Characterization of a salt-responsive promoter from the moss Physcomitrium patens

Engineering stress-responsive gene regulation is critical for developing abiotic stress-tolerant plants. Two native Physcomitrium patens promoters was characterized under osmotic and salt stress, identifying Pp3c20_8210 as a salt-inducible promoter in P. patens that could hold a potential in future synthetic biology applications. We also investigated salt-responsive elements in the native promoters of Physcomitrium patens with long-term monitoring of protein expression under high osmotic and saline stress conditions. To evaluate the role of cis-regulatory elements in salt-inducible gene expression, we analyzed shorter engineered versions of Pp3c20_8210, which displayed continuous salt-responsive expression, even after the targeted deletion of key transcription factor binding sites for Homeodomain, MYB, GATA, and Dof transcription factors. Deletion of Homeodomain and GATA transcription factor binding sites reduced Venus reporter expression, suggesting that they are possibly the primary drivers of the Pp3c20_8210 promoter activity in P. patens. No statistically significant effect on promoter responsiveness was detected following the deletion of MYB and Dof transcription factor binding sites under our experimental conditions, possibly due to compensatory mechanisms from other regulatory elements. These findings lay the foundation for future dissection of salt-responsive promoter architecture and can provide valuable tools for synthetic gene regulation in plants.