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Article

Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice

by
Wangmin Lian
1,†,
Xiaodeng Zhan
1,†,
Daibo Chen
1,
Weixun Wu
1,
Qunen Liu
1,
Yinxing Zhang
1,
Shihua Cheng
1,
Xiangyang Lou
1,
Liyong Cao
1,2 and
Yongbo Hong
1,3,*
1
China National Center for Rice Improvement, State Key Laboratory of Rice Biology and Breeding, Zhejiang Key Laboratory of Super-Rice, China National Rice Research Institute, Hangzhou 311401, China
2
Northern Rice Center, China National Rice Research Institute, Shuangyashan 155600, China
3
National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(5), 1035; https://doi.org/10.3390/agronomy14051035
Submission received: 13 March 2024 / Revised: 26 April 2024 / Accepted: 2 May 2024 / Published: 13 May 2024
(This article belongs to the Special Issue Innovative Research on Rice Breeding and Genetics)
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Abstract

:
Protein ubiquitination is common and crucial in cellular functions, however, little is known about how deubiquitinating enzymes (DUBs) reverse regulate the ubiquitination signaling process. PPPDE family proteins are a novel class of deubiquitinating peptidases with demonstrated deubiquitination/deSUMOylating activities. In this study, we identified 10 PPPDE genes from the rice (Oryza sativa L.) genome unevenly distributed on five chromosomes, where most of these members have not been reported to date. Based on the gene structure, the OsPPPDE family consists of three distinct subgroups within the phylogenetic tree. Cis-element analysis identified light/phytohormone-responsive, development, and abiotic stress-related elements in the promoters of OsPPPDE. Furthermore, we conducted and analyzed the transcript abundance of OsPPPDE under various tissues and stresses using the transcriptome data of 352 samples from the Rice Expression Database and GEO datasets. Moreover, OsPPPDE5 showed differential regulation of its transcript abundance during Cd and drought stress. Collinearity and syntenic analysis of 101 PPPDEs and PPPDE-like proteins in 10 plant genomes indicated that this family is evolutionarily conserved. Domestication analysis suggests that OsPPPDEs may contribute to indica–japonica divergence using the data from the 3K Rice Genome Project. Our study provides a foundation for further study on the function and molecular mechanism of the OsPPPDE gene family.

1. Introduction

The ubiquitin signaling network plays major roles in the regulation of cell cycle progression including RNA metabolism, pathogen response, and programmed cell death (apoptosis) in eukaryotic cells [1,2]. The ubiquitin signaling network is assembled by a triad of ubiquitin (Ub), ubiquitin-activating (E1), ubiquitin-conjugating (E2), ubiquitin ligase (E3), 26S proteasome (26S), and deubiquitinating enzymes (DUBs) [3,4]. Ubiquitination regulates subcellular localization, enzyme activity, and protein interactions, but target protein multiubiquitination is usually the signal for the ubiquitin hydrolysis of proteins [5]. DUBs that can remove ubiquitin tags from substrate proteins are an essential regulatory factor in the Ub-dependent pathway [6]. DUBs have four molecular roles: (1) Processing Ub precursors; (2) generating free Ub monomers from Ub precursors and Ub adducts as well as from the poly-Ub chains produced [7]; (3) recycling of Ub or Ub-oligomers from Ub–protein conjugates targeted for degradation [8]; and (4) preventing the degradation of pre-targeted proteins by releasing intact ubiquitin and targets to conjugates [9]. DUBs are carried out by a classical evolutionary conserved deubiquitinase superfamily known for countering the action of E3 ligases. In plants, all seven known DUB subfamilies have been identified [10], namely Ub-binding protease/Ub-specific protease (UBP/USP), Ub C-terminal hydrolase (UCH), Machado–Joseph domain-containing protease (MJD), ovarian-tumor domain-containing protease (OTU), zinc finger with UFM1-specific peptidase domain protease (ZUFSP), motif interacting with Ub-containing novel DUB family (MINDY), and the JAB1/MPN/MOV34 protease (JAMM) [11,12,13]. The regulation of ubiquitylation is the key to plant growth and development, in which the activities of ubiquitylating enzymes as well as DUBs determine the stability or function of the modified proteins [14]. UBP24 may have a crucial role in responding abscisic acid (ABA) signaling and the drought and salt of Arabidopsis (Arabidopsis thaliana) as ubp24 mutants have been shown to be hypersensitive to ABA and salt stress. In addition, UBP24 can form homodimers and cleave UBQ1 into Ub-monomers [15]. UBP8, in Magnaporthe oryzae, is involved in carbon catabolite repression, appressoria development, melanin formation, and the pathogenicity of M. oryzae [6]. OTLD1, which belongs to the OTU (ovarian tumor protease) deubiquitinase families, increases the cell size and stimulates plant growth due to the transcriptional repression of multiple major genes of Arabidopsis growth and development [16].
The PPPDE superfamily (after Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes) has been identified as putative deubiquitinating isopeptidases and is involved in de-Ub and/or deSUMOylation in most eukaryotic lineages [17]. The PPPDE domain consists of thiol peptidases with a circularly permuted papain-like fold. The secondary structure of PPPDE, which has an α + β fold with five conserved α-helices and six β-strands with the conserved histidine positioned at the N-terminus of strand 2, is similar to the classic papain-like proteases. PPPDE superfamily proteins have a strictly conserved polar residue at the C-terminus to orient the catalytic histidine [17,18]. The PPPDE superfamily is not only found in eukaryotes, but also in dsRNA viruses and a single-stranded DNA virus. Furthermore, phylogenetic tree analysis showed that PPPDE proteins can be divided into three families, in particular, the family 1 PPPDEs, which can be traced back to the last common ancestor of eukaryotes. The presence of PPPDE protein in the early stage of eukaryotic evolution suggests that it may target evolutionarily conserved proteins, and join in the regulation of some crucial cellular processes like other DUBs [17].
Although some researchers think that the function of the PPPDE domain might be partially redundant with that of other DUBs due to repeated gene loss events in evolution, recent studies have indicated that PPPDE is involved in cell cycle regulation, embryonic development, and apoptosis. DeSUMOylating isopeptidase 1 (DeSI1; previously described as PPPDE2), a member of the human and mouse PPPDE peptidase domain superfamily, catalyzed the deSUMOylation of BZEL [19]. In addition, PPPDE1 also has deubiquitinating and deSUMOylating activity [20]. The substrate proteins of PPPDE1 are ribosomal proteins S7 and RPS7, and PPPDE1 could mediate the ubiquitin chain editing of RPS7, stabilizing the RPS7 protein [21,22]. The catalytic domains of animal and plant DUB families show high homology, whereas the regions outside of the catalytic site can vary significantly [14]. In Arabidopsis, Desi1 and AtC3H59 interact in the nucleus to jointly regulate seed germination, seedling development, and seed development [23]. Although a recent study has reported that PICI1 stabilizes OsMETS to promote Met–ethylene biosynthesis, which contributes to broad-spectrum blast resistance [24], the function of OsPPPDE superfamily has still not been reported yet, except for PICI1. The objective of our research was to identify PPPDE genes and investigate the evolutionary pattern and functions of the PPPDE superfamily in rice through a whole genome data and annotation analysis.

2. Materials and Methods

2.1. Identification of OsPPPDE Amino Acid Sequences

Whole genome data and annotation of Oryza sativa Nipponbare were downloaded from the Rice Genome Annotation Project [25]. Hidden Markov model (HMM) analysis results of the PPPDE putative peptidase domain (NO. PF00145) were downloaded from the Pfam database [26]. HMMER 3.0 software [27] was employed to search for members of the OsPPPDE proteins with a cut-off E-value of 1 × 10−5. The obtained proteins were examined for the existence of the PPPDE domain using the SMART online software tool [28]. The molecular weights (MWs) and isoelectric points (pIs) of the members of OsPPPDE proteins were calculated with ExPASy online software [29]. The predicted subcellular localization of proteins were estimated using WolfPsort II [30].

2.2. Gene Duplication, Phylogenetic Relationship, Intron–Exon, and Conserved Motifs Analysis

The reference genome data of nine plants were acquired from Ensembl Plants (https://plants.ensembl.org/index.html) including Arabidopsis thaliana, Brachypodium distachyon, Glycine max, Hordeum vulgare, Oryza rufipogpon, Setaria italica, Sorghum bicolor, Triticum aestivum, and Zea mays. To reveal the collinearity of the OsPPPDE gene family, we used MCScanX to identify homologous gene pairs and syntenic relationships [31]. The location information of all OsPPPDE genes including the exons, introns, and UTR location information on the chromosome was available in the gff3 annotation file of the genome from the Rice Genome Annotation Project [25]. The results of syntenic analysis, chromosomal localization (Figure S1), and gene structure were diagramed using Tbtools [32]. The phylogenetic tree was built with PPPDE amino acid sequences by MEGA-X software (bootstrap for 1000 replicates) [33]. The result of the phylogenetic tree was pictured using ggtree [34]. The full-length protein sequences of the OsPPPDE were used to search for potential conserved motifs using the MEME [35] online webserver with the default parameters (Figure S2).

2.3. Cis-Acting Element Analysis of OsPPPDE Genes Promoter

To search the functional promoter regions, 2 kb sequences located upstream of all OsPPPDE genes were extracted to analyze the cis-element of genes. The PlantCARE database [36] was used to predict the cis-acting elements, which were visualized using gggenes and ggplot2 [37].

2.4. Prediction of Secondary and Tertiary Structures of Proteins

The SOPMA database [38] was used to predict the secondary structures, and the Alphafold2 database [39] was used to predict the tertiary structures. The PPPDE domain structure model (No. 2WP7) was obtained from the protein structure models of the Protein Data Bank (PDB). The 3D structure analysis of OsPPPDE proteins was conducted using PyMOL [40] (Figure S3).

2.5. Expression Analysis of PPPDE in Rice

Gene expression analysis of the rice PPPDE genes was conducted by utilizing the Rice Expression Database (RED) [41] and GEO datasets (GSE118355; GSE126961; GSE81906; GSE81108; GSE84800; GSE131641) and fragments per kilobase millions (FPKM) were downloaded. The rice morphology diagrams and heat map were visualized using gganatogram [42] and ggplot2 [37].
We constructed a co-expression network for the normal type and blast inoculation leaf by calculating the correlation among all rice expressed genes using Spearman’s correlation (Rs). We adjusted the p value with the FDR method for multiplicity and considered Rs > 0.7 and FDR < 0.01 as the significance cut-off point. The PCA results (Figure S4) of all 352 samples from the RED and GEO datasets showed that there was not a significant batch effect among the 30 RNA-Seq projects. The detailed experimental information is summarized in Table S1, and the expression abundance data without the batch effect is recorded in Table S2.

