Genome-Wide Investigation and Characterization of SWEET Gene Family with Focus on Their Evolution and Expression during Hormone and Abiotic Stress Response in Maize

The sugar will eventually be exported transporters (SWEET) family is an important group of transport carriers for carbon partitioning in plants and has important functions in growth, development, and abiotic stress tolerance. Although the SWEET family is an important sugar transporter, little is known of the functions of the SWEET family in maize (Zea mays), especially in response to abiotic stresses. To further explore the response pattern of maize SWEET to abiotic stress, a bioinformatics-based approach was used to predict and identify the maize SWEET gene (ZmSWEET) family. Twenty-four ZmSWEET genes were identified using the MaizeGDB database. Phylogenetic analysis resolved these twenty-four genes into four clades. One tandem and five segmental duplication events were identified, which played a major role in ZmSWEET family expansion. Synteny analysis provided insight into the evolutionary characteristics of the ZmSWEET genes with those of three graminaceous crop species. A heatmap showed that most ZmSWEET genes responded to at least one type of abiotic stress. By an abscisic acid signaling pathway, among which five genes were significantly induced under NaCl treatment, eight were obviously up-regulated under PEG treatment and five were up-regulated under Cd stress, revealing their potential functions in response to abiotic stress. These findings will help to explain the evolutionary links of the ZmSWEET family and contribute to future studies on the functional characteristics of ZmSWEET genes, and then improve abiotic stress tolerance in maize through molecular breeding.


Introduction
Sugar is an important and basic source of carbon to support plant morphogenesis and growth [1]. As a substrate for the production of primary and secondary metabolites, sugar participates in many physiological processes, including energy metabolism and signal transduction, to maintain plant growth and development and respond to stresses [2,3]. Therefore, the transport and distribution of sugar is crucial in plants [4]. Sugar requires carriers to mediate their transport through biofilms, and the transporters include monosaccharide transporters, sucrose transporters, and sugar will eventually be exported transporters (SWEETs) [5]. The SWEET protein family is an important intracellular and intercellular transporter of sugar, and is responsible for the transmembrane transport of sugar for various physiological functions such as providing sucrose for phloem loading, a response to abiotic stress and regulating seed filling, etc. [5][6][7].
SWEET proteins are distinguished by the presence of the MtN3/saliva (MtN3_slv) domain, also termed the PQ-loop repeat. The MtN3_slv domain is made up of three α-helical transmembrane domains . Eukaryotic SWEETs are normally composed of seven TM domains, comprising two tandemly repeated 3-TMs and a single linking TM domain [8].
Maize is one of the most important crops in the world. In addition to being used as food, maize also plays important roles in feed, industry, medical treatment and other aspects [35]. Studies of the gene family are important for the analysis of the origin and prediction of genes functions, and then predict the function of genes [5,29,30]. The SWEET gene family has been identified in many plants [29,30]. However, the functions of SWEET proteins are currently poorly understood in maize, especially in response to abiotic stresses. In the present study, twenty-four SWEET genes were identified by genome-wide analysis in maize and were classified into four clades. Comprehensive analysis of the SWEET genes was performed, including chromosomal distribution, phylogenetic relationships, exon-intron structure, motif composition, gene duplication, and collinearity analysis. The expression patterns of the SWEET genes were analyzed in response to polyethylene glycol (PEG), salt (NaCl), cadmium (Cd) stresses and exogenous ABA treatment. The results provide insights for functional characterization of the proteins and important information for further study of the regulatory mechanism of SWEET family members under abiotic stresses in maize.

Gene Structure and Conserved Motif Analysis of ZmSWEET Gene Family
Structural features of the ZmSWEET genes were analyzed and conserved motifs were detected using the MEME Suite. The number of exons and introns was similar within a clade. Most of the ZmSWEET genes harbored 4-6 exons, but ZmSWEET3b had three exons ( Figure 2). Similar motif compositions in the same clade were indicative of functional similarity among the members of each clade ( Figure 2). The ZmSWEETs in Clades I and II harbored motifs 1, 2, 3, and 5. Clade III ZmSWEETs contained motifs 1, 2, 3, 4, 5, 6, 7, 9, Phylogenetic tree for SWEET proteins of maize, rice, and Arabidopsis. Multiple sequence alignment of the SWEET domains was performed using MUSCLE, and the phylogenetic tree was constructed using MEGA 7.0 with the maximum likelihood method with 1000 bootstrap replicates. The tree was divided into four clades, designated I, II, III, and IV. Proteins of maize, rice, and Arabidopsis are highlighted in black, orange, and blue, respectively.

