Identification and analysis of MATE gene family in wheat
A total of 20 MATE genes were discovered from the wheat genome using homology search and MATE domain analysis, and they were designated TaMATE1–20 depending on their chromosomal position. MATE proteins from wheat encoded by 20 TaMATE genes were sequenced, and the proteins were found to be 202–533 amino acids long, with molecular weights ranging from 21.45 kDa to 58.20 kDa, theoretical isoeletric points (pI) ranging from 5.46 to 9.19, GRAVY ranging from 0.449 to 0.783, and the number of TM regions in TaMATE proteins ranging from 3 to 12. (Table 1). The sequence alignment indicated the presence of a conserved domain (MatE, pfam01554) and numerous residues in the MATEs (Fig. 1 A-B), indicating that the MATE domain organization is highly conserved.
The results of subcellular localization prediction showed that MATE proteins of wheat are well distributed in the plasma membrane (PM), chloroplast (chlo), vacuolar membrane (vacu), nucleus (nucl), endoplasmic reticulum (ER), and mitochondria (mito), golgi body (golg), cytoskeleton (cyto), extracellular and (extr). Surprisingly, 50% of TaMATE proteins were localized to the plasma membrane, 28% to the golgi body, and 9% to the endoplasmic reticulum. However, TaMATE10 was specifically localized to the endoplasmic reticulum and extracellular (Fig. 2)
Chromosome distribution and MATE gene duplication in wheat
The 20 TaMATE genes were found on 3 of the 12 wheat chromosomes (Table 1). Chromosomes 1 and 5 each had six TaMATE genes and chromosome 7 had eight TaMATE genes. Two gene pairs, TaMATE3- TaMATE5 and TaMATE4 -TaMATE6 exhibited tandem duplication, whiles three gene pair, TaMATE9 - TaMATE13, TaMATE10 - TaMATE12, and TaMATE14 -TaMATE19 showed segmental duplication (Table 2). The synonymous rate (Ks) for the segmental duplicated gene ranged from 0.114 to 0.228, with a and divergent time ranging from 8.67 to 17.35 Mya. The Ka/Ks value for the segmental duplicate gene ranged from 0.061 to 0.348, whereas that of the two tandem duplicate genes ranged from 0.093 to 0.095. The Ka/Ks values of the segmental and tandem duplicates were less than 1, indicating that purification selection occurred in these duplicates (Table 2).
Phylogenic analysis and classification
Following multiple sequence alignment, a phylogenetic tree was constructed using the MEGA-X software (v10.0.2) for the 20 TaMATE proteins, 10 AtMATEs, 10 OsMATEs, 10 ZmMATEs, and 10 StMATEss. MATE genes from wheat, rice, Arabidopsis, potato, maize, and rice were classified into four major subfamilies (groups), namely I-IV. Group I had 9 TaMATE genes with 3 OsMATEs, 7 AtMATEs, 3 StMATEs, and 4 ZmMATEs. Group II had 1 TaMATE with 3 ZmMATEs, 2 StMATEs, and 2 OsMATEs. Group III contained 8 TaMATEs with 2 AtMATEs, 4 OsMATEs, 2 ZmMATEs, and 4 StMATEs, while group IV had 2 TaMATEs with 1 AtMATE and 1 StMATE (Fig. 3).
Gene structure and motif analysis of MATE genes in wheat
From the results of the gene structure analysis, the twenty TaMATE genes could be divided into three separate groups (I-III) based on the similarity of their gene sequence. Group I had eight TaMATE genes, Group II had eight TaMATE genes, and Group III had four TaMATE genes (Fig. 4 A). Genes had one exon (5%, TaMATE9), two exons (9%, TaMATE8 and TaMATE17), three exons (5%, TaMATE16), five exons (5%, TaMATE9), six exons (9%, TaMATE15 and TaMATE20), seven exons (14%, TaMATE2, TaMATE13, and TaMATE18), eight exons (48%, TaMATE1, TaMATE3, TaMATE4, TaMATE5, TaMATE10, TaMATE11, TaMATE12, TaMATE114, and TaMATE19), nine exons (5%, TaMATE6) (Fig. 4A).
