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Article

Genome-Wide Identification and Expression Analyses of the PP2C Gene Family in Paulownia fortunei

by
Zhenli Zhao
1,2,
Peiyuan Zhang
1,
Minjie Deng
1,2,
Yabing Cao
1 and
Guoqiang Fan
1,2,*
1
College of Forestry, Henan Agricultural University, Zhengzhou 450002, China
2
Institute of Paulownia, Henan Agricultural University, Zhengzhou 450002, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(2), 207; https://doi.org/10.3390/f14020207
Submission received: 9 December 2022 / Revised: 18 January 2023 / Accepted: 18 January 2023 / Published: 21 January 2023
(This article belongs to the Special Issue Strategies for Tree Improvement under Stress Conditions)
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Abstract

:
We explored the composition and roles of the protein phosphatase 2C (PP2C) family in Paulownia fortunei. The genome P. fortunei harbored 91 PfPP2C genes, encoding proteins with 120–1107 amino acids (molecular weight range, 13.51–124.81 kDa). The 91 PfPP2Cs were distributed in 12 subfamilies, with 1–15 PfPP2Cs per subfamily. The number and types of conserved structure domains differed among PP2Cs, but the distribution of conserved motifs within each subfamily was similar, with the main motif structure being motifs 3, 16, 13, 10, 2, 6, 12, 4, 14, 1, 18, and 8. The PfPP2C genes had 2 to 20 exons. There were ABA-response elements in the promoters of 42 PfPP2C genes, response elements to phytohormones, and stress in the promoters of other PfPP2C genes. A covariance analysis revealed that gene fragment duplication has played an important role in the evolution of the PfPP2C family. There were significant differences in the transcript levels of some PfPP2C genes in P. fortunei affected by witches’ broom (PaWB) and after treatment with rifampicin and methyl methanesulfonate. PfPP2C02, PfPP2C12, PfPP2C19, and PfPP2C80 were strongly related to PaWB. These findings provide a foundation for further studies on the roles of PP2Cs in PaWB.