2.6. Domestication Analysis of OsPPPDE Genes

Single-nucleotide polymorphisms (SNPs) that are located at the flanking regions were identified in 3000 Rice Genome Project [43] (Table S3). Fixation index (FST), nucleotide diversity (π), and Tajima’s D were calculated using a sliding window approach by vcftools [44]. The distribution of FST and π was plotted in sliding windows of 2 kb with a 200-bp step size and the distribution of Tajima’s D was drawn in sliding windows of 2 kb. The linkage disequilibrium (LD) heat map (Figure S5) for OsPPPDE gene and flanking regions in five rice groups were visualized LDheatmap [45].

3. Results

3.1. Identification of OsPPPDE Genes from Rice Genome

To identify the OsPPPDE genes, amino acid sequences of the conserved PPPDE domain (PF05903) were examined through the whole rice genome using HMM. As a result, a total of 10 PPPDE genes were found; three of them had two transcripts. The OsPPPDEs were named as OsPPPDE1 to OsPPPDE10 according to their similarities with the PPPDE domain. Next, we collected more information on the OsPPPDEs including the chromosomal locations, the length of coding sequence (CDS) and amino acid (aa) sequence, the number of exons, the molecular weight (MW), and the isoelectric point (pI) (Table 1). The encoding protein length of these genes varied from 176 to 315 aa, and there was a big difference in their pIs, which ranged between 4.69 and 10.4. Noticeably, the majority (8/10) of them were annotated as ethylene-responsive element-binding protein, whereas the others were not. The OsPPPDEs were located on Chr2, Chr3, Chr4, Chr6, and Chr10. The syntenic relationship analysis showed that OsPPPDE2 and OsPPPDE3 were a collinear pair, which were localized on Chr4 and Chr6, respectively (Figure 1).
To deeply understand the evolutionary relationship of OsPPPDEs, we built the phylogenetic tree with the maximum likelihood method (ML) using amino acid sequences (Figure 2A). The members of OsPPPDEs were grouped into three groups; group I consists of OsPPPDE3, OsPPPDE3, OsPPPDE2, OsPPPDE4.1, OsPPPDE4.2, OsPPPDE1, OsPPPDE7.1, OsPPPDE7.2, OsPPPDE6; group II consists of OsPPPDE10 and OsPPPDE9; and group III consists of OsPPPDE5, OsPPPDE8.1 and OsPPPDE8.2.
We compared the DNA sequence of OsPPPDEs to analyze the composition of introns and exons; most of them contained two to five exons (Figure 2B), and OsPPPDE9 had the longest intron length. We also identified 10 conserved motifs (Figure 2C,D). Motif 1, motif 3, and motif 5 were related to the PPPDE domain, but others had no functional annotation, and motif 1 presented in all OsPPPDE genes.

3.2. Cis-Acting Element Analysis of OsPPPDE Genes Promoter

Since Cis-acting regulatory elements play a key role in the gene regulation of gene expression patterns in response to hormones and environmental stresses [46], we scanned potential cis-acting elements using the PlantCARE database in the upstream regions (2 kb) of OsPPPDEs (Figure 3). In total, forty-five cis-acting elements were predicated in all OsPPPDEs, which could be classified into four groups in light of their putative functions. Light responsive elements included 21 members, which was the largest subdivision including G-box (involved in light responsiveness) and Box 4 (part of a conserved DNA module involved in light responsiveness) as representatives. Regulatory elements related to phytohormone-response comprised 10 components, which involved methyl-jasmonate (MeJA), abscisic acid (ABA), gibberellic acid (GA), salicylic acid (SA), auxin, and flavonoid responsive elements, moreover, abscisic acid responsiveness element (ABRE) was found in eight OsPPPDEs. Core promoter elements such as TATA-box and CAAT-box were identified in all OsPPPDEs. The number of elements on the promoter of OsPPPDE10 was the largest, most of which (72/108) were TATA-box; the number of elements on the promoter of OsPPPDE2 was the least, and the greatest type (10/36) was CAAT-box. Additionally, the stress-related elements were also found in OsPPPDEs, these included drought, salinity, low-temp, and anoxic responsive elements. These results suggest that OsPPPDE genes are extensively involved in regulating different growth and developmental processes and phytohormone responses in rice.

3.3. Prediction of Secondary and Tertiary Structures of OsPPPDEs

Protein structures can provide valuable information that can be used to infer biological processes [47], so we utilized SOPMA and Alphafold2 to predict secondary structure distributions and the 3D structures of OsPPPDEs. All four secondary structure patterns (α-helix, random coil, extend strand, and β-turn) were found in the OsPPPDE proteins (Figure 4). The results revealed that all OsPPPDE proteins were mainly comprised of α-helix and random coil, and the sum of these two patterns accounted for between 68% and 85% of the full length of the protein. Only OsPPPDE5 and OsPPPDE10 had more α-helix than random coil, the rest had more random coil than α-helix. Most OsPPPDE proteins consisted of around 20% extended strands and 6% β-turns.
We further predicted the 3D structures of these OsPPPDE proteins using Alphafold2 (neural network based) modeling (Figure S3). The per-residue confidence score (pLDDT) of the PPPDE domain in the OsPPPDE proteins was high, above 90. The PPPDE domain was usually located at the start of the N-terminus of the OsPPPDEs protein, but it was located in the middle (after residue 40th) of OsPPPDE4, OsPPPDE6, and OsPPPDE7. Although structures other than the PPPDE domain could also be predicted such as α-helix on OsPPPDE5, OsPPPDE8, OsPPPDE9, and OsPPPDE10, most of the remaining pLDDTs were less than 50.

