Classification and Expression Profile of the U-Box E3 Ubiquitin Ligase Enzyme Gene Family in Maize (Zea mays L.)

The U-box E3 (PUB) family genes encode the E3 ubiquitin ligase enzyme, which determines substrate specific recognition during protein ubiquitination. They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristic of PUB gene family in the important staple crop, maize (Zea mays L.). In this study, the PUB gene in maize was aimed to identify and classify through whole-genome screening. Phylogenetic tree, gene structure, conserved motif, chromosome location, gene duplication (GD), synteny, and cis-acting regulatory element of PUB member were analyzed. The expression profiles of ZmPUB gene family in maize during development and under abiotic stress and hormones treatment were analyzed by the RNA-seq data. A total of 79 PUB genes were identified in maize genome, and they were stratified into seven categories. There were 25 pairs of segmental duplications (SD) and 1 pair of tandem duplication (TD) identified in the maize PUB gene family. A close relationship was observed between the monocot plant maize and rice in PUB gene family. There were 94 kinds of cis-acting elements identified in the maize PUB gene family, which included 46 biotic- and abiotic-responsive elements, 19 hormone-responsive elements, 13 metabolic and growth-related elements. The expression profiles of maize PUB gene family showed characteristics of tissue specificity and response to abiotic stress and hormones treatment. These results provided an extensive overview of the maize PUB gene family.


Introduction
The ubiquitin proteasome system (UPS) is an energy dependent system that modulates protein activity and degeneration. It is involved in cellular growth, differentiation and apoptosis, secretion and endocytosis, gene transcription and expression, as well as signal transduction and immune response [1,2]. UPS is composed of the ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. Ubiquitination is a common post-translational modification involving proteins. The process is as follows: E1 binds ubiquitin using a high-energy, thiol-ester bond in an ATP-based system. The E1-ubiquitin complex then interacts with E2 and transfers the active ubiquitin to a cysteine residue in E2. E3 next recognizes the substrate proteins and catalyzes the formation of an isopeptide bond between the C-terminal carboxyl group of ubiquitin and free lysl-amino groups within the protein substrate [3][4][5][6][7]. Finally, the ubiquitinated protein is degraded by 26S protease [1].
The ubiquitin ligase enzymes (E3) determine ubiquitin-specificity via recognition of substrate proteins, and they constitute the largest family among the three enzymes. According to the functional mechanism and subunit composition, E3 can be classified into four distinct categories: homologous to E6-AP COOH-terminus (HECT), U-box, really (PF04564). We further analyzed the gene locus, chromosomal location, sequence length, exon amount, molecular weight (MW), isoelectric point (PI), and subcellular location of all ZmPUB genes (Table S1).
The protein length of ZmPUBs was between 94-1353 amino acids, and the MW was between 10.75-144.72 kDa. The PI range was 4.57 to 9.80. The subcellular localization was estimated using the WoLF PSORT website. Among the 79 ZmPUB proteins, 19 were predicted to be cytoplasmic proteins; 14 were located in the nucleus; 10 were in the plasma membrane; and the rest were localized in various organelles, such as the chloroplast (29), endoplasmic reticulum (3), golgi apparatus (2), mitochondrion (1), and peroxisome (1).
To explore the relationship among different PUB genes, we generated a neighborjoining phylogenetic tree with the U-box domain sequences of ZmPUBs, AtPUBs, and OsPUBs. Based on our analysis, the 79 ZmPUB proteins were stratified into seven categories, namely group I to group VII ( Figure 1). Group IV consisted of the most members, namely 20 ZmPUB proteins, followed by group VII (19), and group II (17). Groups I and V possessed the least amounts of members, including only four and five ZmPUB proteins, respectively. The protein structure domain analysis was carried out using SMART and Pfam databases (Table S1). All ZmPUB proteins harbored the U-box domain. From group I to group VII, the proportion of ZmPUB proteins containing only the U-box domain was 50% (2/4), 11.7% (2/17), 42.8% (3/7), 35% (7/20), 100% (5/5), 0 (0/7), and 89.4% (17/19). We also identified an additional 10 structural domains in the ZmPUB proteins. In group I, the RS4NT or TPR domain was detected in two ZmPUB proteins. In groups II and IV, a total of eight domain types were observed, including the ARM, pkinase-tyr, coil coil region, UFD2P_CORE, USP, TPR, WD40, and KAP domains. In groups V, VI, and VII, there were three major domain types, which were all in the coil coil region, as well as the ZnF_TTF and ARM domains, and they were located in 19 ZmPUB proteins. In maize, most ZmPUB proteins carried only the U-box domain, accounting for 45.6% (36/79) of total proteins. There were similar proportions surveyed in Arabidopsis, rice, barley and citrus, and they were 25.0% (16/64), 25.9% (20/77), 31.3% (21/67), and 30.3% (17/56), respectively [12,13,18,20]. A previous study revealed that the ARM domain was related to substrate recognition during the ubiquitination process, and it is the second populated domain in rice and Arabidopsis [21]. In maize, 25 ZmPUB proteins harbored the ARM domain, and it was similar to rice and Arabidopsis. Additionally, approximately 70-74% of ZmPUB proteins were clustered into three groups (groups II, IV, and VII) in rice, Arabidopsis, and maize ( Figure 1). This suggested that the maize PUB gene family was evolutionarily conserved.

