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Article

Genome-Wide Identification, Characterization, and Expression Analysis of the U-Box Gene Family in Punica granatum L.

by
Lide Chen
†,
Dapeng Ge
†,
Yuan Ren
,
Yuying Wang
,
Ming Yan
,
Xueqing Zhao
and
Zhaohe Yuan
*
Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(2), 332; https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13020332
Submission received: 5 December 2022 / Revised: 13 January 2023 / Accepted: 18 January 2023 / Published: 22 January 2023
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
The ubiquitination pathway is essential for several developmental phases in plants, and the U-box protein family of ubiquitin ligases plays an important role in this process. However, little is known about pomegranate’s PUB genes. In this investigation, the pomegranate U-box gene family was identified using whole-genome sequencing data. We identified a total of 56 members of the pomegranate U-box family based on the U-box domain, and the PgPUBs were classified into four groups. Chromosomal localization, phylogenetic analysis, motif distribution, gene duplications, cis-acting elements, and expression profiling were also investigated. The PgPUB genes were unevenly distributed among the eight pomegranate chromosomes, and collinear duplicated genes were identified between the Arabidopsis thaliana genome and the Punica granatum genome. Furthermore, the gene expression analysis revealed that expression of U-box genes in pomegranate was induced by abiotic stressors. Collectively, our findings provide insight into the U-box gene family and will assist in understanding the functional divergence of U-box genes in Punica granatum L.

1. Introduction

Being sessile organisms, plants must adapt to changing environmental conditions to survive stressful challenges. Plants perceive and transmit internal or external signals, and post-translational modification of proteins exerts essential roles during these processes [1]. Protein post-translational modifications, such as phosphorylation, methylation, ubiquitination, and acetylation, have key but distinct roles in the regulation of protein stability and activity during different stages of plant development and plant–environment interactions [2]. Ubiquitin (Ub), a 76-amino-acid protein, is highly conserved among all eukaryotes [3]. Protein modification by Ub, also referred to ubiquitination, is catalyzed by three sequential ubiquitination enzymes composed of the Ub-activating enzyme (E1), the Ub-conjugating enzyme (E2), and the Ub-protein ligase (E3) [4,5]. E1 transfers ubiquitin to E2, E2 binds the ubiquitin, and E3 removes the ubiquitin molecule from E2 and attaches it to the target substrate, forming a covalent bond between ubiquitin and the target [6]. E3 ubiquitin ligases, one of the three enzymes involved in the ubiquitination process, play an important role in determining ubiquitination specificity and have been shown to function in various biological and abiotic stresses [7,8]. E3s can be classified into different types according to their structure, substrate specificity, and functions. These include HECT (Homologous to E6-associated protein C terminus) E3s, CRL (Cullin-RING ligase) E3s, RING (Really Interesting New Gene) E3s, and U-box ligases [9]. U-box type E3s are characterized by the presence of a specific U-box domain of about 70 amino acids. The U-box motif structure resembles RING-type E3 ligases, but lacks scaffold and zinc-chelating cysteine and histidine [10,11]. The U-box E3 ubiquitin ligase enzyme gene family has been identified in various plants. U-box gene family members have previously been identified—64 in Arabidopsis thaliana [12], 77 in Oryza sativa [13], 62 in Solanum lycopersicum [14], 64 in Medicago truncatula [15], and 125 in Glycine max [16]—indicating that U-box genes are widespread among plants.
In rice, the U-box type E3 ligase OsSPL11 plays a fundamental role in regulating the flowering mechanism, and the flowering time of OsSPL11 mutant plants was delayed under long-day conditions [17]. Furthermore, PUB13, the homologous gene of OsSPL11 in A. thaliana is involved in negative regulation during the flowering time [18,19]. U-box genes have been documented in the regulation of plant growth in response to different stimuli. Jasmonic acid (JA) inhibits root growth, and PUB10 seedlings were hypersensitive to this effect [20]. Many researchers have found that PUB proteins are involved in abiotic stress responses. AtPUB22 and AtPUB23 were found to play combinatory roles in response to drought stress by ubiquitinating cytosolic RPN12a in A. thaliana [21]. In rice, OsPUB15 can regulate cell death and ROS stress [22]. Zhang et al. reported that TaPUB1 overexpression could increase the salt tolerance of transgenic plants during both the seedling and full-grown stages [23].
The evidence presented above suggests that U-box proteins play important roles in plant responses to abiotic stresses. However, until now, U-box gene analysis has not been carried out in pomegranate. Pomegranate is a small deciduous tree or shrub native to Central Asia belonging to the Lythraceae family. It has a high economic value due to its edible fruits, ornamental flowers, and exocarp in Chinese medicines [24,25,26,27]. In the present study, pomegranate U-box genes were identified and analyzed. The findings of this study could improve our understanding of the U-box gene family and provide valuable information for the further functional analysis of U-box E3 ubiquitin ligase in pomegranate.

