Ethylene, a major phytohormone, is one of the simplest organic
molecules with biological activity and controls many aspects of plant
growth and development(1, 2) . The gas is
endogeneously produced during unique developmental stages such as
growth, senescence, and abscission of leaves and flowers, development
and ripening of fruits, and germination of seeds(2) . Ethylene
production is also induced by various stress conditions and chemical
compounds, including wounding, temperature fluctuation, drought,
anaerobiosis, viral infection, elicitor treatment, heavy metal
exposure, or lithium ions(2) . Ethylene serves as a signaling
molecule to initiate and coordinate profound physiological changes and
adaptations throughout the life cycle of a
plant(1, 2) .
Ethylene biosynthesis is stringently
regulated during plant development (2, 3) . The
rate-limiting reaction catalyzed by the enzyme ACC (
)synthase (S-adenosyl-L-methionine
methylthioadenosine-lyase, EC 4.4.1.14) is responsible for the
formation of the immediate ethylene precursor, ACC, from S-adenosylmethionine. The committed step is subject to control
at the transcriptional and post-transcriptional
level(2, 3) . ACC synthase is a short-lived cytosolic
enzyme (4) and is encoded by a highly divergent multigene
family in a number of plant species including zucchini(5) ,
tomato(6) , mung bean(7) , rice(8) , and Arabidopsis thaliana(9, 10) . Each member of
the gene families is differentially expressed during plant development
as well as in response to a distinct subset of environmental and
chemical
stimuli(5, 6, 7, 8, 9, 10) .
For instance, the plant hormone auxin, typified by IAA, is a known
inducer of ethylene production (2) and regulates specific
members of each ACS multigene family in a tissue-specific
manner(5, 8, 10, 11, 12) .
We are interested in elucidating the multiple signal-transduction
pathways leading to ACS gene activation by a diverse group of
inducers, using biochemical, molecular, and reverse genetic approaches.
Our particular goal is to understand how ACS genes are
activated by auxin, by protein synthesis inhibition, and by lithium
ions(6, 8, 10) . Protein synthesis inhibitors
such as CHX have been widely used as a tool to unmask regulatory
mechanisms of early gene activation(13) . Likewise, the lithium
ion is known to interfere with phosphatidylinositol metabolism and
signaling(14) . On the other hand, auxin-inducible ACS genes provide a molecular probe to study mechanisms of auxin
action and the intimate interrelationship of both plant hormones. As a
first step toward this long term goal we have cloned ACS multigene families in tomato(6) , rice(8) , and A. thaliana(10) . We are attempting to develop the
molecular genetic approaches in Arabidopsis, a model organism
for a flowering plant(15) . We have previously identified an
auxin-regulated ACS gene in A. thaliana, ACS4(10) . Here, we report its structure and specific
expression characteristics in response to IAA.
EXPERIMENTAL PROCEDURES
Arabidopsis Strains and Growth Conditions
The
following Arabidopsis strains were used: wild type A.
thaliana (L.) Heynh. ecotype Columbia, and auxin-resistant mutant
lines axr1-12(16) , axr2-1(17) , and aux1-7(18) . Seeds of the auxin-resistant mutant
lines were kindly provided by Mark Estelle (Indiana University). To
grow etiolated seedlings, seeds were surface sterilized for 8 min in 5%
sodium hypochlorite (30% chlorox), 0.1% Triton X-100, excessively
rinsed in distilled water, and plated in Petri dishes onto sterile
filter paper discs on top of 0.7% agar (Bacto-agar, Difco) containing
0.5
Murashige-Skoog salts (Life Technologies, Inc.) at pH 5.6.
After cold treatment at 4 °C for 3 days, the plates were incubated
in the dark at 22 °C for 5-6 days.
