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• W1964226250 abstract "The use of 35S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated asZmSAUR1 (Zea mays SAURs) was expressed inEscherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH2-terminal domain. A synthetic peptide of amino acids 20–45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K d ∼15 nm) in a calcium-dependent manner. Comparison of the NH2-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic α-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH2-terminal regions ofArabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 μm α-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonistN-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction. The use of 35S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated asZmSAUR1 (Zea mays SAURs) was expressed inEscherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH2-terminal domain. A synthetic peptide of amino acids 20–45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K d ∼15 nm) in a calcium-dependent manner. Comparison of the NH2-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic α-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH2-terminal regions ofArabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 μm α-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonistN-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction. calmodulin α-naphthaleneacetic acid polyacrylamide gel electrophoresis polymerase chain reaction small auxin up RNA N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride Plant hormone auxin plays a central role in growth and development by controlling cell division, cell elongation, and cell differentiation (1.Hardin J.W. Cherry J.H. Morre D.J. Lembi C.A. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3146-3150Crossref PubMed Scopus (49) Google Scholar, 2.Guilfoyle T.J. CRC Crit. Rev. Plant Sci. 1986; 4: 247-276Crossref Scopus (92) Google Scholar, 3.Hobbie L. Timpte C. Estelle M. Plant Mol. Biol. 1994; 26: 1499-1519Crossref PubMed Scopus (71) Google Scholar, 4.Abel S. Theologis A. Plant Physiol. 1996; 111: 9-17Crossref PubMed Scopus (584) Google Scholar). Auxin-induced cell elongation, one of the fastest hormonal responses known, has been used widely as a model system to study the mechanism of auxin action (5.Ray P.M. Recent Adv. Phytochem. 1974; 7: 93-122Crossref Scopus (19) Google Scholar, 6.Evans M.L. Annu. Rev. Plant Physiol. 1974; 25: 195-223Crossref Google Scholar, 7.Evans M.L. CRC Crit. Rev. Plant Sci. 1985; 2: 317-365Crossref PubMed Scopus (89) Google Scholar). A vast array of cellular responses to external and internal stimuli such as light and hormones involves Ca2+ as a second messenger (8.Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1987; 6: 47-103Crossref PubMed Scopus (278) Google Scholar, 9.Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1993; 12: 185-211Crossref PubMed Scopus (328) Google Scholar, 10.Bush D.S. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 95-122Crossref Scopus (903) Google Scholar, 11.Trewavas A.J. Malho R. Plant Cell. 1997; 9: 1181-1195Crossref PubMed Scopus (210) Google Scholar, 12.Trewavas A. Plant Physiol. 1999; 120: 428-433Crossref Scopus (150) Google Scholar). Calmodulin (CaM),1 a ubiquitous Ca2+-binding protein in eukaryotes, is a primary intracellular Ca2+ receptor that transduces the second messenger Ca2+ signal by binding to and altering the activity of the variety of other proteins (9.Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1993; 12: 185-211Crossref PubMed Scopus (328) Google Scholar, 13.Roberts D.M. Harmon A.C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 375-414Crossref Scopus (567) Google Scholar, 14.Snedden W.A. Fromm H. Trends Plant Sci. 1998; 3: 299-304Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 15.Zielinski R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 697-725Crossref PubMed Scopus (388) Google Scholar). Recent evidence indicates that there is a close relationship between the mechanism of auxin action and calcium signaling, but the interaction between them has been controversial and is still unresolved (3.Hobbie L. Timpte C. Estelle M. Plant Mol. Biol. 1994; 26: 1499-1519Crossref PubMed Scopus (71) Google Scholar, 16.Reddy A.S.N. Koshiba T. Theologis A. Poovaiah B.W. Plant Cell Physiol. 