The Genome-Wide Identification, Characterization, and Expression Analysis of the Strictosidine Synthase-like Family in Maize (Zea mays L.)

Maize is often subjected to various environmental stresses. The strictosidine synthase-like (SSL) family is thought to catalyze the key step in the monoterpene alkaloids synthesis pathway in response to environmental stresses. However, the role of ZmSSL genes in maize growth and development and its response to stresses is unknown. Herein, we undertook the systematic identification and analysis of maize SSL genes. Twenty SSL genes were identified in the maize genome. Except for chromosomes 3, 5, 6, and 10, they were unevenly distributed on the remaining 6 chromosomes. A total of 105 SSL genes from maize, sorghum, rice, Aegilops tauschii, and Arabidopsis were divided into five evolutionary groups, and ZmSSL gene structures and conserved protein motifs in the same group were similar. A collinearity analysis showed that tandem duplication plays an important role in the evolution of the SSL family in maize, and ZmSSL genes share more collinear genes in crops (maize, sorghum, rice, and Ae. tauschii) than in Arabidopsis. Cis-element analysis in the ZmSSL gene promoter region revealed that most genes contained many development and stress response elements. We evaluated the expression levels of ZmSSL genes under normal conditions and stress treatments. ZmSSL4–9 were widely expressed in different tissues and were positively or negatively regulated by heat, cold, and infection stress from Colletotrichum graminicola and Cercospora zeina. Moreover, ZmSSL4 and ZmSSL5 were localized in the chloroplast. Taken together, we provide insight into the evolutionary relationships of the ZmSSL genes, which would be useful to further identify the potential functions of ZmSSLs in maize.


Introduction
Maize is one of the most widely distributed food crops in the world, with the total area under cultivation only less than wheat and rice.In addition to being eaten, it can also be used to produce feed and ethanol.Crops (as for example maize) are often subjected to various natural disasters (abiotic and biotic stress) during their growth and development stages, which have a great impact on their quality and yield [1].Because plants lack motility, they have evolved complex stress response systems, and a critical step in these responses is signal transduction [1,2].Many membrane-localized receptors sense stress signals or plant hormones (abscisic acid, ABA; ethylene; salicylic acid, SA; and methyl jasmonate, JA) produced by stress treatments and transmit stress signals into the cells through the influx of Ca 2+ and activate downstream genes of protein kinases and transcription factors (TFs).This, in turn, activates the expression of relevant defense genes to enhance plant stress tolerance [2][3][4][5][6].Alkaloids are an important secondary metabolite of plants; their synthesis is regulated by TFs, and they play a significant role in plant resistance to biotic and abiotic stress [7,8].Strictosidine (STR) is a well-known precursor substance that works on the de novo biosynthesis of many plant alkaloids, and strictosidine synthase (SSL, E.C.4.3.3.2.) catalyzes the synthesis of strictosamide (STR) from geraniol diphosphate and tryptamine in cytoplasm [8,9].Previous research has shown that chitooligosaccharides, the only positively charged basic amino oligosaccharides in nature, can induce the expression of STR and other genes, thereby improving the drought tolerance and yield of plants [10][11][12].
The protein sequences of the plant SSL gene family have two conservative motifs: strictosidine synthase (SS) and the SS-like N-terminal domain [8].The SS motif is responsible for the synthesis of STR, and the fold conformation of the SS-like N-terminal domain is similar to the SS motif [8].The sequence of the SSL extracellular motif (containing 400 amino acids) also shares a high similarity to the 100 kDa Drosophila immune-related protein (i.e., hemomucin, an important receptor) [8,9].
Until now, the SSL gene family has been systematically characterized only in Populus trichocarpa (13 members) [8], Arabidopsis thaliana (15 members) [13], and Salix purpurea (22 members) [8].All Arabidopsis SSL members are biotic/abiotic-stress-responsive [9,13].Further functional analysis revealed that AtSSL4-7 displays the enzyme activity necessary to catalyze the synthesis of STR, and the AtSSL4-7 gene is upregulated by treatments of SA, JA, and ethylene [13].The expression level of the PtrSSL2, 6, 8, 10, and 12 genes is elevated by drought and high salt levels, and the PtrSSL2, 7, 8, and 10 genes respond to leaf blight (Alternaria alternata) stress [8].In addition to responding to adversity, five or seven PtrSSL family members were highly expressed in stems or leaves, respectively [8].These results imply that SSL genes play an important role in normal growth and response to biotic/abiotic stresses in Poplar trees.There are a few reports about the numbers and function of SSL genes in other species.In Catharanthus roseus, the SSL gene is highly induced by drought stress, thus conferring plant drought tolerance [10].Tomato miR1916 pairs with STR gene mRNA and negatively regulates its accumulation; the over-expression of miR1916 in tomato plants significantly decreased the drought tolerance of transgenic lines [14].Conversely, the silencing of miR1916 in Solanum lycopersicum increased the expression level of STR-2, some immune-response-related proteins, and MYB transcription factor genes, which, in turn, significantly elevated the tolerance of tomato plant leaves to two common fungi (Phytophthora infestans and Botrytis cinerea) [15].Overall, although several reports indicate that the SSL gene family is related to plant stress responses, the role of ZmSSL genes in maize growth and development or response to biotic/abiotic stresses is far from known.
In the present study, we systematically identified and analyzed the maize SSL gene family members.With the help of homology comparisons using conserved protein motifs, 20 ZmSSL genes were identified; chromosomal localization was also undertaken along with the clarification of phylogenetics and gene structure.Cis-elements located in promoter analysis were also carried out.Moreover, we analyzed the expression level of ZmSSL genes under normal growth and various biotic/abiotic conditions using published transcriptome data and the RT-qPCR method.This work provides new information about the evolutionary relationships of the ZmSSL gene family, which provides a foundation for further detecting their possible biological functions.

