text:
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The key enzyme of sulfate assimilation, adenosine
5¢-phosphosulfate reductase, is regulated by HY5
in Arabidopsis
Bok-Rye Lee†,‡, Anna Koprivova†and Stanislav Kopriva*
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
Received 15 April 2011; revised 18 May 2011; accepted 25 May 2011; published online 4 July 2011.
*For correspondence (fax +44 1603 450014; e-mail firstname.lastname@example.com).
†These authors contributed equally to this work.
‡Present address: Department of Biochemistry and Molecular Biology, Michigan State University, 209 Biochemistry Building, East Lansing, MI 48824-1319, USA.
Plant sulfate assimilation is regulated by demand for reduced sulfur, as is its key enzyme, adenosine
5¢-phosphosulfate reductase (APR). In a genetic screen for mutants lacking this regulation, we identified the
bZIP transcription factor LONG HYPOCOTYL 5 (HY5) as a necessary component of the regulatory circuit.
Regulation of APR activity by the inhibitor of glutathione synthesis, buthionine sulfoximine, or by the
precursor of cysteine, O-acetylserine, was disrupted in the hy5 mutant. When dark-adapted plants were
re-illuminated, the rapid induction of APR1 and APR2 mRNA levels was attenuated in hy5 seedlings, but APR3
regulation was not affected. Chromatin immunoprecipitation revealed that HY5 binds directly to the APR1 and
APR2 promoters but not to the APR3 promoter. Accordingly, the regulation of APR1 and APR2 by
O-acetylserine was disturbed in hy5 roots. HY5 is also important for the coordination of nitrogen and sulfur
assimilation, as, unlike the wild-type, hy5 mutants do not undergo a reduction in sulfate uptake and APR
activity during nitrogen starvation. Altogether, these data show that HY5 plays an important role in regulation
of APR gene expression and plant sulfate assimilation.
Keywords: adenosine 5¢-phosphosulfate reductase, LONG HYPOCOTYL 5, sulfate assimilation, glutathione,
O-acetylserine, nitrate deficiency.
Sulfur is an essential nutrientfor all forms of life. It is present
in the amino acids cysteine and methionine, and is thus an
important component of proteins and peptides. Many
enzymes require sulfur-containing co-enzymes and pros-
thetic groups, such as iron–sulfur centers or thiamine, for
their activity. Plants contain many other organic sulfur
compounds, such as thiols, sulfolipids, glucosinolates or
alliins, that play important roles in the normal plant lifecycle
and in protection against stress and pathogens. The source
of sulfur for plants is inorganic sulfate. In the sulfate
assimilation pathway (Figure 1), sulfate is taken up by
sulfate transporters. It is first activated to adenosine-5-
phosphosulfate (APS) by ATP sulfurylase, and then reduced
to sulfite by APS reductase (APR). Sulfite is reduced to sul-
the amino acid acceptor O-acetylserine (OAS) to form
cysteine. Cysteine can be used directly for synthesis of
proteins and glutathione (GSH), or serve as a donor of
reduced sulfur for synthesis of methionine and various
co-enzymes (Davidian and Kopriva, 2010; Takahashi et al.,
2011). The tripeptide GSH is a major sulfur-containing
metabolite that is important for many cellular processes,
such as maintenance of cellular redox status or defence
against abiotic and biotic stress (Rouhier et al., 2008). In
addition, APS can be further phosphorylated by APS kinase
to 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS), which is
the donor of active sulfate for many sulfation reactions in
secondary metabolism (Mugford et al., 2009).
The key enzyme of the sulfate assimilation pathway, APR,
is highly regulated according to the demand for reduced
sulfur (Figure 1) (Lappartient et al., 1999; Kopriva, 2006;
Davidian and Kopriva, 2010; Takahashi et al., 2011). This
regulation appears to be at the transcriptional level, as APR
activity and mRNA levels of its three isoforms are reduced
during nitrogen or CO2deficiency (Koprivova et al., 2000;
Kopriva et al., 2002), and by exposure to thiol compounds
ª 2011 The Authors
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Page 2
such as cysteine and GSH (Vauclare et al., 2002). Indeed,
Arabidopsis root cultures exposed to thiols have been used
for control flux analysis to confirm the major role of APR in
regulation of sulfate assimilation (Vauclare et al., 2002). On
the other hand, APR is induced during sulfur deficiency, as
well as by high levels of OAS (Neuenschwander et al., 1991).
Another example of demand-driven control is the regulation
of APR by buthionine sulfoximine (BSO), which inhibits the
first enzyme of GSH synthesis. BSO causes GSH depletion
and thus a higher demand for sulfate reduction, such that
APR activity is increased (Hartmann et al., 2004).
While the responses of APR activity and transcript levels
to changes in environmental conditions are well docu-
mented, very little is known about the molecular mechanism
of regulation and signalling pathways. Similarly, only a few
transcription factors involved in the regulation of sulfur
metabolism have been identified. The SULFUR LIMITA-
TION1 (SLIM1) transcription factor has been shown to
up-regulate expression of the sulfate transporter genes
AtSULTR1;1, AtSULTR1;2 and AtSULTR4;2 in response to
sulfate starvation (Maruyama-Nakashita et al., 2006). SLIM1
is also responsible for regulation of genes involved in
glucosinolate degradation and for induction of miR395, but
is not involved in regulation of APR (Maruyama-Nakashita
et al., 2006; Kawashima et al., 2009). In addition, two groups
of R2R3-MYB factors, which control aliphatic and indolic
glucosinolate biosynthesis in Arabidopsis thaliana (Gigo-
lashvili et al., 2007; Hirai et al., 2007), also regulate genes of
sulfate assimilation. Two isoforms of APS kinase, APK1 and
APK2, and two ATP sulfurylase genes, ATPS1 and ATPS3,
are regulated by these MYB factors (Yatusevich et al., 2010).
Beside these two enzymes that provide PAPS for the last
step of glucosinolate synthesis, APR and sulfite reductase
from the reductive part of the pathway are part of the same
regulatory network (Yatusevich et al., 2010). However, the
contribution of the MYB factors to control of primary sulfate
assimilation is unclear, and no other transcription factor
controlling sulfur assimilation, and in particular APR, has
been identified so far.
To identify components of the signalling pathways that
control APR gene expression in response to demand for
reduced sulfur, we performed a genetic screen to identify
genes involved in regulation of APR by BSO. One of mutants
that we identified, BSO insensitive roots (bir24), was found
to possess a nonsense mutation in a gene encoding the bZIP
transcription factor HY5. Here we describe the role of HY5 in
regulation of APR and the sulfate assimilation pathway. We
show that HY5 plays a central role in regulation of APR by
light and also by other conditions.
HY5 is involved in demand-driven regulation of sulfate
To dissect the mechanisms of demand-driven regulation of
sulfate assimilation by BSO, we exploited the well-known
inhibitory effect of BSO on root growth (Vernoux et al., 2000;
Koprivova et al., 2010a,b; Maughan et al., 2010) to perform a
genetic screen for BSO-insensitive mutants. Among the
BSO-insensitive mutants that possessed long roots despite
the presence of BSO in the medium, two mutants, bir24 and
bir25, displayed an additional phenotype, a long hypocotyl
(Figure 2a). Complementation analysis revealed that the
mutants were allelic, and bir24 was selected for further
analysis. Coarse mapping positioned the BIR24 gene on the
top arm of chromosome 5. A gene, LONG HYPOCOTYL 5
(HY5), mutations of which produce the same phenotype as
bir24, is located at this position (At5g11260). Sequencing of
the HY5 gene from the bir24 mutant revealed a G fi A
mutation in exon 3 at position 752 after the ATG, resulting in
a change of Asn138 into a premature stop codon (Figure 2b).
To confirm that the phenotype of bir24 is caused by the
mutation in the HY5 gene, HY5 was expressed in the bir24
mutant under the control of the CaMV 35S promoter. Two
independent complemented lines showed strongly reduced
root growth in the presence of BSO, as for the Col-0 wild-
type (WT) (Figure S1). In addition, a T-DNA insertion line in
the HY5 gene, denoted hy5, was analysed (Figure 2b). In WT
plants, root length was reduced by BSO by 80–90%, but the
root growth inhibition was highly attenuated in both the
bir24andhy5mutants(Figure 2c).Thus,themutationinHY5
is responsible for reduced sensitivity of the bir24 mutant to
BSO. All further experiments were therefore performed with
the T-DNA insertion line hy5.
Figure 1. Pathway of sulfate assimilation and regulation of APR.
The scheme also shows the inhibition of GSH synthesis by BSO, leading to
GSH depletion, and the negative regulation of APR by cysteine and nitrogen
deficiency. Positive regulation of APR by sulfur deficiency, light, O-acetylser-
ine and GSH depletion is indicated by arrows.
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Next we tested whether HY5 affects the demand-driven
regulation of the sulfate assimilation pathway. As BSO
inhibits GSH synthesis, GSH levels were determined in
seedlings grown on 1.25 mM BSO for 6 days. BSO treatment
reduced GSH content in both Col-0 and the hy5 mutant, but
the decrease was more pronounced in Col-0 (89%) than in
the mutant (73%) (Figure 2d). To investigate whether HY5
is also involved in regulation of APR by BSO, the enzyme
activity was measured in these seedlings. As expected, APR
activity in Col-0 was increased by BSO but was not affected
in the hy5 mutant (Figure 2e). Thus HY5 participates in the
response of sulfate assimilation to inhibition of GSH
synthesis by BSO.
As the previous experiments addressed adaptation of the
plants to BSO over a period of 6 days, we further explored
whether HY5 disruption also affects short-term regulation of
APR. When 2-week-old Arabidopsis seedlings were treated
with 1.25 mM BSO for 2 days, APR activity in the roots
increased 2.5-fold in Col-0 but not in hy5 (Figure 3a). Thus,
more general regulation of APR activity was compared
between hy5 and Col-0. Similar to the findings for BSO, 24 h
after transfer to plates supplemented with 1 mM OAS, APR
activity increased fourfold in Col-0 but was not affected in
hy5 (Figure 3b). However, HY5 does not seem to be involved
in all regulatory circuits. Treatment with 1 mM cysteine
strongly reduced APR activity in both genotypes (Figure 3c).