Tissue Treatment
Intact etiolated seedlings
(5-6 days old) were removed from the filter discs and placed in
Petri dishes containing 0.5
Murashige-Skoog salt solution
buffered at pH 5.6 with 0.5 mM MES and supplemented with the
appropriate chemicals. The seedlings were incubated in the dark at room
temperature with shaking (50-100 revolutions/min). Mock control
incubations were supplemented with an equal amount of the solvent used
to prepare the stock solution of the respective chemical. After the
indicated time, aliquots (3-5 g fresh weight) of seedlings were
removed, briefly blotted dry, frozen with liquid nitrogen, and stored
at -80 °C. For the short time course experiment, intact
etiolated seedlings (5-6 days old; 3-5 g fresh weight) were
placed in 50-ml Falcon tubes. After the addition of 15 ml of 0.5
MS salts, 0.5 mM MES, pH 5.6, the tubes were
moderately shaken by hand for the indicated period. The seedlings were
immediately frozen with liquid N
after decanting the
bathing solution and stored at -80 °C.
Plasmids
The following recombinant clones were
used in this study: pAAA1 contains the 8.3-kb EcoRI fragment
of
AT-8 in pUC18.
AT-8 is an Arabidopsis genomic
clone containing the ACS4 gene(10) . pAAA2 was derived
from pAAA1 by deleting the 2.1-kb SacI/EcoRI fragment
by SacI digestion and religation. pAAA3 contains the 2.1-kb EcoRI fragment of
AT-8 in pUC18. pAAA4 was derived from
pAAA3 by deleting the 1.1-kb BamHI/EcoRI fragment by BamHI digestion and religation. pAAA5 was constructed by PCR
using sequencing primers A2A (5`-GAAGCCTACGAGCAAGCC-3`) and T3D
(5`-TTGTGTCTGGGAGGAGAC-3`) as amplimers and
AT-8 phage DNA as the
template. The 0.4-kb PCR product was subcloned into the EcoRV
site of pIC20R. pAAA6 contains a PCR-generated, 1.4-kb ACS4 cDNA insert in the BamHI site of pUC19.
Poly(A)
RNA from IAA/CHX-treated etiolated Arabidopsis seedlings was reverse-transcribed(19) .
The single-stranded cDNAs were used as the template in a PCR (20) with amplimers CDA1, 5`-GGCCGGATCCAA ATG GTT CAA
TTG TCA AGA AAA GC-3`, and CDA2, 5`-GGCCGGATCCA CTA TCG TTC CTC
AGC CTC ACG G-3` (BamHI recognition sites are underlined;
start and stop codons are in bold-face type).
DNA Sequencing
Dideoxy sequencing of
double-stranded DNA of pAAA plasmids was performed with universal and
synthetic primers using [
S]dATP (21) and
the modified T7 DNA polymerase, Sequenase, according to the
manufacturer's instructions (U.S. Biochemicals Corp., Cleveland,
OH). DNA sequences were analyzed with the Sequence Analysis Software
Package of the Genetics Computer Group (University of Wisconsin).
Expression of ACS4 in E. coli
Plasmid pAAA6 was
introduced into Escherichia coli DH5
and
M15[pREP4]. Expression conditions and measurements of ACC
formation were as described previously(6) .
Isolation of Nucleic Acids
Total nucleic acids
were prepared from frozen Arabidopsis tissues. Typically,
etiolated seedlings or other plant material (
3-5 g fresh
weight in 50-ml Falcon tubes) were supplemented with 30 ml of
extraction buffer (1 volume of 200 mM Tris-HCl, pH 7.5, 100
mM LiCl, 5 mM EDTA, 1% SDS, 1%
-mercaptoethanol,
and 1 volume of phenol/chloroform/isoamyl alcohol, 25:24:1) and
macerated with a polytron mixer (Brinkman) at the highest setting for 2
min. The homogenate was centrifuged, and the aqueous phase was
reextracted with an equal volume of phenol/chloroform/isoamyl alcohol.
After a second reextraction with chloroform, the aqueous phase was
brought to 2 M LiCl and incubated overnight at 4 °C.
Nucleic acids were recovered by centrifugation, dissolved in 0.5 ml of
water, and reprecipitated with 2.5 volumes of ethanol after adjusting
the salt concentration to 300 mM sodium acetate, pH 5.5.