2. Materials and Methods

2.1. Genome and Transcriptome Data Sources

The genome-wide data (ASM765513v2) [28] and RNA-Seq data of pomegranate were retrieved from the NCBI database (Table 1). The U-box gene family data of A. thaliana were obtained from The Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/browse/genefamily/plantubox.jsp, accessed on 27 January 2021) [8].

2.2. Identification and Sequence Analysis of U-Box Gene Family

To identify the U-box protein family in pomegranate, the hidden Markov model profile for the U-box domain (PF04564) was retrieved from the Pfam database (version 33.1, http://pfam.xfam.org/, accessed on 4 February 2021). To identify candidate U-box sequences, we used HMMsearch in the HMMER program (version 3.3) with default parameters [32]. All candidates for U-box family members were further detected in the SMART (http://smart.embl-heidelberg.de/, accessed on 4 February 2021) and Pfam databases (version 33.1) [33,34] to eliminate repetition sequences and delete sequences without the U-box domain, confirming that each candidate protein sequence contains U-box domain. The MW (molecular weight), PI (iso-electric point), GRAVY (Grand average of hydropathicity), and number of amino acids of all U-box proteins were calculated through the ProtParam server (https://web.expasy.org/protparam/, accessed on 6 February 2021) with the help of a Python script to perform a batch computation (Python script: https://github.com/haha1s/ProtParam-batch, accessed on 4 February 2021) [35]. Moreover, the BUSCA (Bologna Unified Subcellular Component Annotator, http://busca.biocomp.unibo.it/, accessed on 8 February 2021) [36] web server was used to predict the subcellular protein localization.

2.3. Phylogenetic Analysis and Protein Motif Prediction

The 56 protein sequences of U-box family members were aligned using the MAFFT server (version 7, https://mafft.cbrc.jp/alignment/server/, accessed on 9 February 2021) with default parameters [37]. Phylogenetic analysis was performed by RaxML-NG software (version 1.0.0) with 1000 bootstrap replicates, and the best-fitting substitution model of sequence evolution was calculated with ModelFinder (integrated into IQ-TREE, version 1.6.12) [38,39]. ModelFinder identified LG + F + R5 as the best-fit model according to the Bayesian information criterion. The constructed phylogenetic tree was visualized using iTOL [40]. The MEME Suit program (version 5.1.1, http://meme-suite.org/tools/meme, accessed on 9 February 2021) was used to predict conserved protein motifs of the PgPUBs [41]. A total of 10 motifs and any number of repetitions were used to analyze with other default parameters. TBtools software (version 1.049) was used to illustrate the motif and domain diagrams [42]. Putative cis-acting regulatory DNA elements in U-box genes were identified in the 2 kb upstream region preceding the translation initiation site. Afterwards, promoter sequences were analyzed with the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 February 2021) [43] for the identification of cis-regulatory elements (ABRE, ARE, ERE, GARE motif, P-box, LTR, MBS, and TC-rich repeat).

2.4. Chromosomal Distribution and Gene Duplication

The chromosomal location information related to all 56 U-box genes was obtained from the annotation gff3 file. Finally, TBtools was used to generate the chromosomal distribution of pomegranate U-box genes. Potential duplicated pomegranate U-box genes were identified using the MCScanX software [44]. All pomegranate protein sequences were compared against each other using BLASTp, with e-value < 1e − 10 and five hits for a gene according to the manual of MCScanX. The synteny relationship of U-box genes between pomegranate and A. thaliana was also completed with MCScanX. Finally, the results were depicted using the TBtools software. KaKs Calculator (version 2.0) and ParaAT (version 2.0) software were used to calculate the non-synonymous substitution rates (Ka) values, synonymous substitution rates (Ks), and Ka/Ks ratios for duplicated U-box homologues with an MA (model averaging) method [45,46].

2.5. Gene Expression Analysis

The expression profiles of U-box gene family members in pomegranate were derived from the RNA-Seq data using Kallisto (version 0.44.0) software with the following parameters: -t 4 -b 50 [47]. Kallisto then generated TPM (transcripts per million) values of various genes, and the normalized value of the log2(TPM+1) was considered the expression data. The heatmaps were created using the TBtools software.