Similarly, APR activity was induced by sulfate deficiency to
the same extent in roots of both Col-0 and hy5 (Figure 3d).
However, in agreement with the role of HY5 in light
regulation, illumination of dark-adapted plants for 90 min
resulted in an increase in APR activity in Col-0 but not in hy5
(Figure 3e). Interestingly, APR activity was consistently
higher in the hy5 mutant than in Col-0 under control
conditions. These data thus showed that disruption of HY5
affects regulation of APR under some but not all conditions.
HY5 is involved in light regulation of APR gene expression
HY5 is a bZIP transcription factor and a positive regulator of
photomorphogenesis (Ang et al., 1998). Mutation in HY5
causes a defect in the inhibition of hypocotyl elongation
Figure 2. Mutation in HY5 causes the bir24 phenotype.
(a) Comparison of the phenotypes of the original bir24 and bir25 mutants with
those ofCol-0 andhy5. The plantswere grownfor 6 days onMS/phytagel(top
panel) or MS/phytagel supplemented with 1.25 mM BSO (bottom panel).
(b) The positions of the point mutation in bir24 and the SALK T-DNA insertion
for HY5 are indicated by triangles. Exons are shown as black boxes and
introns as thin lines.
(c) Root length of Col-0, bir24 and hy5 seedlings grown for 6 days on MS/
phytagelplates withorwithout1.25 mM BSO.Valuesaremeans ? SDfromat
least 30 plants.
(d, e) Glutathione (GSH) concentration (d) and adenosine 5¢-phosphosulfate
reductase (APR) activity (e) of Col-0 and hy5 seedlings grown for 6 days on
MS/phytagel plates with or without 1.25 mM BSO.
Values are means ? SD from three independent plant pools. Different letters
indicate significant differences (P < 0.05).
1044 Bok-Rye Lee et al.
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under all light conditions, suggesting that HY5 acts down-
stream of PHYA, PHYB, cryptochromes and UV-B (Ang et
al., 1998; Ulm et al., 2004). HY5 directly binds to the pro-
moters of more than 1000 light-inducible genes (Lee et al.,
2007). An initial experiment indicated that disruption of HY5
affects light induction of APR (Figure 3e). We therefore
tested the role of HY5 in light regulation of APR in detail.
Col-0 and hy5 seedlings were adapted to the dark for 38 h,
exposed to white light, and transcript levels of the three
APR isoforms were determined by quantitative RT-PCR
(Figure 4). Light rapidly increased steady-state transcript
levels for all three APR isoforms in Col-0, but to different
extents. After 90 min illumination, APR1 mRNA levels were
increased approximately fourfold and APR3 mRNA levels
were increased approximately 2.5-fold. However, the level
of APR2 mRNA was increased threefold after 30 min, and
reached 12-fold higher levels after 90 min than that in
controls kept in the dark (Figure 4b). In contrast, in hy5
seedlings, APR1 mRNA was not induced by light at any
time point. The degree of APR2 induction was lower in hy5,
especially at 60 and 90 min. No difference in light regula-
tion of APR3 between hy5 and Col-0 was detected (Fig-
ure 4c).These resultsshow
participate in regulation of APR expression by light, but in
an isoform-specific manner.
To find out what consequences the differential regulation
of APR by light in hy5 has on the the sulfate assimilation
thatHY5 does indeed
pathway, the flux through pathway was determined in dark-
adapted seedlings kept in darkness and re-illuminated for
4 h. Sulfate uptake was not affected by light in either
genotype (Figure 5a). The incorporation of35S into cysteine,
GSH and proteins after 4 h incubation in solution with
[35S]sulfate, as a measure of flux through the pathway, was
(Figure 5b). However, while the flux in Col-0 was increased
3.5-fold in light compared to dark, the increase in hy5 was
only twofold, in agreement with a lower capacity for light
induction of APR mRNA in this mutant.
As HY5 integrates signals of phytochromes and crypto-
chromes, we determined whether specific photoreceptors
are necessaryforthelightinductionofAPR.Thus,regulation
of APR by light was assessed in the phytochrome mutants
phyA and phyB and in the cryptochrome mutants cry1, cry2
and cry3. APR activity did not change by 60 min after
re-illumination of Col-0 or any of the mutants compared to
dark-adapted plants (Figure S2a). However, APR mRNA
levels were induced by light in all genotypes. In dark-
adapted plants, APR1 mRNA levels were significantly lower
in cry1 and cry2 than in Col-0, but they were higher in phyB
and cry2 after re-illumination (Figure S2b). The induction of
APR2 mRNA by light was lower in phyB and cry2 mutants
(four- to fivefold) than in WT (sevenfold), but no substantial
differences were observed for APR3 (Figure S2d). Thus,
while no single photoreceptor is specific for APR regulation
Figure 3. HY5 is involved in the regulation of APR activity.
(a–c) Col-0 and hy5 plants were grown on MS/phytagel plates for 2 weeks and transferred to fresh media with or without (a) 1.25 mM BSO for 2 days, (b) 1 mM OAS
for 1 day, or (c) 1 mM cysteine for 1 day, and APR activity was measured in the roots.
(d) Col-0and hy5 were grown on MS/phytagelplates containing 1500 mM sulfate (control) and 15 mM sulfate (S0) for 2 weeks, and APR activity wasmeasured in the
(e) Col-0 and hy5 seedlings were grown for 7 days on horizontal MS/phytagel plates and kept in darkness for 38 h. Afterwards, the plates were either further kept in
darkness or exposed to light for 90 min. APR activity was measured in whole seedlings.
Values are means ? SD from three pools of ten roots. Different letters indicate values that are significantly different at P < 0.05.
HY5 in control of sulfate assimilation 1045
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by light, PHYB and CRY2 may have more importance for
regulation of APR2.
Involvement of HY5 in regulation of APR by OAS
As preliminary experiments indicated that HY5 is involved
in demand-driven regulation of APR, we compared the
well-known regulation of APR by OAS in hy5 and Col-0. Di-
rect application of OAS solution to the roots led to rapid
induction of mRNA levels for all three APR isoforms in both
genotypes (Figure 6 and Figure S3). However, as with light
treatment, the level of induction differed between hy5 and
Col-0. While APR1 transcript levels in Col-0 roots were
induced fivefold and 3.5-fold at 2 and 4 h, respectively, the
increase in hy5 was significantly lower (3.5- and 2.5-fold,
respectively) (Figure 6a). An even greater difference was
observed for APR2, for which the mRNA levels in OAS-
treated Col-0 roots were 7.5-fold higher than in control roots
after 2 h, but only fourfold higher in hy5 (Figure 6b). As for
light treatment, no difference in regulation of APR3 was
observed (Figure 6c). The lower degree of APR transcript
induction in the hy5 mutant led to a significant difference in
the response of APR activity to OAS treatment in the two
genotypes. Although OAS treatment increased APR activity
Figure 4. Regulation of APR expression by light in Col-0 and hy5 plants.
Col-0 and hy5 seedlings were grown for 7 days on horizontal MS/phytagel
plates and kept in darkness for 38 h. Afterwards, the plates were either further
kept in darkness or exposed to light for 15–90 min. Steady-state transcript
levels of the APR isoforms APR1 (a), APR2 (b) and APR3 (c) were determined
by quantitative RT-PCR in whole seedlings. Closed symbols represent
measurements in plants kept in darkness for an additional 90 min. The
quantitative RT-PCR reactions were performed in triplicate for each of the
three independent biological samples. The values in the dark at time 0 were
set to 1 for both genotypes.
Values are means ? SD from three pools of ten seedlings. Asterisks indicate
hy5 values that are significantly different from those of Col-0 at P < 0.05.
Figure 5. Regulation of sulfate assimilation by light in Col-0 and hy5 plants.
Col-0 and hy5 seedlings were grown for 7 days on horizontal MS/phytagel
plates and kept in darkness for 38 h. Afterwards, seedlings were incubated for
4 h in nutrient solution adjusted to a sulfate concentration of 0.2 mM and
supplemented with [35S]sulfate in the dark, or exposed to light.
(a, b) Sulfate uptake (a) was determined, as well as the flux through sulfate
assimilation (b), measured as35S incorporation into cysteine, glutathione and
Values are means ? SD from three pools of ten seedlings. Different letters
indicate significant differences at P < 0.05.
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in both genotypes between 2 and 7 h (Figure S4), this
increase was more substantial and significant in Col-0 than
in hy5. The maximum level of APR activity induction was
higher, and it was achieved earlier in Col-0 than in hy5 roots
(Figure 6d). Thus, HY5 participates in the demand-driven
regulation of APR by OAS.
HY5 is important for regulation of sulfate assimilation
by nitrogen deficiency
Sulfate assimilation is known to strongly interact with
nitrogen metabolism, and APR is specifically down-regu-
lated in nitrogen-starved plants (Koprivova et al., 2000). In
addition, a recent study found that the HY5 is essential for
induction of a key enzyme in nitrogen assimilation, nitrate
reductase (NR, encoded by Nia genes) (Jonassen et al.,
2008). Therefore, a possible involvement of HY5 in regula-
tion of APR by nitrogen availability was tested. WT plants
and hy5 mutants were subjected to nitrogen deficiency for
6 days, and the mRNA levels of APR isoforms and APR
activity were determined in the shoots (Figure 7 and Figure
S5). Nitrogen deficiency resulted in a slow decrease in APR
transcript levels, particularly APR1 and APR3. No difference
in regulation of APR mRNA levels between hy5 and Col-0
was observed. However, APR activity declined much more
rapidly in shoots of nitrogen-deficient Col-0 than in hy5
(Figure 7d). HY5 is thus important for regulation of APR by
nitrogen deficiency; however, this regulation may not be
directly through transcriptional regulation.
