Identification of MATE Family and Characterization of GmMATE13 and GmMATE75 in Soybean’s Response to Aluminum Stress

The multidrug and toxic compound extrusion (MATE) proteins are coding by a secondary transporter gene family, and have been identified to participate in the modulation of organic acid exudation for aluminum (Al) resistance. The soybean variety Glycine max “Tamba” (TBS) exhibits high Al tolerance. The expression patterns of MATE genes in response to Al stress in TBS and their specific functions in the context of Al stress remain elusive. In this study, 124 MATE genes were identified from the soybean genome. The RNA-Seq results revealed significant upregulation of GmMATE13 and GmMATE75 in TBS upon exposure to high-dose Al3+ treatment and both genes demonstrated sequence homology to citrate transporters of other plants. Subcellular localization showed that both proteins were located in the cell membrane. Transgenic complementation experiments of Arabidopsis mutants, atmate, with GmMATE13 or GmMATE75 genes enhanced the Al tolerance of the plant due to citrate secretion. Taken together, this study identified GmMATE13 and GmMATE75 as citrate transporter genes in TBS, which could improve citrate secretion and enhance Al tolerance. Our findings provide genetic resources for the development of plant varieties that are resistant to Al toxicity.


Introduction
More than 50% of arable land consists of acidic soil worldwide.The severity of acidity is increasing due to multiple factors, including over agriculture, fertilizer utilization, and acid rain caused by air pollution [1].In acidic soils, the presence of soluble aluminum (Al) leads to the formation of toxic Al 3+ from aluminosilicate clays.This toxicity inhibits plant root elongation and growth, impairing water and nutrient uptake, ultimately resulting in low yield and poor growth of the plant [2].Different plant species employ various mechanisms to reduce Al 3+ toxicity and enhance survival under high Al 3+ conditions.Two major mechanisms, namely internal tolerance and exclusion have been extensively studied [3].The internal tolerance mechanism involves detoxifying Al 3+ in the cytosol by forming nontoxic organic acid (OA)-Al complexes and sequestering Al 3+ in vacuoles [4].The exclusion mechanism involves chelating Al 3+ in the rhizosphere using organic acid anions to form nontoxic OA-Al chelates, which limit Al 3+ uptake by roots, preventing Al 3+ interaction with sensitive root sites [5].Under Al stress, plants enhance resistance by upregulating root secretion of OAs such as citrate, malate, and oxalate, which aid in removing Al 3+ from roots.Consequently, a better understanding of the genes involved in plant Al resistance will contribute to the discovery of novel genotypes suitable for cultivation in acidic soils.
In the past decades, numerous genes and signaling pathways have been identified in relation to the exudation of OAs induced by Al stress.For example, the first gene identified was TaALMT1 (aluminum-activated malate transporter) in wheat [6], followed by SbMATE (multidrug and toxic compound extrusion) in sorghum [7] and HvAACT1 (aluminum-activated citrate transported 1) in barley, all of which are responsible for the secretion of OAs in response to aluminum [8].These transporters belong to the MATE family.Since then, multiple MATE genes have been found to participate in the modulation of OAs' exudation for Al resistance, making the MATE family the most widely categorized multidrug efflux transporter family [3].MATE transporters are widely present in bacteria, fungi, plants, and mammals, and they possess a unique structure with 12 transmembrane (TM) helices [9].Homologous genes encoding MATE proteins are required for Al resistance and detoxification and are localized in the root epidermis cells [10].Furthermore, MATE transporters also play a key role in a wide range of biological functions, including the accumulation of alkaloids and flavonoids, iron homeostasis and translocation, plant diseases and virus resistance, and plant hormone signaling [11].
Based on genome-wide analysis, numerous putative MATE transporters have been identified in multiple plant species, including 49 in maize [12], 67 in tomato [13], 56 in Arabidopsis thaliana [14,15], 45 in Oryza sativa [14], and 117 in Glycine max [16].Among these species, Glycine max has the highest number of MATE transporters.As an important oil-bearing crop, soybean is extensively cultivated in acidic soils with a long history.This has led to the development of diverse Al tolerance genes and mechanisms in soybeans.When exposed to Al stress, the roots of soybeans secrete organic acids (OAs) as a means of Al detoxification [17].Tolerant soybean genotypes exhibit higher OA secretion compared to sensitive cultivars.Previous studies have indicated that several MATE genes encode proteins that participate in the Al-induced secretion of OAs in soybeans as a response to Al toxicity.GsMATE from Glycine soja exhibits the highest expression level in roots and enhances resistance to Al stress [3].GmFRD3b has higher expression levels in the iron-efficient cultivar than in the iron-inefficient line, indicating a similar function to AtFRD3 in facilitating the efflux of citrate into the xylem [18].Additionally, another study showed upregulation of GmMATE75 in an Al-tolerant genotype called Jiyu 70, suggesting its involvement in Al resistance [19].Therefore, conducting further studies on GmMATEs from different soybean phenotypes would be valuable in enhancing our understanding of how soybeans cope with Al stress.
The Tamba black soybean (TBS) genotype is renowned for its remarkable Al tolerance attributed to its ability to secrete citrate in response to Al stress [20].In the present study, we aimed to investigate the functions of the MATE gene family in TBS in response to Al stress.We identified the MATE gene family and evaluated the gene expression patterns of these MATE family genes through RNA-seq analysis following Al treatment.Furthermore, we characterized the function and subcellular localization of the sensitive GmMATE genes through citrate transport activity assays and subcellular location detection methods.We also analyzed the phenotypes of Arabidopsis plants overexpressing these genes to gain insights into the mechanisms.Overall, our findings demonstrate which MATE genes are involved in Al tolerance and how they enhance Al tolerance in Glycine max "Tamba".