1988; 29: 1165-1170Google Scholar). The effect of auxin on changes in cellular calcium levels has been obtained using calcium-sensitive fluorescent dyes or Ca2+-sensitive microelectrodes. Felle (17.Felle H. Planta. 1988; 174: 495-499Crossref PubMed Scopus (203) Google Scholar) reported a decrease in free calcium in cells after auxin treatment, whereas Gehring et al. (18.Gehring C.A. Irving H.R. Parish R.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9645-9649Crossref PubMed Scopus (194) Google Scholar) observed an increase in calcium levels after auxin treatment. Depletion of calcium in tissues using calcium chelators and CaM inhibitors has implicated a role for calcium in the auxin signal transduction. Raghothama et al. (19.Raghothama K.G. Mizrahi Y. Poovaiah B.W. Plant Physiol. 1985; 79: 28-33Crossref PubMed Google Scholar) found that CaM antagonists such as chlorpromazine, trifluoperazine, fluphenazine, and W-7 inhibited the auxin-induced elongation of oat and corn coleoptile segments. Gonzalez-Daros et al. (20.Gonzalez-Daros F. Carrasco-Luna J. Calatayud A. Salguero J. del Valle-Tascon S. Physiol. Plant. 1993; 87: 68-76Crossref Scopus (7) Google Scholar) observed that some, but not all, CaM inhibitors tested could inhibit auxin-induced medium acidification by oat coleoptile segments. Similarly, Reddy et al. (16.Reddy A.S.N. Koshiba T. Theologis A. Poovaiah B.W. Plant Cell Physiol. 1988; 29: 1165-1170Google Scholar) observed that the calcium chelator EGTA and calcium channel blocker D-600 inhibited auxin-induced elongation of pea epicotyl segments. Auxin has been linked with Ca2+ transport (21.Kubowicz B.D. Vanderhoef L.N. Hanson J.B. Plant Physiol. 1982; 69: 187-191Crossref PubMed Google Scholar), release of Ca2+ ion from membrane vesicles (22.Drobak B.K. Ferguson I.B. Biochem. Biophys. Res. Commun. 1985; 145: 1043-1047Google Scholar), and phosphatidylinositol hydrolysis (23.Ettlinger C. Lehle L. Nature. 1988; 331: 176-178Crossref PubMed Scopus (169) Google Scholar). It was proposed that calcium acts as a second messenger in the transduction of the hormone signal (8.Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1987; 6: 47-103Crossref PubMed Scopus (278) Google Scholar, 9.Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1993; 12: 185-211Crossref PubMed Scopus (328) Google Scholar, 10.Bush D.S. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 95-122Crossref Scopus (903) Google Scholar, 11.Trewavas A.J. Malho R. Plant Cell. 1997; 9: 1181-1195Crossref PubMed Scopus (210) Google Scholar, 12.Trewavas A. Plant Physiol. 1999; 120: 428-433Crossref Scopus (150) Google Scholar, 23.Ettlinger C. Lehle L. Nature. 1988; 331: 176-178Crossref PubMed Scopus (169) Google Scholar). Auxin-calcium interaction in cellular processes could be regulated through CaM; however, no direct molecular and biochemical evidence for this interaction has been reported so far. Here we report the isolation and characterization of a novel CaM-binding protein that is encoded by a corn homolog ofSAURs (small auxin up RNAs); it is designated asZmSAUR1 (Zea mays SAURs). SAURs belong to one group of the early auxin-response genes (4.Abel S. Theologis A. Plant Physiol. 1996; 111: 9-17Crossref PubMed Scopus (584) Google Scholar). Many early auxin-responsive genes have been cloned and characterized (2.Guilfoyle T.J. CRC Crit. Rev. Plant Sci. 1986; 4: 247-276Crossref Scopus (92) Google Scholar, 4.Abel S. Theologis A. Plant Physiol. 1996; 111: 9-17Crossref PubMed Scopus (584) Google Scholar,24.Theologis A. Huynh T.V. Davis R.W. J. Mol. Biol. 1985; 183: 53-68Crossref PubMed Scopus (236) Google Scholar, 25.Theologis A. Annu. Rev. Plant Physiol. 1986; 37: 407-438Crossref Google Scholar, 26.Key J.L. Bioessays. 1989; 11: 52-58Crossref PubMed Scopus (50) Google Scholar, 27.Guilfoyle T.J. Hagen G. Li Y. Gee M.A. Ulmason T.N. Martin G. Biochem. Soc. Trans. 1992; 20: 97-101Crossref PubMed Scopus (6) Google Scholar). SAURs are one of the gene families in higher plants which have been well characterized. Initially isolated from soybean (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar), SAUR genes have also been characterized from several dicots such as mung bean (29.Yamamoto K.T. Mori H. Imaseki H. Plant Cell Physiol. 1992; 33: 93-97Google Scholar), Arabidopsis (30.Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Crossref PubMed Scopus (113) Google Scholar), and apple (31.Watillon B. Kettmann R. Arredouani A. Hecquet J.F. Boxus P. Burny A. Plant Mol. Biol. 1998; 36: 909-915Crossref PubMed Scopus (22) Google Scholar). In all cases examined, SAUR genes encode short transcripts with highly conserved open reading frames that accumulate rapidly and specifically after auxin treatment. Soybean SAUR gene transcription can be detected as soon as 2.5 min after the application of auxin (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar, 32.McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Crossref PubMed Scopus (177) Google Scholar). We have demonstrated that corn ZmSAUR1 is a rapid auxin-responsive gene as well. The results described here provide direct molecular and biochemical evidence for the involvement of the Ca2+/CaM messenger system in auxin action. 35S-Labeled recombinant CaM was prepared as described (33.Fromm H. Chua N.H. Plant Mol. Biol. Rep. 1992; 10: 199-206Crossref Scopus (60) Google Scholar) using a potato CaM PCM6 cloned into the pET-3b expression vector (34.Takezawa D. Liu Z. An G. Poovaiah B.W. Plant Mol. Biol. 1995; 27: 693-703Crossref PubMed Scopus (92) Google Scholar). The specificity of 35S-labeled CaM was about 0.5 × 106 cpm/μg. A corn root cDNA expression library (λZAP II) prepared in our laboratory was screened using 35S-labeled PCM6 as described (33.Fromm H. Chua N.H. Plant Mol. Biol. Rep. 1992; 10: 199-206Crossref Scopus (60) Google Scholar). Several positive clones were isolated from 2 × 105 recombinant phages. The cDNA clones were sequenced on both strands. DNA sequences were analyzed using GCG version 8.0 and 9.0 software (35.Devereaux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11514) Google Scholar). Corn (Zea mays L. cv. Merit) seeds were sown in plastic trays filled with vermiculite and kept in the dark for about 5 days at 24 °C. The dark-grown coleoptiles were harvested under a dim green light, and 8-mm segments, excluding the 3-mm tip, were excised. Coleoptile segments were transferred into a beaker containing distilled water and kept floating for a 4-h period. Sets of 10 presoaked coleoptile segments were transferred into Petri dishes containing 10 ml of incubation medium consisting of 10 mm KH2PO4(pH 6.3), 1.5% w/v sucrose, 10 mm sodium citrate, and 0.1% v/v ethanol. NAA (Sigma), W-7 (Sigma), and W-5 (Sigma) treatments were carried out as described (19.Raghothama K.G. Mizrahi Y. Poovaiah B.W. Plant Physiol. 1985; 79: 28-33Crossref PubMed Google Scholar). After auxin treatment, the lengths of the coleoptile segments were measured using a ruler under a dim green light, or the samples were frozen in liquid nitrogen for RNA extraction. Corn genomic DNA was extracted as described (36.Yang T. Segal G. Abbo S. Feldman M. Fromm H. Mol. Gen. Genet. 1996; 252: 684-694Crossref PubMed Scopus (50) Google Scholar). 10 μg of DNA was digested with various restriction enzymes, separated by electrophoresis on 0.8% agarose Tris acetate gel, and transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech) with 0.4 m NaOH. Southern blot analysis was carried out as described earlier (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar) using a ZmSAUR1probe covering the complete coding region from nucleotide 37 to 480 (see Fig. 1). Total RNA was isolated from frozen tissue essentially as described (37.Longemann J. Shell J. Willmitzer L. Anal. Biochem. 1987; 163: 16-20Crossref PubMed Scopus (1601) Google Scholar). RNA samples (50 μg) were denatured and separated on 1.5% formaldehyde-agarose gels. After transfer to Hybond N+ filters, the blots were hybridized using 32P-labeled ZmSAUR1 cDNA fragment 37-480 and washed as described earlier (38.Yang T. Lev-Yadun S. Feldman M. Fromm H. Plant Mol. Biol. 1998; 37: 109-120Crossref PubMed Scopus (31) Google Scholar). Blots were stripped and reprobed with a fragment of Arabidopsis 18 S rDNA, accession no. X16077, nucleotides 158–1669. Templates coding for wild type ZmSAUR1 and deletion mΔC and mΔN were produced by PCR amplification from the original cDNA with ZmSAUR1-specific oligonucleotides containing appropriate restriction sites (NdeI at the 5′-end and BamHI at the 3′-end) for cloning into the downstream of the His6 tag in a pET-14b expression vector (Novagen, Inc.). The wild type and deletion mutant proteins of ZmSAUR1 were expressed in Escherichia coli strain BL21(DE3) pLysS according to the method of Studier et al. (39.