1. Introduction

Protein phosphorylation and dephosphorylation are important for the functional expression of proteins and are essential regulatory mechanisms in living organisms [1]. Protein phosphatases catalyze the dephosphorylation of phosphorylated proteins, thereby playing important roles in plants’ responses to abiotic stresses and hormones [2]. Protein phosphatases can be classified according to their substrates into protein tyrosine phosphatases (PTPs), serine/threonine phosphatases (STPs), and dual-substrate PTPs (DSPTPs) [3,4,5]. The STPs can be further divided into phosphoprotein phosphatases (PPP) and protein phosphatase metal-dependent (PPM) proteins according to their crystal structure [6]. The protein phosphatase 2C (PP2C) and phosphopyruvate dehydrogenase phosphatase (PDP) families are the two main PPM families [6]. PP2Cs are Mg2+- or Mn2+-dependent monomeric enzymes that are widely found in Archaea, bacteria, fungi, animals, and plants [7]. Compared with other organisms, plants tend to have more PP2C proteins [8]. Plant PP2C proteins have a specific structural pattern; most have a conserved catalytic region at the C-terminus, while the N-terminus is less conserved, with a variable-length extension region that contains sequences related to intracellular signaling, such as transmembrane and kinase interaction sequences [9]. PP2Cs are widely involved in physiological processes, such as abscisic acid (ABA) signaling and trauma signaling, plant growth and development, and plant disease resistance [10]. Arabidopsis thaliana has 80 PP2C gene family members in 12 subfamilies [11]. The PP2C proteins in subfamily A negatively regulate ABA signaling by binding to the ABA receptor proteins PYR/PYL/RCAR, thereby causing physiological responses, such as inhibition of germination and stomatal closure [12,13]. The AtPP2C proteins in subfamily B play a regulatory role in the mitogen-activated protein kinase (MAPK) pathway [14]. Members of subfamily C are involved in physiological processes such as plant flower organ development [15]. Members of subfamily D in A. thaliana are involved in regulating seed germination in the dark, seed growth, and the ABA signaling pathway by mediating the activity of the plasma membrane H+-ATPase in cells [16,17]. Less is known about the other subfamilies. However, various studies have shown that PP2Cs are also involved in biological responses under abiotic stresses; for example, AtPP2C31 and AtPP2CG1 negatively regulate the response to high salt and low temperatures, respectively, in A. thaliana [18,19].
Previous studies have shown that members of the PP2C family play important regulatory roles in responses to abiotic stresses, such as low temperature, high temperature, and drought, as well as in hormonal regulation. For example, gene microarray expression profiling and real-time fluorescence quantification analyses revealed that members of the subclades C, E, and G of the PP2C family in Vitis vinifera were up-regulated under stress conditions, while members of subclades A, D, F, H, and K were down-regulated [20]. In Broussonetia papyrifera, four members of the 18 BpPP2Cs tested were found to be up-regulated under low temperature (4 °C) [21]. Furthermore, two proteins showed increased phosphorylation levels at 6 h of the low-temperature treatment, demonstrating that PP2Cs are involved in the cold stress response in plants [21]. In Brachypodium distachyum, almost all BdPP2Cs were up-regulated under low-temperature stress (4 °C) [22]. In Oryza sativa, OsPP2C09 mediated ABA desensitization, which contributed to root elongation, under drought stress [23]. Under low-temperature, high-temperature, and drought conditions, 10, 8, and 9 PP2C genes respectively, were found to be continuously up-regulated in Poncirus trifoliata [24]. Six Phyllostachys heterocycla PP2C genes, including PH02Gene33357.t1 and PH02Gene38274.t1, were up-regulated under high-salt conditions (200 mmol·L−1 NaCl) and by ABA (100 μmol·L−1) [25]. In Dendrobium catenatum treated with 20% PEG-6000, 200 mmol·L−1 NaCl, 100 µmol·L−1 ABA, and 200 µmol·L−1 salicylic acid, DcPP2C5, DcPP2C5, DcPP2C5, and DcPP2C5 were up-regulated under drought and salt stress, and DcPP2C20, DcPP2C38, and DcPP2C56 were up-regulated in the roots under ABA and SA treatment [26]. These findings indicated that D. catenatum PP2Cs are not only involved in responses to abiotic stresses, but also in responses to hormones [26].
Paulownia fortunei is a deciduous tree in the genus Paulownia (family Scrophulariaceae) [27]. It is a source of timber and is planted for farmland protection in China [27]. It has fast growth, produces high-quality wood, and shows strong adaptability and resistance [27]. It contributes to alleviating timber shortages, improving the ecological environment, ensuring food security, and improving people’s living standards [27]. However, there are some serious problems in its production, such as the occurrence of witches’ broom disease (PaWB), which increases tree mortality, slows tree growth, and seriously affects the development of the Paulownia industry. Although the genome of P. fortunei has been sequenced [28], the members of the PfPP2C gene family in this species have not yet been reported. In this study, using the PP2C gene sequences from Arabidopsis thaliana as search queries, members of the PfPP2C gene family in P. fortunei were screened and identified using homologous alignment analyses. The genes and their encoded proteins were analyzed using a series of bioinformatic tools. Differences in gene expression between diseased and healthy P. fortunei seedlings were determined by analyses of RNA-Seq data. A preliminary investigation of the expression patterns of PfPP2Cs in P. fortunei under various stress conditions and in PaWB-affected plants reveal potential functions of the PfPP2C family, and provide a theoretical basis for exploring their roles in the development of PaWB.

2. Materials and Methods

2.1. Identification, Physicochemical Properties, and Prediction of Subcellular Localization of PP2C Family Members in P. fortunei

The sequences of Arabidopsis thaliana PP2C proteins were obtained from the TAIR database (https://www.arabidopsis.org/) (accessed on 24 May 2022). The P. fortunei genome database was searched for homologous protein sequences with high structural similarity to the Arabidopsis thaliana PP2C family using BlastP. The hidden Markov model (PF00481) file of the PP2C protein structural domain was downloaded from the Pfam database (http://pfam.xfam.org/) (accessed on 25 May 2022), and then used in Biolinux to search the genome of P. fortunei using hmmersearch. Candidate protein sequences were those with an e-value of ≤10−2. The candidate protein sequences of the PP2C family in P. fortunei were those that were detected in both the BlastP and hmmer analyses. The candidate protein sequences were verified by Pfam, and protein structural domains were identified using SMART (http://smart.embl.de/smart/batch.pl) (accessed on 26 May 2022) and CDD (https://www.ncbi.nlm.nih.gov/cdd) (accessed on 26 May 2022). The protein sequences without PP2C structural domains were removed to obtain the final set of PP2C family members in P. fortunei. The number of amino acids, isoelectric point, and molecular weight of putative PP2C proteins of P. fortunei were predicted using Expasy (https://web.expasy.org/protparam/) (accessed on 22 June 2022). Subcellular localization of the PP2C gene family members by using the Cell-PLoc 2.0 online tool (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) (accessed on 14 January 2023).