Poly(A)
RNAs from larger batches of total nucleic
acids were isolated by affinity chromatography using oligo(dT)
cellulose as described by Theologis et al.(22) .
RNA Hybridization Analysis
Northern analysis was
essentially performed according to Ecker and Davis(23) . Total
nucleic acids were glyoxylated at 50 °C, electrophoresed on 1%
agarose gels, and transferred to GeneScreen membrane (DuPont-NEN).
After baking at 80 °C for 3 h, the membranes were prewashed in 0.1
SSPE, 0.1% SDS at 60 °C for 1 h. Prehybridization was
performed in 50% formamide, 5
SSPE (1
SSPE is 0.18 M NaCl, 10 mM sodium phosphate, pH 7.0, 1 mM EDTA), 5
BFP (1
BFP is 0.02% bovine serum albumin,
0.02% polyvinyl pyrrolidone (M
= 360,000),
0.02% Ficoll (M
= 400,000)), 1% SDS, and
100 µg/ml denatured salmon sperm DNA at 42 °C for 4-6 h.
The hybridization buffer contained in addition 5% dextran sulfate (M
500,000) and the appropriate radioactively
labeled probe. Probes were prepared from DNA restriction fragments by
the random hexamer-primed synthesis method (24) to a specific
activity of
1.0
10
counts/min/µg.
Hybridizations were carried out with radiolabeled probes of 2
10
counts/min/ml hybridization solution at 42 °C for
16-20 h. The membranes were washed in 50% formamide, 5
SSPE, 0.1% SDS at 42 °C for 1 h followed by a final high stringency
wash in 0.1
SSPE, 0.1% SDS at 60 °C for 1 h. The wet
filters were exposed to Kodak XAR-5 film with a DuPont Cronex Lightning
Plus intensifying screen at -80 °C. The autoradiograms were
quantified using an LKB ultrascan laser densitometer (Bromma, Sweden).
After exposure, the probe was removed by rinsing the filters in 0.1
SSPE, 0.1% SDS at 95-100 °C for 15-30 min.
Primer Extension Analysis
Primer extension was
performed essentially according to the method of Boorstein and
Craig(25) . Approximately 5
10
counts/min
of a 5`-end-labeled 29-mer synthetic oligonucleotide, DP1,
complimentary to nucleotides +146 to +174 of the ACS4 gene (Fig.1B) was hybridized at 50 °C for 3 h
to 15 µg of poly(A)
RNA from etiolated Arabidopsis seedlings treated with 20 µM IAA and
50 µM CHX for 2 h. The primer-RNA hybrids were incubated
with 20 units of reverse transcriptase in 50 µl of 20 mM Tris-HCl, pH 8.0, 30 mM NaCl, 100 µM of each
dNTP, 10 mM dithiothreitol, 60 µg/ml actinomycin D for 1 h
at 42 °C. The products were analyzed on a 6% denaturing
polyacrylamide gel.
Figure 1:
Structure of the ACS4 gene. A, gene organization of ACS4 and structure of derived
plasmid constructs. A partial restriction map of clone
AT-8 and
derived plasmids is shown (R, EcoRI; B, BamHI; S, SalI; Sc, SacI).
Both strands of the indicated regions were sequenced by primer walking
as described under ``Experimental Procedures.'' The arrows indicate the extent of each sequence determination. The filled box in
AT-8 is the fragment which hybridizes to
the TZ-region of ASC genes (10) . Filled boxes in the derived pAAA plasmids indicate the coding region of ASC4. The gene organization of ACS4 is given below. Open blocks indicate the 5`- and 3`-untranslated regions. The
exons are shown as filled blocks, and the connecting lines designate the introns. The +1 and arrow indicate the start and direction of transcription in ACS4.
