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

Area of Science:

• Plant molecular biology
• Biochemistry
• Calcium signaling

Background:

• Calmodulin (CaM) is a crucial calcium sensor in eukaryotes.
• Glutamate decarboxylase (GAD) synthesizes gamma-aminobutyric acid (GABA).
• The regulation of GABA synthesis in plants is not fully understood.

Purpose of the Study:

• To isolate and characterize plant calmodulin-binding proteins.
• To investigate the potential role of Ca2+-dependent calmodulin binding in GAD function.
• To explore the regulation of GABA synthesis in plants.

Main Methods:

• Screening of a petunia cDNA expression library using labeled calmodulin.
• Sequence analysis and comparison with known proteins (E. coli GAD).
• Expression of recombinant protein in E. coli and functional assays (GAD activity, calmodulin binding).
• Mapping of the calmodulin-binding domain using truncated protein fragments and affinity chromatography.
• Immunodetection of the plant GAD using specific antibodies.

Main Results:

• A petunia cDNA encoding a Ca2+-dependent calmodulin-binding protein with 67% sequence similarity to E. coli GAD was isolated.
• The recombinant petunia protein exhibited both GAD activity and calmodulin binding.
• E. coli GAD lacked calmodulin-binding activity.
• The calmodulin-binding domain was localized to a carboxyl-terminal extension unique to the plant GAD.
• Plant extracts contained a protein recognized by anti-GAD antibodies, which also bound calmodulin in vitro.

Conclusions:

• This study reports the first isolation of a plant GAD gene and a calmodulin-binding GAD.
• The findings suggest that intracellular Ca2+ signals, mediated by calmodulin, may regulate GABA synthesis in plants.
• The carboxyl-terminal extension is critical for calmodulin interaction and potentially for GAD regulation in plants.