Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit.
BACKGROUND
The ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O2 deficiency and elevated CO2. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca(2+)/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity.


RESULTS
GAD activity in cell-free extracts of apple fruit was stimulated by Ca(2+)/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca(2+)/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca(2+)/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain.


CONCLUSIONS
Plant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30-50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.
Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada. bshelp@uoguelph.ca.
Christopher P
CP
Trobacher
Adel
A
Zarei
Jingyun
J
Liu
Shawn M
SM
Clark
Gale G
GG
Bozzo
Barry J
BJ
Shelp
D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology* / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D027845:Malus / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
D002147:Calmodulin; D010940:Plant Proteins; D005968:Glutamate Decarboxylase