Gene Structure and Conserved Motif Analysis of ZmSWEET Gene Family
Structural features of the ZmSWEET genes were analyzed and conserved motifs were detected using the MEME Suite. The number of exons and introns was similar within a clade. Most of the ZmSWEET genes harbored 4-6 exons, but ZmSWEET3b had three exons ( Figure 2). Similar motif compositions in the same clade were indicative of functional similarity among the members of each clade ( Figure 2). The ZmSWEETs in Clades I and II harbored motifs 1, 2, 3, and 5. Clade III ZmSWEETs contained motifs 1, 2, 3, 4, 5, 6, 7, 9, and 10. Cluster IV ZmSWEETs featured motifs 1, 2, 3, 4, 5, and 6. All ZmSWEETs contained motifs 1 and 2, which are typical motifs of the MtN3_slv domain, and each clade shared similar motif features, further supporting the phylogenetic classification of the SWEET family.

Evolutionary Differentiation and Collinearity Analysis of ZmSWEET Genes
In the present study, only one pair of ZmSWEET genes (ZmSWEET4c/4b) was identified as a tandem duplication and were located on chromosome 5 ( Figure S1). Five pairs of segmental duplication events were identified, comprising ZmSWEET6a/6b, ZmSWEET11a/11b, ZmSWEET12a/12b, ZmSWEET13b/13c, and ZmSWEET17a/17b ( Figure  3 and Table S1). In comparison, seven, four, and four pairs of segmental duplication events were identified among SWEET genes in foxtail millet, rice, and sorghum, respectively (Table S1). Conserved motifs and gene structure analyses of maize SWEETs based on phylogenetic relationships. All motifs were identified with the MEME Suite using the complete amino acid sequences. Exon-intron structure analyses were performed with TBtools. (a) Neighbor-joining tree indicating evolutionary relationships. Clades I, II, III, and IV are indicated in gray, green, blue, and red, respectively. (b) Exon-intron structure. Green boxes, yellow boxes, and black lines indicate the untranslated region, coding sequence, and gene length, respectively. (c) Conserved motifs.

Evolutionary Differentiation and Collinearity Analysis of ZmSWEET Genes
In the present study, only one pair of ZmSWEET genes (ZmSWEET4c/4b) was identified as a tandem duplication and were located on chromosome 5 ( Figure S1). Five pairs of segmental duplication events were identified, comprising ZmSWEET6a/6b, ZmSWEET11a/11b, ZmSWEET12a/12b, ZmSWEET13b/13c, and ZmSWEET17a/17b ( Figure 3 and Table S1). In comparison, seven, four, and four pairs of segmental duplication events were identified among SWEET genes in foxtail millet, rice, and sorghum, respectively (Table S1). The syntenic relationships of the ZmSWEET family were analyzed by constructing six intergenomic collinear maps with three dicotyledons (Arabidopsis, Medicago sativa, and Brassica rapa) and three monocotyledons (Setaria italica, Oryza sativa, and Sorghum bicolor). A syntenic relationship was not detected between the SWEET genes of maize and dicotyledons. In contrast, 17, 15, and 16 pairs of SWEET collinear genes were identified between maize and foxtail millet, rice, and sorghum, respectively ( Figure 4 and Table S2). The syntenic relationships of the ZmSWEET family were analyzed by constructing six intergenomic collinear maps with three dicotyledons (Arabidopsis, Medicago sativa, and Brassica rapa) and three monocotyledons (Setaria italica, Oryza sativa, and Sorghum bicolor). A syntenic relationship was not detected between the SWEET genes of maize and dicotyledons. In contrast, 17, 15, and 16 pairs of SWEET collinear genes were identified between maize and foxtail millet, rice, and sorghum, respectively ( Figure 4 and Table S2).
The evolutionary constraints acting on the duplicated genes were estimated by calculating the rates of synonymous substitution (K s ) and non-synonymous substitution (K a ) and K a /K s ratio for duplicated gene pairs in maize, foxtail millet, rice, and sorghum (Table S1). Values of K a /K s = 1 indicate neutral selection, K a /K s < 1 indicates purifying selection, and K a /K s > 1 indicates positive selection to accelerate evolution. In the four graminaceous crops, KQL05781 and KQL14056 were the only gene pair detected with K a /K s close to 1. Other SWEET gene pairs had undergone purifying selection and the estimated divergence time ranged from 16 to 86 Mya (Table S1). The evolutionary constraints acting on the duplicated genes were estimated by calculating the rates of synonymous substitution (Ks) and non-synonymous substitution (Ka) and Ka/Ks ratio for duplicated gene pairs in maize, foxtail millet, rice, and sorghum (Table  S1). Values of Ka/Ks = 1 indicate neutral selection, Ka/Ks < 1 indicates purifying selection, and Ka/Ks > 1 indicates positive selection to accelerate evolution. In the four graminaceous crops, KQL05781 and KQL14056 were the only gene pair detected with Ka/Ks close to 1. Other SWEET gene pairs had undergone purifying selection and the estimated divergence time ranged from 16 to 86 Mya (Table S1).

