Ethanol suppresses rice seed germination through inhibiting ROS signaling

Ethanol is frequently used not only as priming but also as a solvent to dissolve hardly water-soluble phytohormones gibberellic acid (GA₃) and abscisic acid (ABA) in seed germination. However, the molecular and physiological mechanisms of ethanol's impact on seed germination remain elusive. In this report, we investigated how ethanol affected reactive oxygen species (ROS) during rice seed germination. Ethanol at a concentration of 3.5% (v/v) inhibited 90% seed germination, which was almost reversed by H₂O₂. H₂O₂ contents in embryos were reduced by ethanol after 18 h imbibition. Antioxidant enzymes assays revealed that only superoxide dismutase (SOD) activities in seed embryos were lowered by ethanol, in line with the suppressed mRNA expression of SOD genes during imbibition. Additionally, compared to the mock condition, ethanol increased ABA contents but decreased GA (GA₁ and GA₃) in seed embryos, resulting in disharmonizing GA/ABA balance. Conceivably ethanol induced transcription of OsNCEDs, the key genes for ABA biosynthesis, and OsABA8ox3, a key gene for ABA catabolism. Furthermore, ethanol promoted ABA signaling by upregulating ABA receptor genes and ABA-responsive element (ABRE)-binding protein/ABRE-binding factors during imbibition. Overall, our results demonstrate that lowering of H₂O₂ levels due to suppressed SOD activities in rice germinating seed embryos is the decisive factor for ethanol-induced inhibition of seed germination, and GA/ABA balance and ABA signaling also play important roles in ethanol's inhibitory impact on seed germination.