Genome-Wide Identification and Expression Analysis of the Protease Inhibitor Gene Families in Tomato

The protease inhibitors (PIs) in plants are involved primarily in defense against pathogens and pests and in response to abiotic stresses. However, information about the PI gene families in tomato (Solanum lycopersicum), one of the most important model plant for crop species, is limited. In this study, in silico analysis identified 55 PI genes and their conserved domains, phylogenetic relationships, and chromosome locations were characterized. According to genetic structure and evolutionary relationships, the PI gene families were divided into seven families. Genome-wide microarray transcription analysis indicated that the expression of SlPI genes can be induced by abiotic (heat, drought, and salt) and biotic (Botrytis cinerea and tomato spotted wilt virus (TSWV)) stresses. In addition, expression analysis using RNA-seq in various tissues and developmental stages revealed that some SlPI genes were highly or preferentially expressed, showing tissue- and developmental stage-specific expression profiles. The expressions of four representative SlPI genes in response to abscisic acid (ABA), salicylic acid (SA), ethylene (Eth), gibberellic acid (GA). and methyl viologen (MV) were determined. Our findings indicated that PI genes may mediate the response of tomato plants to environmental stresses to balance hormone signals. The data obtained here will improve the understanding of the potential function of PI gene and lay a foundation for tomato breeding and transgenic resistance to stresses.


Introduction
Protease is one of the most important enzymes involved in various physiological and biochemical processes, such as protein catabolism, signal transduction, digestion, and protein-protein interaction, in all organisms [1]. Proteases are critical for the maintenance and survival of their host organisms, but their high activity may be damaging. Therefore, it is crucial to regulate protease activity for maintaining the cellular homeostasis. Among several methods controlling excessive protease activity, the use of protease inhibitors (PIs) is a promising approach. PIs effectively modulate protease activity by direct interaction and clearing the signal receptor of the proteases [2,3].
PIs are widely distributed in the plant kingdom [4]. Plant PIs, usually small proteins, are present in storage tissues, such as tubers and seeds, but they have also been found in the aerial parts of plants [5]. Plant PIs are localized in plant tissues, including leaves, stems, and developing fruits and seedlings. By analysis of plant PIs development-and tissue-specific expression, their physiological functions have been predicted [6].
with 100 µM ABA, 100 µM GA, 100 µM SA, 1 mM Eth, and 100 µM MV until runoff) simultaneously for gene expression profiling. Untreated plants were used as controls to avoid the effects of biological clock on differential gene expression. After 0, 1, 4, 6, 12, and 24 h of treatment, the materials of leaves were immediately frozen in liquid nitrogen and stored at −80 • C. The RNA isolation and real-time PCR was performed as described previously [39] with gene-specific primers (Supplementary Table S1).

Identification of Tomato PI Genes
The predicted PI genes were identified as follows. First, homologs were searched using "protease inhibitor" as a keyword in the SGN (Sol Genomics Network) tomato database. Afterward, the PI genes were verified and found via the BLASTP search against the SGN and National Center for Biotechnology Information databases. After manually removing the redundant sequences, all these predicted genes were examined for the different PI domains in SMART (version 7, EMBL, Heidelberg, Germany), Pfam (version 32, EMBL-EBI, Cambridgeshire, UK), and InterProScan (version 71, EMBL-EBI, Cambridgeshire, UK), and those without the different PI domains were excluded.

Phylogenetic Analysis
The PI families' protein sequence of tomato (downloaded from SGN) was analyzed using the ClustalX software (version 2.1, UCD, Dublin, Ireland). Based on the results of aa sequence alignment, the neighbor-joining algorithm in MEGA (version 6, Pennsylvania State University, State College, PA, USA) [40] was used to draw the phylogenetic tree. The mode and gap was set to "Poisson correction" and "Pairwise Deletion", respectively, and the verification parameter was Bootstrap = 1000.

Chromosomal Location, Gene Structure, and Sequence Alignment
The PI genes were mapped to tomato chromosomes by identifying their chromosomal positions according to SGN database. The exon and intron structures of SlPIs were generated using the Gene Structure Display Server 2.0 [41] by aligning the CDS sequences with the corresponding genomic DNA sequences from SGN database. Domains were identified using the Pfam and the SMART programs.