Identification of MATE Genes in the Soybean Genome
Through Blast searches and domain (Pfam: PF01554) prediction, 124 soybean MATE genes were ultimately identified.The genes were named GmMATE1-124 according to Liu et al. [16] and their physical location on the chromosome.The proteins encoded by these genes exhibit lengths ranging from 80 to 593 amino acids, molecular weights spanning from 8.71 to 64.28 kD, and predicted isoelectric points varying between 5.13 and 9.70 (Table S1).
A comparison was conducted among soybean MATE protein sequences, along with 45 Arabidopsis and 56 rice MATE protein sequences.The resulting phylogenetic tree categorized all MATE proteins into five subfamilies (Figure 1), with soybean MATE protein family members distributed among all these subfamilies.The fifth subfamily comprised the highest count of soybean MATE proteins, with 40 numbers, while the second and fourth group contains 32 and 31 soybean MATE proteins respectively.exhibit lengths ranging from 80 to 593 amino acids, molecular weights spanning from 8.71 to 64.28 kD, and predicted isoelectric points varying between 5.13 and 9.70 (Table S1).
A comparison was conducted among soybean MATE protein sequences, along with 45 Arabidopsis and 56 rice MATE protein sequences.The resulting phylogenetic tree categorized all MATE proteins into five subfamilies (Figure 1), with soybean MATE protein family members distributed among all these subfamilies.The fifth subfamily comprised the highest count of soybean MATE proteins, with 40 numbers, while the second and fourth group contains 32 and 31 soybean MATE proteins respectively.

Figure 1.
The phylogenetic tree of the MATE (multidrug and toxic compound extrusion) family from Arabidopsis thaliana, Oryza sativa, and Glycine max.The tree was constructed with MEGA 6.0 using the maximum likelihood (ML) method.Bootstrap values in percentages are 1000 replicates.Different subfamilies are highlighted using different colors: group I in red, group II in orange, group III in deep yellow, group IV in green, and group V in blue.

Gene Structure of the GmMATE Genes
The evolutionary progression of a gene family is chiefly evident in the diversity of gene structures and alterations in conserved motifs.Utilizing the MEME online prediction tool, the conserved motifs in soybean MATE proteins were identified, as illustrated in Fig- ure 2A.Ten conserved sequences were detected and denoted as motifs 1-10.The motif sequence was listed in Table S2.The first group of GmMATE proteins had fewer motifs than other groups in general.The majority of the members in groups II-V shared 7-10 motifs.All members of group II were equipped with motif 3. The existence of a protein domain in the MATE genes was searched in the NCBI Conserved Domain Search.As  Figure 1.The phylogenetic tree of the MATE (multidrug and toxic compound extrusion) family from Arabidopsis thaliana, Oryza sativa, and Glycine max.The tree was constructed with MEGA 6.0 using the maximum likelihood (ML) method.Bootstrap values in percentages are 1000 replicates.Different subfamilies are highlighted using different colors: group I in red, group II in orange, group III in deep yellow, group IV in green, and group V in blue.

Gene Structure of the GmMATE Genes
The evolutionary progression of a gene family is chiefly evident in the diversity of gene structures and alterations in conserved motifs.Utilizing the MEME online prediction tool, the conserved motifs in soybean MATE proteins were identified, as illustrated in Figure 2A.Ten conserved sequences were detected and denoted as motifs 1-10.The motif sequence was listed in Table S2.The first group of GmMATE proteins had fewer motifs than other groups in general.The majority of the members in groups II-V shared 7-10 motifs.All members of group II were equipped with motif 3. The existence of a protein domain in the MATE genes was searched in the NCBI Conserved Domain Search.As shown in Figure 2B, all of the identified GmMATE proteins contain MATE-related domains.The exon-intron structure of GmMATE genes was analyzed using the annotation information of the soybean genome (Figure 2C).Members of group II had a smaller number of exons, with approximately 1-3 exons.However, members of groups III, IV, and V typically possessed 6-8 exons, with a few exceptions among those with shorter sequences, which may have a lower number of exons such as GmMATE59 and GmMATE83.Group I members generally included a higher number of exons of over seven.Whereas, an exception was observed in GmMATE71, which contained only four exons.
shown in Figure 2B, all of the identified GmMATE proteins contain MATE-related domains.The exon-intron structure of GmMATE genes was analyzed using the annotation information of the soybean genome (Figure 2C).Members of group II had a smaller number of exons, with approximately 1-3 exons.However, members of groups III, IV, and V typically possessed 6-8 exons, with a few exceptions among those with shorter sequences, which may have a lower number of exons such as GmMATE59 and GmMATE83.Group I members generally included a higher number of exons of over seven.Whereas, an exception was observed in GmMATE71, which contained only four exons.

