Download Involvement of HLS1 in Sugar and Auxin

Plant Cell Physiol. 47(12): 1603–1611 (2006)
doi:10.1093/pcp/pcl027, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Involvement of HLS1 in Sugar and Auxin Signaling in Arabidopsis Leaves
Masa-aki Ohto 1, 4, *, Shingo Hayashi
Kenzo Nakamura 1, 2
2, 5
, Shinichiro Sawa 3, Akiko Hashimoto-Ohta
1
and
1
Division of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki, 444-8585 Japan
Laboratory of Biochemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan
3
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, 113-0033 Japan
2
Sugar regulates a variety of genes and controls plant
growth and development similarly to phytohormones. As part of
a screen for Arabidopsis mutants with defects in sugarresponsive gene expression, we identified a loss-of-function
mutation in the HOOKLESS1 (HLS1) gene. HLS1 was
originally identified to regulate apical hook formation of darkgrown seedlings (Lehman et al., 1996, Cell 85: 183–194). In
hls1, sugar-induced gene expression in excised leaf petioles was
more sensitive to exogenous sucrose than that in the wild type.
Exogenous IAA partially repressed sugar-induced gene expression and concomitantly activated some auxin response genes
such as AUR3 encoding GH3-like protein. The repression and
the induction of gene expression by auxin were attenuated and
enhanced, respectively, by the hls1 mutation. These results
suggest that HLS1 plays a negative role in sugar and auxin
signaling. Because AUR3 GH3-like protein conjugates free IAA
to amino acids (Staswick et al., 2002, Plant Cell 14: 1405–1415;
Staswick et al., 2005, Plant Cell 17: 616–627), enhanced
expression of GH3-like genes would result in a decrease in the
free IAA level. Indeed, hls1 leaves accumulated a reduced level
of free IAA, suggesting that HLS1 may be involved in negative
feedback regulation of IAA homeostasis through the control of
GH3-like genes. We discuss the possible mechanisms by
which HLS1 is involved in auxin signaling for sugar- and
auxin-responsive gene expression and in IAA homeostasis.
Keywords: Arabidopsis — Auxin — HOOKLESS1 — Sugar
signaling.
Abbreviations: ARF, auxin response transcription factors;
AuxRE, auxin response element; HXK, hexokinase; Suc, sucrose.
Introduction
In many plant species, sugar produced in source leaves
is transported via the phloem to sinks such as roots, fruits
and seeds or tubers. Sugar is an essential energy source and
provides carbon needed for plant growth and development.
In addition, sugar has hormone-like functions in regulating
a variety of genes that control growth and development,
e.g. seed germination, post-germinative growth, floral
transition, leaf senescence and development of storage
organs such as seeds or tubers (Bernier et al. 1993, Koch
1996, Jackson 1999, Smeekens 2000, Rolland et al. 2002,
Borisjuk et al. 2004, Gibson 2005, Wingler et al. 2006).
Studies carried out over the past 10 years have begun to
reveal the molecular mechanisms underlying sugar signaling
in higher plants. Genetic screens for Arabidopsis mutants
have provided powerful tools to identify genes involved in
sugar signaling. A number of Arabidopsis mutants have
been isolated based on the negative effects of sugar on seed
germination and post-germinative growth. Many of the
genes identified act in ethylene and ABA pathways,
indicating a complex genetic interactions between ethylene,
ABA and sugar signaling (Gazzarrini and McCourt 2001,
Rolland et al. 2002, Rook and Bevan 2003, Gibson 2005).
Genetic screens based on the expression of sugar-responsive
endogenous genes or reporter transgenes have also been
performed, and mutants with altered expression of sugarresponsive genes have been isolated (Rolland et al. 2002,
Rook and Bevan 2003, Yoine et al. 2006).
Here, we report an unusual sugar response2 (uns2)
mutation in Arabidopsis that causes altered expression of
sugar-responsive genes. uns2 is a new allele of the hookless1
(hls1) mutation that causes defects in apical hook formation
in dark-grown seedlings (Lehman et al. 1996). Apical hook
formation, i.e. differential growth of hypocotyls in darkgrown seedlings, which is regulated by ethylene and
light, coincides with the unequal distribution of auxin that
stimulates cell elongation in hypocotyls (Schwark and
Schierle 1992). Auxin is also known to play a pivotal role
in many aspects of plant growth and development such as
cell cycle control, apical dominance, the differentiation
of root or vascular cells, and cell elongation during
gravitropic and phototropic responses (Estelle and Klee
1994, Chen et al. 1999, Vanneste et al. 2005, Woodward
and Bartel 2005). In dark-grown hls1 seedlings, patterns of
auxin response gene expression were altered (Lehman et al.
1996), suggesting a possible involvement of HLS1 in auxin
4
Present address: Department of Plant Sciences, Plant Reproductive Biology Building, Mail Stop 5, University of California, One Shields
Avenue, Davis, CA 95616, USA.
5
Present address: Medical and Biological Laboratories, Ina, Nagano, 396-0002 Japan.
* Corresponding author: E-mail, [email protected]; Fax, þ1-530-752-2278.
