Mechanism of pod shattering in the forage legume Medicago ruthenica

Pod shattering is a seed dispersal strategy and an important agronomical trait in domesticated crops. The relationship between pod shattering and pod morphology in the genus Medicago is well known; however, the detailed mechanism underlying pod dehiscence in Medicago ruthenica, a perennial legume used for forage production, is unknown. Here, the pod ventral sutures of shatter-resistant and shatter-susceptible M. ruthenica genotypes were examined at 8, 12, 16, and 20 d after flowering. The mechanism of pod shattering was analyzed through microscopic observations, polygalacturonase (PG) and cellulase (CE) activity analyses, and RNA-sequencing (RNA-Seq), and the results were verified via reverse transcriptase-quantitative polymerase chain reaction. Pod shattering at the ventral suture in M. ruthenica occurs via a combination of two mechanisms: degradation of the middle lamella at the abscission layers (ALs) and detachment of lignified cells on either side of the ALs triggered by physical forces. Increased PG and CE activities in the pod ventral suture are essential for AL cell-autolysis in the shatter-susceptible genotype. RNA-Seq revealed that 11 genes encoding PG and CE were highly expressed in the ventral sutures of the shatter-susceptible genotype. The expression levels of auxin biosynthesis-related genes decreased in the AL cells and they were negatively associated with pod dehiscence. These results enhance our understanding of the pod shattering mechanism not only in M. ruthenica but also in other leguminous plants.