The mechanism of GA-induced seed dormancy release via MsGAI1-like-miR159a-MsMYB101 module in Magnolia sieboldii

Magnolia sieboldii K. Koch seeds belong to the morphophysiological dormancy type, and it is extremely difficult to germinate under natural conditions. Gibberellins (GAs) are crucial for facilitating seed dormancy release. However, the regulatory mechanisms by GA-mediated seed dormancy release in M. sieboldii remain unclear. Here, we reveal that exogenous GA₃ could quickly break the physiological dormancy of M. sieboldii seeds through morphological and physiological analyses. To investigate the role of miRNA159s in GA-induced seed dormancy release, MsmiR159a and its target gene MsMYB101 were isolated and characterized. Spatial-temporal expression analyses showed that MsmiR159a and MsMYB101 had opposite expression patterns. A cleavage interaction between MsmiR159a and MsMYB101 was confirmed. Furthermore, we identified GA-INSENSITIVE1-like (MsGAI1-like), a negative regulator of GA signaling pathway, as directly binding to the promoter of miR159a to modulate the expression of MsmiR159a/MsMYB101. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that MsMYB101 directly binds to the promoter of the alpha-amylase gene MsAMY2. Dual-luciferase reporter assay indicated that MsMYB101 positively regulates MsAMY2 expression, suggesting that MsmiR159a-MsMYB101 module contributes to seed dormancy release by influencing starch metabolism. In conclusion, this study elucidates the potential mechanism underlying the response of the MsGAI1-like-miR159a-MsMYB101 network to dormancy release in M. sieboldii seeds. These findings provide new insights into the molecular mechanisms of morphophysiological dormancy seeds.