the wild type. Western-blot
analysis showed that the GAPA protein level was nearly
indistinguishable between the wild-type and the uga
mutants. These results suggested that posttranscriptional control was
involved in the up-regulation of the GAPA protein in the mutants. The
uga1-1 mutation was mapped to the bottom arm of
chromosome V of the Arabidopsis genome.
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INTRODUCTION |
Transcription is one of the primary
steps at which light regulates gene expression in plants (Terzaghi and
Cashmore, 1995
). Two classes of photoreceptors, phytochrome and blue
light/UV-A receptor (cryptochrome), are involved in the regulation of
photosynthetic genes (Batschauer, 1998; Briggs and Huala, 1999; Deng
and Quail, 1999; Fankhauser and Chory, 1999). It has been suggested
that eukaryotic phytochromes are Ser/Thr kinases with a two-component His kinase ancestry (Yeh et al., 1997; Yeh and Lagarias, 1998; Fankhauser and Chory, 1999; Fankhauser et al., 1999). Five phytochrome genes have been identified in Arabidopsis (Clack et al., 1994; Quail et
al., 1995; Quail, 1997). Current evidence indicates that the different
phytochromes may have distinct functions (Quail et al., 1995; Quail,
1997). Genetic and molecular studies have led to the identification of
four blue-light photoreceptors in Arabidopsis (Briggs et al., 2001).
CRY1 (HY4) and CRY2/PHH1 have partial overlapping functions in
promoting anthocyanin formation and inhibiting hypocotyl elongation
(Ahmad and Cashmore, 1993; Ahmad et al., 1995; Lin, 2000), whereas
PHOT1/NPH1 and PHOT2 regulate phototropism, stomatal opening, and
chloroplast movement (Liscum and Briggs, 1995; Briggs and Huala, 1999;
Kinoshita et al., 2001; Sakai et al., 2001). In addition, the mutations
in CRY1 and CRY2 genes affect blue-light-mediated
regulation of photosynthetic gene expression (Ahmad et al., 1995;
Conley and Shih, 1995; Mazzella et al., 2001).
Several mutants affecting the light signal transduction pathway appear
to be defective in genes that encode transcription factors.
PIF3 was found not only to interact directly with PhyB but
also with the promoters of many light-regulated genes (Ni et al., 1999;
Martinez-Garcia et al., 2000). The
hfr1/rsf1/rep1 mutants, on the other
hand, appeared to be specific for PhyA pathway (Fairchild et al., 2000;
Fankhauser and Chory, 2000; Soh et al., 2000). The HFR1 gene
product is a bHLH protein and, therefore, a putative DNA-binding
protein (Fairchild et al., 2000; Soh et al., 2000). It was also found
to interact with PIF3 (Ni et al., 1999; Fairchild et al.,
2000; Martinez-Garcia et al., 2000). Other phytochrome-specific
intermediates have also been cloned via the isolation of mutants. The
HY5 gene product was shown to be a basic Leu Zipper
transcription factor that interacts with light-responsive promoters
(Chattopadhyay et al., 1998b).
A number of cis-acting elements, including GT elements, G boxes, I
boxes, CGF element, and CCA element, have been characterized from
several photosynthetic genes, including RBCS and
LHCB, the nuclear genes encoding the small subunit of
Rubisco and light harvest complex proteins, respectively (Donald and
Cashmore, 1990; Gilmartin et al., 1990; Anderson et al., 1994;
Kenigsbuch and Tobin, 1995; Terzaghi and Cashmore, 1995; Wang et al.,
1997b). Based on in vitro-binding assays, genes that encode GBF,
GT1, and CCA1 factors have been identified in Arabidopsis. A survey of
the Arabidopsis genomic sequences indicated that each of these genes
belongs to a small gene family, with a highly conserved sequence in the
putative DNA-binding domains. To show which member(s) in the gene
family is involved in light regulation, it is essential to establish a
direct link between the in vitro-binding activities and the in vivo
function of transcription activation. This line of evidence is mostly
lacking, with the exception of CCA1, in which it was shown
that transgenic Arabidopsis plants expressing antisense RNA for CCA1
showed reduced phytochrome induction of the endogenous
LHCB1-3 gene (Wang et al., 1997b).
We have been studying light regulation of two nuclear genes
(GAPA and GAPB) that encode chloroplast
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis. In
higher plants, there are two chloroplast GAPDH isozymes, with subunit
structures of A4 and
A2B2, which are key enzymes
in the photosynthetic carbon fixation cycle (Cerff, 1982). In previous
studies, we showed that the expression of these two genes is
coordinately regulated by light at the transcriptional level in tobacco
(Nicotiana tabacum) and Arabidopsis (Shih and
Goodman, 1988; Dewdney et al., 1993). Several cis-acting elements and
their cognate binding factors of both GAPA and
GAPB genes were identified (Conley et al., 1994; Kwon et
al., 1994; Park et al., 1996; Chan et al., 2001). In etiolated seedlings, a short light pulse can induce transient increases of
GAPA and GAPB mRNA levels. However, this
induction cannot be reversed by subsequent far-red light treatment
(Dewdney et al., 1993). These regulatory patterns are distinct from
those of the pea (Pisum sativum) RBCS
genes (Kaufman et al., 1984) and Arabidopsis LHCB genes
(Karlin-Neumann et al., 1988), in which the effect of a short
red light pulse can be reversed by a subsequent far-red light
treatment. Continuous exposure of dark-treated mature plants or
etiolated seedlings to red, blue, or white light is required for
sustained high-level expression of GAPA and GAPB
genes in Arabidopsis, with blue and white light much more efficient
than red light (Dewdney et al., 1993; Conley and Shih, 1995). Our
results indicated that this effect is mediated by a combination of
phytochromes and the blue light photoreceptor encoded by the
CRY1 (HY4) gene (Conley and Shih, 1995). Results
from saturation linker scan mutagenesis of the GAPB promoter
constructs in transgenic Arabidopsis suggest that a single cis-acting
element may respond to more than one photoreceptor (Chan et al.,
2001).
