Nitrogen (N) is the major nutrient factor for plant growth and development, and plants can absorb and utilize inorganic and organic N in soil. Organic N includes amino acids, peptides and proteins (Cai and Aharoni, 2022). Approximately 40% of organic N in soil comprises peptides and proteins, and 45%–50% of soil organic N comprises amino acids (Cao et al., 2016). The concentrations of total amino acids in soil may be as high as 150 μmol/L (Weigelt et al., 2005). Amino acids and their derivatives not only participate in N fluxes both in poor and fertile soils, but also play an important role in the N cycle in plant organs (Inselsbacher and Näsholm, 2012). For plants, amino acids are essential for the activities of enzymes and proteins. Moreover, amino acids are important components of chlorophyll, polyamines, phytohormones, creatinine and nucleotides (Dong et al., 2020; Yahyaoui and Pérez-Frías, 2020). Therefore, these acids play important roles in the entire life cycle of plants, including regulating the defense response and N utilization efficiency, affecting the quality and biomass, changing the root or shoot morphology, and act as precursors of secondary metabolites (Perchlik and Tegeder, 2017; Guo et al., 2020b; Guo et al., 2021).

Generally, proteins and peptides in organic N cannot be directly absorbed by plant roots, while amino acids can be obtained by roots and transported to shoots or leaves through xylem and phloem (Näsholm et al., 2009; Tegeder and Masclaux-Daubresse, 2018). As the level of amino acids in the soil is significantly lower than that of root cells, membrane localized amino acid transporters (AATs) are necessary for the uptake of amino acids from soil (Tegeder, 2014). Currently, plant AATs are divided into three main families: the amino acid/auxin permease (AAAP) family, the amino acid polyamine and choline (APC) transporter family, and the usually multiple amino acids move in and out transporter (UMAMIT) family (Cao et al., 2024). The AAAP family contains eight subfamilies: proline transporters (ProTs), amino acid permeases (AAPs), auxin transporters (AUXs), lysine and histidine transporters (LHTs), aromatic and neutral amino-acid transporters (ANTs), γ-aminobutyric acid transporters (GATs), amino acid transporter-like proteins (ATLs), and vesicular aminergic-associated transporters (VATs) (Yang et al., 2020). The APC family contains three subfamilies: cationic amino acid transporters (CATs), L-type amino acid transporters (LATs), and polyamine H+-symporters (PHSs) (Saha and Gupta, 2022; Du et al., 2024). Among these families, AAP, LHT, and ProT are the most studied AATs in plants (Fan et al., 2023).

AtAAP1 was the first identified amino acid transporter in plants and mediates the transport of neutral and charged amino acids (Perchlik et al., 2014). Overexpression of AtAAP1 significantly increases the transport of chlorantraniliprole–alanine conjugate (Ren et al., 2019). AtAAP2 functions in the long distance transfer of amino acids, and the growth and development of leaves are enhanced in the atapp2 mutant, which in turn increases seed yield and oil content (Elashry et al., 2013). AtAAP3 is mainly expressed in the roots and can transport basic amino acids, such as lysine, histidine, and arginine (Okumoto et al., 2004). The root-knot nematode (RKN) infestation levels and egg mass number in the atapp3 mutant were notably decreased, indicating that AtAAP3 may act as a positive regulator for plants against RKN invasion (Marella et al., 2012). In total, 19 OsAAP genes have been identified in the rice genome. OsAAP1 is located in plasma and nuclear membranes, its overexpression increases tiller numbers and fills grains, while the osaap1 mutant presents the opposite phenotype (Ji et al., 2020). Lysine and arginine can be transported by OsAAP3, overexpression of OsAAP3 increases the accumulation of these amino acids, but reduces the number of tillers and fills grains in transgenic plants (Wei et al., 2021). OsAAP12 is located in the plasma membrane, the number of tillers is decreased in OsAAP12 overexpression plants but increased in osaap12 mutant lines (Jin et al., 2024).