Genetic Structures and Conserved Motifs Analyses of the PUB Gene Family
To better elucidate the association between gene function and evolution, we explored the structural organization and conserved motifs of PUB genes ( Figure 2, Table S2). We observed between 1 to 16 exons, with a mean of 3.61 exons per gene. There were about 37.9% ZmPUB genes with one exon but no intron. The proportions of ZmPUB genes containing two, three, and four exons were 9.8%, 8.8%, and 13.5%, respectively. The largest number of exons was 16, and they were detected in ZmPUB4 and ZmPUB69. In group I, the exon number in ZmPUB genes was between 5-8, with a mean of 6.75. In groups II and IV, we detected the maximum variation of exons, which was 1-16 and 2-9, respectively. In group III, there were 2-4 exons per gene. Around 84-100% of ZmPUB genes from groups V, VI, and VII harbored only one exon. The ZmPUB genes from the same group shared comparable gene structure, thereby suggesting functional conservation among the maize PUB gene family. Additionally, the exon variations in ZmPUB genes may indicate the function diversity of the maize PUB gene family.
We screened ten conserved motifs among the 79 ZmPUB genes (Figure 2, Tables S2 and S3). About 83% of ZmPUB genes contained motifs 1, 3 and 4 simultaneously. Motifs 6, 7, and 9 mainly existed in the ZmPUB genes of group II. The tandem repeats of motifs 2 and 5 were characteristic of ZmPUB genes in group IV, and it likely served a distinct biological function. Motif 8 was prevalent exclusively among groups IV and VI. Motif 10 existed

Genetic Structures and Conserved Motifs Analyses of the PUB Gene Family
To better elucidate the association between gene function and evolution, we explored the structural organization and conserved motifs of PUB genes ( Figure 2, Table S2). We observed between 1 to 16 exons, with a mean of 3.61 exons per gene. There were about 37.9% ZmPUB genes with one exon but no intron. The proportions of ZmPUB genes containing two, three, and four exons were 9.8%, 8.8%, and 13.5%, respectively. The largest number of exons was 16, and they were detected in ZmPUB4 and ZmPUB69. In group Ⅰ, the exon number in ZmPUB genes was between 5-8, with a mean of 6.75. In groups Ⅱ and Figure 1. The phylogenetic tree of the PUB gene family. Multiple sequence alignments of the U-box domain sequences of rice, Arabidopsis, and maize were performed using MEGA 7. A phylogenetic tree was generated via the neighbor-joining technique with 1000 bootstrap values. The PUB genes were then separated into seven distinct categories marked by distinct colors.

Chromosomal Locations, Duplications, and Synteny Analysis of the Maize PUB Gene Family
To examine the chromosomal distribution of ZmPUB genes, we screened the genomic database, based on the DNA sequence of individual ZmPUB genes, and drew the chromosomal location map via the MapChart software ( Figure 3). In total, 77 ZmPUB genes (97.4%) were mapped to 10 chromosomes, and they were unevenly distributed within each chromosome. We identified 10, 12, and 13 ZmPUB genes at chromosomes 1, 4, and 5, respectively. On chromosomes 2, 3, 9, and 10 were identified 9, 8, 8, and 7 ZmPUB genes, respectively. Lastly, only two ZmPUB genes were mapped to the chromosome 8.
maize PUB gene family. Additionally, the exon variations in ZmPUB genes may indica the function diversity of the maize PUB gene family.
We screened ten conserved motifs among the 79 ZmPUB genes (Figure 2, Tables and S3). About 83% of ZmPUB genes contained motifs 1, 3, and 4 simultaneously. Mot 6, 7, and 9 mainly existed in the ZmPUB genes of group Ⅱ. The tandem repeats of mot 2 and 5 were characteristic of ZmPUB genes in group Ⅳ, and it likely served a distin biological function. Motif 8 was prevalent exclusively among groups Ⅳ and Ⅵ. Motif existed in all groups except for groups I and Ⅱ. The conserved motif analysis suggest that the PUB genes reserved the U-box domain and accumulated additional motifs ov the course of evolution. Lastly, similar motifs among ZmPUB genes might suggest t conserved evolutionary relationship and similar biological function. Figure 2. Gene structures and conserved motifs of the maize PUB gene family. A phylogenetic tr of PUB genes. Multiple sequence alignment of the U-box domain sequences in maize was carri out with Clustal W. The neighbor-joining (NJ) tree was generated via MEGA X with 1000 bootstr replicates; The gene structure of PUB genes. The yellow boxes, green boxes, and black lines rep sent exon, intron, and UTR (untranslated region), respectively; The conserved motif of PUB gen MEME analysis revealing the conserved motifs belonging to the maize PUB gene family. The c ored boxes on the right denote 10 motifs.