Promoter Region Analysis of ZmSWEET Gene Family
To analyze the transcriptional regulatory mechanism of the ZmSWEET genes, the cisacting elements in the promoter region of the ZmSWEET genes were identified using the PlantCARE database. The cis-acting elements were identified and classified into three categories, comprising abiotic and biotic stresses, plant hormone response, and plant growth and development ( Figure 5 and Table S3). Among the abiotic and biotic stress elements, two general stress response motifs were detected, namely MYB (CCAAT box) and MYC (CAC ATG box) binding sites, accounting for 29% and 25% of the total number of cisacting elements identified, respectively. Other stress-specific cis-acting elements identified were responsive to injury and pathogens, including W-box, TC-rich repeats, and WUNmotif. Among the plant hormone response elements, the ABRE element involved in ABA response was identified in the promoter regions of 21 ZmSWEET members, the TCA-element involved in salicylic acid response was detected in six ZmSWEETs, and the TGACGmotif involved in methyl jasmonate response was identified in 17 ZmSWEETs. Other elements, such as as-1, which is involved in salicylic acid and oxidative stress response, the CGTCA-motif, which is involved in response to methyl jasmonate, and EREs, which are involved in ethylene reactions, accounted for 18%, 18%, and 4%, respectively, of the total number of cis-acting elements in this category. Twelve cis-acting elements associated with plant growth and development were identified in the ZmSWEET gene promoter regions

Promoter Region Analysis of ZmSWEET Gene Family
To analyze the transcriptional regulatory mechanism of the ZmSWEET genes, the cis-acting elements in the promoter region of the ZmSWEET genes were identified using the PlantCARE database. The cis-acting elements were identified and classified into three categories, comprising abiotic and biotic stresses, plant hormone response, and plant growth and development ( Figure 5 and Table S3). Among the abiotic and biotic stress elements, two general stress response motifs were detected, namely MYB (CCAAT box) and MYC (CAC ATG box) binding sites, accounting for 29% and 25% of the total number of cis-acting elements identified, respectively. Other stress-specific cis-acting elements identified were responsive to injury and pathogens, including W-box, TC-rich repeats, and WUN-motif. Among the plant hormone response elements, the ABRE element involved in ABA response was identified in the promoter regions of 21 ZmSWEET members, the TCA-element involved in salicylic acid response was detected in six ZmSWEETs, and the TGACG-motif involved in methyl jasmonate response was identified in 17 ZmSWEETs.
Other elements, such as as-1, which is involved in salicylic acid and oxidative stress response, the CGTCA-motif, which is involved in response to methyl jasmonate, and EREs, which are involved in ethylene reactions, accounted for 18%, 18%, and 4%, respectively, of the total number of cis-acting elements in this category. Twelve cis-acting elements associated with plant growth and development were identified in the ZmSWEET gene promoter regions ( Figure 5). These results suggested that the ZmSWEET genes might be broadly involved in growth and development, and response to various abiotic stresses in maize.

