Genome-Wide Identiﬁcation and Analysis of the Maize Serine Peptidase S8 Family Genes in Response to Drought at Seedling Stage

: Subtilisin-like proteases (subtilases) are found in almost all plant species and are involved in regulating various biotic and abiotic stresses. Although the literature on subtilases in different plant species is vast, the gene function of the serine peptidase S8 family and its maize subfamily is still unknown. Here, a bioinformatics analysis of this gene family was conducted by describing gene structure, conserved motifs, phylogenetic relationships, chromosomal distributions, gene duplications, and promoter cis-elements. In total, we identiﬁed 18 ZmSPS8 genes in maize, distributed on 7 chromosomes, and half of them were hydrophilic. Most of these proteins were located at the cell wall and had similar secondary and tertiary structures. Prediction of cis-regulatory elements in promoters illustrated that they were mainly associated with hormones and abiotic stress. Maize inbred lines B73, Zheng58, and Qi319 were used to analyze the spatial-temporal expression patterns of ZmSPS8 genes under drought treatment. Seedling drought results showed that Qi319 had the highest percent survival after 14 d of withholding irrigation, while B73 was the lowest. Leaf relative water content (LRWC) declined more rapidly in B73 and to lower values, and the nitrotetrazolium blue chloride (NBT) contents of leaves were higher in Qi319 than in the other inbreds. The qPCR results indicated that 6 serine peptidase S8 family genes were positively or negatively correlated with plant tolerance to drought stress. Our study provides a detailed analysis of the ZmSPS8s in the maize genome and ﬁnds a link between drought tolerance and the family gene expression, which was established by using different maize inbred lines.


Introduction
When plants are exposed to various environmental stimuli, endogenous phytohormones such as melatonin, polyamines (PA), and jasmonic acid (JA) levels are increased to improve the stress tolerance of plants. In addition, the plants' cellular redox homeostasis is disrupted [1][2][3][4], while triggering an endoplasmic reticulum (ER) stress response [5]. Accumulating unfolded or misfolded proteins during ER stress results in the generation of excessive reactive oxygen species (ROS), leading to the oxidation of side chains of amino acid residues [6] and the formation of protein-protein covalent cross-linkage, which can lead to protein inactivation or denaturation [7]. If they are not rapidly degraded, oxidatively modified proteins can undergo direct fragmentation or can form large aggregates due to covalent cross-linking and increased surface hydrophobicity, which lead to cell death [8].
The serine peptidase S8 (subtilisin-like proteinases) family belongs to the subtilases (SBTs) superfamily, which is a widely distributed family of serine proteases and was first found in eukaryotes [9]. Researchers subsequently found nine subtilases in mammals that are involved in the maturation of proteins in animals [10]. It also has been reported that the such as genetic structure analysis, promoter analysis, and protein structure analysis. The expression of these 18 genes in Qi319, Zheng58, and B73 seedlings at different time points before and after drought treatment and the drought tolerance of three maize inbreds were analyzed. Our findings lay the foundation for further evolutionary research on the plant serine peptidase gene family and offer some useful information for the identification of key genes in response to drought stress.

Identification and Classification of SPS8 Proteins in Zea mays
Based on the BLASTP program and the HMM files, a total of 18 non-redundant SPS8 proteins were obtained. According to the physico-chemical characteristics predicted by the Expasy tool, we found that the protein lengths, molecular weights (MWs), and isoelectric points (pI) of the family members showed large variation. The length of the ZmSPS8 gene's coding region varied from 285 bp (ZmSPS8. 3 11.38 (ZmSPS8.1.14). Nine of the SPS8 proteins were hydrophilic, and the other nine proteins were hydrophobic ( Figure S1). According to the subcellular localization predictions, most of the family members were localized in the cell wall, while only two proteins (ZmSPS8.2.0 and ZmSPS8.1.14) were localized in the nucleus. In addition, only one protein (ZmSPS8.3.1) was localized in the cell membrane, and ZmSPS8.1.11 was localized in both the cell wall and cell membrane. All of the molecular characteristics of the SPS8 proteins are listed in Table 1.