Chromosomal Locations, Duplications, and Synteny Analysis of the Maize PUB Gene Fam ily
To examine the chromosomal distribution of ZmPUB genes, we screened the genom database, based on the DNA sequence of individual ZmPUB genes, and drew the chrom somal location map via the MapChart software ( Figure 3). In total, 77 ZmPUB gen (97.4%) were mapped to 10 chromosomes, and they were unevenly distributed with Figure 2. Gene structures and conserved motifs of the maize PUB gene family. A phylogenetic tree of PUB genes. Multiple sequence alignment of the U-box domain sequences in maize was carried out with Clustal W. The neighbor-joining (NJ) tree was generated via MEGA X with 1000 bootstrap replicates; The gene structure of PUB genes. The yellow boxes, green boxes, and black lines represent exon, intron, and UTR (untranslated region), respectively; The conserved motif of PUB genes. MEME analysis revealing the conserved motifs belonging to the maize PUB gene family. The colored boxes on the right denote 10 motifs.
Gene duplication (GD) involves segmental duplication (SD) and tandem duplication (TD), and it promotes the expansion of the gene family. We conducted GD analysis to reveal the expansion process of the maize PUB genes ( Figure 4). We identified 25 pairs (43 ZmPUB genes) of SD and one pair of TD (ZmPUB43/ZmPUB44) (Figures 3 and 4). GD occurred on one or two loci. Using synteny analysis, we identified eight ZmPUB genes (ZmPUB27, 32, 43, 45, 48, 55, 58 and 62) with duplications on two loci, whereas the rest of the genes exhibited duplication on one locus ( Figure 4). To elucidate the evolutionary pressures acting on ZmPUB genes, we computed the Ka and Ks of the duplicated gene pair, which represented nonsynonymous and synonymous substitution rates, respectively ( Table 1). The Ka values were between 0.02-1.00. The Ks values were between 0.17-2.81. The Ka/Ks values of 26 gene pairs were between 0.05 and 0.99, which suggested that these ZmPUB genes evolved under strong purifying selection (Ka/Ks < 1). In addition, we computed the probable dates of the duplication events, and they occurred between 13.06 Mya (Ks = 0.17, Million years ago) and 215.77 Mya (Ks = 2.81, Million years ago), with an average of 93.55 Mya (Ks = 1.21, Million years ago). To further explore the evolutionary associations of the PUB genes in Arabidopsis, maize, and rice, we assessed syntenic interactions among the three species using the software MCScanX ( Figure 5). A total of 64, 79, and 77 PUB genes were used from Arabidopsis, maize, and rice, respectively [12,13]. There were 53 syntenic PUB gene pairs screened between maize and rice, while only 5 syntenic PUB gene pairs were found in Arabidopsis and maize.
tively ( Table 1). The Ka values were between 0.02-1.00. The Ks values were between 0.17-2.81. The Ka/Ks values of 26 gene pairs were between 0.05 and 0.99, which suggested that these ZmPUB genes evolved under strong purifying selection (Ka/Ks < 1). In addition, we computed the probable dates of the duplication events, and they occurred between 13.06 Mya (Ks = 0.17, Million years ago) and 215.77 Mya (Ks = 2.81, Million years ago), with an average of 93.55 Mya (Ks = 1.21, Million years ago). To further explore the evolutionary associations of the PUB genes in Arabidopsis, maize, and rice, we assessed syntenic interactions among the three species using the software MCScanX ( Figure 5). A total of 64, 79, and 77 PUB genes were used from Arabidopsis, maize, and rice, respectively [12,13]. There were 53 syntenic PUB gene pairs screened between maize and rice, while only 5 syntenic PUB gene pairs were found in Arabidopsis and maize.

Expression Profiles of ZmPUB Genes during Root Development
To explore a possible role of ZmPUB genes during root development, we employed the RNA-seq data from the MaizeGDB website, which is a submission by prior study [37]. The RNA-seq data revealed that ZmPUB genes were differentially expressed during root development ( Figure 7, Table S6). Three days after seed sowing, 21 ZmPUBs were upregulated in the differentiation zone of the primary root, whereas, only eight and four ZmPUBs were upregulated in the meristematic zone and stele of the primary root, respectively. Further, 6-7 days after seed sowing, 19 and 18 ZmPUBs exhibited elevated expression in the primary and seminal roots. In the V7 stage, 31, 21, and 2 ZmPUBs were highly expressed in the crown root nodes 1 through 3, crown root node 4, and crown root node 5, respectively. In the V13 stage, a total of 26 ZmPUBs revealed elevated expression in the crown root node 5. Interestingly, we noticed that ZmPUB4, ZmPUB8, ZmPUB16, ZmPUB23, ZmPUB27, ZmPUB35, ZmPUB43, ZmPUB49, ZmPUB66, and ZmPUB78 showed a relatively high expression in varying root locations (FPKM > 10) (Table S6). Among these genes, four ZmPUB genes contained the GA response element, five ZmPUB genes contained the IAA response element, and nine ZmPUB genes contained the meristem expression-related element (Tables S4 and S5). Lastly, seven ZmPUB genes were not at all expressed in the root.

Cis-Acting Regulatory Element Analysis
To better elucidate the transcriptional modulatory pathways associated with ZmPU genes, we performed cis-acting regulatory element analysis with the 1500 bp upstrea development-related elements harbored five seed or endosperm development elements (O 2 -site, GCN4_motif, OCT, RY-element, and MBSI), three meristem expression-associated elements (CAT-box, CCGTCC-box, and HD-Zip 1), two gene expression-related elements (CARE and AT-rich element), one circadian regulatory element (circadian), one root-specific regulatory element (motif I), and one cell cycle regulatory element (MSAlike).