B, DNA sequence of the ACS4 gene including introns and
5`- and 3`-flanking regions. The nucleotide at position +1
corresponds to the transcription initiation site (Fig.2). The
nucleotides upstream from the +1 position are negatively
numbered. The sequence of the mRNA transcribed by the gene is
shown in capital letters. The cDNA sequence is shaded; the remainder of the sequence is in small
letters. The derived amino acid sequence is presented in the one-letter code below the DNA sequence and is
numbered separately. The 11 boxed residues are the invariant
amino acids conserved between ACC synthases and various
aminotransferases(6) . The underlined dodecapeptide
beginning at Ser
is part of the active center of ACC
synthase(37) . The arrow indicates primer DP1 used for
determining the start of transcription (Fig.2). The TATA box
and putative polyadenylation signal sequences are boxed.
Sequence motifs in the ACS4 promoter reminiscent of
functionally defined auxin-responsive elements are underlined.
A dot indicates the EcoRI and BamMI site,
respectively.
Figure 2:
Determination of the transcription
initiation site of ACS4.
P-Labeled primer DP1 (Fig.1B) was hybridized with 15 µg of
poly(A)
RNA from etiolated Arabidopsis seedlings treated with 20 µM IAA and 50 µM CHX for 2 h, and extended with reverse transcriptase (lane
1). Lanes 2-5 are reactions G, A, T, and C,
respectively. The arrow and bold residue represent
the major start site of transcription.
Other Molecular Techniques
Standard molecular
techniques were performed according to Sambrook et
al.(26) .
RESULTS AND DISCUSSION
Isolation and Structural Characterization of the ACS4
Gene
Screening of a
DASH Arabidopsis genomic library
with the ptACS2 cDNA of tomato (6) as a probe under low
stringency hybridization conditions resulted in the isolation of
genomic clones for five ACC synthase genes ACS1-5(10) . The structure and expression
characteristics of ACS2 have been reported(10) . In
this study, we present the structural characterization of the
auxin-responsive gene ACS4 using the previously isolated
AT-8 genomic clone(10) . The 8.3-kb EcoRI fragment of
the
AT-8 clone (Fig.1A) hybridizes to a specific
PCR-generated sequence corresponding to the TZ region of ACC
synthases(8, 10) , indicating that this fragment
contains the ACS4 gene(10) . However, determination of
the orientation of ACS4 by PCR in plasmid pAAA1 reveals that
the 8.3-kb EcoRI fragment contains only part of the gene,
coding for the N-terminal half of ACS4 (Fig.1A). The
2.1-kb EcoRI fragment of
AT-8 in pAAA3 codes for the
C-terminal region and contains 3`-nontranslated sequences of ACS4 (Fig.1A). To verify the immediate contiguity of
both EcoRI fragments, an overlapping fragment was generated by
PCR using
AT-8 phage DNA as the template. The sequence of the
subcloned fragment in pAAA5 is identical with flanking sequences of
pAAA1 and pAAA3 inserts and excludes the possibility of an additional,
closely positioned EcoRI site (Fig.1A). The
sequence of 3438 nt of the ACS4 genomic locus has been
determined and is shown in Fig.1B. The gene consists
of four exons and three introns. The sequence also includes 1.3 kb of
the 5`-flanking region and 0.5 kb of the 3`-nontranslated region. The
predicted coding region of the ACS4 gene consists of 1,422
base pairs. The intron/exon junctions which are typical of donor and
acceptor splice sites (27) have been established by reference
to the sequence of ACS4 cDNA (see below). The number and size
of exons and the location of introns are similar to other ACS genes isolated from zucchini, tomato, rice, and Arabidopsis(5, 6, 8, 10) .
However, the zucchini twin genes CP-ACS1A/1B have five exons (5) , and LE-ACS4 from tomato and VR-ACS4 and VR-ACS5 from mung bean contain only three
exons(6, 7) .
