Stratification overcomes ABA-mediated seed dormancy by uncoupling RGL2/ABI5 inhibition from α-amylase expression

Cold-pretreatment (stratification) is widely employed to overcome seed dormancy and enhance germination, however, its underlying molecular mechanisms remain elusive. We tested the hypothesis that stratification alleviates the repression of α-amylase expression, a critical step for endosperm starch mobilization during germination. Stratification at 4 °C effectively overcame dormancy in wild-type Arabidopsis thaliana, the ABA-catabolism mutant cyp707a2, the GA-insensitive mutant sleepy1, and the cyp707a2 sleepy1 double mutant, but failed to rescue the dormancy phenotype of the GA-biosynthesis mutant ga3ox1. Moreover, stratification markedly promoted starch hydrolysis by increasing both the transcript abundance and the enzymatic activity of α-amylase in freshly harvested seeds of wild-type and in those mutants exhibiting elevated endogenous ABA or treated with exogenous ABA. Concomitantly, stratification substantially suppressed the expression of RGL2, a DELLA protein that represses GA signaling, and ABI5, a key ABA-responsive transcription factor. Collectively, our data indicates that stratification initiates GA biosynthesis, thereby relieving RGL2-mediated repression of α-amylase expression. Secondly, stratification mitigates ABA-imposed inhibition of germination without alleviating the suppressive effect of ABA on post-germinative seedling development.