Expression Profiles of ZmPUB Genes during Root Development
To explore a possible role of ZmPUB genes during root development, we employed the RNA-seq data from the MaizeGDB website, which is a submission by prior study [37]. The RNA-seq data revealed that ZmPUB genes were differentially expressed during root development (Figure 7, Table S6). Three days after seed sowing, 21 ZmPUBs were upregulated in the differentiation zone of the primary root, whereas, only eight and four ZmPUBs were upregulated in the meristematic zone and stele of the primary root, respectively. Further, 6-7 days after seed sowing, 19 and 18 ZmPUBs exhibited elevated expression in the primary and seminal roots. In the V7 stage, 31, 21, and 2 ZmPUBs were highly expressed in the crown root nodes 1 through 3, crown root node 4, and crown root node 5, respectively. In the V13 stage, a total of 26 ZmPUBs revealed elevated expression in the crown root node 5. Interestingly, we noticed that ZmPUB4, ZmPUB8, ZmPUB16, ZmPUB23, ZmPUB27, ZmPUB35, ZmPUB43, ZmPUB49, ZmPUB66, and ZmPUB78 showed a relatively high expression in varying root locations (FPKM > 10) (Table S6). Among these genes, four ZmPUB genes contained the GA response element, five ZmPUB genes contained the IAA response element, and nine ZmPUB genes contained the meristem expression-related element (Tables S4 and S5). Lastly, seven ZmPUB genes were not at all expressed in the root.

Expression Profiles of ZmPUB Genes during Leaf Development
To explore the function of ZmPUBs, we analyzed the expression profiles of ZmPUBs during leaf development, and the gene expression data were provided by a previous study [38]. In the previous study, the fully expanded third leaf of the seeding maize B73 plants was sliced into 15 sections (M1-M15) from the base to the tip, and a gradient gene expression analysis was performed from immature to mature leaf tissue. There were 34 highly expressed ZmPUB genes, which were mainly concentrated within the basal part of the leaf (Figure 8, Table S7). These evidences suggested that these genes may contribute to cell division and elongation. Eight ZmPUB genes were identified, which were highly expressed only in the middle region of the leaf. Three ZmPUB genes, namely ZmPUB1, ZmPUB48, and ZmPUB62, were only upregulated in the tip of the leaf. In addition, we identified ZmPUB43, ZmPUB44, ZmPUB53, ZmPUB54, ZmPUB62, and ZmPUB78 with very high expression (FPKM > 20) in the different parts of the leaf, indicating that these may significantly modulate leaf development. The promoter regions of the highly expressed genes were further analyzed; six genes contained the MeJA, JA, ETH and light-response elements, five genes contained the ABA response elements, five genes contained the meristem expression-related element, and four genes contained the GA response elements (Tables S4 and S5). Therefore, it is our belief that these highly expressed genes in various locations of leaves may be crucial for photosynthesis, differentiation, and hormonal responses. Lastly, we also found that seven ZmPUB genes were not at all expressed in the leaves.   Table S6.  Table S7.

Expression Profiles of ZmPUB Genes during Seed Development
To explore the function of ZmPUB genes during seed development, we gathered the seed development RNA-seq data from prior investigations (Table S8) [39,40]. Overall, 50 ZmPUB genes were expressed during the whole-seed development. Among them, 31 ZmPUB genes were present within the embryo, 9 ZmPUB genes were present in the endosperm, and 12 ZmPUB genes were present in both the endosperm and during embryo development (Figure 9). We also observed multiple highly expressed PUB genes, including ZmPUB3, ZmPUB4, ZmPUB8, ZmPUB23, ZmPUB24, ZmPUB43, and ZmPUB52 (FPKM > 10), which suggested that they may associate with seed development (Table S8). Among these genes, seven genes harbored the ETH and ABA response elements, six genes contained the meristem expression-related element, four genes contained the gliadin metabolic regulatory element, three genes contained the MeJA and JA response elements, two genes contained the GA response elements, one gene contained the IAA response elements, and one gene contained the endosperm formation regulatory element (Tables S4 and S5). In previous studies, hormones were reported as information transfer substances, which served essential functions in crop development, particularly, during grain filling [41][42][43]. This indicates that these highly expressed genes may participate in seed development. Additionally, we also identified 10 ZmPUB genes that were not expressed during seed development.