Determination of the Transcription Initiation Site
The start site of transcription was determined by primer
extension analysis using reverse transcriptase and the primer DP1 that
is complimentary to the 5`-end of the ACS4 mRNA (Fig.2). One major primer extension product of 174 nucleotides
was obtained with poly(A)
RNA from auxin-treated
etiolated Arabidopsis seedlings. These data define the size of
the 5`-nontranslated region of ACS4 mRNA to be 124 nucleotides
long (Fig.1B, Fig. 2). The sequence at position
-35 to -29, TATATAA, qualifies as a TATA box (27) ,
and a CAAT sequence (27) is present further upstream at
position -122 to -119 (Fig.1B). The ACS4 promoter contains four sequence motifs reminiscent of
functionally defined cis-elements of early genes regulated by
auxin in a primary fashion, PS-IAA4/5 from pea (28, 29) and GH3 from soybean (30) (Fig.1B). A comparison in Fig. 3A shows that the sequence motif at position
-411 to -404 of ACS4 resembles the
auxin-responsive element (AuxRE) of the auxin-responsive
domain A in PS-IAA4/5 from pea(28, 29) . An AuxRD A element is present in the promoters of a number of
early auxin-responsive genes, including PS-IAA4/5-related
genes from various plant species, SAUR genes from soybean, OS-ACS1 from rice, T-DNA gene 5 and rol b/c from Agrobacterium(31) . Noteworthy, nucleotides flanking
the AuxRD A-like sequence in ACS4 and rol b/c are remarkably conserved (Fig.3A). Two sequence
motifs of ACS4, at position -462 to -448 and at
position -372 to -359, display a high degree of identity
with the AuxRD B element of the auxin-responsive domain in PS-IAA4/5 and related genes (28, 29, 31) (Fig.3B). The AuxRD B motif functions as an auxin-specific enhancer element
in PS-IAA4/5(28, 29) . In addition, a third
sequence element at position -285 to -271 of ACS4 is strikingly similar to a motif conserved in two independently
acting auxin-responsive elements of the soybean GH3 promoter(30) . The presence of putative auxin-responsive
elements in ACS4 with similarities to functionally defined cis-elements in other early auxin-regulated genes suggests, at
least in part, utilization of analogous trans-acting factors
for signaling auxin-mediated ACS4 gene activation.
Figure 3:
Putative auxin-responsive elements in the
promoter of ACS4. Sequence motifs of the ACS4 promoter (underlined in Fig.1B) are
compared with functionally defined auxin-responsive cis-elements in PS-IAA4/5 from pea (28, 29) and GH3 from soybean(30) .
In addition, similar motifs of selected, early auxin-responsive genes
are compared. Nucleotides conserved between ACS4 sequence
motifs and functionally characterized as well as putative
auxin-responsive elements are shaded. A, comparison with the AuxRD A of PS-IAA4/5(28, 29) . B, comparison with the AuxRD B of PS-IAA4/5(28, 29) . C, comparison
with the conserved motif in two functionally characterized
auxin-responsive elements in GH3(30) . The source of
the sequences is the alignment by Oeller et al.(31) and, for GH3, the report by Li et
al.(30) .
Isolation of ACS4 cDNAs
An ACS4 cDNA was obtained by reverse
transcription-coupled PCR amplification using primers corresponding to
deduced nontranslated genomic sequences and poly(A)
RNA from auxin-treated etiolated Arabidopsis seedlings
as the template. The sequence of the PCR-generated ACS4 cDNA
in pAAA6 comprises 1,422 base pairs and is identical with the deduced
coding region of the genomic ACS4 clone. To determine the
3`-end of the ACS4 gene, the insert of pAAA6 was used as a
probe to screen two Arabidopsis cDNA
libraries(32, 33) . After screening of approximately
800,000 plaques, two hybridizing ACS4 cDNA clones were
purified. However, both cDNAs do not contain poly(A) tails. A second
approach using inverse PCR to clone flanking ACS4 cDNA
sequences repeatedly failed for unknown reasons. Nonetheless, we note
two potential polyadenylation signals, ATTAAA, approximately 210 base
pairs downstream of the translational stop codon. The ACS4 mRNA is predicted to be about 1,800 nt long (5`-untranslated
region, 124 nt; coding region, 1,422 nt; 3`-untranslated region,
250 nt), close to the size of 1,850 nt detected by RNA
hybridization analysis (data not shown).
