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Article

Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean

by
Yuan Zhang
1,2,†,
Le Wang
1,†,
Bao-Hua Song
3,
Dan Zhang
4,* and
Hengyou Zhang
1,*
1
Key Laboratory of Soybean Molecular Design Breeding, State Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA
4
Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
*
Authors to whom correspondence should be addressed.
†
These authors contribute equally to the work.
Agronomy 2024, 14(1), 52; https://doi.org/10.3390/agronomy14010052
Submission received: 2 December 2023 / Revised: 18 December 2023 / Accepted: 21 December 2023 / Published: 23 December 2023
(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)
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Abstract

:
Amino acid permeases (AAPs) play important roles in transporting amino acids in plant species, leading to increased low-nitrogen tolerance, grain yield, or protein content. However, very few AAPs have been characterized in soybean (Glycine max). In this study, we scanned the soybean reference genome and identified a total of 36 AAP genes (named GmAAP). The GmAAPs were phylogenetically divided into three evolutionary clades, with the genes in the same clades sharing similar gene structures and domain organization. We also showed that seventeen GmAAP genes on ten chromosomes were in collinearity, likely due to whole-genome duplication. Further analysis revealed a variety of cis-acting regulatory elements (such as hormone response elements (ABRE, ERE, GARE, P-box, and TGA-element), stress response elements (LTR, MBS, MYB-related components, TC-rich repeats, TCA-element, and WUN-motif), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian) present in the 2 kb region of the GmAAP promoter region, demonstrating functional diversity and expression specificity. RNA-Seq data and quantitative real-time PCR identified five GmAAPs showing differential expression under nitrogen limitation, including GmAAP3, GmAAP5, and GmAAP8 showing downregulation while GmAAP14, GmAAP29 showed upregulation, suggesting their involvement in low-nitrogen stress response. These results provide comprehensive information on soybean AAP genes in nitrogen stress, and provide putative candidates with possible roles in enhancing amino acid delivery to seeds for yield improvement.

1. Introduction

Nitrogen (N) is a major component in protein, nucleic acid, chlorophyll, and ATP, thus making it an essential element for growth and development in plants. Plants utilize the root system to take up inorganic N (i.e., nitrate or ammonium), which is assimilated to amino acids, or take up amino acids from soil [1]. However, soils usually are deficient in N, which is insufficient to maintain plant growth and development [2]. As such, crops require a large amount of N fertilizers to meet the growing demand for productivity in crops such as maize, wheat, and rice [3]. Although N fertilizers can provide season-long, sufficient N for uptake, N fertilizers are not environmentally friendly and can be costly. Overuse of N fertilizers in soil also causes deterioration of soil microbiological properties and function, as well as water pollution [4]. It has been demonstrated that plant species vary greatly in N uptake and usage [5]. This is likely attributed to plants’ ability to evolve an array of molecular mechanisms to optimally manage N acquisition and utilization. It is necessary to reveal these mechanisms and apply this knowledge to breed crop species with improved N uptake and usage.
To achieve increased N uptake under N deficiency, plants respond to N stress by changing the root architecture system (increasing root length and root hair) [6,7,8,9]. Meanwhile, N-related transporters also play an important role in improving N uptake and utilization [10]. Amino acid transporters play an important role in phloem loading and unloading of amino acids [11], of which amino acid permeases (AAPs) are a subfamily of the amino acid transporter family (ATF) and are proton-coupled amino acid transporters with a wide range of substrate specificity. A total of eight AAP genes have been identified in Arabidopsis, and nineteen AAP members have been identified in rice [12,13]. Studies have demonstrated that AAP genes are expressed in a variety of tissues, mainly involved in the loading of xylem and phloem, transport of amino acids from source to sink, and absorption of amino acids [14,15], and play an important role in improving N utilization and yield [16,17]. For example, Arabidopsis mutant ataap2 increased seed yield and N uptake under low- and high-N conditions [18]. Similarly, rice OsAAP3 and OsAAP5 RNAi lines also increased grain yield under sufficient N conditions [19,20]. On the other hand, some AAPs were expressed in specific tissues and had specific functions related to certain traits. AtAAP1 is expressed specifically in cotyledons and endosperm, regulating the transport of amino acids to root cells or developing embryos, and is essential for seed yield and storage protein synthesis [21,22]. AtAAP2, located in the phloem of the whole plant, is key for amino acid transportation from the xylem to the phloem and N and C absorption, while the AtAAP2 T-DNA insertion line reduces N content and storage proteins in individual seeds [18]. The total amino acid concentration in the mutant ataap6 is lower than that of the wild type, and lysine and phenylalanine are reduced [23]. AtAAP8 is expressed in the phloem of the source leaves, the mutant ataap8 shows a decrease in amino acid loading and distribution to sink organs. This results in an increase in the total N of the source leaves but a decrease in nutrient biomass and seed number [24,25]. Similarly in rice, OsAAP1, OsAAP7, and OsAAP16 transport neutral amino acids, while OsAAP3 specifically binds basic amino acids, of which overexpression of OsAAP1 increased the number of tillers and the number of grains per plant; however, in contrast, its knockout reduced the number of tillers and affected the transport and metabolism of N [20,26,27]. Overexpression of OsAAP6 increased grain protein content and improved protein quality [28]. A recent study showed that cucumber CsAAP2 is highly expressed in root vascular cells. Root meristem development was inhibited in the knockout lines, resulting in delayed lateral root initiation and inhibited root elongation [29]. These studies indicate that AAPs are important in the regulation of N uptake and usage, which are agriculturally important traits.
Despite the capacity for N fixation in legumes, legumes overexpressing AAP genes can also show enhance plant performance associated with traits such as increased yields and nutrient quality. For example, overexpression of PsAAP1 in peas shows a higher seed yield than the wild type under different N fertilizer conditions; PsAAP6 is involved in amino acid transport from nodules to other sink organs [30,31]. In broad bean (Vicia faba), VfAAP1 was mainly expressed in roots, stems, and pods, and overexpression of this gene increased storage protein abundance in seeds [32]. Therefore, AAP genes from legumes are also important for enhancing N uptake and transport of organic N, and further improving seed proteins and yield. However, studies of AAP family genes in legumes have not gained the same level of attention as those in cereal crops, and few of them have been characterized.
Soybean (Glycine max) is an important economic and oil crop, which provides approximately 30% of edible oil and 69% of dietary protein worldwide [33]. Because of the higher protein content in soybean seeds, more N uptake is required to ensure normal seed growth in soybean compared to other crops [34]. It has been shown that with N application reaching 60 kg/hm2, soybean yield, pod number per plant, and seed number per unit area can all reach the highest values under field conditions [35]. Soybean amino acid transporters have been globally characterized [36], while AAPs in soybean have yet to be comprehensively investigated. GmAAP6a in soybean selectively transports neutral and acidic amino acids, and the overexpression of GmAAP6a increases low-N tolerance, seed quality, and the source-to-sink transfer of amino acids [37]. This demonstrated its importance in improving the yield and quality of soybean. However, to the best of our knowledge, other than GmAAP6a, few AAPs in soybean have been reported in plant tissues, particularly under low-N conditions. It is necessary to comprehensively characterize the AAP family in soybean, especially their expression in specific tissues and identification of AAP genes responding to low-N stress.
In this study, we identified AAP genes in the soybean genome and comprehensively investigated the characteristics including chromosomal distribution, gene structure, domain organization, cis-acting elements in promoters, and evolutionary relationships. We also investigated the tissue expression patterns and the changes in tissue expression patterns at varying time points under low-N stress, followed by quantitative real-time PCR validation. Our work emphasizes the important role of gene duplication in the expansion of the soybean AAP family. Combining these genes’ expression characteristics and their responses to low-N stress which were not reported previously, this study will provide a basis for further functional analysis of the GmAAPs, as well as an improved understanding of the molecular mechanisms underlying the role of GmAAPs in the regulation of soybean growth and low-N stress adaptation.

