R3-MYB proteins OsTCL1 and OsTCL2 modulate seed germination via dual pathways in rice

Salt and drought stress are common abiotic factors that exert a detrimental influence on seed germination, potentially leading to significantly impaired growth and production in rice. Gaining a comprehensive understanding of the molecular response of seeds to abiotic stress during the germination is of paramount importance. In the present study, we identified two R3-MYB genes in rice, namely OsTCL1 and OsTCL2, and characterized their roles in regulating seed germination under salt and drought stress. Plants with tcl1 and tcl2 mutant alleles exhibited delayed seed germination, particularly under stress conditions. The tcl1 tcl2 double mutant showed an even more pronounced reduction in germination during initial stages of germination, thereby indicating a redundant regulatory function of OsTCL1 and OsTCL2 in seed germination under abiotic stresses. Furthermore, we demonstrated that the transcript levels of several phospholipase D (PLD) genes were downregulated in the tcl1 tcl2 mutant, resulting in a decreased level of the phosphatidic acid (PA) product. Application of 1-butanol, a competitive substrate inhibitor of PLD-dependent production of PA, attenuated the stress response of the tcl1 tcl2 mutant. This suggests that OsTCL1 and OsTCL2 partially modulate seed germination through the PLD-PA signaling pathway. Moreover, there were alterations in the expression of genes involved in abscisic acid (ABA) biosynthesis, metabolism and signaling transduction in the double mutant. These changes affected the endogenous ABA level and ABA response, thereby influencing seed germination. Application of both 1-butanol and ABA synthesis inhibitor sodium tungstate (Na₂WO₄) nearly eliminated the differences in stress response between wild type and the tcl1 tcl2 mutant. This indicates that OsTCL1 and OsTCL2 synergistically coordinate seed germination under abiotic stresses through both the PLD-PA signaling and ABA-mediated pathways.