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Article

Genome Wide Analysis of Amino Acid Transporter Superfamily in Solanum lycopersicum

by
Fatima Omari Alzahrani
Department of Biology, Faculty of Science, Albaha University; Albaha 65527, Albaha Province, Saudi Arabia
Plants 2021, 10(2), 289; https://doi.org/10.3390/plants10020289
Submission received: 24 December 2020 / Revised: 31 January 2021 / Accepted: 2 February 2021 / Published: 3 February 2021
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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Abstract

:
Amino acid transporters (AATs) are integral membrane proteins and have several functions, including transporting amino acids across cellular membranes. They are critical for plant growth and development. This study comprehensively identified AAT-encoding genes in tomato (Solanum lycopersicum), which is an important vegetable crop and serves as a model for fleshy fruit development. In this study, 88 genes were identified in the S. lycopersicum genome and grouped into 12 subfamilies, based on previously identified AATs in Arabidopsis, rice (Oryza sativa), and potato (Solanum tuberosum) plants. Chromosomal localization revealed that S. lycopersicum AAT (SlAAT) genes are distributed on the 12 S. lycopersicum chromosomes. Segmental duplication events contribute mainly to the expansion of SlAAT genes and about 32% (29 genes) of SlAAT genes were found to originate from this type of event. Expression profiles of SlAAT genes in various tissues of S. lycopersicum using RNA sequencing data from the Tomato Functional Genomics Database showed that SlAAT genes exhibited tissue-specific expression patterns. Comprehensive data generated in this study will provide a platform for further studies on the SlAAT gene family and will facilitate the functional characterization of SlAAT genes.

1. Introduction

Tomato (Solanum lycopersicum) has great global commercial importance owing to its nutritional value and serves as a model for fleshy fruit development and as a reference species for plants in the Solanaceae family. Several genome-wide studies have identified 63, 87, 23, 189, and 72 genes encoding amino acid transporters (AATs) in Arabidopsis thaliana, Oryza sativa, Selaginella, Glycine max, and Solanum tuberosum, respectively [1,2,3,4,5]. Although AATs have been studied in several plant species, there is limited information available about AATs in S. lycopersicum.
AATs are integral membrane proteins, which mediate the nitrogen allocation between source and sink [6] in plants. The phloem sap is rich in amino acids and they are responsible for procuring the organic [2]; nitrogen that is necessary for plant growth and development [7]. Furthermore, AATs play fundamental roles in plant physiological processes such as defense against pathogens and resistance to abiotic stresses.
AATs are divided into two families; the first is amino acid/auxin permease (AAAP), which is in turn sub-grouped into eight subfamilies, including amino acid permease (AAP), lysine/histidine transporter (LHT), γ-aminobutyric acid (GABA) transporter (GAT), proline transporter (ProT), like-auxin influx carriers (LAX), aromatic and neutral amino acid transporter (ANT), vesicular aminergic-associated transporter (VAAT), and amino acid transporter-like proteins (ATL) [8]. The second family comprises of amino acid-polyamine-choline (APC) transporters, which in turn is sub-grouped into four subfamilies, including amino acid/choline transporters (ACT), tyrosine-specific transporter (TTP), cationic amino acid transporter (CAT), and polyamine H+ co-transporters (PHS) [9]. In addition, a new subfamily has been added to the AAT superfamily called Usually Multiple Amino Acids Move In and Out Transporters (UMAMIT) [10].
A few studies have functionally characterized the members of the AAT superfamily in plants. Eight AAPs have been identified in Arabidopsis, which are localized in the plasma membrane and involved in the H+-coupled amino acid uptake system [11]. AAP1 and AAP2 in Arabidopsis, the first two AATs to be characterized, have different substrate specificity with high expression in siliques of Arabidopsis, suggesting their involvement in supplying the seeds with organic nitrogen [12]. AAP3, AAP4, and AAP5 were later isolated from Arabidopsis with broad substrate specificities and AAP5 was found to participate in amino acid transport in the developing embryo [13]. Arabidopsis thaliana AAP7 (AtAAP7) is still not functionally characterized. AtAAP6 and AtAAP8 are involved in high-affinity amino acid transport, since AAP8 imports organic nitrogen into developing seeds and AAP6 is expressed in xylem, which has low concentrations of amino acids [14]. Similar to Arabidopsis, eight members of AAPs have been identified in S. tuberosum [5], and 19 have been identified in O. sativa [1]. AAPs in other plant species have also been functionally characterized. For example, AAP1 is expressed in the leaves and is involved in the long-distance transport of amino acids in S. tuberosum [15]. Three primases, including VfAAP1, VfAAP3, and VfAAP4 were isolated from Vicia faba L. and have been shown to transport a broad range of amino acids [16].
Ten LHTs (AtLHT1–10) have been identified in Arabidopsis. They were found to be localized in the plasma membrane and were found to import organic nitrogen into the roots, mesophyll cells [17], and the cells of reproductive floral tissue [18]. AtLHT1 together with AtAAP5 are involved in the uptake of neutral, acidic, and basic amino acids from the soil when amino acid levels in the nutrient solutions (or soil) are low [19,20].
Unlike AAPs and LHTs, the only proteinogenic amino acid transported by ProTs is proline [21]. However, studies have shown that ProTs are responsible for transporting glycine betaine and GABA [22]. Three ProTs (AtProT1, AtProT2, AtProT3) were identified in Arabidopsis and it was shown that despite the similar localization of the three transporters in the plasma membrane and similar affinity to glycine betaine, each transporter has a different role in Arabidopsis [22]. For example, AtProT1 is highly expressed in the phloem, suggesting its involvement in long-distance transport of compatible solutes. AtProT2 is active in the roots, while AtProT3 is active in the epidermal cells of the leaves [22].
ProT1 in S. lycopersicum is a general transporter for compatible solutes [23] and transports proline to roots even under salt-stress conditions [24]. Regarding GATs in Arabidopsis, it was revealed that AtGAT1 has a high affinity to GABA and is localized in the plasma membrane [25]. In addition, it is highly expressed in flowers under elevated GABA conditions, such as wounding and senescence [25].
Aux1 mutant Arabidopsis plants show defects in the root gravitropic response, and AtAUX1 is expressed in the columella, lateral root cap, epidermis, and stele tissues of the primary root [26]. AtAUX1 together with AtPIN2 (auxin exporter) regulate root gravitropism [27], and AtAUX1 promotes lateral root formation and phyllotactic pattern in Arabidopsis [28]. In Arabidopsis, the AUX1 gene belongs to a small family consisting of four members, including AUX1 and three LAX genes (LAX1, 2, and 3) [29], while there are five AUX/LAX transporters in O. sativa [30]. Regarding ANT transporters, only AtANT1 was characterized in Arabidopsis, which transports arginine, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid [31]. Of CAT transporters, nine members have been functionally characterized in Arabidopsis with varying affinities and functions [32]. For example, AtCAT5 is a high-affinity basic amino acid transporter and is involved in reuptake of the leaking amino acids at the leaf margin, while AtCAT2 is involved in long-sought vacuolar amino acid transport [32]. Recently, six CAT members were identified and characterized in Camellia sinensis and their expression was revealed to be sensitive to abiotic stress [33]. In Arabidopsis, a bidirectional amino acid transporter (BAT), which performs both exporting and importing activities, was reported to exhibit transport activity for alanine, arginine, glutamate, and lysine [34].
Since the functions of S. lycopersicum AATs are not fully studied, we aimed to elucidate the entire members of AAT gene superfamily in S. lycopersicum. In this study, a genome-wide identification and phylogenetic analysis of S. lycopersicum AAT (SlAAT) genes were performed for AAT superfamily classification and to explore the evolution of this gene superfamily. In addition, the features of the exon-intron structures, patterns of the conserved motifs, and duplication events, including tandem and segmental duplications within the tomato genome that likely contribute to the expansion of the SlAAT superfamily were explored. The resulting data will be useful in studies on the biological functions of each gene in the SlAAT superfamily.

