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.

中文翻译：

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吡咯啉-5-羧酸盐代谢途径的遗传修饰以增强大米香气

大米 （Oryza sativa L.） 是世界上一半以上人口的主食，其中芳香品种因其独特的香味而备受推崇。这种香气主要归因于 2-乙酰基-1-吡咯啉 （2-AP）。然而，在不影响产量或其他关键农艺性状的情况下提高 2-AP 水平仍然是水稻育种的主要挑战。在这项研究中，我们采用了一种协同遗传策略，通过靶向脯氨酸和 Δ¹-吡咯啉-5-羧酸盐 （P5C） 代谢途径中的关键酶来促进 2-AP 的产生。我们使用 miRNA 诱导的基因沉默 （MIGS） 敲低内源性 OsP5CR （吡咯啉-5-羧酸还原酶） 和 OsP5CDH （吡咯啉-5-羧酸脱氢酶） 基因，导致 P5C 积累增加，并增强 2-AP 合成的通量。此外，细菌反馈不敏感的 proB74-proA 基因的外源过表达进一步增强了脯氨酸和 P5C 积累的水平，从而促进了 2-AP 的产生。值得注意的是，结合 MIGS 介导的 OsP5CR 和 OsP5CDH 敲低与 proB74-proA 过表达的杂交系表现出协同升高的 2-AP 水平。农艺评估证实，这些基因改造不会对与产量相关的性状或谷物质量产生不利影响。我们的研究结果强调了多基因、综合代谢工程方法增强大米香气的潜力，为开发满足市场需求而不牺牲产量的高质量芳香水稻品种提供了一种有前途的策略。