3. Results

3.1. Identification and Characterization of U-Box Gene Family

The identification of U-box genes in pomegranate was completed with the aid of the published genome. Finally, 56 U-box genes were identified/detected from the pomegranate genome (Table 2); these PUBs were renamed based on their location on the chromosome. The length of the PUB protein sequence in the pomegranate varied significantly, ranging from 284 aa (PgPUB55) to 1952 aa (PgPUB11), with an average length of 691 aa. The predicted molecular weight (MW) of the U-box gene family was between 32,078.43~219,216.64 Da, and the ranged of isoelectric point (PI) was from 4.91 (PgPUB2) to 9.08 (PgPUB44). The GRAVY values reflecting the hydropathicity of the proteins were less than 0 for 87% of U-box members, suggesting that most of the proteins are hydrophilic. There were some differences in the chemical and physical properties of the pomegranate U-box proteins. The difference in amino acids mainly caused these differences [48]. The subcellular localization analysis showed that pomegranate U-box proteins could be found in the chloroplast, endomembrane system, extracellular space, nucleus, and plasma membrane. Importantly, 73% of the pomegranate U-box proteins were present in the nucleus, indicating that U-box genes in pomegranate function in the nucleus.

3.2. Phylogenetic Analysis of U-Box Genes in Pomegranate

To investigate the evolutionary relationships among the 56 pomegranate U-box gene family members, the full-length of the U-box’s protein sequences were used to construct a phylogenetic tree (Figure 1 and Table S1). The U-box family in pomegranate was distributed into four classes according to the presence of domains other than the U-box domain. Only Class 1 members contained the U-box domain. PgPUBs of Class 2 possessed armadillo repeats. Class 3 contained protein kinase domain and Class 4 members contained other domains, including RING, S_TKc, TPR, and WD40. The largest group was Class 1 with 25 genes, followed by Class 2 with 20 genes, Class 3 with 3 genes, and Class 4 with 8 genes. Notably, 16 genes (64%) from Class 1 had a closer relationship, suggesting that these proteins originated from a common ancestor. The remaining 36% of U-box-only type members were distributed along with other classes. PgPUB33, PgPUB11, PgPUB10, PgPUB46, and PgPUB32 were found to be closely related to Class 2, while PgPUB26/30, and PgPUB16/24 shared significant similarities with Class 4 and Class 3, respectively. Surprisingly, PgPUB35 of Class 4 showed high distinct similarity with Class 1, unlike other Class 4 U-box genes clustered with its class group.

3.3. Conserved Motifs and Domain Organization Analysis

To gain greater insight into the structural features of pomegranate PUBs, the distribution of conserved motif and protein domain were analyzed. In total, 10 conserved motifs of U-box family members were identified (Figure 2B), and varied from 10 (Motif 5) to 50 (Motif 9) amino acids in length (Table S2). It is worth noting that all 56 PgPUB proteins contain Motif 1, suggesting that it was the main part of the U-box domain (Figure 2B). Notably, all the PgPUBs except for PgPUB30, PgPUB55, and PgPUB26 contained at least four motifs. In general, pomegranate U-box protein members grouped in the same cluster generally possessed similar motif compositions, indicating that they may have similar functions. For example, PgPUB18, PgPUB2, PgPUB3, PgPUB19, PgPUB24, PgPUB16, PgPUB4, and PgPUB25 were clustered into one subgroup, and they all had Motifs 8/9/6/10/1/5/3; respectively with the same motif distribution order. All PgPUB proteins contained one U-box domain, demonstrating a combination of the U-box domain and other domains, such as RING, S_TKc, TPR, and WD40. The U-box domain is distributed at different positions of the 56 identified protein sequences. PgPUB proteins present in one subgroup often share a similar position structure. PgPUB36, PgPUB31, PgPUB13, PgPUB21, PgPUB9, PgPUB31, PgPUB12, and PgPUB37 were in one subgroup, and the U-box domain of these proteins was located at the N-terminal, whereas some other PgPUB proteins had their U-box domain situated at the C-terminal (Figure 2C). Notably, U-box/ARM type proteins were found to contain at least three ARM repeats, and only one U-box/TPR type protein member existed, showing that PgPUB55 possessed a different function from others.

3.4. Cis-Acting Regulatory Element Prediction in Promoter Regions of U-Box Family Members

To understand the possible regulatory role of cis-regulatory elements in PgPUB genes, we analyzed the promoter regions of 56 pomegranate U-box genes. The eight selected stress-responsive cis-elements in the promoter regions of 56 PgPUB genes are shown in Figure 3, including the abscisic acid responsive element (ABRE), ethylene responsive element (ERE), gibberellin-responsive element (GARE-motif, P-box), the anaerobic induction element (ARE), low temperature responsive element (LTR), MYB-binding site (MBS), and defense and stress responsive element (TC-rich). Remarkably, the ABRE element was found in 50 PgPUB gene promoter regions, all except PgPUB33, PgPUB 56, PgPUB37, PgPUB34, PgPUB29, and PgPUB28. Furthermore, the PgPUB46 promoter region contains the maximum number of 13 ABRE motifs, followed by PgPUB40 containing 11 ABRE motifs, indicating that the expressions of PgPUBs were associated with the abscisic acid signal. Additionally, the TC-rich element was found in only 14 PgPUBs promoter regions, implying that the TC-rich element possesses a unique role in the function of these 14 PgPUBs. All PgPUB genes upstream regions except for PgPUB37 had at least two different cis-elements. The cis-element analysis illustrated that these regulatory elements in the PgPUBs promoter sequences are directly correlated with plant development and abiotic stress responses.

