ASGSB 1999 Annual Meeting Abstracts

MOLECULAR EVIDENCE FOR THE INVOLVEMENT OF CALCIUM/CALMODULIN IN AUXIN ACTION. B.W. Poovaiah and T.Yang. Lab. Plant Mol. Biol. & Physiol., Dept of Horticulture, Washington State Univ, Pullman, WA.

Calcium regulates diverse developmental processes in plants through the action of calmodulin (CaM). Earlier investigations in this laboratory have revealed that calcium/CaM are involved in auxin-induced cell elongation (Plant Physiol. 79:28-33, 1985; Plant Cell Physiol. 29:1165-70, 1988). The use of ³⁵S-labeled 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 designed as ZmSAUR1 (Zea mays SAURs) was expressed in E. 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 demonstrated that the CaM-binding site was located at the N-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 (Kd, ~15 nM) in a calcium-dependent manner. Comparison of the N-terminal portion of all the characterized SAURs revealed that they all contain a basic -amphiphilic helix similar to the CaM-binding region of ZmSAUR1. The ZmSAUR1 gene expression began within 10 min, increased rapidly between 10-60 min, and peaked around 60 min following 10 µM -naphthaleneacetic acid (NAA) treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist, W-7, inhibited the auxin-induced cell elongation, but not the auxin-induced expression of ZmSAUR1. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the posttranslational level. These results provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction. 

(Supported by NASA grant NAG-10-0061 and NSF grant MCB 96-30337.)

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