Integrative transcriptomic and metabolomic analyses provide insights into the effects of overexpression and knockout of NtLHT1 in different tissues

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Lysine-Histidine Transporter 1 (LHT1) is a key amino acid transporter involved in plant nutrition, metabolism, leaf development, and abiotic stress tolerance. However, the regulatory mechanisms of NtLHT1 (LHT1 in Nicotiana tobacum ) across different plant organs remain poorly understood. In this study, we utilized NtLHT1 knockout (LHT1-KO), overexpression (LHT1-OE), and wild-type (LHT1-WT) tobacco lines (cv. Honghuadajinyuan) to investigate the organ-specific effects of altered NtLHT1 expression on metabolic and transcriptomic profiles. At the 10-leaf stage, leaves, stems, and roots were collected for transcriptome sequencing (RNA-seq) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)-based metabolomic analysis, with qRT-PCR performed to validate RNA-seq results. Principal component analysis (PCA) revealed that organ identity exerted a stronger regulatory influence on the metabolome and transcriptome than NtLHT1 genetic manipulation. LHT1-OE induced more profound metabolic and transcriptomic perturbations across organs compared to LHT1-KO: in roots, LHT1-OE upregulated pathways related to alkaloid biosynthesis and zeatin biosynthesis; in stems, it enhanced phenylpropanoid and terpenoid metabolic flux; in leaves, it repressed flavonoid and terpenoid biosynthesis by downregulating key structural genes (e.g., ACCT , FPPS, TPS ) and modulated hormone homeostasis by increasing cytokinin (CTK) accumulation and decreasing auxin (IAA) and gibberellin (GA) levels. Collectively, our multi-omics analysis demonstrates that NtLHT1 acts as a “metabolic node” integrating primary and secondary metabolism as well as hormone signaling, exerting organ-specific regulatory effects on tobacco metabolism and gene expression. These findings provide a molecular framework for understanding NtLHT1’s multifunctional roles and offer potential targets for improving agronomic traits (e.g., leaf size, stress tolerance) in tobacco and other crops through genetic manipulation.