Identification and characterization of functional DOG1 residues regulating the abscisic acid response in Arabidopsis

Abscisic acid (ABA) is an important phytohormone regulating seed dormancy and germination. DELAY OF GERMINATION 1 (DOG1) is a pivotal regulator of seed dormancy and regulates the ABA response by binding with ABA HYPERSENSITIVE GERMINATION 1 (AHG1) and heme. However, to date, the molecular function and regulatory mechanisms of DOG1 remain unclear, including the relationship between DOG1 and the ABA response. Here, we investigate the mechanism of DOG1 in the ABA response using RNA sequencing, genetics, and biochemistry experiments. Our data suggest that DOG1 and AHG1 regulate the expression of many common genes, including seed maturation and ABA response. Moreover, DOG1 acts upstream of ABA INSENSITIVE 5 (ABI5) and regulates ABI5 target genes including LATE EMBRYOGENESIS ABUNDANT 1 (EM1) and EM6. We therefore performed a genetic screen to isolate mutants that suppress the ABA hypersensitive phenotype of YFP-DOG1-overexpressing transgenic plants. Ten mutant alleles caused mutations in the DOG1 transgene region, including three premature stop codon mutations and seven single amino acid substitutions. One of these mutants, P178L, which contains an amino acid substitution, abolished the interaction with AHG1 and promoted the dimerization of DOG1. Furthermore, we identify a heme-binding residue, Cys96, that plays an important role in ABA response. Overall, these data suggest that DOG1 and AHG1 regulate ABA response via ABI5 and that the association between heme and AHG1 is critical for the function of DOG1 during the regulation of seed germination.