Phylogenetic Relationships Analysis, SPS8 Conserved Motifs Prediction and Gene Structure Analysis of ZmSPS8
The full-length amino acid sequences of the 18 family members were aligned and used to construct an unrooted phylogenetic tree to analyze their phylogenetic relationships. The result showed that these family members were divided into three groups (class I-class III) with high bootstrap value support, indicating that they have a conserved phylogenetic relationship ( Figure 1A). Therefore, all family members were named according to the evolutionary tree: ZmSPS8.1.1-ZmSPS8.1.14 for class I, ZmSPS8.2 for class II, ZmSPS8.3.1-ZmSPS8.3.3 for class III. The gene structure analysis was performed by TBtools software to further support their phylogenetic relationships ( Figure 1B). The result revealed that the intron number in the genomic sequences of the family members ranged from 0 to 33. There is no intron in four genes (ZmSPS8.1.5, ZmSPS8.1.1, ZmSPS8.1.3, and ZmSPS8.1.2), while ZmSPS8.3.3 contains 33 introns. Ten motifs were identified among the family members through conserved motif analysis by MEME ( Figures 1A and S1). These motifs were named motifs 1-10, motifs 1, 5, and 9, and were all identified as the peptidase_S8 domain (PF00082); motif 7 was the inhibitor_I9 domain (PF05922), motif 6 was the EABR domain; however, the others were unknown domains. All proteins in class I and ZmSPS8.2.1 contained all ten motifs. Except for three proteins (ZmSPS8.1.14, ZmSPS8.1.11, and ZmSPS8.1.9), other SPS8 proteins contained motif 7 which is the inhibitor I9 domain (PF05922). Combining the analysis results of the phylogenetic tree and conserved motifs, we can also find that the ZmSPS8s of the same class contained similar motif compositions, but they differed from different classes, suggesting that different classes have complementary functions, but the same class exhibits redundancy. According to the phylogenetic tree, gene structure, and conserved motif analysis, we found that the gene pair ZmSPS8.1.1-ZmSPS8.1.5 exhibited highly similar conserved motifs and exon-intron organization patterns, suggesting their close relationship.

Chromosomal Distributions, Gene Duplications and Divergence Time
We drew a location map of each family gene on the maize chromosomes to research the genomic distribution of the 18 genes and found that these genes are unevenly distributed on 7 of the 10 chromosomes. There are 4 genes on Chr 7; 3 genes on Chr 2, 5, and 10, respectively; 2 genes on Chr 3 and 4, respectively; only one gene on Chr 6. The chromosome location information is shown in Figure 2. Three duplicated genes were identified based on the amino acid identity >85% and gene alignment coverage >0.75, and they were divided into three groups (ZmSPS8. 3 was distributed on the same chromosome. However, they are genetically separated on the chromosome, implying that tandem duplication events were not involved in the expansion of ZmSPS8 genes, and all the duplicated genes showed segmental duplication. The ratios of Ka/Ks for two of these three groups were less than 1.0 (Table 2), suggesting that these two pairs had evolved mainly under purifying selection. The last pair was under positive selection because the Ka/Ks ratio was greater than 1.0 ( Table 2). The divergence

Chromosomal Distributions, Gene Duplications and Divergence Time
We drew a location map of each family gene on the maize chromosomes to research the genomic distribution of the 18 genes and found that these genes are unevenly distributed on 7 of the 10 chromosomes. There are 4 genes on Chr 7; 3 genes on Chr 2, 5, and 10, respectively; 2 genes on Chr 3 and 4, respectively; only one gene on Chr 6. The chromosome location information is shown in Figure 2. Three duplicated genes were identified based on the amino acid identity >85% and gene alignment coverage >0.75, and they were divided into three groups (ZmSPS8. 3 was distributed on the same chromosome. However, they are genetically separated on the chromosome, implying that tandem duplication events were not involved in the expansion of ZmSPS8 genes, and all the duplicated genes showed segmental duplication. The ratios of Ka/Ks for two of these three groups were less than 1.0 (Table 2), suggesting that these two pairs had evolved mainly under purifying selection. The last pair was under positive selection because the Ka/Ks ratio was greater than 1.0 ( Table 2). The divergence times of

Prediction of the Protein Structure, Signal Peptides and Trans-Membrane Helix
Secondary structure predictions revealed that the SPS8 proteins mainly consisted of alpha helices, extended strands, beta turns, and random coils ( Table 3). The random coils accounted for the largest percentage of the secondary structures, followed by alpha helices and extended strands. In addition, the 3D structure models of all the proteins were predicted using the AlphaFold Protein Structure Database [38]. We found that these family proteins mostly shared similar structures, except for 2 proteins that failed prediction (

