Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated.

A cDNA coding for a 58-kD calcium-dependent calmodulin (CaM)-binding glutamate decarboxylase (GAD) previously isolated in our laboratory from petunia (Petunia hybrida) (G. Baum, Y. Chen, T. Arazi, H. Takatsuji, H. Fromm [1993] J Biol Chem 268: 19610-19617) was used to conduct molecular studies of GAD expression. GAD expression was studied during petunia organ development using the GAD cDNA as a probe to detect the GAD mRNA and by the anti-recombinant GAD serum to monitor the levels of GAD. GAD activity was studied in extracts of organs in the course of development. The 58-kD CaM-binding GAD is expressed in all petunia organs tested (flowers and all floral parts, leaves, stems, roots, and seeds). The highest expression levels were in petals of open flowers. Developmental changes in the abundance of GAD mRNA and the 58-kD GAD were observed in flowers and leaves and during germination. Moreover, developmental changes in GAD activity in plant extracts coincided in most cases with changes in the abundance of the 58-kD GAD. We conclude that the 58-kD CaM-binding GAD is a ubiquitous protein in petunia organs and that its expression is developmentally regulated by transcriptional and/or posttranscriptional processes. Thus, GAD gene expression is likely to play a role in controlling the rates of GABA synthesis during petunia seed germination and organ development.

A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis.

Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.