Identification of the Maize SSL Gene Family
To systematically identify the SSL gene family members in maize (Zea mays L.), sorghum (Sorghum bicolor L.), rice (Oryza sativa L.), and one of the parents of common wheat, Aegilops tauschii (Aegilops tauschii Coss.), a homology analysis was conducted in a recent version of the genomic database of each species using AtSSLs protein sequences [8] and two conserved motifs (PF03088 and PF20067) based on the hidden Markov model (HMM) [8].After taking the intersection of comparison results and removing duplicates, 20, 21, 16, and 33 SSL genes were evaluated in maize, sorghum, rice, and Ae.tauschii, respectively.All of these identified genes were named in the order of the position in their chromosomes (Supplementary Table S1).The maize SSL gene family is unevenly distributed on 6 of the 10 chromosomes with the highest number (10 members) on chromosome 8 and the lowest on chromosomes 4 and 9 (Supplementary Table S1).Like the maize, the other three SSL family members were also unevenly distributed (Supplementary Table S1).The sequence characteristics and predicted subcellular localization of the SSL genes in these four species are shown in Table 1 and Supplementary Table S2.The ZmSSL genes encode proteins of 197-412 amino acids (aa) in length (average: 340 aa), with isoelectric points (PI) ranging from 5.81 to 8.92 (average: 6.57), and protein molecular weights (Mw) of 20.81-46.54kDa (average: 36.52).Among the 21 SbSSL proteins, the average aa, PI, and Mw were 353, 7.23, and 38.23, respectively.Ae. tauschii contains 33 SSL members; the average aa, PI, and Mw were 337, 7.61, and 36.63,respectively.The number of OsSSLs was almost identical to Arabidopsis; the average aa, PI, and Mw were 384, 8.00, and 41.66, respectively.Based on the predicted subcellular localization, ZmSSL genes were located in the chloroplast/thylakoid (14 members), extracellular/cell wall (4 members), plasma membrane (1 member), and endoplasmic reticulum (1 member), respectively (Table 1).The members of the other four species were also distributed in other places, such as the mitochondrion, vascular membrane, and cytosol (Table 1 and Supplementary Table S2).

Phylogenetic Analysis of the ZmSSL Genes' Protein
To examine the evolutionary relationships of SSL genes in maize, sorghum, rice, Ae. tauschii, and Arabidopsis (model species), a maximum likelihood phylogenetic tree was constructed using the protein sequences of the SSL genes listed in Table 1 and Supplementary Table S2.As shown in Figure 1, the 105 SSL members from five species could be divided into five groups (I-V), each group including 32, 11, 26, 29, and 7 SSL genes, respectively.SSL genes were present in Group I and Group II from all five plants, implying that members in these two groups may undergo a similar evolutionary pattern (Figure 1).Meanwhile, there is also an unequal distribution in the evolutionary tree; all AtSSLs were only represented in Groups I and II (14 members in I and 1 member in V) (Figure 1); the SSL genes of four crops were mostly represented in Groups III-V (from the same ancestor) (Figure 1), which might be due to the relatively distant evolutionary relationship between Arabidopsis and the crops.Maize SSL genes were predominantly in Group IV (13 members), and all branches of ZmSSL genes were more adjacent to SbSSL gene branches than to the other two crops (Figure 1).This indicates that maize and sorghum are evolutionary more closely related.
implying that members in these two groups may undergo a similar evolutionary pattern (Figure 1).Meanwhile, there is also an unequal distribution in the evolutionary tree; all AtSSLs were only represented in Groups I and II (14 members in I and 1 member in V) (Figure 1); the SSL genes of four crops were mostly represented in Groups III-V (from the same ancestor) (Figure 1), which might be due to the relatively distant evolutionary relationship between Arabidopsis and the crops.Maize SSL genes were predominantly in Group IV (13 members), and all branches of ZmSSL genes were more adjacent to SbSSL gene branches than to the other two crops (Figure 1).This indicates that maize and sorghum are evolutionary more closely related.