1603
Role of HOOKLESS1 in sugar and auxin signaling
Results
hls1 mutations cause defects in sugar-responsive gene
expression
In order to identify genes that are involved in sugar
signaling, we screened our activation-tagged mutant populations (Suzuki et al. 2004) and isolated putative mutants
with altered levels of amylase activity and/or anthocyanin
accumulation in excised leaf petioles that were supplied
with Suc through the cut edges (Mita et al. 1995). One of
these mutants was named unusual sugar response2 (uns2).
uns2 showed increased levels of amylase activity and
anthocyanin accumulation in Suc-supplied leaf petiole
as compared with the wild type (Fig. 1), implying a
hypersensitivity to exogenous Suc in uns2. We further
discovered that the left border of T-DNA was inserted
650 bp downstream from the stop codon of HLS1 on
chromosome 4 and that uns2 genomic DNA sequence
flanking the right border was identical to that of At4g38540,
a gene located 360 kb downstream from HLS1, suggesting
that a large genomic rearrangement occurs near HLS1 upon
T-DNA insertion that caused a loss-of-function mutation in
the HLS1 gene (data not shown). Indeed, uns2 seedlings
grown in the dark did not form an apical hook, similar to
hls1-1 (Supplementary Fig. S1). Furthermore, strong alleles
of hls1 mutants such as hls1-1, hls1-7 and hls1-9 in the
Columbia ecotype also showed increases in amylase activity
and anthocyanin accumulation in Suc-supplied excised leaf
petioles as compared with wild-type plants (Supplementary
Fig. S2). Crossing a male uns2 with a female hls1-1 and
hls1-7 did not complement the mutations, indicating that
uns2 is an allele of the hls1 mutants (data not shown).
In order to investigate further the effects of the hls1
mutation on sugar-responsive gene expression, we analyzed
sugar-induced and sugar-repressed genes in Suc-supplied
5
3
4
Wt
Wt
uns2
uns2
3
2
1
0
Anthocyanin (relative value)
transport or auxin signaling. Li et al. (2004) identified
suppressor mutations of hls1 that were allelic with lossof-function mutations of the auxin response transcription
factor gene, ARF2. These results showed that ethylene- and
light-regulated differential cell elongation in hypocotyls of
dark-grown seedlings was mediated by regulation of ARF2
in a HLS1-dependent manner.
Here, we show that hls1 mutants exhibit greater
sensitivity to exogenous IAA as judged by the expression of
a selection of auxin response genes in leaves as compared with
the wild type, suggesting that HLS1 plays a negative role in
IAA-inducible gene expression. In addition, sugar-responsive
gene expression in hls1 was more sensitive to exogenous
sucrose (Suc) than the wild type. In this paper, we discuss the
complex mechanisms by which auxin regulates sugarresponsive genes auxin homeostasis. Furthermore, this
paper discusses the involvement of HLS1 in these processes.
Amylase activity
(units / mg protein)
1604
2
1
0
Nt
5% Suc
Nt
5% Suc
Fig. 1 uns2 mutation affects Suc-induced accumulation of
amylase activity and anthocyanin. Leaf-petioles excised from
3-week-old plants grown on 3% Suc were supplied with 5% Suc
for 36 h. Left panel: values are the average of amylase activity
determined in three independent experiments. Right panel: relative
levels of anthocyanin accumulation were determined, and the
value of wild type treated with 5% Suc was set as 1. The average
values were determined from two independent experiments. The
error bars indicate one SD of the mean. Nt represents non-treated
leaves.
leaf petioles. Expression of At-Amy encoding cytosolic
-amylase and CHS encoding chalcone synthase, a ratelimiting enzyme for anthocyanin biosynthesis, was induced
20-fold or more by Suc in a day (Mita et al. 1995, Mita et al.
1997), whereas expression of APS encoding the small
subunit of ADP-glucose pyrophosphorylase, a key enzyme
for starch biosynthesis, was increased by Suc to a much
lower extent than At-Amy and CHS (Sokolov et al. 1998).
Fig. 2 shows that At-Amy and CHS transcripts in the wild
type were induced by Suc in a dose-dependent manner.
In support of the results in Fig. 1, their transcripts levels
were 10–20 times higher in leaf-petioles treated with 5%
Suc than in those not treated with Suc. In hls1 mutants,
At-Amy and CHS transcripts increased in a dosedependent manner, but transcript levels were 3–6 times
higher than those in the wild type. The APS transcript
increased in a dose-dependent manner as well, but to a
lesser extent in both wild type and hls1 mutants as
compared with At-Amy and CHS (Fig. 2). The APS
transcript level was only twice as high in leaf-petioles
treated with 5% Suc than in those without Suc in the wild
type. In hls1mutants, transcript levels were only 1.6- to
1.8-fold higher than in the wild type.
We used a photosynthetic gene, RBCS encoding the
Rubisco small subunit, as representative of sugar-repressed
genes, because it is subject to negative feedback regulation
by sugars (Sheen 1990). In control (0% Suc) leaves, the
RBCS transcript was present at equal levels in hls1 and the
wild type (Fig. 2). Following treatment with Suc, RBCS
Role of HOOKLESS1 in sugar and auxin signaling
8
6
Atb-Amy
Wt
hls1-7
hls1-1
4
2
n.d.
0
6
4
CHS
Wt
hls1-7
hls1-1
2
n.d.
Relative transcripts level
0
2
1.5
APS
Wt
hls1-7
hls1-1
1
0.5
n.d.
0
1.5
RBCS
Wt
hls1-7
hls1-1
1
0.5
n.d.
0
10
HLS1
Wt
hls1-7
hls1-1
8
6
4
2
1605
affecting a common part of the signaling pathways for both
sugar-induced and sugar-repressed gene expression.
HLS1 expression in vegetative and reproductive tissues
was much lower (20-fold less) than that in etiolated
seedlings in the dark (Lehman et al. 1996). hls1-1 and
hls1-7 genes have base substitution mutations in the
transcribed region (Lehman et al. 1996), and hls1-1 and
hls1-7 transcripts were detected by Northern blot hybridization at a size that is identical to that of the wild type
(data not shown). Fig. 2 shows that the levels of hls1-1 and
hls1-7 transcripts were higher than that of the wild type,
suggesting that HLS1 is subject to negative feedback
regulation. A decrease in HLS1/hls1 transcripts by the
presence of Suc was observed only in hls1 mutants,
indicating that HLS1 is a conditional sugar-repressed gene
only when Suc sensitivity is high as a result of the hls1
mutation. However, sugar repression of HLS1 was less than
that for RBCS.