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorf J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5981) Google Scholar). The nucleotide sequence of all cloned fragments derived by PCR amplification was determined after cloning into the pET-14b vector, using oligonucleotides designed for sequencing from both sides of the pET-14b cloning sites as primers. Wild type and truncated ZmSAUR1 proteins were extracted and purified essentially as described (40.Takezawa D. Ramachandiran S. Paranjape V. Poovaiah B.W. J. Biol. Chem. 1996; 271: 8126-8132Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The amount of protein was estimated by the Bradford (67.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211925) Google Scholar) method using a protein assay kit (Bio-Rad). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred onto a polyvinylidene difluoride membrane (Millipore), and treated with 35S-labeled recombinant CaM with 1 mm CaCl2 or 2 mm EGTA as described (40.Takezawa D. Ramachandiran S. Paranjape V. Poovaiah B.W. J. Biol. Chem. 1996; 271: 8126-8132Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The synthetic peptides were prepared using an Applied Biosystems peptide synthesizer 431A in the Laboratory of Bioanalysis and Biotechnology, Washington State University. Samples containing 240 pmol (4 μg) of bovine CaM (Sigma) and different amounts of purified synthetic peptides in 100 mm Tris-HCl (pH 7.2) and either 1 mm CaCl2 or 2 mm EGTA in a total volume of 30 μl were incubated for 1 h at room temperature. The samples were analyzed by nondenaturing PAGE as described (41.Arazi T. Baum G. Snedden W.A. Shelp B. Fromm H. Plant Physiol. 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar). Using the CaM binding screening approach, nine positive clones from a corn root cDNA expression library were obtained. One of these clones had high affinity to CaM. DNA sequencing indicated that the 760-base pair cDNA clone contained a partial coding region and a full 3′-untranslated region plus a poly(A)+ tail. The clone was designated as ZmSAUR1 because it has high homology to soybean SAUR genes. To get the full clone, a cDNA-specific primer as indicated in Fig.1 and the vector specific T3 primer were used for PCR, and the longest amplified fragment was sequenced. The PCR fragment was 60 base pairs longer than the cDNA clone at the 5′-end. An in-frame methionine residue was deduced in the downstream region of an in-frame stop codon (Fig. 1). Thus, the ZmSAUR1cDNA with the full coding region and 3′-untranslated region was obtained. Fig. 1 shows the nucleotide sequence and the deduced amino acid sequence of ZmSAUR1. The cDNA codes for a polypeptide of 147 amino acids flanked by a 320-base pair untranslated region at the 3′-end and a 36-base pair untranslated region at the 5′-end. The calculated molecular mass and the isoelectric point of the ZmSAUR1 polypeptide are 16.6 kDa and 7.22, respectively. The soybeanSAUR genes encode proteins around 10 kDa in size with an isoelectric point between 6 and 7. Like other characterized SAURs (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar, 29.Yamamoto K.T. Mori H. Imaseki H. Plant Cell Physiol. 1992; 33: 93-97Google Scholar, 30.Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Crossref PubMed Scopus (113) Google Scholar, 31.Watillon B. Kettmann R. Arredouani A. Hecquet J.F. Boxus P. Burny A. Plant Mol. Biol. 1998; 36: 909-915Crossref PubMed Scopus (22) Google Scholar), the amino acid sequence does not contain a typical signal sequence, endoplasmic reticulum retention signal, orN-glycosylation signal, suggesting that the ZmSAUR1 protein does not enter the secretory pathway. However, it is possible that the ZmSAUR1 protein is a nuclear protein because it contains two short regions of basic amino acids (amino acids 33–37 and 67–69) that may form a bipartite nuclear localization signal (42.Raikhel N. Plant Physiol. 1992; 100: 1627-1632Crossref PubMed Scopus (191) Google Scholar). The deduced amino acid sequence of ZmSAUR1 is aligned in Fig. 2 with those of soybeanSAUR 10A5, 15 A (32.McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Crossref PubMed Scopus (177) Google Scholar), mung bean SAUR ARG7 (29.Yamamoto K.T. Mori H. Imaseki H. Plant Cell Physiol. 1992; 33: 93-97Google Scholar), and Arabidopsis SAUR-AC1 (30.Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Crossref PubMed Scopus (113) Google Scholar). The size of ZmSAUR1 is larger than other SAURs. However, searching the Arabidopsis genomic sequences, a ZmSAUR1 homolog, SAUR-A2, with an even larger molecular mass, was found (Fig. 2). The difference lies in the NH2-terminal 54 amino acids and about 30 amino acids in the COOH terminus of ZmSAUR1, where soybean and other plant SAURs have less similarity. In contrast, the sequences are highly similar within the central portion (from 55 to 117 in ZmSAUR1) in all SAURs. Between these residues, ZmSAUR1 is 70.6% similar (58.8% identical) to the soybean 10A5 and 72.5% similar (54.9% identical) to ArabidopsisSAUR-AC1. Thus it seems likely that the central conserved portion of these proteins is most important for whatever function they fulfill. To study further the properties of ZmSAUR1, the ZmSAUR1 protein was expressed in E. coli, using the pET-14b expression vector. The recombinant protein was present mainly in the soluble fraction. The following two experiments proved that CaM binds to the ZmSAUR1 protein. First, the 18.8-kDa fusion protein (16.6-kDa ZmSAUR1 plus 2.2-kDa NH2-terminal His6 tag) was purified by CaM affinity chromatography to near homogeneity as judged by SDS-PAGE (data not shown). Second, 35S-labeled CaM binds to ZmSAUR1 protein only in the presence of Ca2+ (Fig.3). After adding 2 mm EGTA, no CaM binding was observed, suggesting that CaM binding to ZmSAUR1 is calcium-dependent. The proteins from E. colitransformed with the pET-14b vector did not show any CaM binding (data not shown). To map the CaM binding region of ZmSAUR1, two mutants were prepared (Fig. 4 A). The mutant mΔC lacks the COOH-terminal 81 amino acid residues, which includes the conserved central portion; the mutant mΔN lacks the NH2-terminal 66 residues. The wild type ZmSAUR1 and the two mutants were expressed in E. coli and purified as described (see “Experimental Procedures”). These proteins were used for35S-CaM binding assays in the presence and absence of Ca2+. The binding of CaM to wild type and mutant mΔC was similar, whereas CaM did not bind to the mutant mΔN (Fig.4 A), indicating that a CaM binding region is restricted to the 66 amino acids of the NH2 terminus, where SAURs showed the least similarity. CaM binding to wild type and mΔC of ZmSAUR1 was prevented by the addition of 2 mm EGTA (data not shown), indicating an absolute requirement of Ca2+ for CaM binding. CaM is a protein capable of recognizing the basic amphiphilic α-helical domain of the target proteins (14.Snedden W.A. Fromm H. Trends Plant Sci. 1998; 3: 299-304Abstract Full Text Full Text PDF Scopus (194) Google Scholar, 41.Arazi T. Baum G. Snedden W.A. Shelp B. Fromm H. Plant Physiol. 1995; 108: 551-561Crossref PubMed Scopus (108) Google Scholar, 47.Snedden W.A. Koutsia N. Baum G. Fromm H. J. Biol. Chem. 1996; 271: 4148-4153Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Helical wheel projection of the peptide sequences predicted that the CaM binding region was restricted further to amino acids 20–45 of ZmSAUR1. The amino acid residues 32–45 formed a typical basic amphiphilic α-helix (Fig. 4 B), similar to CCaMK, a plant Ca2+/CaM-dependent protein kinase (40.Takezawa D. Ramachandiran S. Paranjape V. Poovaiah B.W. J. Biol. Chem. 1996; 271: 8126-8132Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). A peptide with 26 residues corresponding to the amino acids 20–45 was incubated with bovine CaM, and complex formation was assessed by nondenaturing PAGE in the presence or absence of Ca2+. The results showed that the peptide is capable of forming a stable complex with CaM in the presence of Ca2+ (Fig. 4 C) but not in the absence of Ca2+ (data not shown). Several ratios of peptide to CaM were used. In the absence of the peptide, there was a single band reflecting the pure CaM. As the peptide was added, another band of low mobility appeared, representing the peptide-CaM complex. When the ratio of peptide to CaM was equal, the CaM band disappeared, and the intensity of the peptide-CaM complex increased. At a peptide to CaM molar ratio of 1.5, no free CaM was detected. At higher ratios (up to 2.5), no new band appeared on the gel, nor did the peptide-CaM complex band change its intensity, suggesting that multivalent complexes were absent. These observations indicate that the peptide binds to Ca2+/CaM with a 1:1 stoichiometry. Based on the primary structure of SAURs in Fig. 2, it seems that ZmSAUR1 is very divergent isoform with a longer NH2-terminal domain. However, using the