Interestingly, nitrogen deficiency resulted in a strong dark
colouring of Col-0 leaves but not hy5 leaves. Indeed, after
6 days of nitrogen deficiency, the levels of total chlorophyll
in Col-0 were strongly decreased but anthocyanins accumu-
lated (Figure S6). No such changes in pigment levels were
detected in hy5 plants.
also controls other components of the sulfate assimilation
sulfate assimilation pathway was determined. Similar to the
resultsintheshoots,3 daysofnitrogendeficiencyresultedin
(Figure 8a). Under control conditions, sulfate uptake was
lower in hy5 than in Col-0 (Figure 8c). Under nitrogen
deficiency, the uptake was significantly decreased in WT
but not in hy5 mutants (Figure 8c). However, this result was
not corroborated by expression analysis, as, similar to APR,
mRNA levels for the main transporter responsible for uptake
of sulfate into the roots, SULTR1;2, did not differ between
the two genotypes (Figure 8b). Thus, nitrogen deficiency
Figure 6. Regulation of APR by OAS in Col-0 and hy5 plants.
Col-0 and hy5 plants were grown for 2 weeks on vertical MS/phytagel plates. Water (control) or 1 mM OAS solution were pipetted directly onto the roots, and the
plants were incubated for further 24 h.
(a–c) Steady-state transcript levels of the APR isoforms APR1 (a), APR2 (b) and APR3 (c) were determined in the roots by quantitative RT-PCR.
(d) APR activity was measured in the roots at the indicated times after treatment.
Values are means ? SD of relative values in treated (OAS) versus control plants for both genotypes. Asterisks indicate time points at which relative values (OAS
versus control) in hy5 are significantly different from those in Col-0 at P < 0.05. The absolute values are presented in Figures S3 and S4.
HY5 in control of sulfate assimilation 1047
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resulted not only in reduced APR activity but also in signif-
in Col-0 but not hy5 (Figure 8d). The differences between
sulfate uptake, APR activity and flux through the sulfate
assimilation pathway for of Col-0 and hy5 under control
conditions are reflected in the concentrations of sulfur-
containing metabolites. Although sulfate content was lower
in hy5 seedlings than in Col-0, both cysteine and GSH
accumulated in the mutant (Table 1). Thus, HY5 appears to
be important not only for the response of APR to nitrogen
deficiency, but also for the regulation of sulfate uptake and
the flux through the sulfate assimilation pathway.
To test whether HY5 plays a general role in response to
nitrogen deficiency, expression of genes involved in nitrate
uptake and assimilation was compared in nitrogen-deficient
Col-0 and hy5 roots. NRT1;1, NRT2;1, Nia1 and Nia2 were
regulated by nitrogen deficiency in the same way in Col-0
and hy5 (Figure S7). Thus, HY5 appears to be important for
some but not all plant responses to nitrogen deficiency,
namely the regulation of sulfate but not nitrate assimilation.
As HY5 appears to be involved in signalling of nitrogen
status to sulfur assimilation, we tested whether it may also
status to nitrate assimilation. Therefore, regulation of genes
involved in nitrogen assimilation by sulfur deficiency was
compared in WT and hy5. In contrast to nitrogen deficiency,
HY5 was not involved in regulation of APR by sulfate
deficiency (Figure 3d), as APR activity was significantly
increased in both Col-0 and hy5. The transcript levels of
the nitrogen assimilationgenes NRT1;1, Nia1 and Nia 2 were
significantly decreased by sulfur starvation in both hy5 and
Col-0, but NRT2;1 was not affected (Figure S8). Therefore, it
appears that HY5 participates only in signalling from nitro-
gen to sulfur assimilation but not the other way round.
HY5 directly controls APR1 and APR2 transcription
In order to discover whether HY5 directly regulates APR
gene expression by binding to the promoters of the corre-
spondinggenes,chromatin immunoprecipitation (ChIP)was
used. HY5 antisera were used to precipitate chromatin from
Col-0 and hy5 seedlings. Quantitative PCR of DNA isolated
from this chromatin revealed enrichment of the amplifica-
tion in Col-0 compared to the hy5 mutant for promoters of
APR1 and APR2 but not APR3 (Figure 9). As controls, the
sulfur-inducible gene At5g24660 that is not under control of
Figure 7. Regulation of APR by nitrogen deficiency in Col-0 and hy5 plants.
Col-0 and hy5 plants were grown on plates containing nutrient solution with a nitrate concentration of 4 mM for 2 weeks, and transferred either to fresh plates
(control) or to nitrate-free plates (N0). The plants were incubated for further 6 days.
(a–c) Steady-state transcript levels of APR isoforms APR1 (a), APR2 (b) and APR3 (c) were determined in the shoots by quantitative RT-PCR.
(d) APR activity was measured in the shoots at the indicated times after treatment.
Values are means ? SD of relative values in treated (N0) versus control plants for both genotypes. Asterisks indicate time points at which relative values (N0 versus
control) in hy5 are significantly different from those in Col-0 at P < 0.05.
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HY5 (Lee et al., 2007) were used. These showed no signifi-
cant difference in amplification between Col-0 and hy5.
Thus, HY5 directly binds to the promoters of APR1 and
APR2. As we observed differences in regulation of sulfate
uptake by nitrogen deficiency between Col-0 and hy5, we
also tested whether HY5 binds the promoter of SULTR1;2, as
suggested by the global analysis (Lee et al., 2007). Indeed,
the ChIP data indicate that HY5 interacts with the SULTR1;2
promoter and thus affects transcription of another key gene
in controlof sulfateassimilation.
SULTR1;2 mRNA levels were induced by OAS to greater
extent in Col-0 than in hy5 roots (Figure 10). Thus, in addi-
tion to APR, HY5 regulates another key step in sulfate
assimilation, i.e. high-affinity sulfate uptake into the roots.
HY5 is a transcription factor that regulates APR
Very little is known about the molecular mechanisms of
APR.Using agenetic screento searchfor new regulators,we
identified a mutation in the HY5 gene that led to significant
alterations in the mutant response to inhibition of GSH
synthesis. The screen exploited the reduction of root growth
by BSO (Vernoux et al., 2000; Koprivova et al., 2010a,b), and
mutants with long roots under these conditions were iso-
lated.Thesamescreeningconceptled totheidentificationof
a family of three thiol transporters in the chloroplast mem-
brane (Maughan et al., 2010) and a pentatricopeptide pro-
tein involved in splicing of the mitochondrial nad7 transcript
and affecting the function of respiratory complex I (Kopriv-
ova et al., 2010b). Several aspects of the response to BSO
(root growth, GSH content and induction of APR activity) are
affected in the hy5 mutant. Further detailed analysis
revealed that HY5 is important for regulation of APR by light
and by several conditions that alter the demand for reduced
sulfur, such as OAS treatment and nitrogen deficiency. This
function of HY5 is not directly linked with the conditions of
the genetic screen. However, consistently over most exper-
iments, APR activity under control conditions was higher
in hy5 than in Col-0 (e.g. Figure 3). This is reflected in the
increased GSH content in both control and BSO-treated hy5
(Figure 2d and Table 1). The higher GSH level is most
probably the reason why the gene was identified in the
screen,as primaryroot growthisdependenton GSHcontent
(Koprivova et al., 2010a).
HY5 thus can be added to the still very short list of
transcription factors that regulate sulfur metabolism, which
so far consists of SLIM1 and six MYB factors (Maruyama-
Nakashita et al., 2006; Yatusevich et al., 2010). However, in
contrast to these other transcription factors, direct evidence
for the binding of HY5 to APR promoters was obtained
(Figure 9). The ChIP experiment clearly revealed binding of
HY5 to APR1 and APR2 promoters, but such binding was not
confirmed for APR3. The difference in signal strength
between the hy5 mutant and WT was not particularly strong,
Figure 8. Regulation of sulfate assimilation by
nitrogen deficiency in hy5.
Col-0 and hy5 plants were grown on plates
containing nutrient solution with a nitrate con-
centration of 4 mM (control) for 2 weeks before
transfer for a further 3 days to either fresh
control or nitrate-free medium (N0).
(a) APR activity was measured in the roots.
(b) Transcript levels of SULTR1;2 were deter-
mined in the roots by quantitative RT-PCR.
(c, d) The seedlings were incubated for 4 h with
their roots submerged in nutrient solution
adjusted to sulfate concentration of 0.2 mM and
supplemented with [35S]sulfate. Sulfate uptake
(c) was determined as well as the flux through
sulfate assimilation (d), measured as 35S incor-
poration into cysteine, glutathione and proteins.
Values are means ? SD from three independent
pools of eight plants. Asterisks indicate values
that are significantly different from controls at
Table 1 Concentration of sulfur-containing metabolites in hy5 and
Col-0 plants
23.1 ? 0.9
0.11 ? 0.02
0.95 ? 0.08
18.6 ? 3.1*
0.18 ? 0.04*
1.62 ? 0.35*
Col-0 and hy5 seedlings were grown for 14 days on horizontal MS/
determined in three independent pools of ten seedlings. Values are
means ? SD (lmol g FW)1). Asterisks indicate values that are signif-
icantly different between the genotypes at P < 0.05.
HY5 in control of sulfate assimilation 1049
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Page 9
but was comparable to the results obtained by Xi et al.
(2010). It is possible that the signal in hy5 is at least partially
caused by the presence of the related transcription factor
HYH (homologue of HY5; Holm et al., 2002). The results of
ChIP thus confirmed the results of a global identification of
putative HY5 binding sites (Lee et al., 2007), which also
predicted binding of HY5 to APR1 and APR2 promoters but
not APR3. The results also agree well with differential
regulation of individual APR isoforms between hy5 and
Col-0 (Figures 4 and 6). In both the light induction and OAS
treatment experiments, only APR1 and APR2 responded
differently in Col-0 and hy5, whereas APR3 was regulated in
the same way in both genotypes. The direct biochemical
evidence that HY5 binds to APR promoters is significant, as
the evidence for the previously identified factors is only
indirect. SLIM1 was identified based on lack of up-regulation
of a reporter gene expressedfromthe SULTR1;2promoter in
response to sulfur starvation in the slim1 mutant, and the
involvement of MYB factors in APR regulation was deter-
mined by in vitro assay and analysis of plants over-
expressing the corresponding genes (Maruyama-Nakashita
et al., 2006;Yatusevich et al., 2010). In addition to APR, other
genes of the pathway have been predicted to be a target for
HY5, most notably the SULTR1;2 gene, which encodes the
major high-affinity sulfate transporter (Lee et al., 2007).