One explanation for the effects of the hls1 mutation
on sugar-responsive gene expression is that hls1 cells
incorporate more sugars than wild-type cells. We tested
this possibility with plants grown on different concentration
of Suc (Table 1). In both the wild type and hls1, the
accumulation of soluble sugar and starch was increased
by Suc in a dose-dependent manner, but hls1 mutants
accumulated less total carbohydrate (soluble sugar and
starch) than the wild type. For example, the starch level
was higher in hls1 mutants grown on 5% Suc than in the
wild type, possibly due to enhanced expression of APS.
However, the total soluble sugar level was much lower
in hls1 mutants than in the wild type, causing total
carbohydrate levels to be lower in the mutant. These results
do not indicate that hls1 cells are capable of incorporating
more sugars.
n.d.
0
0% Suc
1.7% Suc
5% Suc
Fig. 2 Effects of hls1 mutation on sugar-responsive gene expression. Leaf-petioles excised from 3-week-old plants on 2% Suc were
treated with 0–5% Suc for 24 h and harvested for the isolation of
total RNA. Relative levels of each transcript were determined by
real-time PCR and normalized relative to the ACT2 transcript level.
For At-Amy, CHS and APS, and for RBCS and HLS1, values for the
wild type treated with 5% Suc and with 0% Suc were set at 1,
respectively. All values are averages determined in three independent experiments. Error bars indicate one SD of the mean. n.d., not
determined.
transcript levels decreased in a dose-dependent manner in
hls1, but the decreases were more pronounced in hls1
than in the wild type (Fig. 2). Thus, the hls1 mutation
caused expression of all sugar-responsive genes to be more
sensitive to exogenous Suc, suggesting that HLS1 may
negatively regulate sugar-responsive gene expression by
Effects of the hls1 mutation on auxin-responsive gene
expression
Altered expression of sugar-responsive genes by the
hls1 mutation (Fig. 2) and the regulation of ARF2 by HLS1
during apical hook formation (Li et al. 2004) may suggest
that sugar-responsive gene expression is affected by auxin.
In order to examine this possibility, we analyzed the effects
of exogenous IAA on sugar-responsive gene expression.
We used hls1-7 for further analyses because hls1-7 plants
more closely resembled wild-type plants than hls1-1 plants
with respect to size (data not shown). Since sugar treatment
of excised leaf-petioles has been performed for 24–48 h
(Mita et al. 1997, Masaki et al. 2005, Yoine et al. 2006),
we supplied exogenous IAA to excised leaf petioles
for 24 h. Fig. 3 shows the effects of exogenous IAA on
sugar-responsive gene expression in the wild type and hls1-7.
In the wild type, IAA partially repressed sugar-induced
accumulation of At-Amy, CHS and APS transcripts.
1606
Table 1
Role of HOOKLESS1 in sugar and auxin signaling
Carbohydrate accumulation (mg g fresh weight) in hls1 leaves
Experiment 1
Wild type
hls1-1
Experiment 2
Wild type
hls1-1
Suc in medium
Soluble sugar (A)
Starch (B)
Total carbohydate (A þ B)
1.7%
3.4%
5.1%
1.7%
3.4%
5.1%
1.72
4.01
8.46
1.25
3.00
5.53
0.28
0.58
1.16
0.17
0.43
2.41
1.99
4.59
9.62
1.41
3.42
7.94
1.7%
3.4%
5.1%
1.7%
3.4%
5.1%
1.43
2.25
5.35
1.05
1.73
2.59
0.28
0.51
0.76
0.22
0.36
1.65
1.70
2.76
6.11
1.27
2.09
4.24
Wild type and hls1-1 were grown on 1.7, 3.4 and 5.1% Suc for 3 weeks. Mature leaves were immediately harvested to determine soluble
sugar (glucose, fructose and Suc) and starch accumulation.
Following induction by Suc, the ratios of increased
At-Amy, CHS and APS transcripts in the presence and
absence of IAA were 0.30, 0.65 and 0.79, respectively [values
were determined from relative values in Fig. 3 by
the formula: ratios ¼ (5% Suc/IAA – 0% Suc/IAA)/(5%
Suc – 0% Suc)]. In hls1-7 mutants, IAA partially repressed
induction of At-Amy, CHS and APS transcripts by Suc.
The ratios of At-Amy, CHS and APS transcripts following
Suc treatment in the presence and absence of IAA were 0.50,
0.70 and 0.87, respectively. Thus, the inhibitory effect of
IAA on sugar-induced gene expression occurred to a lesser
extent in hls1 mutants as compared with the wild type,
although the hls1 mutation only partially relieved IAA
repression of Suc-induced gene expression. The effect of the
hls1 mutation was the least pronounced with the APS
transcript, which was similar to the effects of the hls1
mutation on the Suc-increased APS transcript. These
results suggest that HLS1 may be partially involved in the
negative effects of IAA on sugar-responsive gene expression. To test the involvement of HLS1 in auxin signaling,
we examined several early auxin response genes, AUXIN
UPREGULATED1 (AUR1) encoding SAUR-AC1-like protein, AUR3 encoding GH3-like protein, and IAA2 in
addition to HAT2 encoding a protein of the HD-Zip II
subfamily which was also activated rapidly by exogenous
auxin (Sawa et al. 2002). AUR1 and AUR3 transcripts were
highly induced by IAA in both the wild type and hls1, but
transcript levels were 2-fold higher in hls1-7 as compared
with the wild type (Fig. 4). In contrast, HAT2 and IAA2
transcripts increased to a lesser extent than those of AUR1
and AUR3 in the wild type (Fig. 4). In hls1-7, HAT2
transcripts were several-fold higher than those in the wild
type in the absence and presence of IAA, whereas increases
in IAA2 transcripts by IAA were very weakly affected by the
hls1 mutation. These results indicate that HLS1 is involved
in auxin-regulated expression of some, but not all, auxin
response genes. In the presence of Suc, the levels of AUR3
transcript, but not of the other three transcripts, was clearly
affected by the hls1 mutation.