2. Materials and Methods

2.1. Identification and Physicochemical Properties of the GmAAP Gene Family

In this study, the reference genome sequence and GFF3 annotation file (Glycine max Wm82.a2.v1) of soybean were downloaded from the JGI Phytozome v13 (https://phytozome-next.jgi.doe.gov/, accessed on 1 July 2023) [38]. Using Arabidopsis AAP proteins as query sequences, the BLAST GUI Wrapper in TBtools version 2.012 [39] was used to search transmembrane amino acid transporter domains against the protein sequences of the soybean genome (the E-value was set to 1 × 10−35 to determine the significant hits). The resulting putative AAP proteins were further confirmed manually using the NCBI Conservative Domain Database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 5 July 2023) [40]. The physicochemical properties of GmAAP proteins were analyzed with the Protein Parameter Calc of TBtools version 2.012 [39], including number of amino acids, molecular weight (kDa), theoretical pI, instability index, and grand average of hydropathicity.

2.2. Phylogenetic Tree Construction, Chromosome Localization, and Collinearity Analysis

Clustal W version 2.1 was used to compare the full-length amino acid sequences of AAPs in soybean, Arabidopsis, and rice with default parameters [12,13]. MEGA 11 was used to analyze phylogeny using the neighbor-joining (NJ) method with the following parameters: Poisson model, pairwise deletion, and 1000 bootstrap replications, with the result being modified by iTOL v6 (https://itol.embl.de/, accessed on 27 July 2023) [41]. Information about gene location and tandem replication of the GmAAP gene family was shown using the program Gene Location Visualize from GTF/GFF of Tbtools. Collinearity analysis was performed using the MCScanX algorithm [42]: BLASTP matches are sorted according to gene positions. If consecutive BLASTP matches have a common gene and its paired genes are separated by fewer than five genes, these matches are collapsed using a representative pair with the smallest BLASTP E-value. Dynamic programming is employed to find the highest-scoring paths. GmAAP genes falling in the identified collinear blocks were considered segmental events, while closely adjacent homologous GmAAP genes were considered to represent tandem duplication events, based on the identification standards in MCScanX. The result of collinearity was shown by the program Advanced Circos in TBtools version 2.012. The ratio of synonymous mutation to non-synonymous mutation (Ka/Ks) between gene pairs was calculated using the the Simple Ka/Ks Calculator in TBtools version 2.012.

2.3. Analysis of Conserved Motif, Domain, and Gene Structure of the GmAAP Proteins

Conserved motifs of the GmAAP proteins were analyzed through MEME Suite 5.5.5 (https://meme-suite.org/meme/doc/meme.html, accessed on 26 August 2023) with the default parameters, in which the site distribution was patterned on zoops and the number of motifs was set to fifteen [43]. Domain analysis was performed by NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 26 August 2023) with default parameters. Data on gene structure were retrieved from the downloaded GFF3 file, and all results were visualized with Gene Structure View (Advanced) in TBtools version 2.012.

2.4. Promoter Analysis

According to methods of analyzing promoters of the gene family Tian et al. and Wu et al. [44,45], the upstream 2000 bp base sequences of the soybean GmAAP genes were extracted in the program GXF Sequence Extract of TBtools. PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 26 August 2023) was applied to predict cis-regulatory elements [46]. The resulting data were visualized with Basic Biosequence View in TBtools.

2.5. Plant Materials and Treatment

Soybean seeds (variety Dongnong 50) were germinated in vermiculite, following which seedling roots were washed with tap water and cotyledons were removed before seedlings were transferred to plastic boxes containing a foam board with 40 holds for hydroponics. The hydroponic solution containing 0.25 mM KH2PO4, 2.5 mM Ca(NO3)2, 2.5 mM KNO3, 1 mM MgSO4·7H2O, 0.082 mM Fe-EDTA, 0.02313 mM H3BO3, 0.00038 mM ZnSO4·7H2O, 0.00157 mM CuSO4·5H2O, 0.00009 mM (NH4)6Mo7O24·4H2O, 0.00457 mM MnCl2·4H2O, and 0.000567 mM Na2MoO4·H₂O was used as the normal N (NN) condition to represent controls. The N content of low-nitrogen hydroponic solution (10% of that of normal N hydroponic solution) was used as low-N (LN) treatment, in which the missing Ca2+ and K+ were replaced by CaCl2 and K2SO4, respectively. The hydroponic solution was changed every two days. Plants were grown in artificial climate chamber RXZ-436 (Jiangnan Instrument, Ningbo, Zhejiang, China) with a 16 h light/8 h dark photoperiod, 25 °C, 60% RH, 30,000 Lux. Once the plants reached seedling stages with the first fully developed triple compound leaf, the seedlings cultured in NN solution were transferred to LN solution for LN treatment [47]. At 12 and 24 h post-treatment, the roots were harvested individually and were flash-frozen in liquid N and stored at −80 °C until RNA isolation. The roots from two or three plants were pooled as one replicate, and the experiment was set up for three biological repetitions.

2.6. RNA Extraction and Quantitative Real-Time PCR

RNA was extracted using an ultrapure RNA kit (CWBIO, Beijing, China). The NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel were used to test the quality of extracted RNA. Then, the first-strand cDNA was synthesized according to the instructions of the reverse transcription kit (TransGen Biotech, Beijing, China). Primers were designed by Primer Premier 5, and their specificity was checked by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 25 September 2023). qRT-PCR reactions were performed on a LightCycler® 480 (Roche, Switzerland). The reaction volume was 20.0 μL, including 10.0 μL of 2 × PerfectStart® Green qPCR SuperMix, 0.4 μL of each primer (10 μM), 1.0 μL template (10× diluted cDNA liquid), and 8.2 μL nuclease-free water. The thermal cycling program was 94 °C for 30 s; 40 cycles of 94 °C for 5 s; and 60 °C for 30 s. Expression of the genes was calculated using the 2−ΔΔCT method [48], and the soybean TUB4 gene (GenBank accession no. NM_001252709) was used as the internal reference gene.

2.7. Statistic Analysis

This qRT-PCR used three biologicals and three technical replicates. The Student’s t-test (* a = 0.05, ** a = 0.01) was performed using GraphPad Prism version 9.5.1 for Windows, GraphPad Software, San Diego, CA, USA (www.graphpad.com, accessed on 25 September 2023).

