ASC4, a Primary Indoleacetic Acid-responsive Gene Encoding 1-Aminocyclopropane-1-carboxylate Synthase in Arabidopsis thaliana
1995; Elsevier BV; Volume: 270; Issue: 32 Linguagem: Inglês
10.1074/jbc.270.32.19093
ISSN1083-351X
AutoresSteffen Abel, Minh Dang Nguyen, William Chow, Athanasios Theologis,
Tópico(s)Plant Gene Expression Analysis
Resumo1-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. 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. Ethylene, a major phytohormone, is one of the simplest organic molecules with biological activity and controls many aspects of plant growth and development(1Abeles F.B. Morgan P.W. Saltveit Jr., M.E. Ethylene in Plant Biology. Academic Press, New York1992: 1-296Crossref Google Scholar, 2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar). 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(2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar). 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(2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar). Ethylene serves as a signaling molecule to initiate and coordinate profound physiological changes and adaptations throughout the life cycle of a plant(1Abeles F.B. Morgan P.W. Saltveit Jr., M.E. Ethylene in Plant Biology. Academic Press, New York1992: 1-296Crossref Google Scholar, 2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar). Ethylene biosynthesis is stringently regulated during plant development (2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar, 3Kende H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 283-307Crossref Scopus (1030) Google Scholar). The rate-limiting reaction catalyzed by the enzyme ACC 1The abbreviations used are: ACC1-aminocyclopropane-1-carboxylic acidIAAindole-3-acetic acidACSACC synthase2,4-D2,4-dichlorophenoxyacetic acidPAAphenylacetic acidα-NAAnaphthalene-1-acetic acidBAbenzyl adenineCHXcycloheximideMES2-(N-morpholino)ethanesulfonic acidntnucleotidesPCRpolymerase chain reactionkbkilobase(s). 1The abbreviations used are: ACC1-aminocyclopropane-1-carboxylic acidIAAindole-3-acetic acidACSACC synthase2,4-D2,4-dichlorophenoxyacetic acidPAAphenylacetic acidα-NAAnaphthalene-1-acetic acidBAbenzyl adenineCHXcycloheximideMES2-(N-morpholino)ethanesulfonic acidntnucleotidesPCRpolymerase chain reactionkbkilobase(s). 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(2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar, 3Kende H. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 283-307Crossref Scopus (1030) Google Scholar). ACC synthase is a short-lived cytosolic enzyme (4Kim W.T. Yang S.F. Plant Physiol. 1992; 100: 1126-1131Crossref PubMed Scopus (36) Google Scholar) and is encoded by a highly divergent multigene family in a number of plant species including zucchini(5Huang P.L. Parks J.E. Rottmann W.H. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7021-7025Crossref PubMed Scopus (108) Google Scholar), tomato(6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar), mung bean(7Botella J.R. Schlagenhauser C.D. Arteca J.M. Arteca R.N. Phillips A.T. Gene (Amst.). 1993; 123: 249-253Crossref PubMed Scopus (42) Google Scholar), rice(8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar), and Arabidopsis thaliana(9Van Der Straeten D. Rodrigues-Pousada R.A.R. Villarroel R. Hanley S. Goodman H.M. Van Montagu M. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 9969-9973Crossref PubMed Scopus (85) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). 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(5Huang P.L. Parks J.E. Rottmann W.H. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7021-7025Crossref PubMed Scopus (108) Google Scholar, 6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar, 7Botella J.R. Schlagenhauser C.D. Arteca J.M. Arteca R.N. Phillips A.T. Gene (Amst.). 1993; 123: 249-253Crossref PubMed Scopus (42) Google Scholar, 8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar, 9Van Der Straeten D. Rodrigues-Pousada R.A.R. Villarroel R. Hanley S. Goodman H.M. Van Montagu M. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 9969-9973Crossref PubMed Scopus (85) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). For instance, the plant hormone auxin, typified by IAA, is a known inducer of ethylene production (2Yang S.F. Hoffman B.E. Annu. Rev. Plant Physiol. 1984; 35: 155-189Crossref Google Scholar) and regulates specific members of each ACS multigene family in a tissue-specific manner(5Huang P.L. Parks J.E. Rottmann W.H. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7021-7025Crossref PubMed Scopus (108) Google Scholar, 8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar, 11Nakajima N. Mori H. Imaseki H. Plant Cell Physiol. 1990; 31: 1021-1029Google Scholar, 12Nakagawa N. Mori H. Yamazaki K. Imaseki H. Plant Cell Physiol. 1991; 32: 1153-1163Google Scholar). 1-aminocyclopropane-1-carboxylic acid indole-3-acetic acid ACC synthase 2,4-dichlorophenoxyacetic acid phenylacetic acid naphthalene-1-acetic acid benzyl adenine cycloheximide 2-(N-morpholino)ethanesulfonic acid nucleotides polymerase chain reaction kilobase(s). 