Properties of ACS4 and Expression in E. coli
Conceptual translation of the coding region of ACS4 mRNA yields a polypeptide of 474 amino acid residues (53,795 Da,
pI 8.2), close to the size of other ACC synthase isoenzymes from
various plant species(34) . The ACS4 protein also contains all
the conserved regions found in other ACS isoenzymes as well as the 11
invariant amino acids conserved between ACC synthases and
aminotransferases (Fig.1B). The dodecapeptide sequence
characteristic of the pyridoxal phosphate binding site is also present (Fig.1B). The ACS4 amino acid identity to other ACS
isoenzymes varies from 49 to 73%(35) . ACS4 is most similar in
primary sequence to Arabidopsis ACS5, winter squash CM-ACS2,
apple MS-ACS1, and to tomato LE-ACS3(35) . Together with the
rice OS-ACS1(8) , these ACS isoenzymes comprise a major lineage
in the ACC synthase phylogenetic tree(35) . Interestingly, most
of the genes in this lineage are auxin regulated in vegetative tissues
indicating a striking correlation between their phylogenetic
relationship and their pattern of expression (8, 35) . Authenticity of the polypeptide encoded by ACS4 mRNA was
confirmed by expression experiments in E. coli. Plasmid pAAA6
contains in pUC19 a PCR-generated cDNA corresponding to the coding
region of ACS4 mRNA, fused to the N terminus of the
-galactosidase gene in sense orientation. Transformants bearing
the pAAA6 plasmid produce 2.8 nmol of ACC/10
cells after 4
h of incubation in the presence of 1 mM
isopropyl-
-D-thiogalactoside. E. coli cells
containing only pUC19 do not accumulate detectable ACC.
Specificity of ACS4 mRNA Accumulation
We have previously shown that ACS4 is differentially
expressed in mature Arabidopsis plants. ACS4 mRNA is
detectable by RNA blot analysis in roots, leaves, and flowers
only(10) . Interestingly, ACS4 transcript accumulation
is inducible by auxin. In this study, we have analyzed the auxin
response of ACS4 in detail.
Response to IAA and CHX
The effect of IAA and of the
protein synthesis inhibitor CHX was examined (Fig.4). Treatment
of 5-day-old intact etiolated Arabidopsis seedlings with 20
µM IAA for 1 h increases the steady-state ACS4 mRNA level about 10-fold, relative to untreated or mock-treated
seedlings (compare lane 3 with lanes 1 or 2). The response to auxin is highly potentiated (about
50-fold) by 50 µM CHX (compare lane 3 with lane 5). Application of 50 µM CHX alone has the
same effect (compare lane 3 with lane 4). At this
concentration, CHX effectively prevents protein biosynthesis in Arabidopsis seedlings (data not shown). These results qualify ACS4 as a primary response gene which activation is
independent of de novo protein synthesis. Induction of gene
expression by protein synthesis inhibition can be explained by
transcriptional activation via depletion of a short-lived repressor
polypeptide and/or by mRNA stabilization(13, 36) .
Both effects have been described for CHX-mediated PS-IAA4/5 gene activation. (
)
Figure 4:
Specificity of ACS4 mRNA
expression. Total RNA (25 µg) from 6-day-old etiolated seedlings
treated for 1 h with various chemicals and conditions, if not otherwise
indicated, were hybridized with the
P-labeled DNA insert
of pAAA6. The lanes are: 1, untreated; 2 and 13, control-treated; 3 and 14, 20 µM IAA; 4, 50 µM CHX; 5, 20 µM IAA + 50 µM CHX after 30 min of pretreatment
with 50 µM CHX only; 6, 20 µM 2,4-D; 7, 20 µM
-NAA; 8, 20 µM PAA; 9, 20 µML-tryptophane; 10, wounding; 11, 0.5 M sorbitol; 12, heat treatment at 42 °C for 15 min followed by 45 min
recovery at room temperature; 15, 20 µM abscisic
acid; 16, 20 µM gibberellic acid; 17, 20
µM BA; 18, 20 µM IAA + 20
µM BA; 19, 20 µM IAA + 20
µM BA + 50 mM LiCl; 20, 50 mM LiCl; 21, 10 parts/million ethylene; 22,
N
; 23, air control.
