Genome-Wide Identification and Functional Analysis of Lysine Histidine Transporter (LHT) Gene Families in Maize

Amino acid transporters (AATs) are essential membrane proteins that transfer amino acids across cells. They are necessary for plant growth and development. The lysine histidine transporter (LHT) gene family in maize (Zea mays) has not yet been characterized. According to sequence composition and phylogenetic placement, this study found 15 LHT genes in the maize genome. The ZmLHT genes are scattered across the plasma membrane. The study also analyzed the evolutionary relationships, gene structures, conserved motifs, 3D protein structure, a transmembrane domain, and gene expression of the 15 LHT genes in maize. Comprehensive analyses of ZmLHT gene expression profiles revealed distinct expression patterns in maize LHT genes in various tissues. This study's extensive data will serve as a foundation for future ZmLHT gene family research. This study might make easier to understand how LHT genes work in maize and other crops.


Introduction
Maize (Zea mays) is a significant and widely produced crop throughout the world. Maize is vital for food and nutritional security as well as economic viability, particularly in resource-scarce smallholder communities [1]. Additionally, it is one of the world's most significant cereal crops and serves as a model system for genetic research [2]. Maize relies heavily on both inorganic (nitrate and ammonium) and organic (amino acids, peptides, and proteins) nitrogen sources in the soil/media [3,4]. Nitrogen (N) is a critical nutrient for plant growth since it is required in a range of compounds in various forms and is primarily found in plants as amino acids. Amino acids are necessary for the formation of enzymes and proteins and the metabolism and structure of plants [5]. Inorganic nitrogen (nitrate and ammonium) can be converted in roots and leaves to amino acids [6]. Proteolytic enzymes require amino acids (AAs) for their production. Amino acids also create polyamines, nucleotides, creatinine, and glucosamine [7]. Furthermore, they are the precursors of nucleic acids, phytohormones, and chlorophyll. ey are also called the low-nitrogen transfer modes [8]. ey are also precursors of secondary metabolites, which are essential for plant growth and development. Abiotic and biotic stresses are also discussed concerning amino acids [9]. Plants get nitrogen from amino acids and uptake from soil by the roots and transport to leaves as well as to other organs via the phloem. e xylem carries amino acids in roots. Membrane transporter proteins are necessary for root cells to uptake amino acids [10].
Candidates could be from the LHT family, described as a lysine and histidine selective transporter which includes high affinity neutral and acidic amino acid transporters [18]. Studies on LHT members revealed their ability to transport a broad spectrum of amino acids. LHT genes have been found in roots, leaves, and flowers. ey can mediate amino acid uptake from the soil and transport and partition inside plants [19]. According to functional tests of 53 putative Arabidopsis AATs, LHT1 has a higher affinity for amino acids than AAP subfamily transporters. In Arabidopsis, a single LHT1 mutation stops plant growth and affects amino acid intake and distribution. To better understand the N cycle in plants, we may overexpress LHT1 in nitrogen-scarce plants [20,21]. AtLHT6 is required for the uptake of acidic amino acids, glutamine, alanine, and maybe phenylalanine by root cells [18]. Ten Arabidopsis LHT genes are involved in AA import into tapetum cells, trichomes, and have a role in organic N transfer for pollen production [22].
ere are six Arabidopsis LHT transporters found in male and female floral tissue: AtLHT1, 2, 4, 5, and 6. ey are also needed for proper sexual plant reproduction. Also, AtLHT4 and AtLHT7 may be involved in anther and pollen development [23]. A qRT-PCR investigation identified OsLHT1 expression in rice root, stem, flag leaf, sheath, and immature panicle. OsLHT1 is restricted to the plasma membrane in rice protoplasts, consistent with its amino acid transporter activity. e loss of OsLHT1 function significantly reduced rice growth and fertility [19].
LHTs' strong affinity and substrate selectivity for neutral and acidic amino acids were likely due to their gene function in plant progenitors. Studies on Arabidopsis LHT localization and expression suggest that LHTs are important in seed plant sexual reproduction [23]. Phylogenetic research guides the evolutionary genesis of amino acid transporters in maize [5,24]. Phylogeny and conserved domains identified genes with similar biological roles and close evolutionary links [25][26][27][28]. Its functional structure is determined by the number of introns in a gene, and the transmembrane helices decide on its tertiary structure in a protein [25,29]. e goal of this study was to do a genome-wide identification and phylogenetic analysis of ZmLHT genes and investigate the evolution of this gene family. In addition, the characteristics of exon-intron structures, patterns of conserved motifs, 3D protein structures, transmembrane domains, and expression patterns were also investigated. e information gathered will aid research into the biological functions of the LHT gene family in maize.