Gene Location of the GmMATE Genes on the Chromosome
The 124 GmMATE genes have been mapped to all 20 soybean chromosomes (Figure 3).However, their distribution across individual chromosomes appears to be uneven.Chromosome 9 exhibits the highest density of GmMATE genes, with a total of 12 members spanning from GmMATE 46 to GmMATE 57.Meanwhile chromosomes 2, 10, and 18 also have a high density of MATE genes, each harboring nine GmMATE genes.In contrast, chromosomes 4, 14, and 15 contain a lower number of GmMATE genes, with only four identified on each chromosome.

Gene Location of the GmMATE Genes on the Chromosome
The 124 GmMATE genes have been mapped to all 20 soybean chromosomes (Figure 3).However, their distribution across individual chromosomes appears to be uneven.Chromosome 9 exhibits the highest density of GmMATE genes, with a total of 12 members spanning from GmMATE 46 to GmMATE 57.Meanwhile chromosomes 2, 10, and 18 also have a high density of MATE genes, each harboring nine GmMATE genes.In contrast, chromosomes 4, 14, and 15 contain a lower number of GmMATE genes, with only four identified on each chromosome.

The Expression of GmMATE Genes on TBS under Al 3+ Stress
We identified the GmMATE genes in the transcriptome data to assess their expression patterns under Al stress.The expression heatmap of these genes is presented in Figure 4A.According to the expression profiles of the GmMATE genes across different treatments, the GmMATE genes were classified into nine clusters with time series analysis (Figure 4B).We focused on clusters 1 and 9, which showed a tendency of low expression in the NC, AC, and LD treatments, and high expression in the HD treatment.The expression patterns of genes within clusters 1 and 9 are depicted in Figure 4C.Noteworthily, within the two clusters, GmMATE13 and GmMATE75 demonstrated significant upregulation, with fold changes of 6.5 and 245, respectively, in the HD treatment compared to the NC treatment.

The Expression of GmMATE Genes on TBS under Al 3+ Stress
We identified the GmMATE genes in the transcriptome data to assess their expression patterns under Al stress.The expression heatmap of these genes is presented in Figure 4A.According to the expression profiles of the GmMATE genes across different treatments, the GmMATE genes were classified into nine clusters with time series analysis (Figure 4B).We focused on clusters 1 and 9, which showed a tendency of low expression in the NC, AC, and LD treatments, and high expression in the HD treatment.The expression patterns of genes within clusters 1 and 9 are depicted in Figure 4C.Noteworthily, within the two clusters, GmMATE13 and GmMATE75 demonstrated significant upregulation, with fold changes of 6.5 and 245, respectively, in the HD treatment compared to the NC treatment.

Character of GmMATE13 and GmMATE75 Genes in TBS
The full-length coding sequences (CDS) of GmMATE13 and GmMATE75 were amplified using cDNA from TBS.Sequencing results indicated that the full-length CDS of GmMATE13 and GmMATE75 are 1503 bp and 1674 bp, respectively.The sequence analysis revealed that the molecular formula of GmMATE13 protein is C 2457 H 3941 N 607 O 682 S 20 , with a total of 7707 atoms.The molecular weight is 53.5 kD, and the isoelectric point (PI) is 7.72.The amino acid composition is characterized by a high proportion of leucine (13.6%) and proline (10.4%), and a low proportion of cysteine (0.4%).The instability index (31.05< 40) indicates that GmMATE13 is a stable protein.The SOPMA software (https: //npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 6 December 2022) predicted the secondary structure of this protein, where α-helices account for 57.00%, extended chains account for 12.00%, β-turns account for 5.40%, and irregular coils account for 25.6%.

Character of GmMATE13 and GmMATE75 Genes in TBS
The full-length coding sequences (CDS) of GmMATE13 and GmMATE75 were amplified using cDNA from TBS.Sequencing results indicated that the full-length CDS of GmMATE13 and GmMATE75 are 1503 bp and 1674 bp, respectively.The sequence analysis revealed that the molecular formula of GmMATE13 protein is C2457H3941N607O682S20, with a total of 7707 atoms.The molecular weight is 53.5 kD, and the isoelectric point (PI) is 7.72.The molecular formula of the GmMATE75 protein is C 2763 H 4426 N 702 O 760 S 23 , with a total of 8674 atoms.The molecular weight is 60.4 kD and the PI is 9.54.The amino acid composition of GmMATE75 includes a higher proportion of leucine (12.6%) and proline (11.1%), and a lower proportion of cysteine (0.7%).The instability index is 28.14 < 40, indicating that GmMATE75 is a stable protein.The SOPMA software (https://npsa-prabi. ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 6 December 2022) predicted the secondary structure of this protein, where α-helices account for

The Expression of GmMATE13 and GmMATE75 upon Al 3+ Stress
The qRT-PCR results showed significant upregulation of both GmMATE13 an GmMATE75 under Al stress (Figure 6A,B).The two genes demonstrated significant higher expression levels in response to high Al 3+ concentration treatment (above 50 μM Al 3+ ), as compared to their expression levels in low Al 3+ concentration treatment (25 μM Al 3+ ).Specifically, GmMATE13 showed the highest expression level at 50 μM Al 3+ trea ment (Figure 6A), while GmMATE75 showed the highest expression level at 75 μM A treatment (Figure 6B).In terms of treated time gradient of 0-24 h, both GmMATE13 an GmMATE75 expression increased with Al 3+ treatment time.GmMATE13 reached its hig est expression level at 12 h (Figure 6C), while GmMATE75 reached its highest expressio level at 18 h (Figure 6D).Both GmMATE13 and GmMATE75 showed the highest expressio level under Al treatment compared to other metal ion treatments.Additionall GmMATE13 was upregulated in response to Ga, Fe, Cu, and Gr treatments (Figure 6E while GmMATE75 was upregulated in response to Ga, Fe, and Gr treatments (