Expression Profiles of ZmPUB Genes during Abiotic Stress
To elucidate changes in the transcriptome response to abiotic stress, RNA-seq was carried out in maize under control and stress conditions [44]. Under cold stress (7 • C for 16 h), 31 ZmPUB genes were markedly elevated in maize seedlings relative to control, 12 of which showed high expression (fold change >1). ZmPUB45 was the most up-regulated gene (fold change >36). Twenty-four ZmPUB genes were down-regulated in maize seedlings under cold stress. Among them, ZmPUB29, ZmPUB61, and ZmPUB73 displayed the most down-regulated expression, which were 31%, 24%, and 35% of control, respectively ( Figure 10, Table S9). Under heat stress (50 • C for 4 h), sixteen ZmPUB genes were highly expressed in maize seedlings relative to control, seven of which showed extremely high expression (fold change >1). ZmPUB30 showed the highest up-regulated expression, which was up-regulated by 12.5 times. Forty ZmPUB genes were down-regulated, nineteen of which were significantly down-regulated by more than 50% ( Figure 10, Table S9). Under salt stress, twenty-six ZmPUB genes were up-regulated in maize seedlings, and among them, eight genes were up-regulated by more than 1-fold, and ZmPUB45 and ZmPUB57 were the most up-regulated genes, with increases by 25.5 and 84.8 times, respectively. Thirty-two genes were down-regulated in maize seedlings as compared to controls, and four genes (including ZmPUB1, ZmPUB27, ZmPUB40, and ZmPUB78) showed the most down-regulated expression, with reductions of 67%, 69%, 82%, and 63%, respectively ( Figure 10, Table S9). Under UV stress, thirty-nine ZmPUB genes were up-regulated, and seven genes were up-regulated more than 1-fold; among them, ZmPUB27 and ZmPUB45 had the most up-regulated expression, which were 5.18 and 19.8 times, respectively. Twentyfive ZmPUB genes were down-regulated, four of which were down-regulated by more than 50% ( Figure 10, Table S9). In addition, nine ZmPUB genes, including ZmPUB7, ZmPUB9, ZmPUB12, ZmPUB28, ZmPUB41, ZmPUB56, ZmPUB74, ZmPUB77, and ZmPUB79, were not influenced by various abiotic stressors.  Table S8.  Table S9.
Drought stress affected plants particularly during reproduction. To investigate the potential role of ZmPUB genes in drought stress, we collected the RNA-seq data of drought-exposed and appropriately watered fertilized ovary and basal leaf meristem tissue ( Figure 10, Table S9) [45]. Under drought stress, 36 up-regulated and 30 down-regulated genes were screened in the ovary, among which 41 genes were altered more than 50%. ZmPUB49 was the gene with the maximum change in expression, which was up-regulated by 6.5 times. There were 32 up-regulated and 30 down-regulated genes in the basal leaf meristem following drought stress, among which 20 genes were altered more than 50%. Four genes (including ZmPUB2, ZmPUB31, ZmPUB57, and ZmPUB78) exhibited the maximum alterations in expression, which increased by 14.0, 5.3, 5.6, and 8.0 times, respectively. Lastly, nine genes were not expressed under drought stress in the ovary or basal leaf meristem tissue.

Expression Profiles of ZmPUB Genes during Hormonal Treatment
To further elucidate whether hormone affect ZmPUBs expression, we conducted RNAseq under the hormone treatments (ABA, IAA, and GA). Based on our observations, 42 (53.16%) ZmPUB genes were detected in the leaf tissue (FPKM > 1) ( Figure 11, Table S10). Thirty-nine ZmPUB genes responded to the hormones ABA, IAA, and GA but with variable degrees of sensitivity. Under ABA treatment, 35 ZmPUB genes were significantly altered in expression pattern, and among them, 13 genes were down-regulated, 20 genes were upregulated, and 2 genes were down-regulated at the early stage (3 or 6 h) and up-regulated at the late stage (12 h). Under IAA treatment, 15 ZmPUB genes were altered, among which 5 were reduced, and 10 were augmented. Under GA treatment, 17 ZmPUB genes were altered, 6 of which were significantly diminished at one stage, and 11 were significantly elevated at one or two stage. As a result, we identified eight ZmPUB genes that showed the highest degree of hormone-induced expression, and they were ZmPUB1, ZmPUB4, ZmPUB43, ZmPUB45, ZmPUB57, ZmPUB64, ZmPUB68, and ZmPUB78, respectively. These evidences suggested that these ZmPUB genes contributed to the hormone-responsive network.
among which 5 were reduced, and 10 were augmented. Under GA treatment, 17 ZmPUB genes were altered, 6 of which were significantly diminished at one stage, and 11 were significantly elevated at one or two stage. As a result, we identified eight ZmPUB genes that showed the highest degree of hormone-induced expression, and they were ZmPUB1, ZmPUB4, ZmPUB43, ZmPUB45, ZmPUB57, ZmPUB64, ZmPUB68, and ZmPUB78, respectively. These evidences suggested that these ZmPUB genes contributed to the hormone-responsive network. Figure 11. Expression profile of ZmPUB genes following hormone treatment. The white column represents control. The light gray column represents the ABA treatment. The dark gray column represents the IAA treatment. The black column represents the GA treatment. The level of significance (p < 0.05) among the different treatments is indicated by different letters. The original expressions of individual ZmPUB genes are summarized in supplementary materials: Table S10.