helical wheel projection method, we found that the NH2-terminal portions in all of the SAURs listed in Fig. 2 as well as other SAURs in the data base can form a basic amphiphilic α-helix, suggesting that SAURs in general are CaM-binding proteins. To prove this, two SAURs, 10A5 and SAUR-AC1 from soybean and Arabidopsis,respectively, were selected for further analysis. Both amino acids 9–22 in soybean 10A5 and 3–16 in ArabidopsisSAUR-AC1 formed a basic α-amphiphilic helix, just like ZmSAUR1 (Fig.5 A). Two peptides, corresponding to 2–24 of 10A5 and 2–19 of SAUR-AC1, were synthesized, and a similar gel mobility shift assay was used to study the CaM binding efficiency. In the presence of calcium, like the peptide from ZmSAUR1, both peptides formed a stable complex with CaM, visualized as a larger size band instead of the smaller size band of CaM itself (Fig.5 B). Moreover, the two peptides bind to Ca2+/CaM with a 1:1 stoichiometry, too; however, only one CaM band was detected in the presence of EGTA (data not shown). CaM binding affinity of ZmSAUR1 was studied using different concentrations of 35S-labeled CaM. To eliminate nonspecific CaM binding, bovine serum albumin was used as a negative control. The average background count was subtracted from the counts of ZmSAUR1 protein samples when calculating the specific binding. Binding of labeled CaM to ZmSAUR1 saturated at concentrations above 100 nm (Fig. 6), indicating the presence of a saturable high affinity binding site in ZmSAUR1. From Scatchard plot analysis of the saturation curve, the dissociation constant (K d) of CaM for ZmSAUR1 was estimated to be about 15 nm. The binding of CaM to ZmSAUR1 was blocked completely in the presence of 2 mm EGTA. Scatchard analysis also indicated that ZmSAUR1 has a single CaM binding site (Fig. 6). Most studies of SAUR gene expression in soybean have been conducted using etiolated elongating hypocotyl sections that respond rapidly to auxin treatment (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar, 32.McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Crossref PubMed Scopus (177) Google Scholar). An analogous system in corn is the etiolated coleoptile segments. Corn coleoptile segments floated in 10 μm NAA solution showed a significant increase in length from that of control without application of NAA (Fig.7 A). After about a 2-h incubation, the elongation of coleoptiles was detectable. After 16 h, the coleoptile elongation increased by more than 50%; however, coleoptiles in the medium without the NAA application elongated only about 5%. To study the auxin induction kinetics of ZmSAUR1expression, corn coleoptile segments were collected at different times after incubation in the medium with 10 μm NAA for RNA preparation. Northern analyses indicated that the level ofZmSAUR1 is undetectable if NAA was not applied (Fig.7 B). Treatment with NAA led to a significant induction of the ZmSAUR1 with a size of ∼ 0.8 kilobase, which coincides with the cDNA size of ZmSAUR1. The induction began within 10 min, a sharp increase occurred between 20 and 60 min, with half-maximal after 30 min and saturation in 60 min. This kinetics of auxin induction is similar to Arabidopsis SAUR-AC1 (30.Gil P. Liu Y. Orbovic V. Verkamp E. Poff K.L. Green P.J. Plant Physiol. 1994; 104: 777-784Crossref PubMed Scopus (113) Google Scholar), in contrast to soybean SAUR mRNAs, in which the induction happened in 2.5 min and peaked at 15 min (28.McClure B.A. Guilfoyle T.J. Plant Mol. Biol. 1987; 9: 611-623Crossref PubMed Scopus (191) Google Scholar, 32.McClure B.A. Hagen G. Brown C.S. Gee M.A. Guilfoyle T.J. Plant Cell. 1989; 1: 229-239Crossref PubMed Scopus (177) Google Scholar). This demonstrates that ZmSAUR1 is indeed an early auxin-responsive gene. Earlier studies in our laboratory revealed that W-7, a CaM antagonist, inhibited auxin-induced coleoptile elongation. However, its structural homolog W-5 (same as W-7 but lacking a Cl), which is 10 times less active as a CaM antagonist with the same membrane affinity, did not inhibit cell elongation significantly (19.Raghothama K.G. Mizrahi Y. Poovaiah B.W. Plant Physiol. 1985; 79: 28-33Crossref PubMed Google Scho" @default.
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• W1964226250 title "Molecular and Biochemical Evidence for the Involvement of Calcium/Calmodulin in Auxin Action" @default.
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