The Expression of GmMATE13 and GmMATE75 upon Al 3+ Stress
The qRT-PCR results showed significant upregulation of both GmMATE13 and Gm-MATE75 under Al stress (Figure 6A,B).The two genes demonstrated significantly higher expression levels in response to high Al 3+ concentration treatment (above 50 µM Al 3+ ), as compared to their expression levels in low Al 3+ concentration treatment (25 µM Al 3+ ).Specifically, GmMATE13 showed the highest expression level at 50 µM Al 3+ treatment (Figure 6A), while GmMATE75 showed the highest expression level at 75 µM Al 3+ treatment (Figure 6B).In terms of treated time gradient of 0-24 h, both GmMATE13 and GmMATE75 expression increased with Al 3+ treatment time.GmMATE13 reached its highest expression level at 12 h (Figure 6C), while GmMATE75 reached its highest expression level at 18 h (Figure 6D).Both GmMATE13 and GmMATE75 showed the highest expression level under Al treatment compared to other metal ion treatments.Additionally, GmMATE13 was upregulated in response to Ga, Fe, Cu, and Gr treatments (Figure 6E), while GmMATE75 was upregulated in response to Ga, Fe, and Gr treatments (Figure 6F).Furthermore, under Al stress, GmMATE13 was upregulated in the roots, stems, and leaves of the TBS, while it was downregulated in the cotyledons (Figure 6G).On the other hand, GmMATE75 was only upregulated in the roots under Al stress, with no change in expression in other tissues (Figure 6H).
Furthermore, under Al stress, GmMATE13 was upregulated in the roots, stems, and leaves of the TBS, while it was downregulated in the cotyledons (Figure 6G).On the other hand, GmMATE75 was only upregulated in the roots under Al stress, with no change in expression in other tissues (Figure 6H).(E,F) Expression level of GmMATE13 and GmMATE75 treated with different metal ions for 24 h.CK was the untreated group.(G,H) Expression pattern of GmMATE13 and GmMATE75 in different plant organs, including roots (R), stems (S), leaves (L), and cotyledons (C).Data shown as mean ± SEM of n = 3 independent experiments and was analyzed using one-way ANOVA with Dunnett's multiple comparisons test.Different letters above columns indicate significance at p < 0.05.

Subcellular Localization of MATE Proteins
It has been reported that most MATE proteins in plants are localized on cell membranes [21].After the transient expression of GmMATE13-eGFP and GmMATE75-eGFP plasmids in Nicotiana benthamiana, green fluorescence with confocal microscopy was found to be concentrated on the cytoplasmic membrane, indicating that both GmMATE13 and GmMATE75 proteins are localized on the plasma membrane of plant cells (Figure 7).

Subcellular Localization of MATE Proteins
It has been reported that most MATE proteins in plants are localized on cell membranes [21].After the transient expression of GmMATE13-eGFP and GmMATE75-eGFP plasmids in Nicotiana benthamiana, green fluorescence with confocal microscopy was found to be concentrated on the cytoplasmic membrane, indicating that both GmMATE13 and GmMATE75 proteins are localized on the plasma membrane of plant cells (Figure 7).

Screening and Al Tolerance Identification of Arabidopsis Mutant Complementation Plants
RT-PCR analysis confirmed the complementary expression of GmMATE13 and GmMATE75 in the Arabidopsis mutant atmate (Figure 8A).The relative root elongation, root tip tissue staining, and citrate secretion were examined under Al stress.Arabidopsis complementation plants overexpressing GmMATE13 or GmMATE75 exhibited significantly higher relative root elongation compared to the atmate mutant but lower than the WT (Figure 8B,C).Staining with Evans blue and Chrome azurol S indicated reduced root tip damage and decreased absorption of Al 3+ in the Arabidopsis complementation plants compared to the atmate mutant (Figure 8D).Citrate secretion under Al stress showed a similar pattern to root elongation, with significantly higher levels in Arabidopsis complementation plants overexpressing GmMATE13 or GmMATE75 compared to the atmate mutant (Figure 8E).

Screening and Al Tolerance Identification of Arabidopsis Mutant Complementation Plants
RT-PCR analysis confirmed the complementary expression of GmMATE13 and Gm-MATE75 in the Arabidopsis mutant atmate (Figure 8A).The relative root elongation, root tip tissue staining, and citrate secretion were examined under Al stress.Arabidopsis complementation plants overexpressing GmMATE13 or GmMATE75 exhibited significantly higher relative root elongation compared to the atmate mutant but lower than the WT (Figure 8B,C).Staining with Evans blue and Chrome azurol S indicated reduced root tip damage and decreased absorption of Al 3+ in the Arabidopsis complementation plants compared to the atmate mutant (Figure 8D).Citrate secretion under Al stress showed a similar pattern to root elongation, with significantly higher levels in Arabidopsis complementation plants overexpressing GmMATE13 or GmMATE75 compared to the atmate mutant (Figure 8E).

