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In this study, a total of 48 PUB genes were explored from the tomato genome and were renamed as SlPUB1-SlPUB48 based on their chromosomal positions from Chromosome 1−12 (Chr1−12). For these genes, we explored various gene properties including, chromosomal distribution, coding sequence (CDS) length (bp), and five different types of protein properties which include, protein length[26], molecular weight (MW) kDa, isoelectric point (PIs), grand average of hydropathicity (GRAVY), and subcellular prediction for each of PUBs protein (Dataset 1). The PUB genes in tomato were randomly distributed on the 1−12 chromosomes of the tomato genome, except none of the genes were detected on Chr8 and Chr10. The CDS length of SlPUB ranged from 831−3,141 bp (SlPUB32-SlPUB27), the protein length ranged from 276−1,046 aa (SlPUB32-SlPUB27). Similarly, the MW ranged from 31.77−118.442 kDa (SlPUB32-SlPUB14), the PIs 5.2−9.12 (SlPUB15-SlPUB2), and GRAVY −0.528 (SlPUB32) to 0.137 (SlPUB22). The observed variability among SlPUB genes specifically for GRAVY analysis revealed most of them were hydrophilic and only four genes i.e., SlPUB11, SlPUB7, SlPUB25, and SlPUB22 were hydrophobic having positive values. Also, the subcellular localization analysis predicted that the maximum PUB genes were involved in the nucleus, cytoplasm, chloroplast, plasma membrane, vacuole, and endoplasmic reticulum.
Phylogenetic characterization, motif and gene structure analysis of PUB genes in tomato
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To explore the evolutionary relationships, we constructed a neighbor-joining phylogenetic tree by utilizing the protein sequences i.e., 48 from tomato and 64 from Arabidopsis. The results revealed that PUB genes were clustered into seven subgroups (Fig. 1) and show the uneven distribution of SlPUB when compared with AtPUB. Notably, group V was observed with the most number of genes (29), followed by group VII and VI with eight and six genes respectively. Based on the group-wise comparison of SlPUB, group V and VI were scattered in the phylogenetic tree and showed relatively close genetic relationships with Arabidopsis.
Figure 1.
Phylogenetic relationships of the PUB genes between tomato and Arabidopsis. The seven different subgroups of PUBs are indicated in different colors.
To better understand the conservation and diversification among PUBs, we analyzed the conserved motifs and gene structure. The motifs analysis was explored by the MEME program, and motifs ranged from 1−10 for SlPUBs. The results showed that several motifs were widely dispersed, however, motifs 1 and 4 were highly conserved amongst almost all the members of PUBs, suggesting their common evolution and the rest of PUB members indicated slightly structural diversification among the SlPUB gene family (Fig. 2a). Again, the MEME server was explored for the PUB protein LOGOS of these motifs. Motif6 was found with the highest number (100) of consensus sequences, while motif4 with a fewest number (50) of sequences (Supplemental Fig. S1). The gene structure analysis showed similar patterns to the motif composition, most of the PUBs generally exhibited similar gene structure in terms of their lengths (Fig. 2b). However, two of them showed contrasting results such as SlPUB8 and SlPUB9. These results suggested that most of the PUBs carry similar features and slight differentiation in their sources of functions during the process of evolution.
Figure 2.
(a) Motif structures and (b) gene structures of PUBs in tomato. The different motifs (Motif 1 − Motif 10) are displayed in different colors. The gene structures of PUBs are based on the coding sequences (CDS) and untranslated region (UTR) which are shown in yellow and green.
Gene collinearity, duplication, and Ka/Ks analysis of PUB
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The chromosomal mapping of 48 PUB genes in tomato from Chr1-Chr12 was drawn by using TBtools software. All the PUB genes were distributed across the tomato genome except Chr8 and Chr10 as shown in Fig. 3. The distribution of the number of genes varies from chromosome such as the maximum amount of genes (7) was identified on Chr1 and Chr2, followed by Chr4 possessing six genes, while five of each gene were located on Chr5, Chr9 and Chr11. Thus, these results unveiled their uneven patterns among PUB members in the tomato genome. Collinearity relationships between PUB genes were also demonstrated by drawing circos using the TBtools software. Among SlPUB, high conservation of nucleotide was detected and a total of 11 pairs were detected as collinear with possible interaction with each other and another family. These collinear pairs include: SlPUB6-SlPUB15, Solyc01g099290-SlPUB38, SlPUB8-SlPUB9, SlPUB12-SlPUB17, SlPUB18-SlPUB25, SlPUB19-SlPUB26, SlPUB23-SlPUB46, Solyc06g008710-SlPUB38, SlPUB31-SlPUB43, SlPUB34-SlPUB45, and SlPUB41-SlPUB43. The two unknown genes (Solyc01g099290 and Solyc06g008710) were identified as cullins genes based on their domain analysis, and these genes are recognized for their vital role in the process of ubiquitination in combination with diverse cellular processes[31]. Additionally, the gene duplication investigation displayed that the maximum of the genes (26) were dispersed type followed by whole-genome duplication (WGD) or segmental (19), and tandem (3) as shown in Fig. 3.