Prediction of the Protein Structure, Signal Peptides and Trans-Membrane Helix
Secondary structure predictions revealed that the SPS8 proteins mainly consisted of alpha helices, extended strands, beta turns, and random coils ( Table 3). The random coils accounted for the largest percentage of the secondary structures, followed by alpha helices and extended strands. In addition, the 3D structure models of all the proteins were predicted using the AlphaFold Protein Structure Database [38]. We found that these family proteins mostly shared similar structures, except for 2 proteins that failed prediction (

Cis-Element Analysis of ZmSPS8 Genes in Maize
Little evidence indicates that ZmSPS8 genes play important roles in responses to otic stresses [39]. To explore the possibility of ZmSPS8 genes involved in stress respon the presence of abiotic stress-related cis-elements in 18 genes was investigated. We fo

Cis-Element Analysis of ZmSPS8 Genes in Maize
Little evidence indicates that ZmSPS8 genes play important roles in responses to abiotic stresses [39]. To explore the possibility of ZmSPS8 genes involved in stress responses, the presence of abiotic stress-related cis-elements in 18 genes was investigated. We found that the cis-acting regulatory elements in ZmSPS8 genes promoter regions were mainly divided into the following four categories: light, hormone, stress responsive, and growth and metabolic responsive elements ( Figure 4). These stress-responsive cis-elements in promoters were involved in drought, low-temperature, defense mechanism, and anaerobic condition. In addition, cis-elements related to salicylic acid (SA), methyl jasmonate (MeJA), gibberellins (GA), and auxin (IAA) were also identified. Almost all ZmSPS8 gene promoters contain cis-elements associated with drought stress, suggesting that the ZmSPS8 gene family may be involved in regulating drought response. that the cis-acting regulatory elements in ZmSPS8 genes promoter regions were mainly divided into the following four categories: light, hormone, stress responsive, and growth and metabolic responsive elements ( Figure 4). These stress-responsive cis-elements in promoters were involved in drought, low-temperature, defense mechanism, and anaerobic condition. In addition, cis-elements related to salicylic acid (SA), methyl jasmonate (MeJA), gibberellins (GA), and auxin (IAA) were also identified. Almost all ZmSPS8 gene promoters contain cis-elements associated with drought stress, suggesting that the ZmSPS8 gene family may be involved in regulating drought response.

Expression Patterns of ZmSPS8 Genes at Different Developmental Stages
To explore the possible functions of ZmSPS8 genes, the expression patterns of the 18 family members were analyzed in 23 tissues from various development stages and organs by using publicly available transcript data [40]. Some genes showed detectable expression levels in most of the 23 tissues and developmental stages with different expression patterns, and some genes showed no expression ( Figure 5). Practically, we noted that ZmSPS8. 3

Expression Patterns of ZmSPS8 Genes at Different Developmental Stages
To explore the possible functions of ZmSPS8 genes, the expression patterns of the 18 family members were analyzed in 23 tissues from various development stages and organs by using publicly available transcript data [40]. Some genes showed detectable expression levels in most of the 23 tissues and developmental stages with different expression patterns, and some genes showed no expression ( Figure 5). Practically, we noted that ZmSPS8.3.3 had different degrees of expression levels in all stages. ZmSPS8.1.3 and ZmSPS8.1.1 only showed expression at 12 DAP (days after pollination) endosperm, while ZmSPS8.1.2 and ZmSPS8.1.14 only showed expression at mature pollen. Some ZmSPS8 genes showed similar expression patterns that reflected their close relationships, especially for three pairs of genes (ZmSPS8.1.3 and ZmSPS8.1.1, ZmSPS8.1.2 and ZmSPS8.1.14, ZmSPS8.1.13, and ZmSPS8.1.12), which might suggest they play the same functions in plant growth and development.

Drought Tolerance Test of Qi319, Zheng58 and B73 Seedlings
We conducted a drought tolerance test on seedlings of three maize inbred lines Qi319, Zheng58, and B73. These three maize lines all grew normally under well-watered conditions, and drought stress was imposed by continuously withholding water from soil-grown plants. As shown in Figure 6, under well-watered conditions, the growth of all three inbred plants was similar and healthy, while after 7 days of drought stress treatment (the soil relative water content (SRWC) 9.0%.), all leaves of the B73 severely curled and wilted, the leaves of Zheng58 began to curl down from the base of the blade, but Qi319 showed still vigorously. After 10 days of drought treatment (SRWC 8.1%.), Zheng58 leaves were too severely withered to stand up, the Qi319 leaves also showed withered and drooped, while the whole plants of the B73 showed totally withered. All leaves of the three inbred lines curled and wilted seriously after 14 days of drought treatment ( Figure 6A). The SR (survival rate) of plants was measured 7 days after re-watering; the survival rates of Qi319, Zheng58, and B73 were 62.5%, 56.25%, and 17.18%, respectively, and those of well-watered plants were 100% ( Figure 6D). The morphological changes of the plants with increasing drought treatment time are shown in Supplementary Figure S2.