Indeed, the ChIP analysis confirmed interaction of HY5 with
the SULTR1;2 promoter (Figure 9), and the regulation of this
gene by OAS was affected in the hy5 mutant (Figure 10).
However, only minor alterations in sulfate metabolism, such
as theconcentrationof sulfur-containingcompoundsorAPR
activity level, were detected in hy5 mutants under normal
conditions. Thus, similar to SLIM1 and the MYB factors
(Maruyama-Nakashita et al., 2006; Yatusevich et al., 2010),
HY5 affects multiple genes of the pathway,but appearsto be
responsible only for certain aspects of regulation.
HY5 is involved in regulation of APR by light
Our results clearly show that HY5 is important for light reg-
ulation of APR. We found that APR1 transcript levels were
not increased in hy5 seedlings in the first 90 min of re-illu-
mination of dark-adapted plants, and APR2 was induced
more slowly in the mutant (Figure 4). The results, including
thegreaterdifferencebetweenhy5andWTfor APR1than for
APR2, agree well with the results of ChIP, which confirmed
binding of HY5 to APR1 and APR2 promoters, and with the
results of Lee et al. (2007), who identified more putative HY5
binding sites in the APR1 promoter than in the APR2 pro-
moter, and no binding sites in the APR3 promoter. Obvi-
ously, however, HY5 is not the only factor involved in
regulation of APR by light, as APR2 and APR3 mRNA levels
were also increased in hy5. The differences in APR transcript
Figure 9. HY5 binds to the promoters of APR1
and APR2.
The binding of HY5 to promoters of APR iso-
forms was determined by chromatin immuno-
precipitation. Chromatin was isolated from Col-0
and hy5 seedlings and subjected to immunopre-
cipitation with antisera against HY5. The precip-
itated DNA was purified and subjected to
quantitative real-time RT-PCR using primers
specific for promoters and coding regions of
APR1, APR2 and APR3, and to the promoter of
SULTR1;2. Amplification with primers for the
promoter of At5g24660 was used as a negative
control. The PCR results were standardized using
primers against a Mu-like retrotransposon. Sig-
nificant differences (P < 0.05) between Col-0 and
hy5 are indicated by asterisks.
Figure 10. Regulation of SULTR1;2 by OAS in Col-0 and hy5 plants.
Col-0 and hy5 plants were grown for 2 weeks on vertical MS/phytagel plates.
Water (control) or 1 mM OAS solution were pipetted directly onto the roots,
and the plants were incubated for further 24 h. Steady-state transcript levels
of SULTR1;2 were determined in the roots by quantitative RT-PCR.
Values are means ? SD of relative values in treated (OAS) versus control
plants for both genotypes. Asterisks indicate time points at which relative
values (OAS versus control) in hy5 are significantly different from those of
Col-0 at P < 0.05.
1050 Bok-Rye Lee et al.
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The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1042–1054
Page 10
levels led to a difference in induction of APR activity, which
was not affected by re-illumination of dark-adapted hy5
mutants but was induced in Col-0. Correspondingly, the flux
through sulfate assimilation was inducedby light to a higher
level in Col-0 than in hy5. As APR activity in hy5 under nor-
malgrowth conditionswasconsistentlyhigherthan inCol-0,
this difference inactivity after a short lightinduction must be
complemented after light onset by the action of alternative
transcription factors.
Our results on light regulation of APR agree well with
previous evidence for regulation of sulfate assimilation by
light. APR has previously been shown to be regulated by
light and induced by illumination conditions similar to this
study in mature Arabidopsis plants (Kopriva et al., 1999). In
both studies, APR2 was induced more strongly and rapidly
than the other isoforms. APR1 and APR3 were regulated
more strongly and rapidly in the seedlings than in mature
plants (Figure 4 and Kopriva et al., 1999). APR activity and
flux through the sulfate assimilation pathway in Lemna
minor are also induced by light (Neuenschwander et al.,
1991). In addition, ATP sulfurylase activity increased with
irradiation in oats (Avena sativa), barley (Hordeum vulgare)
and maize (Zea mays), and decreased when inhibitors of
photosynthetic electron transfer were added to nutrient
solution (Passera et al., 1989). Interestingly, HY5 was
recently shown to regulate the key enzyme of nitrate
assimilation, nitrate reductase (Jonassen et al., 2008). The
nitrate assimilation pathway in plants has many common
features with sulfate assimilation, and the two pathways are
highly coordinated (Brunold, 1993). NR activity and protein
levels show a similar diurnal rhythm to APR, with a peak
to APR, NR mRNA levels increase at the end of the dark
period. Our results indicate that the pathways may even
share the mechanisms of regulation of their key genes.
Indeed, Jonassen et al. (2008, 2009) reported that loss-of-
function mutants of HY5 and HYH genes disturbed light
induction of NR, similar to the attenuation of light regulation
of APR in hy5.
HY5 connects nitrogen and sulfur assimilation
Sulfate assimilationis strongly interconnected with nitrogen
metabolism. Deficiency of one element represses the other
pathway (Takahashi and Saito, 1996; Kim et al., 1999;
Koprivova et al., 2000). APR expression and activity have
been shown to be down-regulated by nitrogen starvation in
various systems (Yamaguchi et al., 1999; Koprivova et al.,
2000), while sulfur deficiency results in a reduction of NR
activity and accumulation of amino acids (Reuveny et al.,
1980; Migge et al., 2000; Prosser et al., 2001).
We clearly show that nitrogen and sulfur assimilation are
connected through HY5. Compared to Col-0, APR activity
was less reduced in hy5 under nitrogen deficiency in both
leaves and roots (Figures 7 and 8). Importantly, the hy5
mutation affected not only APR. The uptake of sulfate and
flux through sulfur assimilation were also not reduced by
nitrogen deficiency in the hy5 mutant. However, the reduc-
tion in flux in Col-0 appears to be caused by reduced sulfate
unaffected by nitrogen deficiency, despite lower APR activ-
ity. Thus, under nitrogen limitation, sulfate uptake appears
to exert greater control over sulfate assimilation than APR.
In hy5, the relative flux is slightly higher than in Col-0 under
both control and N-limited conditions, corresponding to the
higher APR activity (Figure 3) and agreeing with the higher
thiol content in hy5 (Table 1). The lower sulfate content in
hy5 appears to reflect both this increased flux and the lower
sulfate uptake capacity under control conditions. In addition
to the substantial alteration in the response of sulfate
assimilation to nitrogen deficiency, another well-described
response, accumulation of anthocyanins (Diaz et al., 2006)
was disrupted in hy5 plants. This agrees with previous
reports showing that induction of anthocyanin synthesis by
cytokinins or chilling is impaired in hy5 mutants (Vanden-
bussche et al., 2007; Zhang et al., 2011). Interestingly, the
function of HY5 in regulation of response to nitrogen
deficiency does not include nitrate assimilation (Figure S7).
Down-regulation of nitrate transport and nitrate reductase
under these conditions must thus be achieved by another
regulatory network, possibly including the lateral organ
boundary domain (LBD) family of transcription factors
(Rubin et al., 2009). The existence of multiple regulatory
circuits in response to nitrogen limitation is thus similar to
the response to sulfur deficiency, in which SLIM1 controls
the regulation of sulfate transporters but not APR (Maruy-
ama-Nakashita et al., 2006).
HY5 appears to connect nitrate and sulfate assimilation
unidirectionally. Although this factor is important for regu-
lation of nitrate reductase by light (Jonassen et al., 2008,
2009), no difference in regulation of nitrate uptake and
assimilation in response to sulfur deficiency was observed
between hy5 and Col-0. As regulation of APR by sulfur
limitation was also not affected in the hy5 mutant, HY5
probably plays no role in regulation of the sulfur starvation
response. The search for components of signalling of sulfur
status to nitrogen assimilation thus continues.
HY5 and regulation of sulfate assimilation
HY5 has been described as transcription factor controlling
(Ang et al., 1998; Ulm et al., 2004; Lee et al., 2007). In
accordance with these results, we showed that it is impor-
tant for light inductionof APR1 and APR2.However, our data
suggest that HY5 is also involved in demand-driven meta-
bolic regulation of sulfate assimilation. It is possible to
speculate that, in hy5 mutants, a light (and HY5-)-dependent
component of OAS or nitrogen-deficiency signalling cas-
cades is disrupted, and thus the HY5 involvement in such
HY5 in control of sulfate assimilation 1051
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The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1042–1054
Page 11
regulation is only indirect and not through direct transcrip-
tional regulation of APR. This may be true for the nitrogen
deficiency response: despite evidence for direct interaction
of HY5 with the promoters of APR1, APR2 and SULTR1;2
(Figure 9), no differences in corresponding transcript levels
in nitrogen-deficient hy5 plants were detected. However,
this explanation does not seem likely for the regulation by
OAS. If the role of HY5 were only indirect, all three APR
isoforms would be affected, but only APR1 and APR2 regu-
lation was disrupted in hy5.
Thus, in conclusion, we have shown that HY5 is involved
in the regulation of APR gene expression. HY5 appears
to integrate signals from several conditions representing
demand-driven regulation of sulfate assimilation: the reduc-
tion of GSH content by BSO, nitrogen deficiency, light and
O-acetylserine (Figure 11). In addition, HY5 is involved in
regulation of sulfate uptake by OAS and nitrogen deficiency.
HY5 affects these key steps in sulfate assimilation both
directly at the transcriptional level and indirectly by inter-
fering with so far unknown signalling components. HY5 can
thus be considered as a new regulator of the pathway, and
as a link between nitrogen and sulfate assimilation.