Structural Analysis of ZmSSL Genes
To further explore the ZmSSL family members, we evaluated the gene structure and conserve motifs of ZmSSL genes.The gene exon-intron structure/distribution analysis indicated that, except for ZmSSL4/5/6/20 (Group II), where the number of exons and introns varies from each other, the same group displays structural similarities; ZmSSL7 (Group I) contained 2 exons and 1 intron; four Group II members contained 3, 4, or 6 exons and 2, 3, or 5 introns, respectively; two Group III members (ZmSSL8 and ZmSSL9) included 3 exons and 2 introns; and all of Group IV (13 members) contained only 1 exon and no introns (Figure 2).Ten conserve protein motifs (amino acid numbers ranging from 15 to 80) were identified in ZmSSL genes using MEME (Figure 2).Further analysis revealed that there were similar sequences and numbers of motifs in the same subgroups (indicating close evolutionary branches), while there were some differences between the different subgroups (Figures 1 and 2).For example, ZmSSL1-3 contained motifs 1-8 (Figure 2).The most conserved motif, motif 10, was only located in ZmSSL5 and ZmSSL6 (Figure 2).ZmSSL7 and ZmSSL4 were from different groups, but the motif they contained was the same (Figure 2), probably due to those genes originating from the same branch (Figure 1).Some ZmSSL genes contained different motifs while some included the same motifs, suggesting that the functions of these genes may be different or redundant (Figure 2).Overall, these results indicated that the same group of ZmSSL genes shows a similar structure and this is consistent with the phylogenetic results presented in Figure 1.
distinguish each group.

Structural Analysis of ZmSSL Genes
To further explore the ZmSSL family members, we evaluated the gene structure and conserve motifs of ZmSSL genes.The gene exon-intron structure/distribution analysis indicated that, except for ZmSSL4/5/6/20 (Group II), where the number of exons and introns varies from each other, the same group displays structural similarities; ZmSSL7 (Group I) contained 2 exons and 1 intron; four Group II members contained 3, 4, or 6 exons and 2, 3, or 5 introns, respectively; two Group III members (ZmSSL8 and ZmSSL9) included 3 exons and 2 introns; and all of Group IV (13 members) contained only 1 exon and no introns (Figure 2).Ten conserve protein motifs (amino acid numbers ranging from 15 to 80) were identified in ZmSSL genes using MEME (Figure 2).Further analysis revealed that there were similar sequences and numbers of motifs in the same subgroups (indicating close evolutionary branches), while there were some differences between the different subgroups (Figures 1 and 2).For example, ZmSSL1-3 contained motifs 1-8 (Figure 2).The most conserved motif, motif 10, was only located in ZmSSL5 and ZmSSL6 (Figure 2).ZmSSL7 and ZmSSL4 were from different groups, but the motif they contained was the same (Figure 2), probably due to those genes originating from the same branch (Figure 1).Some ZmSSL genes contained different motifs while some included the same motifs, suggesting that the functions of these genes may be different or redundant (Figure 2).Overall, these results indicated that the same group of ZmSSL genes shows a similar structure and this is consistent with the phylogenetic results presented in Figure 1.

Chromosomal Localization and Collinearity Analysis of ZmSSL Genes
Based on the annotation of the maize genome, we mapped the physical location of ZmSSL genes on maize chromosomes.As shown in Figure 3A, ZmSSL1-3 located in Chr 1 and ZmSSL12-19 located in Chr 8 were both tandem repeats.Further collinearity analysis of the duplication events of the ZmSSL gene using MCScanXs showed that no segmental duplications (homologous genes) were obtained (Figure 3B).These results

Chromosomal Localization and Collinearity Analysis of ZmSSL Genes
Based on the annotation of the maize genome, we mapped the physical location of ZmSSL genes on maize chromosomes.As shown in Figure 3A, ZmSSL1-3 located in Chr 1 and ZmSSL12-19 located in Chr 8 were both tandem repeats.Further collinearity analysis of the duplication events of the ZmSSL gene using MCScanXs showed that no segmental duplications (homologous genes) were obtained (Figure 3B).These results indicated that tandem duplication is an important event in the evolution of the maize SSL gene family.
To further evaluate the evolutionary relationship of ZmSSL genes, a covariance analysis was performed.A total of 19 homologous pairs were identified between maize and sorghum, rice, Ae. tauschii, and Arabidopsis (8, 6, 5, and 0 collinear genes, respectively) (Figure 4).This indicates that the SSL family is conserved during the evolution process of explored grass crops.
indicated that tandem duplication is an important event in the evolution of the maize SSL gene family.
To further evaluate the evolutionary relationship of ZmSSL genes, a covariance analysis was performed.A total of 19 homologous pairs were identified between maize and sorghum, rice, Ae. tauschii, and Arabidopsis (8, 6, 5, and 0 collinear genes, respectively) (Figure 4).This indicates that the SSL family is conserved during the evolution process of explored grass crops.