Figure 3.
Chromosomal locations of the PUB genes in the tomato genome from Chr1-Chr12. The collinear genes are presented inside the circle in purple. The different types of duplication such as dispersed, segmental, and tandem are marked in red, green, and blue.
The synonymous with non-synonymous mutation analysis was also performed among the different types of gene duplications in tomato i.e., dispersed, tandem, and WGD or segmental. In the process of evolution, genes are typically associated with diverse kinds of selection pressure that include, purifying selection (Ka/Ks < 1), positive selection (Ka/Ks > 1), and neutral selection (Ka/Ks = 1)[32]. A possible combination of 23 pairs was selected for Ka/Ks calculation based on their duplication types i.e., 13 pairs of dispersed, one tandem, and nine pairs of WGD or segmental as described in Table 1. The results have shown that the maximum of gene pairs having a Ka/Ks ratio of less than 1.00, from three different types of duplication. These analyses signifying that purifying selection and reducing divergence occurred among them. Only three pairs of dispersed genes that include, SlPUB16-SlPUB22, SlPUB30-SlPUB32, and SlPUB36-SlPUB37 were perceived with greater than 1.00 values, suggesting positive selection.
Table 1. Gene duplications of PUB genes in tomato with outlier Ka/Ks values.
Gene 1 Gene 2 Ks Ka Ka/Ks Selection pressure Gene duplications SlPUB3 SlPUB4 0.74 0.59 0.80 Dispersed Purifying SlPUB5 SlPUB7 0.68 0.62 0.92 Dispersed Purifying SlPUB10 SlPUB11 0.65 0.59 0.90 Dispersed Purifying SlPUB13 SlPUB14 0.80 0.59 0.73 Dispersed Purifying SlPUB16 SlPUB22 0.58 0.61 1.06 Dispersed Positive SlPUB24 SlPUB27 0.70 0.68 0.98 Dispersed Purifying SlPUB28 SlPUB29 0.88 0.63 0.71 Dispersed Purifying SlPUB30 SlPUB32 0.44 0.45 1.03 Dispersed Positive SlPUB33 SlPUB35 0.88 0.62 0.70 Dispersed Purifying SlPUB36 SlPUB37 0.46 0.67 1.45 Dispersed Positive SlPUB39 SlPUB40 0.78 0.65 0.83 Dispersed Purifying SlPUB42 SlPUB44 0.72 0.53 0.73 Dispersed Purifying SlPUB47 SlPUB48 0.75 0.61 0.82 Dispersed Purifying SlPUB20 SlPUB21 0.22 0.11 0.52 Tandem Purifying SlPUB1 SlPUB6 0.63 0.46 0.74 WGD or Segmental Purifying SlPUB8 SlPUB9 0.65 0.05 0.07 WGD or Segmental Purifying SlPUB12 SlPUB15 0.74 0.61 0.82 WGD or Segmental Purifying SlPUB17 SlPUB18 0.76 0.65 0.86 WGD or Segmental Purifying SlPUB19 SlPUB23 1.08 0.55 0.51 WGD or Segmental Purifying SlPUB25 SlPUB26 0.76 0.66 0.87 WGD or Segmental Purifying SlPUB31 SlPUB34 0.77 0.62 0.80 WGD or Segmental Purifying SlPUB38 SlPUB41 0.79 0.61 0.77 WGD or Segmental Purifying SlPUB43 SlPUB45 0.80 0.64 0.80 WGD or Segmental Purifying Transcriptional analysis of PUB genes in multiple tissues and developmental stages in tomato
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Three major Illumina RNA-seq datasets of various multiple tissues were utilized from cultivated tomato (Solanum lycopersicum cv. Heinz) and the wild relative Solanum pimpinellifolium[33,34], and the Micro-Tom (MT) (unpublished data). The ten tissues from the cultivated tomato include bud, flower, leaf, roots, 1 cm, 2 cm, 3 cm, mature green, breaker, and breaker+ 10 fruits. From the wild relative, eight tissues and organs, which included anthesis flower (0DPA), 10 d post-anthesis (DPA) fruit, 20 DPA fruit, ripening fruit (33 DPA), mature leaves (ML), whole root (WR), young flower buds (YFB), and young leaves[30], were selected for analysis. The data from our unpublished RNA-seq data of the model variety Micro-Tom (MT) was also utilized for four different tissues at various stages such as root 30, 45, and 85 days post germination (DPG), stem 30, 45, and 85 DPG, leaf 30, 45, and 85 DPG, and flower 30 and 45 DPG. Several developmental stages like, 10 DPA (55 DPG), 20 DPA (65 DPG), immature green (IMG) at 75 DPG, mature green (MG) at 80 DPG, Br (85 DPG), Br3 (88 DPG), Br7 (92 DPG), Br10 (95 DPG) and Br15 (100 DPG).