Plant material and growth conditions
In this study, Arabidopsis thaliana ecotype Col-0 was used as the
wild-type (WT). The homozygous T-DNA insertion line SALK_
056405, in which the HY5 gene (At5g11260) is disrupted, was
obtained from the Nottingham Arabidopsis Stock Centre. The
insertion mutants phyA (SALK_122430C), cry1 (SALK_042397C),
cry2 (N3732) and cry3 (SALK_122430C) were obtained from the
Nottingham Arabidopsis Stock Centre, and phyB was provided by
Phil Wigge (Cell & Developmental Biology, John Innes Centre,
Norwich, UK). Plants were grown on vertical plates with Murashige
& Skoog medium without sucrose (MS), supplemented with 0.5%
Phytagel (Sigma, http://www.sigmaaldrich.com/). The plates were
placed in a controlled environment room at 20?C under a 16 h light/
8 h dark cycle. For light condition experiments, plants were grown
on the same plates placed horizontally for 7 days. For other treat-
ment conditions, plants grown vertically for 2 weeks were trans-
ferred to fresh plates with or without the addition of 1.25 mM BSO,
1 mM OAS or 1 mM cysteine. Alternatively, for the time course
experiment, OAS treatment was performed by pipetting 1 mM OAS
solution directly onto the roots, with controls receiving the same
volume of water. For nitrogen and sulfate deficiency treatment,
nitrogen-deficient and sulfur-deficient media were produced by
exchanging all nitrates and sulfates, respectively, for chlorides.
Enzyme assays
APR activity was determined as the production of [35S]sulfite,
assayed as acid-volatile radioactivity formed in the presence of
[35S]APS with dithioerythritol as reductant (Koprivova et al., 2008).
The protein concentrations were determined using a Bio-Rad pro-
tein kit (http://www.bio-rad.com) with BSA as a standard. To cal-
culate relative activity, individual values for treated plants were
divided by the mean value for control plants at the same time point.
Quantitative RT-PCR
Total RNA was isolated by standard phenol/chlorophorm and LiCl
precipitation. First-strand cDNA was synthesized from 1 lg total
RNA using a QuantiTect reverse transcription kit (Qiagen, http://
www.qiagen.com), which includes a DNase step to remove possible
DNA contamination. Quantitative real-time RT-PCR was performed
using gene-specific primers (Table S1) and the fluorescent inter-
calating dye SYBR Green (Applied Biosystems, http://www.
appliedbiosystems.com/) in an OPTICON2 continuous fluorescence
detector (Bio-Rad) for 2 min at 95?C, followed by 40 cycles con-
sisting of 15 sec at 95?C, 15 sec at 60?C and 30 sec at 72?C, then
10 min at 72?C, followed by standard dissociation protocol to
ensure that each amplicon was a single product. All quantifications
were normalized to ubiquitin UBQ10. The RT-PCR reactions were
performed in triplicate for each of three independent samples. To
calculate relative expression (treatment/control), individual values
for treated plants were divided by the mean value for control plants
at the same time point.
HPLC analysis of low-molecular-weight thiols
The analysis of cysteine and GSH was performed as described
previously(Koprivova et al.,2008)using10–20 mgofplantmaterial,
with derivatization using monobromobimane and separation of the
conjugates by HPLC.
Determination of flux through sulfate assimilation
The flux through sulfate assimilation was measured as incorpora-
tion of35S from [35S]sulfate into thiols and proteins essentially as
described by Kopriva et al. (1999) and Vauclare et al. (2002). For the
light experiment, Col-0 and hy5 plants were grown for 1 week on
vertical MS/phytagel plates and transferred to the dark for 38 h. The
plants were transferred in darkness into 24-well plates containing
2 ml MS nutrient solution adjusted to a sulfate concentration of
0.2 mM and supplemented with 5.6 lCi [35S]sulfate (Hartmann
Analytic, http://www.hartmann-analytic.de) to a specific activity of
1860 kBq nmol)1sulfate,and incubatedeitherindarknessorinlight
for 4 h. For the nitrogen experiment, the plants were grown for
2 weeks and subjected to nitrogen deficiency for 3 days, before
transfer into [35S]sulfate-containing nutrient solution and incuba-
tion in light for 4 h. After incubation, the seedlings were washed
Figure 11. Scheme of involvement of HY5 in regulation of sulfate assimila-
HY5 directly (solid line) controls the transcriptional regulation of APR1 and
APR2 by light, and of APR1, APR2 and SULTR1;2 by OAS, and indirectly
(dashed line) regulates APR activity and sulfate uptake during nitrogen
1052 Bok-Rye Lee et al.
ª 2011 The Authors
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Page 12
three times with 2 ml non-radioactive nutrient solution, carefully
blotted with paper tissue, weighed, transferred into 1.5 ml tubes,
and frozen in liquid nitrogen. Quantification of35S in plant material
and in various sulfur-containing compounds was performed exactly
as described by Mugford et al. (2011).
Chromatin immunoprecipitation
Col-0 and hy5 seedlings were grown for 7 days on horizontal MS/
phytagel plates. ChIP was performed as described by Sorefan et al.
(2009). Approximately 500 mg seedlings were fixed with formal-
dehyde, and the chromatin was isolated. Aliquots corresponding to
approximately 200 mg tissue were incubated with 25 ll antisera
against HY5 (provided by Xing-Wang Deng, Department of Molec-
ular, Cellular and Developmental Biology, Yale University, New
Haven, CT). Dynabeads/Protein A (100-02D, Invitrogen, http://
www.invitrogen.com) was used to isolate the protein–DNA com-
plexes. After digestion with proteinase K, the DNA was purified
using a DNA purification kit (Qiagen) and subjected to quantitative
PCR using primers against promoter regions of the genes encoding
the three APR isoforms and SULTR1;2 (Table S2). The primers were
positioned to be within 100 bp from putative HY5 binding sites as
determined by genome-wide analysis (Lee et al., 2007). As controls,
the DNA was amplified using primers for coding regions of APR
isoforms and for the promoter of a gene not connected to the HY5
network, At5g24660. AMu-like retrotransposon (Table S2)wasused
as an internal control for normalization (Sorefan et al., 2009).
Determination of chlorophyll and anthocyanins
Chlorophyll was extracted using 80% ethanol and levels were
measured spectrophotometrically at 645 and 663 nm. Anthocyanin
quantification was performed as described by Deikman and Ham-
mer (1995) after extraction in 18% 1-propanol/1% HCl. Absorbance
at 535 and 650 nm of the extracts was determined, and the amount
of anthocyanins is shown as (A535–A650) per g fresh weight.
S.K.'s work is supported by the UK Biotechnology and Biological
Sciences Research Council (BBSRC). We thank P. Wigge (Cell &
Developmental Biology, JohnInnesInstitute,Norwich, UK)forphyB
seeds, and X.-W. Deng (Department of Molecular, Cellular and
Developmental Biology, Yale University, New Haven, CT) for the
HY5 antiserum. The help of T. Girin with the chromatin immuno-
precipitation experiment is greatly acknowledged.
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Complementation of the bir24 phenotype using HY5.
Figure S2. Regulation of APR by light in photoreceptor mutants.
Figure S3. Regulation of APR mRNA levels by OAS in Col-0 and hy5
Figure S4. Regulation of APR activity by OAS.
Figure S5. Regulation of APR activity by nitrogen deficiency.
Figure S6. Changes in pigments due to nitrogen deficiency.
Figure S7. Regulation of nitrate assimilation by nitrate deficiency in
hy5 plants.
Figure S8. Regulation of nitrate assimilation by sulfate deficiency in
hy5 plants.
Table S1. Primers used for quantitative RT-PCR.
Table S2. Primers used for ChIP.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
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delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
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Ulm, R., Baumann, A., Oravecz, A., Ma ´te ´, Z., Ada ´m, E., Oakeley, E.J., Scha ¨fer,
E. and Nagy, F. (2004) Genome-wide analysis of gene expression reveals
function of the bZIP transcription factor HY5 in the UV-B response of Ara-
bidopsis. Proc. Natl Acad. Sci. USA, 101, 1397–1402.
D. and Ahmad, M. (2007) HY5 is a point of convergence between crypto-
chrome and cytokinin signalling pathways in Arabidopsis thaliana. Plant J.
49, 428–441.
Vauclare, P., Kopriva, S., Fell, D., Suter, M., Sticher, L., von Ballmoos, O.,
Kra ¨henbuhl, U., Op den Camp, R. and Brunold, C. (2002) Flux control of
sulfate assimilation in Arabidopsis thaliana: adenosine 5¢-phosphosulfate
reductase is more susceptible to negative control by thiols than ATP sul-
furylase. Plant J. 31, 729–740.
Vernoux, T., Wilson, R.C., Seeley, K.A. et al. (2000) The ROOT MERISTEM-
LESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent
pathway involved in initiation and maintenance of cell division during
postembryonic root development. Plant Cell, 12, 97–109.
Xi, W., Liu, C., Hou, X. and Yu, H. (2010) MOTHER OF FT AND TFL1 regulates
seed germination through a negative feedback loop modulating ABA sig-
naling in Arabidopsis. Plant Cell, 22, 1733–1748.
Yamaguchi, Y., Nakamura, T., Harada, E., Koizumi, N. and Sano, H. (1999)
Differential accumulation of transcripts encoding sulfur assimilation
enzymes upon sulfur and/or nitrogen deprivation in Arabidopsis thaliana.
Biosci. Biotechnol. Biochem. 63, 762–766.
Yatusevich, R., Mugford, S.G., Mattewman, C., Gigolashvili, T., Frerigmann,
H., Delaney, S., Koprivova, A., Flu ¨gge, U. and Kopriva, S. (2010) Genes of
primary sulfate assimilation are part of the glucosinolate biosynthetic
network in Arabidopsis thaliana. Plant J. 62, 1–11.
Zhang, Y., Zheng, S., Liu, Z., Wang, L. and Bi, Y. (2011) Both HY5 and HYH are
necessary regulators for low temperature-induced anthocyanin accumu-
lation in Arabidopsis seedlings. J. Plant Physiol. 168, 367–374.