3.4. Expression Pattern and Co-Expression Network of OsPPPDEs

To comprehensively investigate the expression at the mRNA level of OsPPPDEs, we collected the RNA-Seq data of 142 normal treatment samples from RED and GEO datasets (see the “Section 2”). The expression levels of the rice PPPDE genes varied significantly in different tissues (Figure 5A and Figure S4B). Among nine tissues, the average OsPPPDE gene expression was the highest in the panicle, followed by the root, and the lowest was in the pistil (Figure 5A). Moreover, the variation in the expression levels was the largest in the leaves for all OsPPPDE genes compared to the other tissues (Figure S4B). OsPPPDE3, OsPPPDE6, and OsPPPDE10 exhibited a moderate expression level in the anther and panicle, whereas they were negligibly expressed in the leaf, shoot, and root. Additionally, OsPPPDE5 was expressed highly in most tissues, except aleurone. These tissue expression patterns suggest that OsPPPDEs played an influential role in rice development.
Furthermore, the expression patterns of rice PPPDE genes under 30 treatment conditions were also investigated from the RED and GEO datasets (Figure 5B). Results showed that the expressions of OsPPPDEs showed a difference under biotic/abiotic stresses, except for OsPPPDE3 and OsPPPDE10. For instance, OsPPPDE2, OsPPPDE5, and OsPPPDE9 had upregulated expression under drought stress. OsPPPDE5 was highly induced to express in cadmium (Cd)-treated shoots and roots. Both OsPPPDE7 and OsPPPDE9 significantly changed after blast inoculation. Furthermore, OsPPPDE5 was also strongly upregulated by phytohormones like ABA and GA. These results illustrate that the rice PPPDE genes play an important role in response to diverse abiotic stresses and phytohormone responses.
As OsPPPDE9 (PICI1) contributes to broad-spectrum blast resistance [24], we constructed the co-expression network of PPPDEs in two types of rice leaf, of which one was for normal leaves, and the other was after blast inoculation (Figure 5C). We built the co-expression network by calculating Rs and FDR among all of the rice expressed genes. There were 14,687 and 12,193 high-confident co-expressed gene pairs identified in the normal treatment leaf and inoculated leaf, respectively. Among them, a total of 9015 gene pairs were shared in two treatments. Taken together, our results suggest functional renewing of the PPPDE network in biotic stress.

3.5. Domestication of OsPPPDEs in Rice

To characterize the genetic variation of the OsPPPDE genes, 734 SNPs were identified in the 3000 Rice Genome Project [43]. As shown in Table S3, all members of the OsPPPDE genes had more than 50 SNPs in the gene and flanking regions except for OsPPPDE2, of which the SNP number was 13. The number of SNPs in the flanking region, both the 5′-flanking region and 3′-flanking region, was larger than that in the gene region. The promoter of OsPPPDE1 had the largest number of SNPs (59) compared to others, while OsPPPDE4 had the largest number of SNPs (49) in the 3′-flanking region. OsPPPDE2 and OsPPPDE7 had three and five SNPs in the 3′-flanking region, respectively, of which the number of SNPs was much smaller than the others. We also analyzed the LD pattern using 734 SNPs (Figure S5). This result showed that the OsPPPDE4 locus was a strong LD region in all subgroups, especially in japonica cultivars.
Moreover, we examined the evolutionary relationship by estimating the level of fixation index (FST), nucleotide diversity (π), and Tajima’s D (Figure 6). Pairwise measurements of FST showed that population differences were higher between japonica and indica in the OsPPPDE7 gene and flanking regions, but strikingly weaker differences were found at the OsPPPDE1 and OsPPPDE4 loci. The OsPPPDE3, OsPPPDE5, and OsPPPDE10 gene regions had low mean FST. The nucleotide diversity of five groups (japonica, indica, admix, ecotypes from Aus, Boro, and Rayada and aromatic varieties from Basmati and Sadri) was calculated (Figure 6B). All groups exhibited similar patterns of nucleotide diversity in all OsPPPDE genes. Nucleotide diversity of the indica cultivars was higher than that of japonica in most OsPPPDE genes except for OsPPPDE8, and a low π was detected of japonica and indica in OsPPPDE7 and OsPPPDE9. The trend in all OsPPPDE genes of Tajima’s D was similar to that of π (Figure 6C). Signals of artificial selection were found in all OsPPPDE genes. For instance, these had Tajima’s D of OsPPPDE9, which were positive in aus, bas, and admix accessions and negative in the japonica and indica cultivars.