Drought Tolerance Test of Qi319, Zheng58 and B73 Seedlings
We conducted a drought tolerance test on seedlings of three maize inbred lines Qi319, Zheng58, and B73. These three maize lines all grew normally under well-watered conditions, and drought stress was imposed by continuously withholding water from soilgrown plants. As shown in Figure 6, under well-watered conditions, the growth of all three inbred plants was similar and healthy, while after 7 days of drought stress treatment (the soil relative water content (SRWC) 9.0%.), all leaves of the B73 severely curled and wilted, the leaves of Zheng58 began to curl down from the base of the blade, but Qi319 showed still vigorously. After 10 days of drought treatment (SRWC 8.1%.), Zheng58 leaves were too severely withered to stand up, the Qi319 leaves also showed withered and drooped, while the whole plants of the B73 showed totally withered. All leaves of the three inbred lines curled and wilted seriously after 14 days of drought treatment ( Figure  6A). The SR (survival rate) of plants was measured 7 days after re-watering; the survival rates of Qi319, Zheng58, and B73 were 62.5%, 56.25%, and 17.18%, respectively, and those of well-watered plants were 100% ( Figure 6D  Leaf RWC (relative water content) of the tested plants was assayed. The leaf RWC of all plants decreased to varying degrees with the imposition of drought stress. As shown in Figure 6C, the leaf RWC of B73 decreased by 36.4% after 10 days of drought treatment, and Qi319 and Zheng58 decreased by 12.1% and 19.9%, respectively. The result indicated that the water-retaining capacity of B73 was weaker than Qi319 and Zheng58. Drought stress has been reported to accelerate the accumulation of ROS, and the overproduction of ROS in plants can cause chlorosis and cell death. To investigate whether chlorotic and cell death were observed at the adaxial side of the tested plants, the superoxide radicals of the tested leaves were stained by NBT. Our results showed that B73 accumulated more O 2 ·− than Qi319 and Zheng58 at 7 days, 10 days, and 14 days after drought treatment ( Figure 6B). Plants 2023, 12, x FOR PEER REVIEW 10 of 18 Leaf RWC (relative water content) of the tested plants was assayed. The leaf RWC of all plants decreased to varying degrees with the imposition of drought stress. As shown in Figure 6C, the leaf RWC of B73 decreased by 36.4% after 10 days of drought treatment, and Qi319 and Zheng58 decreased by 12.1% and 19.9%, respectively. The result indicated that the water-retaining capacity of B73 was weaker than Qi319 and Zheng58. Drought stress has been reported to accelerate the accumulation of ROS, and the overproduction of ROS in plants can cause chlorosis and cell death. To investigate whether chlorotic and cell death were observed at the adaxial side of the tested plants, the superoxide radicals of the tested leaves were stained by NBT. Our results showed that B73 accumulated more O2 .− than Qi319 and Zheng58 at 7 days, 10 days, and 14 days after drought treatment (Figure 6B).

Expression Analysis of ZmSPS8 Genes under Drought Treatment
In order to analyze if SPS8 proteins were involved in drought tolerance regulation, we used qRT-PCR to detect the expression of the ZmSPS8 genes in the three inbred lines before and after drought treatment. As shown in Figure 7, the expression of ZmSPS8.3.3, ZmSPS8.3.1, ZmSPS8.1.7, and ZmSPS8.1.9 in the three maize lines was increased or decreased compared to that of untreated, but their expression levels varied among the three maize inbred lines; the highest expression was in Qi319, followed by Zheng58, and the lowest level of expression was in B73. However, exactly the opposite was observed in ZmSPS8.1.13 and ZmSPS8.1.4: the highest expression levels of them were in B73, and the lowest expression was in Qi319. In addition, expression levels of ZmSPS8.1.14 and ZmSPS8.3.2 were significantly down-regulated throughout the experiment time, and those of ZmSPS8.2.0 and ZmSPS8.1.10 were significantly up-regulated only in Qi319. Notably, some genes were differentially expressed at only a single time point. For example, ZmSPS8.1.6 expression was significantly up-regulated at 10 days, but significantly down-