2. Results

2.1. Identification of SlAATs in the S. lycopersicum Genome

Using ATTs of Arabidopsis thaliana, O. sativa, and S. tuberosum as queries in BLAST search at SGN with ‘amino acid transporter’ and ‘amino acid permease’ as keywords, we identified 88 members of SlAATs. All the retrieved protein sequences of SlAATs were subjected to InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/ (accessed on 31 January 2021)), and all the candidate proteins were found to contain AAT domain(s). Information about locus identity number of SlAATs assigned by SGN (Solanaceae Genomics Network, http://solgenomics.net/SOL (accessed on 31 January 2021)), the given nomenclature of S. lycopersicum AATs, number of intron(s) in the SlAAT genes, length of the ORF for SlAATs, protein characterization of SlAATs (amino acid length, MW, pI), and genomic location are presented in Table 1. The intron number ranged from 1 to 13 and the length of the ORF for SlAATs ranged from 267 to 3376 bp. It was observed that each subfamily member shared a nearly similar gene structure and intron numbers. For example, members of ProT subfamily contained six introns and an ORF length of 1473–1828 bps. The intron-exon regions in each genomic sequence of SlAAT are illustrated in Figure 1.
The number of transmembrane regions (Table 1), which were predicted by the TMHMM Server, were found to range from 2 to 18 and some members of each subfamily of SlAATS shared a similar number of transmembrane regions. Using the information available in the SGN about SlAATs, it was determined that SlAAT genes are distributed in all the 12 chromosomes in S. lycopersicum (Figure 2). An uneven distribution of SlAAT genes on the 12 chromosomes was observed; for example, chromosome 2 contained 13 SlAAT genes, while chromosome 7 contains four SlAAT genes. The gene duplication data obtained from PGDD (Plant Genome Duplication Database, http://chibba.agtec.uga.edu/duplication (accessed on 31 January 2021)) (S. lycopersicum vs. S. lycopersicum) revealed that 29 SlAAT genes originate from the duplication events and there are 16 gene pairs of SlAATs (Table 2). Examining the 16 gene pairs according to the criterion of tandem duplication, all the duplication events were characterized to be segmental duplication. Thus, the divergence time ranged from 39.70 to 240.07 May. According to the phylogenetic relationships, the proteins of duplicated genes are close to each other and have higher sequence similarity; for example, the sequence similarity between SlLAX2 and SlLAX5 was found to be 93.15%. Only in two cases, the similarity between the proteins of duplicated genes was found to be below 50%; the similarity between SlLHT1 and SlLHT10 was 38.84%, and that between SlCAT1 and SlCAT4 was 39.19%. To study the selection pressure among the SlAAT duplicated gene pairs, Ka/Ks was calculated (Table 2) and the results revealed that they evolved under purifying selection (Ka/Ks < 1).

