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.
Baum
G
G
Department of Plant Genetics, Weizmann Institute of Science, Israel.
Lev-Yadun
S
S
Fridmann
Y
Y
Arazi
T
T
Katsnelson
H
H
Zik
M
M
Fromm
H
H
D002147:Calmodulin / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D018076:DNA, Complementary; D004533:Egtazic Acid / Q000494:pharmacology; D005968:Glutamate Decarboxylase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008969:Molecular Sequence Data; D010936:Plant Extracts; D030821:Plants, Genetically Modified / Q000254:growth & development* / Q000378:metabolism*; D011485:Protein Binding; D014268:Trifluoperazine / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002147:Calmodulin; D002614:Chelating Agents; D018076:DNA, Complementary; D010936:Plant Extracts; D014268:Trifluoperazine; D018698:Glutamic Acid; D004533:Egtazic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase