Figure 5

Coregulation of CrMPK3 expression, kinase activity and expression of MIA genes in C. roseus leaves upon wounding, UV, and MeJA treatment. 6–8 weeks old C. roseus plants were subjected to wounding, UV treatment and MeJA application and leaves harvested at indicated time intervals. (A) Northern blot analysis of CrMPK3. Lowermost panel shows 28 S rRNA as loading control. (B) CrMPK3 activity assay in response to wounding, UV, MeJA treatments. Total protein extracts (200 μg) was subjected to immunoprecipitation with the 4 G10, phosphotyrosine antibody. The immunoprecipitated complex was electrophoresed on SDS 10%(w/v) polyacrylamide gel and immunoblot was performed using anti-CrMPK3. MeJA mock treatment was performed by applying solvent only (ethanol) in the similar fasion to that of MeJA. ‘CBB’ shows representative equal protein loading controls. (C) Quantitative RT-PCR analysis of key MIA pathway genes, Tdc, Str, D4h and Dat. Expression levels were normalized against expression of C. roseus Actin gene as an internal control and are shown relative to untreated control plants. The relative level of each gene in control plants at time 0 was standardized as 1. Values are presented as the mean and the errors bars indicate standard deviation of triplicate samples. The experiments were repeated three times with similar results.