1054 Bok-Rye Lee et al.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 1042–1054


text:
Here's a taste of what TheBody.com has to offer on this topic:
Ask the Experts
IS MASTURBATIN (MASTURBATION, 2010) (MASTURBATION SIDE EFFECTS, 2010) (MASTURBATION AND STD/HIV, 2010)dear doc.. i am a 16 year old guy who is very concerned about this situation. for the past few months i have been masturbating a lot and i have also found acne on my whole face and back. is there anything i can do tohelp this...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...Bobmasterbation (MASTURBATION SIDE EFFECTS, 2009) Mar 11, 20091.I have been masterbating for 5 years now atleast 10-12 times a week is it true that if you masterbate too much you get blind?Response from Dr. FrascinoHi,Well, believe it or not,... Read more »
Masturbation Effects (MASTURBATION SIDE EFFECTS, 2011)I have read quite a bit of information on your site but was looking for a more specific answer to... Read more »
Doctor, you changed my life. Please post this for others. (MASTURBATION SIDE EFFECTS, 2009)Hello doctor. my story goes like this.I'm a 20 year old male. When I was 11 or something, i started... Read more »
Stopping the Spread of HIV...antiretroviral drugs (ARVs). This is called "post exposure prophylaxis" or PEP. You would take two or three medications for several weeks. These drugs can decrease the risk of infection, but they have some serious side effects. Fact Sheet 156 ... Read more »
HIV Testing 101...nervous, scared or even a little paranoid when you think you've put yourself at risk for HIV. An HIV test -- taken at least six weeks after the risky event -- can answer that question. Approved HIV tests are extremely reliable. There's even a... Read more »
Answering Questions About Sex...your inquiries in or check my questions archive in my website www.riverhuston.com . Here are a couple of questions from this week: Is it normal for a 16-year old guy to jerk-off five times a day? Let's start with normal . There has never... Read more »
Safer Sex Guidelines...to have sex with you. Some people got HIV from their steady partners who were unfaithful "just once." Even people who got a negative test result might be infected. They might have been infected after they got tested, or they might have gotten... Read more »
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31 days test
Posted by Roper2008, 1 Reply...on neck area, fatigue, aching body all over and a very high fever!!! this happened in the space of one week, not over several weeks!! So I went to get tested, it was at 32 days. I was sure it would be positive but it was negative. They told me... Read more »
Re: In need of some advice!
Posted by maiden, 1 Reply...recently she started sneezing a lot, then followed by diarrea and fever. He on the other hand got rashes at his chest but after a week, it faded away. it could be due to the massage oil. He told me that as the girl was massaging him, one of her... Read more »
it looks like HIV finally got me
Posted by uglyduck, 4 Replies...Not, my test is not positive (not yet)... But my symptoms look way to obvious. 2 or 3 weeks post exposure Joint Paints (knees, wrists, pelvis, legs, arms, toes and fingers) Back Pain (Burning Sensation -> my back Read more »
usual questions...pls comfort me
Posted by Anonymous, 2 Replies...you say could help me prepare for the days ahead... how much comfort should i draw from a 46 day negative test (6 and a half weeks)? what stds should i check for, if i have an eyesore and a rash on my back? or should i test at all, if my risk... Read more »
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subset: commoncrawl


text:
Kathy Liu
The Last Whispers of Life
Heaven above, hell below.
I looked in the mirror and ugly showed.
Punishing myself and living in hell.
Wished I was gone, flashes before your eyes,
but they said you didn't know I existed, no matter how hard I try.
Left alone with tears, anger, hate, and what else?
Living in long nightmare,
And I can't end this dream.
The pain of the past will always remain,
Living in the shadows, like broken glass.
If everyone felt pain and sadness like I do,
the world would be scarred.
I never knew
My heart was stolen,
by all the hatred I had and have,
Scars, secrets, lies spread.
Nothing to see, now I'm blind.
Nothing to feel,
my heart was stolen for me.
If you listen closely you'll hear a cry for love, who will that be?
If you can hear me at all, speak up please.
Because hell is empty,
all the demons are here,
Now I know where I stand,
Staring back at the ground.
Seeing my reflection from my puddle of broken dreams.
I've come to figure out there was a seed.
Ready to bloom, in need to love.
And when that seed is ready to come out,
out of the shadows into the light,
you will see what you have not seen before,
It hasn't been the true me all along.
Topic of this poem: Depression
Kathy Liu's Other Poems
Related Poems
Read this poem in other languages
This poem has not been translated into any other language yet.
I would like to translate this poem »
Comments about this poem (The Last Whispers of Life by Kathy Liu )
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(4 April 1928 - 28 May 2014)
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(19 January 1809 - 7 October 1849)
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(10 December 1830 – 15 May 1886)
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- The Road Not Taken, Robert Frost
- Still I Rise, Maya Angelou
- If, Rudyard Kipling
- Daffodils, William Wordsworth
- If You Forget Me, Pablo Neruda
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- Stopping by Woods on a Snowy Evening, Robert Frost
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text:
Call for Music Writers... Rock, Indie, Hip-hop, R&B, Electronic, Americana, Metal, World and More
By Nikki Tranter
The Travel Channel's World Poker Tour has helped make poker the hottest obsession around.
© 1999-2015 PopMatters.com. All rights reserved.PopMatters.com™ and PopMatters™ are trademarksof PopMatters Media, Inc.PopMatters is wholly independently owned and operated.


text:
Ivory Travertine Laminate Floor Planks
Just wanted to update all of you. I finally had the Ivory Travertine laminate put down in 3 bathrooms and really, really like it!! At first, I didn't think I liked it because it just looked darker than I had imagined it to be. After all bathrooms were done, I was very pleased.
Even though all 3 of my bathrooms have different color schemes, the ivory travertine seems to just work with many colors. I was amazed at that because I was thinking I would have to paint and change some things in at least 1 of the bathrooms. But, I don't have to:)
About the only disappointment was it was cheaper a few weeks before I purchased it. I could have saved $120. Oh well. I'm still happy!
I'm glad you are enjoying it, but laminate in a bathroom? Laminate is not suited to wet locations. I hope that you are the only adult occupant and you are extremely vigilant about shower leaks and not leaving wet towels or rugs down on it.
Oops, I meant to say Ivory Travertine Vinyl Floor Planks;) Sorry about that.


text: Stacey Yap (Sociology) and Khuan Chong (Political Science) had a conference with the Faculty of Arts and Social Science at Universiti Tunku Abdul Rahman (UTAR) on August 1-‐2 in Kampar, Malaysia. The agenda included topics related to faculty and student exchanges. Also, the meetings that followed saw the presentation of 3 CDs that covered the topics of: "Tin and the Malaysian Communists," "The Role of Women Dulangs in Tin Mining," and "Tin Mining and the Growth of Malaysia." These works came about from the suggestions made to the Social Science and Journalism Departments of UTAR in 2012, when Stacey and Khuan visited the University and attended the establishment of the Tin Museum in 2012. The collaboration of PSU and UTAR to produce such quality works set the foundation for future academic exchanges.


text:
Enterovirus Spreads Among US Kids
September 10, 2014|
A previously rare respiratory virus is landing hundreds of children in hospitals.
Daily News Roundup
September 10, 2014|
A previously rare respiratory virus is landing hundreds of children in hospitals.
September 10, 2014|
World Health Organization officials say intervention efforts must be scaled up at least three-fold.
September 9, 2014|
Human trials are now underway.
September 8, 2014|
The Federal Court of Australia rejected an appeal of a ruling that allows companies to patent isolated human genes.
September 8, 2014|
The US Food and Drug Administration last week approved the first of a new type of immunotherapy that aims to pit a patient's own immune system against her cancer.
September 2, 2014|
Genetic analysis reveals the history of the earliest human migrations in the region.
August 28, 2014|
Updated guidelines expand upon existing policy to include more kinds of data.
August 27, 2014|
Researchers link a gene already tied to alcohol dependence with a neurotransmitter involved in anxiety and relaxation.
August 26, 2014|
Algorithms that map social media posts and mobile phone data can help researchers track epidemics.
August 22, 2014|
Monkeys infected with lethal doses of Marburg virus were rescued by an experimental siRNA-based therapeutic, a study shows.


text:
An article posted today on Tampa Bay Online suggests likely Republican presidential nominee Mitt Romney could pick Michigan Gov. Rick Snyder as his running mate for November's election.
The article -- which is placed in the opinion section but carries no byline other than "TBO.com staff" -- bases this theory on the hotel assignments for state delegations coming to the Republican National Convention.
The Florida delegation got a hotel assignment that puts them 40 miles and a two-hour bus ride away from Tampa, where the convention is being held.
Meanwhile, the articles says: "The RNC (and to a large degree the Romney campaign) is loving Michigan, though. Detroit, Michigan's largest city, is home to GM, the once-American company. Today, GM stands for Government Motors, and Detroit is the armpit of America. Once the fourth-largest U.S. city, Detroit is now ninth, and flight from the city continues. Michigan as a whole is overly reliant upon unionized, low-skilled industries. Built on a near-century-old economic model, Michigan's economy is broken for sure.
Michigan hasn't voted for the Republican nominee for president since 1988, when George H.W. Bush faced Michael Dukakis. But there is a reason Michigan got the second-best hotel assignment: Gov. Rick Snyder. My bet is he's Romney's man for vice president.


text:
Rancher knowledge: an untapped resource
Land management and conservation agencies have traditionally performed rangeland management activities based predominately on technical information resulting from scientific research. However, rancher experience and knowledge is an untapped resource that could help broaden the scope of these activities and lead to more sustainable land management.
Read more Read it in print


text:
Weekend Box Office Report: 'Godzilla' is King of the Box Office
Everyone expected 'Godzilla' to take the number one spot at the box office this weekend, but few people saw it doing business that rivals 'Captain America: The Winter Soldier' and 'The Amazing Spider-Man 2.' 'Godzilla' didn't just win the box office this weekend, it conquered it, proving that after all of these years, the king of the monsters is still a massive draw.
