Abstract
The use of (35)S-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 as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia 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 NH(2)-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) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis 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 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF148498
We report the isolation and characterization of a novel CaM-binding protein that is encoded by a corn homolog of small auxin up RNAs (SAURs); it is designated as ZmSAUR1 (Zea mays SAURs)
We have demonstrated that corn ZmSAUR1 is a rapid auxin-responsive gene as well
Summary
CaM, calmodulin; NAA, ␣-naphthaleneacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SAUR, small auxin up RNA; W-5, N-(6-aminohexyl)-1naphthalenesulfonamide hydrochloride; W-7, N-(6-aminohexyl)5-chloro1-naphthalenesulfonamide hydrochloride. Calmodulin Binds to SAUR Proteins bean [29], Arabidopsis [30], and apple [31]. SAUR genes encode short transcripts with highly conserved open reading frames that accumulate rapidly and after auxin treatment. Soybean SAUR gene transcription can be detected as soon as 2.5 min after the application of auxin [28, 32]. 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 Ca2ϩ/CaM messenger system in auxin action
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