On the other hand, application of exogenous IAA
caused decreases in RBCS transcript to some extent in
both the wild type and hls1, but decreases in the transcript
appeared to be more significant in hls1 than in the wild
type (Fig. 3). The negative effects of exogenous IAA on
photosynthetic gene expression confirm previous observations that auxin repressed photosynthetic gene expression in
Arabidopsis cotyledons of dark-grown seedlings (Gil et al.
2001). Negative effects of IAA on the levels of HLS1/hls1
transcripts were observed only in hls1 mutants, which
were similar to the negative effects of Suc on the hls1
transcript (Fig. 3).
Involvement of HLS1 in auxin homeostasis
Auxin-inducible expression of AUR3 encoding
GH3-like protein is up-regulated by the hls1 mutation
(Fig. 4). Even in the absence of exogenous IAA, AUR3
transcripts were approximately four times higher in hls1
than in the wild type, with relative values representing
AUR3 transcripts in the absence or presence of Suc in Fig. 4
being 0.011 and 0.049, or 0.020 and 0.077 in the wild type
and hls1-7, respectively. Staswick et al. (2002, 2005)
reported that the AUR3 protein and six other GH3
homologs exhibited IAA adenylation activity, an initial
step in the conjugation of free IAA to amino acids,
Role of HOOKLESS1 in sugar and auxin signaling
4
Atb-Amy
3
Wt
3
hls1-7
2
1607
AUR1
Wt
hls1-7
2
1
1
0
0
6
AUR3
CHS
3
Wt
4
2
hls1-7
1
Relative transcripts level
2
Relative transcripts level
0
2
APS
Wt
hls1-7
1
RBCS
4
3
HAT2
Wt
hls1-7
2
1
1.5
Wt
hls1-7
1
1
IAA2
Wt
hls1-7
0.5
0.5
0
0
6
0
0
0
1.5
Wt
hls1-7
0%Suc
HLS1
Wt
hls1-7
4
2
0
0%Suc
0%Suc
+ IAA
5%Suc
5%Suc
+ IAA
0%Suc
+ IAA
5%Suc
5%Suc
+ IAA
Fig. 4 Effects of hls1 mutation on IAA-responsive gene expression. Relative levels of each transcript were determined by realtime PCR and normalized relative to the ACT2 transcript level. First
strand cDNAs used to generate the data shown in Fig. 3 were used
for this analysis. Values for the wild type treated with 50 mM IAA in
the absence of Suc were set at 1. All values are averages of three
independent experiments. The error bars indicate one SD of
the mean.
Fig. 3 Effects of IAA on sugar-responsive gene expression.
Leaf-petioles excised from 3-week-old plants on 2% Suc were
treated with 0 or 5% Suc in the absence or presence of 50 mM IAA
for 24 h and harvested for the isolation of total RNAs. Relative
levels of each transcript were determined by real-time PCR and
normalized relative to the ACT2 transcript level. For At-Amy, CHS
and APS, and for RBCS and HLS1, values for wild type treated with
5% Suc and with 0% Suc were set at 1, respectively. All values are
averages of three independent experiments. Error bars indicate one
SD of the mean.
IAA level is altered by the hls1 mutation, we measured the
free IAA level in hls1-7. Table 2 shows that the free IAA
level in hls1-7 leaves was approximately 67% of that in
the wild type. The lower free IAA level in hls1 relative to
the wild type may explain why mutant plants exhibit low
auxin phenotypes such as decreased apical dominance
(Lehman et al. 1996) and small rosette leaves (Table 2)
(Ouyang et al. 2000).
suggesting its role in a negative feedback regulation of auxin
levels. Similarly, activation of GH3-like genes by an
elevated level of free IAA may cause a decrease in free
IAA levels because GH3 proteins cause conjugation to
IAA–amino acid. In order to test the possibility that the free
Discussion
HLS1 regulates sugar-responsive gene expression
HLS1 was originally identified as a gene that regulates
differential growth of hypocotyls in dark-grown seedlings
1608
Table 2
Role of HOOKLESS1 in sugar and auxin signaling
hls1 leaves contained reduced levels of free IAA
Wild type
hls1-7
hls1-1
Free IAA
(ng g1 fresh weight)
Leaf weight
per plant (mg)
5.0 0.5
3.4 0.5
n.d.
40.5 4.9 (14)
27.5 4.2 (10)
19.4 5.1 (10)
Plants were grown on 3% Suc for 3 weeks, and mature leaves were
used to determine levels of free IAA. Values SD for free IAA
levels are the average determined in five independent experiments.
The mean values SD for total weights of the third to sixth leaves
per plant were determined in one of the experiments. Differences
between values for the wild type and each hls1 mutant are
significant at the 50.005 level. Numbers in parentheses indicate
the number of plants used to measure leaf weight. n.d.; not
determined.
