**Background:** Nitrogen (N) is an essential nutrient for crop growth, but approximately 50% of applied N is lost from soil, leading to environmental pollution and economic costs. Cereal nitrogen use efficiency (NUE) averages only about 33%, indicating substantial N fertilizer loss. MicroRNAs (miRNAs) are known to regulate gene expression in response to abiotic stresses including N deficiency. Indian dwarf wheat (Triticum sphaerococcum Perc.) is a landrace cultivated in the Indian subcontinent with potential for better N uptake capacity. This study aimed to identify Indian dwarf wheat genotypes with contrasting NUE and N deficiency tolerance (NDT) traits and characterize N-responsive miRNAs through miRNAseq analysis.
**Methods:** Three experiments were conducted: (1) Field screening of 11 Indian dwarf wheat genotypes (S1–S11) and one bread wheat genotype (BT-Schomburgk, BTS) under N-sufficient (N120, 120 kg/ha N) and N-deficient (N0, no applied N) conditions during rabi seasons 2018 and 2019 at ICAR-IARI, New Delhi. The average native soil N content was 175 kg/ha in the N-field. (2) Hydroponic evaluation of three selected genotypes (BTS, S2, S3) grown for 30 days under optimum N (7.5 mM nitrate) and low N (0.05 mM nitrate) conditions in controlled environment growth chambers. Measurements included growth parameters, root architecture (using Win-RHIZO software), photosynthetic pigments, anthocyanin content, nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) activities, tissue nitrate content, and IAA content and localization. (3) miRNAseq analysis of leaf and root tissues from genotypes BTS and S3 at 7, 14, and 21 days after treatment using Illumina HiSeq, with differential expression analysis using EdgeR.
**Key Results:** Under field N deficiency, leaf area was reduced by approximately 47% and total chlorophyll content by 50% compared to N-sufficient conditions. Anthocyanin content increased under N deficiency, with Indian dwarf wheat genotypes S4 (24%), S5 (50%), and S6 (67%) showing significantly higher accumulation. Based on NUE and N starvation response, genotypes BTS (high NUE), S2 (high NUE), and S3 (low NUE but not affected by N level) were selected for further study. In hydroponics, N deficiency reduced leaf area by 31% and shoot dry weight by 25%, while root dry weight increased by 2.6%. Genotype BTS showed an 88% increase in total root length and 89% increase in lateral root length under N deficiency. Shoot nitrate content declined by approximately 95% across all genotypes under N deficiency. NR activity was fourfold lower in roots than leaves; BTS recorded the highest leaf NR activity under N-sufficient conditions. GS activity approximately doubled under N deficiency. GOGAT activity in leaves decreased by 55% under N deficiency, while root GOGAT activity increased by 2.5%. BTS showed the highest leaf GDH activity under N deficiency (13.6-fold higher than N-sufficient). miRNAseq analysis identified approximately 50 differentially expressed miRNAs under N deficiency. Target genes of these miRNAs belonged to four main functional classes: Kinases (Ser-Thr, wall-associated), N metabolism (purine metabolism, amino acid transporter, Cys peptidase, metalloendopeptidase, NiR, NR, proteolysis, proteasome system), secondary metabolism (CYP450, PPO, terpene synthase), dirigent proteins (lignin biosynthesis), and transcription factors (F4, WRKY, ARF, GRAS, MYB-like SPL, NAC). Specific miRNAs including tae-miR1137a, tae-miR1137b-5p, tae-miR1122b-3p, tae-miR1120b-3p, tae-csmR156-1, miR160, miR167a, miR159b, miR171a, miR396-5p, miR444b, miR1125, miR1136, and miR1222 were differentially regulated.
**Clinical Implications:** This study provides insights into miRNA-mediated regulatory networks underlying NUE in wheat, identifying potential biotechnology targets for improving nitrogen efficiency. The coordinated signal transduction network involving miRNAs targeting N metabolism, secondary metabolism, and N signal perception pathways could be leveraged for developing wheat varieties with enhanced NUE, reducing fertilizer requirements and associated environmental impacts. The identified contrasting genotypes (high NUE BTS and low NUE S3) serve as valuable genetic resources for further molecular breeding efforts.