The Cis-Elements Analysis of ZmSSL Gene Promoter Regions
Using PlantCARE to analyze the promoter regions of the ZmSLL genes, we identified a large number of cis-elements involved in the development and hormones/abiotic/biotic stress responses in the promoter regions of most ZmSSL genes (Figure 5, Supplementary Table S3).These included light-and development-relative elements (CAT-box, HD-Zip1, CIRCADIAN, G-box, AE-box, CCAAT-box, etc.), ABA-responsive motifs (ABRE and

The Cis-Elements Analysis of ZmSSL Gene Promoter Regions
Using PlantCARE to analyze the promoter regions of the ZmSLL genes, we identified a large number of cis-elements involved in the development and hormones/abiotic/biotic stress responses in the promoter regions of most ZmSSL genes (Figure 5, Supplementary Table S3).These included light-and development-relative elements (CAT-box, HD-Zip1, CIRCADIAN, G-box, AE-box, CCAAT-box, etc.), ABA-responsive motifs (ABRE and ABRE4/3a), salicylic acid-relative elements (TCA-element), IAA response motifs (AuxRRcore and TGA-element), GA elements (P-box), MYB and MYC binding site (MBS and MYC) elements involved in drought response, TC-rich elements involved in defense and stress responses, low-temperature-responsive elements (LTR and WRE3), and STRE for antioxidant responses.Overall, these results suggested that ZmSSL genes may participate in maize growth, development, and responses to a stressful environment.S3.

Expression Patterns of ZmSSL Genes in Different Maize Tissues under Normal Conditions
To explore the expression patterns of ZmSSL genes in maize tissues under normal conditions, published transcriptome sequencing data [16] (Supplementary Table S4) from 15 different maize tissues (defined by different developmental periods), including ear, embryo, endosperm, stalk, leaf, root, shoot apical meristem (SAM), and tassel, were used  S3.S4.

Expression Patterns of ZmSSL Genes under Abiotic/Biotic Stress Conditions
To explore the expression patterns of ZmSSL genes in maize tissues under abiotic and biotic stresses, published transcriptome sequencing data (Supplementary Table S5) for drought [17], heat or cold [18], and two fungal infections (Colletotrichum graminicola and Cercospora zeina) [19,20] were used to analyze and build an expression heat map (Figure 7).None of the 20 ZmSSL genes were induced during 1 h of dehydration stress (Figure 7), even though the promoter region was rich in drought-responsive elements (Figures 5 and  7).ZmSSL5 was induced after 24 h of C. graminicola infection (Figure 7), indicating it may be involved in maize responses to fungal infection.Both 50 °C for 4 h and 5 °C for 16 h clearly downregulated the expression of ZmSSL4, ZmSSL5, and ZmSSL7-9 (Figure 7).ZmSSL4 was downregulated by C. zeina infection while ZmSSL5 was upregulated, implying that it had the opposite effect in C. zeina infections (Figure 7).Similar to the tissue expression results (Figure 6), none of the 13 Group IV members or ZmSSL20 responded to abiotic/biotic stresses, indicating these genes may be responsive to other stresses or are otherwise pseudogenes.S4.

Expression Patterns of ZmSSL Genes under Abiotic/Biotic Stress Conditions
To explore the expression patterns of ZmSSL genes in maize tissues under abiotic and biotic stresses, published transcriptome sequencing data (Supplementary Table S5) for drought [17], heat or cold [18], and two fungal infections (Colletotrichum graminicola and Cercospora zeina) [19,20] were used to analyze and build an expression heat map (Figure 7).None of the 20 ZmSSL genes were induced during 1 h of dehydration stress (Figure 7), even though the promoter region was rich in drought-responsive elements (Figures 5 and 7).ZmSSL5 was induced after 24 h of C. graminicola infection (Figure 7), indicating it may be involved in maize responses to fungal infection.Both 50 • C for 4 h and 5 • C for 16 h clearly downregulated the expression of ZmSSL4, ZmSSL5, and  S5).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