(Lehman et al. 1996, Li et al. 2004). Here we show that
loss-of-function hls1 mutants are hypersensitive to Suc for
the expression of sugar-responsive genes in mature leaves
(Figs. 1, 2). How does HLS1 regulate the expression of
sugar-responsive genes? One possibility was that the hls1
mutation affects the uptake of sugars into cells. However,
determination of glucose, fructose, Suc and starch levels in
hls1 mutants grown on 1.7–5.1% Suc indicated that hls1
cells did not accumulate sugars more actively than wild-type
cells (Table 1), suggesting that the hls1 mutation affects
a sugar signaling pathway. We show here that both sugar
induction of At-Amy, CHS and APS expression and sugar
repression of RBCS expression were enhanced by the hls1
mutation (Fig. 2), suggesting that HLS1 may control a
common step in the sugar signaling pathways for sugarinduced genes and sugar-repressed genes (Fig. 5). Jang
and Sheen (1997) proposed that there were at least three
sugar signaling pathways: (i) a hexokinase (HXK)dependent pathway in which HXK acts as a sugar sensor;
(ii) a glycolysis-dependent pathway where certain metabolites downstream of hexose phosphorylation could serve
as signals; and (iii) an HXK-independent pathway possibly
involving a sugar receptor on the plasma membrane.
HXK is a branch point of the HXK-dependent pathway
and the glycolysis-dependent pathway. Sugar-repressed
genes, such as RBCS and CAB, are known to be regulated
under the control of the HXK-dependent pathway
(Jang and Sheen 1994, Jang et al. 1997). Sugar-induced
expression of At-Amy, CHS and APS genes is under the
control of the glycolysis-dependent pathway (Ohto et al. in
preparation). Therefore, although more direct evidence is
needed to elucidate how HLS1 controls sugar-responsive
gene expression at the molecular level, one possible
mechanism is that HLS1 may control HXK activity,
a common step of the HXK-dependent pathway and the
glycolysis-dependent pathway.
Auxin
Sugar
HLS
RBCS
Atb-Amy
CHS
APS
1
AUR1
AUR3
HAT2
IAA2
Fig. 5 A model to explain HLS1 function in sugar- and IAAresponsive gene expression in leaves. HLS1 works with auxin to
affect both sugar-induced and sugar-repressed gene expression
negatively. HLS1 also negatively regulates auxin-induced expression of some, but not all, auxin response genes. Auxin homeostasis
is maintained, in part, through negative feedback regulation in
which HLS1 is involved in the control of AUR3, a GH3-like gene.
HLS1 negatively regulates auxin-responsive gene expression
The hls1 mutation enhances auxin-induced expression
of several auxin response genes such as AUR1 encoding
SAUR-AC1-like protein, AUR3 encoding GH3-like protein
and HAT2 encoding a protein of the HD-Zip II subfamily,
but not IAA2 (Fig. 4). These findings indicate a negative
role for HLS1 in auxin-induced expression of some auxin
response genes (Fig. 5).
Expression of early auxin response genes is regulated
positively and negatively by auxin response transcription
factors (ARFs) that bind to auxin response elements
(AuxREs) in the upstream regions of auxin response genes
(Ulmasov et al. 1999a, Ulmasov et al. 1999b, Hagen and
Guilfoyle 2002, Liscum and Reed 2002). ARF5–ARF8
activate and ARF1–ARF4 and ARF9 repress the activity of
auxin-responsive synthetic promoters containing AuxREs
(Tiwari et al. 2003). ARF1–ARF4 and ARF6–ARF10
are uniformly expressed in cultured cells, roots, rosette
leaves, cauline leaves and flowers, and ARFs transcripts
(ARF1, ARF2 and ARF4–ARF8) were unchanged in
response to exogenous auxin (Ulmasov et al. 1999a,
Hagen and Guilfoyle 2002).
Li et al. (2004) proposed that ARF2 negatively
regulates apical hook formation in a HLS1-depenedent
manner. Consistent with this interpretation, results
obtained in mature leaves suggest that ARF activators or
repressors may require HLS1 to negatively regulate AUR1,
AUR3 and HAT2 in a HLS1-dependent manner. For
example, ARF activators that are negatively regulated by
HLS1 may regulate AUR1, AUR3 and HAT2. In this
model, HLS1 decreases ARF protein levels by decreasing
the stability of ARF activators or increasing the stability
of Aux/IAA proteins that repress ARF activators
Role of HOOKLESS1 in sugar and auxin signaling
(Hagen and Guilfoyle 2002). Alternatively, HLS1 may
increase ARF repressors that regulate AUR1, AUR3
and HAT2. On the other hand, it is not clear why the
hls1 mutation did not affect IAA-induced IAA2 expression significantly. The reason may be due to the difference
in tissue-specific expression patterns of HLS1 and
IAA2, since IAA2 showed tissue-preferential expression
in leaves (Marchant et al. 2002). However, cell- or tissuespecific expression of HLS1 in leaves has not been
reported, yet.
The hls1 mutation causes altered expression of sugarresponsive genes (Figs. 1, 2), which also implies that sugarresponsive gene expression is regulated by HLS1 through
its role in auxin signaling. HLS1 encodes a protein
homologous to N-acetyltransferase (Lehman et al. 1996).
N-Acetyltransferases transfer the acetyl group from acetyl
coenzyme A to the amino group of proteins, peptides and
small molecules. N-terminal acetylation of proteins is
known to affect protein function and stability (Kendall
et al. 1990). Li et al. (2004) proposed that HLS1 may
acetylate ARF2 or other components to regulate the
stability of ARF2. Since as many as 50–80% of eukaryotic
proteins are N-terminally acetylated (Driessen et al. 1985),
it may be possible that HLS1 has a wide spectrum of
substrates, including ARF(s), to regulate sugar signaling.