AtLHT1 was the first identified transporter of the lysine and histidine transporter family. AtLHT1 exhibits high expression in roots, flowers, and siliques. Overexpression of AtLHT1 improves N efficiency and promotes plant growth, whereas disruption of AtLHT1 affects amino acid uptake and inhibits plant growth (Hirner et al., 2006). In addition, AtLHT1 is associated with the transport of 1-aminocyclopropane-1-carboxylic acid, an important precursor of ethylene (Shin et al., 2015). AtLHT4 has strong expression in anthers, which suggests that AtLHT4 may be involved in anther and pollen development (Rabby et al., 2022). There are six OsLHT genes (OsLHT1-OsLHT6) in the rice genome, and the most studied member is OsLHT1. OsLHT1 is strictly located in the plasma membrane and participates in the transport of Asparagine (Guo et al., 2020b). Knockout of OsLHT1 decreases the concentration of amino acid in xylem sap, and the translocation of amino acid in the shoot is restricted, resulting in reduced plant height, stem length, and grain yield (Guo et al., 2020a). Notably, loss of function of OsLHT1 increases the expression of defense-responsive genes and produces more salicylic acid and jasmonic acid. Therefore, rice blast disease resistance is improved in OsLHT1 mutants (Guo et al., 2023). OsLHT5 was localized in the cytosol and plasma membrane. The expression of OsLHT5 was downregulated when treated with PEG and NaCl, which suggests that OsLHT5 may play a role in abiotic stress response in rice (Fan et al., 2023). There were 23 NtLHT genes (NtLHT1-NtLHT23) in tobacco, and the overexpression of NtLHT1 accelerated leaf senescence and affected leaf morphology. Moreover, the expression of NtLHT1 was increased under abiotic stress, and the germination rate in NtLHT1-overexpressing plants was significantly higher than in ntlht1 mutants, which suggests that NtLHT1 may be involved in abiotic stress tolerance in tobacco (Xing et al., 2024). NtLHT22 was localized in the plasma membrane. The amino acid content was significantly altered in NtLHT22-overexpressing plants and ntlht22 mutants, which implies that NtLHT22 participates in amino acid homeostasis in tobacco (Li Z. et al., 2022).

Lonicera macranthoides Hand.-Mazz. is a species of the Caprifoliaceae family, also known as “shanyinhua” or “mountain honeysuckle” (Liu et al., 2024). It is widely planted in Southwestern China, including Hunan Province, Guizhou Province, and Chongqing City. The fresh and dried flower buds of Lonicera macranthoides are used as important ingredients in traditional Chinese medicine to treat and prevent fever, furuncles, inflammation, and cardiovascular diseases (Pan et al., 2021). The LHT gene family is important for plant growth, development, and quality. In this study, we have systematically and comprehensively identified the LHT gene family in L. macranthoides. The physicochemical characteristics, phylogenetic evolutions, gene architecture, chromosomal localization, gene collinearity, and cis-acting elements in the LmLHT gene family were analyzed. Additionally, the expression patterns of LmLHT genes in different tissues and abiotic stresses were examined using quantitative RT-PCR (qRT-PCR). Our research provides useful insights for future functional analysis of LmLHT.

N, including organic N and inorganic N, is a core factor in determining plant yield and quality. In most plants, N is exchanged and transported primarily in the form of amino acids. For this reason, amino acids have multiple functions in plant growth and development and are involved in various biological processes. These functions include regulating N metabolism and protein synthesis, altering root and shoot architecture, and acting as signal molecules to defend against biotic and abiotic stresses (Dong et al., 2024). AATs mediate the transport of amino acids from soil and the translocation to different tissues in plants. The AAAP superfamily is an important part of AATs, and the LHT subfamily is widely studied and functionally characterized by AAAP (Wang et al., 2019). Based on the whole genome sequencing and bioinformatics analysis, LHTs were identified in many species, including Arabidopsis (10 genes), rice (6 genes), tobacco (23 genes), and maize (15 genes) (Li Z. et al., 2022; Rabby et al., 2022), while the members and functions of LHTs are much less known in medicinal plants.

L. macranthoides is a famous traditional medicinal plant that is widely cultivated in Southwestern China. It has been reported that L. macranthoides plays a unique role in defending against animal and human viruses, such as the H1N1 flu virus, SARS coronavirus, and COVID-19. L. macranthoides contains a variety of biologically active compounds, including chlorogenic acid, phenolic acids, flavonoids, and organic acids (Tang et al., 2021). Increasing evidence suggests that amino acids and their derivatives possess antioxidant capacity, and the content of amino acids may provide an important contribution to the pharmacological effects of L. macranthoides (Long et al., 2024). The homeostasis and metabolism of amino acids are closely associated with AATs. However, AATs have yet to be isolated and characterized in L. macranthoides. In this study, we conducted a comprehensive identification and classification of the LHT subfamily of AATs in L. macranthoides.

We identified 11 LHT genes in the L. macranthoides genome, more than the number previously found in rice and Arabidopsis but less than that in tobacco and maize. The difference in the number of LmLHTs compared to other plants may be related to genome size, gene duplication, polyploidization events, and evolutionary history (Chen et al., 2025). Physiochemical properties, such as molecular weight, isoelectric point, amino acid composition, instability index, GRAVY index, and predicted subcellular localization were similar in LmLHT proteins (Table 2), which suggested that LmLHT proteins were relatively conserved during evolution. However, there are also some subtle differences. For example, the number of transmembrane helices in LmLHT proteins varied greatly (Supplementary Figure S1). Phylogenetic analysis of LHT proteins from L. macranthoides, tobacco, rice, and Arabidopsis indicated that these family members were categorized into two subgroups, and subgroup I possesses more LHT proteins than subgroup II (Figure 1), which is consistent with previous studies on tobacco regarding the LHT family (Li Z. et al., 2022). We also found that the distribution of OsLHTs and AtLHTs in the subgroups differs from previous studies, which may be attributed to the differences in bootstrap parameters (Wang et al., 2019). The exon–intron organization in most LmLHT family members was similar (Figure 2B), while the number of motifs in the LmLHT family members were ranged from 9 to 14 (Figure 2C). Therefore, these results imply that the number and type of motifs in LmLHT family members may be closely related with function conservation and diversification.

