ANTHOCYANIDIN REDUCTASE promotes physical dormancy in <i>Medicago truncatula</i> seeds

Physical dormancy, or hardseededness, refers to a type of dormancy in which seeds cannot germinate due to seed coat impermeability. Physical dormancy broadly exists in seed plants, especially in leguminous species, and plays an essential role in maintaining the durability of natural seed banks. However, physical dormancy restricts the utilization of leguminous seeds in agricultural production. Seeds of the leguminous model plant Medicago truncatula show typical physical dormancy. In this study, we report a function of anthocyanidin reductase (ANR) in controlling M. truncatula seed physical dormancy. The ANR gene was mostly highly expressed in the M. truncatula seed coat. Loss-of-function mutations in ANR resulted in the absence of hardseededness, allowing the seed to absorb water quickly without scarification. The content of glycolipids, especially MDGD, was significantly decreased in the seed coat of the anr mutant. A large increase in the levels of the most abundant flavonoids (flavonoid-3-O-glucosides) was also observed in the anr mutant seed coat. Knockout of the upstream genes transparent testa 8 (TT8), flavonoid 3',5'-hydroxylase 1 (F3'5'H1), or anthocyanidin synthase (ANS) hindered flavonoid-3-O-glucoside accumulation. Accordingly, mutating these genes in the anr background (anr tt8, anr f3'5'h1, and anr ans) restored seed physical dormancy. These results indicate that proanthocyanidins are not directly associated with hardseededness. Rather, an excessive accumulation of flavonoid-3-O-glycosides and a reduction in lipids are associated with seed physical dormancy. This study provides information regarding the molecular and biochemical mechanisms underlying physical dormancy.