Discussion
The PUB genes are common among mammals, plants, and microorganisms, and they encode the PUB ligase enzyme, a key enzyme in ubiquitin proteasome-mediated degradation [8]. The genome-wide analysis of PUB genes was performed in numerous species, and they were found to play essential roles in modulating plant development and response to biotic, abiotic, and hormonal stressors [1,11,22,23]. Herein, we screened 79 ZmPUB genes in the maize genome and designated ZmPUB1 through ZmPUB79 based on their location on the chromosome.
Phylogenetic analysis revealed that these ZmPUB genes can be clustered into seven categories. Similar results were found in rice, soybean, barley, citrus, and banana (Musa paradisiaca), wherein the genes were classified into 6-8 groups using phylogenetic analysis [15,16,18,46]. Further analysis revealed that the 36 ZmPUB genes encoded proteins that harbor only the U-box domain, and 43 ZmPUB genes encoded proteins that contain the U-box domain, along with additional 10 domains, including the ARM repeat (25), coil coil region(10), pkinase-tyr (9), TPR (2), UFD2P_CORE (2), ZnF_TTF (1), WD40 repeat (1), KAP (1), RS4NT (1), and USP (1) domains (Table S1). In Arabidopsis, 16 AtPUB proteins harbor only the U-box domain, whereas 48 AtPUB proteins contain both the U-box domain and several other domains, such as ARM repeat (27), kinase (14), WD40 repeat (2), MIF4G (2), cyclophilin (1), UFD2 (1), and TPR domains (1) [13]. In rice, the domain composition of PUB proteins are similar to Arabidopsis; all OsPUB proteins contain the U-box domain, and 57 OsPUB proteins contain six types of domains except MIF4G [12]. In higher eukaryotes, genomic evolution can lead to the gene expansion and functional differentiation of gene families. Being the core domain of the PUB gene family, the U-box domain is evolutionarily conserved. Meanwhile, there are plenty of other domains observed in numerous species, which might introduce functional diversity to the PUB genes.
In eukaryotes, genes are usually composed of exons, introns and untranslated regions (UTRs) at the 5 and 3 ends. The structural organization is closely related to gene function and family evolution [47]. In the maize PUB gene family, there was a wide variation in the number of exons; the mean number of exons per gene was 3.61, and it ranged from 1 to 16. Around 37.97% of the ZmPUB genes contained only one exon and no intron (Figure 2, Table S2). In prior investigations, about 1 to 17 exons were identified in the PUB genes of tomato and Medicago, with an average of four exons per gene, and the PUB genes with only one exon and no intron accounted for 34.14% and 40.32% of all PUB genes [17,21]. The structural organization of PUB genes are similar in different species. It is suggested that the PUB gene family is evolutionarily conserved. Exons possess the core information needed by the cell to synthesize protein, whereas introns protect the coding proteins from randomly generating deleterious mutations [48]. In the PUB gene family, numerous intronless genes were reported in multiple species, such as tomato, grape, citrus, Medicago, and Chinese cabbage [14,17,18,21,49], suggesting a strong structural integrity of PUB genes. Furthermore, the conserved motif distributions suggested a strong structural and functional similarity among maize PUB genes ( Figure 2, Table S2). For instance, motifs 1, 3, and 4 are highly conserved and exist in almost all PUB genes. We also observed some unique features in some groups of the maize PUB gene family. Motifs 5 and 2 tandem sequences were mainly located in group IV. Motifs 6, 7, and 9 were the exclusive feature of group II. These analyses are highly beneficial to scientists who are interested in the evolution, structure, and function of the PUB gene family.
Gene duplication (GD), mutation, and natural selection are the major sources of new genes and functions, and they provided a basis for biodiversity [50]. Gene duplication and syntenic analysis identified 25 pairs (43 ZmPUB genes) of SD and one pair of TD (Zm-PUB43/ZmPUB44) in the maize PUB gene family (Table 1). GD is speculated to be the major contributor of expansion and diversity in the maize PUB gene family. We next evaluated the domain composition of the maize duplicate genes. New domains were introduced to the newly duplicated genes; five genes were added to the ARM domain (ZmPUB10, ZmPUB19, ZmPUB39, ZmPUB43, and ZmPUB63), two genes to the coil coil region (ZmPUB62 and Zm-PUB8), and one gene to the KAP domain (ZmPUB49) ( Table 1 and Table S1). The addition of new domains to the duplicated genes may have introduced diversification of function to PUB genes. The maize PUB gene family was predicted to harbor GD events around 13-84 Mya and 129-215 Mya (Table 1). Based on our analysis, there were approximately 0.05-0.99 synonymous substitutions per site, which indicated that the maize PUB gene family evolved under purifying selection, whereby deleterious mutations were eradicated while the core functional domains were conserved [51]. In addition, the maize PUB genes displayed a stronger association with rice than Arabidopsis ( Figure 5). This suggested that rice is a more suitable reference for the functional study of the maize PUB gene family.
Cis-acting regulatory element analysis identified 94 kinds of elements in the promoter sequences of the maize PUB gene family. These were 19 hormone-response elements, 46 biotic-and abiotic-response elements, 13 metabolism-and development-related elements, and 13 unknown function elements (Tables S4 and S5). Similar element patterns were reported in tomato, citrus, and Medicago, which suggested a common relationship between PUB genes and stresses as well as hormonal and developmental modulatory mechanisms in plants [17,18,52].
Emerging evidences revealed that the PUB genes played essential functions in modulating plant development and in developing tolerance to abiotic stresses such as cold, heat, salt, and drought [53][54][55][56]. Using available transcriptome data and RNA-seq analysis under various treatments, we examined the expression profile of the maize PUB genes in this research. Our work could provide new clues to the biological function of PUB genes in maize.