Effect of IAA Analogs
The effect of
various IAA analogs on steady-state levels of ACS4 mRNA is
shown for 20 µM 2,4-D (lane 6), 20 µM
-NAA (lane 7), 20 µM PAA (lane
8), and 20 µML-tryptophane (lane
9). As compared with the response to 20 µM IAA
(compare lane 1 or lane 2 with lane 3; or lane 13 with lane 14), the synthetic auxin 2,4-D and
the natural auxin
-NAA are similar effective to induce
accumulation of ACS4 transcripts. The weak natural auxin PAA
is less effective, and the structural analog tryptophane has no effect.
Effect of Other Plant Hormones
Other plant
hormones such as 20 µM abscisic acid (lane 15),
20 µM gibberellic acid (lane 16), 20 µM BA (lane 17), or 10 parts/million ethylene (lane
21) do not increase ACS4 mRNAs above control levels (as
compared with mock treatment in lane 13, or with an air
control in lane 23 for the ethylene treatment). Furthermore, a
combination of 20 µM IAA with 20 µM BA
neither augments nor attenuates the response to auxin (compare lane
13 with lane 18).
Effect of Various Stress Conditions
The effect of
several stress conditions on ACS4 gene expression was studied.
Wounding (lane 10), osmotic shock (lane 11), heat
shock (lane 12), and anaerobiosis (lane 23) fail to
induce ACS4 transcript accumulation. LiCl has no effect,
neither when applied alone (lane 20) nor in combination with
20 µM IAA and 20 µM BA (lane 19).
Taken together, these results are very similar to the expression
profile of early auxin-inducible IAA genes of Arabidopsis(
)and demonstrate specificity of ACS4 gene
expression for auxin and protein synthesis inhibitors such as CHX. A
similar expression characteristics has been described for CM-ACS2 from winter squash(12) , OS-ACS1 from
rice(8) , and for auxin-inducible LE-ACS genes from
tomato. (
)
Kinetics of ACS4 mRNA Accumulation
We monitored ACS4 mRNA accumulation in intact
etiolated seedlings for up to 8 h after addition of 20 µM IAA (Fig.5). The ACS4 gene responds rapidly to
exogenous auxin by increasing transcript levels within the first 30 min
of treatment (Fig.5A). ACS4 mRNA accumulate
steadily and reach a 20-fold higher steady-state level after 8 h of
exposure to IAA (Fig.5A). A short time course
experiment reveals first detectable increases in ACS4 mRNA
after 25 min of auxin treatment (Fig.4B). A comparable
induction kinetics in response to IAA has been reported for CM-ACS2 which transcripts start to accumulate 20 min after auxin addition (12) and for the auxin-inducible LE-ACS genes in
tomato.
Figure 5:
Kinetics of ACS4 mRNA
accumulation in response to IAA. Intact etiolated Arabidopsis seedlings (5 day old) were incubated in the presence of 20
µM IAA. After the indicated periods, total RNA was
isolated and separated in a 1% agarose gel (25 µg for each time
point), transferred to a nylon membrane, and hybridized with the
P-labeled DNA insert of pAAA6 or with a
P-labeled 17 S rDNA insert. The results are shown
graphically relative to the mRNA level of the zero time control
(arbitrary value of ``1''). The original autoradiogram is
also shown.
Dose Response to IAA
Next, we studied the effect of different IAA concentrations
on the expression of ACS4. A dose-response curve ranging from
1
10
to 5
10
M of IAA was obtained after an auxin exposure of intact
seedlings for 2 h (Fig.6). The ACS4 dose-response
curve shows a characteristic bimodal shape. Optimal ACS4 transcript accumulation occurs at 1
10
M of IAA. Higher IAA concentrations are suboptimal, and
IAA at 5
10
M completely inhibits ACS4 mRNA accumulation. However, IAA concentrations as low as
100 nM are sufficient to elevate basal ACS4 mRNA
levels. A similar biphasic dose-response curve has been described for a
number of other primary auxin-responsive genes, such as IAA genes in Arabidopsis seedlings,
PS-IAA4/5 in pea protoplasts(28) , GH1 in soybean
seedlings(38) , or the SAUR promoter-GUS transgene in
tobacco(39) . Interestingly, the bimodal response of
auxin-induced gene expression is paralleled by biphasic characteristics
of other auxin-elicited processes such as stem elongation, stomatal
openening, or potassium influx into stomatal guard cells(40) .