Discussion
Aluminum toxicity is one of the primary limiting factors for crop growth and yield in acidic soils.The secretion of organic acids, such as citrate, in the root system is a wellknown mechanism for Al tolerance in plants [22].Many studies have shown that the secretion of OAs is mediated by anion channels and transport proteins located on the plasma membrane.Multidrug and toxic compound extrusion transporters (MATE) are commonly found in plant cells, predominantly in cell membranes, where they serve a crucial function in expelling plant secondary metabolites and toxic compounds [23].Over the past few decades, many members of the MATE gene family associated with aluminum tolerance have been identified, including the sorghum SbMATE gene [24], barley HvAACT gene [8], Arabidopsis AtMATE gene [25], maize ZmMATE gene [26], rice bean VuMATE gene [27], rice OsFRDL4 gene [28], and peanut AhFRDL1 gene [29].These genes have been shown to

Discussion
Aluminum toxicity is one of the primary limiting factors for crop growth and yield in acidic soils.The secretion of organic acids, such as citrate, in the root system is a wellknown mechanism for Al tolerance in plants [22].Many studies have shown that the secretion of OAs is mediated by anion channels and transport proteins located on the plasma membrane.Multidrug and toxic compound extrusion transporters (MATE) are commonly found in plant cells, predominantly in cell membranes, where they serve a crucial function in expelling plant secondary metabolites and toxic compounds [23].Over the past few decades, many members of the MATE gene family associated with aluminum tolerance have been identified, including the sorghum SbMATE gene [24], barley HvAACT gene [8], Arabidopsis AtMATE gene [25], maize ZmMATE gene [26], rice bean VuMATE gene [27], rice OsFRDL4 gene [28], and peanut AhFRDL1 gene [29].These genes have been shown to play important roles in citrate secretion and genetic transformation of these genes can enhance the secretion of citrate in plant root tips, thereby alleviating aluminum toxicity.
MATE transporters have been found in both prokaryotic and eukaryotic organisms, exhibiting a distinctive topology [30].However, the MATE family were conserved between dicots and monocots [19].Referring to Duan et al. [21], Blast searches were conducted using the MATE genes from two extensively studied plants, Arabidopsis thaliana and Oryza sativa, as query sequences to identify GmMATE genes in the present study.In comparison to a previous study on MATE genes in soybeans that identified 117 GmMATE genes [16], our research has identified an expanded set of 124 GmMATE genes.The seven additional GmMATE genes, namely GmMATE118-124, are situated on chromosomes 5, 6, 8, 11, 12, 16, and 18, respectively, and they share MATE or MATE-like domains.The functional characterization of these genes awaits further investigation and could provide valuable insights in future studies.
The GmMATE genes were identified in the transcriptome profile of Tamba black soybeans to assess the relation of the expression of GmMATE genes and the Al tolerance of TBS.Among the GmMATE genes in TBS, GmMATE13 and GmMATE75 exhibited significant upregulation in response to high-dose Al 3+ treatment, with fold changes of 6.5 and 245, respectively, compared to the NC treatment.They were proposed to function as important plasma-membrane-localized citrate transporters in TBS.Both genes were cloned and characterized, revealing a high degree of homology with GsFRD3, VuMATE1, VuMATE2, and GsDTX42.VuMATE1 and VuMATE2 are Al-activated citrate transporters that conferred Alinduced citrate efflux in Arabidopsis [31,32].Additionally, subcellular localization showed that GmMATE13 and GmMATE75 were localized on the plasma membrane, consistent with the localization of EcMATE1 in Eucalyptus camaldulensis [33], OsFRDL4 in rice [28], and ZmMATE1 in maize [26], which verified their potential functioning as Al-activated citrate transporters.They were mainly expressed in plant roots upon Al 3+ treatment.GmMATE13 was upregulated in roots, stems, and leaves, while GmMATE75 was only upregulated in roots, indicating the expression of GmMATE13 participates in more pathways in addition to responses to Al stress and GmMATE75 is closely related with the modulation of Al tolerance in roots.These findings are consistent with the MATE expression patterns in rice bean and buckwheat [32,34].Furthermore, GmMATE13 and GmMATE75 showed the highest relative expression levels at 12 h and 18 h, respectively.Similarly, PtrMATE1 was induced after 12 h of Al stress, while PtrMATE2 was induced after 24 h in poplar.The different response times of the two MATE genes may suggest the synergistic secretion of citrate by these citrate channel proteins to adapt to Al stress [17].These findings also imply the coordinated action of GmMATE13 and GmMATE75 in TBS to cope with Al stress.
Numerous studies have shown that overexpression of MATE genes can enhance citrate secretion in plants under Al stress and alleviate Al toxicity.For example, overexpression of the BoMATE and GmMATE2 genes can increase citrate secretion in Arabidopsis thaliana and tobacco, respectively [35].Additionally, in Arabidopsis mutants, overexpression of the AhFRDL1 gene can restore citrate secretion and iron transport [29].In the present study, transgenic complementation experiments in the Arabidopsis mutant atmate demonstrated that both the GmMATE13 and GmMATE75 genes mediated the secretion of citrate and improved Al tolerance.Furthermore, research by Liu et al. found that GmMATE13 and GmMATE75 are involved in response to Al stress [16].Subsequently, Zhou et al. discovered that overexpressing GmMATE75 in Arabidopsis resulted in increased citrate secretion under Al stress, thus alleviating the inhibition of root elongation caused by Al [19].Wang et al. found that the overexpression of GmMATE13 significantly increased citrate secretion in soybean hairy roots [36].These results further demonstrate the involvement of GmMATE13 and GmMATE75 in citrate secretion.In summary, our results indicated both GmMATE13 and GmMATE75 are citrate transporter proteins located on the cytoplasmic membrane.Overexpression of both genes in the mutant Arabidopsis can alleviate Al-stress-induced root tip impairment.This discovery provides valuable gene resources for breeding plant varieties suitable for growth in acidic soils.
In summary, GmMATE13 or GmMATE75 are proposed as key participants in conferring aluminum tolerance in TBS.They are citrate transporters at the plasma membrane.The expression levels of GmMATE13 and GmMATE75 are significantly upregulated under Al stress, which contribute to inducing the production of citrate efflux.The tolerance of plants to aluminum toxicity could therefore increase.The two GmMATE genes show differential expression responses under different durations of aluminum stress, indicating potential synergistic actions of their encoded proteins in adapting to aluminum stress.

