Optimization of Nitrogen Application and Root Biomass Modulates 2-Acetyl-1-Pyrroline Biosynthesis in Fragrant Rice

The rice root system mediates nutrient uptake while adapting to tillage, management, and environmental changes. While optimized nitrogen (N) supply is known to enhance 2-acetyl-1-pyrroline (2-AP) biosynthesis in fragrant rice, the underlying mechanisms linking nitrogen availability, root development, and their combined effects on physiological processes and aroma formation remain unclear. To address this knowledge gap, we conducted a pot experiment employing two fragrant rice cultivars (Huahangxiangyinzhen and Qingxiangyou19xiang) under three nitrogen regimes (0, 1.5, and 3.0 g N pot^-1, recorded as LN, MN, and HN, respectively), and the root zone was isolated by enclosing it in a specialized bag to set contrasting root growth conditions (root-restricted and root-unrestricted, recorded as RR and UR, respectively). The results showed that root restriction increased the average 2-AP content by 22.90% despite reducing root biomass by approximately 50%, compared to unrestricted roots. Under nitrogen-containing treatments, root-restricted and root-unrestricted rice showed, respectively, an average 6.43% and 5.23% higher 2-AP content compared to nitrogen-free treatment. The structural equation modeling analysis illustrated that nitrogen metabolism physiology and photosynthetic physiology directly or indirectly significantly affected the accumulation of 2-AP. Random forest analysis further revealed that physiological changes at maturation substantially contributed to 2-AP synthesis. Therefore, this study advances the theoretical understanding of how nutrient assimilation processes and root morphology interact to modulate 2-AP biosynthesis in scented rice cultivars.