Objectives Nitrate nitrogen is the primary nitrogen source for dryland plants, NRT2/3 plays a crucial role in plant nitrate absorption and the regulation of nitrogen uptake. We analyzed the members of lettuce NRT2/3 family using bioinformatics, studied their physicochemical properties, and gene structure evaluation, and assessed the functions of select NRT2/3 genes utilizing VIGS technology.

Methods The NRT2/3 proteins from Arabidopsis and rice, as well as the annotated NRT proteins from lettuce were downloaded from the NCBI website. Bioinformatics methods were utilized to identify the members of the lettuce NRT2/3 family, and a multi-sequence alignment of the NRT2/3 proteins from lettuce and Arabidopsis was conducted using DNAMAN software. Furthermore, a multiple sequence alignment of the NRT2/3 proteins from lettuce, rice, and Arabidopsis was conducted using MEGA11 software, followed by the construction of a phylogenetic tree. Then, their coincidences of the common NRT2/3 proteins were proved in physicochemical properties, functional domains, conserved motifs, hydrophilicity, cis-acting elements, and gene structure, using online software. The chromosome localization, the secondary structure, phosphorylation sites, and transmembrane regions of lettuce NRT2/3 family proteins were predicted. Drawing from our team’s prior transcriptome sequencing findings, the NRT2/3 genes, which had comparatively high expression levels, were targeted for expression analysis: 1) The seedlings of the two lettuce varieties grown to 20 days were planted on substrates and irrigated by NO₃⁻ 0, 0.35, 0.7, and 11.5 mmol/L solutions for the invetigated of expression levels. 2) The expression levels of these genes in the two lettuce varieties were reduced using TRV-VIGS transient silencing method, subsequently, the lettuce seedlings were grown in normal nitrogen nutrient solutions for 21 days, and then invetigated the expression levels, N uptakes and growth indexes.

Results This study identified a total of 8 lettuce NRT2/3 family members, consisting of 7 LsaNRT2 proteins and 1 LsaNRT3.1 protein. These members were unevenly distributed across 4 chromosomes. Their physicochemical properties, functional domains, transmembrane regions, phosphorylation sites, cis-acting elements, and gene structure, et al. basically conformed to the common characteristics of the NRT2/3 family. The transcriptome sequencing results showed that among the 8 identified genes, LsaNRT2.4 and LsaNRT3.1 had notably high expression levels in leaves of the two lettuce varieties. LsaNRT2.4 and LsaNRT3.1 genes responded to low NO₃⁻ concentrations to a certain extent. Following VIGS silencing, the expression levels of LsaNRT2.4 and LsaNRT3.1 in lettuce leaves were significantly reduced, as were the plant height, leaf length, leaf width, chlorophyll-a content, and leaf nitrate content. Notably, the nitrate content in leaves of lettuce (TRV:: LsaNRT3.1) was significantly lower than that of lettuce (TRV:: LsaNRT2.4). Observations of the phenotype revealed that lettuce silenced using VIGS displayed symptoms like smaller leaves and withered leaf tips, and the lettuce (TRV:: LsaNRT3.1) exhibited a more pronounced effect.

Conclusions Most characteristics of lettuce NRT2/3 family members align with the common traits of plant NRT2/3 family, with LsaNRT2.4 and LsaNRT3.1 genes showing significantly higher expression levels in the leaves compared to the other 6 genes; LsaNRT2.4 and LsaNRT3.1 play pivotal roles in lettuce nitrate absorption, and responsed to deficient and low NO₃⁻ stress very quickly, especially in their expressions in roots. The increased expressions of LsaNRT2.4 and LsaNRT3.1 genes, improved the N uptake and growth of lettuce.