Our findings that exogenous IAA partially repressed sugarinduced gene expression and that the IAA repression
was less pronounced in hls1 than in the wild type (Fig. 3)
may suggest that the negative effect of IAA on sugar
signaling is mediated at least in part by ARF(s) in a
HLS1-dependent manner.
Role of HLS1 in IAA homeostasis
The activities of group II GH3-like proteins from
Arabidopsis including AUR3 that conjugate free IAA with
amino acid (Staswick et al. 2002, Staswick et al. 2005)
suggest a negative feedback regulation of auxin levels.
Activation of GH3-like genes by elevated levels of free
IAA increases production of IAA–amino acids, resulting
in a decrease in the effective IAA concentration. Both an
enhancement of AUR3 expression (Fig. 4) and reduction in
the free IAA level (Table 2) in hls1 mutant leaves suggest
an involvement of HLS1 in the negative feedback regulation
of free IAA level through its role in controlling GH3-like
genes (Fig. 5). Tian et al. (2004) reported that overexpression of ARF8 increased gene expression for group II
GH3-like proteins and decreased the free IAA level to 80%
of that in the wild type in light-grown Arabidopsis seedlings.
Thus, auxin levels are regulated at least in part through
the activities of genes encoding GH3-like proteins that
are in turn regulated by ARF activators and repressors,
and by HLS1.
1609
Materials and Methods
Plant materials and growth conditions
hls1-1, hls1-7 and hls1-9 in the Columbia ecotype were
obtained from the Arabidopsis Biological Resources Stock Center
(Ohio State University, Columbus, OH, USA). uns2 in the
Wassilewskija (Ws) ecotype was isolated from a population of
plants mutagenized with T-DNA (Suzuki et al. 2004). uns2 seeds
are immediately available upon request. Sterilized seeds were
imbibed, then treated in the dark at 48C for 4 d before sowing.
Plants cultured in vitro were grown on medium that contained 2%
(w/v) Suc, unless otherwise indicated, 0.3% (w/v) Gellan Gum,
2.5 mM MES-KOH (pH 5.7), 1 MS salts and B5 vitamins at 228C
under continuous light. The light intensity during plant growth was
50 mmol m2 s1. For apical hook analyses, seeds sown on medium
that contained 2% (w/v) Suc, 1.2% agarose, 1 MS salts and B5
vitamins were illuminated for 12 h to accelerate germination.
Plates were subsequently placed vertically in the dark for 4 d.
Treatment of excised leaf-petioles with Suc, and determinations of
amylase activity and anthocyanin
For experiments using leaf-petioles treated with Suc, the third
to sixth rosette leaves were excised with a sharp razor blade from
3-week-old plants grown on 2% Suc, unless otherwise indicated.
The cut edges of petioles were immersed in a sterile solution of
sugar and incubated at 228C under continuous light. To determine
levels of amylase activity and anthocyanin, tissues from 10–15
plants were combined before the extraction procedures. Protein
extractions and the determination of both protein levels and total
amylase activities were performed as described previously (Mita
et al. 1995). One unit of total amylase activity was defined as the
amount of enzymes that released 1 mmol of reducing sugar per min
under the assay conditions. Extraction and determination of
anthocyanin were carried out as described previously (Mita et al.
1997).
Quantification of free IAA, soluble sugars and starch
Plants were grown on 3% Suc for 3 weeks, and mature
leaves at the third to sixth leaf position were used to determine free
IAA, soluble sugar and starch levels. Quantification of the free
IAA level was performed as described previously (Woodward et al.
2005). Determination of soluble sugars and starch was performed
as described by Mita et al. (1997).
RNA preparation and expression analysis
Total RNA isolation and Northern blot hybridization were
performed as described previously (Mita et al. 1995). For
quantitative analyses of transcript levels by real-time PCR, total
RNA was treated with DNase I and subsequently purified
with an RNeasy RNA purification column (Qiagen, Hilden,
Germany). First strand cDNA was synthesized from 2.5 mg of
the column-purified total RNA with oligo(dT)20 primers using
SUPERSCRIPT III (Invitrogen, Carlsbad, CA, USA) and diluted
with 10 vols of water. Real-time PCR was performed in duplicate
on a 25 ml reaction mixture containing 5 ml of diluted cDNA
solution, 12.5 ml of a Cybergreen dye set (Bio-Rad, Hercules, CA,
USA), and 0.5 ml of each primer (final concentration of 200 nM).
PCR was initiated with denaturation at 958C for 5 min, followed
by 40 cycles of denaturation at 958C for 30 s, annealing at 628C for
30 s and extension at 728C for 30 s. The comparative threshold
cycle method was used to determine the relative mRNA levels.
ACT2 was used as an internal reference, and transcripts levels
1610
Role of HOOKLESS1 in sugar and auxin signaling
were presented as values relative to the control treatment.
