Background: Raising nitrogen use efficiency of crops by improving root system architecture is highly essential not only to reduce costs of agricultural production but also to mitigate climate change. The physiological mechanisms of how biochar affects nitrogen assimilation by crop seedlings have not been well elucidated.Results: Here, we report changes in root system architecture, activities of the key enzymes involved in nitrogen assimilation, and cytokinin (CTK) at the seedling stage of cotton with reduced urea usage and biochar application at different soil layers (0–10 cm and 10–20 cm). Active root absorption area, fresh weight, and nitrogen agronomic efficiency increased significantly when urea usage was reduced by 25% and biochar was applied in the surface soil layer. Glutamine oxoglutarate amino transferase (GOGAT) activity was closely related to the application depth of urea/biochar, and it increased when urea/biochar was applied in the 0–10 cm layer. Glutamic-pyruvic transaminase activity (GPT) increased significantly as well. Nitrate reductase (NR) activity was stimulated by CTK in the very fine roots but inhibited in the fine roots. In addition,AMT1;1, gdh3, andgdh2were significantly up-regulated in the very fine roots when urea usage was reduced by 25% and biochar was applied.Conclusion: Nitrogen assimilation efficiency was significantly affected when urea usage was reduced by 25% and biochar was applied in the surface soil layer at the seedling stage of cotton. The co-expression ofgdh3andgdh2in the fine roots increased nitrogen agronomic efficiency.The synergistic expression of the ammonium transporter gene andgdh3suggests that biochar may be beneficial to amino acid metabolism.

Biochar enhancement of nitrogen efficiency in crops is highly essential not only to reduce costs of agricultural production but also to conserve resources, lower energy consumption for products of these fertilizers, strengthen soil health, and eventually helps in slowing climate change; however nitrogen efficiency physiology by biochar effects is not clear. Here, we reported on the morphological, nitrogen metabolism and cytokinin, at seedling stage, under different layers of biochar and limited urea conditions grown in soil culture. Expression profile of miRNAs and AOB was further studied in fine and medium roots. It showed active root absorption area, fresh weight, and nitrogen agronomic efficiency responded significantly under biochar and reduction by 20% urea condition in the surface soil layer. Also, NR and GPT activity in fine roots remarkably increased with cytokinin, but decreased significantly in medium roots, meanwhile both NR and GDH activity did so. GOGAT activity was to be dependent with biochar and urea locations. In addition, AMT1;1, gdh3 and gdh2 in fine roots showed their up-regulation with reduction 20% urea and biochar. It revealed that co-expression of gdh3 and gdh2 in fine roots significantly affected nitrogen assimilation under reduction 20% urea with biochar on surface soil at seedling stage. Highlights: The co-expression of ammonium transporter gene and GDH induced by biochar effect improves nitrogen efficiency and seedling growth. These data emphasizes the importance of effects of cytokinin on nitrate reductase activity closely related to the position under biochar condition, which is a key element of enhancement nitrogen assimilation efficiency in cotton seedling. Biochar addition applied into 0 to 10cm soil had a more positive effect on seedling growth than that into 10 to 20cm soil layers.