3. Results

3.1. Identification and Physicochemical Properties of the GmAAP Gene Family

With the gene search strategy, a total of 36 GmAAP putative genes were identified in the soybean reference genome. After confirmation of GmAAPs, we named them GmAAP1 to GmAAP36 according to the physical location on 20 chromosomes (Table 1). The analysis showed that the number of amino acids in these GmAAP proteins ranged largely from 101 (GmAAP35) to 531 (GmAAP15), which resulted in a large variation in molecular weight from 11.13 kDa (GmAAP35) to 58.47 kDa (GmAAP15). The grand average of hydropathicity was greater than zero, indicating that all members of the GmAAPs were hydrophobic proteins. Theoretical pI values of GmAAP proteins were more than 7, with eight exceptions including GmAAP1 (5.18), GmAAP3 (6.82), GmAAP6 (6.31), GmAAP15 (6.69), GmAAP17 (5.95), GmAAP26 (6.71), GmAAP32 (5.76), and GmAAP35 (5.05). In addition, the instability index for GmAAP35 was 24.29, which was the smallest among all GmAAP proteins, and conversely, GmAAP25 ranked the highest in instability index (45.58). However, GmAAP1, GmAAP24, GmAAP25, and GmAAP26 belonged to the labile protein because their instability index was greater than 40.

3.2. Chromosomal Localization and Phylogeny of the GmAAP Gene Family

According to the chromosomal locations, the 36 GmAAPs were identified on thirteen chromosomes and distributed unevenly (Figure 1). No GmAAPs were identified in the remaining seven chromosomes 1, 3, 7, 9, 15, 16, and 20. Specifically, chromosome 6 contained the largest number (6) of GmAAP members, followed by chromosomes 4 (5) and 18 (4), and chromosomes 2, 8, and 17, each containing three GmAAP genes. The rest of the chromosomes contained only one GmAAP gene. Interestingly, nine AAP genes were distributed in tandem on three chromosomes, including GmAAP6 and GmAAP7 on chromosome 4, GmAAP10 and GmAAP11 on chromosome 6, GmAAP14 and GmAAP15 on chromosome 6, and GmAAP32, GmAAP33 and GmAAP34 on chromosome 18. These findings are similar to those observed in wheat with twenty-six tandemly duplicated AAP members, and in rice with ten tandemly duplicated AAPs [12,44].
To gain insight into the function of GmAAPs, a rootless phylogenetic tree comprising AAPs from soybean, Arabidopsis, and rice was constructed because of relatively intensive studies in these plant species [12,17,27]. The tree was constructed with 63 AAP proteins including 36 soybean GmAAPs, 19 rice OsAAPs, and 8 Arabidopsis AtAAPs (Figure 2). The tree showed that the 63 AAP proteins were divided into four groups according to the topology, each containing 26 (I), 18 (II), 15 (III), and 4 (IV) proteins, among which the 36 GmAAP proteins were distributed in I, II and III. Group IV consisted of only four AAP members that were all from rice (OsAAP4, OsAAP5, OsAAP6, OsAAP17), suggesting that they might play roles different from other AAP genes. The more closely related proteins are likely to have similar functions in phylogenetic trees [49]. For example, the GmAAP29 in group II is a homolog of AtAAP6, with demonstrated functions in increasing the size of seeds of mutant ataap6 [23], and has been reported to be associated with source-sink transport and improved total N content, soluble protein content, and free amino acids in seeds [37]. Similarly, GmAAP5 and GmAAP12, which are clustered on the same branch with GmAAP29, might share common functions.
AtAAP3 and AtAAP5 are distributed in the same branch in group I, and reportedly transport basic amino acids [17]. Four GmAAP genes (GmAAP1/8/13/22/28/35) in the same branch likely function similarly to AtAAP3 and AtAAP5 in the regulation of amino acid transport [17]. In group III, the function of AtAAP7 has yet to be revealed [13]; whether the GmAAPs in this group are involved in amino acid transport or possess unreported functions merits further investigation. In group IV, the RNAi lines for OsAAP5 in Japonica increased tillering numbers and grain yield, while OsAAP5 overexpression had the opposite effect [19]. No GmAAPs were identified in this group, likely because the clustered rice AAPs played species-specific functions.

3.3. Gene Structure and Conserved Domain of GmAAP Gene Family

Composed of 36 GmAAP proteins, the phylogenetic tree was clustered into three groups (I, II, III) (Figure 3), which was consistent with the topological structure of the above-mentioned three-species phylogenetic tree (Figure 2). We further investigated if the tree topology is associated with protein motif or gene structure. After investigation, we found that the GmAAP members exhibit large variation in the number of exons, ranging from two to eight, while the majority of them (53%) carried seven exons. This pattern is likely conserved within plant species because nearly half of AAP genes (6 of 15) in Nicotiana tabacum contain seven exons, and over half of AAPs (18 AAPs of 34 AAPs) from Brassica napus contain six exons; 5 members of the 8 Arabidopsis AAP genes contained six exons [50,51,52].
We also observed that genes that were clustered in the same group, particularly those in pairs in the tree, were highly similar in their exon–intron organization, despite the variation in the length of the intron. For example, GmAAP4 and GmAAP11 from group II both contained seven exons, and they were clustered into the same branch. Similar patterns were also seen for GmAAP5 and GmAAP12, and GmAAP25 and GmAAP30. We also noticed some exceptions with significantly reduced numbers of exons. For example, both GmAAP1 and GmAAP35 in group I and GmAAP26 in group II contain only three exons, with all three proteins having less than 200 amino acids, making them dramatically shorter than other GmAAPs that contain an average of 400 amino acids (Table 1).
In addition, domain analysis showed that all the genes of the GmAAP proteins carried the Aa_trans domain, a transmembrane region specifically belonging to transmembrane amino acid transporters. To further investigate the protein sequence features of GmAAPs, we examined the conserved motifs and the organization within the family. In total, 15 motifs were identified and designated motifs 1 to 15 (Figure 3), with motif sequences shown in Table 2. Interestingly, only GmAAP20 and GmAAP21 were found to contain all of the 15 motifs, while 30 of 36 (83.3%) GmAAP proteins contained 7 motifs. To identify their potential functions, we searched the sequences in the InterProScan and the results showed that motifs 1, 2, 3, 5, and 6 were transmembrane domains with transmembrane transporter activity, while the other ones were unannotated. Regarding motif distribution, motif 1 was identified in all but three GmAAPs (GmAAP1, GmAAP23, and GmAAP35), suggesting that motif 1 is essential for the majority of GmAAPs, but it is likely not required for the function of the three GmAAPs. In contrast, motif 2 was identified in nearly all GmAAPs except for GmAAP26. Some 28 of 36 (77.8%) GmAAP proteins contained motif 3, motif 5, and motif 6, suggesting the importance of these conserved motifs for GmAAPs. However, there is no strong correlation between the numbers of motifs and exons, such as the GmAAP31 and GmAAP36 containing four motifs and six motifs, respectively, while both contain eight exons. Notably, phylogenetically close genes are similar in gene structure and conserved motifs, and therefore likely function similarly.