Identification of the ZmLHT Gene Family Members in Maize.
e LHT proteins were found in maize using an HMM (hidden Markov model) profile of AtLHT protein conserved domains based on the PFAM, CDD, and SMART databases. Briefly, a local database based on protein sequences was generated using the TBtools software after downloading the maize genome (B73 RefGen v4) sequence from the maize database (http://www.maizesequence.org/index.html). e known fifteen Arabidopsis LHT protein sequences were downloaded from TAIR (http://www.arabidopsis.org) and utilized as queries in a local BLASTP search against the maize protein database with an e-value of 1e-5 and a criterion of up to 50% identity. Furthermore, all Arabidopsis LHT protein sequences were aligned for multiple sequence alignment using ClustalX 2.1 (https:// clustalx.software.informer.com/2.1/), and the results were used to search the maize database using the online HMMER search engine. Manual editing was used to compare and parse the results of the HMMER search tools and BLASTP. e PFAM database (http://pfam.xfam.org/), the NCBI conserved domain database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/), and the SMART database (http://smart.embl-heidelberg.de/) were used to eliminate redundant sequences by testing for the presence of ZmLHT (PF01490) conserved. To confirm the conserved domains in the remaining sequences, Inter-Pro-Scan (http://www. ebi.ac.uk/interpro/scan.html) was utilized. ese designations were given to maize genes that were close to Arabidopsis genes. e physical and chemical properties of the ZmLHT proteins were examined using the ProtParam service (http://web.expasy. org/protparam/). To estimate the subcellular localization of ZmLHT proteins, the ProtComp server (http://linux1.softberry. com/) was utilized [11,30,31].

Phylogenetic Tree Construction.
e ZmLHT protein sequences generated from the maize genome were aligned using ClustalX (version 2.1). e phylogenetic tree of ZmLHT proteins was then generated using the MEGAX software by comparing AtLHT proteins using the maximum likelihood (ML) technique and 1000 bootstrap replicates [11,30].

Gene Structures and Conserved Motifs Analysis.
e webbased GSDS server (http://gsds.cbi.pku.edu.cn/) was used to investigate the gene structure of ZmLHT proteins, and the conserved domains of the ZmLHT proteins were characterized using the Pfam (http://pfam.xfam.org/) and TBtools software.

Prediction of ree-Dimensional Modeling.
e Phyre2 website (http://www.sbg.bio.ic.ac.uk/phyre2) was used to create the three-dimensional structure of representative ZmLHT proteins in order to investigate structural modifications and their effects on the functions of maize ZmLHT family members [11]. 2 Genetics Research

Transmembrane Domain Prediction.
e crystal structures of transmembrane topology were constructed using the online tool HMMTOP (http://www.sacs.ucsf.edu/cgi-bin/ hmmtop.py), and the transmembrane helices of the ZmLHT proteins were calculated manually [32].