2.2. Phylogenetic Analysis and Classification of the SlAATs

The phylogenetic tree, which that was constructed after the alignment of AAT amino acid sequences of S. lycopersicum, O. sativa and Arabidopsis thaliana, revealed that the 88 SlAATs could be clustered into 12 clades (Figure 3). These SlAATs can be classified into two main superfamilies: AAAP and APC. The AAAP family can be classified into eight subfamilies: AAP, LHT, GAT, ProT, LAX, ANT, VAAT, and ATL. On the other hand, the APC family can be classified into four subfamilies according to phylogenetic tree: ACT, TTP, CAT, and PHS.
Motif analysis using MEME showed that each subfamily has a similar motif and nearly the same their number (Figure 4). For example, all members of the AAP subfamily were found to have the same motifs. In addition, some motifs are more common in one family than the other. For example, motif 1, 2, and 5 are likely to be more common in the AAAP family than the APC family. On the other hand, some motifs were specific to one subfamily; for example, motif 17 was specific to the LAX subfamily and motif 19 was specific to the ATL subfamily. Analysis of the transmembrane region conservation, revealed that most of the transmembrane regions were highly conserved. The alignment of AAP amino acid sequences and the transmembrane conserved region are illustrated as an example in Figure 5.

2.3. Expression Analysis of SlAAT Genes Based on RNA-Seq Data

The RNA-Seq data from various tissues during vegetative and reproductive developmental stages of the tomato cultivar S. lycopersicon ‘Heinz’ and the wild relative S. pimpinellifolium was used to study the expression of SlATT genes (Figure 6). According to the column dendrogram, it was observed that there is a similar expression pattern between the leaves of both pimpinellifolium and S. lycopersicon ‘Heinz, S. lycopersicon ‘Heinz 1 cm fruit, S. lycopersicon ‘Heinz 2 cm fruit, S. lycopersicon ‘Heinz 3 cm fruit, and S lycopersicon ‘Heinz mature green fruit, S. pimpinellifolium immature green fruit, S. pimpinellifolium breaker fruit, S. lycopersicon ‘Heinz breaker fruit, and S. lycopersicon ‘Heinz breaker+ 10 fruit, S. lycopersicon ‘Heinz unopened flower buds, and S. lycopersicon ‘Heinz fully opened flowers. The expression of SlLHT3, SlANT5, SlLHT5, SlVAAT7, and SlCAT2 was detected to be high only in S. lycopersicon ‘Heinz unopened flower buds and extremely low in other examined parts of S. pimpinellifolium and S. lycopersicon ‘Heinz tomato plants. In addition, six genes, including SlAAP8, SlLHT4, SlLHT12, SlLHT13, SlAAP5, and SlAAP3 showed high expression levels in S. lycopersicon ‘Heinz unopened flower buds and fully opened flowers compared to other organs of both S. pimpinellifolium and S. lycopersicon ‘Heinz tomato plants.
The expression of SlLHT9, SlCAT9, SlCAT10, and SlATL5 was considerably high in both S. lycopersicon ‘Heinz unopened flower buds and S. pimpinellifolium leaves but was low in other examined organs. The expression of SlTTP1, SlLAX5, SlVAAT10, SlCAT3, and SlVAAT2 appeared to be upregulated only in the leaves. Interestingly, SlVAAT9, SlLHT8, SlGAT3, SlCAT6, and SlCAT5 were observed to be highly expressed only in the roots, while the expression in other organs was downregulated. The expression of genes of the AAP family: SlAAP2, SlAAP3, SlAAP4, SlAAP5, SlAAP6, SlAAP7, and SlAAP8 was observed to be high in some organs such as S. lycopersicon ‘Heinz unopened flower buds and S. lycopersicon ‘Heinz fully opened flowers, but low in the other examined organs. Six out of 13 genes of the LHT subfamily (SlLHT1, SlLHT2, SlLHT6, SlLHT7, SlLHT8, and SlLHT10) showed high expression in the roots. The high expression of ProT genes was observed in different organs; for example, SlProT1 and SlProT2 were highly expressed in the flowers and in buds respectively, while SlProT3 and SlProT4 were highly expressed in both S. lycopersicon ‘Heinz leaves and roots. The genes of LAX subfamily were highly expressed in the roots except SlLAX3, which showed a high expression in the fruits (S. lycopersicon ‘Heinz breaker fruits, S. pimpinellifolium breaker fruits, S. pimpinellifolium immature green fruits, S. lycopersicon ‘Heinz mature green fruits, and S. lycopersicon ‘Heinz 3 cm fruits). The expression of SlBAT2 and SlBAT3 was the same in all the examined organs. Two genes of the TTP subfamily (SlTTP1 and SlTTP4) exhibited a similar expression pattern: high in the leaves and low in other organs.

2.4. Syntenic Analysis

Syntenic analysis performed among the AATs in S. lycopersicum, Arabidopsis thaliana, and O. sativa demonstrated that the S. lycopersicum AATs are orthologs of a number of O. sativa and Arabidopsis thaliana AAT genes (Figure 7 and Figure 8). For example, SlLAT5 and SlLAT6 genes are orthologs of OsLAT7 (LOC_Os01g61044.1), and StLAX2 is an ortholog of OsAUX3 (LOC_Os03g14080), whereas StLAX4 is an ortholog of OsAUX1 (LOC_Os01g63770). Syntenic AAT gene pairs between S. lycopersicum and Arabidopsis thaliana, and between S. lycopersicum and O. sativa are presented in Supplementary Tables S1 and S2.