Expression Profiles of ZmSWEET Genes in Different Tissues and Expression Analysis of ZmSWEET Genes in Response to Abiotic Stresses and Exogenous ABA
To explore the functions of ZmSWEET genes, transcriptome analysis was performed to investigate the expression of ZmSWEET genes in different tissues at different developmental stages of maize ( Figure 6). ZmSWEET1a/4b/15b/4c/15a were expressed in almost all tissues analyzed. ZmSWEET4a/13a/3a/13b/1b/13c/17b were highly expressed in vegetative organs. ZmSWEET14a/14b/16/11a/11b were significantly expressed in the seed.
ZmSWEET6b/12b were weakly expressed in all tissues. These findings suggested that the ZmSWEET genes play diverse roles in the growth and development of maize.
Genes 2022, 13, 1682 9 of 18 ( Figure 5). These results suggested that the ZmSWEET genes might be broadly involved in growth and development, and response to various abiotic stresses in maize.

Expression Profiles of ZmSWEET Genes in Different Tissues and Expression Analysis of ZmSWEET Genes in Response to Abiotic Stresses and Exogenous ABA
To explore the functions of ZmSWEET genes, transcriptome analysis was performed to investigate the expression of ZmSWEET genes in different tissues at different developmental stages of maize ( Figure 6). ZmSWEET1a/4b/15b/4c/15a were expressed in almost all tissues analyzed. ZmSWEET4a/13a/3a/13b/1b/13c/17b were highly expressed in vegetative organs. ZmSWEET14a/14b/16/11a/11b were significantly expressed in the seed. ZmSWEET6b/12b were weakly expressed in all tissues. These findings suggested that the ZmSWEET genes play diverse roles in the growth and development of maize. To investigate ZmSWEET gene expression in response to abiotic stresses, the transcript abundance of the ZmSWEET genes was analyzed by qRT-PCR (Figure 7). Under salt stress, the transcription of five ZmSWEET genes (ZmSWEET1a/4a/6a/6b/13a) was significantly induced, whereas seven genes (ZmSWEET4b/11b/12a/14a/14b/15a/15b) were signif- To investigate ZmSWEET gene expression in response to abiotic stresses, the transcript abundance of the ZmSWEET genes was analyzed by qRT-PCR (Figure 7). Under salt stress, the transcription of five ZmSWEET genes (ZmSWEET1a/4a/6a/6b/13a) was significantly induced, whereas seven genes (ZmSWEET4b/11b/12a/14a/14b/15a/15b) were significantly inhibited. Under drought stress, the transcription of ZmSWEET4c/14b/15a was significantly increased. Under Cd stress, the transcript abundance of ZmSWEET1a was initially increased and thereafter decreased, and the transcript abundance of ZmSWEET3b/11a/13b/17a was distinctly increased. Abscisic acid is an important phytohormone involved in plant response to abiotic stresses. Under exogenous ABA treatment, the transcription of 16 ZmSWEET genes was induced, of which five genes (ZmSWEET1a/3b/12a/14a/15b) were significantly induced. Overall, the expression of 23 ZmSWEET genes showed different alterations under the abiotic stresses and exogenous ABA treatment, and some genes were affected on multiple treatments ( Figure S2).

Discussion
Crop growth and development are hampered by abiotic stresses, which affect c yields [36,37]. Sugar metabolism is an important physiological activity and is involve the plant response to diverse environmental factors [3]. SWEET proteins are among carriers of sugars that influence sugar transport and distribution in the plant [5]. In current study, 24 ZmSWEET genes were identified in the maize genome, and one n gene (ZmSWEETb-2) was identified on chromosome 3 from the results of a previous st [18]. However, Zm00001d009365 was not identified in the present study, probably beca of the use of different versions of maize genomic data. In addition, 17, 21, 23, and SWEETs were identified in Arabidopsis, rice, sorghum, and foxtail millet, respectiv which was consistent with the results of previous studies [12,29,36]. All evidence s gested that graminaceous species contain a similar number of SWEET genes.
The present phylogenetic analysis revealed that SWEET family genes (compris ZmSWEETs, AtSWEETs, and OsSWEETs) could be divided into four clades (Figure The SWEET proteins from maize and rice tended to cluster together, whereas prot from Arabidopsis clustered separately in the phylogenetic tree, suggesting that SWE genes may have mainly evolved after monocotyledon-dicotyledon divergence and be the differentiation of graminaceous species. The distinct clades within the gene fam may adopt distinct roles during evolution to maximize the flexibility of environme adaptation [5]. The SWEET proteins of Clades I and II preferentially transport hexo (such as glucose and fructose), those of Clade III are effective sucrose transporters, acco ing to studies of Arabidopsis and rice, and the proteins of Clade IV are localized to vesicular membrane and regulate fructose inflow and efflux [5]. The MtN3_slv domai the signature domain of the SWEET family of proteins and is represented by two p