The conserved motifs of the TaMATE proteins were identified using the MEME program, and the motif sequences and annotations were further predicted by Pfam online server. The results showed that 10 putative conserved motifs were identified in most of the TaMATE proteins (Table 3, Fig. 4 B and C). The conserved motifs varied in length from 15 to 50 amino acids, with putative TaMATE domains predicted in conserved motifs 1, 2, 3, 4, 5, 6, and 8 of the TaMATE proteins (Table 4). Motifs 1, 2, 3, 4, 5, 6, and 8, which contained MATE domains, were found to be highly conserved in the 20 TaMATE proteins. Twelve (12) TaMATEs (60%) contained all ten motifs, 4 TaMATEs (20%) possessed nine motifs, 3 TaMATEs (15%) possessed five motifs, and 1 TaMATE (5%) possessed four motifs.
Promoter cis-acting regulatory element analysis and 3-dimensional modelling
Cis-acting regulatory elements (CAREs) serve as specific binding sites for transcription factors and so play a crucial role in regulating genes involved in the growth, differentiation, and development of organisms, including plants. The number of cis-acting regulatory elements found in the putative TaMATE genes ranged from 56 (2%, TaMATE9) to 173 (6%, TaMATE6) (Fig. 5 A). According to their functional annotations, the CAREs were divided into three groups: growth and development responsive elements (15%), phytohormone responsive elements (46%), and environmental stress responsive cis elements (39%) (Fig. 5 B). Furthermore, the growth and development responsive elements were CAT-box, ARE, AACA_motif, O2-site, NON-box, RY-element, GCN4_motif, GC-motif, HD-Zip 1, which were mostly involved in meristem expression, anaerobic induction, endosperm-specific negative expression, zein metabolism regulation, meristem specific activation, seed-specific regulation, endosperm expression, anoxic specific inducibility, and differentiation of the palisade mesophyll cells, respectively (Fig. S1). CGTCA-motif, TGACG-motif, TGA-element, ABRE, TATC-box, TCA-element, P-box, GARE-motif, and AuxRR-core elements in group II were implicated in the response to plant hormones such as gibberellin, auxin, abscisic acid, and methyl jasmonate (Fig. S1), whereas In group III, MBS, 3-AF1 binding site, G-box, G-Box, Sp1, CGTCA-motif, LTR, ACE, TC-rich repeats, GT1-motif, and MRE were implicated in low-temperature, defense and stress, and light responses (Fig. S1).
MATE proteins were predicted to have secondary structures, such as α-helix, TM helix, and coil structures (Fig. 6 and Table S2). The multiple α-helix structures ensured that MATE proteins were transported across the membrane in an efficient and stable manner. Most MATE proteins had comparable three-dimensional structures, and the closer the evolutionary relationship between genes, the more similar the three-dimensional (secondary) structures of the proteins were, as was the case with TaMATE10, 11, 12, 13, and 14 (Fig. 6 and Table S9).
Gene ontology (GO) analysis and miRNA targets
The analysis of MATE GO annotations showed three elements of functional classification: biological process (BP), molecular functions (MF), and cellular component (CC) (Fig. 7). The 20 MATE genes were involved in all the GO functional annotation. For BP, MF, the MATE genes were mainly involved in transmembrane transport, detoxification, response to toxic substances, and export of toxic substances across the plasma membrane (Fig. 8 A). For MF, the MATE genes were involved in activities such as transmembrane transport, xenobiotic transport, secondary active transport, and antiporter activity (Fig. 8 B). The 20 MATEs annotated to the CC were shown to function in cellular membrane (Fig. 8 C).
The predicted miRNA target regions of the MATE genes were identified using the psRNATarget online server, and 299 miRNAs targeting the MATE genes were identified (Fig. 8 A and Table S2). Tae-miR5175e was the miRNA that highly targeted MATE genes (Fig. 8 B). TaMATE11, 16, 13, and 14 are the genes highly targeted by the miRNAs (Fig. 8 B). The discovered miRNAs were involved in either cleavage (Fig. 8 C) or translation inhibition (Fig. 8 D). Among them, TaMATE11 showed the highest number of predicted miRNAs involved in cleavage inhibition (11%), whereas MATE7 showed the highest name of predicted miRNAs involved in translation inhibition (22%) (Fig. 8C-D).
