Identification of the MATE genes in the rice genome
Via homology searches and domain (Pfam: PF01554) prediction, 46 genes encoding specific MATE proteins were ultimately identified in the rice genome. The genes were named OsMATE1-OsMATE46 according to their physical location on the chromosome. The length of the proteins encoded by these genes is between 370 and 598 aa, the molecular weight ranges from 39.41 to 61.65 kD, and the predicted isoelectric point ranges from 5.01 to 11.98. Most of the proteins are neutral or partially alkaline. The protein subcellular localization prediction results indicate that rice MATE proteins are distributed on the endoplasmic reticulum (Table1).
Chromosome distribution and replication pattern of the OsMATE genes
The results of the OsMATE gene chromosome mapping show that 46 MATE genes are distributed across the 12 chromosomes of rice, but the distribution is uneven. Among them, chromosome 3 contains the largest number of MATE genes—a total of 10, and only one of the MATE genes is on chromosome 5 (Figure 1A). There are 3 pairs of tandem repeat OsMATE genes (OsMATE21 and OsMATE22, OsMATE39 and OsMATE40, and OsMATE41 and OsMATE42) in rice, which are located on chromosomes 6, 10 and 11, respectively, and there is a high similarity between the protein sequences within each gene cluster. In addition, 6 pairs of fragment repeat genes were detected (Figure 1B). Taken together, these results indicate that tandem repeats and fragment replication contribute to the expansion of the rice MATE gene family.
Phylogenetic analysis of the MATE family
To study the phylogenetic relationship of rice MATE proteins, a phylogenetic tree was constructed using the MATE protein sequences of four different species (rice, corn, cotton and soybean) (Figure 2). According to the topology of the evolutionary tree, 46 rice MATE proteins could be grouped into four groups. The first group contains the largest number of MATE proteins, a total of 18, followed by the second group, which contains 13 MATE proteins. According to the phylogenetic relationship of the protein sequences, the functions of plant MATE proteins with known functions can be used to predict the functions of rice MATE proteins.
The first group contains 18 rice MATE proteins and several known genes, including AT3G59030 (AtTT12), AT4G25640 (AtFFT) and others. The function of the known MATE transporters in this branch suggests that members of the MATE subfamily I may be involved in the transport and accumulation of plant flavonoids, anthocyanins or alkaloids. The second group contains 13 rice MATE proteins. According to genes with known functions, the members of MATE subfamily II mainly transport multiple complexes. The third group includes nine MATE proteins. The members of this group of known MATE proteins has many different functions, including disease resistance, organogenesis, iron ion homeostasis regulation, and leaf senescence. The fourth group contains 6 MATE proteins. OsMATE4 and OsMATE9 have been found to participate in the secretion of citric acid in the root tips or in the transport of metal ions, indicating that these proteins are likely to participate in the physiological process of metal ion detoxification.
Gene structure and conserved motifs of the OsMATE gene family
The evolution of a family is mainly manifested as the diversity of gene structures and changes in conserved motifs. To better understand the structure of the rice MATE genes, the exon-intron structure of the OsMATE genes was analyzed using the annotation information of the rice reference genome (Figure 3B). The OsMATE genes were found to contain 1 to 14 exons, which is similar to the clustering results of the evolutionary tree. Genes in the same group often have similar structures but vary in their length of introns. Most genes in group I contain 7 or 8 exons, but OsMATE30 and OsMATE31 contain only 3 exons; moreover, the intron length of the genes in this group varies greatly. The genes in group II have 6-8 exons, but the length of their introns is shorter than that of many genes in group I. The group III genes have the fewest number of exons, with only 1 or 2, and the length of the exons is longer than that of the members in the other three subgroups. The group IV genes have the largest number of exons—7~13. The MEME online prediction tool was used to identify the conserved motifs in the rice MATE proteins (Figure 3C). A total of 10 conserved sequences (motifs 1~10) were identified. The results showed that all the rice MATE proteins contained at least 2 conserved motifs, and most MATE proteins (54%) contained all the conserved motifs. Most proteins in subfamilies I, II, and III contain similar types and numbers of conserved motifs, but there are significant differences from the proteins in the fourth group. The MATE proteins in the fourth group contained only 2 to 3 conserved motifs, and the number of motifs was significantly lower than the number of proteins in the first three groups (Figure 3C). These findings are similar to the prediction results of the conserved motifs of the MATE proteins in soybean [29], which may indicate that the function of the protein in the fourth group is more differentiated than that of the other three groups of members.