RT-qPCR Validation of ZmSSL Genes under Abiotic/Biotic Stresses
To further analyze the expression levels of ZmSSL genes under abiotic/biotic stresses, we treated maize B73 to heat, cold, C. graminicola, and C. zeina stresses, and performed an RT-qPCR to explore the transcript level of ZmSSL4-9.The expression of four genes (ZmSSL4, 7, 8, 9) was notably downregulated at 42 °C (Figure 8A).ZmSSL8 and ZmSSL9 both positively responded to cold stress (Figure 8B).The results after C. graminicola infection indicated that ZmSSL5 and ZmSSL8 were upregulated after 24 and 48 h postinoculation (hpi), suggesting they may be involved in the response process (Figure 8C).Like the RNA-seq data (Figure 7), ZmSSL4 and ZmSSL5 displayed the opposite expression pattern under C. zeina infection (Figure 8D).Overall, six ZmSSL genes showed a diverse expression model under different biotic/abiotic stresses and may have different functions during these different stresses.S5).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

RT-qPCR Validation of ZmSSL Genes under Abiotic/Biotic Stresses
To further analyze the expression levels of ZmSSL genes under abiotic/biotic stresses, we treated maize B73 to heat, cold, C. graminicola, and C. zeina stresses, and performed an RT-qPCR to explore the transcript level of ZmSSL4-9.The expression of four genes (ZmSSL4, 7, 8, 9) was notably downregulated at 42 • C (Figure 8A).ZmSSL8 and ZmSSL9 both positively responded to cold stress (Figure 8B).The results after C. graminicola infection indicated that ZmSSL5 and ZmSSL8 were upregulated after 24 and 48 h post-inoculation (hpi), suggesting they may be involved in the response process (Figure 8C).Like the RNA-seq data (Figure 7), ZmSSL4 and ZmSSL5 displayed the opposite expression pattern under C. zeina infection (Figure 8D).Overall, six ZmSSL genes showed a diverse expression model under different biotic/abiotic stresses and may have different functions during these different stresses.

Subcellular Localization of ZmSSL4 and ZmSSL5
To further explore the functions of the ZmSSL gene family, ZmSSL4 and ZmSSL5, which exhibited opposite response patterns under C. zeina infection, were chosen to be evaluated in terms of their subcellular localization.As shown in Figure 9, in contrast to the localization of GFP control in maize mesophyll protoplasts, the ZmSSL4-GFP and ZmSSL5-GFP signals both overlapped with the fluorescence of chloroplasts (ChI), indicating that both ZmSSL4 and ZmSSL5 were located in chloroplasts.

Subcellular Localization of ZmSSL4 and ZmSSL5
To further explore the functions of the ZmSSL gene family, ZmSSL4 and ZmSSL5, which exhibited opposite response patterns under C. zeina infection, were chosen to be evaluated in terms of their subcellular localization.As shown in Figure 9, in contrast to the localization of GFP control in maize mesophyll protoplasts, the ZmSSL4-GFP and ZmSSL5-GFP signals both overlapped with the fluorescence of chloroplasts (ChI), indicating that both ZmSSL4 and ZmSSL5 were located in chloroplasts.

Subcellular Localization of ZmSSL4 and ZmSSL5
To further explore the functions of the ZmSSL gene family, ZmSSL4 and ZmSSL5, which exhibited opposite response patterns under C. zeina infection, were chosen to be evaluated in terms of their subcellular localization.As shown in Figure 9, in contrast to the localization of GFP control in maize mesophyll protoplasts, the ZmSSL4-GFP and ZmSSL5-GFP signals both overlapped with the fluorescence of chloroplasts (ChI), indicating that both ZmSSL4 and ZmSSL5 were located in chloroplasts.