Expression Analysis of ZmSPS8 Genes under Drought Treatment
In order to analyze if SPS8 proteins were involved in drought tolerance regulation, we used qRT-PCR to detect the expression of the ZmSPS8 genes in the three inbred lines before and after drought treatment. As shown in Figure 7, the expression of ZmSPS8.3.3, ZmSPS8.3.1, ZmSPS8.1.7, and ZmSPS8.1.9 in the three maize lines was increased or decreased compared to that of untreated, but their expression levels varied among the three maize inbred lines; the highest expression was in Qi319, followed by Zheng58, and the lowest level of expression was in B73. However, exactly the opposite was observed in ZmSPS8.1.13 and ZmSPS8.1.4: the highest expression levels of them were in B73, and the lowest expression was in Qi319. In addition, expression levels of ZmSPS8.1.14 and Zm-SPS8.3.2 were significantly down-regulated throughout the experiment time, and those of ZmSPS8.2.0 and ZmSPS8.1.10 were significantly up-regulated only in Qi319. Notably, some genes were differentially expressed at only a single time point. For example, ZmSPS8.1.6 expression was significantly up-regulated at 10 days, but significantly down-regulated expression was observed at 14 days in Qi319 and Zheng58. ZmSPS8.1.8 expression was significantly up-regulated at 7 days in all inbred lines, but this gene could still remain up-regulated only in Qi319 from 7 to 10 days after drought treatment. The remaining five genes among the family members did not have detectable expression, indicating that they might be pseudogenes or are not expressed in the leaves at the maize seedling stage. These findings suggested the important roles of ZmSPS8 genes in response to drought stress, but they may have different regulatory mechanisms (Figure 7). sion was significantly up-regulated at 7 days in all inbred lines, but this gene could still remain up-regulated only in Qi319 from 7 to 10 days after drought treatment. The remaining five genes among the family members did not have detectable expression, indicating that they might be pseudogenes or are not expressed in the leaves at the maize seedling stage. These findings suggested the important roles of ZmSPS8 genes in response to drought stress, but they may have different regulatory mechanisms (Figure 7).

Discussion
Plant subtilases are a very diverse and widely distributed subtilisin-like family of serine proteases. They have been reported to be involved in the breakdown and replacement of proteins or protein complexes, protein post-translational processing, plant reproductive organ development, physiological modification of cell walls, abiotic and biotic responses, and apoptosis [9,[19][20][21]. Plants use escape strategies, including premature senescence and leaf reduction, to cope in response to abiotic stresses such as drought and heat. The current study found that chloroplast degradation and proteolysis are implicated in the senescence processes of plants, and genes involved in these biological processes show increased expression. This suggests that proteases play crucial roles in abiotic stress responses in plants. [41][42][43]. To date, however, there have been few reports on the potential biological function of the serine peptidase S8 subfamily in maize. In this article, we identified and characterized 18 maize SPS8 family genes and speculated on their potential effect on drought.
These 18 SPS8 family genes showed different chromosome distribution and the coded proteins showed various pI, stability, and subcellular location prediction. The prediction results showed that most of the serine peptidase S8 family members localized to the cell wall, and a few were predicted to localize to the cell membrane and nucleus. It