In addition to the identification of cis-acting elements, we are
interested in obtaining mutations that affect light regulation of
GAPA and GAPB genes. Although a variety of
photomorphogenic mutants are available in Arabidopsis, most of these
mutants are defective in early steps in light-signaling pathways or are
not defective in GAP gene expression (Conley and Shih, 1995;
M.-C. Shih, unpublished data). Therefore, we used a negative selection scheme to isolate regulatory mutants that are defective in light activation of the GAPA gene. Here, we report the
characterization of two of these mutants. Our results indicated that
these two mutations affect very downstream steps in light signal
transduction pathways leading to the activation of the GAPA gene.
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RESULTS |
Selection of Mutants Affecting GAPA Gene
Expression
In the presence of allyl alcohol, wild-type plants with functional
ADH enzyme will die because of the conversion of allyl alcohol to toxic
aldehyde by ADH. In contrast, plants without functional ADH can survive
allyl alcohol treatment. Negative selection schemes using ADH as a
selectable marker were used to isolate aar mutants, which
are defective in hypoxic induction of ADH (Conley et al.,
1999), and cue mutants, which are defective in controlling the expression of LHCB3 (Li et al., 1995; Lopez-Juez et al.,
1998). We designed a similar selection scheme to isolate regulatory
mutants that are defective in light activation of the GAPA gene.
In the current scheme, we first transformed an Arabidopsis
ADH null mutant, adh1-2, with a construct that
puts ADH and 
-glucuronidase (GUS) coding sequences under the control
of separate GAPA promoters (see "Materials and Methods"
for details). Several independent transgenic lines that have GUS and
ADH activity were obtained. In all of these lines, the expression of
ADH and GUS transgenes was regulated by light
similar to that of the GAPA gene. One of these lines, AG-5G,
was chosen for mutagenesis. In 5-d-old etiolated AG-5G seedlings, the
accumulation of ADH activity reached a steady-state level after 12 to
24 h of white light treatment, similar to that of the endogenous
GAPA gene (Dewdney et al., 1993; Conley and Shih, 1995).
Titration experiments indicated that 7.5 mM allyl alcohol is needed to cause 100% lethality of the 24-h light-treated seedlings.
To obtain mutants that underexpress ADH, a total of 50,000 M2 seeds of AG-5G were germinated on filter
papers in the dark for 5 d and then subjected to 24 h of
white light treatment. The filters were then transferred onto medium
containing 7.5 mM allyl alcohol. After 2 h, filters
were moved onto a fresh agar medium. The surviving plants, which must
have lacked ADH activity, were assayed for GUS activity in leaves.
Among the 99 plants that survived allyl alcohol selection, 77 were GUS
positive and 22 were GUS negative. Only seven of the latter mutants
survived long enough to produce seeds, whereas the other 15 died or
failed to set seeds after transfer to the soil. The lethality could be
because of mutations in essential genes or the occurrence of multiple
mutations in these plants. The surviving
adh
gus plants,
designated as uga (underexpressor of GAPA), are
presumably defective in regulatory genes that control the expression of
GAPA. We characterized two of these mutants,
ugab3 and ugab9, as described below.
Table I showed that the
F1 progeny from crosses between line AG-5G and
each of the two uga mutants had low GUS activity
(GUS), indicating that all of them exhibited
the mutant phenotype. These results suggested that both
ugab3 and ugab9 mutations are dominant. However,
the F2 progeny from both crosses deviated
significantly from the expected 1:3 ratio. GUS+
and GUS plants in the F2
progeny from the cross between AG-5G and ugab3 showed a 1:6
ratio, whereas the cross between AG-5G and ugab9 produced a
1:8 ratio. One possible explanation for this observation could be that
the presence of the transgene resulted in the expression of ADH at
abnormally high level in leaves. This may have caused a high rate of
lethality to the individual plants that show the wild-type phenotype,
hence yielding a lower than expected wild-type progeny. Table I also
shows that the F2 progeny of the
ugab3 × ugab9 cross gave a
GUS+:GUS ratio of 0:132,
i.e. all the F2 progeny were mutant. This
indicated that the mutations in b3 and b9 belonged to the same
complementation group. The ugab3 and ugab9
mutants were hence renamed uga1-1 and uga1-2,
respectively.
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Table I.
Genetic analysis of uga mutants
GUS enzymatic assays were used to assess GUS+ and
GUS phenotype. F1 and F2 progeny
with GUS activity comparable with the homozygous AG-5G line as shown in
Fig. 1B were assigned as GUS+, whereas those with GUS
activity similar to homozygous mutants were assigned as
GUS.
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Effects of uga Mutations on the Expression of
GAPA
To quantify the effect of uga mutations on
the expression of GAPA::GUS and
GAPA::ADH transgenes, we compared the
levels of ADH and GUS activities in wild-type AG-5G and uga
mutants in 5-d-old etiolated seedlings subjected to 24 h of white
light treatment. Both mutants showed a moderate reduction in ADH and
GUS activity compared with the AG-5G line (Fig.