Discussion
Crop growth and development are hampered by abiotic stresses, which affect crop yields [36,37]. Sugar metabolism is an important physiological activity and is involved in the plant response to diverse environmental factors [3]. SWEET proteins are among the carriers of sugars that influence sugar transport and distribution in the plant [5]. In the current study, 24 ZmSWEET genes were identified in the maize genome, and one new gene (ZmSWEETb-2) was identified on chromosome 3 from the results of a previous study [18]. However, Zm00001d009365 was not identified in the present study, probably because of the use of different versions of maize genomic data. In addition, 17, 21, 23, and 24 SWEETs were identified in Arabidopsis, rice, sorghum, and foxtail millet, respectively, which was consistent with the results of previous studies [12,29,36]. All evidence suggested that graminaceous species contain a similar number of SWEET genes.
The present phylogenetic analysis revealed that SWEET family genes (comprising ZmSWEETs, AtSWEETs, and OsSWEETs) could be divided into four clades (Figure 1). The SWEET proteins from maize and rice tended to cluster together, whereas proteins from Arabidopsis clustered separately in the phylogenetic tree, suggesting that SWEET genes may have mainly evolved after monocotyledon-dicotyledon divergence and before the differentiation of graminaceous species. The distinct clades within the gene families may adopt distinct roles during evolution to maximize the flexibility of environmental adaptation [5]. The SWEET proteins of Clades I and II preferentially transport hexoses (such as glucose and fructose), those of Clade III are effective sucrose transporters, according to studies of Arabidopsis and rice, and the proteins of Clade IV are localized to the vesicular membrane and regulate fructose inflow and efflux [5]. The MtN3_slv domain is the signature domain of the SWEET family of proteins and is represented by two predicted motifs, motif 1 and motif 2. The ZmSWEET family members frequently contained motifs 1, 2, 3, 4, and 5, indicating that these motifs are conserved within the ZmSWEET family and may be associated with the basic function of the protein. Motif 6 was detected in Clades III and IV, motifs 7, 9, and 10 were present in Clade III, whereas motif 8 was detected only in Clade II. The motif analysis showed that conserved domains, such as MtN3_slv, were widely present in all members, and unique motifs were detected in each clade (Figure 2). These results implied that, while SWEET genes transport sugar, the substrates may differ between each clade.
The evolution of gene function in higher plants suggests that each isoform is physiologically differentiated with regard to expression sites and regulatory modalities, thus helping the organism to adapt to different environments. SemiSWEETs, the bacterial homologs of SWEETs, are among the smallest transporter proteins known. In both SWEETs and SemiSWEETs, a collection of three TMHs combine to produce a single MtN3 unit [38,39], and the doubling of TMHs enhances the ability of the SWEET protein to transport sucrose. Currently, there are two hypothesized mechanisms to explain the evolution of SWEETs. The first hypothesis suggests that SWEET proteins arose through the replication and fusion of hemiglycan chains (containing three TMHs) [38]. The second hypothesis suggests that SWEET proteins were derived from the fusion of archaeal and bacterial hemiglycan chains [40]. Internal replication of 3-TMH genes must have occurred early in evolution to produce new proteins with 7-TMHs able to translocate greater amounts of sucrose [38]. Therefore, the current ZmSWEETs in the maize genome with 6-7 TMHs imply that the replication and fusion of SWEETs may have occurred in the early genome.
For the gene family expansion, three predominant mechanisms are whole-genome duplication (WGD) events, tandem duplication (TD), and chromosomal fragment duplication (SD) [41]. Among these mechanisms, WGD is an important mechanism that provides potential for the evolution of novel gene functions to enable new adaptations for physiological processes, such as plant tolerance of biotic and abiotic stresses, and generation of gene functional diversity, which in turn affects the evolutionary process of species. As a graminaceous crop, maize has undergone four WGD events (i.e., τ-, σ-, ρ-, and mWGD) of which ρ-WGD was graminaceous-specific and occurred~95-115 Mya [42]. Maize underwent an additional species-specific WGD event (mWGD,~26 Mya) [42]. In addition to WGD events, tandem and segmental duplications have played a critical role in the expansion of gene families [43]. In the present study, several gene duplication events were identified, including one tandem duplication and five segmental duplication events. The segmental duplication gene pairs ZmSWEET6a/6b, ZmSWEET11a/11b, and ZmSWEET13b/13c contained similar motif architectures. The tandem repeat gene pair ZmSWEET4b/4c differed somewhat with ZmSWEET4c missing motif 3. The motif structures of the remaining two segmental duplication gene pairs (ZmSWEET12a/12b and ZmSWEET17a/17b) differed slightly from one another, suggesting that the motif structures may have changed during gene duplication. SWEET genes with different structural domains may have diverged in function during replication. Thus, tandem duplication and segmental duplication are the major forms of SWEET family expansion in maize. The K a /K s ratio of 10 pairs of ZmSWEET genes indicated that all had undergone purifying selection. In addition, 17, 15, and 16 maize genes showed collinearity with the foxtail millet, rice, and sorghum genomes, respectively. In contrast, no gene collinearity was observed between maize and Arabidopsis. The results of collinearity analysis suggested that the expansion of the SWEET gene family may have mainly occurred after the divergence of monocotyledons and dicotyledons, and before the divergence of graminaceous species. This conclusion is consistent with previous evolutionary analyses.
The expression data showed different expression patterns of ZmSWEET genes in different tissues. ZmSWEET1a/4b/4c/15a/15b were significantly expressed in almost all tissues analyzed, ZmSWEET1b/3a/4a/13a/13b/13c were substantially expressed in vegetative organs, and ZmSWEET11a/11b/14a/14b/16 were mainly expressed in the endosperm of the seed, indicating that the genes play important roles in sugar transport in different maize tissues ( Figure 6). The changes in expression during organ development also suggested that different ZmSWEET genes function at different stages of organ development. The expression of ZmSWEET genes under several abiotic stresses was analyzed by qRT-PCR. Some ZmSWEET genes were significantly induced by high salinity, PEG, or Cd stress. The results implied that certain ZmSWEET genes were induced to modify sugar distribution in response to abiotic stresses in maize. A number of ZmSWEET genes were induced by exogenous ABA treatment and abiotic stress, such as ZmSWEET1a/4c/14b/15b/16/17a, suggesting that these genes might be under the regulation of the ABA signaling pathway to respond to abiotic stresses. However, several genes were induced by abiotic stress but not by exogenous ABA treatment, such as ZmSWEET4a/6a/6b/15a, which implied that these genes were independent of the ABA signaling pathway.
Overall, 24 ZmSWEET genes have been identified in the maize genome. These genes function as sugar transporters and play essential roles in maize growth and development as well as in response to biotic and abiotic stresses. In this study, we analyzed the evolutionary relationships and expression patterns of the ZmSWEET genes. The results provide insight into the potential functions and characteristics of the ZmSWEET genes. The information will contribute to future studies on the biological roles of the ZmSWEET genes in maize.