Discussion
Plant subtilases are a very diverse and widely distributed subtilisin-like family of serine proteases. They have been reported to be involved in the breakdown and replacement of proteins or protein complexes, protein post-translational processing, plant reproductive organ development, physiological modification of cell walls, abiotic and biotic responses, and apoptosis [9,[19][20][21]. Plants use escape strategies, including premature senescence and leaf reduction, to cope in response to abiotic stresses such as drought and heat. The current study found that chloroplast degradation and proteolysis are implicated in the senescence processes of plants, and genes involved in these biological processes show increased expression. This suggests that proteases play crucial roles in abiotic stress responses in plants. [41][42][43]. To date, however, there have been few reports on the potential biological function of the serine peptidase S8 subfamily in maize. In this article, we identified and characterized 18 maize SPS8 family genes and speculated on their potential effect on drought.
These 18 SPS8 family genes showed different chromosome distribution and the coded proteins showed various pI, stability, and subcellular location prediction. The prediction results showed that most of the serine peptidase S8 family members localized to the cell wall, and a few were predicted to localize to the cell membrane and nucleus. It implies that they may be involved in various cell wall-related physiological and biochemical processes. It is reported that plants invest available resources into root growth to explore residual water in the soil and reduce shoot growth when exposed to drought stress [44], and that process is accompanied by cell wall synthesis and remodeling [45]. Another study reported some cell wall synthesis-related genes in soybean leaf under drought and flooding conditions using RNA-seq, and these genes were up-regulated under drought stress and down-regulated under flooding stress, which indicated that cell wall modification may be a protective strategy against drought stress [46]. Laura et al. [47] found that the cell wall integrity sensor THESEUS1 modulates the mechanical properties of walls, turgor loss point, ABA biosynthesis (ABA is essential for plant adaptation to drought stress and has therefore been investigated extensively in guard cells), and ABA-controlled processes in Arabidopsis thaliana, which further showed that responses to drought depend on the presence of a functional cell wall. A tissue-specific NAC gene called necrotic upper tips1 (nut1) was associated with secondary cell wall formation in the protoxylem. When this gene was lost, protoxylem vessels became thinner, resulting in the defective formation of secondary cell walls. Ultimately, it led to a block in water transport and reduced drought tolerance [48]. Xingming et al. [49] studied a rice drought-tolerant gene DROUGHT1(DROT1), which is specifically expressed in vascular bundles. Its protein is primarily located on the periphery of the cell, especially on the cell wall, and may specifically promote cellulose synthesis under drought stress to protect cell wall integrity. Over-expression of DROT1 in rice plants showed improved drought tolerance, and when knocked out, the plants exhibited significantly reduced drought resistance.
Cis-elements in gene promoters are important for transcriptional regulation. For our promoter analysis result, a series of binding sites of transcription factors related to plant growth and development were identified in the promoters of these 18 genes. For example, light-responsive elements presented in the promoters of all these genes, stress-responsive cis-elements, including ABA-responsive (ABRE), drought-responsive (MBS), and low temperature-responsive (LTRE) were also found in the promoters of these 18 genes. Almost all family members contain MBS or ABRE, or both, suggesting the important roles of 18 genes in drought stress responses.
Abiotic stress such as drought reduces plant growth and survival. In our study, three maize inbred lines (Qi319, Zheng58, and B73) were selected. Drought stress for 7 days exhibited deleterious effects on B73 which caused significant leaf curling and wilted with 9% SRWC. While SRWC dropped to 8.1%, Qi319 leaves started to curl, and Zheng58 already showed severe leaf curling. In addition, plenty of studies have also used plant SR to evaluate drought tolerance: Overexpression of ZmDREB2.7 in Arabidopsis resulted in rising survival rates through acquired drought treatment [35] and the transgenic maize with enhanced ZmVPP1 gene expression showed an increased survival rate relative to the B73 inbred line (CK) under water-deficit conditions [40]. Interestingly enough, the transgenic plants of the negative regulator of drought tolerance GhWRKY33, OsJAZ1, or AtPUB19 reduced their SR compared with the wild type (WT) [50][51][52]. In addition to SR, leaf RWC is also positively correlated with drought resistance ability and can truly reflect the degree of water deficit in plants under drought stress, the higher Leaf RWC, the stronger the drought tolerance, and conversely, the weaker the drought tolerance [53]. According to the results of Leaf RWC, LRWC declined more rapidly in B73, such that it was 45% at 10 d, and 42% at 14 d, whereas it remained higher in Zheng58 and Qi319, such that in Qi319 LRWC was 55% at 14 d ( Figure 6C). Thus, we conclude that B73 is a drought-sensitive line, which is in good agreement with other literature reports [36,40]. Likewise, histochemical staining with NBT also indicated that the ROS-scavenging capacity of B73 is weaker than that of Zheng58 and Qi319. Taken together, we conclude that Qi319 is the most tolerant inbred, followed by Zheng58, and B73.
It has been documented that drought-tolerant inbred lines, such as CIMBL55, 92, 70, and CML118, have significantly higher expression of ZmNAC111 than drought-sensitive inbred lines (B73, Mo17) with lower expression of ZmNAC111 [36]. In our research, we found the spatial-temporal expression of four members of the serine peptidase S8 gene family (ZmSPS8.3.3, ZmSPS8.3.1, ZmSPS8.1.9, ZmSPS8.1.7) was consistent with ZmNAC111 gene expression results. This finding indicated that these genes expression positively correlated with maize drought tolerance. Moreover, another gene negatively associated with maize drought tolerance was ZmPP2C-A10, which was used as a reference gene in our study. The expression of ZmPP2C-A10 was up-regulated in both drought-sensitive and drought-tolerant inbreds, but the up-regulated levels of this gene were significantly different, with higher levels of ZmPP2C-A10 in sensitive inbreds than those in tolerant ones [54]. The expression of ZmSPS8.1.13 and ZmSPS8.1.4 was consistent with ZmPP2C-A10 gene expression, suggesting these two genes negatively correlated with maize drought toler-ance. Finally, based on the published RNA-seq data, we found that part of the S8 gene family genes was not expressed in all of the 23 tissues and organs including ZmSPS8.1.6, ZmSPS8.1.7, ZmSPS8.1.9, ZmSPS8.1.11, and ZmSPS8.2.0 while four of them were demonstrated to have significant changes in the expression levels under drought stress in qRT-PCR analysis results. The possible reasons are essentially two-fold: one is that part of the genes is drought induced, so they are upregulated; the other is that the inbred line used for the public data was B73, and the expression of genes may vary in different inbred lines.