To represent the tissue-specific expression of 48 SlPUB genes, a heatmap was generated on RPKM values. Results of Heinz and MT data showed an almost identical expression pattern and slight inconsistency with its wild relative such as SlPUB4-SlPUB19, SlPUB22, SlPUB24, SlPUB26, SlPUB28, SlPUB30-SlPUB39, SlPUB41-43, SlPUB47, and SlPUB48 significantly expressed in both of them (Figs 4a, 5a & 5b) and Dataset 2 and 3. While, the wild relative showed that SlPUB4-SlPUB17, SlPUB19, SlPUB22, SlPUB24, SlPUB26, SlPUB28, SlPUB30-SlPUB34, SlPUB36-SlPUB39, SlPUB41-43, SlPUB47 and SlPUB48 were significantly expressed entirely in the tissues, indicating their foremost contribution in tissue-specific response and development (Fig. 4b) and Dataset 4. The remaining PUB genes showed either very low, moderate, and even a few of them were not expressed in or several selected tissues, suggesting their slightly partial response in tomato.
Figure 4.
Expression profiling of the 48 differentially expressed genes in multiple tissues based on RPKM values, including (a) Solanum lycopersicum cv. Heinz and (b) the wild relative Solanum pimpinellifolium. The brown bars represent up-regulated genes and dark blue bars represent down regulation.
Figure 5.
Expression profiling of the 48 differentially expressed PUB genes from MT data based on RPKM values, including (a) different developmental stages and (b) different organs. Red bars represent up-regulated genes and blue bars represent down-regulation.
qRT-PCR analysis of PUB genes in different tissues, and their response to salt and cold stress
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The expression profiling of 30 PUB genes was randomly selected and then quantified by qRT-PCR in five different tissues including root, stem, leaf, flower bud, and flower (Fig. 6a). As shown in Fig. 6c, the same genes were tested in response to salt and cold stress, as well as control (CK). In most tissues, the majority of genes were highly expressed, and only a few genes were down-regulated such as SlPUB3, SlPUB23, SlPUB25, and SlPUB47. Similarly, findings showed that most PUB genes expressed distinctly in response to both stresses, implying that these genes can play a variable role in tomato plant resistance. When it came to salt stress, for example, most of the genes were significantly up-regulated, especially SlPUB10, SlPUB33, SlPUB30, SlPUB43, SlPUB5, SlPUB13 and SlPUB31. When compared to low temperature, SlPUB10, SlPUB43, SlPUB13 and SlPUB31 were significantly up-regulated. Several genes, including SlPUB3, SlPUB16, and SlPUB34, were down-regulated in both stress conditions. In response to both treatments, the rest of the genes showed either poor or moderate expression. Additionally, we also performed principal component analysis (PCA) to gain deeper insights into their involvement in different tissues and against abiotic stress. For different tissues, PCA analysis of PUBs transcripts showed 39.37% variation in PC1 and 31.06% variation in PC2, with 67.03% in PC1 accounting for 32.07% of total variation in PC2 for stress conditions (Fig. 6b).
Figure 6.
Relative expressions of SlPUBs (a) and (c) in five different tissues including root, stem, leaf, flower, and flower bud, and two stress conditions i.e., salt (200 mM) and cold stress (4 °C). The principal component analysis for (b) different tissues and (d) stress conditions.
