PP no 74

Abstract

529 examine the quaternary complex, LexA±AP3 and PI were expressed on the bait vector, and GAL4 AD-AG and/or SEP3-MIK were expressed on the prey vector. When two genes were expressed on the same vector, they were both driven by ADH1 promoters. Amino-acid residues 1±167 and 1±171 were used for the truncated AP1-MIK and SEP3-MIK proteins, respectively. Other processes and the colony-lift b-gal assays were performed in accordance with the manufacturer's instructions (Clontech). Immunoprecipitation For immunoprecipitation experiments, radiolabelled AP1 or SEP3 were mixed with haemagglutinin (HA)-tagged proteins and precipitated with anti-HA antibody. Precipitated AP1 and SEP3 were separated by SDS±PAGE and detected by radio-imaging analyser, BAS2000 (Fuji®lm). Other procedures were done as described 7,12. Transactivation assay For yeast, MADS proteins cDNAs were fused in-frame to GAL4 DNA-binding domain on pAS2-1 (Clontech) and transformed into the yeast strain YRG-2 (UAS::lacZ, Stratagene). AP1-K2C (residues 125±256) and SEP3-K2C (128±257) were used as truncated MADS proteins. Yeast cells were grown at 22 8C overnight, and the b-gal activity was assayed at 30 8C using o-nitrophenyl-b-D-galactopyranoside. For onion epidermal cells, 35S promoter-driven MADS cDNAs that express native MADS proteins (effector) and CArG::LUC (reporter) were co-transfected into onion epidermal cells by using a particle delivery system (Bio-Rad). CArG::LUC has seven repeats of MADS protein binding consensus sequence 29 , 59-GGGGTGGCTTTCCTTTTTTGG TAAATTTTGGATCC-39 (CArG box is underlined), upstream of the 35S minimal promoter (-30). 35S::Renilla luciferase (RLUC) was used for the internal control. LUC assays were conducted using Dual-luciferase reporter system (Promega). Other procedures were done as described 30. Plant material Arabidopsis Columbia ecotype was used for Agrobacterium-mediated vacuum transformation 31. Plant crossing was carried out by manual cross-pollination. The presence of the transgenes was con®rmed by PCR. AP3::GUS plants have a 600-base-pair region of the AP3 promoter 16. Staining for GUS activity was done as described 16. Cryo-scanning electron micrograph We used a Hitachi S-3500N scanning electron microscope equipped with a cryo-stage. For observation and photography, the stage was chilled at-20 8C and the natural scanning electron microscopy (SEM) mode (70 Pa) was used with a 25-kV accelerating voltage.