Figure 4

Characterization of atocd mutants and over-expression lines. A. Schematic representation of AtOCD to show the position of T-DNA insertions. Black bar indicates coding region while unfilled bars represent 5′ and 3′ untranslated regions. B. Semi-quantitative RT-PCR to show the AtOCD transcript accumulation in WT, mutants and over-expression lines. Actin2 gene is used as a positive control. C. Quantitative real time PCR of AtOCD in mutant and overexpression lines. Error Bars indicate S.E. of triplicate samples. Panels at the top of the figure show western blotting of wild type and AtOCD overexpression plants to detect the C-terminal FLAG tag. All lanes were on the same gel and are separated for clarity of presentation. The band shown migrated at the expected molecular weight for the AtOCD-FLAG fusion protein (37.7 kD). Right panel of the figure shows the wild type and atocd mutant results on an expanded scale to show clearly the greatly reduced mRNA level in the atocd muants. D. Relative expression of AtOCD in two independent AtOCD RNAi lines. RNA was isolated from unstressed seedlings.