3. Discussion

AATS have been identified in several plant species such as Arabidopsis thaliana, O. sativa, and S. tuberosum. However, ATTs in S. lycopersicum have not been identified so far. In this study, 88 AATs were identified in S. lycopersicum and divided into two superfamilies based on their similarity with previously identified AATs in Arabidopsis thaliana, O. sativa, and S. tuberosum plants [1,5]. Interestingly, the number of members in the AAP subfamily are the same (8 members) as that in Arabidopsis, S. tuberosum, and S. lycopersicum but in O. sativa, this number is more than double (19 members). This expansion in the members of AAP subfamily in O. sativa may be due to segmental and tandem duplication events [1]. However, the number of members in the same subfamily varies between species. For example, there are six members in the LHT subfamily in O. sativa, ten in Arabidopsis, 11 in S. tuberosum, and 13 in S. lycopersicum. In fact, the largest subfamily in S. lycopersicum is LHT and the smallest subfamilies are ACT and GAT with only three members each (Table 1). Our results of the phylogenetic analysis were in agreement with those of O. sativa, Arabidopsis, and S. tuberosum. The SlAATs were divided into two main clades: AAAP and APC superfamilies (Figure 3). Similar to Arabidopsis thaliana, O. sativa, and S. tuberosum, the chromosomal mapping of S. lycopersicum AAT genes showed that they are distributed throughout the 12 chromosomes, and most SlAAT genes were localized on chromosome 2 and the least were found on chromosome 7 (Figure 2). In the current study, we found that segmental gene duplication within the tomato genome mainly contributed to the expansion of SlAAT superfamily in S. lycopersicum, and no tandem duplication was found on the gene pairs. This is not the case for O. sativa, since segmental and tandem duplication contributes equally to the expansion of the AAT superfamily [1] and in S. tuberosum, only tandem duplication contributes greatly to the expansion of the AAT superfamily [5]. Examination of the expression patterns of the segmental duplicated genes of SlAAT superfamily within tomato genome, showed that each gene exhibits a distinct expression pattern. This may suggest that segmental duplication events in the SlAAT superfamily in S. lycopersicum do not have overlapping functions and therefore may contribute to the functional divergence of the duplicated genes.
The analysis of the expression patterns of SlAAT genes may provide useful information for determining their function. In the current study, expression analysis of SlAAT genes based on RNA-Seq data showed that a number of genes were expressed in specific tissues and developmental stages. As seen in Figure 6, the expression profiles of SlAAT genes that were highly expressed may be associated with specific organs, including leaves, root, and flowers, but not fruits.
The genes encoding SlLAX1, SlLAX2, SlLAX4, and SlLAX5 are specifically expressed in roots. Syntenic analysis revealed that StLAX1 is an ortholog of OsAUX1 and OsAUX2, which both showed high expression in the roots. In addition, StLAX1 is an ortholog of AtAUX1 (AT2G38120), which is known to be primarily expressed in the roots [37]. Further, the expression of SlLHT2, SlCAT5, and SlProT4 were observed to be high in the roots. Syntenic analysis showed that SlLHT2, SlAAP2, and SlProT2 are orthologs of AtLHT1, AtAAP5, and AtProT2, respectively, which are reported to play a role in amino acid uptake by the roots [20,38,39].
In addition, SlCAT6 expression was high in both S. lycopersicon ‘Heinz roots and S. lycopersicon ‘Heinz fully opened flowers. AtCAT6, was revealed to be involved in amino acid uptake into the sink cells of flowers and root primordia [40]. The expression of SlAAP8, the ortholog of AtAAP6, was high in S. lycopersicon ‘Heinz unopened flower buds and S. lycopersicon ‘Heinz fully opened flowers. However, AtAAP6 expression is mainly present in the xylem parenchyma cells [41].
The expression patterns of SlAAT genes elucidated in the current study provide foundation for further investigation of the AATs to completely understand their importance and roles in plant physiology and their contribution to plant productivity. Moreover, as AATs are usually induced by abiotic stresses such as salinity and drought [21], it is important to perform further studies on their interaction with the environment to evaluate the plant performance under stress conditions.

4. Materials and Methods

4.1. Identification of AAT Genes in S. lycopersicum

To identify the AATs in S. lycopersicum, those in Arabidopsis, O. sativa, and S. tuberosum [1,5] were used as queries in Basic Local Alignment Search Tool (BLAST) in the SOL Genomics Network (SGN; http://solgenomics.net/ (accessed on 31 January 2021)) against the S. lycopersicum genome and protein sequence database (version SL4.0) with default settings. The databases were also queried for homologs using ‘amino acid transporter’ and ‘amino acid permease’ as keywords. The redundant sequences were removed, and the remaining protein sequences were submitted to InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/ (accessed on 31 January 2021)) to scan for AAT domains. Information about gene structure (length, number of introns, length of the open reading frame (ORF), locus accession, amino acid length, and genomic location were all acquired from SGN. Molecular weight (MW) and the theoretical isoelectric point (pI) were predicted using the Compute/Mw tool (http://web.expasy.org/ (accessed on 31 January 2021)). The gene structure of SlAATs were also analyzed using Gene Structure Display Server (GSDS) (http://gsds.gao-lab.org/index.php (accessed on 31 January 2021)) [42]. The TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 31 January 2021)) was used to predict the putative transmembrane regions in each SlAAT protein [43]. In order to display the gene structure of the exons and introns of SlAATs, the genomic and cDNA sequences of each SlAAT identified in this study were retrieved from the SGN and used as queries in the GSDS (http://gsds.cbi.pku.edu.cn/ (accessed on 31 January 2021)).The nomenclature of S. lycopersicum AATs was assigned according to chromosome order and considering their phylogenetic relationship. However, SlLax1, SlLax2, SlLax3, SlLax4, and SlLax5 have been previously named [44].

