Fig. 4. Requirement for a functional cis-element and ATHB6 for ABA-mediated promoter activation in Arabidopsis protoplasts. (A) The reporter constructs (wt and mutant) contain four tandemly orientated copies of the ATHB6-binding sequence (CAATTATTA, oligo A) or the point-mutated sequence (CAATTGTTA, oligo B), respectively, fused upstream to the CaMV 35S core promoter (35S core) that controls expression of firefly luciferase (LUC). They are represented schematically together with the effector constructs that allow constitutive expression of ATHB6 and ΔATHB6 under control of the 35S promoter (35S). In ΔATHB6, the α-helix 3 (amino acids 96–117) of the homeodomain (HD) essential for DNA binding was deleted. The positions of HD helices are indicated by shaded boxes. (B) The ATHB6-dependent activation of reporter constructs was tested in a transient gene expression system. Transfected Arabidopsis protoplasts were incubated for 24 h in the presence (+) or absence (–) of 30 µM ABA and analysed for expression of the LUC reporter. Co-transfection of a constitutively expressed aequorin gene allowed normalization of expression in independent experiments. The induction of LUC activity from wild-type and mutant reporter constructs by different effectors is shown relative to the expression from wild-type reporter in the absence of ectopically expressed effector. The data reflect the results of three independent transfections ± SD.