Discussion
The SSL gene family is thought to be playing a significant role in plant responses to biotic/abiotic stresses [8,9].Although many types of SSL genes have been identified in various plants through bioinformatics analysis, such as Arabidopsis [13], Catharanthus roseus [10,21], and Solanum lycopersicum [15], no relevant reports about the family membership and function analysis of ZmSSL genes have been undertaken in maize and other crops (sorghum, rice, and Ae.tauschii).In the current study, using AtSSLs and the conserved HMM motif of SSL protein as a query to blast genome data, a total of 20, 21, 16, and 33 SSL genes were identified in maize, sorghum, rice, and Ae.tauschii, respectively (Table 1 and Supplementary Table S2).ZmSSL genes were unevenly distributed on chromosomes 1, 2, 4, 7, 8, and 9 (Supplementary Table S1 and Figure 3A).Meanwhile, the family members of the other three plants were also unevenly distributed on chromosomes (Supplementary Table S1) indicating that the chromosomal distribution of SSL genes may be evolutionarily consistent in different species.Replication events play an important role in the amplification of gene family members during plant evolution; if the spacing between two homologous genes is less than five genes it is called a tandem repeat, and if the spacing between two homologous genes is greater than five genes, it is called segmental repeat [22][23][24].In the present study, ZmSSL1-3 located in chromosome 1 and ZmSSL12-19 in chromosome 8 were both tandem repeats and no segmental repeat was founded in the maize genome (Figure 3A,B).Meanwhile, both ZmSSL1-3 and ZmSSL12-19 showed almost no expression under normal or stress conditions (Figures 6-9), suggesting that tandem repeats played a significant role in expanding family members in the evolution of maize SSL genes and might be leading to a loss of gene function.
To understand the evolutionary relationship of ZmSSL genes, we used 105 SSL sequences from maize, sorghum, rice, Ae. tauschii, and Arabidopsis (15 members) to build a phylogenetic tree.The results showed that these SSL genes are divided into five groups (Figure 1).Except for Group V, each group contained ZmSSL genes, with Group IV having the highest number (13 members).TBtools were used to analyze the structure of the ZmSSL genes and the conserved motif/domain of amino acid sequences.As shown in Figure 2, ZmSSL genes in the same evolutionary group contained similar numbers of exons, introns, and protein motifs, which showed high correlations with the evolutionary analysis results.A recent study indicated that the SSL family members of Populus trichocarpa (13 members), Arabidopsis thaliana, and Salix purpurea (22 members) are divided into four groups based on the neighbor-joining (NJ) method of constructing a phylogenetic tree, and each group includes both SSL genes from three species, indicating that those from herbaceous and woody SSL genes might undergo relatively consistent evolutionary progress [8].In our work, only Group I and Group II contained SSL genes from all five plants and all AtSSL genes were mainly included in Group I (Figure 1).This result suggested that SSL genes from Arabidopsis and crops may undergo different evolutionary patterns.Further cross-species collinearity analysis revealed that maize shared more homologous pairs with sorghum, rice, and Ae.tauschii than with Arabidopsis (Figure 4), suggesting that the SSL family in our explored crops is more conserved during evolution.
The analysis of cis-acting elements in promoters can provide a basis for the functional study of genes [25].Existing studies have shown that there is an important correlation between the cis-acting elements of the promoter region and the expression level of specific genes [26,27].Multiple cis-acting elements involved in plant growth, development, hormones, and abiotic/biotic stress response have been found in the promoter region of most ZmSSL genes (Figure 5 and Supplementary Table S3), suggesting that ZmSSL genes might play a significant role in maize adaptations to the environment.Based on the predicted results of cis-acting elements, we used published transcriptome data [17][18][19][20] (Supplementary Tables S4 and S5) (including different maize tissues and different abiotic/biotic stresses) to evaluate the expression pattern of 20 ZmSSL genes, and the results showed that the Group IV maize SSL gene members were not expressed in different tissues and were not significantly induced by stress treatment (Figures 6 and 7).The gene density analysis of chromosomes indicated that the Group IV ZmSSL genes both showed tandem repeats in the genome (Figures 1 and 3A).Perhaps multiple copy events during evolution led to the promoter regions of these genes being disrupted, resulting in a loss of expression of these ZmSSL genes during maize growth and development.The other six ZmSSL members displayed diverse expression patterns in different tissues, and some of them were positively and negatively induced by heat, cold, and infection with C. graminicola and C. zeina fungus (Figures 6 and 7), indicating that ZmSSL4-9 may be playing different roles in maize development and stress responses.Further RT-qPCR analysis confirmed that ZmSSL4, 7, 8, and 9 were downregulated by heat, while ZmSSL8 and ZmSSL9 were upregulated by cold (Figure 8).ZmSSL5 and ZmSSL8 both positively responded to the C. graminicola infection while ZmSSL4 and ZmSSL5 showed opposite expression levels during C. zeina infection (Figure 8).Heat and cold are the two main adverse factors leading to reduced maize production.C. graminicola leads to anthracnose in maize [28] and C. zeina causes maize gray leaf spot [29], both economically significant diseases worldwide.These results indicated that ZmSSL genes may play an important role in the growth and development of maize.However, the specific function of the ZmSSL genes needs to be further researched.
ZmSSL4, ZmSSL5, and AtSSL5-7 were both in Group II (Figure 1).A previous study showed that AtSSL5-7 is positively modulated by SA, JA, and pathogen infection [9], while in this study, ZmSSL4 and ZmSSL5 displayed the opposite response under C. zeina stress (Figure 8).To further explore the function of ZmSSL4/5, we evaluated the localization of ZmSSL4 and ZmSSL5 in maize B73 mesophyll protoplasts.Based on the predicted results of subcellular protein localization, ZmSSL4 and ZmSSL5 were located in the plasma membrane and chloroplast/thylakoid, respectively (Table 1).As shown in Figure 9, both of these two proteins were located in chloroplasts.This result suggests that chloroplasts might the main place for ZmSSL genes to perform their function.This hypothesis needs to be confirmed through further investigation of ZmSSL genes in maize.

