ICE1 and ZOU determine the depth of primary seed dormancy in Arabidopsis independently of their role in endosperm development

Seed dormancy is a widespread and key adaptive trait that is essential for the establishment of soil seed banks and prevention of preharvest sprouting. Herein we demonstrate that the endosperm-expressed transcription factors ZHOUPI (ZOU) and INDUCER OF CBF EXPRESSION1 (ICE1) play a role in determining primary dormancy depth in Arabidopsis. We show that ice1 or zou increases seed dormancy and the double mutant has an additive phenotype. The increased dormancy is associated with increased ABA levels, and can be separated genetically from their role in endosperm maturation, because loss of ABA biosynthesis or DELAY OF GERMINATION 1 reverses the dormancy phenotype without affecting the aberrant seed morphology. Consistent with these results, ice1 endosperms had an increased capacity for preventing embryo greening, a phenotype previously associated with an increase in endospermic ABA levels. Although ice1 changes the expression of many genes including some in ABA biosynthesis, catabolism and/or signalling, only ABA INSENSITIVE 3 is significantly misregulated in ice1 mutants. We also demonstrate that ICE1 binds to and inhibits expression of the ABA INSENSITIVE 3. Our data demonstrate that Arabidopsis ICE1 and ZOU determine the depth of primary dormancy during maturation independently of their effect on endosperm development.