4.2. Chromosomal Mapping of SlAAT Genes and Gene Duplication

The approximate locations of SlAAT genes were identified using the available information at SGN, and mapped onto the 12 corresponding S. lycopersicum chromosomes using MapChart software [45].
The Plant Genome Duplication Database (PGDD) (http://chibba.agtec.uga.edu/duplication (accessed on 31 January 2021)) was used to download the gene pairs of the S. lycopersicum genome and the related information about synonymous substitution rate (Ks) and nonsynonymous substitution rate (Ka) was also obtained. Genes distributed nearby and separated by five or fewer genes was selected as the criterion for considering tandem duplicates. Divergence time (Mya, million years ag) of the gene pairs was estimated using the following equation: T = Ks/2x (x = 6.56 × 10−9), in which x is the mean synonymous substitution rate for tomato [46].
The sequence similarity between the proteins of duplicated genes was calculated using the sequence manipulation suite programs (https://www.bioinformatics.org/sms2/ident_sim.html (accessed on 31 January 2021)).

4.3. Syntenic Analysis

A multiple collinear scanning toolkit (MCScanX) [47] was used to perform an examination of syntenic regions between the AATs of S. lycopersicum, Arabidopsis thaliana, and O. sativa and the result was plotted using Dual Synteny Plotter software [48].

4.4. Phylogenetic Analysis and Sequence Alignment

In order to construct the phylogenetic tree, the multiple sequence alignment AAT proteins of S. lycopersicum, O. sativa, and Arabidopsis thaliana was performed using MEGA 6.0 software, with the default settings of MUltiple Sequence Comparison by Log-Expectation (MUSCLE) alignment [49]. The phylogenetic tree was constructed using the maximum likelihood method with the following settings: test of phylogeny: bootstrap method; substitution type: amino acid, rates among sites, uniform rates; missing data treatments: use all sites; ML heuristic method: nearest-neighbor-interchange (NNI); initial tree for ML: make initial automatically tree; branch swap filter: none; number of threads: 3. For maximum likelihood analysis, the best model of protein evolution was determined in MEGA6 using the ‘find best DNA/protein models’ tool. The best model was the ‘Whelan and Goldman’ (WAG) model [35]. The robustness of the analyses was examined using 500 bootstrap replicates [50]. To analyze the transmembrane region conservation, the amino acid sequences of SlAATs were aligned using MUSCLE with default settings in Jalview version 2 [51].
The motifs of SlAAT proteins were identified using the MEME tool (http://meme-suite.org/tools/meme (accessed on 31 January 2021)) with default settings, except that the maximum number of motifs was defined as 20.

4.5. Expression Analysis of SlAAT Genes Based on RNA-Seq Data

Expression profiles of the SlAAT genes were obtained from RNA sequencing (RNA-Seq) data from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/ (accessed on 31 January 2021)) using the transcriptomic analysis of various tissues in the S. lycopersicon ‘Heinz and the wild relative S. pimpinellifolium. The search was performed using the locus name as a query. The heatmap was generated using ComplexHeatmap version 2.2.0 package in R [52].