Plant Materials and Stress Treatments
The maize B73 inbred line was used in this research.Surface-sterilized B73 seeds were planted in nutrient soil and grown under a 16 h/8 h day/night photoperiod at 25 • C in a greenhouse.Two-week-old (V3 stage) maize seedlings were used to assess abiotic stress.For either heat or cold stress, the seedlings were put in a 42 • C or 4 • C growth chamber for 0, 2, 4, or 8 h.There were three replicates for each treatment and three pots of seedlings for each replicate.Two fungal pathogens, Colletotrichum graminicola and Cercospora zeina, were used to infect the leaf of B73 (V12 growth stage) with the brush method [19,20].Inoculated (1 × 10 6 conidia per mL) leaf material (two leaves per plant) was harvested at four time points (0 hdpi, 12 hdpi, 24 hdpi, and 48 hdpi) from three biological replicates.The fungus was prepared as described [19,20].Leaves treated with water were used as controls.The maize leaves were harvested after stress treatments and then immediately frozen in liquid nitrogen and stored at −80 • C until use.

Phylogenetic and Structural Analysis of ZmSSL Genes
The identified SSL protein sequences from maize, sorghum, rice, Ae. tauschii, and Arabidopsis were aligned using Clustal X (Version 1.83) [30].The aligned results were imported into MEGA7 (Version 7.0) to build a phylogenetic tree using the maximum likelihood method and 1000 bootstrap replicates.The conserved motifs of ZmSSL genes were evaluated with MEME (Version 5.5.0)[31] and visualized using TBtools v1.120 [32].The exon-intron structures of ZmSSL genes were obtained and visualized with TBtools v1.120.

Transcriptome Data Analysis
The RNA-seq data used for the tissue-specific expression analysis of ZmSSL genes under normal conditions were obtained in the published research [16].The published RNA-seq data for heat/cold [18], drought [17], and fungal infection [19,20] were used to evaluate the ZmSSL gene expression levels under stress.Heatmap construction was based on the fragments per kilobase per million (FPKM) mapped reads values of published RNA-seq data and visualized with TBtools [32].Different periods of tissues and stresses are labeled in Supplementary Tables S4 and S5.

RT-qPCR Analysis of Gene Expression
Total leaf RNA extraction was performed following a published protocol [35].Total RNA (1 µg) was used for first-strand cDNA synthesis using a cDNA Kit (CWBIO, Beijing, China).The products were diluted with RNase-free water and used for the template.The RT-qPCR Mix (20 µL) included 6 µL cDNA, 2 µL RNase-free water, a total of 2 µL of each primer, and 10 µL SYBR Mix (Thermo Fisher Scientific, Waltham, MA, USA).A PCR was performed on a CFX96 Real-Time System (Bio-Rad, Hercules CA, USA) and the procedure was as follows: 95 • C for 5 min, followed by 44 cycles of denaturation at 95 • C for 15 s, annealing at 60 • C for 30 s, then melting curves were performed using 65-95 • C in the final step.ZmActin1 (GRMZM2G126010) was used as the internal control.All primers used in the RT-qPCR are listed in Supplementary Table S6.

Subcellular Localization
For the subcellular localization of ZmSSL4 and ZmSSL5, the CDS of ZmSSL4 and ZmSSL5 were amplified without the stop codon using primers ZmSSL4-F/R and ZmSSL5-F/R (Supplementary Table S6).Amplicons were ligated into the pROKII plasmid [35,36].An empty pROKII vector was used as a control.ZmSSL4/5-GFP plasmid DNA (20 µg) was transformed into maize mesophyll protoplasts following a published protocol [35,37].The protoplasts were incubated at 25 • C for 17 h.The GFP and chlorophyll autofluorescence signals were observed with a scanning confocal microscope (Andor Revolution WD, https: //andor.oxinst.com/).

Statistical Analysis
Statistical analysis was performed using SPSS 19.0.Significance (p < 0.05; p < 0.01) was assessed by using the Student's t test.