Gene Expression Analysis.
For the expression-based classification of ZmLHTs, gene expression data from a variety of organs at various stages of development, including the leaf, internodes, root, reproductive tissues (tassel, silk, and cob), seed, embryo, and endosperm were analyzed. eir gene expression data retrieved from MaizeMine (http:// maizemine. rnet. missouri. edu: 8080/maizemine/begin.do), a publicly accessible transcriptome resource [33]. e heat map was created using log2 transformed FPKM (fragments per kilobase per million) values to show expressions. e FPKM values were determined using Edge R, an R program.
e TBtools software generated the heat maps for the FPKM values dataset [34].

Identification and Characterization of the Members of LHT Gene Families in Maize.
A total of 15 LHT proteins were identified in the maize genome by sequence validation and designated ZmLHT1-ZmLHT15 (Table 1). e biochemical and physiological characteristics of all translated proteins are summarized in Table 1. e amino acids length in maize LHT proteins ranged from 160 (ZmLHT15) to 904 (ZmLHT1). e molecular weights of the ZmLHT proteins ranged from 18.27 (ZmLHT15) to 100.65 (ZmLHT1) kD.
e pI values of all ZmLHT proteins exceeded 7, indicating that they were all basic proteins. GRAVY scores of all proteins were positive, indicating that they were hydrophobic. All ZmLHT proteins are expected to be localized to the plasma membrane on a subcellular level.

Phylogenetic Analysis of the Members of LHT Gene
Families in Maize. Based on the results of phylogenetic analysis, 15 LHT proteins were classified into three subcategories ( Figure 1). e three subcategories are A, B, and C. Group A included 11 ZmLHTs (1-10 and 15). Only ZmLHT12 was classified as a member of group B, whereas the remaining three ZmLHTs (11,13, and 14) were classified as members of group C. Maize LHT proteins belonging to subclasses A, B, and C may share functional similarities with AtLHT1,2,5,10; AtLHT3, 6,8,9; and AtLHT4,7 proteins; respectively. e HMM model's encoded genes did not agree with the phylogenetic analyses. is could be because the algorithms utilized in the two models are different. Both, however, account for the functional characteristics of the LHT proteins, which are connected with distinct clades of Arabidopsis genes.

Gene Structures and Conserved Motif Analysis of LHT Gene Families in Maize.
e emergence of multigene families has had a significant impact on the diversity of gene architecture. Exon-intron structures vary among ZmLHTs, as illustrated in Figure 2. Under different cluster groups, the number of introns in the ZmLHT gene family varies from two (ZmLHT2,14, and 15) to ten (ZmLHT1) (Figure 2). e number of introns in ZmLHT group A ranges from two (ZmLHT2,14) to ten (ZmLHT1). As a member of group B, the ZmLHT12 has six introns. e number of introns in ZmLHT group C varies from two (ZmLHT2) to four (ZmLHT11,13). e intron-exon organization and phylogenetic tree demonstrated that members of the same group had similar gene structures.

ree-Dimensional Modeling of the LHT Gene Family in
Maize. After predicting the gene structures of ZmLHTs, we analyzed secondary protein structures to predict the rearrangements of structures and the nature of polypeptide bonds present in ZmLHTs proteins. As per three-dimensional protein structure analysis, all ZmLHT proteins had numerous alpha helices, transmembrane helix, and coil topologies (Figure 4). In the ZmLHTs, the percentage of alpha helix ranged from 63% (ZmLHT11) to 81% (ZmLHT15). e fraction of transmembrane helices in ZmLHTs ranged from 50% (ZmLHT11) to 68% (ZmLHT12). All proteins had a 100% confidence level (Table S1). On the other hand, in Arabidopsis LHT (AtLHT) protein, the percentages of alpha helix ranged from 64% to 72%. Compared to AtLHT, the percentage of alpha helix in ZmLHTs is higher up to 81% (ZmLHT15). However, the overall percentages of alpha helix are identical between AtLHT and ZmLHT proteins. Similarly, fraction of transmembrane helices in ZmLHTs and AtLHTs are identical to each other (Table S2), which summarized that the protein structure of ZmLHT and AtLHT are almost similar on the basis of secondary structure of proteins.