1-aminocyclopropane-1-carboxylic acid indole-3-acetic acid ACC synthase 2,4-dichlorophenoxyacetic acid phenylacetic acid naphthalene-1-acetic acid benzyl adenine cycloheximide 2-(N-morpholino)ethanesulfonic acid nucleotides polymerase chain reaction kilobase(s). 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(6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar, 8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). Protein synthesis inhibitors such as CHX have been widely used as a tool to unmask regulatory mechanisms of early gene activation(13Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (942) Google Scholar). Likewise, the lithium ion is known to interfere with phosphatidylinositol metabolism and signaling(14Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3287) Google Scholar). 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(6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar), rice(8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar), and A. thaliana(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). We are attempting to develop the molecular genetic approaches in Arabidopsis, a model organism for a flowering plant(15Meyerowitz E.M. Cell. 1989; 56: 263-269Abstract Full Text PDF PubMed Scopus (225) Google Scholar). We have previously identified an auxin-regulated ACS gene in A. thaliana, ACS4(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). Here, we report its structure and specific expression characteristics in response to IAA. The following Arabidopsis strains were used: wild type A. thaliana (L.) Heynh. ecotype Columbia, and auxin-resistant mutant lines axr1-12(16Lincoln C. Britton J. Estelle M. Plant Cell. 1990; 2: 1071-1080Crossref PubMed Scopus (548) Google Scholar), axr2-1(17Pickett F.B. Wilson A.K. Estelle M. Plant Physiol. 1990; 94: 1462-1466Crossref PubMed Scopus (245) Google Scholar), and aux1-7(18Wilson A.K. Pickett F.B. Turner J. Estelle M. Mol. & Gen. Genet. 1990; 222: 377-383Crossref PubMed Scopus (362) Google Scholar). 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. 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 N2 after decanting the bathing solution and stored at −80°C. The following recombinant clones were used in this study: pAAA1 contains the 8.3-kb EcoRI fragment of λAT-8 in pUC18. λAT-8 is an Arabidopsis genomic clone containing the ACS4 gene(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). 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 λAT-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 λAT-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(19Jones M.D. Methods Enzymol. 1993; 218: 413-419Crossref PubMed Scopus (13) Google Scholar). The single-stranded cDNAs were used as the template in a PCR (20Compton T. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, New York1990: 39-45Google Scholar) 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). Dideoxy sequencing of double-stranded DNA of pAAA plasmids was performed with universal and synthetic primers using [35S]dATP (21Sanger F. Nicken S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52232) Google Scholar) 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). Plasmid pAAA6 was introduced into Escherichia coli DH5α and M15[pREP4]. Expression conditions and measurements of ACC formation were as described previously(6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar). 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%β-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.(22Theologis A. Huynh T.V. Davis R.W. J. Mol. Biol. 1985; 183: 53-68Crossref PubMed Scopus (236) Google Scholar). Northern analysis was essentially performed according to Ecker and Davis(23Ecker J.R. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5202-5206Crossref PubMed Google Scholar). 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 (Mr = 360,000), 0.02% Ficoll (Mr = 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 (Mr 500,000) and the appropriate radioactively labeled probe. Probes were prepared from DNA restriction fragments by the random hexamer-primed synthesis method (24Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16562) Google Scholar) to a specific activity of ~1.0 × 109 counts/min/μg. Hybridizations were carried out with radiolabeled probes of 2 × 106 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 was performed essentially according to the method of Boorstein and Craig(25Boorstein W. Craig E. Methods Enzymol. 1989; 180: 347-368Crossref PubMed Scopus (141) Google Scholar). Approximately 5 × 104 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. Standard molecular techniques were performed according to Sambrook et al.