Plant Molecular Biology 37: 967–975, 1998.
© 1998 Kluwer Academic Publishers. Printed in Belgium.
Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by
calcium/calmodulin and differ in organ distribution
Moriyah Zik, Tzahi Arazi, Wayne A. Snedden
and Hillel Fromm
DepartmentofPlantSciences,Weizmann Instituteof Science, Rehovot, 76100 Israel (
Present address: Department of Botany, University of Toronto, Toronto, Ontario, Canada
Received 22 September 1997; accepted in revised form 25 February 1998
Key words: baculovirus, calcium, calmodulin, GABA (γ-aminobutyric acid), Sf9 insect cells
The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated
GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The
recombinant proteins bound [
S] calmodulin (CaM) in the presence of calcium, and a region of 30–32 amino
acids from the C-terminal of each isoform was sufﬁcient for CaM binding when fused to glutathione S-transferase.
Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors.
Recombinantproteins were partially puriﬁedby CaM afﬁnity chromatographyand were found to exhibitglutamate
decarboxylase activity, which was dependent on the presence of Ca
/CaM at pH 7.3. Southern hybridizations
with GAD gene-speciﬁc probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2.
Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in
roots, leaves, inﬂorescence stems and ﬂowers. Our study provides the ﬁrst evidence for the occurrence of multiple
/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis
GAD activity involves modulation of isoform-speciﬁc gene expression and stimulation of the catalytic activity of
GAD by calcium signalling via CaM.
Glutamate decarboxylase (GAD) catalyzes the con-
version of glutamate to γ-aminobutyric acid (GABA).
The occurrence of GAD activity in plants has been
known for about half a century . GAD activity
and GABA have since been suggested to be associated
with various physiological responses including regu-
lation of cytosolic pH [8, 17, 22], carbon ﬂuxes into
the tricarboxylicacid cycle , nitrogen metabolism,
transport and storage [5, 11, 14, 19], and deterrence
of insects . The possibility of GABA functioning
as a signalling molecule in plants, similar to its role in
vertebrates and invertebrates, remains to be tested.
Studies of the function of GAD and its regulation
in plants have been stimulated by cloning of the petu-
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers U10034 (GAD1) and U49937 (GAD2).
nia GAD gene and characterization of the encoded
enzyme as a calcium (Ca
(CaM)-bindingprotein . Subsequently,it was found
that GAD activity in extracts of Vicia faba , soy-
bean , petunia , rice  and asparagus  is
modulated by Ca
/CaM. Detailed molecular analy-
sis of the CaM-binding domain (CaMBD) of petunia
GAD  and the characterization of the puriﬁed re-
combinant petunia GAD as a Ca
enzyme  provided a working model to explain the
rapid stimulation of GAD activity, in response to var-
ious stress situations which elicit changes in cytosolic
concentrations.Evidence to supportthis hypoth-
esis was provided by demonstrating that stimulation
of GAD activity in response to anoxia  and cold 
/CaM. Moreover, the rapid response to
cold (within minutes) did not seem to involve changes
in cytosolic pH , indicating that changes in pH are
not required for GAD activation in certain stress situ-