1A). When 4-week-old light-grown plants
were assayed for reporter gene activities, the difference between the
wild type and the mutants was far more dramatic. As shown in Figure 1B,
the two mutants exhibited 30% and 23% of the wild-type level of
steady-state ADH activity, whereas their GUS activity was reduced to
3% and 5% of that in the wild type. These results indicated that both
mutants are impaired in the expression of both the ADH and
GUS reporter genes, especially so in mature plants.
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Figure 1.
Effects of uga mutations on
GAPA::ADH and
GAPA::GUS transgenes. GUS and ADH
activities of AG-5G and uga mutants in 5-d-old etiolated
seedlings, greening seedlings, and 4-week-old plants were determined as
described in "Materials and Methods." A, Unit of ADH enzyme is
defined as an increase in A340 of 1 min mg
protein1. B, GUS activity is expressed as pmol
4-methylumbelliferone min1
mg1 protein. The data presented are the average
of three independent treatments. Plants grown at different times were
used for replicated treatments. For each treatment, a total of about
500 plants was pooled and used for protein extracts preparation. Error
bars = SDs.
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To determine the effects of uga mutations on the expression
of the endogenous GAPA gene, mRNA levels from 5-d-old
light-grown Arabidopsis seedlings were compared with those from 5-d-old
etiolated seedlings. The results from one set of representative
northern blots were illustrated in Figure
2A. These experiments were repeated three
times and the resulting blots were quantified using the GAPA
mRNA levels from light-grown AG5G as 100% (Fig. 2B). In greening seedlings, GAPA mRNA levels in uga1-1 and
uga1-2 were 2- to 3-fold lower than the level in AG-5G (Fig.
2).
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Figure 2.
Effects of uga mutations on the
expression of GAPA in seedlings. A, Total RNAs from
seedlings grown in continuous light (L) or complete darkness (D) for
5 d were isolated and analyzed by northern-blot analysis.
Representative data are from gels loaded with 5 µg RNA
lane1 and probed with radiolabeled
GAPA or TUB. B, Each northern-blot analysis was
repeated three times using RNA samples from plants grown at different
times. Relative densitometric values were obtained by first taking the
ratio of GAPA signal intensity over that of the
corresponding TUB signal for each lane, and then dividing that by the
ratio to obtain obtained for light-grown AG-5G. Therefore, relative
densitometric value for light-grown AG5G is taken as 1. Error bars = SDs.
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To determine the effects of uga mutations on the
expression of GAPA in mature plants, mRNAs from light-grown
4-week-old plants were compared with those of plants that were light
grown for 4 weeks and then dark adapted for 5 d (Fig.
3A). The quantification data showed that
levels of GAPA mRNA in uga1-1 and
uga1-2 were more than 20-fold lower than that of AG-5G in
3-week-old plants (Fig. 3B). Consistent with our prior results (Dewdney
et al., 1993; Conley and Shih, 1995), the data also showed that there was barely detectable GAPA mRNA in both etiolated seedlings
(Fig. 2) and dark-adapted mature plants (Fig. 3). The combined results demonstrated that uga1-1 and uga1-2 mutations
affect the expression of both the endogenous GAPA gene and
the GAPA::GUS and
GAPA::ADH transgenes. Therefore, it is
likely that these mutations are defective in a regulatory gene that
controls the expression of GAPA. However, the observation
that the uga mutations had more severe effects on the mRNA
levels of GAPA in mature plants than in seedlings suggested
that the transcription complexes required for GAPA
activation are not identical in these two stages.
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Figure 3.
Effects of uga mutations on the
expression of GAPA in mature plants. A, Total RNAs from
light-grown 4-week-old plants (L) were compared with those of plants
that were light grown for 4 weeks and then dark adapted for 5 d
(D) were isolated and analyzed by northern-blot analysis.
Representative data are from gels loaded with 5 µg RNA
lane1 and probed with radiolabeled
GAPA or TUB. B, Each northern-blot analysis was
repeated three times and quantified as described in Figure 2. The
average densitometric value for light-grown AG5G is taken as 1. Error
bars = SDs.
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Effects of uga Mutations on the Expression of Other
Light-Regulated Genes
Because GAPA and GAPB gene products
constitute subunits of the GAPDH holoenzyme (Cerff, 1982), it is
reasonable to expect that these two genes are controlled by the same
regulatory mechanism. Therefore, we compared the GAPB mRNA
levels between wild-type and the two uga mutants in mature
light-grown and dark-adapted plants by northern-blot analysis (Fig.
4A). Surprisingly, the levels of
GAPB mRNA in uga1-1 and 1-2 were similar to that
of wild type (Fig. 4, A and B). Next, we determined the effects of uga mutations on the expression of two other carbon fixation
genes, TIM and FBA. We found that the kinetics of
mRNA accumulation for these two genes during light induction were
identical to those of GAPA and GAPB (M.-C. Shih,
unpublished data). However, no significant difference in the
transcription of these genes was observed between the uga
mutants and AG-5G (Fig. 4).
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Figure 4.
Effects of uga mutations on the
expression of other light-regulated genes in mature plants. A,
Northern-blot analyses of RNAs from 4-week-old light-grown (L) or
dark-adapted (D) AG-5G, uga1-1, and uga1-2 were
performed as described in Figure 3 with radiolabeled GAPB,
TIM, FAB, LCHB, GAPC,
CHS, and TUB probes. B, Each northern-blot
analysis was repeated three times and quantified. The average
densitometric value of each gene from light-grown AG5G is taken as 1. Error bars = SDs.
