[Genome-wide identification and stress response analysis of sugarcane glutamine synthetase gene family]

Glutamine synthetases (GSs) are central enzymes in plant nitrogen assimilation, governing nitrogen uptake, transport, and remobilization. This study systematically identified the GS gene family members in sugarcane (Saccharum spontaneum), analyze the sequence features and spatiotemporal expression profiles of SsGSs, and mined candidate genes functionally linked to high nitrogen use efficiency (NUE), aiming to furnish gene resources for enhancing NUE of sugarcane through molecular breeding. We employed S. spontaneum and its elite hybrid progeny as model materials. First, nitrogen source preference was rigorously assessed through ¹⁵N isotope tracing. Subsequently, genome-wide identification of SsGSs was conducted via integrated bioinformatic pipelines. Leveraging multi-omics evidence-including transcriptome profiling, RT-qPCR, and subcellular localization assays-we comparatively analyzed the expression dynamics of the two core members, SsGS1 and SsGS2, across diverse tissue samples, developmental stages, and abiotic stress conditions. The ¹⁵N tracer experiments revealed a pronounced preference of sugarcane for ammonium (NH₄⁺) over nitrate as a nitrogen source. A total of 13 SsGSs were identified and phylogenetically classified into three conserved subfamilies: GS1a, GS1b, and GS2. Promoter cis-acting element analysis demonstrated significant enrichment of elements particularly those responsive to abscisic acid and methyl jasmonate. Subcellular localization confirmed cytoplasmic targeting of SsGS1 and chloroplastic localization of SsGS2. RT-qPCR quantification showed that SsGS1 exhibited relatively high expression in roots and senescent leaves and was dose-dependently upregulated as the NH₄Cl supply increased. In contrast, SsGS2 displayed internode-specific high expression and exhibited robust responses upon exposure to H₂O₂ and ethephon. Collectively, these data indicate that SsGS1 likely functions in root-localized ammonium assimilation and nitrogen translocation, whereas SsGS2 appears to coordinate carbon-nitrogen metabolism in internodes and contribute to abiotic stress responses. This work delivers a curated set of high-priority candidate genes for mechanism dissection of NUE improvement in sugarcane and marker-assisted breeding.