Cis-Element Prediction for PI Gene Promoter
The PI sequences were used as BLASTN (version 2.40, NCBI, Bethesda, MD, USA) search queries against the tomato whole genome scaffolds at the SGN website. The promoter sequences (3.0 kb upstream of 5 UTR) of all identified PI genes were submitted to the PlantCARE (Plant Cis-Acting Regulatory Element) database for cis-element prediction.

Tissue Expression Analysis of SlPIs of Tomato
The IDs of SlPI genes were searched in the online NexGenEx-Tom database [42] to investigate their expression patterns. Data showing the SlPI expression in 10 different tissues/stages, including leaf, root, flower, flower bud, 1, 2, and 3 cm fruit, mature green fruit, breaker fruit, and fruit at 10 days were obtained from two biological replicates. The gene expression level was defined on the basis of the iTAG mRNA loci and normalized using reads per kilobase per million (RPKM) for each tissue/stage, and the log2 transformation was selected from the platform [42].

Expression Pattern of SlPI Genes Under Different Stress Conditions
Microarray analysis was performed to gain insight into the expression profiles of the SlPI gene families under different environmental stresses. Whole genome microarray data for various environmental stresses, such as drought, salt, high temperature, B. cinerea, and tomato spotted wilt virus (TSWV)) were obtained from the TFGD (Tomato Functional Genomics Database) database. The array platforms for microarray data included the TOM2 oligo and the Affymetrix genome arrays. For the TOM2 oligo array, the probe sets of the SlPI genes were identified through BlastN analysis in the database "TOM2 oligo sequences". For the Affymetrix genome array, the probe sets of the SlPI genes were identified through BlastN analysis in the database "Affy target sequences". The average value was considered for SlPI genes with more than one probe set. The expression values of SlPI genes that were upregulated or downregulated by more than two-fold with p < 0.05 were considered as differently expressed.

Identification of PI Genes in Tomato
In this study, 55 PI genes in the tomato genome cDNA release 2.40 were identified (Table 1). These genes were named as SlPI01 to SlPI55 on the basis of their distributions and relative linear orders among their respective chromosomes. To further understand PI proteins, the amino acid (aa) length, molecular weight, isoelectric point, hydrophilic coefficient, and subcellular localization prediction of 55 PI proteins were analyzed ( Table 1). The lengths of SlPI proteins varied from 77 aa residues (SlPI04, SlPI29, and SlPI30) to 603 aa residues (SlPI25), with an average length of 179 aa. Most of the lengths of the PI proteins were between 100 and 300 aa. Molecular weight ranged from 8 kDa (SlPI04) to 68 kDa (SlPI25), and the isoelectric point varied from 4.64 (SlPI30) to 10.11 (SlPI47). Most tomato PI proteins (72.7%) were predicted to be in the extracellular matrix, but some proteins may be located in other subcellular compartments, such as the cytoplasm (14.5%), chloroplasts (10.9%), and vacuoles (1.8%).
PIs with single inhibitor domains are dubbed simple inhibitors, and those with multiple inhibitor domains are called complex inhibitors. All 55 SlPI proteins are simple inhibitors. Moreover, PI proteins with the same domain were similar and concentrated. For example, all potato_inhibit domains were in the C-terminal. In summary, the diversity of domain type caused the functional diversity of PI in tomato. To explore the phylogenetic relationship and divergence of the PI families in tomato, a phylogenetic tree was constructed using the MEGA6 in accordance with the aligned 55 PI protein sequences. According to the phylogenetic analysis, the tomato PI families were divided into seven families, including Potato Inhibitor I, Potato Inhibitor II, Cysteine proteinase inhibitor, Serine protease inhibitor, Soybean trypsin inhibitor (Kunitz), carboxypeptidase A inhibitor, and Peptidase inhibitor I9 ( Figure 2). Except for SlPI17 and SlPI18, the PI proteins located in the same subfamily had the same domain. The potato inhibitor I subfamily was the largest with 20 members, whereas the potato inhibitor II, cysteine PI, s serpin, and STI had 12, 7, 4, and 10 members, respectively.