Plant Materials and Treatments
The autogamous maize cultivar "inbred line" was used in the study. Seeds were preserved in our laboratory and incubated in the seedling culture room of the Laboratory of Plant Physiology and Germplasm, Shenyang Agricultural University, from August to December 2020. Seeds of uniform size and full grains were selected. The seeds were disinfected with 75% ethanol and washed with distilled water after 1 min to remove the residual ethanol. The cleaned seeds were evenly sown in seedling pots, covered with vermiculite to a depth of 2 cm, and irrigated with distilled water to allow the vermiculite to absorb sufficient water. The seeds germinated after approximately 3 days and the seedling roots protruded from the bottom grid of the pots. Hoagland's nutrient solution (pH 6.0) was added to the basal tray in which the seedling pots were placed to ensure that the roots could access the nutrient solution. The nutrient solution was replaced every 3 days until the seedlings attained the three-leaf stage [44]. The three-leaf seedlings were then treated with drought, salt, and Cd stresses and exogenous ABA by application of halfstrength Hoagland's nutrient solution supplemented with 20% PEG for drought stress treatment, 200 mol/L NaCl for salt stress treatment, 40 mg/L CdCl 2 for Cd stress treatment, or 100 µmol/L ABA. The uppermost mature leaves were collected at 0, 6, 12, and 24 h after initiation of the stress treatment, with three biological replicates at each time point. The sampled leaves were snap-frozen in liquid nitrogen and stored in an ultra-low-temperature refrigerator at −80 • C.

