Figure 6.

Functional analysis of Lys to Arg substitution mutants of OsGAPDH1. (A) Transactivation function of indicated single, double or triple substitution mutants compared to wild type OsGAPDH1 in transient transfection assays. (B) Effect of the substitutions on nuclear accumulation of OsGAPDH1-GFP in transfected rice protoplasts. The first panel shows relative transfection efficiency of each construct tested by immunoblots with anti GFP. Coomassie staining was used for loading controls. The lower panels show nuclear accumulation of the substitution mutants relative to wild type OsGAPDH1 revealed by immunoblots using anti-GFP. Anti-H3 was used to control loadings of nuclear proteins. The ImagJ software was used to quantify the signals indicated on the right. (C) In vitro enzymatic activities of the substitution mutants relative to the wild type OsGAPDH1 protein. The wild type and mutants were produced as GFP fusion proteins in tobacco cells and purified with anti-GFP resins. (D) Enzymatic activity of tobacco cell-produced OsGAPDH1-GFP after incubation with E. coli-produced OsSRT1-GTS or GST alone. Significant differences are indicated by **P value < 0.01.