Complementation assay in Arabidopsis

For details regarding plasmid construction, see Methods S1. All constructs were introduced into Agrobacterium strain GV3101 (Hood et al., ¹⁹⁹³), and Arabidopsis svp‐41 mutant plants (Hartmann et al., ²⁰⁰⁰) were transformed using the floral dip method (Clough & Bent, ¹⁹⁹⁸). T1 independent lines were BASTA selected, lines showing a Mendelian segregation (3 : 1) were retained, and two to three homozygous single‐copy T3 lines from independent T1 lines were selected for further use. Seeds were stratified on soil in the dark at 4°C for 7 d. Plants were grown under controlled conditions at 22°C with a long‐day (LD) photoperiod (16 h : 8 h, light : dark). Arabidopsis Columbia‐0 (Col‐0) was used as the wild‐type (WT). Flowering time was scored by counting total leaf number (cauline and rosette leaves) of at least 10 plants per genotype. The number of days from germination to bolting (elongation of the first internode by 0.5 cm) and to the opening of the first flower were recorded. Total RNA was isolated from 7‐d‐old manually dissected leaves and shoot apices using the RNeasy Mini Kit (Qiagen). Real‐time polymerase chain reaction was performed as previously described. PP2A was used as a reference gene.