The following primer sets were used:ACT2 [At3g18780, GI
(GenBank Gene Info Identifier number): 1669386], 50 -CTG
TTGACTACGAGCAGGAGATGGA-30 and 50 -GACTTCTGG
GCATCTGAATCTCTCA-30 ; APS (At5g48300, GI: 1575753),
50 -GTCAAGATCATAAACAGCGACAACG-30 and 50 -GGGAT
TAAGGCGTCTTTGATAACC-30 ; At-Amy (At4g15210, GI:
166601), 50 -CATGAGATTGTGCCGTTGAA-30 and 50 -TCGGT
TTCAGAGTCCCACTT-30 ; AUR1 (At2g18010, GI: 20197593/
T27K22.12), 50 -GAAGATGATCTTCCTCAAGACGTGC-30 and
50 -GGAGCAATGTTTGGAACTCGGAG-30 ; AUR3 (At4g37390,
GI: 4468801/F6G17.40), 50 -GAGCTTCACACCTATCATGG
AGCTG-30 and 50 -CTAAAACCGCACATCATCCGGTTG-30 ;
CHS (At5g13930, GI: 166669), 50 -GGAGGAAGTCAGCTA
AGGATGGTG-30 and 50 -AGGCAAGCGTTCTGTTTAGA
GAGG-30 ; HAT2 (At5g47370, GI: 527626), 50 -AGAGGCTATG
GAGCTTCGAACTCTC-30 and 50 -CCTGTGATTGTGGTGAT
GGTTCG-30 ; HLS1 (At4g37580, GI: 1277089), 50 -TCTCATTT
CCACACTCACCTTCCTC- 30 and 50 -CTCTCTAACCACCGT
CATGTTTTGG-30 ; IAA2 (At3g23030, GI: 2598931), 50 -GCA
ATGGCGTACGAGAAAGTCAAC-30 and 50 -CATCACGAGTT
TCCTCAAATAGACG-30 ; RBCS (At5g38410, At5g38420,
At5g38430, GI: 2264320/MXI10.13–MXI10.15), 50 -GTTGAAGA
ATGCAAGAAGGAGTACC-30 and 50 -GGCTTGTAGGCAAT
GAAACTGATG-30 .
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.oxford
journals.org.
Acknowledgments
The authors are grateful to Dr. Joseph Ecker (Salk Institute,
La Jolla, CA, USA) for his generous gift of full-length HLS1
cDNA. We are grateful to Drs. John Harada (University of
California, Davis) and Kiyoshi Onai (Nagoya University) and
Mr. Hironaka Tsukagoshi (Nagoya University) for their useful
comments. Thanks are also due to the Arabidopsis Biological
Resource Center (Ohio State University) for materials and to
Ms. Etsuko Aoki, Ms. Yasuko Furukawa (National Institute for
Basic Biology) and Mr. Kensuke Yamazaki (Yakult) for their
excellent technical assistance. This work was supported in part by
grants from the Ministry of Education, Culture, Sports, Science
and Technology of Japan to M.O. and from the Japan Society for
the Promotion of Science to K.N.
References
Bernier, G., Havelange, A., Houssa, C., Petitjean, A. and Lejeune, P. (1993)
Physiological signals that induce flowering. Plant Cell 5: 1147–1155.
Borisjuk, L., Rolletschek, H., Radchuk, R., Weschke, W., Wobus, U. and
Weber, H. (2004) Seed development and differentiation: a role for
metabolic regulation. Plant Biol. 6: 375–386.
Chen, R., Rosen, E. and Masson, P.H. (1999) Gravitropism in higher
plants. Plant Physiol. 120: 343–350.
Driessen, H.P., de Jong, W.W., Tesser, G.I. and Bloemendal, H. (1985)
The mechanism of N-terminal acetylation of proteins. CRC Crit. Rev.
Biochem. 18: 281–325.
Estelle, M. and Klee, H.J. (1994) Auxin and cytokinin in Arabidopsis.
In Arabidopsis. Edited by Meyerowitz, E.M. and Somerville, C.R.
pp. 555–580. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
Gazzarrini, S. and McCourt, P. (2001) Genetic interactions between ABA,
ethylene and sugar signaling pathways. Curr. Opin. Plant Biol. 4:
387–391.
Gibson, S.I. (2005) Control of plant development and gene expression by
sugar signaling. Curr. Opin. Plant Biol. 8: 93–102.
Gil, P., Dewey, E., Friml, J., Zhao, Y., Snowden, K.C., Putterill, J.,
Palme, K., Estelle, M. and Chory, J. (2001) BIG: a calossin-like protein
required for polar auxin transport in Arabidopsis. Genes Dev. 15:
1985–1997.
Hagen, G. and Guilfoyle, T. (2002) Auxin-responsive gene expression:
genes, promoters and regulatory factors. Plant Mol. Biol. 49: 373–385.
Jackson, S.D. (1999) Multiple signaling pathways control tuber induction in
potato. Plant Physiol. 119: 1–8.
Jang, J.C., Leon, P., Zhou, L. and Sheen, J. (1997) Hexokinase as a sugar
sensor in higher plants. Plant Cell 9: 5–19.
Jang, J.C. and Sheen, J. (1994) Sugar sensing in higher plants. Plant Cell 6:
1665–1679.
Jang, J.-C. and Sheen, J. (1997) Sugar sensing in higher plants. Trends Plant
Sci. 2: 208–214.
Kendall, R.L., Yamada, R. and Bradshaw, R.A. (1990) Cotranslational
amino-terminal process. Methods Enzymol. 185: 398–407.
Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 509–540.
Lehman, A., Black, R. and Ecker, J.R. (1996) HOOKLESS1, an ethylene
response gene, is required for differential cell elongation in the
Arabidopsis hypocotyl. Cell 85: 183–194.
Li, H., Johnson, P., Stepanova, A., Alonso, J.M. and Ecker, J.R. (2004)
Convergence of signaling pathways in the control of differential cell
growth in Arabidopsis. Dev. Cell 7: 193–204.
Liscum, E. and Reed, J.W. (2002) Genetics of Aux/IAA and ARF action in
plant growth and development. Plant Mol. Biol. 49: 387–400.
Marchant, A., Bhalerao, R., Casimiro, I., Eklof, J., Casero, P.J.,
Bennett, M. and Sandberg, G. (2002) AUX1 promotes lateral root
formation by facilitating indole-3-acetic acid distribution between sink
and source tissues in the Arabidopsis seedling. Plant Cell 14: 589–597.