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Because combinatorial cis-acting elements are required to confer light
responsiveness of light-regulated promoters in plants (Terzaghi and
Cashmore, 1995; Puente et al., 1996; Chattopadhyay et al., 1998a), it
is possible that uga mutations affect the expression of
genes from different metabolic pathways. Therefore, we
performed northern-blot analyses for three other genes, including
GAPC, LHCB3, and CHS (Feinbaum and
Ausubel, 1988; Yang et al., 1993; Li et al., 1995), which are
known to be regulated by light. In addition to light, the transcription
of GAPC, which encodes the C subunit of GAPDH, could also be
regulated by Suc (Shih and Goodman, 1988; Yang et al., 1993). As shown
in Figure 4, A and B, there were no observable differences in the mRNA
levels of these genes in either light-grown or dark-adapted mature
plants between the two uga mutants and the AG-5G line. These
data suggested that the uga mutations specifically affect
the expression of GAPA.
Biochemical Characterization of uga Mutants
Because the GAPA mRNA levels decreased drastically in
both uga1-1 and uga1-2 mutants, we decided to
examine whether the chloroplast GAPDH activity in these mutants was
similarly affected. As seen in Figure 5,
the two mutants showed only slightly lower chloroplast GAPDH activities
compared with AG-5G. This result suggested that posttranscriptional
regulation of GAPA mRNA or posttranslational modification of
the GAPDH enzyme could have occurred to compensate for the reduced
GAPA mRNA level in the uga mutants. To
distinguish between these possibilities, western-blot analysis was
performed to quantify the protein levels of the A and B subunits (Fig.
6). The data showed that there were
similar amounts of A and B polypeptides in leaf extracts from wild
type, uga1-1, and uga1-2. These findings suggested that translational control of GAPA must have
occurred in the uga mutants to compensate for their reduced
levels of GAPA mRNA.
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Figure 5.
Chloroplast GAPDH activity of mature plants.
Chloroplast GAPDH activity of 5-week-old plants from AG-5G and
uga mutants was assayed as described by Cerff (1982). Each
reading was obtained from the pooling of the aerial portions of 10 individual plants per line. The data shown are the average of two
independent measurements from plants grown at different times. Specific
activity is calculated as the rate of decrease of
A366 per milligram protein extract. Error
bars = SDs.
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Figure 6.
Western analysis of GAPA and GAPB in AG-5G and
uga mutants. Total cellular proteins were isolated from
leaves of mature AG-5G, uga1-1, and uga1-2
plants. Ten-microgram proteins from each sample were subjected to
western-blot analysis using rabbit antibody raised against the GAPDH
A2B2 tetramer. The arrows
indicate the positions of A and B subunits of the chloroplast
GAPDH.
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The uga1-1 Mutation Maps to the Bottom Arm of
Chromosome V
We used the simple sequence length polymorphism (SSLP) mapping
method (Bell and Ecker, 1994) to determine the chromosomal location of
the uga1-1 mutation. The transgenic line AG-5G, the parental
strain of uga mutants, is derived from Columbia ecotype (Col-O). We performed crosses between uga1-1 and Landsberg
erecta (Ler) to generate F2
progeny as mapping populations. To score F2
progeny, we needed a suitable marker. Unfortunately, uga
mutants lack any visible phenotype and the cross to Ler
resulted in a loss of one or two copies of the transgene in some of the
F2 progeny. As a result, GUS activity could not
be used as a scoreable phenotype. However, knowing that the
GAPA mRNA level differs by almost 20-fold between wild type
and uga1-1 (Fig. 3), we used the GAPA mRNA levels to assess the genotype of the F2 progeny.
Dot-blot analyses were used to compare GAPA mRNA levels of a
population of F2 progeny. We isolated total RNA
from leaves of 94 F2 progeny of
uga1-1 × Ler. Genomic DNA was isolated from
each of these plants by the method of Edwards et al. (1991). RNA from F2 progeny was subjected to slot-blot analyses
using a P32-labeled cDNA fragment of
GAPA as the hybridizing probe. We found that 20 of 94 F2 progeny had GAPA mRNA levels
comparable with those of wild type and that the remaining 74 samples
had very low levels of GAPA mRNA (Fig.
7A). Because of the dominant nature of
uga1-1 mutation, the 74 plants with low mRNA levels should be either UGA1+/uga1-1 or
uga1-1/uga1-1 and the 20 plants with high
GAPA mRNA should be homozygous
UGA1+. As opposed to the skewed ratios
obtained using GUS expression as the phenotypic marker, the RNA dot
blot gave a 1:3.3 ratio, which was close to the expected 1:3 ratio.
This confirmed that the uga1-1 mutation is a dominant,
monogenic mutation.
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Figure 7.
SSLP analyses of uga1-1 × Ler F2 progeny. A, Dot-blot analysis
was performed as described in "Materials and Methods" to compare
GAPA mRNA levels of 94 F2 plants. RNA
from AG-5G line was loaded on two corners of the filter (A1 and H12) to
be used as a quantification standard. B, Gel electrophoresis of PCR
products for the ciw9 SSLP marker. Genomic DNA from
UGA3+/UGA3+
F2 plants was used in PCR with ciw9 as the primer
pair. PCR conditions were identical to those described by Bell and
Ecker (1994). Lanes 1 through 3 were PCR products from reactions using
genomic DNA from (1) Ler (2), Col-O, and (3) AG-5G, respectively, as
templates.