Characterization of putative cis-regulatory elements in the promoter regions of OsMATE genes
Cis-acting regulatory elements in the promoter regions play important roles in the plant response to stress. Using the PlantCARE database, we identified 11 putative stress-responsive cis-acting elements 1500 bp upstream of these OsMATE genes, including ABREs (ABA-responsive elements), TGACG motifs, CGTCA motifs (which are involved in the MeJA response), LTRs (low-temperature-responsive elements), MYBs, MBSs (MYB-binding sites), TCA elements (which are involved in salicylic acid responsiveness), TC-rich repeats (defense- and stress-responsive elements), WUN motifs (wound-responsive elements), GARE motifs (gibberellin-responsive elements) and AREs (anaerobic-responsive elements). The elements associated with the highest number of stress response elements within the OsMATE gene family are abscisic acid stress-related regulatory elements (ABREs) and drought-related elements (MYBs, MBSs) (Figure 4). ABA is synthesized mainly in response to drought and high salinity stress. Among the elements, the number of defense- and stress-related response elements (TC-rich repeats) is the smallest. In addition, there are regulatory elements related to anaerobic stress (AREs), low-temperature response elements (LTRs), and hormone response elements (TGACG motifs, CGTCA motifs, GARE motifs and TCA elements, etc). These results showed that the rice MATE genes and stress-related response elements are relatively complete, but the type and number of stress-related elements contained in each MATE gene promoter differ, indicating that members of the rice MATE gene family can respond differently to different stresses.
Expression patterns of OsMATE genes in different tissues
Via RNA-seq data, heat maps of 43 mate genes represented by FPKM values in different tissues and organs were constructed. A heatmap of gene expression was generated from a representative sample of 10 different organs (Figure 5). All OsMATE genes were expressed, while a few (MATE4, MATE32, MATE38 and MATE45) were expressed only in one tissue or organ. The number of gene families with members expressed in the leaves is the largest. Eleven genes, MATE1, MATE5, MATE14, MATE20, MATE24, MATE25, MATE26, MATE31, MATE34, MATE37 and MATE40, were expressed in all of the tissues and showed constitutive expression. Some OsMATE genes showed similar expression patterns in various tissues. MATE41, MATE42 and MATE44, which were placed in the first group in the phylogenetic analysis, showed relatively high expression levels in the shoots. MATE2 and MATE39 of the second gene family in the phylogenetic analysis were highly expressed in embryos, and MATE19 and MATE45 exhibited high expression levels in the pistil. The expression in different plants parts is closely related to the functions of genes.
Expression analysis of the rice MATE genes in response to abiotic stress
Crop production and yield quality in most farmlands are severely affected by salt and drought stresses. To further explore the expression changes in MATE genes in response to various abiotic stresses, including salt and drought, we randomly used eight OsMATE genes from the four phylogenetic groups. qRT-PCR was used to measure the transcript levels of the OsMATE genes. The expression levels of the MATE genes under salt and drought stresses varied among the eight members (Figure 6). MATE42 and MATE46 were downregulated after treatment. The remaining OsMATE genes were upregulated under salt stress, but the changes were not as extreme as those under drought stress. The expression of four genes (MATE4, 34, 16 and 45) reached the highest level for 24 h after salt stress, but the expression of two genes increased sharply for 3-6 hours after salt stress. The OsMATE genes were sensitive to drought stress, with none being downregulated. Notably, all genes presented their highest expression levels for six hours after drought treatment. There were different responses and regulatory mechanisms of the MATE family members under various abiotic stress conditions.