3.5. Chromosomal Distribution and Synteny Analysis of U-Box Family Members

A total of 56 genes were unevenly localized on eight chromosomes of pomegranate, with each chromosome containing 4–10 U-box members (Figure 4A). Chromosome 5/6 contained the largest number of U-box genes (10 U-box members), while Chromosomes 1 and 8 both had only four U-box genes. Additionally, Chromosome 1 had the longest length, while only four genes were found on it, presenting the lowest distribution density. Moreover, no positive correlation was detected between chromosome length and the number of U-box genes.
The collinear relations among the U-box genes were evaluated using the MCScanX program. A total of 11 duplicated U-box gene pairs comprised of 17 PgPUBs were located within collinear blocks on all pomegranate chromosomes (Figure 4A). PgPUB18, PgPUB27, PgPUB37, PgPUB4, and PgPUB9 all had two paralogous copies in the pomegranate genome, while the remaining PgPUBs contained only one copy. Furthermore, the collinear relationships between the orthologous U-box genes of pomegranate and A. thaliana were investigated to explore the potential evolutionary history’s clues (Figure 4B). A total of 28 PgPUB genes showed syntenic relationships with 41 AtPUB genes. In total, 46 pairs of orthologous PUB genes were identified between two species, and three PUB genes (PgPUB12, PgPUB25, PgPUB27) were found to be associated with three syntenic gene pairs, respectively, indicating that these genes may have played essential functions during evolution. Figure 4B also shows that multiple AtPUBs in A. thaliana only had one copy in pomegranate, except for AtPUB22, AtPUB23, AtPUB27, AtPUB28, and AtPUB33, of which two copies were found. To make further inferences of the evolutionary information of the pomegranate U-box gene family, the nonsynonymous (Ka) and synonymous (Ks) substitution ratio calculations were performed (Table S3). In general, the Ka/Ks value may illustrate positive selection (Ka/Ks > 1), neutral selection (Ka/Ks = 1), and purifying selection (Ka/Ks < 1) during evolution history. All duplicated pairs of PgPUB genes and orthologous U-box gene pairs had Ka/Ks < 1, indicating that the pomegranate U-box gene family in the P. granatum genome may have experienced intense selective purification pressure during long-term evolution.

3.6. Expression Patterns of U-Box Family

For the pomegranate U-box family expression analysis, the RNA-Seq data across five different tissues (flower, root, leaf, seed, fruit) were used. The TPM values represent the relative expression of each U-box genes (Table S4). The expression heatmaps were used to visualize the tissue-specific expression patterns of the U-box in pomegranate (Figure 5). It was noteworthy that the patterns of U-box gene expression were completely different in all five tissues. During flower development, most of the U-box genes from Cluster 1 exhibited an increased expression trend (Figure 5A). These up-regulated PUB genes in pomegranate might function in flower growth. FMF (functional male flowers) are female-sterile flowers that fail to set fruit and eventually drop. The U-box genes of Cluster 2 showed higher expression during the flower F1 stage (3.0–5.0 mm functional buds), indicating that these genes might be related to FMF formation (Figure 5A). U-box type E3 ligases were also demonstrated to act as regulators in seed germination and leaf senescence processes by mediating ABA signaling or biosynthesis [49]. Figure 5B shows that the Cluster 1 U-box genes had lower expression in the seed germination S1 stage, while these genes’ expression was up-regulated during the later periods (S3, S4 stages). These genes likely play a role in mediating seed germination. Differential expression between root and leaf was observed under salt stress. Most of the U-box genes in Cluster 1 showed an increased expression level in the roots of pomegranates, and multiple U-box genes from Cluster 2 were up-regulated in the leaves (Figure 5C). PgPUB16, PgPUB34, PgPUB13, and PgPUB5 showed higher expression patterns in L2 (6d NaCl stress), indicating that these genes probably had conserved functions in plant salt stress response pathways. Pomegranate is endemic to subtropical climates and fruits can develop chilling injury symptoms when exposed to unfavorably low temperatures [50,51]. In order to assess the role of the pomegranate U-box family in the responses to low temperatures, we surveyed the expression of PgPUB genes in pomegranate fruit at four different time points with published RNA-Seq data (Figure 5D). PgPUB19, PgPUB55, PgPUB54, PgPUB56, PgPUB39, PgPUB56, PgPUB35, and PgPUB6 were up-regulated under LTC (low-temperature conditioning) treatment. With 2 weeks of cold storage at 1 °C, PgPUB32, PgPUB38, PgPUB12, PgPUB22, PgPUB17, PgPUB37, PgPUB42, PgPUB24, PgPUB46, PgPUB27, PgPUB26, PgPUB 7, and PgPUB16 had obviously higher expression in pomegranate fruit (Figure 5D, C3). PgPUB1, PgPUB48, PgPUB11, and PgPUB25 were up-regulated under C4 treatment (LTC + 2 weeks at 1 °C). None of the four genes (PgPUB14, PgPUB44, PgPUB45, PgPUB9) showed expression patterns in the pomegranate fruit under different treatments. In summary, the present findings indicate that U-box proteins may play critical roles in abiotic stress response and plant development in pomegranate.