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Next, we performed PCR analysis of the 20 UGA1+/UGA1+
plants using 14 primer pairs corresponding to 14 SSLP markers that span the Arabidopsis genome over its five chromosomes, with at least one
marker on each arm (see "Materials and Methods" for the list). Our
results showed that the markers on chromosomes I through IV had no
association with the Ler/Ler ecotype. However, in
the case of the marker ciw9 that lies on the bottom arm of chromosome
V, 18 of 20 F2 progeny had a
Ler/Ler ecotype at this locus, indicating that
the UGA1 gene was linked to this marker (Fig. 7B).
Confirming the linkage of the UGA1 gene to this marker, it
was found that the ciw10 marker, which is also located on the bottom
arm of chromosome V, was also linked to the UGA1 gene, but
not as tightly. Here, 11 of 20 samples showed the
Ler/Ler ecotype (data not shown). The
uga1-1 mutation, therefore, is mapped to the bottom arm of chromosome V in the Arabidopsis genome in the vicinity of the ciw9
marker (at 88 cM).
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DISCUSSION |
We have identified two allelic mutations that affect the
expression of the GAPA gene in Arabidopsis. Our results
showed that the mRNA levels of both
GAPA::GUS and
GAPA::ADH transgenes and the endogenous
GAPA gene in light-grown uga1-1 and
uga1-2 mutants are greatly reduced. One possible explanation
for this observation is that the effect of uga mutations on
light induction of the GAPA gene is mediated at the
transcriptional level. However, there were examples that light affects
mRNA stability and this effect often involved the 5'- or
3'-untranslated region (UTR) sequences (Dickey et al., 1998; Anderson
et al., 1999). The fact that the GAPA::GUS and
GAPA::ADH transgenes in the AG5G line
contain all or part of the 5'-UTR of GAPA (see "Materials
and Methods") raised the possibility that the uga
mutations might affect the mRNA stability of GAPA. We are in
favor of the first interpretation, because results from our nuclear
run-on experiments indicated that light effect on the steady-state
GAPA mRNA level occurred mainly at the transcription level
in both tobacco and Arabidopsis (Shih and Goodman, 1988; see also
supplemental data). In addition, we have identified two
cis-acting elements that are required for light induction of
GAPA by deletional analyses of promoter constructs in
transgenic plants (Conley et al., 1994; Park et al., 1996). A
combination of these two elements could confer light responsiveness on
a basal promoter that was not regulated by light (Park et al., 1996).
Results from our genetic analysis showed that both uga1-1
and uga1-2 mutations are dominant (Table I). With
some exceptions, e.g. shy2 (Kim et al., 1996; 1998), most
photomorphogenic mutants that have been isolated, such as
phyA, phyB, red1, fhy1,
fhy3, and cue1, are all recessive (Parks and
Quail, 1993; Whitelam et al., 1993; Li et al., 1995; Wagner et al.,
1997). There are a few ways to explain how this dominant phenotype
could occur. First, the UGA1 gene in its normal wild-type
state could function as a positively acting intermediate in the
signaling pathway leading to the light-activated transcription of
GAPA. This would mean that the uga mutant gene
product must act in a dominant negative manner. One possibility is that
the resulting functional UGA1 gene product is a multimeric
protein comprising several subunits of the UGA1 gene
product. The binding of a mutated subunit could cause the entire
protein structure to lose its function and, therefore, fail to effect
the light-activated transcription of GAPA. Alternatively, we
could propose that the UGA1 gene product in its wild-type
state normally represses GAPA transcription. Along with the
action of other positively acting transcription factors, the
UGA1 gene product would maintain an acceptable level of
GAPA mRNA under a given set of environmental conditions.
Repression could be achieved either by interaction with other
light-signaling molecules in the pathway or by direct interaction with
the GAPA promoter. The mutation could have resulted in a
much tighter interaction and, therefore, a more dramatic repression effect.
It should be pointed out that, although the RNA dot blot of
the F2 progeny generated from the mapping cross
(uga1-1 × Ler) showed the expected 1:3
segregation ratio (Fig. 7A), the data obtained from
F2 progeny of the backcross
(uga1-1 × AG-5G) showed a 1:6 ratio as determined by
the GUS assay (Table I). One possible explanation for this abnormal
segregation ratio in the latter cross is that the AG-5G line contains
the GAPA::ADH transgene. The regulation
of endogenous ADH levels in a plant is tightly controlled in terms of
tissue specificity and in its response to hypoxia and other
environmental stresses (Dolferus et al., 1994; Chung and Ferl,
1999; Conley et al., 1999; Ellis et al., 1999). In AG-5G, however,
where the ADH gene is driven by a GAPA promoter,
the ADH activity is expressed about 70-fold higher than that in the Col
wild-type plant (C.S. Chan and M.-C. Shih, unpublished data).
Furthermore, its expression occurs throughout the entire plant instead
of being tissue specific, which could have resulted in physiological
abnormality in AG-5G. In fact, AG-5G plants were observed as
slow-growing compared with the true wild type, Col. Assuming that the
overexpression and misexpression of ADH in AG-5G had
increased lethality, the introduction of uga mutations,
which decrease ADH expression, might have actually increased
the survival rate of AG-5G.