Transmembrane Domains in LHT Gene Family in Maize.
e ZmLHT proteins have multiple transmembrane (TM) helices in their transmembrane domains. Except for ZmLHT14 and 15, which had six and four transmembrane helices but extended intracellular sequences at the N-terminus and C-terminus, all LHT proteins had 8-12 transmembrane helices ( Figure 5). Transmembrane segments were found to span the full length of all LHT proteins. e transmembrane domains of the ZmLHT proteins range in length from 160 (ZmLHT15) to 904 (ZmLHT1) ( Table S3).

Expression Analysis of Maize LHT Genes in Different
Tissues and Developmental Stages. e expression patterns of ZmLHTs were investigated using RNA-seq data retrieved from an online database from specific tissues at distinct developmental stages. On the heat map, the normalized log2 (FPKM) value was displayed ( Figure 6). Based on their expression patterns, the ZmLHTs were divided into three groups. Except for embryo and endosperm tissues, the single (ZmLHT2) gene in the first cluster exhibited a relatively high expression level and was stably expressed in almost all tissues at different developmental stages. e eight (ZmLHT4,7,8, and 11-15) genes in the second cluster were expressed in low amounts in most tissues where expression level of ZmLHT10 is near to zero. However, a few, such as ZmLHT11 and 14, were slightly expressed in all tissues except seed and leaves, respectively, while ZmLHT13 were slightly expressed in primary root zones. e other five (ZmLHT1,3,5,6, and 9) genes in the third cluster showed a wide range of spatiotemporal expression patterns, with ZmLHT1 and 5 being highly expressed in the leaves, and ZmLHT3 being highly expressed in the roots. Furthermore, ZmLHT1 was found to be slightly expressed in two or more tissues, such as leaves, internodes, and roots; ZmLHT2 was found to be moderately expressed in leaves, internodes, roots, and reproductive tissues. On the other hand, ZmLHT3 was found to be highly expressed in roots compared to leaves.

Discussion
Amino acid transporters (AATs) transport and distribute several types of amino acids in plants, and they are key targets for crop development [35]. AATs have been found   mays have yet to be discovered. In this study, 15 LHTs were identified in Z. mays that contained AAAP super families based on their resemblance to previously reported AATs in Arabidopsis thaliana, O. sativa, and S. tuberosum plants [36,37]. Surprisingly, the number of members in the AAP subfamily is the same in Arabidopsis, S. tuberosum, and S. lycopersicum (8), but it is more than double in O. sativa (19 members). e increase of AAP subfamily members in O. sativa could be the result of tandem and segmental duplication events [36]. e total number of members in a same subfamily varies by species. For example, the LHT subfamily has six members in O. sativa, ten in Arabidopsis, 11 in S. tuberosum, 13 in S. lycopersicum [38], and 15 in Z. mays (Table 1). Different subfamilies showed an abundant variety in subcellular localization, such as AAP, LHT, AUX, GAT, and ProT were found on the plasma membrane, whereas TTP, ACT, ATLb, and ANT were located on the vacuole membrane. However, few members of the same subfamily displayed diverse subcellular localization, such as the ATLa subfamily genes positioned on both the plasma membrane and vacuole; the CAT and PHS subfamilies placed on the plasma, chloroplast, and vacuole [34]. In this study, all 15 LHT genes were located on the plasma membrane (Table 1). Previous research recommended that motifs 1-4 function as a transmembrane region containing transmembrane transporter activity. e existence of the same type of conserved motif recommends that members of the same family may perform similar activities [39]. In this study, we found a total of 13 unique motifs and all ZmLHTs have at least 1-4 motifs except ZmLHTs11, 13, 14, and 15. Motif analysis revealed that the identified ZmLHTs exhibit transmembrane transporter activity. e multiple alpha helices structures during three-dimensional protein modeling guide the functional efficiency and evolutionary origin of a protein.