Gene family expansion is a common event during plant evolution, which in turn increases the gene copies for plants. Therefore, the diversity and quantity of gene families were more complex through gene duplication (Hong et al., 2024). Gene duplication events are well documented in legumes (Chen et al., 2025), grapes (Guo et al., 2025), Arabidopsis (Cheng et al., 2016), etc. Chromosome localization analysis showed that chromosome 4 contained more LmLHT genes (Figure 3), and this distribution pattern may be related to gene duplication. Moreover, we found that tandem and segmental duplication events occurred in the L. macranthoides genome. In addition, 12 gene pairs between L. macranthoides and tobacco, and 6 gene pairs between L. macranthoides and Arabidopsis were observed in the multi-collinearity analysis, while only 2 gene pairs were observed in L. macranthoides and rice (Figure 4). Thus, we speculated that the collinearity gene pairs between L. macranthoides and other plant species were mainly established after the differentiation of monocotyledonous and dicotyledonous plants.

Cis-acting elements are the non-coding DNA sequences identified in the promoter of genes, the types and number of cis-acting elements are closely related to gene function (Li et al., 2021). Promoter analysis of the LmLHT genes revealed that the most abundant cis-acting elements were associated with light response, suggesting that the expression of these genes might be regulated by light stimuli. Phytohormones play multiple roles in various biological processes and signal transduction, and cis-acting elements involved in various phytohormone responses were found in the LmLHT genes. At the same time, biotic and abiotic stress-related cis-acting elements also exist in the promoter of the LmLHT genes (Figure 5). Therefore, the abundant of cis-acting elements in the LmLHT genes promotor may contribute significantly to their functional diversity.

The expression patterns of genes in different plant tissues are helpful for their functional characterization. We analyzed the expression levels of the LmLHT genes in different parts and organs. More than half of the LmLHT genes showed high expression levels in the root. LmLHT6/LmLHT10 are highly expressed in the leaf and LmLHT2/LmLHT11 exhibited constitutive expression in the tested tissues. In addition, the expression level of LmLHT5/LmLHT6/LmLHT8 in the flower is lower than in other tissues (Figure 6). These findings indicated that different LmLHT genes may have identical or reverse functions during growth and development. The expression of AATs in plants could be significantly influenced by abiotic stresses (Tian et al., 2020). In the present study, we found that four and eight LmLHT genes responded to cold and drought stresses, respectively. Cis-acting elements related to cold stress response were not detected in the promoters of LmLHT3/LmLHT9, and a drought-inducibility element was not identified in the promoter of LmLHT11. However, the expression of LmLHT9 and LmLHT11 was increased under cold and drought treatments. In addition, the expression of LmLHT3 was decreased with cold and drought stresses (Figure 7). These results indicate that the expression of LmLHT3/LmLHT9/LmLHT11 under cold or drought stresses may not been directly related to the cis-acting elements on their promoters. Instead, it may be indirectly influenced by transcription factors or epigenetic regulation. Previous studies indicated that overexpression of the light-responsive transcript factor SlBBX20 increased the expression of low-temperature responsive genes in tomato (Ma et al., 2025). The bHLH transcript factor MYC2 is a core regulator of the JA signal pathway, which participates in repeat dehydration stress in Arabidopsis (Liu et al., 2016). The exogenous application of ABA altered DNA methylation and stress-related gene expression in grape, thereby affecting fruit ripening (Li et al., 2024). Therefore, the complex expression patterns of the LmLHT genes indicate that L. macranthoides might have developed specialized regulatory mechanisms to adapt to environmental stresses.

In summary, a systematic study was performed to identify and characterize the LHT family genes in L. macranthoides. The LmLHT genes were studied in terms of their physicochemical characteristics, evolutionary relationships, gene structure, conserved motif, chromosomal locations, collinearity, and cis-acting elements, which provide insights into the evolutionary history of this family in L. macranthoides. The expression patterns of LmLHT genes in different tissues and under cold and drought stresses were complex, suggesting that LmLHT genes play important roles in various biological processes. Overall, this study not only provides useful information for a comprehensive understanding of LmLHT genes, but also lays a solid foundation for the further application of these genes.