4. Discussion

Protein ubiquitination is an essential post-translational modification in eukaryotic cell biology. The E3 ubiquitin ligase is a crucial aspect of the ubiquitination cascade and regulates the ubiquitination of the substrates [52]. The U-box E3s are defined by a conserved U-box domain, which contains about 70 amino acids that are present from yeast to humans. The PUB gene family has been identified and analyzed in some plants [14,15,16]. However, systematic research on the U-box gene family in Punica granatum L is inadequate.
Pomegranate is an ancient fruit widely consumed worldwide fresh or as juice. A genome-wide analysis of the PUB genes in pomegranate would improve our understanding of gene expression and regulatory function in this gene family. In the present study, a total of 56 putative PUB genes in pomegranate were identified and characterized in detail (Table 2). The number of PgPUB genes in pomegranate was less than that in soybean and Chinese cabbage, possibly resulting from different genome sizes [5,16]. We also analyzed the physical and chemical properties of all 56 PgPUBs, providing necessary information about these PUB genes.
The classification of U-box proteins differs from that of the other gene families. It is based not only on U-box homology but also on domains other than the U-box domain present in these proteins [53]. In accordance with the previous study [14], we classified the 56 U-box proteins into four classes. According to the phylogenetic analysis of pomegranate PgPUB, among four classes, the U-box E3 family showed the closest phylogenetic relationship due to the identical domains.
The domain pattern analysis of the study revealed that all identified U-box proteins possessed a typical U-box domain. The U-box motif in pomegranate was found in combination with various domains, including ARM repeats, the Pkinase domain, WD40 repeats, and the TPR domain. We also found that the U-box-only type of PUB class was the largest subgroup, with 25 genes. Furthermore, motif composition analysis revealed that different conserved motifs were present in each pomegranate U-box protein. The pomegranate U-box family members belonging to the same clades usually share similar motif compositions and domain structures, indicating that they may have similar functions. The analysis showed the conservation in the evolution of the pomegranate U-box gene family. The cis-regulatory sequences are linear nucleotide fragments of non-coding DNA, which control gene expression at all the developmental stages [54]. By the plantCARE analysis [43], many cis-acting elements were found from the promoter regions of the PgPUBs. Eight selected cis-acting elements were visualized, showing the distribution difference of these elements in promoter regions. The analysis of cis-acting elements involved in the low temperature and abscisic acid responses proves that the PgPUB genes are related to abiotic stress responses in the pomegranate. Thus, these results will help further understand the various functional role of PgPUB genes under complex abiotic stress conditions.
According to the results of a chromosomal investigation, the U-box genes in pomegranate are spread unevenly across the genome. There were 4, 5, 8, 8, 10, 10, 7 and 4 U-box genes on Chromosomes 1–8, respectively. In the present study, we studied orthologous PUB gene groups between pomegranate and A. thaliana. Gene duplication was found to play a pivotal role in the expansion of the U-box gene family in pomegranate, as 11 paralogous gene pairs were identified in the PUB proteins of pomegranate. The Ka/Ks ratios of the 11 PgPUB pairs were < 1, indicating that this gene family had undergone purifying selection rather than positive selection, and suggesting that the PgPUBs were highly conserved.
Previous studies have reported that PUB genes play an essential role in the process of stress responses and development in plants [23,55,56]. The elucidation of U-box gene expression in different tissues of pomegranate could provide clues about their functions. Therefore, we performed an in silico analysis of 56 PgPUBs expression patterns using RNA-Seq data. It was found that the PgPUBs showed expression in different tissues of pomegranate. During flower development, the expression of 56 pomegranate U-box genes are not the same. PgPUB27 and PgPUB42 showed higher expression in the earlier stage of flower development, while they were down-regulated afterwards. We also found that PgPUBs were differentially expressed under seed germination, as well as in cold and salt stresses conditions.
Although the analysis of PgPUBs should be further explored, the preliminary genome-wide analysis of these genes provides insight into the potential functional roles of pomegranate U-box genes. The U-box E3 gene family is rarely studied in the pomegranate plant, and our analysis provides an overall contribution to knowledge of the U-box E3 gene family in pomegranate.