Expression level analysis of MATE genes in wheat under drought, heat, and salinity stress
To investigate whether abiotic stress affects the expression levels of TaMATE genes, we chose seven TaMATE genes (from each subfamily, depending on gene structure and phylogenetic analysis) after analyzing the phylogenetic tree and promoters, and evaluated their relative expression levels in leaf tissue after drought, heat, and salinity stress for 5, 10, and 15 d using qRT-PCR (Fig. 9). The results of qRT-PCR analysis revealed that, when compared to control (CK), the time for TaMATE genes to reach a higher expression level in the same tissue was comparable, i.e., the period for most genes to reach the highest expression level in leaf tissues was 15 d, under these three abiotic stress conditions. TaMATE1, TaMATE2, and TaMATE18 expression levels rose 3.8-fold, 3.4-fold, and 2.5-fold, respectively, after 15 days of drought stress treatment, compared to the control. Similarly, TaMATE1 and TaMATE18 were the most highly expressed genes under heat stress, with 3.7-fold and 2.9-fold increases in expression, respectively. Additionally, when exposed to salt stress, the expression of TaMATE2 and TaMATE18 increased 3.6-fold and 2.4-fold, respectively, compared to the control (Fig. 9). TaMATE18, on the other hand, was the most expressed gene after 15 days of drought, heat, and salt stress, indicating that the expression patterns of genes in the same subfamily might differ considerably even under the same stress treatment conditions. The findings revealed that some genes had a greater and distinct expression pattern when subjected to certain stress conditions and the expression pattern of most of TaMATE genes, for example, followed a trend of 5 d > 10 d > 15 d.
In addition, the relative expression levels of the six TaMATE genes were utilized in a clustering analysis, with the results shown in a heat map (Fig. 10). The six TaMATE genes were classified into distinct groups based on the findings under drought, heat, and salinity stress conditions. Under drought stress, group I exhibited a lowly upregulated gene (TaMATE13), but group II had upregulated genes (TaMATE1, 10, 18, 2, and 14). (Fig. 10 A). TaMATEs were categorized into one distinct group under heat stress, and among the genes in this group, TaMATE1 and TaMATE14 were the most highly expressed (Fig. 10 B).
Furthermore, the relative expression values of the seven TaMATE genes were used in a clustering analysis and the results displayed in a heat map (Fig. 10). From the results, the seven TaMATE genes were classified into distinct groups under drought, heat, and salinity stress conditions. Under drought stress, group I had gene (TaMATE13), which was lowly upregulated, while group II had genes (TaMATE1, 10, 18, 2, and 14) which were upregulated (Fig. 10 A). Under heat stress, TaMATEs were classified into one distinct group and among the genes in this group, TaMATE1 and TaMATE14 were the highly expressed genes (Fig. 10 B). Similarly, under salt stress, TaMATE14, 1, and 13 comprised group I, and these genes were weakly expressed, whereas TaMATE13, 2, 10, and 18 comprised group II, and these genes were highly expressed (Fig. 10 C).
Physiological response to drought and heat stress in wheat
“Geumgangmil”, a commonly cultivated wheat genotype was used to assess the impact of drought and heat stress on wheat. According to the findings, treatments (drought, heat, and salt stress) caused significant alterations in the physio-biochemical indicators evaluated when compared to the control. However, the magnitude of change in these parameters differed among stress treatment (Fig. 11). For example, after 15 d of stress treatment, a significant 16%, 6%, and 11% decrease in RWC was observed under drought, heat, and salt stress conditions, compared to control (Fig. 11 A). The EL values increased by 61% (under drought stress), 65% (under heat stress), and 75% (under salinity stress) (Fig. 11 B). Furthermore, the MDA levels increased by 50% (under drought stress), 55% (under heat stress), and 62% (under salt stress) (Fig. 11 C), where a significant 35%, 52%, and 73% increase in proline content were observed under drought, heat, and salt stress treatment conditions, respectively (Fig. 11 D). The RWC, EL, MDA, proline, and the expression pattern of putative MATE genes (TaMATE1, TaMATE2, TaMATE10, TaMATE13, TaMATE14, and TaMATE18) were significantly affected by genotype (G), treatments (T), and their interactions (G × T) under drought, heat, and salt stress conditions, according to the two-way analysis of variance (ANOVA) (Table S3-5).
Correlation analysis between TaMATE gene expression and physiological stress indicators
Pearson’s correlation analysis was done to examine the relationship between physiological indicators and the expression of putative TaMATE genes. From the results, the expression of most of the TaMATEs was associated (negative or positive) with the physiological stress indicators (Table S6-S8). For example, under drought stress conditions, RWC, EL, and proline were strongly associated with TaMATE1 and TaMATE18 expression, while MDA was correlated with TaMATE1 expression (Table S6). EL was strongly associated with TaMATE1 and TaMATE10 expression and proline with TaMATE10 expression under heat stress conditions (Table S7), whereas under salt stress conditions, RWC and EL were associated with TaMATE1 and TaMATE18 expression, whereas proline level was correlated with TaMATE18 expression (Table S8).