Transcriptome and Metabolome Reveal the Molecular Mechanism of Barley Genotypes Underlying the Response to Low Nitrogen and Resupply

**Background:** Nitrogen is a critical mineral element for plant growth, but excessive nitrogen fertilizer use causes environmental pollution and reduces crop quality. Improving nitrogen use efficiency (NUE) in crops like barley is therefore important. Barley ranks fourth globally in cereal crop production and area, yet molecular mechanisms underlying its tolerance to low nitrogen stress remain poorly understood. This study aimed to investigate the transcriptomic and metabolomic responses of two barley genotypes with contrasting NUE under low nitrogen and nitrogen resupply conditions.

**Methods:** Two barley genotypes—nitrogen-efficient W26 and nitrogen-sensitive W20—were grown hydroponically. Seedlings were treated with low nitrogen (LN, 0.4 mM) for 3 days and 18 days, then half of the LN-treated plants received resupplied nitrogen (RN, 2 mM) from day 18 to 21. Biomass (shoot and root dry weight) and nitrogen content were measured at 3, 18, and 21 days. RNA-seq was performed on 72 samples (2 genotypes × 2 parts (leaves and roots) × 2 treatments × 3 time points × 3 biological replicates) using Illumina NovaSeq 6000, generating 497.49 Gb of raw data. Metabolite profiling was conducted on 144 samples (6 biological replicates) using UHPLC-MS/MS. Differentially expressed genes (DEGs) were identified using DESeq2 with thresholds |log2(FoldChange)| ≥ 1 and adjusted p ≤ 0.05. Differentially expressed metabolites (DAMs) were selected based on VIP ≥ 1.0, |log2(FoldChange)| ≥ 1, and p ≤ 0.05. KEGG and GO enrichment analyses were performed, and qRT-PCR validated five genes per tissue.

**Key Results:** After 21 days of low nitrogen, NUE was 87.54% for W26 and 61.74% for W20. Under LN, W26 maintained biomass better than W20; W20 biomass decreased by 17.77% after 18 days of LN compared to CK, while W26 showed no significant difference. Shoot nitrogen content in W20 dropped from 39.04 to 35.95 mg·plant⁻¹·g⁻¹ after 3 days of LN, whereas W26 showed no significant change. After 18 days, shoot nitrogen content decreased by 3.69 mg·plant⁻¹·g⁻¹ in W26 and 6.66 mg·plant⁻¹·g⁻¹ in W20. In leaves, 7926 DEGs were identified in W26 and 7537 in W20; in roots, 6579 DEGs in W26 and 7128 in W20. Metabolite analysis revealed 458 DAMs in W26 leaves and 425 in W20 leaves; 486 DAMs in W26 roots and 368 in W20 roots. KEGG joint analysis of DEGs and DAMs showed that glutathione (GSH) metabolism was significantly enriched in leaves of both genotypes. In leaves, GSH, amino acids, and amides were the main DAMs; in roots, GSH, amino acids, and phenylpropanes predominated. Key nitrogen metabolism genes identified included nitrate transporters (HORVU.MOREX.r3.6HG0543390, HORVU.MOREX.r3.6HG0543380), ammonium transporter (HORVU.MOREX.r3.5HG0530810), nitrate reductase (HORVU.MOREX.r3.6HG0541410), and glutamine synthetase (HORVU.MOREX.r3.6HG0613270). Plant hormones 3-indole butyric acid (IBA) in leaves and 3-indolepropionic acid and brassinolide (BR) in roots were also identified as candidate DAMs.

**Clinical Implications:** While this is a plant biology study without direct clinical applications, the findings have significant agricultural implications. Identifying the molecular basis of NUE in barley—particularly the role of glutathione metabolism and specific nitrogen transporter genes—provides targets for breeding or engineering barley varieties with improved nitrogen efficiency. This could reduce reliance on nitrogen fertilizers, mitigating environmental pollution and lowering production costs. The candidate genes and metabolites identified here offer a foundation for future functional validation studies aimed at developing more sustainable barley cultivation practices.