5. Conclusions

In this study, the AAT gene superfamily in S. lycopersicum was identified and analyzed using a broad range of bioinformatic tools. The SGN was first used to retrieve the nucleotide and amino acid sequences of the SlAATs to obtain general information and sequence characterization of SlAATs. Chromosomal localization and gene duplication events in SlAATs were demonstrated. In addition, the phylogenetic relationships and protein motifs of SlAAts were determined. The expression profiles of SlAAT genes in various tissues of S. lycopersicum were elucidated using RNA-Seq data in the Tomato Functional Genomics Database. This study will facilitate further investigation of the SlAAT gene superfamily and the functional characterization of the members of SlAATs. Further analysis of expression profiles under abiotic stress conditions will reveal their potential roles in response to environmental stress.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/10/2/289/s1, Table S1: Syntenic AAT gene pairs between Solanum lycopersicum and Arabidopsis thaliana. Table S2: Syntenic AAT gene pairs between Solanum lycopersicum and Oryza sativa.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Exon-intron structures of the identified SlAAT genes. The graphic representation of the optimized gene model displayed using Gene Structure Display Server (GSDS) (for interpretation of the references to colors in this figure, the reader is referred to the web version of this article). Green boxes represent exons. The black solid lines connecting two exons represent introns, and the blue boxes represent upstream/downstream region of SlAAT genes. AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carrier; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
Figure 1. Exon-intron structures of the identified SlAAT genes. The graphic representation of the optimized gene model displayed using Gene Structure Display Server (GSDS) (for interpretation of the references to colors in this figure, the reader is referred to the web version of this article). Green boxes represent exons. The black solid lines connecting two exons represent introns, and the blue boxes represent upstream/downstream region of SlAAT genes. AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carrier; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
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Figure 2. Chromosomal localization of SlAAT genes. Respective chromosome numbers are indicated at the top of each bar. Gene duplication events of SlAAT genes marked in red. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
Figure 2. Chromosomal localization of SlAAT genes. Respective chromosome numbers are indicated at the top of each bar. Gene duplication events of SlAAT genes marked in red. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.) AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
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Figure 3. Phylogenetic tree of protein sequences of the AATs of Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana generated using MEGA 6.0 software with the Maximum Likelihood method and Whelan and Goldman model [35]. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the SlAATs analyzed. The tree was divided into 12 subgroups, marked by different color backgrounds. AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter; JTT, Jones–Taylor–Thornton. Dashed lines: AAAP family, Solid lines: APC family.
Figure 3. Phylogenetic tree of protein sequences of the AATs of Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana generated using MEGA 6.0 software with the Maximum Likelihood method and Whelan and Goldman model [35]. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the SlAATs analyzed. The tree was divided into 12 subgroups, marked by different color backgrounds. AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter; JTT, Jones–Taylor–Thornton. Dashed lines: AAAP family, Solid lines: APC family.
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Figure 4. Protein motifs of SlAATs. Each colored box represents a specific motif in the protein identified using the MEME motif search tool (For interpretation of the references to colors in this figure, the reader is referred to the web version of this article.) AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
Figure 4. Protein motifs of SlAATs. Each colored box represents a specific motif in the protein identified using the MEME motif search tool (For interpretation of the references to colors in this figure, the reader is referred to the web version of this article.) AAP, amino acid permease; ANT, aromatic and neutral amino acid transporter; ATL, amino acid transporter-like proteins; BAT, bidirectional amino acid transporter; CAT, cationic amino acid transporter; GAT, γ-aminobutyric acid transporter; LAT, L-type amino acid transporter; LAX, like-auxin influx carriers; LHT, lysine/histidine transporter; ProT, proline transporter; TTP, tyrosine-specific transporter; VAAT, vesicular aminergic-associated transporter.
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Figure 5. Multiple sequence alignment and TM region of SlAAPs with the default color scheme used for alignments in Clustal X [36]. The TM regions are marked by red. (For interpretation of the references to colors in this figure, the reader is referred to the web version of this article.) TM, transmembrane; AAP, amino acid permease.
