Physical dormancy of seed is an adaptive trait that widely exists in higher plants. This kind of dormancy is caused by a waterimpermeable layer that blocks water and oxygen from the surrounding environment and keeps embryos in a viable status for a long time. Most of the work on hardseededness has focused on morphological structure and phenolic content of seed coat. The molecular mechanism underlying physical dormancy remains largely elusive. By screening a large number of Tnt1 retrotransposontagged Medicago truncatula lines, we identified nondormant seed mutants from this model legume species. Unlike wild-type hard seeds exhibiting physical dormancy, the mature mutant seeds imbibed water quickly and germinated easily, without the need for scarification. Microscopic observations of cross sections showed that the mutant phenotype was caused by a dysfunctional palisade cuticle layer in the seed coat. Chemical analysis found differences in lipid monomer composition between the wild-type and mutant seed coats. Genetic and molecular analyses revealed that a class II KNOTTED-like homeobox (KNOXII) gene, KNOX4, was responsible for the loss of physical dormancy in the seeds of the mutants. Microarray and chromatin immunoprecipitation analyses identified CYP86A, a gene associated with cutin biosynthesis, as one of the downstream target genes of KNOX4. This study elucidated a novel molecular mechanism of physical dormancy and revealed a new role of class II KNOX genes. Furthermore, KNOX4-like genes exist widely in seed plants but are lacking in nonseed species, indicating that KNOX4 may have diverged from the other KNOXII genes during the evolution of seed plants.S eed dormancy is a complex adaptive trait of higher plants that allows embryos to survive extended periods of unfavorable environmental conditions (1). Over the course of evolution, plant seeds have evolved diverse dormancy forms in response to various climates to maintain viability (2). Physical dormancy, also termed hardseededness, is an exogenous dormancy caused by a water-impermeable hard seed coat. Physical dormancy plays a key role in protecting seeds against microbial attacks and predators and maintaining seed banks in soils (3-5). This type of dormancy has been found in at least 17 plant families and is very common in legume species (2, 6, 7). Seed exhibiting physical dormancy is characterized by specific morphological features, including a water-impermeable palisade cell layer covered by intact cuticles in the hard seed coat. This structure blocks water and oxygen from its surrounding environment and preserves seeds in a viable status for a long time, sometimes for more than hundreds of years (8). Another common type of seed dormancy is physiological dormancy, which is caused by endogenous factors (e.g., abscisic acid) in embryos. Most molecular studies on seed dormancy have focused on physiological dormancy, using model species Arabidopsis thaliana (9, 10).However, A. thaliana seed is water-permeable, and thus not a suitable model to study physical dor...