Chromosomal Localization, Gene Structure, and Duplication of SlPIs
The PI genes in tomatoes were unevenly distributed across chromosomes and mostly existed in the form of gene clusters. The tomato PI genes were mapped with the published chromosomes of tomato genome to determine their genomic distribution ( Figure 3). The chromosomal localizations of two SlPIs, namely, SlPI01 and SlPI02, were uncertain, and the other SlPIs were distributed in 10 chromosomes. Notably, these SlPI genes were unevenly distributed in chromosomes. High-density regions harboring PIs were discovered in chromosomes 3, 4, 9, and 11. Chromosome 9 contained the most PI genes. Only one PI gene (SlPI25) was found in chromosome 5, whereas two PI genes were found in chromosomes 1, 6, 7, 8, and 10.    As shown in Figure 4, the tomato PI gene exon-intron organizations were diverse. Most SlPI genes (94.54%) contained 1-3 exons. SlPI05, SlPI01, and SlPI02 contained 4, 5, and 5 exons. Out of the 55 SlPIs, 21 (38.19%) had three exons, and 18 (32.73%) had no intron. Furthermore, the members of a subfamily showed structural similarities. All PI genes in the STI subfamily (SlPI06, SlPI07, SlPI08, SlPI09, SlPI10, SlPI17, SlPI18, SlPI26, SlPI27, and SlPI55) had no intron.

Cis-Elements in the Promoters of Tomato PI Genes
The gene transcription levels were regulated by the interaction of transcription factors with the cis-acting element in the upstream promoter sequences. Therefore, studying the cis-element in the promoter of PI genes in the tomato may help explore the function of SlPI genes. The promoter regions of 55 SlPI genes were analyzed using the online software Plant CARE (Supplementary Table S3). A total of 43 cis-elements were identified in more than 20 PI genes ( Table 2 and Supplementary Table S4). Among the 43 cis-elements, the functions of 22 cis-elements were not annotated. By excluding the common cis-elements, such as the TATA-box and CAAT-box, the 19 remaining cis-elements can be divided into four groups. Eight cis-elements, namely, Box4, G-box, G-Box, TCT-motif, GT1-motif, GATA-motif, I-box, and chs-CMA1a, were light-responsive. Five cis-elements, including ABRE, CGTCA-motif, TGACG-motif, TCA-element, CAT-box, and five stress-related elements, including ARE, WUN-motif, TC-rich repeats, MBS, and LTR, were related to hormones. Furthermore, the CAT-box, the only element in group 4, was related to meristem expression. Notably, many cis-elements related to abiotic stress in plants were identified at the promoter of PI gene (Figure 5), and the promoter of 52 PI genes had ABRE (cis-element involved in the ABA responsiveness), indicating that PIs played an important role in abiotic stress resistance via ABA response.
Under TSWV infection, four SlPI genes (SlPI17, SlPI18, SlPI31, and SlPI55) were upregulated in tomato roots, three genes (SlPI05, SlPI09 and SlPI10) showed no response, and the expressions of the remaining genes were downregulated. In the leaves of tomato, four genes (SlPI05, SlPI07, SlPI17, and SlPI31) were upregulated, and the expression levels of the remaining genes were downregulated ( Figure 6D). The transcriptions of three genes (SlPI06, SlPI26, and SlPI27) were upregulated in all tested samples due to the wound and the invasion of Botrytis cinerea ( Figure 6E). In the mature green and ripe red fruits, most of the SlPI genes displayed a stronger expression in fruits wound-inoculated with B. cinerea than in wounded fruits. Interestingly, eight genes (SlPI11, SlPI12, SlPI13, SlPI14, SlPI15, SlPI16, SlPI51, and SlPI52) displayed differential expression between the mature green and ripe red fruits.