Identification of MATE Genes in Soybean and Their Molecular Characteristics
The genome sequences and the annotation file of soybean (Glycine max Wm82.a2.v1) were downloaded from Phytozome (https://phytozome-next.jgi.doe.gov/,accessed on 20 December 2023).The 56 and 45 MATE gene family members in Arabidopsis thaliana and Oryza sativa were used as seed sequences to search the genome of soybeans for MATErelated protein sequences with the threshold E-value setting to ≤1 × 10 −5 [21].The obtained sequences were checked in the NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 12 January 2024) and Pfam (http: //pfam.xfam.org/,accessed on 12 January 2024) to detect the existence of conserved MATE protein domains.The protein length (number of amino acids), molecular weight, and theoretical isoelectric point were computed by ExPASy (https://web.expasy.org/protparam/, accessed on 20 January 2024) [37].

Phylogenetic Analysis of MATE Gene Family
The phylogenetic tree of protein sequences encoded by MATE family genes in A. thaliana, O. sativa, and G. max was constructed in the MEGA 6.0 software (Koichiro Tamura, Japan) with the maximum likelihood (ML) algorithm under 1000 bootstrap tests.The protein sequence used in constructing the phylogenetic tree is listed in Table S3.

Gene Structure, Motif Analysis, and Chromosomal Location
Conserved motifs within the MATE proteins were identified using the Multiple Em for Motif Elicitation (MEME, https://meme-suite.org/,accessed on 12 January 2023).The maximum number of motifs were set to ten.The gene structure and chromosome location of each GmMATE gene was analyzed and illustrated in TBtools (Guangzhou, China) with the annotation file of G. max Wm82.a2.v1.

Plant Culture and Al 3+ Treatments
TBS seeds were sterilized, rinsed, and then incubated on moistened filter paper in the dark at 25 • C for germination.The seedlings were cultured in 8 L aquariums with 1/2 Hoagland's nutrient solution (pH 6.0), with a light cycle of 14 h light/10 h dark (200 µmol photons m −2 •s −1 ) at a temperature of 27/22 • C (day/night).The nutrient solution was refreshed every two days.After the true leaf was fully expanded, the seedlings were transferred into 0.5 mM CaCl 2 solution (pH 4.3) pretreated for 24 h.Then, the seedlings were transferred and cultured in the solution with 10 and 50 µM AlCl 3 (pH 4.3, 0.5 mmol/L CaCl 2 ) for 3 d.After treatment, root apices (0-2 cm) were excised and immediately frozen in liquid nitrogen before isolating the total RNA.Each treatment was replicated three times.The seedlings without CaCl 2 and AlCl 3 treatments and those only treated with CaCl 2 were used as the neutral and acidic controls.
Arabidopsis wild type and Al sensitivity mutant atmate were utilized for transfection GmMATE genes and evaluating the response of GmMATE genes to Al stress.Arabidopsis seeds were placed on 1/2 Murashige and Skoog (MS) agar medium and kept in darkness at 4 • C for 2 days.Subsequently, the Arabidopsis seedlings were transferred to fresh 1/2 MS medium containing a specific concentration of Al and cultured for several days at 22 • C under long-day conditions until all the samples were collected.The collected plant materials were instantly put into liquid nitrogen and stored at −80 • C for RNA isolation.

RNA Extraction and Transcriptome Sequencing
Total RNA was extracted using the RNAi so Plus kit (Takara, Dalian, China) according to the manufacturer's description and the concentration and integrity of RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, USA) and the 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively.Samples with high RNA integrity and OD260/280 (above eight) were selected for library construction [38].The whole library was finally sequenced with the Illumina HiSeq platform (Novogene, Beijing, China).

Detecting MATE Family Gene Expression via RNA-Seq and Cluster Analysis
The transcription sequence of GmMATE genes were used in discovering MATE gene expression in TBS.Gene expression levels were estimated by fragments per kilobase of transcript per million fragments (FPKM) mapped.The expression level of MATE family genes was evaluated among different treatment groups, namely neutral control (NC), acidic control (AC), low dose (LD), and high dose (HD) of AlCl 3 (10 and 50 µmol/L).
The Mfuzz R package (version 2.62) was employed to cluster the expression levels of all MATE genes.This package is based on Fuzzy C-Means Clustering, which is initially used to analyze the time trend of gene expression in data with time series characteristics [39].This cluster analysis was performed on the transcriptome data to comprehend the dynamic expression patterns of the biological molecules with the associated functions.C-means clustering was calculated using the reads per million (FPKM) fragments of the neutral and acidic controls and two concentrations of Al 3+ treatment.