The PUB genes were known to regulate development of roots, stems, leaves, flowers, and fruits in plants [17,57]. Herein, we identified different expression profiles of maize PUB genes during the root development (Figure 7, Table S6). In total, 10 genes (ZmPUB4, ZmPUB8, ZmPUB16, ZmPUB23, ZmPUB27, ZmPUB35, ZmPUB43, ZmPUB49, ZmPUB66, and ZmPUB78) were highly expressed at certain stages of root development (FPKM > 10). Further analysis revealed that GA, IAA, and meristem expression-related elements were present in the promoter of these genes (Tables S4 and S5). The root apical meristem-specific genes played essential roles in root development during early somatic embryogenesis [58]. IAA strongly promoted the development of adventitious roots, lateral roots, root hairs, and primary roots. GA determined cell growth polarity in the root cortex of maize [59][60][61]. It is suggested that these genes participate in root morphogenesis. Six highly expressed genes were observed during leaf development, and they were ZmPUB43, ZmPUB44, ZmPUB53, ZmPUB54, ZmPUB62, and ZmPUB78, respectively ( Figure 8, Table S7). We screened the MeJA, JA, ETH, and light-response elements in the promoters of these genes (Tables S4 and S5). MeJA, JA, and ETH regulated plant growth. The enhanced endogenous MeJA levels induced morphological alteration within leaves in the transgenic soybean plants [62]. JA and ETH may positively regulate cotton leaf senescence [63]. Leaf is an important photosynthetic organ in plants, and light is known to modulate plant morphogenesis and the leaf ultrastructure [64]. The presence of MeJA, JA, ETH, and light-response elements provided evidence that these genes may be involved in leaf development. Similarly, seven highly expressed genes (ZmPUB3, ZmPUB4, ZmPUB8, ZmPUB23, ZmPUB24, ZmPUB43, and ZmPUB52) were spotted during seed development, four of which harbored the gliadin metabolic regulatory element (O 2 -site) in their promoter regions ( Figure 9, Table S8). Gliadin is the most abundant storage protein in maize seed, and it is critical for the nutritional profile of maize seeds [65]. Based on our gene expression analysis, these genes were ubiquitously expressed at 2-14 days of whole-seed development. This suggested that they may participate in seed development by modulating protein accumulation within the endosperm.
ZmPUB1 and OsPUB75 were homologous genes. OsPUB75 encoded a cytosolic RINGtype E3 ubiquitin ligase, which was a crucial negative modulator of abiotic stress. Under salinity and mannitol stress, OsPUB75 was transcriptionally repressed in Arabidopsis [66]. In maize, ZmPUB1 displayed an expression profile similar to OsPUB75, and it was downregulated by 26-83% under heat, salt, UV, drought, and ABA treatments (Tables S9 and S10). This suggested that ZmPUB1 may participate in the negative regulation of abiotic stresses and hormonal responses. ZmPUB27 was homologous with AtPUB16 and OsPUB3. In Arabidopsis, AtPUB16 participated in the GA pathway, which favored self-pollination [67]. In maize seedling, a similar ZmPUB27 up-regulation was observed at 3 and 12 h after GA treatment. This indicated that ZmPUB27 may contribute to the GA response. In rice, OsPUB3 was a positive regulator of cold stress, and its over-expression in transgenic plants showed enhanced tolerance to cold stress compared to wild-type plants [53]. ZmPUB27 expression was elevated under conditions of cold, drought, and UV, and it was reduced after heat and salt treatment. This suggested that ZmPUB27 was involved in abiotic stresses. ZmPUB30 and ZmPUB57 were a pair of duplicated genes, and they were homologous to OsPUB5, At-PUB18, and AtPUB19. In rice, OsPUB5 encoded a copper (Cu) transporter 6 protein, which was highly expressed in the node, and it blocked the cadmium (Cd) upward transport [68]. AtPUB18 and AtPUB19 were positively regulated by ABA and salt, and their double mutants could not respond well to ABA and the salt-based suppression of seed germination relative to wild-type plants [69]. Under cold, heat, salt, and ABA treatments, ZmPUB30 and ZmPUB57 were up-regulated by 0.5-12.5 or 3.67-84.82 times. The conserved motif analysis revealed that ZmPUB57 contained more of motifs 2, 5, and 8 than ZmPUB30. This may greatly enhance the stress response ability of ZmPUB57. ZmPUB31 and ZmPUB45 were a pair of duplicated genes. Under cold, heat, salt, drought, UV, and ABA stresses, ZmPUB31 and ZmPUB45 were up-regulated by 0.19-3.8 and 3.1-36.6, respectively (Tables S9 and S10). MeJA and JA were naturally occurring physiologically active materials that respond to exoteric stimulations by transmitting stress signals and activating the stress-resistant genes in plants [70][71][72]. Compared to ZmPUB31, there were more JA and MeJA response elements in the promoter sequence of ZmPUB45 (Tables S4 and S5). This may enhance the responsive capacity of ZmPUB45 under environmental stress. OsPUB45 was homologous with Zm-PUB31 and ZmPUB45. In rice, OsPUB45 was up-regulated many-folds under salt, drought, and cold stresses [73]. We observed a similar expression pattern between ZmPUB45 and OsPUB45, which indicated that these two genes may possess comparable functions. Zm-PUB43 was the homologous gene of OsPUB8. In rice, OsPUB8 was markedly up-regulated during drought stress [74]. ZmPUB43 was up-regulated by 29-36% in the maize ovary and meristem under drought stress (Table S9). Further analysis revealed that there were significant alterations in expression of ZmPUB43 after salt, UV, and ABA treatments. ABA was critical for multiple biological processes, such as seed dormancy, germination, and adaptive responses to abiotic stresses [75,76]. This suggested that ZmPUB43 may contribute to abiotic stress responses via the ABA pathway. ZmPUB78 was homologous to AtPUB11. AtPUB11 was up-regulated under drought and ABA treatments, and its silent mutant was highly tolerant to drought stress compared to the wild type [77]. In maize, the expression levels of ZmPUB78 were up-regulated by 2.8-8.0 fold after ABA and drought treatments, which suggested that ZmPUB78 may negatively regulate the ABA-mediated drought response. In contrast, ZmPUB78 was down-regulated by the cold, heat, and salt stresses, thus indicating that ZmPUB78 may also be involved in muti-abiotic stresses. Above all, these results provided insights into the possible biological functions of the maize PUB gene family. Our extensive analyses aided in the selection of specific PUB genes for additional functional research so as to improve the genetic agronomic characteristics and strengthen environmental resistance within maize.

