Fig. 1. miR164 overexpression phenocopies the cuc1 cuc2 double mutant. (A) The 2x35S::miR164A and 2x35S::miR164B constructs contain 1049 bp and 1021 bp of genomic DNA centred on the two predicted pre-miR164 precursors, respectively (black box) (Reinhart et al., 2002) under the control of the Cauliflower mosaic virus double 35S promoter and upstream of the Cauliflower mosaic virus 35S terminator. The 2x35S::erGFP construct was used as a control. (B) Floral phenotype of wild-type and transgenic lines. (1) Wild-type plants have unfused sepals and expanded petals and stamens. (2,3) 2x35S::miR164A or 2x35S::miR164B flowers have fused sepal margins. (3) In lines with strong phenotypes, petals and stamens did not expand. (4) However, dissection of the flower reveals petals and stamens which cannot grow out of the fused sepals, resembling cuc1 cuc2 double mutant flowers (Aida et al., 1997). The degree of sepal fusion (5) and the petal number (6) was scored in 2x35S:: erGFP, 2x35S::miR164A and 2x35S::miR164B primary transformants. The degree of fusion is expressed on a scale ranging from 0 for normal sepals (as represented in 1) to 16 for the strongest sepal fusion phenotype (as shown in 3). Data represent mean values for 10 flowers per line. (C) Seedling phenotype of wild-type and transgenic lines. (1) Wild-type seedlings have aligned cotyledons. (2-5) 2x35S::miR164A or 2x35S::miR164B seedlings have misaligned (2), partially fused cotyledons (3) or cup-shaped cotyledons (4) and petiole fusion revealed by dissection (5). (D) miR164 is overexpressed in 2x35S::miR164A and 2x35S::miR164B lines. (1) MIR164A and MIR164B predicted hairpin precursors (Reinhart et al., 2002). In each precursor, the nucleotides corresponding to miR164 are red and those corresponding to the other strand resulting from RNase III-mediated cleavage, miR164A^* or miR164B^*, are blue. (2) Detection of miR164 and control 5S RNA in apices of wild-type plants, 2x35S::erGFP and representative 2x35S::miR164A and 2x35S::miR164B lines showing either a weak (W), intermediate (I) or strong (S) flower phenotype. The normalised ratio between miR164 and 5S RNA expression level is indicated. The migration of 21 and 24 nucleotides DNA primers is indicated. (3) Detection of miR164A^*, miR164B^* and miR164 and control 5S RNA, in wild type and in representatives of strong 2x35S::erGFP, 2x35S::miR164A and 2x35S::miR164B lines. Each inset (right) represents the hybridization signal obtained under identical conditions with 100 pg of DNA oligonucleotides corresponding to miR164A^*, miR164B^* and miR164.