3.6. Synteny Analysis, Phylogenetic Analysis, and Motif Composition of the PPPDEs in Ten Plants

To clarify the evolutionary relationship of PPPDE proteins, we traced the syntenic relationship between OsPPPDE and homologs in seven monocotyledons (brachypodium, barley, wild rice, millet, sorghum, wheat, maize) and two dicotyledons (Arabidopsis, soybean). In the above species genomes, nine, six, ten, nine, ten, twenty-two, twelve, four, and six homologous genes were detected successively (Figure 7). The results indicated that the relationship between OsPPPDEs and PPPDEs in wheat, wild rice, maize, and sorghum was close. OsPPPDE2, OsPPPDE3, and OsPPPDE6 showed the most homolog pairs with other plants. There were twelve, eight, five, and five homolog gene pairs identified between rice and wheat, maize, wild rice, and sorghum, respectively. These results imply that these genes may be conserved during evolution in the PPPDE gene family.
To further study the evolutionary diversification of PPPDE, we also constructed a neighbor-joining (NJ) tree of 101 PPPDE full length amino acid sequences from ten species using MEGA-X. All members of PPPDE could be divided into six groups labeled clade 1 to 6 (Figure 8A). In detail, OsPPPDE8.1, OsPPPDE8.2, and OsPPPDE5 were in clade 1, OsPPPDE9 and OsPPPDE10 were in clade 2, OsPPPDE6 was in clade 3, OsPPPDE7.1 and OsPPPDE7.2 were in clade 5, and OsPPPDE1, OsPPPDE2, OsPPPDE4.1, and OsFTIP4.2 were in clade 6. We also observed that compared to other monocots, in all clades, wild rice PPPDEs were closer to rice PPPDE proteins. These results indicate that OsPPPDEs originated from wild rice, and there were no obvious genetic differentiations.
We used the MEME program to predict 10 conserved motifs in 101 PPPDE proteins, and these predicted motifs were annotated with the InterProScan program (Figure 8B and Figure S2). Based on the motif analysis, all of the PPPDE proteins contained motifs 1, 3, 4, and 5, except for clade 1. Moreover, the protein in clade 1 included motif 7 and motif 8, while the others did not. Clade 2 also detected subgroup-specific motifs: motif 9 and 10. Motif 1, motif 7, and motif 8 were related to the PPPDE domain, but the others had no functional annotation. In addition, the orthologous gene showed similar motif compositions, indicating that there were no functional differences among them.

4. Discussion

The PPPDE superfamily, which is conserved in eukaryotes including humans, is a deubiquitinase (DUB). Members of this family have been identified as putative deubiquitinating isopeptidases and are involved in deubiquitination and/or deSUMOylation in mammals [21]. In previous research, PPPDE1 was overexpressed in hepatocellular carcinoma (HCC), which is a key modulator of the p53 protein and its downstream pathway [22]. However, the function of PPPDEs has been reported in Arabidopsis and rice, and the biological information in most crop species remains uncertain. AtC3H59 regulates cell division by interacting with the PPPDE family protein Desi1 through its WD40 domain [23]. The deubiquitinase PICI1 contains a PPPDE domain as an immunity hub for PTI and ETI in rice [24]. The PPPDE structure domain, therefore, may be involved in plant cell division and the immune response. In this study, ten members in OsPPPDE family were characterized.

4.1. The Gene Structure and Domains in OsPPPDEs Are Conserved

The gene structure analysis of the OsPPPDEs disclosed that these genes had multiple introns, with two to five (Figure 2B). However, another study asserted that plants retained intronless or less introns genes to manage stress conditions during evolution [48]. In our study, there were three genes that had intron retention due to alternative splicing. Intron retention also plays an significant part in the post-transcription of a variety of stressful conditions [49]. The PPPDE domains in PPPDE1 and OsPPPDE9 were mediated by the communication with other factors including p53 in humans and OsMETS [22,24]. PPPDE1 and OsPPPDE9 play an essential role in the ubiquitin signaling network. Only the amino acid sequences of the PPPDE domain were conserved among the rice PPPDE proteins (Figure 2B and Figure S3), and these results provide clues for analyzing the functions of other members of the OsPPPDE family. We found that there were collinearity relationships between OsPPPDE2 and OsPPPDE3, which means that the OsPPPDEs family has experienced gene expansion events driven by segmental duplication. Furthermore, phylogenetic analysis categorized all OsPPPDE proteins into three distinct lineages differing from the exon–intron association and motif arrangement (Figure 2). Both group Ⅰ and Ⅱ members in rice were the ethylene-responsive element-binding protein.

4.2. OsPPPDEs Are Universally Induced by Biotic and Abiotic Stress

Through the gene expression profiling of 352 rice samples from 30 projects, it was found that most OsPPPDE family members were differentially expressed in different treatments and regions (Figure 5). For example, OsPPPDE5 is highly induced in dry leaves and cadmium-treated roots [50]. OsPPPDE5 is a thioredoxin (TRX) family protein. The redox regulation of TRX is a specific control point for signal transduction pathways related to plant growth and stress response [51]. Therefore, OsPPPDE5 may play an important role in response to drought and cadmium stress.
Gene expression is regulated by the complex interaction of many cis-acting elements and trans-acting factors involved in various pathways [52]. Notably, promoter analysis of the OsPPPDE gene revealed the presence of various stress response elements such as light response, phytohormone response, cold response, defense and stress responses, and anaerobic inducible elements (Figure 3). In a previous study, OsPPPDE9 played a key role in broad-spectrum blast resistance mediated by the nucleotide binding domain and leucine-rich-repeat containing receptors (NLRs). The model of OsPPPDE9 deubiquitinate OsMETS mediates the methionine-ethylene cascade used in both PTI and ETI, which is ETI–PTI. Meanwhile, NLRs in the plant immune system such as PigmR protect OsPPPDE9 from effector-mediated degradation [24]. PPPDEs are likely to respond to abiotic and biological stresses by deubiquitination. The seven OsPPPDE promoters also included multiple MYB binding sites (MBS), suggesting that some of these may be regulated by MYB transcription factors. In this study, multiple OsPPPDEs were found to be upregulated under drought stress conditions (Figure 5B). OsPPPDE5, a thioredoxin protein, was significantly upregulated under Ca treatment, while the others were not. Pleiotropic effects of PPPDE on seed germination, seedling development, and seed development were found in previous studies [23]. Furthermore, we found that some meristem expression and cell cycle regulation elements were also found in some OsPPPDE gene promoters (Figure 3). The expression level of rice PPPDE was significantly different in different tissues and developmental stages. This suggests that the OsPPPDE gene may be involved in plant growth and cell differentiation.

