Fig. 5. (A) Comparison of MPK6 activity in Arabidopsis wild-type (WT), ctr1, etr1-1, ein2, ein3-1 and hyperactive SIMKK plants in the absence or presence of ACC. The A.thaliana Columbia lines were treated for 0 or 10 min with 1 mM ACC. Upper panel: MPK6 activity was detected by immunocomplex kinase assays using MPK6-specific antibody. Lower panel: MPK6 protein amounts were detected by immunoblotting the extracts used for the immunocomplex kinase assays with MPK6 antibody. (B) Ethylene-induced gene expression in wild-type and hyperactive SIMKK plants. Detached leaves were kept in culture medium for 2 h before treatment with 1 mM ACC. After 0, 6 and 48 h, total RNA was extracted from leaves, separated by denaturing agarose electrophoresis, blotted onto nylon filters and probed with ³²P-labeled DNA fragments of ERF1, PDF1.2, GST2 and CHI-B. An actin gene fragment was used as a constitutive control. (C) Quantification of ERF1, PDF1.2, GST2 and CHI-B gene expression levels in untreated wild-type and hyperactive SIMKK plants. Gene expression levels were calculated from three independent RNA gel blots by PhosphorImager analysis and normalized to the respective actin expression levels.