Like Sitting in the Middle of the Movie Theater? That's Gonna Cost You Extra Now
Movie theaters tickets have gotten steadily more expensive over the years, but this takes the proverbial cake: a Canadian theater chain has announced a new program where moviegoers will have to pay a surcharge to sit in the middle seats of each row. You'll soon have to pay an additional $2-$3 to sit in the "prime" seats, ensuring that you'll have the (supposed) best view of the screen and that you'll be isolated from all of the riffraff in the cheap seats.
'Batman vs. Superman' Director Zack Snyder Teases the New Batmobile
This is serious business, so let's get down to brass tacks: 'Batman vs. Superman' director Zack Snyder took to Twitter to tease his film's take on the Batmobile, AKA, Batman's personal vehicle, AKA, one of the most famous vehicles in all of popular culture. Snyder shared the first image of the iconic automobile, mostly covered by a tarp, with a message suggesting that a full reveal could come as soon as tomorrow.
Weekend Box Office Report: 'The Other Woman' Defeats 'Captain America 2′
After nearly a month on top of the box office, 'Captain America: The Winter Soldier' got taken down by a bunch of funny ladies. 'The Other Woman' effortlessly took the top spot, sending Marvel's latest superhero adventure to the number two slot and making the other new releases of the week look bad.
Our Nightmares Are Coming True: 'It's a Small World' and 'Peeps' Movies in the Works
When you follow movie news as closely as we do, you learn to roll your eyes at terrible ideas and move on. There's not point in getting worked up when a truly awful project is announced because getting worked up over every bad idea that comes out of Hollywood would mean getting worked up every single day of the year and we just don't have the energy to deal with getting worked up so often (whew).
But, today is a special day. Today, they've gone and announced movies based on Disneyland's 'It's a Small World' ride and popular Easter candy, Peeps Marshmallow Treats. Today, they have gone too far.
Weekend Box Office Report: 'Captain America 2′ Runs Laps Around 'Rio 2′
This weekend saw a neck-and-neck race for the first spot at the box office, with the animated 'Rio 2' offering strong competition for last week's champion, 'Captain America: The Winter Soldier.' When the dust, debris and feathers settled, the one starring the superhero took the first slot, but there is no shame in a second place that strong.
Weekend Box Office Report: 'Captain America 2′ Throws His Mighty Shield, Wins the Box Office, Etc.
Like 'Iron Man 3' and 'Thor: The Dark World,' the question was never whether or not 'Captain America: The Winter Soldier' would take the number one spot at the box office, but rather how much it would earn while sitting pretty at the top of the list. Also, like its "Phase 2" predecessors, the second Steve Rogers adventure proved that people are still punch-drunk in love with the Avengers characters.
Klingon for Rosetta Stone Might Be the Best April Fool's Day Prank
If you're a particularly extreme 'Star Trek' geek and have been looking for a new way to celebrate your fandom, look no further than Nerdist and Think Geek's newest venture: Rosetta Stone software for learning the harsh, bloodthirsty language of the Klingon empire! On hand to sell the product to you is 'Star Trek: The Next Generation' and 'Star Trek: Deep Space Nine' star Michael Dorn, who explains all of the details. Order now and you'll soon be able to threaten your friends and loved ones with the galaxy's most terrifying language! All you need is to pay the low, low price of $269.99
Weekend Box Office Report: 'Noah' and Russell Crowe Flood 'Sabotage' and Arnold Schwarzenegger
Insert your own "when it rains, it pours" joke right here. Darren Aronofsky's instantly controversial biblical epic 'Noah' overcame mixed buzz to win the box office this weekend, catering to both religious audiences and film buffs who like weird movies from unique, picky directors. But all was not well for everyone -- the latest film from a certain Hollywood action legend got washed away in 'Noah''s success.
Weekend Box Office Report: 'Divergent' Leaves 'Muppets Most Wanted' in the Dust
If the main goal of 'Divergent' was to snag a large portion of the audience for 'The Hunger Games,' it looks like it succeeded. Although it didn't reach the absurd heights of Katniss' two movies, Shailene Woodley's dystopian adventure effortlessly nabbed the number one spot and, unless something bad happens next week, announced the arrival of a new big franchise.
Weekend Box Office Report: 'Need For Speed' Sputters at the Box Office
If the goal for 'Need For Speed' was to replicate the success of the 'Fast and Furious' films, then the film's opening weekend is not promising. Opening in third place, the video game adaptation was overtaken by two of last week's releases, making this potential franchise D.O.A.
Weekend Box Office Report: '300: Rise of an Empire' Conquers All
After seven years of waiting, no one knew if a sequel to '300' would do the business of its predecessor. And now we have a definitive answer: yes. '300: Rise of an Empire' effortlessly took the number one spot at the box office, proving that America still loves shirtless Greeks murdering each other in slow motion.


text:
Hotel Industria
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Travel Blogs from Brescia
Not so glamorous Bergamo
Leaving Milano, we started the day with Bergamo which is an ancient town about 40 km east of Milan we've been hearing a lot about. I have to admit that it wasn't as great as I thought it would be. Maybe it's just me setting my expectation too high.
The old town (citta alta) is up on the hill and we had to take ...
Bergamo and the Alps
... buses to get to our hostel. After about an hour that included bus rides and lots of stairs, we arrived at the hostel, where we dropped off our stuff and left to explore the city.
We first went to try to figure out the busses and trains (Abby wanted to go to Pisa the next day) in the lower city. However, we were at a loss and gave up on that idea. We headed to the Citta Alta, which is the upper medieval city ...
Desanzano & Sirmione
... the old castle
gates and another bar or two. At the end of a lovely day, we hiked
back to the hotel and sat on the roof top watching the sun drop in
the sky while drinking Prosecco. Our evening meal was in the pizza
restaurant near the hotel again. Unfortunately the friendly waiter
was not there again.
Having waited ten years
to see the Italian lakes, we were not disappointed. What a wonderful
Boat Trips on Lake Garda
... sorts
arrived to take us across to the western shore and Limone, supposedly
named after lemons – thought to be the first place in Europe that
they were introduced. Everywhere was covered with lemons, either
growing or in shop windows. Real, fake, lemon derived, lemon
obsessed. Lovely never the less.
Our next ferry was a
paddle steamer affair, running on diesel rather than coal now. It
was packed with passengers so we stood outside to ...
Four Lakes
... final hour of riding took us to Sirmione at the southern end of Lake
Garda. Our hotel proved very comfortable with a lake view of sorts
and good facilities. The walk into the town was slightly longer than
expected but mostly along the lake front which made it pleasant. Our
evening meal at a nearby pizza restaurant proved to be a good
recommendation by the hotel staff. The food was very good and the
restaurant deservedly popular with the locals. ...


text:
Yellow Swiss Dot Washi Tape
Swiss Dot Washi Tape Yellow
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text:
52nd Academy Awards
From Wikipedia, the free encyclopedia
|52nd Academy Awards
|April 14, 1980
|Dorothy Chandler Pavilion, Los Angeles
|Johnny Carson
|Howard W. Koch
|Marty Pasetta
|Best Picture
|Kramer vs. Kramer
|Most awards
|Kramer vs. Kramer (5)
|Most nominations
|All That Jazz and Kramer vs. Kramer (9)
|TV in the United States
|3 hours, 12 minutes
The 52nd Academy Awards were presented April 14, 1980, at the Dorothy Chandler Pavilion in Los Angeles. The ceremonies were presided over by Johnny Carson who, in noting the long duration of the production, joked that President Jimmy Carter was working hard for their "release" from the ceremonies, a clear reference to the Iranian hostage crisis.
Among the nominees for Best Supporting Actor were 8 year-old Justin Henry—the youngest Best Supporting Actor nominee ever—and 79 year-old Melvyn Douglas. This was the largest age difference between two competing actors in Oscar history until 2013.
Winners are listed first and highlighted in boldface.[1][2]
Academy Honorary Awards[1][edit]
- Hal Elias
- Alec Guinness
Medal of Commendation[1][edit]
- John O. Aalberg, Charles G. Clarke and John G. Frayne
Jean Hersholt Humanitarian Award[1][edit]
Irving G. Thalberg Memorial Award[1][edit]
|Wayne, PatrickPatrick Wayne
|Explains the voting rules to the public
|Leachman, ClorisCloris Leachman
|Presenters of the award for Best Supporting Actress
|Lemmon, JackJack Lemmon
|Miller, AnnAnn Miller
|Presenters of the award for Best Art Direction and Best Set Decoration
|Rooney, MickeyMickey Rooney
|Parton, DollyDolly Parton
|Presenters of the Music Awards
|Vereen, BenBen Vereen
|Fairbanks Jr., DouglasDouglas Fairbanks Jr.