4.3. Evolutionary Conservation of PPPDEs in Crops

Collinear analysis of PPPDEs and PPPDE-like proteins indicated that the number of PPPDE gene families was evolutionarily conserved, as many PPPDE orthologous pairs existed in the other nine crops and were similar in both monocotyledonous and dicotyledonous plants. The most orthologous pairs were found in wheat, with 22 pairs, suggesting that whole-genome duplication is extremely important for the expansion of PPPDE gene family members [53]. The researchers analyzed the PPPDE genes of Arabidopsis and found that AtPPPDEs can be divided into three groups, similar to our results [23]. In a multispecies phylogenetic tree, genes in a subgroup often have similar functions. In these studies, phylogenetic analyses of the PPPDE family in rice and other species were performed using their amino acid sequences. The results showed that the monocotyledonous and dicotyledonous orthologs clustered in the same clade (Figure 8A). MEME analysis revealed that 10 conserved motifs were identified in the PPPDE protein. The structures of PPPDE proteins in the phylogenetic group had similar motifs (Figure S2). Previous studies revealed that genes typically undergo tandem repeat events to amplify gene family members during the evolutionary process [54]. In this study, gene duplication was identified between OsPPPDE2 and OsPPPDE3 (Figure S1) as these two genes had collinear gene pairs with the other nine species (Figure 7). These results suggest that PPPDEs amplify gene family members by tandem repeat events in most crops, and that the PPPDE genes are more primitive than the divergence between monocots and dicots.

4.4. OsPPPDEs Alleles Confer Indica–Japonica Divergence

Asian cultivated rice is thought to have been domesticated from wild rice species such as Oryza rufipogon and Oryza nivara [43,55]. The genome of wild rice is more diverse than cultivated rice, suggesting that various wild rice species are valuable resources for rice genetic improvement. OsPPPDEs showed significant collinearity with those in wild rice. The amount and chromosomal location of the PPPDE protein in wild rice were similar to those in cultivated rice. All OsPPPDEs paired with homologous chromosomes in wild rice (Figure 7). One study showed that PICI1, a member of the rice PPPDE gene family, plays a role in indica–japonica domestication, and further found that the promoter of PICI1 in japonica had a higher transcriptional activity than indica after chitin treatment [24]. Our results suggest that selection signals are present in most members of the OsPPPDE family (Figure 6), indicating that OPPPDEs play a role in indica–japonica differentiation.
From the 3K Rice Genome Project and Rice Expression Database and GEO database, 10 OsPPPDE genes were identified and characterized using a variety of biological information software. OsPPPDE5 was related to drought tolerance, Cd and rice blast stress showed upregulated expression under comprehensive analysis (Figure 3A). Furthermore, three genes including OsPPPDE3/6/9 were downregulated, indicating that these genes may negatively regulate abiotic and biotic stresses. Taken together, these results provide valuable information in understanding the rice PPPDE gene family. It follows that we plan to combine a variety of rice SNPs datasets and use wet lab experiments to validate the PPPDE family functions to determine the deubiquitination function of various haplotypes under abiotic and biotic stresses.

5. Conclusions

This study investigated the genome-wide identification, structure, expression pattern, domestication, and evolution analysis of PPPDE genes in rice. The motifs of OsPPPDEs were highly conserved throughout the evolutionary history of rice. Most OsPPPDE genes were highly expressed in the panicle, roots, and leaves, and OsPPPDE5 may be involved in the response to drought and cadmium stresses. Selection signals were present in most members of the OsPPPDE family, suggesting that OPPPDEs may play a role in indica–japonica differentiation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14051035/s1, Figure S1: Syntenic analysis of OsPPPDE genes; Figure S2: MEME domain analysis of PPPDE genes in different plants; Figure S3: The 3D structure modeling of OsPPPDE proteins and PPPDE domain; Figure S4: The expression levels of the rice PPPDE genes; Figure S5: LD heat map for OsPPPDE gene and flanking regions in 5 rice groups; Table S1: The detailed experiment information from RED and GEO datasets; Table S2: The expression abundance data without the batch effect; Table S3: SNPs of of OsPPPDE genes and flanking regions.

Author Contributions

Conceptualization, X.L. and Y.H.; methodology, W.L. and Y.H.; software, W.L.; writing-original draft preparation, W.L. and Y.H.; writing-review and editing, X.L. and Y.H.; supervision, D.C., W.W., Q.L. and Y.Z.; project administration, S.C. and L.C.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The project was supported by the Nanfan special project, CAAS, (Grant Number: ZDXM2317; Grant Number YDLH2302), the China Agriculture Research System (Grant Number CARS-01-03), the Zhejiang Provincial Key Special Projects (Grant Number 2021C02063-1), and the Major Special Project of Guangxi Science and Technology (Grant Number AA23062015). We also acknowledge the Natural Science Foundation of China (31961143016, 31701338), the National Key Research and Development Program (2018YFD0100806), and the Strategic Science and Technology Innovation Cooperation Project of the Ministry of Agriculture and Rural Affairs (2020FYE0202300).