3.4. Collinearity Analysis of the GmAAP Gene Family

As we observed, the majority of GmAAPs were clustered in pairs in the phylogenetic tree (Figure 3), so we questioned if these paired GmAAPs are in collinearity within the genome. The collinearity analysis led to the identification of a total of 26 pairs of homologous GmAAP genes in soybean (Table 3). Based on the inter-correlation among the paired genes, these homologous genes could be divided into four groups on ten chromosomes (2, 4, 5, 6, 8, 11, 12, 14, 17, and 18). As shown in the Circos map in Figure 4, the paired GmAAPs are linked with solid lines, with four different colors representing the collinear ones, and reside in the duplicated regions in the genome [53]. The four colored lines include the red group consisting of GmAAP6, GmAAP8, GmAAP9, GmAAP13, GmAAP14 and GmAAP16; the blue group consisting of GmAAP2, GmAAP17, GmAAP23, and GmAAP32; the orange group of GmAAP20 and GmAAP21; and the green group of GmAAP4, GmAAP5, GmAAP12, GmAAP25 and GmAAP29. Therefore, these homologous GmAAPs likely originated from duplication events on the 10 chromosomes, which could be a major mechanism for GmAAP family expansion [54], similar to other gene families. For example, 94% (279 of 296) of AAT genes were generated by whole-genome duplication in wheat [44], whereas 55.30% (47 of 85) of AAT genes were duplicated genes in rice [12].
Next, we calculated the Ka/Ks values for the paired genes to study the evolutionary selection pressure of the GmAAP gene family. The result showed that Ka/Ks values of all the colinear GmAAPs were smaller than 1.0 (Table 3). Ka/Ks < 1 indicates that these genes have undergone purifying (stable) selection, and Ka/Ks > 1 at specific sites indicates that these genes are under positive or Darwinian selection [55]. This result suggests that the GmAAP gene family might have been selected for purification during evolution.

3.5. Cis-Regulatory Elements in Promoters

Cis-acting regulatory elements (CREs) in promoters play important roles in transcriptional regulation [56]. To identify CREs for GmAAPs, we investigated CREs upstream in the first 2000 bp of GmAAPs according to the Wm82 reference genome. In total, we identified 13 cis-regulatory elements with diverse function annotations for all the members (Figure 5). They were divided into four categories based on their putative functions, including hormone response elements (ABRE, ERE, GARE, P-box, and the TGA-element), stress response elements (LTR, MBS, MYB-related components, TC-rich repeats, the TCA-element, and WUN-motif), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian). Specifically, GmAAP4 promoter region contains the most cis-regulatory elements (24), followed by GmAAP19 and GmAAP21 (22), GmAAP11 (20), and in contrast, only 2 cis-regulatory elements were identified in the promoter region of GmAAP1. Notably, promoter regions for all soybean AAP genes contained MYB-related elements (Myb, MYB recognition site, MYB). In addition, we also observed that four elements, including abscisic acid response elements (ABRE), ethylene response elements (ERE), salicylic acid response elements (TCA-element), and trauma response elements (WUN-motif), were commonly identified in the promoter regions of over half of the GmAAPs, and their distribution in the region varied greatly (Figure 5). This indicated that the GmAAP family likely plays functional roles within these pathways in response to abiotic stressors such as drought, salt, cold, and heat [57].

3.6. GmAAPs Display Clear Tissue Expression Patterns

To gain insight into the expression of GmAAPs, we examined expression levels in different tissues (RT (roots), STEM (stems), LF (leaf), FLUB (floral bud)), and developing seeds at different stages (GLOB (globular stage), HRT (heart stage), COT (cotyledon stage), EM (early maturation stage), MM (mid maturation stage), LM (late maturation stage), DS (dry seed), GLOB_ES (globular stage endosperm), HRT_ES (heart stage endosperm), COT_ES (cotyledon stage endosperm)) by re-analyzing the RNA-Seq data (seedgenenetwork.net, accessed on 26 October 2023). The expression data were illustrated using a heatmap that was executed using the R package pheatmap version 1.0.12 (Figure 6). We observed that the GmAAPs showed significant differential expression in the investigated tissues. Based on this pattern, the GmAAPs could be classified into three groups. Group 1 contains 10 members that were highly expressed in roots and stems, with GmAAP21 showing the strongest expression in roots and stems. Three GmAAPs, GmAAP5, GmAAP12, and GmAAP29 (Figure 2), were highly expressed in floral bud [37]. Group 2 consists of twelve members, of which ten members exhibited negligible to low expression in the investigated tissues, except for GmAAP19 and GmAAP30, which were highly expressed in endosperm. In Group 3, fourteen members were mainly expressed in developing seeds and LF and FLUB, of which GmAAP6 was the most highly expressed in all tested tissues except for endosperm, and is similar to the homologous gene AtAAP2, which was located in the phloem of whole plants [18].

3.7. Expression Patterns of GmAAP Genes under Low Nitrogen

To investigate whether some GmAAPs showed responses to low-N (LN) stress, we grew soybean plants in normal N (7.5 mM) and low-N (0.75 mM) hydroponic solutions and compared the transcriptomic profiles in the roots of soybean seedlings after the treatment at 12 h and 24 h [47]. Surprisingly, we observed that the majority (23) of GmAAPs showed little to no expression in roots, including GmAAP9, GmAAP12, GmAAP19, GmAAP24, GmAAP25, GmAAP26, GmAAP33, and GmAAP34 (Figure 7a). Among these genes, the expression of GmAAP3 and GmAAP5 was the highest in roots at both time points (12 h and 24 h), followed by GmAAP13 and GmAAP14. Compared with the control, the expression of GmAAP3 was downregulated after LN stress. Additionally, GmAAP5, GmAAP6, GmAAP8, and GmAAP23 were slightly sensitive to N deficiency. In contrast, GmAAP14 and GmAAP29 were both induced by LN at different degrees, which indicated that GmAAP14, similar to GmAAP29, might be involved in the regulation of soybean adaptation to LN stress [37]. In addition, GmAAP2, GmAAP13, GmAAP18, GmAAP20, GmAAP21, and GmAAP27 were expressed to some extent in roots, but showed insensitivity to LN stress.
To verify the results, we examined the expression levels of five genes (GmAAP3, GmAAP5, GmAAP8, GmAAP14, GmAAP29) which showed differential expression in the transcriptomic data using quantitative RT-PCR (qRT-PCR), with RNA samples utilized in RNA-Seq. Primers for qRT-PCR are shown in Table S1. Overall, we observed that three of the selected GmAAPs were significantly downregulated approximately two-fold (by 1.48–3.06 times) after LN stress (Figure 8), of which, GmAAP3 consistently showed downregulation during both 12 h and 24 h after LN stress compared with NN (normal N). GmAAP5 and GmAAP8 displayed significant downregulation at 12 h only, suggesting that both genes might be important during early response. In contrast, GmAAP14 and GmAAP29 were both consistently upregulated significantly after LN at 12 h and 24 h. The qRT-PCR verified the transcriptomic results, suggesting that those with consistent changes in expression during LN treatment might play important roles in LN response, and merit further investigation.