5. Conclusions

To our knowledge, this is the first comprehensive genome-wide analysis of the U-box gene family in pomegranate. Here, we identified 56 U-box genes in pomegranate, unevenly distributed on eight chromosomes. The presence of predicted conserved motifs and domains, chromosomal and subcellular localization, and synteny analyses all contributed to a better understanding of the structure and putative functions of PgPUBs. Our results showed that the U-box gene family of pomegranate was principally influenced by purifying selection. The expression patterns of the PgPUBs in different tissues were analyzed, providing valuable data/insight to understand the involvement of these genes in pomegranate growth. Our findings represent a foundation for the exploration of functional roles of U-box genes in pomegranate.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agronomy13020332/s1, Table S1: 56 PgPUB protein sequences, Table S2: Analysis of conserved motifs in PgPUBs, Table S3: Ka-Ks calculation of each duplicated pair of U-box genes, Table S4: List TPM values of U-box gene in pomegranate different tissues.

Author Contributions

Conceptualization, L.C., D.G. and Z.Y.; methodology, L.C. and Z.Y.; formal analysis, D.G.; investigation, D.G., Z.Y. and Y.R.; writing—original draft preparation, L.C. and D.G.; writing—review and editing, L.C., D.G., Y.R., Y.W., X.Z., M.Y. and Z.Y.; supervision, Z.Y.; funding acquisition, X.Z. and Z.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31901341) and the Priority Academic Program Development of Jiangsu High Education Institutions [PAPD].