Multiple Alignments and Phylogenetic Analyses
Multiple sequence alignments were performed using the U-box domain sequences of the 219 PUB proteins (79 ZmPUBs, 77 OsPUBs, and 64 AtPUBs) using MEGA 7 [78]. A phylogenetic tree was generated using the neighbor-joining (NJ) method and the following parameters: Poisson correction, complete deletion, and 1000 bootstrap replicates, and visualization was done in the EvolView v2 software [79,80]. The Oryza sativa and Arabidopsis thaliana PUB protein sequences were obtained from the NCBI website (https://www.ncbi. nlm.nih.gov/ (accessed on 6 May 2022)), as reported in previous studies [12,13].

Chromosomal Mapping, Gene Duplication (GD), and Synteny Analyses
The gene lengths and position data were acquired from the maize genome (B73 RefGen_v4). Next, MapChart was employed for the construction of the chromosomal localization map [83]. To assess GD events, the coding sequences of PUB genes from Arabidopsis, maize, and rice were aligned in BLASTp using an E-value < 1 × e −10 cut-off. MCScanX was employed to assess GD events and examine syntenic association between different species, and visualization was performed in Circos [84,85]. The Kaks_Calculator computed the nonsynonymous (Ka) and synonymous (Ks) substitution rates of duplicated gene pairs, and the approximate time of GD was estimated as follows: T = (Ks/2λ) × 10 −6 , where λ = 6.5 × 10 −9 [86].

Cis-Acting Regulatory Element Analysis
We downloaded the 1500 bp upstream flanking sequences from the transcription start site of each ZmPUB gene. Then the cis-acting regulatory element analysis was carried out in PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 May 2022)) [87].
Zea mays cv. B73 was employed for the examination of gene profile in response to multiple hormonal treatments. All seeds were cultivated in commercial soil at 28 • C in a photoperiod of 16 h light/8 h dark. Three-leaf stage seedlings were treated with 100 µmol/L ABA, 100 µmol/L IAA, and 100 µmol/L GA in 0.1‰ Tween-20, respectively. The controls were treated with water in 0.1‰ Tween-20. The leaves were harvested at 0, 3, 6, 12, and 24 h after hormonal treatments. Three replicates of three plants were used in each treatment. All samples were flash-frozen in liquid nitrogen prior to storage in −80 • C until subsequent analysis.
Total RNA extraction employed Trizol (Invitrogen, Carlsbad, CA, USA). The quality of RNA was assessed via the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcripts were enrich-fragmented into small pieces, which were then converted to cDNA. The cDNA library was generated with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA), and sequencing was completed with Illumina Novaseq 6000 by Gene Denovo Biotechnology Co., (Guangzhou, China). To quantify gene abundance, we filtered reads and mapped the clean reads to the reference genome with the fastp, HISAT2. 2.4, and StringTie v1.3.1 softwares [89][90][91]. Finally, the FPKM (fragment per kilobase of transcript per million mapped reads) gene values were computed in the RSEM software [92].

Statistical Analyses
To compare the variation of gene expression, SPSS 12.0 (SPSS Inc., Chicago, IL, USA) was employed to carry out the least significant difference (LSD) test. Data processing and visualization were done with GraphPad Prism 5. Individual data were presented as mean ± standard error (SE) of three experimental replicates.

Conclusions
Herein, we conducted an extensive analysis of PUB genes in maize. We identified 79 ZmPUB genes, which were stratified into 7 categories. Each group exhibited similar exonintron structures and motif compositions. Using gene duplication and synteny analysis of PUB genes, we obtained important clues regarding the evolutionary profiles of maize PUB genes. PUB genes strongly regulated plant development as well as response to abiotic stresses and hormones. Our phylogenetic and gene expression analyses provided useful information for enhancing our comprehension of the biological roles of the PUB genes in maize.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11192459/s1, Table S1: Information of 79 ZmPUB genes (protein length, molecular weight, isoelectric point, CDS length, subcellular location, and domain structures); Table S2: Motifs of 79 ZmPUB genes; Table S3: Ten motifs identified in the maize PUB gene family; Table S4: Cis-regulatory element analysis in ZmPUB genes; Table S5: Information of the cis-regulatory elements; Table S6: Expression profile of ZmPUB genes in root development; Table S7: Expression profile of ZmPUB genes in leaf development; Table S8: Expression profile of ZmPUB genes in seed development; Table S9: Expression profile of ZmPUB genes in abiotic stress; Table S10: Expression profile of ZmPUB genes in hormone treatment. Data Availability Statement: All data analyzed during this study are included in this article and displayed in supplementary materials.

Acknowledgments:
We thank Liancheng Liu and Yunbin Wang for providing technical assistance in bioinformatics.

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

PUB
The U-box E3 ZmPUB The maize PUB SD Segmental duplication TD Tandem duplication GD Gene duplication UPS The ubiquitin proteasome system E1 The ubiquitin-activating enzyme E2 The ubiquitin-conjugating enzyme E3 The ubiquitin ligase enzyme HECT Homologous to E6-AP COOH-Terminus