Figure 5. Multiple sequence alignment and TM region of SlAAPs with the default color scheme used for alignments in Clustal X [36]. The TM regions are marked by red. (For interpretation of the references to colors in this figure, the reader is referred to the web version of this article.) TM, transmembrane; AAP, amino acid permease.
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Figure 6. Expression patterns of genes in various tissues of Solanum lycopersicon ‘Heinz and their orthologues/paralogs in the wild relative S. pimpinellifolium, which were retrieved by the Locus identity number of SlAATs included in Table 1. The data for RNA sequencing were obtained from a public database (the Tomato Functional Genomics Database), and were analyzed and generated using ComplexHeatmap version 2.2.0 package in R.
Figure 6. Expression patterns of genes in various tissues of Solanum lycopersicon ‘Heinz and their orthologues/paralogs in the wild relative S. pimpinellifolium, which were retrieved by the Locus identity number of SlAATs included in Table 1. The data for RNA sequencing were obtained from a public database (the Tomato Functional Genomics Database), and were analyzed and generated using ComplexHeatmap version 2.2.0 package in R.
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Figure 7. Syntenic analysis between AAt regions of Solanum lycopersicum and Arabidopsis thaliana. Gray lines in the background indicate collinear blocks between Solanum lycopersicum and Arabidopsis thaliana, while red lines highlight syntenic AAT gene pairs.
Figure 7. Syntenic analysis between AAt regions of Solanum lycopersicum and Arabidopsis thaliana. Gray lines in the background indicate collinear blocks between Solanum lycopersicum and Arabidopsis thaliana, while red lines highlight syntenic AAT gene pairs.
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Figure 8. Syntenic analysis between AAt regions of Solanum lycopersicum and Oryza sativa. Gray lines in the background indicate collinear blocks between Solanum lycopersicum and Oryza sativa, while red lines highlight syntenic AAT gene pairs.
Figure 8. Syntenic analysis between AAt regions of Solanum lycopersicum and Oryza sativa. Gray lines in the background indicate collinear blocks between Solanum lycopersicum and Oryza sativa, while red lines highlight syntenic AAT gene pairs.
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Table
Table 1. The general information and sequence characterization of 88 SlAAT genes.
Table 1. The general information and sequence characterization of 88 SlAAT genes.
Gene aLocus bGene StructureORF (bp) eProtein fTMgGenomic Location
Length (bp) cIntron dSize (aa)MW (d)pI
AAP group
SlAAP1Solyc01g10680040156170446551,289.809.1210ch01:94562712..94558698
SlAAP2Solyc06g06011026165202547151,696.388.2210ch06:38031501..38033508
SlAAP3Solyc11g00507018175143147652,595.658.8111ch11:61813..63629
SlAAP4Solyc04g07705046706193148152,581.078.6610ch04:62016870..62021539
SlAAP5Solyc07g06600037433143511611,948.895.772ch07:67550704..67554446
SlAAP6Solyc07g06601028636178845650,495.358.7810ch07:67553145..67556007
SlAAP7Solyc07g06602043076162447051,989.109.0211ch07:67557049..67561355
SlAAP8Solyc12g08819067296146748854,251.368.849ch12:63632109..63638837
LHT group
SlLHT1Solyc01g11198027964208556661,689.619.339ch01:98115988..98118783
SlLHT2Solyc02g09386030437161244950,376.268.759ch02:54561545..54564587
SlLHT3Solyc02g093870572013252583894,214.158.6818ch02:54566638..54572357
SlLHT4Solyc03g11104023496132344049,255.269.499ch03:61718834..61721182
SlLHT5Solyc04g01159035707160943949,164.789.139ch04:4020984..4024553
SlLHT6Solyc04g07956050004213452557,829.749.409ch04:63983135..63988134
SlLHT7Solyc04g08222023685137745850,211.228.7410ch04:65965546..65967913
SlLHT8Solyc05g00970036037163443548,676.178.968ch05:3896183..3899785
SlLHT9Solyc05g01453026945134444750,114.239.2810ch05:8389595..8392288
SlLHT10Solyc10g05574034594141647152,538.139.1311ch10:57311236..57314694
SlLHT11Solyc10g05575041974149149655,434.488.668ch10:57325181..57329377
SlLHT12Solyc12g0278801027466322024,854.869.345ch12:27932977..27934003
SlLHT13Solyc12g027890720160320022,806.755.832ch12:27934068..27934787
GAT group
SlGAT1Solyc03g071840744145615117,089.449.843ch03:18908947..18909690
SlGAT2Solyc08g08208058066169945450,022.088.6810ch08:64977392..64983197
SlGAT3Solyc11g06680064996136545449,886.578.9810ch11:52596193..52602691
ProT group
SlProT1Solyc03g09639046606182844148,541.109.3410ch03:58462282..58466941
SlProT2Solyc03g09638089976164243948,640.159.1411ch03:58427179..58436175
SlProT3Solyc05g05282042606153044148,418.759.2510ch05:62989652..62993911
SlProT4Solyc05g05283060546147345249,478.009.3311ch05:62996789..63002842
AUX/LAX group
SlLAX1Solyc09g01438037736186241146,352.539.1910ch09:6008319..6012091
SlLAX2Solyc01g11131035427183849455,728.038.8610ch01:97592414..97595955
SlLAX3Solyc11g0133102400NA172747053,305.459.0810ch01:29201232..29203317
SlLAX4Solyc10g0767904547NA145848554,557.537.6210ch10:59744696..59751060
SlLAX5Solyc10g0552604758NA147349055,344.708.8810ch10:56522860..56529521
ANT group
SlANT1Solyc00g00713015001150042146,305.958.5111ch02:6739403..6740902
SlANT2Solyc02g08251012781127842546,696.397.5411ch02:46198820..46200097
SlANT3Solyc02g08252012601126042045,887.145.259ch02:46200905..46202164
SlANT4Solyc03g03209012841128442746,877.547.5411ch03:4609738..4611021
SlANT5Solyc10g04818012841128442746,395.875.4311ch10:44437584..44438867
ATLa group
SlATL1Solyc02g06568034305172146350,340.188.1711ch02:36842710..36846139
SlATL2Solyc02g08940040764179446049,949.968.1411ch02:51230438..51234513
SlATL3Solyc03g11735014191141947251,228.095.3511ch03:66495836..66497254
SlATL4Solyc04g07746047881160245149,071.427.1311ch04:62369207..62373994
SlATL5Solyc05g05230021524134343848,192.597.6311ch05:62572297..62574448