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflicts of interests and that our research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

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Figure 1. Chromosomal localization of the OsPPPDE genes in the rice genome. The left is the chromosome number. The right of the chromosomes marked the OsPPPDE genes. The scale bar shows the length of the chromosomes.
Figure 1. Chromosomal localization of the OsPPPDE genes in the rice genome. The left is the chromosome number. The right of the chromosomes marked the OsPPPDE genes. The scale bar shows the length of the chromosomes.
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Figure 2. Phylogenetic and structural analysis of the OsPPPDE genes and encoded proteins in rice. (A) Unrooted phylogenetic tree of OsPPPDEs. Group 1, group 2, and group 3 are displayed in blue, green, and grey colors, respectively. (B) Gene structure of the OsPPPDEs. Green shows untranslated regions (UTRs); yellow shows coding sequence (CDS); lines show introns. The scale bar estimates the length of the genomics DNA sequence of OsPPPDEs at the bottom. (C) Motif distribution of OsPPPDEs. Each motif is represented by a specific color. The scale bar at the bottom indicates the length of the motif protein sequence. (D) Motif sequences identified in the OsPPPDE proteins. The height of different amino acids represents the repeatability. The scale bar at the bottom represents the amino acid with a large proportion of each motif site.
Figure 2. Phylogenetic and structural analysis of the OsPPPDE genes and encoded proteins in rice. (A) Unrooted phylogenetic tree of OsPPPDEs. Group 1, group 2, and group 3 are displayed in blue, green, and grey colors, respectively. (B) Gene structure of the OsPPPDEs. Green shows untranslated regions (UTRs); yellow shows coding sequence (CDS); lines show introns. The scale bar estimates the length of the genomics DNA sequence of OsPPPDEs at the bottom. (C) Motif distribution of OsPPPDEs. Each motif is represented by a specific color. The scale bar at the bottom indicates the length of the motif protein sequence. (D) Motif sequences identified in the OsPPPDE proteins. The height of different amino acids represents the repeatability. The scale bar at the bottom represents the amino acid with a large proportion of each motif site.
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Figure 3. Cis-acting element distribution and its function in all OsPPPDE genes. (A) Distribution of cis-acting elements in upstream (2 kb) of the OsPPPDE gene family. The specific colored boxes show different promoter components. (B) Heat diagram of the cis-acting element. The numbers and colors in the grid indicate the number of components. The line at the bottom of figure divides elements into light responsiveness, phytohormone responsiveness, plant growth and development, and abiotic stresses.
Figure 3. Cis-acting element distribution and its function in all OsPPPDE genes. (A) Distribution of cis-acting elements in upstream (2 kb) of the OsPPPDE gene family. The specific colored boxes show different promoter components. (B) Heat diagram of the cis-acting element. The numbers and colors in the grid indicate the number of components. The line at the bottom of figure divides elements into light responsiveness, phytohormone responsiveness, plant growth and development, and abiotic stresses.
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Figure 4. Secondary structure of OsPPPDE proteins. (A) Percentage of each secondary structure relative to the full length of the protein. (B) Different rendering types of the secondary structure of OsPPPDE. The specific colors show four types: blue is α-helix, purple is random coil, red is extended strand, and green is β-turn. (C) Scores of four secondary structures on protein sequences. Color annotations are the same as before.
Figure 4. Secondary structure of OsPPPDE proteins. (A) Percentage of each secondary structure relative to the full length of the protein. (B) Different rendering types of the secondary structure of OsPPPDE. The specific colors show four types: blue is α-helix, purple is random coil, red is extended strand, and green is β-turn. (C) Scores of four secondary structures on protein sequences. Color annotations are the same as before.
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Figure 5. Global disruption of the OsPPPDE co-expression network in rice. (A) The rice morphology diagrams represent the average expression level of OsPPPDE genes in various tissues of rice, with red indicating a higher expression level and blue indicating a lower expression level. (B) Multiconditional expression level of OsPPPDE genes. Color annotations are the same as before. (C) Schematic representation of the OsPPPDE co-expression network in the normal treatment leaves (blue) and leaves after blast inoculation (red). Colored ovals indicate the number of co-expression pairs detected in normal leaves and leaves inoculated with blast.
Figure 5. Global disruption of the OsPPPDE co-expression network in rice. (A) The rice morphology diagrams represent the average expression level of OsPPPDE genes in various tissues of rice, with red indicating a higher expression level and blue indicating a lower expression level. (B) Multiconditional expression level of OsPPPDE genes. Color annotations are the same as before. (C) Schematic representation of the OsPPPDE co-expression network in the normal treatment leaves (blue) and leaves after blast inoculation (red). Colored ovals indicate the number of co-expression pairs detected in normal leaves and leaves inoculated with blast.