Replication Events and K a /K s Analysis of SWEET Genes
Maize SWEET genes were examined for relationships within genomes and between single or multiple intergenomic covariates using MCScanX (http://chibba.pgml.uga.edu/ mcscan2/MCScanX.zip accessed on 6 May 2022) [59]. Multiple mechanisms may lead to gene family expansion and doubling, including whole-genome duplication or polyploidization, tandem duplication, segmental duplication, transposon-mediated transposon duplication, and retroposition.
To explore the selection pressure on the maize SWEET family, the ratio of K a (nonsynonymous substitution rate) and K s (synonymous substitution rate) was calculated using ClustalW (https://www.genome.jp/tools-bin/clustalw accessed on 7 May 2022). The time of occurrence of segmental duplication events for homologous genes was calculated as where λ is the rate of molecular substitution in grasses (6.5 × 10 −9 ), and expressed as a million years ago (Mya) [60].

Quantitative Real-Time PCR Analysis
Total RNA isolation and quantitative real-time PCR (qRT-PCR) analysis were performed to analyze the expression of maize genes under salt, drought, and Cd stress and exogenous ABA treatment [61]. A total of 23 maize SWEET homologous genes were used for the analysis. Total RNA from plant leaves was extracted using TRIzol Reagent (CW Biotech) and subjected to DNase I treatment to remove genomic DNA contamination. The RNA concentration was determined by means of a BioDrop ultra-micro ultraviolet nucleic acid assay. First-strand cDNA was synthesized from 1 µg total RNA using the UEIris II RT-PCR system. The qRT-PCR assays were performed using a real-time PCR analyzer (Bio-Rad, Applied Biosystems PCR, SCILOGEX Gradient Thermal Cycler PCR Instrument TC1000-G). Each reaction mixture contained 10 µL of 2× SYBR ® Green Pro Taq HS Premix, 1.0 µL cDNA sample, 0.4 µL forward primer (final concentration 10 µM), and 0.4 µL reverse primer (final concentration 10 µM) in a final volume of 20 µL. The thermal-cycling protocol was as follows: 95 • C for 5 min, then 45 cycles of 95 • C for 15 s and 60 • C for 1 min. Melting curve analysis was used to verify the specificity of the reaction. Three technical replicates of each cDNA sample were analyzed. The Zm00001d013367 genes were selected as an internal control to normalize the transcript levels of ZmSWEET genes. The relative gene expression levels were calculated using the 2 −∆∆Ct method (Table S6). The normalized data were processed with TBtools and plotted as a heatmap to visualize the changes in SWEET gene expression (https://github.com/CJ-Chen/TBtools accessed on 19 March 2022) [62]. The number of genes with relative gene expression greater than 2 and relative gene expression less than 0.5 under the four treatments were shown by using the online site Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/ accessed on 22 August 2022) to create venny diagrams. All primer pairs were designed with Primer (v5.0) software (http://www.broadinstitute.org/ftp/pub/software/Primer5.0/ accessed on 1 December 2021) and are listed in Table S7.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13101682/s1, Figure S1: Chromosomal distribution of ZmSWEET genes. Tandem repeat genes are labeled in green. Figure S2: Venny diagram of the ZmSWEET relative expression under four treatments. a The number of ZmSWEET gene is induced under four treatments. b The number of ZmSWEET gene is inhibited under four treatments. Blue ovalshaped, yellow oval-shaped, green oval-shaped and red oval-shaped indicate NaCl treatment, PEG treatment, Cd treatment and ABA treatment, respectively; Table S1: Interchromosomal relationships of SWEET genes in maize, foxtail millet, rice, and sorghum. Table S2: Syntenic gene pairs between maize and foxtail millet, rice, and sorghum. Table S3: Distribution of cis-acting elements in the promoter region of maize SWEET genes. Table S4: The identification of SWEET members in rice, foxtail millet and sorghum. Table S5: Analysis and distribution of conserved motifs in maize SWEET proteins. Table S6: Quantitative results of the ZmSWEET under PEG, salt (NaCl), cadmium (Cd) stresses and exogenous ABA treatment. Table S7