Expression of SlPI Genes in Different Tomato Tissues
The expression levels of SlPI genes were analyzed in 10 different tissues, including leaf, root, flower, flower bud, 1, 2, and 3 cm fruit, mature green fruit, breaker fruit, and fruit at 10 days, at different developmental stages to obtain their expression patterns in different tissues of tomato and in the developmental stages of the fruit. As shown in Figure 7, some SlPIs, including SlPI03, SlPI13, SlPI22, and SlPI46, were highly expressed in all tissues/stages. By contrast, eight genes, including SlPI4, SlPI19, SlPI21, SlPI30, SlPI32, SlPI40, SlPI49, and SlPI52, showed low expression levels in all the tissues/stages. The expression patterns of the other SlPIs showed different patterns of temporal and tissue-specific expressions. In addition, 17 genes, including SlPI01, SlPI16, SlPI17, SlPI18, SlPI25, SlPI28, SlPI33, SlPI34, SlPI45, SlPI37, SlPI38, SlPI39, SlPI41, SlPI42, SlPI44, SlPI54, and SlPI55, were strongly expressed in the flower bud. Specifically, the mRNAs of SlPI1, SlPI29, and SlPI48 accumulated in the root and flower but decreased during fruit development. Moreover, three genes, namely SlPI28, SlPI34, and SlPI35, showed similar expression patterns with a higher expression at the flower and fruit than the other tissues/stages. Figure 7. Heat map of the expression patterns of SlPIs in 10 tissues/stages. The RNA-seq expression data of 10 tissues were used to reconstruct the expression patterns of SlPI genes. The samples were obtained from the leaf, root, flower, flower bud, 1, 2, and 3 cm fruit, mature green fruit, breaker fruit, and fruit at 10 days. Heat map is presented in green/black/red colors that represent low/medium/high expression, respectively.

Analysis of PI Gene Expression in Tomato Under Various Hormone Induction and Oxidation
Plant hormones play central roles in plant adaptability under various environments [43], and most abiotic stresses directly or indirectly lead to the rapid accumulation of toxic products, such as free radicals and reactive oxygen species, which cause oxidative stress [44]. To further analyze the function of this gene families, quantitative RT-PCR was used to investigate the transcriptional profiles of PI genes in tomato under ABA, Eth, SA, GA, and oxidative stress (methyl viologen, MV). The transcription levels of PI genes under different treatments were analyzed by qRT-PCR. To avoid the influence of photoperiod, the expression of PI genes was eliminated for 0 h. The expression profiles of all PI genes in tomato were hard to describe. Therefore, four representative members (SlPI3, SlPI4, SlPI54, and SlPI55) were assessed from four PI gene families (Figures 1 and 8).  Table S1).
As shown in Figure 8, SlPI03 and SlPI54 decreased immediately after reaching the peak, whereas the expression levels of SlPI04 and SlPI55 increased first and then slowly decreased under ABA treatment. Under SA treatment, the expression levels of all three PI genes (SlPI03, SlPI04, and SlPI54) increased, but that of SlPI55 decreased. Notably, the SlPI54 levels dramatically increased by approximately 30-fold after 1 h of Eth treatment, which indicated that Eth can promote the expression of SlPI54. After GA induction, SlPI54 and SlPI55 reached the maximum level at 4 h, whereas SlPI03 and SlPI04 were expressed very similarly, with high expression at 4 and 12 h. After 24 h of MV treatment, the expression levels of all four PI genes were almost zero, indicating that MV treatment inhibited the expression of most PI genes.
In short, hormones can significantly regulate the expression of PI genes, indicating that the PI gene plays an important role in abiotic response.

Evolution of the SlPI Gene Families
Gene duplication is one of the main forces driving the evolution of the genetic system and genome [45]. The chromosome mapping and the genome distribution of SlPI genes indicate that tandem and/or polyploid replication based on the tomato genome annotation database may contribute to the amplification of the SlPI genes. In this study, chromosomal localization reveals that 55 PI genes are unevenly distributed on 10 chromosomes of tomato, except for the unclear chromosome mapping of SlPI01 and SlPI02. Many genes share the same functional domains and similar structure. Therefore, repetitive events have likely contributed to the diversity of the tomato SlPI gene families.
In this study, a tandem repeat gene is defined as a gene with an adjacent gene sharing the same domains and no more than one intermediate gene. A total of 29 genes are involved in tandem gene duplication ( Figure 3). As shown in Figure 3, three groups of SlPI genes (SlPI 06/ SlPI07/ SlPI08/ SlPI09/ SlPI10, SlPI11/ SlPI12/ SlPI13/ SlPI14/SlPI15/SlPI16, and SlPI32-SlPI45) can be identified as tandem duplication genes. These genes belong to the STI, potato inhibitor II, and potato inhibitor I families, respectively, and are located in chromosomes 3 and 9. Hence, tandem duplication has significant contributions to the expansion of the SlPI gene families in tomato.