4. Discussion

Previous studies have shown that the AAP family plays important roles in transporting amino acids, abiotic stress response, and improving yield and seed quality in crops [22,28,32,37,58]. For example, in rice, OsAAP6 is highly expressed in seeds, and its transgenic plants show higher expression levels and produce larger amounts of grain storage proteins [28]. Arabidopsis AtAAP1 is important for the absorption of glutamic acid and neutral amino acids; meanwhile, AtAAP1 plays an important role in endosperm absorption of amino acids, synthesis of storage proteins, and seed yield [22,58]. In legumes, VfAAP1, a broad bean amino acid permease, was reported to improve the nitrogen state of plants and increase the protein content of seeds by increasing the nitrogen absorption capacity of seeds [32]. In addition, GmAAP6a, an N deficiency-responsive amino acid permease, was reported to transport neutral and acidic amino acids in soybean [37]. Overexpression of GmAAP6a enhanced tolerance to low N and increased N export from source and imports into sink leaves under low-N conditions [37]. Meanwhile, the contents of total N, soluble proteins, and total amino acids are all significantly elevated in GmAAP6a transgenic lines grown under both conditions [37]. These studies suggest that soybean AAPs are also important for enhancing nutrients and yield, and therefore deserve further investigation.
Due to the importance of transporting nutrients and improving crop yield and quality [22,28,32,37,58], the AAP gene family has been surveyed at a genome-wide level in several plant species, such as Arabidopsis thaliana [50], Oryza sativa [12], Nicotiana tabacum [51] and Brassica napus [52]. A recent study performed a comprehensive analysis of 35 AAP genes in soybeans [36]. In this study, we expanded the prior analysis by systematically analyzing GmAAPs in the soybean genome. This included gene structure analysis, gene collinearity analysis, promoter analysis, and expression comparisons of different tissues at different developmental stages, as well as the dynamic changes in expression after LN challenges. We revealed that the promoter regions of GmAAPs contain various cis-acting elements, and six GmAAPs responded to low N. This result showed that GmAAPs might play important roles in nitrogen transport and improving crop yield and quality [28,37].

4.1. GmAAP Gene Family Expression Analysis

Cis-acting elements have demonstrated importance in the production of transgenic crops, and promoters are powerful tools to ensure the accurate expression of genes. Thus, the analysis of promoters greatly contributes to understanding gene regulation and plant development [59]. Studies have demonstrated that manipulation of cis-acting elements in promoter regions can be used for crop improvement. For example, Ruan et al. used the P1BS motif (GaATATtC) to modify the promoters of Transporter Traffic Facilitator 1 (PHF1), and they obtained phosphate-efficient transgenic plants with significantly increased yield under low-phosphorus conditions [60]. In this study, we revealed that the soybean AAP gene family contains a variety of cis-acting elements in the promoter regions. GmAAPs promoter elements include hormone response elements (ABRE, etc.), stress response elements (MYB, etc.), tissue expression elements (GCN4-motif), and physiological regulatory elements (circadian).
In contrast to the promoter distribution of the Arabidopsis AAP gene family [50], not all soybean AAP genes contain endosperm tissue expression elements or circadian regulatory elements. Studies have shown that AtAAP1 and AtAAP8 can regulate the absorption of amino acids in endosperm [22,24,25]. We found that GmAAP19 was highly expressed in endosperm using RNA-Seq data (seedgenenetwork.net, accessed on 26 October 2023) (Figure 6), which suggests that this gene may be involved in the transfer of amino acids from the endosperm into the embryo, and may influence seed development [61]. In the GmAAP members with circadian elements, we found that GmAAP2 and GmAAP29 were regulated by circadian rhythm only (Figure 7b) using RNA-Seq data (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA369113, accessed on 26 October 2023), suggesting that these genes may respond to a circadian clock to regulate plant growth and development [62]. Expression patterns of soybean AAP genes have not been reported during seed development [36]. In this study (Figure 6), we found that the expression of GmAAP6 was relatively high in developmental seeds at their full growth stage, and GmAAP2 expression was higher at the early stage only. In addition, we also found that GmAAP21 was largely expressed in roots and stems, far exceeding those of the remaining genes in the GmAAP family. We found that GmAAP6 and GmAAP21 were both highly homologous to AtAAP2 in Arabidopsis (Table 1). In a previous study, AtAAP2, located in the phloem of the whole plant, played a role in amino acid transport from the xylem to the phloem, amino acid distribution to the embryo, and absorption of N and C supply [18]. In soybean, the high expression of GmAAP6 and GmAAP21 in roots, stems, and seeds, respectively, implies that these genes may be of importance for the source and sink transport of amino acids and distribution to seeds. It is worth noting that GmAAP6 responded to LN stress (Figure 7a), suggesting that GmAAP6 may be a promising candidate gene within the AAP family for improving yield in soybean.
In this study, promoter regions of all GmAAP members contain MYB-related components (Figure 5). Studies have shown that MYB is involved in plant adaptation to low-nitrogen stress. For instance, LjMYB101 is expressed specifically in Lotus seeds, and its presumed soybean homolog GmMYB101 regulates flavonoid biosynthesis in nitrate starvation response [63]. In Setaria italica, SiMYB3 regulates root development by modulating the synthesis of plant root auxin to adapt to low-N stress [64]. In chrysanthemums, overexpression of CmMYB42 increases the expression of genes involved in lignin and phenylalanine synthesis, NO3− content, and assimilation enzyme activity in nitrogen metabolism [65]. These results suggest that the GmAAP gene family may play important roles in regulating soybean adaptation to low-N stress.

