|Date||Jun 11, 2010 15:30 – 17:00|
Title: "SEED DORMANCY IN ARABIDOPSIS REQUIRES BINDING OF MULTIPLE ISOFORMS OF THE DOG1 PROTEINS"
Speaker: Kazumi Nakabyashi, Ph.D (Max-Planck-Institute for Plant Breeding Research, Cologne, Germany)
DELAY OF GERMINATION 1 (DOG1) was identified as a major determinant of natural variation for seed dormancy between the accessions Ler and Cvi of Arabidopsis thaliana. The gene was cloned and encodes a protein of unknown function. DOG1 is predominantly expressed in seeds. Mutant alleles of DOG1 are completely non-dormant implicating that DOG1 is absolutely required for the induction of seed dormancy.
DOG1 is alternatively spliced into five different transcripts, resulting in three protein isoforms. We have taken a transgenic complementation approach to check the functionality of these isoforms and found that none of the single isoforms driven by the native promoter was able to complement the non-dormant phenotype of the dog1 mutant. However, transformants that can produce all three isoforms do complement, suggesting that multiple isoforms would be necessary for the DOG1 function.
A yeast-two-hybrid assay revealed that all three forms of the DOG1 protein from alternative splicing are able to bind to each other. Using a combination of truncated DOG1 proteins and alanine-scanning, we successfully identified a single alanine-substitution that significantly reduced binding-efficiency. We tested if the DOG1 genomic fragment with this substitution was able to complement the dog1 mutant, and found that transformants with the substitution-mutation are less dormant than the ones with the wild-type genomic fragment. All together, our data suggest that self-binding of multiple isoforms is necessary for the full function of DOG1 protein in dormancy induction in Arabidopsis.