Expression of the SlPI Gene Families
The SlPI genes are significantly induced under biotic stresses (B. cinerea and TTSWV) ( Figure 6D,E), confirming the role of PI in defense against pests and pathogens [26,[46][47][48]. Despite substantial efforts in projecting PIs as a valuable weapon against pests, the insect insensitivity and efficient tradeoff tactic has resulted in its commercial failure [49]. However, our study shows that the SlPI genes are significantly induced by abiotic stresses, including drought, salt, and heat ( Figure 6A-C). These findings, together with our results, led us to explore the PI genes' role in abiotic stress.
In the natural environment, plants are constantly challenged by various biological and abiotic stresses. Genes integrate environmental stress signals (such as hormones) when plants encounter adverse ambiance. Plant hormones play a vital role in the ability of plants to encounter abiotic stresses by mediating growth, development, nutrient allocation, and source/sink transitions [43]. The plant hormones ABA, Eth, SA, and GA are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses [50]. For example, ABA regulates plant response against several abiotic stresses, such as drought, salt, and cold stresses. Furthermore, ABA plays a negative regulatory function in balancing Arabidopsis resistance response to a necrotrophic fungal pathogen Plectosphaerella cucumerina, which naturally colonizes a broad range of Arabidopsis accessions [51,52]. Our results show that the expression of some SIPIs can be influenced by ABA, Eth, SA, GA, and MV ( Figure 8). The cis-elements of each PI gene promoter have been analyzed to further clarify the relationship between PI gene expression and hormones ( Table 2). The ABRE, a cis-element involved in ABA responsiveness, is found in the promoter of 52 PI genes. The TCA-element, a cis-element involved in SA responsiveness, is found in the promoter of 23 PI genes. ARE, a cis-element that is essential for anaerobic induction, is found in 49 SlPIs. These conditions are consistent with the expression of PI that can be induced by ABA, SA, and MV ( Figure 8). Hence, SlPIs may integrate hormone signals to modulate the plant resistance to biotic or abiotic stresses.
A spatiotemporal regulation of the SlPI gene families is observed at various stages of tissue development. Two genes (SlPI3 and SlPI13) are highly expressed in all studied tissues, suggesting their possible relationship to the specific housekeeping activities of tomato cells. Moreover, some SlPI genes are highly or preferentially expressed, showing tissue-and development-specific expressions (Figure 7). For example, the expression levels of SlP41, SlP43, and SlPI45 in flower bud are higher than those in other tissues. The expression levels of SlPI25 and SlPI54 in roots and leaves are higher than those in other tissues. All these genes are expressed preferentially in tissue development and may affect the growth and development of tomatoes ( Figure 7). Thus, their functions are worthy of further study. In addition, the expression behaviors of some SlPI genes vary in different tissues and stages, implying that SlPI proteins may play multiple roles.