4.2. GmAAP Gene Family Responses to Low-Nitrogen Stress

In a recent study, Nezamivand-Chegini et al. analyzed the response mechanism of soybean to N deficiency by transcriptomics and metabolomics. In that study, the high-affinity nitrate transporters were upregulated in roots under low-N conditions [66]. In contrast, in our study, we identified additional amino acid permeases that were involved in low-nitrogen response in soybean. Previous studies have shown that AAP genes play an important role in increasing protein and yield in legumes, especially GmAAP6a (GmAAP29 in this study) in soybean [30,32,37]. GmAAP6a has been reported important for adapting to low-N stress and improving N use efficiency and seed quality traits (increasing plant biomass and soluble protein content). This is because increase of transport of amino acids from source to sink in the lines of overexpression of GmAAP6a under low-N conditions [37]. In peas, Garneau et al. inserted a high-affinity yeast methionine/cysteine transporter gene into the pea leaf phloem and seed cotyledons, and the methionine transport from source to sink was increased. Meanwhile, nitrogen and carbon assimilation and allocation of source and sink were all up-regulated. This results in increased plant biomass and seed yield [67]. In maize, a recent study showed that Thp9-T, a key superior variant controlling high protein in wild maize, encodes asparagine synthetase 4 and increases N use efficiency. After introducing Thp9-T into maize inbred line B73, the seed protein content and the nitrogen content in the roots, stems, and leaves were increased [68]. These show that it is very important to improve the N use efficiency for increasing crop protein. Hence, we speculated that enhanced expression of GmAAP14 might function similarly to GmAAP29 (Figure 8). Overexpression of GmAAP14 may improve the transport of amino acids from root to sink organ, increase plant tolerance to low N and N use efficiency, and improve yield and protein content in seeds. Further gene cloning followed by functional validation experiments is needed to confirm this hypothesis. Compared with up-regulated GmAAPs, GmAAP3, GmAAP5, and GmAAP8 show a downregulated trend at 12 h. In Arabidopsis, a previous study showed that nitrogen limitation adaptation (NLA) protein abundance was decreased under low-N conditions, which resulted in decreased negative regulation of NRT1.7 (nitrate transporter) and enhanced N remobilization from source to sink [69]. Here, the downregulation of GmAAP3, GmAAP5, and GmAAP8 indicates that they may participate in low-N response to N remobilization through negative regulation. Understanding N assimilation and distribution is a strategy for increasing crop yield [70]. Therefore, transcriptional levels of glutamine synthetase genes must be tested to confirm this hypothesis in the lines of mutant gmaap3, gmaap5, and gmaap8 under low-N conditions [71].
In addition, we also found that eight GmAAP genes (GmAAP9, GmAAP12, GmAAP19, GmAAP24, GmAAP25, GmAAP26, GmAAP33, and GmAAP34) were not expressed in roots; however, they were expressed to varying degrees in other tissues (Figure 6). Like the Arabidopsis AAP gene family, AtAAP1 was expressed in seeds and involved in amino acid transport to the embryo [22]; AtAAP2 was located in the phloem of the whole plant and regulated amino acid transport from xylem to phloem [18]; AtAAP3 was present in the root phloem and transported neutral and basic amino acids [17]; AtAAP6 was expressed mainly in sink tissues (roots, sink leaves) and the xylem parenchyma cells and might transport amino acids from the xylem to the phloem [13]; and AtAAP8, expressed in the phloem of the source leaves, participated in the allocation of amino acids to sink organs [24]. As such, the GmAAP gene family may be involved in amino acid regulation in different tissues. For example, GmAAP12, homologous to AtAAP6, was expressed highly in roots, stems, and floral buds (Figure 6, Table 1). This was similar to AtAAP6 [13]. However, GmAAP25/26, homologous to AtAAP8, was expressed highly in seeds (Figure 6, Table 1), different from AtAAP8 in the source leaves [24]. Therefore, understanding the LN response patterns of AAP expressions in stems, leaves, flowers, seeds and other tissues is of great significance to fully understand the nitrogen regulation mechanisms of the AAP gene family in soybean.
A limitation of this study is that we only applied a single genotype in the investigation of low-N responsiveness; it is worth investigating the responsive patterns of GmAAPs in soybeans with different low-N responsiveness, which could lead to a more comprehensive understanding of GmAAPs expression in response to low-N stress. Further, the present study focuses on the responsiveness within roots that could respond to low N directly. It is also worth simultaneously examining gene expression in above-ground tissues to gain insight into the systemic responsive patterns of GmAAPs from roots to shoots.

5. Conclusions

In this study, we performed a promoter analysis and expression pattern analysis of the low-N stress of the soybean AAP family, and performed a detailed investigation of their phylogenetic relationship, chromosomal distribution, gene structure, motif compositions, and tissue expression patterns. The results provide a comprehensive understanding of GmAAPs and a set of low-N responsive GmAAPs that are likely involved in N deficiency adaptation or seed quality traits (such as protein content) improvement. Further investigation may focus on the systematic expression in other tissues, and determine the functions through genetic engineering approaches such as Cas9-based gene editing. Regarding transgenic lines, we should also investigate in detail how N and amino acids are transported through roots and shoots and delivered to sink tissues, and how AAPs affect plant growth or seed protein content.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14010052/s1, Table S1: Primers used for qRT-PCR.

Author Contributions

Conceptualization, H.Z.; Methodology, L.W.; Software, Y.Z.; Validation, Y.Z.; Formal analysis, Y.Z.; Investigation, Y.Z. and L.W.; Writing—original draft, Y.Z.; Writing—review & editing, B.-H.S., D.Z. and H.Z.; Visualization, L.W.; Project administration, H.Z.; Funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Natural Science Foundation of Heilongjiang Province of China (JQ2022C005); the National Natural Science Foundation of China (32272176, 32272171); the Innovation Team Project of Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences (2022CXTD03); the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA28030101-3); and the Open Research Fund of the Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences (E1296004); Major National Science and Technology Projects during the 14th Five Year Plan period (2023ZD04069).

Data Availability Statement

Data presented in this paper is contained within the article and Supplementary Materials.

