Overexpression of a high affinity nitrate transporter NtNRT2.1 significantly improves the nicotine content in Nicotiana tabacum

Nicotine is a crucial alkaloid in tobacco, contributing to 90 % of the total alkaloids. It also acts as a key indicator of the quality of flue-cured tobacco. Typically, nitrate (NO 3 - ) plays a crucial role in the growth and development of plants, and its limited availability has been shown to restrict crop productivity. In this study, we treated tobacco plants with NO 3 - and ammonium (NH₄ ⁺ ), and found that tobacco growth is more dependent on NO 3 - . In particular, exogenous NO 3 - application promoted tobacco growth, while also increases nicotine content. Additionally, nicotine content with the nitrate treatment was 5–10 times higher than that of ammonium treatment. Upon further analysis of five tobacco nitrate transporter family (NtNRT2) members, we found that NtNRT2.1 induced by low nitrate, and that the transcription level of NtNRT2.1 is 8–10 times higher than that of other treatments. Likewise, we found that under NO 3 - treatment, the overexpression (OE) of NtNRT2.1 significantly increases NO 3 - uptake in the root. However, under low nitrate conditions, the NO 3 - uptake rate in the OE lines was twice as high as the WT, as well as nicotine content in shoot. Our RT-qPCR analysis also showed that the transcription level of nicotine synthesis and transport relate enzyme genes in WT were approximately 50 % lower than the OE lines with the different nitrate treatments. In summary, our work provides a theoretical basis to develop new high-nicotine tobacco varieties through optimized nutrient utilization. • The growth of Nicotiana tabacum (cultivar, K326) relies more on NO 3 - -N compared to NH 4 + -N. • Tobacco treated with NO 3 - contains higher levels of nicotine. • Overexpression of the tobacco nitrate transporter gene NtNRT2.1 significantly enhances NO 3 - uptake in tobacco. • Overexpression of NtNRT2.1 increases nicotine content by boosting the expression of genes related to nicotine synthesis and transport.