Classification and Function of PI Genes in Tomato
In this study, the 55 SlPI proteins of tomato, which contain only one inhibitor domain of PIs, are simple inhibitors. Therefore, they can be easily divided into different families, including Potato inhibitor I, Potato inhibitor II, STI (Kunitz) family, Cysteine proteinase inhibitor, serpin, carboxypeptidase A inhibitor, and peptidase inhibitor I9 families, according to the domain and sequence similarity ( Figure 2). These families in PI proteins confer diverse functions, suggesting the multiple roles the PI protein play in plants.
Potato inhibitors I and II are two typical chymotrypsin and trypsin inhibitors [53,54]. Wounding and UV exposure increases the expression of these two inhibitors in tomato and potato leaves [55,56]. Potato PI I and potato PI II are involved in plant defense against herbivorous animals [55]. Among the 55 PI genes of tomato, 22 belong to the potato PI I family (SlPI04, SlPI17, SlPI18, SlPI30-SlPI45, and SlPI47-49), and 12 belong to the potato PI II family (SlPI02, SlPI11-SlPI16, SlPI29, and SlPI51-54).
In addition, the WUN-motif, a wound response cis-element, is found in the promoters of all these PI genes, except SlPI29, SlPI36, and SlPI37. This finding suggests that these PI genes in tomatoes may play an important role in plant wounds and insect resistance.
The Kunitz gene family is a complex family with various PIs and inhibits serine, cysteine, and other hydrolases [57]. The Kunitz PIs reversibly interact with their target proteases to form stable complexes and inhibit their catalytic activity in a competitive or noncompetitive manner [58]. Animal Kunitz PIs are extensively studied and proven to be involved in various physiological processes, such as inflammatory processes [59], thrombosis [60], AIDS [61][62][63], and fungal infections [64]. The family has also been studied in different plants, but most of the work is focused on their defenses in insect attacks because their gene expression levels are upregulated by wounds and insect feeding [65]. Notably, the Kunitz family plays an important role against lepidopteran and coleopteran pests in various plants, including poplar [66], rice [67], tomato [68], potato, and tobacco [69]. In our study, SlPI06-SlPI10, SlPI26, SlPI27, and SlPI55 belong to the Kunitz gene family and may provide defense against herbivores. SlPI55 is only specifically expressed in flowers and fruits ( Figure 6). Hence, SlPI55 may enhance resistance to biotic stresses at the reproductive stage.
Most plant cysteine PIs, which specifically inhibit the activity of cysteine proteases and are found in plants, insects, and vertebrates, are part of the cystatin superfamily [32]. The cystatin from plants has been classified in a new subfamily (called phytocystatins) due to its special pattern [70]. Although cystatins have been identified in many plant species, only a few cystatins have been well characterized [35]. Some cystatins can prevent the premature proteolytic degradation of newly formed storage proteins during seed development [71][72][73] and reduce the adverse reactions caused by the release of cysteine proteases to the outside of the cells [74]. These PIs also play an important role in plants against herbivorous pests, fungal and nematodes and defense [75][76][77][78][79]. In addition, the overexpression of two cystatins, atCYSa and atCYSb, increases Arabidopsis resistance to drought, cold, high salt, and oxidative stresses [80]. Among the 55 PI genes of tomato, SlPI01, SlPI03, SlPI05, SlPI19, SlPI20, SlPI25, and SlPI46 belong to the family of cysteine PIs and may have similar functions. Notably, SlPI03 is induced by ABA, SA, Eth, GA, and MV (Figure 7), implying that SlPI03 plays an important role in tomato resistance to abiotic stress.
The serpin family, the most widely distributed family in PIs (300-500 aa in size), have been identified in viruses, bacteria, plants, microorganisms, and other organisms [9,[81][82][83]. Many serpins in animals are involved in the regulation of blood pressure, coagulation, thrombosis, and proteolytic activity associated with inflammatory responses and cell death [84]. However, information about the biological function of serpins in plants is limited. Considering the functional diversity of animal serpins, plant serpins may have a range of functions. Serpins comprise small-to medium-sized gene families comprising 5-10 members [85]. In this study, the tomato serpin superfamily has four members (SlPI21, SlPI22, SlPI23, and SlPI24), which is consistent with the small gene family mentioned above. Plant serpins are confounding inhibitors, indicating that serpins can target multiple proteases [86]. The function of serpins is associated with cell death by inhibiting endogenous and exogenous proteases for pest management [87,88]. Serpins are potential signaling molecules involved in the regulation of programmed cell death (PCD) or defense pathways [89]. PCD plays a key role in plant response to stress, including responses to hypoxia, shadowing, extreme temperatures, drought, and oxidative stress. Therefore, four SlPI genes (SlPI21, SlPI22, SlPI23, and SlPI24) may play important roles in abiotic stress. The understanding of the function of plant serine PIs is far from enough and presents exciting challenges for the future.
The carboxypeptidase A inhibitor family is a family of proteins represented by the potato carboxypeptidase inhibitor (PCI), a metallocarboxypeptidase inhibitor. Previous reports have focused on the response of PCI to insect damage or other mechanical damage [90]. For example, carboxypeptidase inhibitors can prevent pest invasion by hydrolyzing pest digestive proteins [91]. In potato leaves and tomatoes, the carboxypeptidase inhibitor can be induced and used to resist aggressive herbivores [92]. In this study, SlPI28 belongs to the family of carboxypeptidase A inhibitors, and WUN-motif, a cis-element in wound response, is found in the promoter of SlPI28. Therefore, SlPI28 is very likely to be involved in plant defense against herbivores. More recently, two homologs of PCI from tomato (TCMP-1 and TCMP-2) have been shown to be involved in fruit development. Compared with the wild type lines, transgenic lines form flowers earlier and produce fruits earlier [93]. Interestingly, SlPI28 is poorly expressed before flowering, and its expression increases after flowering, reaching the highest level in flower buds. The spatiotemporal expression pattern of SlPI28 indicates its role in flower/fruit development. However, a link between the regulation of fruit growth and the inhibitory activity of metallocarboxypeptidases is difficult to determine. Further studies on gene function can provide a better understanding of how genes confer to stress and regulate plant development.
SlPI50 contains the propeptide domain of the N-terminal peptidase belonging to the subtilisin of the MEROPS family S8A. This domain is also found in members of the MEROPS PI family I9 (peptidase B inhibitor family). This family of PIs is highly specific and inhibits the subtilisin family [94]. The majority of inhibitors of this family, such as the propeptide of subtilisin BPN from Bacillus amyloliquefaciens, Saccharomyces cerevisiae proteinase B inhibitor 2, and Pleurotus ostreatus proteinase A inhibitor 1, are found in bacteria and fungi [95,96]. Recently, subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana has been identified as a member of the I9 inhibitor family [97]. SPI-1 plays a role in cold stress, senescence, and seed development in Arabidopsis [97]. In addition, various hormone-related cis-elements, including ABRE (ABA-responsive), CGTCA-and TGACG-motifs (MeJA-responsive), and TGA-element (auxin-responsive), were found in the promoter of SlPI50. Hence, SlPI50 may play an important role in abiotic stresses by integrating hormone signals.