SlPUB10 can effectively regulate tomato's tolerance to low temperature and salt stress
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Due to the relatively high expression of SlPUB10 in roots and leaves, and the significant increase in expression after low temperature and salt treatment, we conducted further research on this gene. The localization of protoplasts shown that SlPUB10 were expressed in the nucleus and cytoplasm (Fig. 7a). In the leaves of mock-treated plants, there was no genotypic effect on the activity of any of reactive oxygen species (SOD) or Peroxidase (POD) (Fig. 7b). This was not the case when the plants were challenged by salt and cold stress: here, the genotypic ranking for SOD and POD was OX>WT>RNAi when treated by salt or low temperature. In the salinity treatment, the activity of the two enzymes were higher than CK. The determination was consistent with the relative expression of SlPUB10 in transgenic lines and wild-type[24] (Fig. 7c). After salt or cold treatment, the overexpression lines of SlPUB10 did not show any difference from those before treatment, while RNAi lines showed more severe wilting than the WT (Fig. 7d), which suggested that SlPUB10 can enhance the salt tolerance and cold resistance of plants.
Figure 7.
Gene function verification of SlPUB10. (a) The proteins’ subcellular locazation in Arabidopsis protoplasts. (b) The activity of SOD and POD in leaves of wild-type (WT) and transgenic tomato lines after salt (200 mM NaCl) and cold treatment (4 °C) for 4 h. The P values indicate the results from pairwise comparisons of one-way ANOVA tests. Different letters represent a significant difference at P < 0.05. (c) The relative expression level of SlPUB10 in transgenic lines. (d) Phenotypes of WT and transgenic lines after 48 h of salt (200 mM NaCl) and cold treatment (4 °C).
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Herein, we identified 48 PUB genes in the tomato genome that were further characterized into seven different groups based on their phylogenetic analysis. We carried out a thorough genome-wide investigation search of the tomato genome for PUB genes identification by several bioinformatics tools, such as the physicochemical properties, gene structure, and motif composition, gene duplication, gene collinearity analysis, evolutionary rates, expression profiling, and functional evolution. Gene duplication analysis proposed mainly three different types among PUB genes i.e., segmental, tandem, and dispersed. Among these duplications notably, the dispersed genes contributed most to the expansion of PUB in tomato. The transcriptional analysis demonstrated significant changes specifically their likely diversification and fates of duplicated gene pairs during the process of evolution. The common fates among these genes were predicted to be involved during their functional or subfunctional conservation and neo-functionalization. These analyses, based on the PUB gene family, lay a solid basis for future investigation and their functional characterization in tomato.
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About this article
Cite this article
Liu G, Hu Q, Zhang J, Li E, Yang X, et al. 2023. Genome-wide identification of the plant U-box (PUB) gene family and their global expression analysis in tomato (Solanum lycopersicum). Vegetable Research 3:16 doi: 10.48130/VR-2023-0016
Genome-wide identification of the plant U-box (PUB) gene family and their global expression analysis in tomato (Solanum lycopersicum)
- Received: 28 November 2022
- Accepted: 13 March 2023
- Published online: 16 May 2023
Abstract: Plant ubiquitination plays an important role in protein post-translational changes that occur in a wide variety of eukaryotic organisms and regulate a broad range of biological processes. However, little is known about the evolutionary relationships, gene duplication, and functional evolution of PUB genes in tomato. Herein, we explored 48 PUB genes in the tomato genome which were further classified into seven major groups based on their sequences and relative genetic similarities with Arabidopsis. Gene structure analysis suggested that most of the PUBs carry similar features and with slight differentiation in their sources of functions during the process of evolution. Collinear analysis showed a high degree of conservation among SlPUBs, with a total of ten pairs identified as collinear with potential interactions with each other and another family. Based on their duplication forms, a total of 23 pairs were chosen for Ka/Ks calculation: 13 pairs of dispersed, one tandem, and nine pairs of WGD or segmental. The majority of the gene pairs from the three forms of duplication had a Ka/Ks ratio of less than 1.00, indicating purifying selection and reduced divergence after duplication. Only three pairs i.e., SlPUB16-SlPUB22, SlPUB30-SlPUB32, and SlPUB36-SlPUB37 were perceived by higher than 1.00 values, signifying positive selection. In addition, three major RNA-seq datasets analyses from the cultivated and its relatives, as well as Micro-Tom (MT), revealed their highly tissue-specific role, with the expression correlation analysis of duplicated pairs indicating highly putative neo-functionalization as compared to functional conservation or sub-functionalization after duplication. SlPUB10 was screened out and preliminary verified as a key gene in salt and cold stress response with validation of transgenic strains. These results are indicative of their response during the gene duplication and evolution process, while further functional validation step is required to determine their specific role.