Specificity of the uga Mutations
Among a number of light-regulated genes examined here,
uga1-1 and uga1-2 affect only the expression of
GAPA. This was seen in the dramatic reduction in the
steady-state GAPA mRNA level in mature plants (Fig. 3),
whereas little or no effect was seen in the expression of
GAPB and several other light-regulated genes, namely
LHCB3, FBA1, and TIM (Fig. 4).
Furthermore, we found that several photomorphogenetic phenotypes,
including hypocotyl length, chlorophyll content, and chloroplast,
appeared to be normal in the uga mutants (data not shown).
In contrast, in most other light-signaling mutants, more than one gene
or class of genes is affected. For example, the cue1 mutant
(now known to be a mutation in the PPT gene), which was
isolated based on defective LHCB3-promoter driven reporter activity, was not only defective in endogenous
LHCB3 transcription but also in the transcription
of RBCS and RBCL (Li et al., 1995). The
psi2 mutant, which was isolated based on elevated LHCB2-LUC (luciferase) activity, was found to be
hypersensitive in LHCB1,
LHCB2, CHS, and RBCS
expression when compared with the wild-type equivalent (Genoud et al.,
1998). However, we cannot eliminate the possibility that other genes
that have not been examined in this study are unaffected by the
uga mutations. The implications of this specificity are
severalfold. First, the UGA1 gene is likely to lie
downstream in the light-signaling pathway leading to the
transcriptional activation of GAPA. The second implication
is that although GAPA and GAPB may be
coordinately regulated at the transcriptional level (Dewdney et al.,
1993), there probably exist distinct portions of their pathways that are independent of each other.
Importance of Translational Control of the GAPA Protein
Although the uga mutants showed drastic reduction in
steady-state GAPA mRNA levels, the uga mutants
appeared to survive very well, even though the A4
isozyme accounts for 80% of the total chloroplast GAPDH activity in
the plant. The assay of chloroplast GAPDH enzyme activities in
5-week-old plants revealed that chloroplast GAPDH activity was only
modestly reduced in the mutants compared with AG-5G (Fig. 5).
Furthermore, western-blot analysis also revealed that the
GAPA protein levels in the uga mutants were
indistinguishable from that in AG-5G (Fig. 6). These results suggested
that step(s) between the end of transcription and the completion of
translation is the critical step in determining the final GAPDH levels
in the uga mutants.
There are two ways in which such posttranscriptional regulation could
have been achieved. First, one can envision a system whereby
differential rate of transcription does not play any significant role
in the regulation of the final GAPA protein levels. This could occur if the GAPA mRNA were always made in excess of
what is required by the cell. Alternatively, because GAPA
mRNA degradation is relatively fast (Dewdney et al., 1993; Conley and
Shih, 1995), it is possible that the differences in transcription rates
between AG-5G and the uga mutants may have little
significance. Instead, translational control led to nearly equal levels
of the GAPA protein between wild type and mutants. We could
not argue in favor of either model based on our current results.
Many nuclear genes in plants are regulated at the posttranscriptional
level (Gallie and Bailey-Serres, 1997). In Arabidopsis, ACS5, which encodes 1-aminocyclopropane-1-carboxylic
acid synthase, is shown to be regulated posttranscriptionally (Woeste
et al., 1999). The cytokinin-inducible soybean (Glycine
max) CIM1 gene is regulated by the stability of the
CIM1 mRNA rather than by the CIM1 transcriptional
level per se (Downes and Crowell, 1998). In a study of the thylakoid
peptide plastocyanin and the Rieske polypeptides, mRNA transcript
levels may have increased 10-fold upon illumination, but association of
transcripts with polysomes only increased 2- to 3-fold, suggesting that
mRNA uptake into polysomes is an important step of posttranscriptional
control (Palomares et al., 1993). In the case of the proton-ATPase
gene, regulation by translation rate in response to developmental and environmental cues is signified by the presence of a long 5'-UTR that
contained an upstream open reading frame (Michelet et al., 1994). There
is a 47-bp UTR in the GAPA transcript, suggesting translation as a possible mechanism of control. As reviewed by Bailey-Serres (1999), translation of mRNA is emerging as an important mode of gene regulation where initiation is frequently the step at
which regulation is achieved. Some features that influence translation
rate include the interactions between the 5' and 3' ends of the
message, and variation in the cap-binding protein of which there are
three types in Arabidopsis, as triggered by developmental and other
environmental cues (Gallie and Bailey-Serres, 1997).
| |
MATERIALS AND METHODS |
Generation of Transgenic Arabidopsis Plants
An Arabidopsis adh1-2 mutant in a Col background
obtained from Dr. Dan Voytas (Department of Genetics, Iowa State
University) was used as the starting strain. A binary construct
carrying two consecutive reporters, ADH and GUS, each driven by the
GAPA promoter, was constructed as follows. The
GAPA::GUS/pBI101, which linked about a 1-kb promoter sequence and the complete 47-bp 5'-UTR of GAPA to the GUS coding sequence (Conley et al., 1994),
was used as the starting plasmid. A DNA fragment that contains the
1,045 to +30 of GAPA was generated by PCR and linked
to a DNA fragment containing the complete coding sequence of
ADH. The
GAPA::ADH DNA fragment was then
cloned into the BamHI site of the
GAPA::GUS/pBI101. The resulting
pBI101 derivative was mobilized into Agrobacterium tumefaciens by triparental mating (Bevan, 1984)
and then transformed into the adh1-2/adh1-2 starting
strain using the floral dip method as described by Clough and Bent
(1998). T1 progeny containing at least one copy of the
transgene were selected by kanamycin resistance. Each transgenic line
was carried on to the T2 generation, where a transgenic
line with a single transgene insertion was selected based on a 3:1
segregation of the kanamycin resistance phenotype at the T2
generation. Within the transgenic T2 population, a
homozygous line, designated as AG-5G, was selected based on a 4:0
segregation pattern at the F3 and then again at the
F4 generation. The bulked seeds of this line constitute the
parental strain, which has an
adh1-2/adh1-2/Col background and carries
two reporters, ADH and GUS, each driven by the GAPA promoter.