Conclusions
The maize serine peptidase S8 family was identified and characterized in our study. Eighteen genes were obtained, and among them, five members were involved in segmental duplication. Phylogenetic relationship analysis clearly divided the family members into three groups. These 18 genes were all distributed on 7 chromosomes, and half of them were hydrophilic Most of these subtilases were located on the cell wall and had similar secondary and tertiary structures. Prediction of cis-regulatory elements in promoters illustrated that they were mainly associated with hormones and abiotic stress responses. The drought tolerance of B73, Zheng58, and Qi319 was tested at the seedling stage. The results suggested that Qi319 was the most tolerant inbred, and we also found that ZmSPS8.3.3, ZmSPS8.3.1, ZmSPS8.1.9, and ZmSPS8.1.7 were positively correlated with maize drought tolerance, while ZmSPS8.1.13 and ZmSPS8.1.4 were negatively correlated with maize drought tolerance. Our study first links the changes in serine peptidase S8 family members' expression under drought treatment with the strength of drought tolerance in maize inbred lines, which could provide a scientific foundation for a comprehensive understanding of the maize serine peptidase S8 family and provide new clues to drought tolerance breeding.

Identification and Characterization of ZmSPS8 Genes in Maize
To identify peptidase-encoding proteins in the maize genome, we downloaded genome sequences of maize from the MaizeGDB database (https://www.maizegdb.org/) (accessed on 1 December 2021). Hidden Markov model (HMM) profiles of the Peptidase_S8 domain (PF00082) were initially obtained from the Pfam database (http://pfam.xfam.org/) (https:// www.maizegdb.org/) (accessed on 3 December 2021) [55] and were used for HMMSEARCH in the maize proteome with an E-value equal to 1 × 10 −10 . We searched maize SPS8 proteins using the Basic Local Alignment Search Tool Algorithm program (BLASTP), with the published Zea mays peptidase protein sequences used as query sequences. BLASTP is based on the protein homology alignment with an E-value equal to 1 × 10 −10 . To validate the accuracy of these candidate genes, we applied Web CD-Search Tools and SMART to confirm the domain in maize SPS8 proteins. After removing the redundant sequences, 18 members were used for further analysis. The ExPASy (https://web.expasy. org/protparam/) (3 December 2021) and Plant-mPLoc online tools (Plant-mPLoc server (sjtu.edu.cn)) (3 December 2021) were used to calculate physico-chemical characteristics including molecular weight, isoelectric point, instability index, aliphatic index, grand average of hydropathicity, etc.

Phylogenetic Analysis of SPS8 Proteins
To analyze the evolutionary relationships of maize SPS8 proteins, the full-length amino acid sequences of these 18 proteins from maize were used to construct a phylogenetic tree with the MEGA-X (Molecular Evolutionary Genetics Analysis) software. All the sequences were obtained from PLAZA 4.0 (https://bioinformatics.psb.ugent.be/plaza/versi-ons/ plaza_v4_monocots/) (accessed on 5 December 2021). The phylogenetic tree was built using the maximum likelihood method and the Jones-Taylor-Thorton (JTT) model with 1000 bootstrap replicates.

Distribution of ZmSPS8 Genes on Chromosomes and Their Duplications and Divergence Time
Maize genome annotation information was obtained from the gff3 file (Zea mays. Zm-B73-REFERENCE-NAM-5.0.51.gff3), including gene location and gene structure. The ZmSPS8 genes' location was displayed on corresponding maize chromosomes by the TBtools software v0.667 (https://github.com/CJ-Chen/TBtools) (accessed on 9 December 2021). To confirm the gene duplications, 18 CDS sequences were blast searched against each other by using blastn of NCBI. At least 85% amino acid identity and 75% gene alignment coverage were needed (E-value < 1 × 10 −10 ). To reckon the divergence of homologous genes and the selective pressure against the duplicated genes, TBtools software (v0.667) was applied to calculate the Ks (synonymous) and Ka (nonsynonymous) replacement rate per site between the members of each gene pair. Ks values > 2.0 must be discarded to avoid the risk of substitution saturation. The divergence times (T) of the gene pairs were estimated by the formula: T = Ks/2λ, with the divergence rate λ = 1.5 × 10 −8 .

