Expression data from stratified anac089-2 mutant and wild-type seeds, ABA-treated.

Through a genetic screening in 3μM (+)-S-ABA, we have isolated the anac089-2 mutant, which was obtained from fast neutron-mutagenized Col-0 M2 seeds. This mutant showed ABA-insensitive phenotypes in the transition from dormancy to germination. anac089-2 mutant line was subjected to mapping based on Col/Ler polymorphisms. This mutation was independently mapped to the top arm of Chromosome 5. The anac089-2 mutation is located in a 25 kb region between CAPS markers MWD9/EcoRV and MWD9-25.3/AccI. This 25 kb region contains 8 genes, and sequencing of these genes revealed that anac089-2 mutant contained a mutation in the At5g22290. This gene encodes ANAC089, a nuclear localized NAC transcription factor, with the deduced protein of 340 amino acids. Southern blot analysis suggests that anac089-2 mutation is a ~700 bp deletion that includes the C-terminal region. ANAC089 is involved in an ABA signaling pathway during seed germination and stress responses (Osuna et al., 2011). To analyze ANAC089 regulation of gene expression in a developmental time window of ABA responsiveness following breakage of seed dormancy (Lopez Molina et al 2001, 2002), anac089-2 mutant and wild-type Col-0 (background) seeds were stratified in water (4ºC, dark) during 3 days, and then transfered to light (22ºC, percival) in presence of 5μM ABA for 3 hours. Three biological replicates were performed in order to get consistent results.