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Figure 6. Natural variation in OsPPPDE genes contributes to subspecies divergence. (A) FST of OsPPPDE genes and flanking regions between japonica and indica. The line at the bottom of the figure shows the gene structure: red is 5′UTR; green is CDS; blue is 3′UTR. (B) Nucleotide diversity (π) of OsPPPDE genes and flanking regions in japonica (GJ), indica (XI), and admix ecotypes from Aus, Boro, and Rayada (aus) and aromatic varieties from Basmati and Sadri (bas). (C) Tajima’s D values of the OsPPPDE genes and flanking regions in 5 rice groups.
Figure 6. Natural variation in OsPPPDE genes contributes to subspecies divergence. (A) FST of OsPPPDE genes and flanking regions between japonica and indica. The line at the bottom of the figure shows the gene structure: red is 5′UTR; green is CDS; blue is 3′UTR. (B) Nucleotide diversity (π) of OsPPPDE genes and flanking regions in japonica (GJ), indica (XI), and admix ecotypes from Aus, Boro, and Rayada (aus) and aromatic varieties from Basmati and Sadri (bas). (C) Tajima’s D values of the OsPPPDE genes and flanking regions in 5 rice groups.
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Figure 7. Syntenic analysis of PPPDE genes between rice and nine representative plant species. Gray line represents the collinear pair of genes of rice, the red line shows the collinear pairs of PPPDE genes. The gray lines at the bottom indicate the collinear blocks within the rice and other plant genomes. The red lines indicate the pairs of PPPDE genes. The results of the syntenic analysis between rice and the model plants including Arabidopsis, brachypodium, soybean, barley, wild rice, millet, sorghum, wheat, and maize.
Figure 7. Syntenic analysis of PPPDE genes between rice and nine representative plant species. Gray line represents the collinear pair of genes of rice, the red line shows the collinear pairs of PPPDE genes. The gray lines at the bottom indicate the collinear blocks within the rice and other plant genomes. The red lines indicate the pairs of PPPDE genes. The results of the syntenic analysis between rice and the model plants including Arabidopsis, brachypodium, soybean, barley, wild rice, millet, sorghum, wheat, and maize.
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Figure 8. Phylogenetic analysis of PPPDE proteins. (A) Evolution relationships among ten plant species. The tree was constructed with MEGA-X software using the neighbor-joining method (bootstrap = 1000 replicates). The tree divided the PPPDE proteins into 6 classes. (B) Conserved motif sequences in the PPPDE proteins of plants. MEME database was used to identify the motifs.
Figure 8. Phylogenetic analysis of PPPDE proteins. (A) Evolution relationships among ten plant species. The tree was constructed with MEGA-X software using the neighbor-joining method (bootstrap = 1000 replicates). The tree divided the PPPDE proteins into 6 classes. (B) Conserved motif sequences in the PPPDE proteins of plants. MEME database was used to identify the motifs.
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Table 1. Information of the OsPPPDE family genes identified in rice genome.
Table 1. Information of the OsPPPDE family genes identified in rice genome.
Common NameRGAP-IDRAP-IDChrCDS Length (bp)Exon NumberProtein Length (aa)Protein MW (Da)pIPredicted Subcellular LocalizationAnnotationRef.
OsPPPDE1LOC_Os06g36490.1Os06t0560400-01Chr.6651421723,557.165.34cyto, nuclethylene-responsive element-binding protein
OsPPPDE2LOC_Os04g46290.1Os04t0548000-01Chr.4624420822,712.35.89cyto, nuclethylene-responsive element-binding protein
OsPPPDE3LOC_Os02g43840.1Os02t0655500-01Chr.2615420522,535.325.36cyto, chlo, nuclethylene-responsive element-binding protein
OsPPPDE4.1LOC_Os03g01130.1Os03t0100900-01Chr.3753425126,965.14.89mito, chlo, nuclethylene-responsive element-binding protein
OsPPPDE4.2LOC_Os03g01130.2Os03t0100900-02Chr.3738424626,491.624.97mito, chlo, vacuethylene-responsive element-binding protein
OsPPPDE5LOC_Os02g56900.1Os02t0814000-01Chr.2759325327,286.976.12cyto, chlo, nucl_plas, nucl, plas, mitothioredoxin family protein[24]
OsPPPDE6LOC_Os06g08360.1Os06t0182100-00Chr.6795226527,149.5710.4chloethylene-responsive element-binding protein
OsPPPDE7.1LOC_Os03g10200.1Os03t0198500-01Chr.3744424827,976.249.57nucl, mito, chlo, plasethylene-responsive element-binding protein
OsPPPDE7.2LOC_Os03g10200.2Os03t0198500-02Chr.3528317620,110.329.44mito, cyto_mito, chlo, nuclethylene-responsive element-binding protein
OsPPPDE8.1LOC_Os10g33350.1 Chr.10945531533,625.288.83mito, chlo_mito, chloendo-beta-N-acetylglucosaminidase
OsPPPDE8.2LOC_Os10g33350.3Os10t0472400-01Chr.10822427428,803.756.23cyto, nucl, cysk_nuclendo-beta-N-acetylglucosaminidase
OsPPPDE9LOC_Os10g38970.1Os10t0533900-01Chr.10741424727,221.356.53nucl, chlo, cyto, protolastsethylene-responsive element-binding protein[24]
OsPPPDE10LOC_Os06g01780.1Os06t0107000-01Chr.6822427429,485.424.69nucl, chloethylene-responsive element-binding protein
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Lian, W.; Zhan, X.; Chen, D.; Wu, W.; Liu, Q.; Zhang, Y.; Cheng, S.; Lou, X.; Cao, L.; Hong, Y. Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice. Agronomy 2024, 14, 1035. https://doi.org/10.3390/agronomy14051035

AMA Style

Lian W, Zhan X, Chen D, Wu W, Liu Q, Zhang Y, Cheng S, Lou X, Cao L, Hong Y. Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice. Agronomy. 2024; 14(5):1035. https://doi.org/10.3390/agronomy14051035

Chicago/Turabian Style

Lian, Wangmin, Xiaodeng Zhan, Daibo Chen, Weixun Wu, Qunen Liu, Yinxing Zhang, Shihua Cheng, Xiangyang Lou, Liyong Cao, and Yongbo Hong. 2024. "Genome-Wide Identification and Characterization of the PPPDE Gene Family in Rice" Agronomy 14, no. 5: 1035. https://doi.org/10.3390/agronomy14051035

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