Figure 6:
Dose-response of ACS4 to IAA.
Intact etiolated Arabidopsis seedlings (5 day old) were
treated with various concentrations of IAA for 2 h. Total nucleic acids
were isolated and separated in a 1% agarose gel (25 µg for each IAA
concentration), transferred to a nylon membrane, and hybridized with
the
P-labeled DNA insert of pAAA6 or with a
P-labeled 17 S rDNA insert. The results are shown
graphically relative to the mRNA level of the no auxin control
(arbitrary value of ``1''). The original autoradiogram is
also shown.
Expression of ACS4 in Auxin-resistant Mutants
Expression of ACS4 was studied in the Arabidopsis auxin-resistant mutant lines axr1-12(16) , axr2-1(17) , and aux1-7(18) . These mutants have a pleiotropic
though auxin-related phenotype and are cross-resistant to several other
plant hormones, including ethylene (41) . Intact etiolated wild
type and mutant seedlings were treated in the presence or absence of 20
µM IAA for 2 h (Fig.7). Relative to the expression
in mock-treated and auxin-treated wild type tissue, steady-state ACS4 mRNA levels are severely reduced (greater than 10-fold)
in respectively treated axr1-12 and axr2-1 seedlings. In contrast to axr1-12 seedlings, auxin
inducibility of ACS4 mRNA accumulation in axr2-1 plants is abolished. Unlike in the axr1-12 and axr2-1 mutant lines, ACS4 gene expression is
only modestly affected in aux1-7 seedlings (Fig.7). ACS4 transcript levels are 1.5-fold reduced,
however, auxin inducibility is retained. A similar defective expression
in these three mutant lines has been described for members of the IAA multigene family
and for the SAUR-AC1 gene(42, 43) . Inhibition of three classes of
genes with different auxin-responsive elements supports the previous
notion that the mutations act early in an auxin reponse pathway and
probably affect general components in hormone signaling(41) .
In view of the specificity of ACS4 induction for auxin,
defective expression in auxin-resistant mutant plants suggests a role
of ACS4 in auxin action and demonstrates the interrelationship
of both plant hormones. This interpretation is corroborated by the
observation that expression of auxin-regulated LE-ACS genes is
defective in the diageotropica mutant of tomato.
The diageotropica mutant expresses only low levels of an
auxin-binding protein which presumably is an auxin
receptor(44) .
Figure 7:
Expression of ACS4 mRNA in
etiolated auxin-resistant seedlings. Intact etiolated Arabidopsis seedlings (5 day old), wild type or the mutant lines axr1-12, axr2-1, or aux1-7, were incubated in the presence (+) or
absence(-) of 20 µM IAA for 2 h. Total RNA was
isolated, separated (25 µg) in a 1% agarose gel, transferred to a
nylon membrane, and hybridized with the
P-labeled DNA
insert of pAAA6 or with a
P-labeled 17 S rDNA
insert.
CONCLUSIONS
We have shown that ACS4 of A. thaliana is an early auxin-responsive gene which is activated by the
hormone in a primary fashion. The expression characteristics of ACS4 are intriguingly similar with the hormonal response of
early IAA genes in Arabidopsis.
Consistently with this observation is the presence of four
putative auxin-responsive cis-elements in the ACS4 promoter that are similar to functionally characterized cis-elements of early auxin-inducible
genes(28, 29, 30, 31) . The
specificity of induction for auxin treatment and the defective
expression of ACS4 in auxin-resistant Arabidopsis mutants qualify ACS4 as a probe to study molecular
aspects of the intimate interrelationship of auxin and ethylene action.