Conclusions
In this study, 20 SSL genes were identified in maize through homologous alignment.Their chromosomal localization, phylogenetic relationship, gene and protein structure, ciselements of promoter regions, and expression patterns under normal or stress conditions were further analyzed.The 20 ZmSSL genes were unevenly distributed on chromosomes.The phylogenetic analysis indicated that ZmSSL genes were located in four groups and members in each group exhibited the same gene and protein structure.Tandem duplications may be the main reason leading to the increasing copy number of ZmSSL genes in the maize genome, which may be leading to gene functional redundancy or no function.Moreover, ZmSSL genes had a high degree of conservatism during the evolution of our explored gramineae crops (sorghum, rice, and Ae.tauschii).Furthermore, six members of the ZmSSL genes, ZmSSL4-9, displayed wide expression in different tissues under normal conditions and were positively or negatively modulated by abiotic (heat and cold) and biotic (C.graminicola and C. zeina infection) stresses.This work may provide useful information to further evaluate the biological functions of ZmSSL genes.

Figure 2 .
Figure 2. Gene exon-intron structure and conserved protein motifs of ZmSSL genes.UTR and CDS are shown as colorful boxes and introns as lines.Ten conserved motifs with common sequences are also shown in differently colored boxes.The clustering of ZmSSL genes was based on the phylogenetic tree shown in Figure 1.

Figure 2 .
Figure 2. Gene exon-intron structure and conserved protein motifs of ZmSSL genes.UTR and CDS are shown as colorful boxes and introns as lines.Ten conserved motifs with common sequences are also shown in differently colored boxes.The clustering of ZmSSL genes was based on the phylogenetic tree shown in Figure 1.

Figure 3 .
Figure 3. Chromosomal distribution and collinearity analysis of ZmSSL genes.(A) Twenty ZmSSL genes located in six chromosomes.Red represents tandem repeat.The scale bar on the left shows the mega base (Mb).(B) The schematic diagram of the relationship of ZmSSL genes between chromosomes.The heatmap in the inside circles indicates chromosome gene density.The gray lines in the background indicate collinear gene pairs in the maize genome.

Figure 3 .
Figure 3. Chromosomal distribution and collinearity analysis of ZmSSL genes.(A) Twenty ZmSSL genes located in six chromosomes.Red represents tandem repeat.The scale bar on the left shows the mega base (Mb).(B) The schematic diagram of the relationship of ZmSSL genes between chromosomes.The heatmap in the inside circles indicates chromosome gene density.The gray lines in the background indicate collinear gene pairs in the maize genome.

17 Figure 4 .
Figure 4.The collinearity diagram of SSL genes between maize and four other plants (sorghum, rice, Ae. tauschii, and Arabidopsis).The gray line in the background represents the collinear gene pairs between the two species, and the collinear genes are connected with a red line.

Figure 4 .
Figure 4.The collinearity diagram of SSL genes between maize and four other plants (sorghum, rice, Ae. tauschii, and Arabidopsis).The gray line in the background represents the collinear gene pairs between the two species, and the collinear genes are connected with a red line.

17 Figure 5 .
Figure 5. Predicted cis-elements in promoter regions of ZmSSL genes.A promoter region of about 2-kb upstream of ZmSSL genes are used for analysis.Different colors represent different genes.The cis-element counts are listed in Supplementary TableS3.

Figure 5 .
Figure 5. Predicted cis-elements in promoter regions of ZmSSL genes.A promoter region of about 2-kb upstream of ZmSSL genes are used for analysis.Different colors represent different genes.The cis-element counts are listed in Supplementary TableS3.

Figure 6 .
Figure 6.Tissue-specific expressions of ZmSSL genes.The heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values of published RNA-seq data.Different tissues of maize are labeled in the figure.SAM: shoot apical meristem.Data links are shown in Supplementary TableS4.

Figure 6 .
Figure 6.Tissue-specific expressions of ZmSSL genes.The heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values of published RNA-seq data.Different tissues of maize are labeled in the figure.SAM: shoot apical meristem.Data links are shown in Supplementary TableS4.

Figure 7 .
Figure 7.The heat map of ZmSSL genes under abiotic and biotic stresses.Different stresses are labeled within the figure.The data (except drought stress) were processed from three replicates of published transcriptomic data (Supplementary TableS5).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

Figure 7 .
Figure 7.The heat map of ZmSSL genes under abiotic and biotic stresses.Different stresses are labeled within the figure.The data (except drought stress) were processed from three replicates of published transcriptomic data (Supplementary TableS5).Heatmap construction is based on the fragments per kilobase per million mapped reads (FPKM) values.

Figure 9 .
Figure 9. Subcellular localization of ZmSSL4 and ZmSSL5.The GFP or ZmSSL4/5:GFP fusion expression vectors were transformed into maize B73 mesophyll protoplasts using the PEG method.The GFP and chlorophyll autofluorescence (ChI) signals are labeled green and red, respectively.A bright field image is shown in the third column.The overlay is shown in the fourth column.The empty GFP vector was used as a control.GFP: green fluorescent protein.Bars = 10 µm.

Table 1 .
Characteristics of SSL genes in maize.