Acknowledgments

We thank Ping Yates from the Department of Biological Sciences, University of North Carolina at Charlotte, for proofreading the article, and Hong Zhai from the Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences for her generous help with the hydroponic assay.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Location distribution of the soybean amino acid permeases (AAP) gene family on chromosomes. The blue-colored blocks represent tandemly duplicated genes. Thirteen chromosomes containing GmAAP genes are displayed.
Figure 1. Location distribution of the soybean amino acid permeases (AAP) gene family on chromosomes. The blue-colored blocks represent tandemly duplicated genes. Thirteen chromosomes containing GmAAP genes are displayed.
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Figure 2. Phylogenetic trees of 63 AAPs in three species. All AAP protein sequences were downloaded from Phytozome v13. The tree was conducted based on the full-length amino acid sequences using MEGA 11 by the neighbor-joining method with 1000 bootstrap replicates. Bootstrap values are shown in percentage terms on the branches. Bootstrap values (1000 repetitions) less than 50 are not shown. The 63 AAP proteins were divided into four groups according to the topology. Group I—green region, containing 13 GmAAPs, 8 OsAAPs, and 4 AtAAPs. Group II—blue region, containing 11 GmAAPs, 4 OsAAPs, and 3 AtAAPs. Group III—orange region, containing 11 GmAAPs, 3 OsAAPs, and 1 AtAAP. Group IV—red region, containing 4 OsAAPs. Stars represent AAPs from soybeans, triangles represent AAPs from rice, and solid circles represent AAPs from Arabidopsis.
Figure 2. Phylogenetic trees of 63 AAPs in three species. All AAP protein sequences were downloaded from Phytozome v13. The tree was conducted based on the full-length amino acid sequences using MEGA 11 by the neighbor-joining method with 1000 bootstrap replicates. Bootstrap values are shown in percentage terms on the branches. Bootstrap values (1000 repetitions) less than 50 are not shown. The 63 AAP proteins were divided into four groups according to the topology. Group I—green region, containing 13 GmAAPs, 8 OsAAPs, and 4 AtAAPs. Group II—blue region, containing 11 GmAAPs, 4 OsAAPs, and 3 AtAAPs. Group III—orange region, containing 11 GmAAPs, 3 OsAAPs, and 1 AtAAP. Group IV—red region, containing 4 OsAAPs. Stars represent AAPs from soybeans, triangles represent AAPs from rice, and solid circles represent AAPs from Arabidopsis.
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Figure 3. Conserved motif, domain, and gene structure of the soybean AAP gene family. (a): According to the phylogenetic relationships, the GmAAP proteins clustered into three major phylogenetic subgroups (I, II, III). (b): The putative conserved motifs, motifs 1–15, are represented by different colored boxes at the top of the figure. (c,d): domains and the exon/intron structures in GmAAP genes are shown, respectively, from left to right in the figure. The relative position and size of the domain and exon can be estimated using the scale at the bottom. Red roundrect, blue roundrect, yellow boxes, and green boxes represent the domain Aa_trans, the domain Sdac, UTR, and exons, respectively.
Figure 3. Conserved motif, domain, and gene structure of the soybean AAP gene family. (a): According to the phylogenetic relationships, the GmAAP proteins clustered into three major phylogenetic subgroups (I, II, III). (b): The putative conserved motifs, motifs 1–15, are represented by different colored boxes at the top of the figure. (c,d): domains and the exon/intron structures in GmAAP genes are shown, respectively, from left to right in the figure. The relative position and size of the domain and exon can be estimated using the scale at the bottom. Red roundrect, blue roundrect, yellow boxes, and green boxes represent the domain Aa_trans, the domain Sdac, UTR, and exons, respectively.
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Figure 4. Analysis and illustration of collinearity of the soybean AAP gene family. The colored lines in the inner circle represent 26 homologous gene pairs. I—20 assembled chromosomes of soybean; II—GC ratio line map; and III—gene density heatmap. Red lines indicated high gene density in the chromosomes, and blue lines indicated low gene density in the chromosomes. The different colored lines instruct AAP syntenic regions on the GmAAP gene family. All GmAAP genes were shown in the outermost ring, and bold font indicated collinear genes. The gray line represented other gene syntenic regions in the soybean genome.
Figure 4. Analysis and illustration of collinearity of the soybean AAP gene family. The colored lines in the inner circle represent 26 homologous gene pairs. I—20 assembled chromosomes of soybean; II—GC ratio line map; and III—gene density heatmap. Red lines indicated high gene density in the chromosomes, and blue lines indicated low gene density in the chromosomes. The different colored lines instruct AAP syntenic regions on the GmAAP gene family. All GmAAP genes were shown in the outermost ring, and bold font indicated collinear genes. The gray line represented other gene syntenic regions in the soybean genome.
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Figure 5. Predicted cis-regulatory element in the 2000 bp region upstream of the start codon (ATG) of each GmAAP gene. (a): The phylogenetic tree of the GmAAP genes. The phylogenetic tree was clustered into three groups (I, II, III) (b): Cis-regulatory elements are represented with colored boxes. The scale below the figure indicates the relative position of each cis-element relative to the start codon, ATG. These elements were divided into four types including stress-responsive elements (LTR, MBS, MYB-related components, TC-rich repeats, the TCA-element, and WUN-motif), hormone-responsive elements (ABRE, ERE, GARE, P-box, and the TGA-element), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian).
Figure 5. Predicted cis-regulatory element in the 2000 bp region upstream of the start codon (ATG) of each GmAAP gene. (a): The phylogenetic tree of the GmAAP genes. The phylogenetic tree was clustered into three groups (I, II, III) (b): Cis-regulatory elements are represented with colored boxes. The scale below the figure indicates the relative position of each cis-element relative to the start codon, ATG. These elements were divided into four types including stress-responsive elements (LTR, MBS, MYB-related components, TC-rich repeats, the TCA-element, and WUN-motif), hormone-responsive elements (ABRE, ERE, GARE, P-box, and the TGA-element), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian).
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Figure 6. Expression pattern of the GmAAP genes in different tissues. The data were obtained from the RNA-Seq data (seedgenenetwork.net, accessed on 26 October 2023). The heatmap was executed by the R package pheatmap version 1.0.12. Different colors in the heatmap represent gene relative expression levels, as shown in the scale bar at the top right of the figure. Abbreviations: RT—roots; STEM—stems; LF—leaf; FLUB—floral bud; GLOB—globular stage; HRT—heart stage; COT—cotyledon stage; EM—early maturation stage; MM—mid-maturation stage; LM—late maturation stage; DS—dry seed; GLOB_ES—globular stage endosperm; HRT_ES—heart stage endosperm; COT_ES—cotyledon stage endosperm.
