Phenotypic Analysis and Spatiotemporal RNA‐Seq Reveal Key Phenes and Regulatory Network Cascades Under Low Nitrogen Stress in Lettuce

ABSTRACT The biological processes underlying nitrogen uptake and utilization represent targets for the genetic improvement of lettuce nitrogen use efficiency (NUE) to increase productivity and counter nitrogen pollution. This study investigated the growth performance and phenotypic traits of 12 lettuce ( Lactuca sativa ) varieties under low nitrogen (LN) conditions. Spatial and time‐resolved RNA‐seq of the LN‐tolerance lettuce variety was conducted to explore the molecular mechanisms of the LN response. The results revealed that leaf area, root diameter, root‐to‐shoot ratio, root hair length, and density are key phenes that respond to LN stress. Transcriptome analysis revealed heterogeneous gene expression in roots and shoots under LN conditions. In roots, NRT2.1 , NRT2.4 , and NRT3.1 were upregulated, whereas NRT2 . 4 and NRT3 . 1 were downregulated in shoots. GDU1 and GDU2 were upregulated in shoots, whereas GDU2 was downregulated in roots, indicating nitrogen redistribution. In shoots, key transcription factors included MYB , ERF , GTE9 , bHLH , and HSF ‐ A7a , whereas MYB , WRKY17 , Trihelix ‐ GT ‐ 3a , HSF ‐ A4c , ERF , and MIKC _ MADS ‐2 were important regulators in roots, offering potential targets for improving NUE in lettuce. The time‐ordered gene co‐expression network revealed tissue‐specific regulatory cascades under LN conditions. Specifically, roots continuously regulate metabolic processes and nutrient absorption, while shoots transition from early‐stage nutrient uptake to later‐stage photosynthesis and cell wall organization, reflecting distinct adaptive strategies. These phenotypic traits and nitrogen‐responsive genes provide valuable targets for breeding high‐NUE lettuce varieties, offering opportunities to increase nitrogen use efficiency, promote sustainable agriculture, and reduce nitrogen pollution.