Figure 2. Expression levels and activity of ZmSO in SO-modified tobacco plants.

(A) Transcription levels of the ZmSO-His₆ in wild-type tobacco plants and six homozygous over-expression (OE) lines (named OE-1, OE-3, OE-4, OE-7, OE-9, and OE-10). ZmSO transcripts detected by semi-quantitative RT-PCR were present in the OE lines, but not in the wild-type plants. Moreover, the ZmSO transcript level was highest in OE-7 among the six over-expression lines. (B) Western blot analysis of wild-type and OE lines (OE-3 and OE-7). Proteins (20 µg per lane) were fractionated by 12.5% SDS-PAGE and immunobloted with histidine tag-specific antibody. The protein level of ZmSO-His₆ was also relatively higher in OE-7 than that in OE-3. (C) Total SO activity in leaf extracts from wild-type and OE lines measured by kinetic assays. The total SO activity was significantly higher in the leaf extracts of OE-3 and OE-7 than in wild-type plants. Kinetics of SO activity was assayed using the ferricyanide reduction technique. The steady-state kinetics of ferricyanide reduction was followed spectrophotometrically at 420 nm using 10 g of protein extract.