|Presenter of the Jean Hersholt Humanitarian Award
|Hays, RobertRobert Hays
|Presenters of the award for Best Costume Design
|McNichol, KristyKristy McNichol
|Fawcett, FarrahFarrah Fawcett
|Presenters of the award for Best Visual Effects
|Russell, HaroldHarold Russell
|Khambatta, PersisPersis Khambatta
|Presenters of the Documentary Awards
|Shatner, WilliamWilliam Shatner
|Hutton, LaurenLauren Hutton
|Presenters of the Short Subjects Awards
|Savalas, TellyTelly Savalas
|Gere, RichardRichard Gere
|Presenter of the Medal of Commendation
|Presenters of the award for Best Foreign Language Film
|Valenti, JackJack Valenti
|Steiger, RodRod Steiger
|Presenters of the award for Best Sound Mixing
|Kellerman, SallySally Kellerman
|Douglas, KirkKirk Douglas
|Presenter of the Irving G. Thalberg Memorial Award
|Curtis, Jamie LeeJamie Lee Curtis
|Presenters of the award for Best Cinematography
|Hamilton, GeorgeGeorge Hamilton
|Kelly, GeneGene Kelly
|Presenters of the award for Best Original Song
|Newton-John, OliviaOlivia Newton-John
|Derek, BoBo Derek
|Presenters of the award for Best Film Editing
|Reeve, ChristopherChristopher Reeve
|Matthau, WalterWalter Matthau
|Presenters of the award for Best Supporting Actor
|Minnelli, LizaLiza Minnelli
|Hoffman, DustinDustin Hoffman
|Presenter of the Honorary Award to Alec Guinness
|Hawn, GoldieGoldie Hawn
|Presenters of the award for Best Director
|Spielberg, StevenSteven Spielberg
|Simon, NeilNeil Simon
|Presenter of the Writing Awards
|Mirisch, WalterWalter Mirisch
|Presenter of the Honorary Award to Hal Elias
|Fonda, JaneJane Fonda
|Presenter of the award for Best Actor
|Dreyfuss, RichardRichard Dreyfuss
|Presenter of the award for Best Actress
|Heston, CharltonCharlton Heston
|Presenter of the award for Best Picture
|Mancini, HenryHenry Mancini
|Musical arranger
|Kermit the Frog
|"Rainbow Connection" from The Muppet Movie
|Miss Piggy
|Williams, PaulPaul Williams
|Moore, DudleyDudley Moore
|"Song from 10 (It's Easy to Say)" from 10
|Reddy, HelenHelen Reddy
|Manchester, MelissaMelissa Manchester
|"Through the Eyes of Love" from Ice Castles and "I'll Never Say Goodbye" from The Promise
|O'Connor, DonaldDonald O'Connor
|"Dancin' on the Silver Screen"
|Warwick, DionneDionne Warwick
|"It Goes Like It Goes" from Norma Rae
|Academy Award Chorus
|"That's Entertainment"
Multiple nominations and awards[edit]
These films had multiple nominations:
- 9 nominations: All That Jazz, Kramer vs. Kramer
- 8 nominations: Apocalypse Now
- 5 nominations: Breaking Away
- 4 nominations: The China Syndrome, Norma Rae, The Rose
- 3 nominations: 1941, La Cage aux Folles, Star Trek: The Motion Picture
- 2 nominations: 10, Alien, ...And Justice for All, Being There, The Black Hole, The Black Stallion, A Little Romance, Manhattan, The Muppet Movie, Starting Over
The following films received multiple awards.
- 5 wins: Kramer vs. Kramer
- 4 wins: All That Jazz
- 2 wins: Apocalypse Now, Norma Rae
See also[edit]
- 37th Golden Globe Awards
- 1979 in film
- List of Academy Awards ceremonies
- 22nd Grammy Awards
- 31st Primetime Emmy Awards
- 32nd Primetime Emmy Awards
- 33rd British Academy Film Awards
- 34th Tony Awards
- ^ a b c d e The Official Acadademy Awards® Database
- ^ "The 52nd Academy Awards (1980) Nominees and Winners". oscars.org. Retrieved 2011-10-07.


text:
A New Angle on Part-Time Students
July 10, 2007
The Department of Education's National Center for Education Statistics has gone beyond comparing full- and part-time undergraduate students by examining differences within the part-time undergraduate student group. According to the new report Part-Time Undergraduates in Postsecondary Education: 2003-04, about 25 percent of part-time undergraduates in 2003-04 showed some characteristics typically associated with full-time students. These "part-time students who looked like full-time students" were traditional college age, graduated from high school with a regular diploma, and received financial help from their parents to pay for college. When compared to other part-time students, they were more likely to be white, have parents with higher degrees, come from wealthier families, and have expectations to earn an advanced degree.
The report also provides demographic information on part-time undergraduates in general as well as distinctive comparisons between part-time and full-time undergraduate students. These findings include:
- Part-time students represented 37 percent of the undergraduate enrollment in all degree-granting postsecondary institutions in fall 2004
- Part-time undergraduates were more likely to be older, female, Hispanic, financially independent, and first-generation students compared to full-time undergraduates
- More than 80 percent of part-time undergraduates worked while enrolled in college with more than half working full time compared to 23 percent of full-time undergraduate students who worked full time
For additional findings, including an analysis of students who switch between part-time and full-time enrollment status, visit the NCES Web site to download the full report.
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Creative Outdoor Distributor Collapsible Wagon
$59.99 $99.99*
Save $40.00 (40%)
Product Information:
Whether you're towing sand toys or garden tools, the Creative Outdoor Distributor® Collapsible Wagon is convenient for hauling a wide variety of cargo. This folding wagon is constructed with a strong steel frame and durable canvas fabric that can handle loads up to 120 lbs. Plastic wheels can roll on a variety of surfaces to get your gear where it needs to go. After your beach day or tailgate party is over, fold the wagon for storage.
- Folding wagon
- Great for yardwork, picnics, the beach, and tailgating
- Built with sturdy and strong steel frame
- Made with durable canvas
- Easy to set up and load
- Conveniently folds for storage and travel
- Travel sleeve for transport and storage
- Wheel material: Plastic
- Wheel dimensions: 7.5" x 7.5" x 1.5"
- Capacity: 120 lbs
- Open dimensions: 20" x 36.5" x 24"
- Collapsed dimensions: 20" x 7.5" x 40"
- Wagon weight: 19 lbs.
- Manufacturer's warranty: 90 days
- Style: 900121
- Creative Outdoor Distributor
Country of Origin: ChinaWeb ID: 34683346
Customer Reviews:
Creative Outdoor Distributor Collapsible Wagon 108 108
Great Love the wagon we are getting ready for the beach the summer. Ordering was easy and shipping was quick March 5, 2015
Great Product! The Creative Outdoor Distributor Collapsible Wagon is such a versatile utility to add to your toolset. Very sturdy design that gives the impression it'll last for a very long time. Excellent construction that reflects the everyday usage you can get out of this wagon. Greatest buy in a long time! March 2, 2015
Love this wagon! Wagon is great. Easy to maneuver even full of Girl Scout cookies! Only down side is we broke the clip to hold the handle up when not in use. The plastic is not good in the cold. February 25, 2015
A little disappointed I like the wagon, the velcro on the front and back is coming undone and we have to continue to squeeze the velcro back together again. I thought the wagon would be a little more durable. But it works for now. February 24, 2015
My New Little Red Wagon I bought this wagon so that I could bring groceries into the house in one trip. I have only used it once, but I am looking forward to saving lots of steps. February 24, 2015
Great value I haven't opened the product but for the price I'm sure it's a great value February 24, 2015
The product works well but has a horrible odor A musty, moth ball smell filled my car during transport, stunk up my garage after opening it and continues to have this odor weeks after purchasing it. I went back to the store to find out if it was just mine but all the other boxes had the same odor. It works well, collapses and hauls a bunch of sporting equipment. February 22, 2015
We love it. It is. very useful especially for older seniors. We love it, used it to load our RV. It is very useful especially for seniors. February 17, 2015
This product is good, but tricky putting back into sleve Bought it for the beach and Mardi Gras and has little e up to its end of the deal so far.. when I bought it all the boxes had holes in it where the Handle holder pushed through the box. February 17, 2015
All good except the smell is horrible Great wagon performs as expected but it has a horrible chemical smell that permeates everything it touches Can't transport inside the vehicle because of this Otherwise would have gave higher rating February 17, 2015


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I was using single sim mobile earlier and using whatspp with that no and data plan of that network. Now I have swithed to dual sim mobile. I want to use data plan of new sim and want to continue whatsapp with old no. But I am not able to do so. Kindly help.
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Franchitti rebounded from a crash in morning practice, which damaged the right front suspension of his Dallara chassis, to claim his first pole at Detroit, and 31st career IndyCar pole, all with Honda power.
Rookie Conor Daly, who started 31st, drove a solid race in the No. 41 ABC Supply Honda to finish 22nd.
A.J. Foyt: "Who would have thought Franchitti last year would have come up and win the race?"
Last year marked the return of manufacturer competition to the IZOD IndyCar Series and Indianapolis, after six years of Honda supplying engines to the full Indy car field.
Welcome to Indiana-ville - Math teacher Jay Alley breaks down the numbers for the Indianapolis 500
Rookie Conor Daly was okay while rookie Carlos Munoz again topped the charts. Tomorrow is Fast Friday and perhaps the expected veterans will be the ones to set the hot laps.
In his 26th year working as a credentialed media member at the Greatest Spectacle in Racing, Jay Alley will share his thoughts and photos with Motorsport.com for the 97th running of the Indianapolis 500.
Brazilian dance moves on the streets in Sao Paulo made for an interesting samba.
Even with their families and their fans supporting them, the three Brazilians were not on the IndyCar podium.
At last-lap, last-turn pass by James Hinchcliffe preventing Takuma Sato from winning his second consecutive IndyCar Series race Sunday in Sao Paulo, Brazil.
Lisa Davidson's take on the IndyCar race in Brazil – the third street circuit in the new season.
If you missed the Sao Paulo IndyCar 300 you just might be kicking yourself.
James Hinchcliffe and Takuma Sato raced to the finish line on the streets of Sao Paulo, Brazil. Both drivers had already noticed victories on street circuits this year in the IZOD IndyCar Series.
Takuma Sato earned his first IndyCar victory on the street circuit in Long Beach and now he will start 12th on Sunday on the Sao Paulo street circuit.
Dario Franchitti: "We're in a good position for the start, and it should be another exciting race here tomorrow."
The IZOD IndyCar teams arrived in the largest city of Brazil for this weekend's race on the street circuit in São Paulo
Takuma Sato comes to Sao Paulo as one of Indy's favorite pilots after winning at the Long Beach street circuit.
Lisa Davidson's take on last weekend's IndyCar contest at the legendary Grand Prix of Long Beach
A.J. Foyt requested to have the surgery on his back moved up a day in order to recover in time for the Indy 500
California: Ocean breezes, suntan lotion, palm trees, the Queen Mary, yachts, sailing, beaches, and surfing... plus the famous hairpin and the sounds of racing engines on the legendary street circuit in Long Beach!
The IZOD IndyCar Series now heads to South America for its first international race of 2013, the May 5 Itaipava Sao Paulo Indy 300 in Sao Paulo, Brazil.
Takuma Sato previous best finish was second at Edmonton 2012.
The final battle on the streets of Long Beach saw A.J. Foyt Racing back in the winner circle. The celebration will last for a bit of time as Sato became the first Japanese driver to capture an IndyCar race.
Takuma Sato continued his run of strong early-season performances.