Metabolomic and transcriptomic insights into nitrogen-responsive flavonoid biosynthesis in red lettuce varieties

Nitrogen (N) is an important macronutrient for plant growth and development, significantly influences nitrogen use efficiency (NUE) and modulates metabolic pathways when applied at varying concentrations. Despite its importance, the mechanistic effects of N on biomass accumulation and polyphenolic biosynthesis in two distinct red lettuce cultivars (Lactuca sativa L. cv. H20K7044 and H20K3447) under differential N concentrations remain unexplored. This study employed integrated metabolomics and transcriptomic analyses to elucidate these dynamics. This study used three different N treatments including high_N (10 mmol L−1), low_N (2.5 mmol L−1) and normal_N (5 mmol L−1) concentrations. Elevated N supply (10 mmol L−1) enhanced growth parameters in H20K7044, including chlorophyll a, b, total chlorophyll contents and fresh/dry biomass, whereas H20K3447 exhibited a higher nitrogen balance index (NBI) under high N conditions. Intriguingly, H20K7044 showed high relative flavonol contents under low N treatment. By mapping polyphenolic metabolic pathways, we identified biosynthetic modification reactions through a multi-omics framework, correlating transcriptomic profiles with metabolite networks across N treatments. Comparative analysis revealed cultivar-specific divergence in secondary metabolism regulation. Key structural genes in flavonoid biosynthesis: PAL (phenylalanine ammonia lyase), CHI (chalcone isomerase), ANS (anthocyanidin synthase), DFR (dihydroflavonol 4 reductase), FLS (flavonol synthase), and F3′H (flavonoid 3′-hydroxylase) were significantly upregulated under low N conditions in both red lettuce cultivars. Metabolomic profiling highlighted differential accumulation of polyphenolic derivatives, including kaempferol 4′-glucoside, quercetin 3-O-(6″-acetyl-glucoside), cyanidin-3-O-glucuronide, and epicatechin-3-O-gallate, which varied with N availability and genotypic background. This study establishes a novel framework for interrogating N-mediated regulation of polyphenolic biosynthesis, linking transcriptional activity to metabolic flux in red lettuce cultivars. The findings underscore the utility of multi-omics integration in dissecting genotype-environment interactions, offering actionable insights for optimizing N fertilization strategies to enhance phytochemical richness and nutritional quality in lettuce cultivation. Such advances hold promises for precision agriculture and enabling tailored nutrient management to improve crop resilience and values.