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree analysis of 56 U-box genes in pomegranate using the maximum-likelihood method with 1000 bootstrap values. The numbers indicate the bootstrap values as a percentage of trees obtained from 1000 replicates. The different colors indicate different U-box classes.
Figure 1. Phylogenetic tree analysis of 56 U-box genes in pomegranate using the maximum-likelihood method with 1000 bootstrap values. The numbers indicate the bootstrap values as a percentage of trees obtained from 1000 replicates. The different colors indicate different U-box classes.
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Figure 2. An analytical view of the pomegranate U-box gene family. (A) Phylogenetic tree of the 56 PgPUB family members. (B) The conserved motifs in the PgPUB proteins; different colored boxes indicates different motifs. (C) Schematic representation of domain structures; different colored boxes indicates different domains.
Figure 2. An analytical view of the pomegranate U-box gene family. (A) Phylogenetic tree of the 56 PgPUB family members. (B) The conserved motifs in the PgPUB proteins; different colored boxes indicates different motifs. (C) Schematic representation of domain structures; different colored boxes indicates different domains.
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Figure 3. Cis-regulatory elements analysis of PgPUB family members. Promoter sequences (2000 bp) of 56 PgPUB genes analyzed by PlantCARE. The number in the box indicates the frequency of cis-elements in the promoter region.
Figure 3. Cis-regulatory elements analysis of PgPUB family members. Promoter sequences (2000 bp) of 56 PgPUB genes analyzed by PlantCARE. The number in the box indicates the frequency of cis-elements in the promoter region.
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Figure 4. Visualization of collinear U-box gene family. (A) Schematic representations for the chromosomal localization and interchromosomal relationships of pomegranate U-box genes. The red curve line indicates the U-box gene replications, with the gene name in red color and the gray curve lines indicate the collinear blocks among different chromosomes of pomegranate. (B) Synteny analysis of U-box genes between pomegranate and A. thaliana. The red lines highlight the syntenic U-box gene pairs between pomegranate and arabidopsis, with Pgchr and Atchr representing the chromosomes of pomegranate and arabidopsis.
Figure 4. Visualization of collinear U-box gene family. (A) Schematic representations for the chromosomal localization and interchromosomal relationships of pomegranate U-box genes. The red curve line indicates the U-box gene replications, with the gene name in red color and the gray curve lines indicate the collinear blocks among different chromosomes of pomegranate. (B) Synteny analysis of U-box genes between pomegranate and A. thaliana. The red lines highlight the syntenic U-box gene pairs between pomegranate and arabidopsis, with Pgchr and Atchr representing the chromosomes of pomegranate and arabidopsis.
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Figure 5. Expression profiles of pomegranate U-box genes in 20 transcriptome libraries. (A) Heatmap of PgPUBs in the pistil with different stages of flower development. (B) Heatmap of PgPUBs in four seed germination stages. (C) Heatmap of PgPUBs in the leaf and root under salt stress. (D) Heatmap of PgPUBs in fruits exposed to LTC (low-temperature conditioning) and cold storage.
Figure 5. Expression profiles of pomegranate U-box genes in 20 transcriptome libraries. (A) Heatmap of PgPUBs in the pistil with different stages of flower development. (B) Heatmap of PgPUBs in four seed germination stages. (C) Heatmap of PgPUBs in the leaf and root under salt stress. (D) Heatmap of PgPUBs in fruits exposed to LTC (low-temperature conditioning) and cold storage.
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Table
Table 1. Information of RNA-Seq data in pomegranate.
Table 1. Information of RNA-Seq data in pomegranate.
Accession
No.		Sample
Name		CultivarsSample
Type		NoteLibrary
Layout		InstrumentReference
SRR5446598B1‘Tunisia’Floral
pistil		3.0–5.0 mm
bisexual buds 		PAIREDIllumina HiSeq 2500[29]
SRR5446595B2‘Tunisia’Floral
pistil		5.1–13.0 mm
bisexual buds		PAIREDIllumina HiSeq 2500[29]
SRR5446592B3‘Tunisia’Floral
pistil		13.1–25.0 mm
bisexual buds		PAIREDIllumina HiSeq 2500[29]
SRR5446607F1‘Tunisia’Floral
pistil		3.0–5.0 mm
functional buds 		PAIREDIllumina HiSeq 2500[29]
SRR5446604F2‘Tunisia’Floral
pistil		5.1–13.0 mm
functional buds 		PAIREDIllumina HiSeq 2500[29]
SRR5446601F3‘Tunisia’Floral
pistil		13.1–25.0 mm
functional buds		PAIREDIllumina HiSeq 2500[29]
SRR11268902S1‘Tunisia’SeedGermination
stage1		PAIREDHiSeq X Ten
SRR11268897S2‘Tunisia’SeedGermination
stage 2		PAIREDHiSeq X Ten
SRR11268894S3‘Tunisia’SeedGermination
stage 3		PAIREDHiSeq X Ten
SRR11268891S4‘Tunisia’SeedGermination
stage 4		PAIREDHiSeq X Ten
SRR7187397R0‘Taishanhong’Root0d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR7187384R1‘Taishanhong’Root3d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR7187385R2‘Taishanhong’Root6d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR7187388L0‘Taishanhong’Leaf0d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR7187390L1‘Taishanhong’Leaf3d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR7187394L2‘Taishanhong’Leaf6d NaCl stressPAIREDIllumina HiSeq 4000[30]
SRR6024695C1‘Wonderful’FruitsImmediately after
harvest		PAIREDIllumina HiSeq 2000[31]
