Figure 2

Molecular and biochemical characterisation of oat -knockout mutants. A: Schematic representation of the exon-intron structure of δOAT (At5g46180) with the T-DNA insertion points in oat1 and oat3. Thick green bars indicate exons, thin green bars indicate introns. The thick red bars indicate the part of the mRNA used as probe for northern blotting. B: PCR with two gene-specific primers and one primer complementary to the T-DNA left border identified homozygous plants. Appearance of two T-DNA specific bands (indicated by arrowheads) indicated an inverted tandem repeat of the T-DNA. C: Northern blot with the δOAT-specific probe on wildtype, oat mutants and δOAT-GFP transgenic plants. D: The same membrane re-probed with a P5CS1-specific probe. E: EtBr staining of the corresponding RNA-gel to demonstrate equal loading. F: OAT activity in whole plant extracts. OAT activity is expressed in arbitrary units of P5C produced per mg total protein during 20 min. Error bars indicate SD of triplicate assays, the whole experiment was repeated with similar results from independent samples.