Masaki, T., Tsukagoshi, H., Mitsui, N., Nishii, T., Hattori, T.,
Morikami, A. and Nakamura, K. (2005) Activation tagging of a gene
for a protein with novel class of CCT-domain activates expression of
a subset of sugar-inducible genes in Arabidopsis thaliana. Plant J. 43:
142–152.
Mita, S., Murano, N., Akaike, M. and Nakamura, K. (1997) Mutants of
Arabidopsis thaliana with pleiotropic effects on the expression of the gene
for -amylase and on the accumulation of anthocyanin that are inducible
by sugars. Plant J. 11: 841–851.
Mita, S., Suzuki-Fujii, K. and Nakamura, K. (1995) Sugar-inducible
expression of a gene for -amylase in Arabidopsis thaliana. Plant Physiol.
107: 895–904.
Ouyang, J., Shao, X. and Li, J. (2000) Indole-3-glycerol phosphate,
a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan
biosynthetic pathway in Arabidopsis thaliana. Plant J. 24: 327–334.
Rolland, F., Moore, B. and Sheen, J. (2002) Sugar sensing and signaling in
plants. Plant Cell 14: S185–S205.
Rook, F. and Bevan, M.W. (2003) Genetic approaches to understanding
sugar-response pathways. J. Exp. Bot. 54: 495–501.
Sawa, S., Ohgishi, M., Goda, H., Higuchi, K., Shimada, Y., Yoshida, S. and
Koshiba, T. (2002) The HAT2 gene, a member of the HD-Zip gene
family, isolated as an auxin inducible gene by DNA microarray screening,
affects auxin response in Arabidopsis. Plant J. 32: 1011–1022.
Schwark, A. and Schierle, J. (1992) Interaction of ethylene and auxin in the
regulation of hook growth I. The role of auxin in different growing
regions of the hypocotyl hook of Phaseolus vulgaris. J. Plant Physiol. 140:
562–570.
Sheen, J. (1990) Metabolic repression of transcription in higher plants. Plant
Cell 2: 1027–1038.
Smeekens, S. (2000) Sugar-induced signal transduction in plants. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 51: 49–81.
Sokolov, L.N., Dejardin, A. and Kleczkowski, L.A. (1998) Sugars and light/
dark exposure trigger differential regulation of ADP-glucose
Role of HOOKLESS1 in sugar and auxin signaling
pyrophosphorylase genes in Arabidopsis thaliana (thale cress). Biochem. J.
336: 681–687.
Staswick, P.E., Serban, B., Rowe, M., Tiryaki, I., Maldonado, M.T.,
Maldonado, M.C. and Suza, W. (2005) Characterization of an
Arabidopsis enzyme family that conjugates amino acids to indole-3acetic acid. Plant Cell 17: 616–627.
Staswick, P.E., Tiryaki, I. and Rowe, M.L. (2002) Jasmonate response
locus JAR1 and several related Arabidopsis genes encode enzymes of the
firefly luciferase superfamily that show activity on jasmonic, salicylic,
and indole-3-acetic acids in an assay for adenylation. Plant Cell 14:
1405–1415.
Suzuki, T., Inagaki, S., Nakajima, S., Akashi, T., Ohto, M., et al. (2004) A
novel Arabidopsis gene TONSOKU is required for proper cell arrangement in root and shoot apical meristems. Plant J. 38: 673–684.
Tian, C., Muto, H., Higuchi, K., Matamura, T., Tatematsu, K., Koshiba, T.
and Yamamoto, K.T. (2004) Disruption and overexpression of auxin
response factor 8 gene of Arabidopsis affect hypocotyl elongation and root
growth habit, indicating its possible involvement in auxin homeostasis in
light condition. Plant J. 40: 333–343.
Tiwari, S.B., Hagen, G. and Guilfoyle, T. (2003) The roles of
auxin response factor domains in auxin-responsive transcription. Plant
Cell 15: 533–543.
1611
Ulmasov, T., Hagen, G. and Guilfoyle, T.J. (1999a) Dimerization and DNA
binding of auxin response factors. Plant J. 19: 309–319.
Ulmasov, T., Hagen, G. and Guilfoyle, T.J. (1999b) Activation and
repression of transcription by auxin-response factors. Proc. Natl Acad.
Sci. USA 96: 5844–5849.
Vanneste, S., Maes, L., De Smet, I., Himanen, K., Naudts, M., Inze, D. and
Beeckman, T. (2005) Auxin regulation of cell cycle and its role during
lateral root initiation. Physiol. Plant. 123: 139–146.
Wingler, A., Purdy, S., MacLean, J.A. and Pourtau, N. (2006) The role of
sugars in integrating environmental signals during the regulation of leaf
senescence. J. Exp. Bot. 57: 391–399.
Woodward, A.W. and Bartel, B. (2005) Auxin: regulation, action, and
interaction. Ann. Bot. 95: 707–735.
Woodward, C., Bemis, S.M., Hill, E.J., Sawa, S., Koshiba, T. and
Torii, K.U. (2005) Interaction of auxin and ERECTA in elaborating
Arabidopsis inflorescence architecture revealed by the activation tagging
of a new member of the YUCCA family putative flavin monooxygenases.
Plant Physiol. 139: 192–203.
Yoine, M., Ohto, M., Onai, K., Mita, S. and Nakamura, K. (2006) The lba1
mutation of UPF1 RNA helicase involved in nonsense-mediated mRNA
decay causes pleiotropic phenotypic changes and altered sugar signaling
in Arabidopsis. Plant J. 47: 49–62.
(Received September 3, 2006; Accepted October 16, 2006)