GmMATE13 and GmMATE75 Expression
After 2 weeks of cultivation, TBS seedlings were pre-treated with 0.5 mmol/L CaCl 2 (pH 4.5) for 24 h.To test the sensitivity of MATE genes to Al 3+ concentration, the seedlings were transferred and cultured in the solution with gradient concentrations of 0, 25, 50, 75, and 100 µM AlCl 3 (pH 4.3, 0.5 mmol/L CaCl 2 ) for 24 h.Root tips were collected to determine the expression level of MATE genes.Moreover, to analyze the temporal expression pattern of MATE genes in response to Al stress, the seedlings were transferred to a solution of 50 µmol/L AlCl 3 (pH 4.3, 0.5 mmol/L CaCl 2 ) for 24 h, and the root tips were obtained from different time points at 3, 6, 9, 12, 15, 18, 21, and 24 h.To determine the impact of other metal ions on GmMATE gene expression, the seedlings were transferred to solutions containing various metal ions, including 50 µmol/L AlCl 3 , 5 µmol/L CuCl 2 , 50 µmol/L FeCl 3 , 50 µmol/L Ga(NO 3 ) 3 , 50 µmol/L GrCl 3 , 50 µM La(NO 3 ) 3 , and 50 µmol/L MnCl 2 cultured for 24 h, respectively.Root tips were collected for measuring the expression of GmMATE 13 and GmMATE 75.Three biological replicates were performed for each treatment.Furthermore, to evaluate the organ-specific expression patterns of MATE genes, different parts of TBS seedlings, including root tips (R), stems (S), leaves (L), and cotyledons (C) were collected after treatment with 50 µM AlCl 3 solution for 24 h.Three biological replicates were performed for each treatment.
Total RNA extraction was the same as described above.Complementary DNA (cDNA) was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, Japan) following the manufacturer's instructions.Real-time quantitative PCR was performed following the manufacturer's instructions.The primers designed for GmMATE13 and GmMATE75 were based on their coding regions.Real-time PCR was performed to amplify the PCR products of the GmMATE13 and GmMATE75 PCR products.The primers used are listed in Table S4.The mRNA abundance was calculated according to the 2 −∆∆Ct method, with the expression levels normalized to the internal reference gene 40S rRNA gene (XM_0035498336.4).

Subcellular Localization of GmMATE Genes
To determine the subcellular localization of GmMATE13 and GmMATE75 proteins, their coding sequences (CDS) were cloned into the pBI121-eGFP vector with a CaMV 35S promoter.The GFP control and GmMATE expression vectors were transiently transformed into Nicotiana benthamiana with the Agrobacterium-mediated transient expression system.Confocal microscopy (Zeiss, LSM 900, Jena, Germany) was used to capture GFP fluorescence and bright-field images, confirming the subcellular location of GmMATE proteins.

Heterologous Expression of GmMATEs in the Arabidopsis Al Sensitivity Mutant Atmate
The Arabidopsis Al sensitivity mutant atmate was used to create GmMATE transgenic lines expressing GmMATE13 and GmMATE75.The CDS of the genes were cloned into a pCXSN vector with a CaMV 35S promoter.Positive colonies were identified using colony PCR and Sanger sequencing.Agrobacterium strain K599 was used to transfer the expression vectors into atmate.Two independent transgenic lines for each gene were obtained and named GmMATE13-CE1, GmMATE13-CE2, GmMATE75-CE1, and GmMATE75-CE2.

Al Resistance Analysis in Transgenic Atmate
The transgenic atmate lines, along with wild type (WT) and atmate plants, were tested for aluminum (Al) resistance.Two-week-old seedlings were pre-treated with 0.5 mM CaCl 2 and then exposed to 0 (control) and 50 µM AlCl 3 for 7 days.Root tips were collected for analysis of relative root elongation (RRE), Evans blue staining, and Chrome Azurol S staining.RRE% = (Al-treated root length − Al untreated root length)/(Control untreated root length − Control treated root length).Evans blue staining was used to assess plant cell injury and activity under acidic conditions.Root tips were stained with a 0.25% Evans blue solution.Chrome azurol S staining was performed to measure the accumulation of Al.Root tips were stained with a 0.1% Chrome azurol S solution.Stained roots were observed and captured using a dissecting microscope.

Statistical Analysis
Statistical analyses were performed using SPSS 22.0.One-way ANOVA and Student's t-test were used to compare significant differences among different groups, with p < 0.05 considered statistically significant.Results were presented as mean ± SEM (standard error of the mean).

Conclusions
The expression patterns of MATE genes in Tamba black soybean exhibit varied responses to aluminum stress.Among them, two MATE genes, GsMATE13 and GsMATE75, were found to be significantly upregulated under aluminum stress.Based on the results of protein structure, phylogenetic analysis, gene expression, protein localization, and overexpression validation in Arabidopsis, these two proteins were identified as citrate transporters located on the plasma membrane, participating in the efflux of citrate ions and alleviating aluminum toxicity in plants.This may be one of the reasons for aluminum tolerance in Tamba black soybean plants.Subsequent studies will involve knockout and overexpression of the MATE genes in Tamba black soybean to determine their roles in aluminum tolerance.