SlATL6Solyc06g05079030394164743547,661.315.9111ch06:33581820..33584858
SlATL7Solyc06g05080018954144343847,942.318.5411ch06:33590884..33592778
SlATL8Solyc09g059850946136312013,678.254.194ch09:56191346..56192291
SlATL9Solyc09g059880554235111612,971.296.223ch09:56307952..56308505
ATLb group
SlVAAT1Solyc03g01316022472139342647,036.678.3411ch03:47050218..47052464
SlVAAT2Solyc03g01344041583118334638,031.138.448ch03:44654581..44658738
SlVAAT3Solyc03g06303031676116823525,699.164.695ch03:33033162..33036328
SlVAAT4Solyc03g078150729411217752557,412.834.8910ch03:50131086..50138379
SlVAAT5Solyc05g050440615156017118,934.735.285ch05:60609933..60610547
SlVAAT6Solyc05g05397016911118635238,856.009.1210ch05:63976214..63977904
SlVAAT7Solyc06g061260582NA58219321,758.229.564ch06:39263684..39264265
SlVAAT8Solyc06g0612702147163021023,054.887.585ch06:39266520..39268666
SlVAAT9Solyc09g09838016872132944248,416.478.7911ch09:72214957.72216643
SlVAAT10Solyc10g084830403110159052957,492.885.0510ch10:64225452..64229482
SlVAAT11Solyc11g008440342710166855560,735.135.839ch11:2646308..2649734
CAT group
SlCAT1Solyc02g03751016,32214221259863,275.376.4515ch02:30972606..30988927
SlCAT2Solyc02g07027018841179159664,632.508.1514ch02:40050344..40052227
SlCAT3Solyc02g07028046972199153257,650.948.8814ch02:40054985..40059681
SlCAT4Solyc02g08185019611161848352,994.828.5410ch02:45639037..45640997
SlCAT5Solyc08g0778101575168722826,105.169.035ch08:61724201..61725775
SlCAT6Solyc08g07782028044179754660,681.698.7211ch08:61726678..61729481
SlCAT7Solyc10g01860092287171056960,434.616.3413ch10:8819787..8829014
SlCAT8Solyc10g081460780113195365067,933.206.5814ch10:62511363..62519163
SlCAT9Solyc11g00671037204175558463,360.167.0215ch11:1308228..1311947
SlCAT10Solyc12g0113701854NA185461767,422.938.8413ch12:4203735..4205588
SlCAT11Solyc12g09638023562180059965,926.198.6414ch12:65323438..65325793
ACT group
SlBAT1Solyc03g0630502544380126629,148.756.025ch03:33065762..33068305
SlBAT2Solyc05g00876027666140727529,637.367.716ch05:2991698..2994463
SlBAT3Solyc05g00877077247105132135,247.125.284ch05:2993981..300170
PHS group
SlLAT1Solyc01g0059003862284027430,206.826.182ch01:590446..594307
SlLAT2Solyc01g0059201719NA171947552,971.878.153ch01:600898..602616
SlLAT3Solyc01g0340801413NA141347052,665.678.7610ch01:35838791..35840203
SlLAT4Solyc01g1118001623NA162346151,325.786.009ch01:97973675..97975297
SlLAT5Solyc02g02162021,558123376988107,982.657.1111ch02:23288582..23310139
SlLAT6Solyc02g07029016,229123251979107,388.675.5411ch02:40062787..40079015
SlLAT7Solyc08g00554049711159353058,240.507.6412ch08:397461..402431
SlLAT8Solyc08g07571064572187253458,793.388.5610ch08:59838882..59845338
SlLAT9Solyc08g0781001407NA140746851,710.245.8110ch08:61950808..61952214
SlLAT10Solyc09g0924201786185828532,004.778.785ch09:71520932..71522717
SlLAT11Solyc10g04964019291147349054,492.655.4611ch10:46312657..46314585
TTP group
SlTTP1Solyc04g04934028877177550555,314.728.6411ch04:41501638..41504524
SlTTP2Solyc07g032290664156418720,294.915.843ch07:37107752..37108415
SlTTP3Solyc10g07847016821154251354,551.239.1411ch10:60286147..60287828
SlTTP4Solyc11g072170848213149449753,505.376.0010ch11:55401477..55409958
SlTTP5Solyc12g027810267NA267889313.278.552ch12:27130397..27130663
a Systematic designation given to Solanum lycopersicum AATs in this study. b Locus identity number of SlAATs assigned by SGN. c Full length of SlAAT genes obtained from SGN. d Number of introns in SlAAT genes obtained from SGN. e Length of the open reading frame of the SlAAT genes. f Protein characterization of SlAATs: size obtained from SGN, pI, and MW obtained from ExPASy. g Number of TM segments possessed by SlAAT proteins, predicted by the TMHMM Server version 2.0. AAT, amino acid transporter; ORF, open reading frame; bp, base pair; aa, amino acids; MW, molecular weight; pI, isoelectric point; TM, transmembrane; NA, not available; Sol Genomics Network, SGN.
Table
Table 2. The gene duplication data obtained from the Plant Genome Duplication Database (PGDD).
Table 2. The gene duplication data obtained from the Plant Genome Duplication Database (PGDD).
Duplicated Gene PairsKaKsKa/KsTime (Mya *)Percent Similarity %
SlAAP1/SlAAP40.24251.62120.149581123.6078.47
SlAAP4/SlAAP80.13670.94320.14493271.9087.50
SlLHT1/SlLHT70.42911.58580.270589120.7059.44
SlLHT1/SlLHT100.49852.23040.22350317038.84
SlLHT2/SlLHT90.26632.04840.130004156.1378.98
SlProT2/SlProT30.19320.69760.2769553.1783.03
SlLAX1/SlLAX40.0510.84220.06055664.1980.37
SlLAX2/SlLAX50.04890.90880.05380769.2793.15
SlANT1/SlANT20.13760.60570.22717546.1785.88
SlANT2/SlANT40.11820.7690.15370658.63 88.29
SlATL1/SlATL20.08070.52080.15495439.70 89.85
SlATL5/SlATL60.32821.9490.168394148.55 71.62
SlCAT1/ SlCAT40.80153.14970.254469240.07 39.19
SlLAT2/ SlLAT30.15910.94930.16759772.36 86.97
SlLAT5/ SlLAT60.06850.60990.11231346.49 91.33
SlLAT7/ SlLAT80.14380.78380.18346559.740 86.03
Mya, million years ago.
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Omari Alzahrani, F. Genome Wide Analysis of Amino Acid Transporter Superfamily in Solanum lycopersicum. Plants 2021, 10, 289. https://doi.org/10.3390/plants10020289

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Omari Alzahrani F. Genome Wide Analysis of Amino Acid Transporter Superfamily in Solanum lycopersicum. Plants. 2021; 10(2):289. https://doi.org/10.3390/plants10020289

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Omari Alzahrani, Fatima. 2021. "Genome Wide Analysis of Amino Acid Transporter Superfamily in Solanum lycopersicum" Plants 10, no. 2: 289. https://doi.org/10.3390/plants10020289

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