Since monosaccharides such as glucose and fructose are essential for metabolism, storage, and transport, MST is crucial to the processes of carbon partitioning and abiotic stress response in plants [12, 26, 29]. Genome-wide analysis of the MST gene family has been widely carried out in many species. Fifty-three, sixty-four, and sixty-nine genes have been identified in Arabidopsis, rice, and sorghum respectively [26, 29]. However, the MST gene family has not been identified in maize. In this study, 66 MST genes were identified in the maize genome, and 77 MST family genes were identified in millet (Table 1 and Additional file 1). By the phylogenetic tree results, ZmMST genes were further divided into 7 subfamilies [30]. Each subfamily of MST was shown to be specific in the differentiation and evolution of the different Gramineae based on our findings that the number of genes in each subfamily of MST varied in maize, rice, sorghum, and millet. It was reported that the STP subfamily is the largest subfamily in rice. The ERD subfamily is the largest subfamily in Arabidopsis [26]. In maize, the largest subfamily is also the STP family, which indicates that the MST family has species-specific subfamily expansion in different plants. These expansions may be caused by gene duplication events, which may play a key role in the evolution of the MST gene family.
The previous study proved that most genes in the Arabidopsis genome were produced by species-specific expansion of the gene family [31]. Maize underwent three genome-wide replication events occurred, including approximately 110 million years ago before the differentiation of monocotyledons and dicotyledons, before the emergence of Gramineae 50 million years ago, and the genome-wide replication event after the differentiation of maize and sorghum 12 million years ago [32, 33]. Three ways of gene family expansion and doubling were found: whole-genome duplication (WGD), tandem duplication (TD), and segmental duplication (SD) [34]. WGD is a massive chromosome doubling event that increases the dose of all genes of a species at once, resulting in a large number of chromosomally doubled segments retained in the genome. Tandem duplication occurs frequently in chromosomal recombination domains, where members of tandemly duplicated gene families are typically tightly aligned on the same chromosome, forming a cluster of genes with related sequences and functions [35]. Segmental duplication occurs when duplicated genes are distant or even located on different chromosomes. In this study, multiple gene replication events were identified, including eight pairs of tandem replication gene pairs (ZmSTP5/6, ZmSTP21/22, ZmPMT5/6, ZmPMT11/12, ZmPMT12/13, ZmPMT15/14, ZmPMT15/16, and ZmERD3/4) with highly similar sequences in adjacent positions of chromosomes and twelve pairs of segmental duplication genes (ZmSTP12/5, ZmSTP5/14, ZmSTP8/17, ZmSTP14/21, ZmSTP12/14, ZmSTP12/21, ZmPMT8/14, ZmPMT14/3, ZmPMT1/18, ZmpGlcT1/4, ZmERD2/5, ZmERD6/3). The results showed that gene tandem duplication and chromosome segmental duplication are the main forms of monosaccharide transporter replication in maize.
Gene family expansion and doubling can provide new adaptability for plant growth and development to resist biotic and abiotic stress, leading to gene functional diversity, and affecting the evolution process of species [36]. Ka/Ks analysis was used to determine the relative divergence time and whether the functional differentiation of replication genes was subject to selection pressure. According to previous studies, Ka > > Ks or Ka/Ks > > 1, Ka = Ks or Ka/Ks = 1, Ka/Ks or Ka/Ks < < 1, and Ka/Ks or Ka/Ks < < 1 denoted that the gene was susceptible to positive selection, neutral evolution, and purifying selection, respectively [37, 38]. In this study, the Ka/Ks of all the duplicated gene pairs were calculated, and most of them showed less than one, indicating that they were subjected to purifying selection in the process of evolution. Five gene pairs subjected to strong positive selection were ZmSTP5/ZmSTP12, ZmERD2/ZmERD5, and ZmERD3/ZmERD6 for segmental duplication and ZmSTP5/ZmSTP6 and ZmSTP3/ZmSTP4 for tandem duplication. This indicated that they were positively selected and rapidly evolved genes in a short period, and gene functions may have diverged. Of these three gene pairs belong to the ERD subfamily, suggesting that the ERD subfamily may be more important for maize to respond to environmental change. Synteny analysis was performed to analyze the expansion of the MST family between species. There were 47, 41, and 33 collinear gene pairs identified between maize and sorghum, foxtail millet and rice, respectively. Only one collinear pair between maize and alfalfa (ZmpGlcT4/AES80568) and ZmpGlcT4 also formed collinear pairs with SORBI_3003G084000 and Os01g0133400 between maize and sorghum, rice, respectively. These results showed a closer evolutionary relationship between the two species containing more collinear gene pairs, and most of the no-collinear genes may be produced in earlier replication events.
Gene structure and conserved motif analyses were performed to further explore the evolutionary relationship in the MST family of maize. MST genes could be divided into seven subfamilies, and the PMT, STP, and TMT subfamilies contained fewer exons and simpler gene structures, while the pGlcT, ERD, INT, and VGT subfamilies had more exons and more complex gene structures, and gene structures were conserved in the same subfamily (Fig. 3). Motifs 1–6 were present in almost all MST family members and were vital for transport function and membrane localization, suggesting that these motifs were highly conserved domains during the evolution of the MST family. Other motifs existed in different subfamilies, indicating that these subfamily members transport different substrates. In addition, STP subfamily members contained all motifs except for motif 11, which implied a broader transport capability for different substrates. The effect of genes on plants depends not only on the function of genes themselves, but also on the regulation of gene expression. The cis-elements in the promoter region were involved in the regulation of the gene expression, and therefore the cis-element analysis was important for the preliminary prediction of gene expression. The type and number of cis-elements in promoters of MST family genes were identified in this study. The ABA response element ABRE belongs to the phytohormone response element and exists in most MST member promoter regions, meaning that the expression of MST members might be involved in the ABA signaling pathway. Additionally, many stress response elements were also found in the promoter region of MST members, such as DRE, MYB, and MYC, suggesting that MST family genes have an important role in maize response to environmental stresses.
To further investigate the functional response to environmental stresses, the expression mode of MST family genes in the maize seedling stage was detected under different stress treatments. The expression of half of MST genes was significantly induced under PEG treatment, while fewer genes were induced by salt and Cd stresses (Fig. 7A, B, and C). Meanwhile, the response of each subfamily in MST to different stresses was also different. Most of the PMT and ERD members responded to osmotic stress, and all ERD members were induced to expression by Cd stresses. The expression of most members was not induced in the STP subfamily under osmotic or ionic stresses. The expression patterns of MST members in response to stress treatments suggested that each subfamily might have different roles in balancing maize growth and development and responding to abiotic stress, implying that the members in each subfamily had also shown functional divergence during evolution and family expansion. ABA is a very important phytohormone in the plant response to abiotic stresses, and many stress response genes are regulated by ABA signaling [39]. In this study, the expression levels of some MST family members were induced by both ABA treatment and drought stress, indicating that these members might respond to stress in the ABA signaling pathway, while others were only induced by drought stress, meaning that they might respond to stress in the non-ABA signaling pathway.
Together, we have identified maize MST family genes. Our findings could contribute to future research on maize MST family genes and provide the foundation for additional investigation of the fundamental functions of this significant monosaccharide transporter family. These findings provide insight into the possible roles of genetic improvement in the capacity of maize to respond to abiotic challenges and may be used to identify relevant candidate MST family genes for functional research.