Characterization of OsNrx3i1, the first intron of rice OsNrx3 gene, as a candidate intron-mediated enhancement element for synthetic promoter engineering.

Intron-mediated enhancement (IME) is a potent endogenous mechanism for boosting gene expression, yet few IME elements function efficiently across distantly related plant species. Here, we characterize the first intron of the rice OsNrx3 gene (OsNrx3i1) as a candidate IME element. OsNrx3i1 significantly enhanced gene expression in rice protoplasts and stable transgenic plants, and also showed detectable activity in the dicot Nicotiana benthamiana in transient assays. The enhancement effect was position-dependent, requiring insertion within the 5' transcribed region, and functionally distinct from typical enhancers. We further identified a repressive upstream open reading frame (uORF) in its native sequence; disruption of this uORF and embedding the intron into an optimized 5' UTR scaffold, unlocked its full potential. Benchmarking against established IME elements (cat-1 intron, AtTub6i1, OsSodCc1i1) revealed that OsNrx3i1 performs competitively in monocots, but all tested introns exhibited context-dependent and host-specific efficacy. When engineered into minimal synthetic promoters, OsNrx3i1 increased their activity by up to 65-fold in rice protoplasts. This work presents OsNrx3i1 as a promising and engineerable genetic module, providing a foundation for its future exploration and application in plant synthetic biology.