Figure 2 .
Figure 2. Gene structure, domain, and conserved motif analysis of GmMATE genes.(A) Distributions of conserved motifs in GmMATE genes.The sequence information of the motifs was listed in Table S2.(B) MATE and MATE-like domains of GmMATE genes.(C) Introns and exons on GmMATE genes.

Figure 2 .
Figure 2. Gene structure, domain, and conserved motif analysis of GmMATE genes.(A) Distributions of conserved motifs in GmMATE genes.The sequence information of the motifs was listed in Table S2.(B) MATE and MATE-like domains of GmMATE genes.(C) Introns and exons on GmMATE genes.

Figure 4 .
Figure 4.The expression of GmMATE genes in Tamba black soybean under Al stress.(A) The heatmap of MATE gene expression in neutral control (NC), acidic control (AC), low dose (LD, 10 μmol/L), and high dose (HD, 50 μmol/L) AlCl3 groups.(B) Time series analysis of GmMATE genes under Al stress.(C,D) Heatmap of the expression of genes belonging to cluster 1 and cluster 9.

Figure 4 .
Figure 4.The expression of GmMATE genes in Tamba black soybean under Al stress.(A) The heatmap of MATE gene expression in neutral control (NC), acidic control (AC), low dose (LD, 10 µmol/L), and high dose (HD, 50 µmol/L) AlCl 3 groups.(B) Time series analysis of GmMATE genes under Al stress.(C,D) Heatmap of the expression of genes belonging to cluster 1 and cluster 9.

Figure 5 .
Figure 5.The structure and phylogenetic analysis of the GmMATE13 and GmMATE75 proteins TBS. (A) Predicted 3D structure of GmMATE13.(B) Predicted 3D structure of GmMATE75.(C) Ph logenetic tree of GmMATE13 and GmMATE75 proteins.

Figure 5 .
Figure 5.The structure and phylogenetic analysis of the GmMATE13 and GmMATE75 proteins in TBS.(A) Predicted 3D structure of GmMATE13.(B) Predicted 3D structure of GmMATE75.(C) Phylogenetic tree of GmMATE13 and GmMATE75 proteins.

Figure 6 .
Figure 6.GmMATE13 and GmMATE75 expression pattern upon Al stress.(A,B) Relative expression of GmMATE13 and GmMATE75 treated with gradient AlCl3 concentration for 24 h.(C,D) Relative expression of GmMATE13 and GmMATE75 treated with 50 μM AlCl3 at various times of exposure.(E,F) Expression level of GmMATE13 and GmMATE75 treated with different metal ions for 24 h.CK was the untreated group.(G,H) Expression pattern of GmMATE13 and GmMATE75 in different plant organs, including roots (R), stems (S), leaves (L), and cotyledons (C).Data shown as mean ± SEM of

Figure 6 .
Figure 6.GmMATE13 and GmMATE75 expression pattern upon Al stress.(A,B) Relative expression of GmMATE13 and GmMATE75 treated with gradient AlCl 3 concentration for 24 h.(C,D) Relative expression of GmMATE13 and GmMATE75 treated with 50 µM AlCl 3 at various times of exposure.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 10 of 18 n = 3 independent experiments and was analyzed using one-way ANOVA with Dunnett's multiple comparisons test.Different letters above columns indicate significance at p < 0.05.

Figure 7 .
Figure 7. Subcellular localization of the GmMATE13 and GmMATE75 proteins; the images were captured with Zeiss LSM 900 confocal microscopy and the scale bar was 20 μm.

Figure 7 .
Figure 7. Subcellular localization of the GmMATE13 and GmMATE75 proteins; the images were captured with Zeiss LSM 900 confocal microscopy and the scale bar was 20 µm.

Figure 8 .
Figure 8.The function of GmMATE13 and GmMATE75 on Al resistance.(A) Expression of GmMATE13 and GmMATE75 in WT and two complementary lines (CE1 and CE2) were detected by RT-PCR.(B) Phenotypic analysis of WT, atmate, GmMATE13-CE, and GmMATE75-CE, scale bar was 1 cm.(C) Relative root elongation of the plants treated with 50 μM AlCl3 for 7 days.(D) Evans blue and Azurol S staining of the root tips treated with 50 μM AlCl3 for 24 h, scale bar was 0.5 mm.(E) Citrate secretion of the Arabidopsis roots treated with 50 AlCl3 Al for 24 h.Data shown as mean ± SEM of n = 4 independent experiments and was analyzed using one-way ANOVA with Dunnett's multiple comparisons test.Different letters above columns indicate significance at p < 0.05.

Figure 8 .
Figure 8.The function of GmMATE13 and GmMATE75 on Al resistance.(A) Expression of GmMATE13 and GmMATE75 in WT and two complementary lines (CE1 and CE2) were detected by RT-PCR.(B) Phenotypic analysis of WT, atmate, GmMATE13-CE, and GmMATE75-CE, scale bar was 1 cm.(C) Relative root elongation of the plants treated with 50 µM AlCl 3 for 7 days.(D) Evans blue and Chrome Azurol S staining of the root tips treated with 50 µM AlCl 3 for 24 h, scale bar was 0.5 mm.(E) Citrate secretion of the Arabidopsis roots treated with 50 AlCl 3 Al for 24 h.Data shown as mean ± SEM of n = 4 independent experiments and was analyzed using one-way ANOVA with Dunnett's multiple comparisons test.Different letters above columns indicate significance at p < 0.05.