(26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Screening of a λDASH Arabidopsis genomic library with the ptACS2 cDNA of tomato (6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar) as a probe under low stringency hybridization conditions resulted in the isolation of λ genomic clones for five ACC synthase genes ACS1-5(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). The structure and expression characteristics of ACS2 have been reported(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). In this study, we present the structural characterization of the auxin-responsive gene ACS4 using the previously isolated λ AT-8 genomic clone(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). The 8.3-kb EcoRI fragment of the λ AT-8 clone (Fig. 1A) hybridizes to a specific PCR-generated sequence corresponding to the TZ region of ACC synthases(8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar), indicating that this fragment contains the ACS4 gene(10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). 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 λ 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 λ 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 (27Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3283) Google Scholar) 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(5Huang P.L. Parks J.E. Rottmann W.H. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7021-7025Crossref PubMed Scopus (108) Google Scholar, 6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar, 8Zarembinski T.I. Theologis A. Mol. Biol. Cell. 1993; 4: 363-373Crossref PubMed Scopus (111) Google Scholar, 10Liang X.-W. Abel S. Keller J.A. Shen N.F. Theologis A. Proc. Natl. Acad. Sci U. S. A. 1992; 89: 11046-11050Crossref PubMed Scopus (186) Google Scholar). However, the zucchini twin genes CP-ACS1A/1B have five exons (5Huang P.L. Parks J.E. Rottmann W.H. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7021-7025Crossref PubMed Scopus (108) Google Scholar), and LE-ACS4 from tomato and VR-ACS4 and VR-ACS5 from mung bean contain only three exons(6Rottmann W.H. Peter G.F. Oeller P.W. Keller J.E. Shen N.F. Nagy B.P. Taylor L.P. Campbell A.D. Theologis A. J. Mol. Biol. 1991; 222: 937-961Crossref PubMed Scopus (262) Google Scholar, 7Botella J.R. Schlagenhauser C.D. Arteca J.M. Arteca R.N. Phillips A.T. Gene (Amst.). 1993; 123: 249-253Crossref PubMed Scopus (42) Google Scholar). 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 (27Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3283) Google Scholar), and a CAAT sequence (27Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3283) Google Scholar) 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 (28Ballas N. Wong L.-M. Theologis A. J. Mol. Biol. 1993; 233: 580-596Crossref PubMed Scopus (133) Google Scholar, 29Ballas N. Wong L.-M. Ke M. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3483-3487Crossref PubMed Scopus (76) Google Scholar) and GH3 from soybean (30Liu Z.-B. Ulmasov T. Shi X. Hagen G. Guilfoyle T.J. Plant Cell. 1994; 6: 645-657Crossref PubMed Scopus (203) Google Scholar) (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(28Ballas N. Wong L.-M. Theologis A. J. Mol. Biol. 1993; 233: 580-596Crossref PubMed Scopus (133) Google Scholar, 29Ballas N. Wong L.-M. Ke M. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3483-3487Crossref PubMed Scopus (76) Google Scholar). 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(31Oeller P.W. Keller J.A. Parks J.E. Silbert J.E. Theologis A. J. Mol. Biol. 1993; 233: 789-798Crossref PubMed Scopus (69) Google Scholar). 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 (28Ballas N. Wong L.-M. Theologis A. J. Mol. Biol. 1993; 233: 580-596Crossref PubMed Scopus (133) Google Scholar, 29Ballas N. Wong L.-M. Ke M. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3483-3487Crossref PubMed Scopus (76) Google Scholar, 31Oeller P.W. Keller J.A. Parks J.E. Silbert J.E. Theologis A. J. Mol. Biol. 1993; 233: 789-798Crossref PubMed Scopus (69) Google Scholar) (Fig. 3B). The AuxRD B motif functions as an auxin-specific enhancer element in PS-IAA4/5(28Ballas N. Wong L.-M. Theologis A. J. Mol. Biol. 1993; 233: 580-596Crossref PubMed Scopus (133) Google Scholar, 29Ballas N. Wong L.-M. Ke M. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3483-3487Crossref PubMed Scopus (76) Google Scholar). 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(30Liu Z.-B. Ulmasov T. Shi X. Hagen G. Guilfoyle T.J. Plant Cell. 1994; 6: 645-657Crossref PubMed Scopus (203) Google Scholar). 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 (28Ballas N. Wong L.-M. Theologis A. J. Mol. Biol. 1993; 233: 580-596Crossref PubMed Scopus (133) Google Scholar, 29Ballas N. Wong L.-M. Ke M. Theologis A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3483-3487Crossref PubMed Scopus (76) Google Scholar) and GH3 from soybean(30Liu Z.-B. Ulmasov T. Shi X. Hagen G. Guilfoyle T.J. Plant Cell. 1994; 6: 645-657Crossref PubMed Scopus (203) Google Scholar). In addition, similar motifs of selected, early auxin-responsive genes are compared. Nucleotides conserved
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