Fig. 1

Molecular features of rice GAD3. a Alignment of C-terminal regions of putative GADs from plants. Red and blue colors indicate identical amino acids and similar amino acids, respectively. Trp (W) and Lys (K) essential for in vitro binding to CaM (Arazi et al. 1995) are indicated by asterisks and a thick line, respectively. The positions of two pseudosubstrate residues (E476 and E480 in PhGAD) (Yap et al. 2003) are indicated by black circles. Os: Oryza sativa, Ph: Petunia hybrida, Sl: Solanum lycopersicum. PhGAD (L16797), OsGAD1 (AB056060), OsGAD2 (AB056061), OsGAD3 (AK071556), SlGAD1 ((AB359913), SlGAD2 (AB359914), SlGAD3 (AB359915). b In vitro Ca²⁺/CaM binding ability of recombinant OsGAD3. Coding sequence for Ca²⁺/CaMBD of OsGAD3 was ligated in-frame with a thioredoxin-coding sequence in an expression vector. Expression of recombinant protein was induced in E. coli for purification with an affinity column. The resulting fusion protein (Fr.1) subjected to a CaM-agarose resin separation; effluent fractions (Fr. 2–5) and eluted fractions with EGTA (Fr. 6–8) were all analyzed using 5–20% SDS-polyacrylamide gel electrophoresis to detect protein bands with Coomassie Brilliant Blue staining. As a control, an intact expression vector was used (Fr. 9: vector-encoding protein, Fr. 10–13: effluent fractions, Fr.14–16: eluted fractions). c In vitro enzyme assays for recombinant OsGAD3 and OsGAD3ΔC. The GAD assay was performed to measure GAD production, as described in Akama and Takaiwa (2007). –Ca²⁺/CaM; without Ca²⁺/CaM, +Ca²⁺/CaM; with 0.5 mM Ca²⁺ and 0.1 μM bovine calmodulin (Sigma). Data present the mean ± standard deviation of three independent experiments for both