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JXB Advance Access originally published online on June 11, 2007
Journal of Experimental Botany 2007 58(10):2699-2707; doi:10.1093/jxb/erm120
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© The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells

Kazuhito Akama1,* and Fumio Takaiwa2

1Department of Biological Science, Shimane University, Nishikawatsu 1060, Matsue, Shimane 690-8504, Japan
2Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

* To whom correspondence should be addressed. E-mail: akama{at}life.shimane-u.ac.jp

Glutamate decarboxylase (GAD) converts L-glutamate to {gamma}-aminobutyric acid (GABA), which is a non-protein amino acid present in all organisms. Plant GADs carry a C-terminal extension that binds to Ca2+/calmodulin (CaM) to modulate enzyme activity. However, rice possesses two distinct types of GAD, OsGAD1 and OsGAD2. Although they both have a C-terminal extension, the former peptide contains an authentic CaM-binding domain (CaMBD), which is common to dicotyledonous plants, while the latter does not. Therefore, the role of the C-terminal extension in functional expression of OsGAD2 was investigated. An in vitro enzyme assay using recombinant OsGAD2 proteins revealed low activity in the presence or absence of Ca2+/CaM. However, a truncated version of GAD2 (OsGAD2{Delta}C) had over 40-fold higher activity than wild-type GAD at physiological pH. These two DNA constructs were introduced simultaneously into rice calli via Agrobacterium to establish transgenic cell lines. Free amino acids were isolated from several lines for each construct to determine GABA content. Calli overexpressing OsGAD2 and OsGAD2{Delta}C had about 6-fold and 100-fold the GABA content of wild-type calli, respectively. Regenerated OsGAD2{Delta}C rice plants had aberrant phenotypes such as dwarfism, etiolated leaves, and sterility. These data suggest that the C-terminal extension of OsGAD2 plays a role as a strong autoinhibitory domain, and that truncation of this domain causes the enzyme to act constitutively, with higher activity both in vitro and in vivo.

Key words: Agrobacterium, amino acid, calmodulin, GABA, GAD, {gamma}-aminobutyric acid, glutamate decarboxylase, overexpression, rice

Received 17 April 2007; Accepted 1 May 2007


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