SRR6024697C2‘Wonderful’FruitsLTC treatmentPAIREDIllumina HiSeq 2000[31]
SRR6024699C3‘Wonderful’Fruits2 weeks of cold
storage at 1 °C		PAIREDIllumina HiSeq 2000[31]
SRR6024701C4‘Wonderful’FruitsLTC + 2 weeks
at 1 °C		PAIREDIllumina HiSeq 2000[31]
Table
Table 2. Characteristics of the U-box gene family in pomegranate.
Table 2. Characteristics of the U-box gene family in pomegranate.
Gene
Name		Protein IDChromosomal
Position		Size (aa)MW (Da)PIGRAVYSubcellular
Localization
PgPUB1XP_031377184.1chr1:2947864-–295232072979,787.026.65−0.25Nucleus
PgPUB2XP_031377001.1chr1:44172533–4418532277887,360.64.91−0.532Nucleus
PgPUB3XP_031402583.1chr1:44186135–4419228476085,622.544.94−0.57Nucleus
PgPUB4XP_031377406.1chr1:52940497–5294448480591,804.836.31−0.452Nucleus
PgPUB5XP_031384103.1chr2:3755632–375868864470,869.917.53−0.156Nucleus
PgPUB6XP_031384353.1chr2:10835456–108429731349149,031.65.82−0.174Chloroplast
PgPUB7XP_031382118.1chr2:14312179–1431726375684,350.436.74−0.317Nucleus
PgPUB8XP_031384418.1chr2:30479382–3048273162667,839.015.77−0.218Nucleus
PgPUB9XP_031379370.1chr2:39234572–3923640942046,459.3590.005Nucleus
PgPUB10XP_031386023.1chr3:6739180–674340374983,936.926.29−0.255Nucleus
PgPUB11XP_031386836.1chr3:7122525–71323371952219,216.65.85−0.309Extracellular space
PgPUB12XP_031389245.1chr3:11307289–1130897942145,666.868.850.081Nucleus
PgPUB13XP_031385069.1chr3:11584904–1158665139844,396.325.73−0.048Nucleus
PgPUB14XP_031385043.1chr3:25161277–2516287044549,557.26.26−0.143Chloroplast
PgPUB15XP_031386047.1chr3:38561364–3856479768775,352.045.46−0.3Nucleus
PgPUB16XP_031384546.1chr3:38592909–3859731082393,294.766.77−0.384Nucleus
PgPUB17XP_031389227.1chr3:39375519–393797221041114,621.76.11−0.196Nucleus
PgPUB18XP_031391630.1chr4:666345–67108374684,562.075.07−0.481Nucleus
PgPUB19XP_031391943.1chr4:674315–680627911101,269.15.79−0.35Chloroplast
PgPUB20XP_031392831.1chr4:6839162–684093346050,247.248.920.045Nucleus
PgPUB21XP_031394767.1chr4:17640750–1764271841844,633.426.460.162Nucleus
PgPUB22XP_031393056.1chr4:32045714–3204871271978,882.88.25−0.037Chloroplast
PgPUB23XP_031391114.1chr4:38020685–380263551011113,047.95.77−0.093Nucleus
PgPUB24XP_031392769.1chr4:38931551–3893667982793,788.416.1−0.45Plasma membrane
PgPUB25XP_031393410.1chr4:39793459–3979804681891,700.166.67−0.459Nucleus
PgPUB26XP_031398020.1chr5:1314957–13207031029116,6565.44−0.207Nucleus
PgPUB27XP_031395601.1chr5:2529317–253080341245,466.068.530.104Nucleus
PgPUB28XP_031398133.1chr5:2533727–253542943248,666.627.17−0.045Nucleus
PgPUB29XP_031395775.1chr5:5898712–590386982589,125.025.85−0.271Nucleus
PgPUB30XP_031396537.1chr5:6124869–613232159665,099.277.63−0.541Cytoplasm
PgPUB31XP_031397642.1chr5:6258825–626151045449,543.715.970.128Nucleus
PgPUB32XP_031397910.1chr5:7601217–760366968875,002.678.88−0.023Chloroplast
PgPUB33XP_031398091.1chr5:9932189–993500644448,413.655.49−0.106Nucleus
PgPUB34XP_031397089.1chr5:14587663–145925471014111,988.45.59−0.054Endomembrane system
PgPUB35XP_031395361.1chr5:28638947–2864086337240,265.738.950.067Nucleus
PgPUB36XP_031399446.1chr6:614826–61652041946,306.385.76−0.018Nucleus
PgPUB37XP_031402878.1chr6:885626–88747842646,705.746.07−0.019Nucleus
PgPUB38XP_031402110.1chr6:19025201–1902843265670,796.776.14−0.141Nucleus
PgPUB39XP_031402746.1chr6:21000873–210051281028114,180.26.27−0.179Chloroplast
PgPUB40XP_031401235.1chr6:22423862–2242627568976,335.558.46−0.008Chloroplast
PgPUB41XP_031400912.1chr6:23386124–2339079279388,102.575.67−0.349Nucleus
PgPUB42XP_031402432.1chr6:23903047–239069321030114,136.65.95−0.121Nucleus
PgPUB43XP_031401153.1chr6:24733246–2473595929432,684.98.92−0.074Chloroplast
PgPUB44XP_031400164.1chr6:25957839–2595940941646,219.59.08−0.077Nucleus
PgPUB45XP_031400178.1chr6:25963675–2596510041346,889.988.76−0.077Nucleus
PgPUB46XP_031407430.1chr7:1054126–105653969776,458.118.4−0.018Chloroplast
PgPUB47XP_031405927.1chr7:1171657–117404253258,012.458.2−0.217Chloroplast
PgPUB48XP_031403345.1chr7:3813240–381488144248,985.145.65−0.164Nucleus
PgPUB49XP_031404116.1chr7:11882839–1188464641546,318.058.95−0.042Nucleus
PgPUB50XP_031407385.1chr7:20996670–210030361468163,030.85.76−0.206Endomembrane system
PgPUB51XP_031407253.1chr7:25091236–2509398966474,079.525.64−0.258Nucleus
PgPUB52XP_031406559.1chr7:25178052–2518244481289,340.785.31−0.021Nucleus
PgPUB53XP_031373608.1chr8:2760493–276269368875,204.038.46−0.023Nucleus
PgPUB54XP_031373317.1chr8:24301646–2430417445850,457.088.02−0.206Nucleus
PgPUB55XP_031373647.1chr8:24309251–2431325128232,078.435.71−0.503Nucleus
PgPUB56XP_031407822.1chr8:26638009–2664069652757,628.747.08−0.239Nucleus
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Chen, L.; Ge, D.; Ren, Y.; Wang, Y.; Yan, M.; Zhao, X.; Yuan, Z. Genome-Wide Identification, Characterization, and Expression Analysis of the U-Box Gene Family in Punica granatum L. Agronomy 2023, 13, 332. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13020332

AMA Style

Chen L, Ge D, Ren Y, Wang Y, Yan M, Zhao X, Yuan Z. Genome-Wide Identification, Characterization, and Expression Analysis of the U-Box Gene Family in Punica granatum L. Agronomy. 2023; 13(2):332. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13020332

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Chen, Lide, Dapeng Ge, Yuan Ren, Yuying Wang, Ming Yan, Xueqing Zhao, and Zhaohe Yuan. 2023. "Genome-Wide Identification, Characterization, and Expression Analysis of the U-Box Gene Family in Punica granatum L." Agronomy 13, no. 2: 332. https://0-doi-org.brum.beds.ac.uk/10.3390/agronomy13020332

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