Genetics Research
e stable transmembrane helices indicate the functional integrity of proteins [33]. e analyzed three-dimensional structure of ZmLHT proteins with multiple alpha and transmembrane helices indicate the functional integrity and transport efficiency of the LHT proteins.
Transmembrane domain guides to evaluate the internal membrane protein structure and the full topology of protein. It also helps to predict genome analysis such as structural variation and gene expression [36]. e ZmLHTs hydrophobic transmembrane domain was predicted, and the family members had 4-21 transmembrane helices. e transmembrane structure can be classified into three categories based on the position of the N/C terminal: the N/C terminals of ZmLHT2-8 and 14 were all predicted as cytoplasmic and containing 6-10 TMs; the N/C terminals of ZmLHT15 were predicted as extracellular and containing 4 TMs; and the N/C terminals of the other ZmLHT proteins were predicted both as extracellular and cytoplasmic containing 9-21 TMs, including ZmLHT1 and 9-13. Our analysis results highlighted that all ZmLHTs are located in the plasma membrane that represents all LHTs might responsible for the transportation of amino acid from cell wall to inside the cell [23]. e study of ZmLHT gene expression patterns may yield important information for determining their likely function [40,41]. e current 10  study found that a number of genes were expressed in specific tissues and developmental stages based on RNA-Seq data analysis of ZmLHT genes. As shown in Figure 6, the expression profiles of ZmLHT genes that were found to be highly expressed may be linked to certain organs such as leaves, roots, internodes, and reproductive cells. According to prior research, AtLHT1 is essential for amino acid intake and mesophyll production in roots, whereas AtLHT2 is primarily involved in amino acid movement and allocation in floral organs [42,43]. Another study discovered that AtLHT1 and AtLHT6 are highly expressed in roots and play a role in amino acid intake from the soil [44]. CsLHT1, CsLHT2, and CsLHT6 are likely involved in the root uptake of amino acids from the soil in the tea plant [45]. SiLHT2, 5, and 10 play critical roles in the transport and distribution of amino acids in the leaf; SiLHT11 uptakes amino acids in the root; and SiLHT9 participates in the long-distance transportation of amino acids from the root to the leaf in foxtail millet [34]. e current study's spatiotemporal expression analysis revealed that some of the discovered genes are selectively expressed in leaves, internodes, roots, and reproductive organs. ZmLHT2 is abundant in leaves, internodes, and roots. Furthermore, ZmLHT1, 3, 5, 6, and 9 are substantially expressed in leaves but only modestly so in internodes. In summary, the expression patterns suggest that ZmLHTs may play an important role in amino acid intake in various tissues and developmental stages of maize. e current study's elucidation of the ZmLHT genes would pave the way for more research in AATs to better understand their importance and roles in plant physiology as well as in yield.

Conclusion
To summarize, this was the first comprehensive and systematic study of the LHT gene family in maize. e maize genome contains a total of 15 ZmLHT genes. Following that, in-silico analysis and expression analysis were used to deduce the probable functions of these genes in maize growth, development, and functional advancement. e findings indicate that the ZmLHT genes are involved in multiple aspects of nitrogen metabolism and might have an effect on maize growth and development. ese findings pave the way for additional research into the molecular mechanisms behind the uptake, assimilation, and transport of nitrogen and amino acids in maize.

Data Availability
e data which support the results of this study are available in databases described in the manuscript and from the corresponding authors upon request.

Conflicts of Interest
e authors declare that they have no competing interests.

Authors' Contributions
Md. Numan Islam and Md. Golam Rabby designed this study. Md. Golam Rabby and Md. Mostafa Kamal performed computational and data analyses and analyzed the data. Md. Numan Islam and Md. Munnaf Hossen wrote the manuscript and revised. All authors read and approved the final manuscript.

Supplementary Materials
Supplementary