Genetic Modifications of the Pyrroline-5-Carboxylate Metabolic Pathway to Intensify Rice Aroma

Rice (Oryza sativa L.) is a staple food for more than half of the world's population, with aromatic varieties especially prized for their distinctive fragrance. This aroma is primarily attributed to 2-acetyl-1-pyrroline (2-AP). However, enhancing 2-AP levels without compromising yield or other key agronomic traits remains a major challenge in rice breeding. In this study, we adopted a synergistic genetic strategy to boost 2-AP production by targeting key enzymes in the proline and Δ¹-pyrroline-5-carboxylate (P5C) metabolic pathway. We used miRNA-induced gene silencing (MIGS) to knock down the endogenous OsP5CR (Pyrroline-5-carboxylate reductase) and OsP5CDH (Pyrroline-5-carboxylate dehydrogenase) genes, resulting in increased P5C accumulation and enhanced flux toward 2-AP synthesis. Additionally, exogenous overexpression of bacterial feedback-insensitive proB74-proA genes further enhanced the levels of proline and P5C accumulation, thereby promoting 2-AP production. Notably, crossbred lines combining MIGS-mediated knockdown of OsP5CR and OsP5CDH with proB74-proA overexpression exhibited synergistically elevated 2-AP levels. Agronomic evaluations confirmed that these genetic modifications did not adversely affect yield-related traits or grain quality. Our findings highlight the potential of a multigene, integrative metabolic engineering approach to enhance rice aroma, offering a promising strategy for developing high-quality aromatic rice cultivars that meet market demands without sacrificing yield.