Expression Patterns of ZmSPS8 Genes in Different Tissues and Organs
To explore the expression patterns of ZmSPS8 genes in maize, the publicly available transcriptome data published by Walley et. al. [60] 23 different developmental stages were collected from MaizeGDB (https://www.maizegdb.org/) (accessed on 11 December 2021). Gene expression levels were expressed as fragments per kilobase of transcript per million fragments mapped (FPKM). We visualized the expression data using standard heat maps.

Plant Materials and Stress Treatments
Maize inbred lines Qi319, Zheng58, and B73 were selected for this study. All three of these inbred lines are parents of a high-yielding hybrid and are frequently used in maize genetics research. Seeds of inbred lines were surface-sterilized, washed with sterile water three times, and germinated in vermiculites until the coleoptile grew to about 2 cm in length. The seeds with consistent germination were selected. Twenty seeds per pot (pot measuring 23 cm in diameter and 17 cm in height) were planted in the greenhouse, each pot was filled with 1 kg of soil, and 16 seedlings were retained one week after seeding emergence. Seedlings were well watered until they reached the three-leaf stage, then drought stress was imposed by withholding water, except for control plants that were watered as usual. Three replicates were set for two treatments: well-watered (WW) and water-stressed (WS). After two weeks of drought stress, all the drought-stressed plants were re-watered. Final survival was analyzed 7 days after rewatering (survival rate calculation formula: survival rate (SR%) = survival quantity/total quantity × 100%). At days 0, 3, 7, 10, and 14 after drought treatment, the third true leaf of each seedling (three leaves from each replicate,) was harvested and flash frozen by liquid nitrogen and stored at −80 • C before RNA isolation. To measure leaf relative water content (RWC), three leaves were removed from each replicate, the 7 cm mid-section was cut from each leaf, and the fresh weight (FW) was measured. This excised section was then placed in a sterile tube containing 10 mL tap water, capped, and left at 25 • C for 12 h. After this time, the leaf sections were blotted carefully, and turgid weight (TW) was measured. The sections were then dried for 12 h at 80 • C in the drying oven for the dry weight (DW). RWC (%) was calculated as (FW-DW/TW-DW) 100%.

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA was extracted using Trizol reagent (Mona, China). First-strand cDNA was synthesized by the HiScript III All-in-one RT SuperMix Perfect for qPCR(Vazyme, Nanjing, China). 2 × RealStar Green Fast Mixture with ROX II (GenStar, Fuzhou, China) was used for real-time fluorescence quantitative analysis. Fourteen specific primers for ZmSPS8 genes were synthesized at the Tsingke Biotechnology Company (Beijing, China) ( Table S1). The PCR program was 2 min at 95 • C, followed by 40 cycles of 15 s at 95 • C, 30 s at 60 • C, and 30 s at 72 • C. The specificity of the reactions was confirmed by the machine standard melt curve method. β-actin served as an internal control.

NBT Staining Assay
We used histochemical staining to detect superoxide in situ in accordance with a previously published report [39,47]. Leaf segments of 2 cm were cut from the middle part of the third leaves of maize plants at days 0, 3, 7, 10, and 14 after drought treatment (the third true fully expanded leaf of each seedling was used, three replicates were used). All the leaves were vacuum infiltrated with NBT solution (0.5 mg/mL) in the dark for 2 h at 25 • C. Then, the leaves were boiled in 100% ethanol for about 30 min to remove the chlorophyll before imaging. Seedlings that grew under normal water conditions were used as controls.

Statistical Analysis
Data were analyzed using SPSS v22.0 (SPSS, Chicago, IL, USA), and a one-way ANOVA was used to assess the significance of the experimental results. All data are presented as the means ± standard error of the mean. Differences were considered significant at a p-value of <0.05 (*), <0.01 (**), or <0.001 (***).

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12020369/s1, Figure S1: Schematic of ZmSPS8 conserved motifs. Ten conserved motifs were identified using MEME; Figure S2: The morphological changes of three maize lines with increasing drought treatment time; Table S1: Primer sequences for qRT-PCR analysis.