Mutagenesis and Generation of M2 Progeny
Twenty thousand seeds of the AG-5G line were subjected to ethane
methane sulfonate mutagenesis according to the method described by
Somerville and Ogren (1982), with a few modifications. In brief, seeds
were soaked in 0.1 mM ethane methane sulfonate for 16 h with rocking at room temperature, washed with 100 mM
sodium thiosulphate, and rinsed thoroughly with water. Seeds were then
treated at 4°C for 3 d before being sown onto soil at a density
of 0.5 cm2 and maintained in a growth chamber at 22°C.
M1 plants were carried on to the next generation by
selfing, after which M2 seeds were harvested into four
separate pools.
Allyl Alcohol Selection
M2 seeds were imbibed on Whatman No. 1 filter paper
(Whatman, Clifton, NJ) soaked in 3.5 mL of Murashige and Skoog
liquid medium containing 2% (w/v) Suc in glass petri dishes at
a density of about 500 seeds per plate, carefully spread out using a
sterile plastic pipette tip. Control plates containing AG-5G seeds as well as seeds of the adh1-2 mutant were similarly
prepared to be later used for comparison of lethality in allyl alcohol.
Seeds were cold incubated at 4°C in the dark for 3 d and then
transferred to a dark growth chamber at 22°C for 5 d. The
etiolated seedlings were then subjected to 24 h of white light
treatment as described in the following section. We found that the
expression of the GAPA gene reached a maximal level
after 24 h of white light treatment (Dewdney et al., 1993; Conley
and Shih, 1995). The filters were then transferred onto medium
containing 7.5 mM allyl alcohol in Murashige and Skoog + 2% (w/v) Suc. This concentration was the minimum concentration
of allyl alcohol that would cause >99% lethality to AG-5G seedlings.
After 2 h, the filters were transferred onto fresh agar Murashige
and Skoog medium containing 2% (w/v) Suc. Allyl alcohol
resistant mutants were isolated on d 4 to 5. These allyl alcohol
resistant plants were then subjected to GUS histochemical staining over
a 24-h staining period.
Light and Growth Conditions
Plants on soil were kept at 22°C under 16-/8-h light/dark
cycle. White light was provided by three cool-white 35-W fluorescent lamps at 50 µmol m2s1. Blue light was
used at 5.5 µmol m2s1 supplied by four
fluorescent lamps with a blue plexiglas 3-mm filter (Rohm-Haas no.
2423, Ditric Optics, Hudson, MA) as previously described (Conley
and Shih, 1995). In the case of etiolated seedlings, about 200 (for
enzyme assays) or 400 (for RNA extraction) seeds were imbibed on 3.5 mL
of Murashige and Skoog + 2% (w/v) Suc liquid medium soaked on
two pieces of Whatman No. 1 filter paper in a glass petri dish,
vernalized for 3 d, and then transferred to a light-proof
incubator at 22°C for etiolation.
RNA Isolation and Northern-Blot Analyses
Total RNA was isolated by the Triazol LS method (Life
Technologies/Gibco-BRL, Cleveland) using about 50 to 200 mg of
plant tissue for each extraction. Northern analysis was performed as previously described (Conley and Shih, 1995). Five micrograms of RNA
was loaded per lane, unless otherwise stated. Gels were blotted
overnight onto nylon Hybond N+ membranes (Amersham
Pharmacia Biotech, Piscataway, NJ) with 10× SSC as the transfer
buffer. The GAPA, GAPB,
GAPC, RBCS, LCHB, and
TUB cDNA probes were as described by Conley and Shih
(1995). The TIM fragment was excised by
SalI and NotI resulting in a 1.4-kb fragment; FBA1 was 1.1 kb in length after excision by
HindIII. Normalization for loading was accomplished by
stripping the original probe off the filter by dipping in deionized
water at 80°C and checking for counts using a Geiger counter. The
filter was then reprobed with TUB, the transcription of
which is unaffected by light (Conley and Shih, 1995). The bands on the
autoradiograph of each northern were quantified using Scion Image
version 1.62 software (National Institutes of Health, Bethesda,
MD). Relative mRNA levels were then determined by taking the
ratio of the band intensity specific for the gene probe of interest
minus the background intensity to that for TUB.
Enzymatic Assays
GUS enzyme assay and histochemical staining were performed as
described by Jefferson et al. (1987). ADH enzyme assays were performed
as described by Xie and Wu (1989). This assay uses ethanol as the
substrate and measures the production of NADH. Measurement of NADH
formation was performed in a DU 64 spectrophotometer (Beckman Instruments, Fullerton, CA). A unit of ADH is defined as the
production of 1 nmol of NADH min1 mg1
protein. Chloroplast GAPDH assays were performed as described by Cerff
(1982). To determine chloroplast-specific GAPDH activity, NADPH was
used as the starting cofactor instead of NADH. Specific activity is
calculated as the rate of decrease of A366
per milligram protein extract.