Conclusions
In this study, the PI gene families were classified and named, and 55 PI genes in the tomato genome were comprehensively analyzed through phylogenetic relationships, gene structure, conserved domains, cis-elements, and expression analysis. SlPI genes were characterized by integrating genome organization, comprehensive sequence, conserved domain, gene structure, cis-element prediction, and expression profile analysis of 10 different tissues by using RNA-seq and under different stresses (heat, drought, salt, TSWV, and B. cinereal) by using microarray atlas. The transcription of some SlPIs was induced by hormones, indicating the vital functions of these PI genes in physiological activities. The research results lay the foundation for genetic evolution analysis and functional research. The discovery of new genes related to plant resistance is important for accelerating the cloning of stress resistance genes in tomato.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4425/11/1/1/s1, Figure S1: Amino acid sequence comparison of SlPI genes, Table S1: List of primers used for RT-PCR, Table S2: The  information of the conserved domains, Table S3: Cis-elements identified in the promoters of SlPI genes, Table S4: Cis-elements identified in the promoters of more than 20 SlPI genes, Supplementary Table S5: Information on probe sets used for microarray expression analysis.
Author Contributions: The work presented here was carried out in collaboration between all authors. "methodology, J.L.; software, W.Y. and Y.F.; validation, Q.Y., W.Y. and Y.F.; data curation, Y.F., C.C., and W.Y.; Writing-Original draft preparation, Y.F. and W.Y.; Writing-Review and editing, J.L. and Y.F.; Supervision, J.L.; Project administration, J.L." All authors read and approved of the final manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was sponsored by grants National Natural Science Foundation of China (No. 31872123), Natural Science Foundation of Chongqing, China (cstc2019jcyj-msxmX0333) and College students' innovation and entrepreneurship projects Chongqing (S201910635019).

Conflicts of Interest:
The authors declare no conflict of interest.