©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ASC4, a Primary Indoleacetic Acid-responsive Gene Encoding 1-Aminocyclopropane-1-carboxylate Synthase in Arabidopsis thaliana
STRUCTURAL CHARACTERIZATION, EXPRESSION IN ESCHERICHIA COLI, AND EXPRESSION CHARACTERISTICS IN RESPONSE TO AUXIN (*)

(Received for publication, April 18, 1995)

Steffen Abel (§) Minh D. Nguyen William Chow Athanasios Theologis (¶)

From thePlant Gene Expression Center, Albany, California 94710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J. A., Shen, N. F., and Theologis, A.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 µM of IAA. However, IAA concentrations as low as 100 nM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1.

The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.


INTRODUCTION

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 (^1)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(2) 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 DH5alpha 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% beta-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(r) = 360,000), 0.02% Ficoll (M(r) = 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(r) 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 10^9 counts/min/µg. Hybridizations were carried out with radiolabeled probes of 2 10^6 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 10^4 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 beta-galactosidase gene in sense orientation. Transformants bearing the pAAA6 plasmid produce 2.8 nmol of ACC/10^8 cells after 4 h of incubation in the presence of 1 mM isopropyl-beta-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. (^2)


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 alpha-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(2); 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 alpha-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 alpha-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(^3)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. (^4)

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.^4


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 10M 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 10M of IAA. Higher IAA concentrations are suboptimal, and IAA at 5 10M 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,^3PS-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^3 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.^4 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.^3 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.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9316475 (to A. T.) and United States Department of Agriculture Grant 5335-21430-003-00D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U23482[GenBank], ACS4 (genomic) and U23481[GenBank], patACS4 (cDNA).

§
Supported by postdoctoral fellowships from Boehringer Ingelheim Fonds, Germany, and United States Department of Agriculture Grant 5335-21430-002-00D.

To whom correspondence should be addressed: Tel.: 510-559-5911; Fax: 510-559-5678; Theo{at}mendel.berkeley.edu.

^1
The abbreviations used are: ACC, 1-aminocyclopropane-1-carboxylic acid; IAA, indole-3-acetic acid; ACS, ACC synthase; 2,4-D, 2,4-dichlorophenoxyacetic acid; PAA, phenylacetic acid; alpha-NAA, naphthalene-1-acetic acid; BA, benzyl adenine; CHX, cycloheximide; MES, 2-(N-morpholino)ethanesulfonic acid; nt, nucleotides; PCR, polymerase chain reaction; kb, kilobase(s).

^2
T. Koshiba and A. Theologis, unpublished data.

^3
Abel, S., Nguyen, M., and Theologis, A. (1995) J. Mol. Biol., in press.

^4
K. Kawakita and A. Theologis, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Mark Estelle for seeds of auxin-resistant mutant lines, Tom Zarembinski for preparation of Fig.1B, and Ron Wells for editing the manuscript.


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