Western-Blot Analysis
The aerial portions of 10 to 15 plants from each mutant line
were harvested and homogenized in liquid nitrogen using a mortar and
pestle after which cold homogenization buffer (15 mM HEPES [pH 7.6], 40 mM KCl, 5 mM MgCl2,
1 mM dithiothreitol, and 0.1 mM
phenylmethylsulfonyl fluoride) was added at 10 mL g1 of
fresh tissue. A 10-fold volume of 4 M ammonium sulfate was added drop-wise with stirring. The mixture was then centrifuged at
20,000 rpm in a SW41 swing bucket rotor (30,000g) at
4°C for 30 min. The supernatant was filtered through a Miracloth
(Calbiochem, La Jolla, CA) after which freshly ground ammonium
sulfate was added slowly at 0.33 g mL1 to
precipitate proteins. Proteins were then spun down at 19,000 rpm in an
SW41 at 4°C for 30 min and resuspended in 1 mL of buffer (20 mM HEPES [pH 7.6], 40 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 10% [w/v]
glycerol). Aliquot (400 µL) of the extract was desalted using
ultra-free low-bind (10-kD cutoff) filter apparatus (no. UFC3LGC00,
Millipore, Bedford, MA). Ten micrograms of protein per sample
was used for western analysis as previously described (Wang et al.,
1997a) using the semiwet transfer system. The membrane was
incubated with a 1:3,000 (w/v) dilution of the rabbit antibody
generated against the GAPDH A2B2 isozyme in
blocking buffer at room temperature with swirling for 1 h. Under
these conditions, this antibody reacts specifically with A and B
subunits (Wang et al., 1997a). The bands were visualized with
ECL western-blotting detection solution (Amersham-Pharmacia Biotech) and quantified with National Institutes of Health
Scion Image software version 1.62.
Mapping Cross and F2 Progeny
The uga1-1 mutant was crossed to the
Ler wild type. The F1 seeds resulting from
this cross were grown and selfed to produce F2 seeds. The
homozygous recessive F2 progeny resulting from the mapping
cross were selected based on the results from the RNA dot-blot analysis.
For dot blot analysis, a 9- by 12-cm nylon Hybond N+
membrane (Amersham) was prewet in 10× SSC, blotted dry on Whatman No. 1 filter paper, and assembled on the dot blot apparatus (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's
manual. The 96 wells were then rehydrated by the addition of 500 µL
of 10× SSC into each well and applying vacuum until dry. Three
micrograms of RNA per sample derived from F2 progeny of the
mapping cross was subjected to alkaline denaturation by the addition of
500 µL of ice-cold 10 mM NaOH and 1 mM EDTA
and kept on ice. A total of 94 samples of the F2 progeny
were dot blotted onto the nylon membrane together with the AG-5G RNA
sample dotted at the top left and bottom right corners as positive
controls. Hybridization with the GAPA probe was as
described above for northern analyses. The F2 individuals
that were homozygous recessive for the uga phenotype
(wild type for GAPA mRNA expression) were then matched to the corresponding numbered plant material reserved for DNA isolation. DNA was isolated using the method of Edwards et al. (1991).
PCR of SSLP Markers
About 1 to 10 ng of template DNA was used for PCR using standard
reaction conditions provided by Promega (Madison, WI) at 2.5 mM of MgCl2. Three control tubes using DNA
isolated from wild-type Ler, wild-type Col, and the
AG-5G line were set up and run concurrently with the F2
samples for band size comparison. The primers used for amplifying SSLP
markers are as follows: chromosome I, ciw12 and nga111; chromosome II,
ciw2, ciw3, and nag168; chromosome III, nag162 and nga6; chromosome IV,
ciw5, ciw7, and nga1107; and chromosome V, CTR1, ciw8, ciw9, and ciw10
(Lukowitz et al., 2000). Typically, the annealing temperature was set
at 2°C above the higher melting temperature (Tm) of the two
primers if they were less than 2°C apart from each other. If the Tms
of the two primers were more than 2°C apart, the average between the
two Tms was used as the annealing temperature. The PCR cycles are 30 cycles of 95°C, 1 min; 55°C (or other annealing temperature), and 1 min; 72°C, 1 min; followed by 5-min extension at 72°C. About 10 µL of the PCR reaction was resolved on a NuSieve GTG 4%
(w/v) agarose gel in 1× Tris-acetate EDTA buffer. Each tier on
the DNA agarose gel was run with three control lanes, which carried the respective PCR products of Ler, Col, and AG-5G for band
size comparison. F2 wild-type recessive progeny from the
mapping cross could then be scored as Ler, heterozygous,
or Col ecotypes on the agarose gels for each SSLP marker.
Statistical Analysis
All comparisons between data of mutants versus AG-5G were done
by ANOVA one-way analysis with Bonferroni's method, whereas phenotypic
ratios of genetic crosses were tested by Chi square analysis. The
SigmaStat software (SPSS Sciences, Chicago) was used in each case.
Distribution of Materials
Upon request, all novel materials described in this publication
will be made available in a timely manner for noncommercial research purposes.
We thank Drs. Erin Irish and Jonathan Poulton for comments on
the manuscript. We thank the Arabidopsis Biological Resources Center
(Ohio State University, Columbus) for providing cDNA clones of
FBA and TIM.
Received April 30, 2002; returned for revision May 20, 2002; accepted July 12, 2002.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.007849.
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ABSTRACT
INTRODUCTION