Figure 6. Expression pattern of the GmAAP genes in different tissues. The data were obtained from the RNA-Seq data (seedgenenetwork.net, accessed on 26 October 2023). The heatmap was executed by the R package pheatmap version 1.0.12. Different colors in the heatmap represent gene relative expression levels, as shown in the scale bar at the top right of the figure. Abbreviations: RT—roots; STEM—stems; LF—leaf; FLUB—floral bud; GLOB—globular stage; HRT—heart stage; COT—cotyledon stage; EM—early maturation stage; MM—mid-maturation stage; LM—late maturation stage; DS—dry seed; GLOB_ES—globular stage endosperm; HRT_ES—heart stage endosperm; COT_ES—cotyledon stage endosperm.
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Figure 7. (a) Expression profiling of the GmAAP genes in soybean roots under CK (normal N, 7.5 mM) and LN (low N, 0.75 mM) conditions at 12 h and 24 h after treatment. (b) Expression pattern of the GmAAP genes in the circadian time course of soybean unifoliolate leaves. The data were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA369113, accessed on 26 October 2023) and reanalyzed to obtain the transcriptome of Williams 82 under constant light conditions. ZT—Zeitgeber time. The red dotted boxes indicate that the genes with circadian elements are regulated by circadian rhythm. The heatmaps were executed by the R package pheatmap version 1.0.12.
Figure 7. (a) Expression profiling of the GmAAP genes in soybean roots under CK (normal N, 7.5 mM) and LN (low N, 0.75 mM) conditions at 12 h and 24 h after treatment. (b) Expression pattern of the GmAAP genes in the circadian time course of soybean unifoliolate leaves. The data were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA369113, accessed on 26 October 2023) and reanalyzed to obtain the transcriptome of Williams 82 under constant light conditions. ZT—Zeitgeber time. The red dotted boxes indicate that the genes with circadian elements are regulated by circadian rhythm. The heatmaps were executed by the R package pheatmap version 1.0.12.
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Figure 8. Relative transcript levels of the GmAAP genes in soybean roots under CK (NN) and LN conditions at 12 h and 24 h after treatment. Soybean TUB4 (GenBank accession no. NM_001252709) was used as an internal control. Data are the mean ± SE of three biological replicates. Asterisks indicate statistically significant differences compared with CK based on Student’s t-test (* a = 0.05, ** a = 0.01). No significant differences were not labeled.
Figure 8. Relative transcript levels of the GmAAP genes in soybean roots under CK (NN) and LN conditions at 12 h and 24 h after treatment. Soybean TUB4 (GenBank accession no. NM_001252709) was used as an internal control. Data are the mean ± SE of three biological replicates. Asterisks indicate statistically significant differences compared with CK based on Student’s t-test (* a = 0.05, ** a = 0.01). No significant differences were not labeled.
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Table 1. Soybean GmAAP family and physicochemical properties.
Table 1. Soybean GmAAP family and physicochemical properties.
Gene NameGene IDNumber of Amino AcidsMolecular Weight (kDa)Theoretical pIInstability IndexGrand Average of HydropathicityArabidopsis Orthologs
GmAAP1Glyma.02G19010010912.045.1844.250.48AtAAP3
GmAAP2Glyma.02G30350046151.138.8336.960.48AtAAP7
GmAAP3Glyma.02G30370047752.176.8232.810.50AtAAP7
GmAAP4Glyma.04G08660047151.639.3535.870.54AtAAP8
GmAAP5Glyma.04G08820047953.298.9334.040.35AtAAP6
GmAAP6Glyma.04G20910048753.826.3134.670.48AtAAP2
GmAAP7Glyma.04G20920048653.298.7637.460.39AtAAP3
GmAAP8Glyma.04G24640048753.618.3932.030.38AtAAP3
GmAAP9Glyma.05G19460048453.158.9139.180.43AtAAP2
GmAAP10Glyma.06G08820047051.849.2433.450.56AtAAP1
GmAAP11Glyma.06G08830047151.579.2935.340.53AtAAP8
GmAAP12Glyma.06G09020047953.328.9532.800.34AtAAP6
GmAAP13Glyma.06G11640048753.568.3933.220.40AtAAP3
GmAAP14Glyma.06G15660046951.408.6535.120.45AtAAP3
GmAAP15Glyma.06G15670053158.476.6938.510.56AtAAP2
GmAAP16Glyma.08G00240042546.869.0034.930.35AtAAP2
GmAAP17Glyma.08G33660032736.375.9538.010.41AtAAP7
GmAAP18Glyma.08G33680046151.008.9433.250.50AtAAP7
GmAAP19Glyma.10G25530046250.759.0629.350.45AtAAP6
GmAAP20Glyma.11G10700051356.989.2736.800.37AtAAP2
GmAAP21Glyma.12G03200051357.059.3234.540.39AtAAP2
GmAAP22Glyma.13G03160047952.738.7931.990.46AtAAP3
GmAAP23Glyma.14G01010044949.207.6533.070.57AtAAP7
GmAAP24Glyma.14G01030037841.818.7340.960.39AtAAP7
GmAAP25Glyma.14G14420047252.009.5645.580.55AtAAP8
GmAAP26Glyma.14G14440017619.776.7143.410.55AtAAP8
GmAAP27Glyma.14G14470046050.279.0036.940.65AtAAP8
GmAAP28Glyma.14G15350047952.578.7433.740.47AtAAP3
GmAAP29Glyma.17G19200047052.048.7731.150.42AtAAP6
GmAAP30Glyma.17G19250046951.509.5338.380.50AtAAP8
GmAAP31Glyma.17G21280023725.667.9335.440.60AtAAP7
GmAAP32Glyma.18G07170046250.595.7634.230.49AtAAP7
GmAAP33Glyma.18G07180046150.829.0136.900.49AtAAP7
GmAAP34Glyma.18G07190046150.848.9932.970.48AtAAP7
GmAAP35Glyma.18G13670010111.135.0524.490.71AtAAP3
GmAAP36Glyma.19G05430030634.208.8836.800.52AtAAP7
Table 2. The motif sequences identified in this study.
Table 2. The motif sequences identified in this study.
MotifsSequencesDomain
1JEIQDTJKSPPPENKTMKKASLISIAVTTFFYLLCGCFGYAAFGBDTPGNAa_trans
2SNPYMILFGIVZILLSQIPBFHNLWWLSIVAAIMSFTYSFIGLGLGIAKVAa_trans
3GFGFYEPYWLIDIANACIVIHLVGAYQVYSQPJFAFVEKWASKRWPBSDFAa_trans
4YTSNLLADCYRTPDPVTGKRNYTYMDAVRSYLG
5PFFNDILGLLGAJGFWPLTVYFPVEMYISQKKIPKWSSKWIAa_trans
6TASAHIITAVIGSGVLSLAWAIAQLGWIAGPAVMAa_trans
7GLVQYJNLYGVAIGYTITASISMMAIKRSNCY
8GTVTEAEKVWRVFQALGBIAFAYSYSTIL
9IPGFPPYNLNLFRLVWRTIYVILTTVIAM
10QQSGSKCYDDDGRLKRTGT
11LQILSFACFJVSLAAAVGSIAGIVLDLKK
12HKSGHEAPCKF
13ENGRFKGSLTG
14YKPFKTKY
15RSRTLPSRIHQGIIEERHDVRPYLQVEVRPNNIQTETZAMN
Table 3. Homologous gene pairs, Ka, Ks, and Ka/Ks values of the GmAAP gene family.
Table 3. Homologous gene pairs, Ka, Ks, and Ka/Ks values of the GmAAP gene family.
Gene PairsKaKsKa/Ks
GmAAP12~GmAAP250.433.000.14
GmAAP13~GmAAP140.231.600.14
GmAAP13~GmAAP160.231.920.12
GmAAP14~GmAAP160.200.520.38
GmAAP17~GmAAP230.180.390.46
GmAAP17~GmAAP320.030.080.35
GmAAP2~GmAAP320.321.310.25
GmAAP2~GmAAP170.301.230.24
GmAAP20~GmAAP210.010.080.14
GmAAP23~GmAAP320.190.500.37
GmAAP25~GmAAP290.442.300.19
GmAAP4~GmAAP290.381.940.20
GmAAP5~GmAAP120.010.130.05
GmAAP5~GmAAP250.432.890.15
GmAAP6~GmAAP80.241.350.18
GmAAP6~GmAAP130.231.690.14
GmAAP6~GmAAP90.160.520.31
GmAAP6~GmAAP140.160.430.38
GmAAP6~GmAAP160.180.470.39
GmAAP8~GmAAP90.211.500.14
GmAAP8~GmAAP140.241.310.18
GmAAP8~GmAAP130.020.100.24
GmAAP8~GmAAP160.251.630.15
GmAAP9~GmAAP130.211.800.12
GmAAP9~GmAAP160.030.110.28
GmAAP9~GmAAP140.170.610.28
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Zhang, Y.; Wang, L.; Song, B.-H.; Zhang, D.; Zhang, H. Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean. Agronomy 2024, 14, 52. https://doi.org/10.3390/agronomy14010052

AMA Style

Zhang Y, Wang L, Song B-H, Zhang D, Zhang H. Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean. Agronomy. 2024; 14(1):52. https://doi.org/10.3390/agronomy14010052

Chicago/Turabian Style

Zhang, Yuan, Le Wang, Bao-Hua Song, Dan Zhang, and Hengyou Zhang. 2024. "Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean" Agronomy 14, no. 1: 52. https://doi.org/10.3390/agronomy14010052

APA Style

Zhang, Y., Wang, L., Song, B. -H., Zhang, D., & Zhang, H. (2024). Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean. Agronomy, 14(1), 52. https://doi.org/10.3390/agronomy14010052

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