2.2. Chromosomal Localization and Phylogenetic Analysis of PP2C Genes in P. fortunei

The genome annotation files were downloaded from the P. fortunei genome database. Information about chromosome length and the location of PP2C genes on chromosomes was extracted from the P. fortunei genome annotation files using TBtools software. The Map MG2C online tool (http://mg2c.iask.in/mg2c_v2.0/) (accessed on 10 July 2022) was used to map the distribution of Paulownia PP2C genes on chromosomes. The PP2C protein sequences were downloaded from the A. thaliana genome website and the Poncirus trifoliata protein sequence file was downloaded from the citrus genome database (http://citrus.hzau.edu.cn/) (accessed on 19 July 2022). The amino acid multiple sequence alignment analysis of PP2C proteins from A. thaliana, P. trifoliata, and P. fortunei was performed using MEGA-X software, and the phylogenetic tree was constructed using NJ in MEGA-X software, with the bootstrap value set to 1000 and other parameters set to default values.

2.3. Conserved Structural Domains, Conserved Motifs, and Gene Structure Analysis of PP2C Family Members in P. fortunei

The online tool Pfam search (http://pfam.xfam.org/search#tabview=tab1) (accessed on 17 August 2022) was used to identify conserved structural domains in P. fortunei PP2C proteins, and the results were visualized using TBtools software. The conserved motifs of PP2C protein sequences were analyzed using the online tool MEME (https://meme-suite.org/meme/tools/meme) (accessed on 7 August 2022), with the number of motifs set to 20, and the results were visualized using TBtools software. The structure of each member of the PP2C gene family, based on its coding sequence, was analyzed online by GSDS (http://gsds.gaolab.org/) (accessed on 12 July 2022).

2.4. Analysis of Promoter Cis-Acting Elements and Covariance in Members of the PP2C Family in P. fortunei

TBtools software was used to extract the 2000-bp upstream sequence of the start codon of each PP2C gene as the promoter sequence. The cis-acting elements in the promoters of PP2C genes were detected using PlantCARE online software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 27 August 2022), and the results were visualized using TBtools software [29]. Fasta Satas, File Merge For MCScanX, Text Block Extract, and Filter tools in TBtools were used to obtain files with information about chromosome length and associations between gene family members of P. fortunei. Table Row Extract or the Filter tool was used to obtain gene ID display files. The Advanced Circos tool was used to conduct the P. fortunei PP2C gene family covariance analysis.

2.5. Expression Analysis of PP2C Genes in P. fortunei

The materials used to generate RNA-Seq data were healthy P. fortunei (PF) and infected P. fortunei (PFI) grown in the intelligent greenhouse of the Paulownia Institute of Henan Agricultural University. At the age of 3 months, seedlings with the same growth status were selected and treated with rifampicin (Rif) at 30 mg/L or with methyl methanesulfonate (MMS) at 20 mg/L. Leaves of five seedlings were randomly taken after 5, 10, 15, and 30 d of treatment, snap-frozen in liquid nitrogen, and stored at −80 °C. This experiment was conducted with three biological replicates, using healthy seedlings in normal culture and seedlings with PaWB as controls. Affymetrix GeneChip 16K gene IDs with identical sequences were retrieved using the PP2C nucleic acid sequence of P. fortunei as a probe, and then the RNASeq data for the PP2C genes of P. fortunei (PF and PFI) treated or not with Rif or MMS were extracted, log2-transformed using Excel, and used to generate a heat-map using TBtools software.

3. Results

3.1. Identification and Physicochemical Properties of the PP2C Family in P. fortunei

A total of 91 PP2C genes were identified in the P. fortunei genome. The protein sequences were extracted using TBtools software. Redundant sequences were manually deleted, and the remaining sequences were further validated using tools at the Pfam, SMART, and CDD databases. The 91 PP2C genes of P. fortunei were finally identified and named PfPP2C1 through PfPP2C91 (Table 1). The lengths of the putative proteins encoded by the PfPP2C genes ranged from 120 aa (PfPP2C61) to 1107 aa, with an average length of 432.39 aa; the predicted molecular weight ranged from 13.51 kDa to 124.81 kDa (average, 47.62 kDa); and the theoretical isoelectric point ranged from 4.60 to 9.51. In total, 20 of the PfPP2C proteins were predicted to be basic (including PfPP2C01 and PfPP2C90) and the remaining 71 were predicted to be acidic (including PfPP2C03 and PfPP2C47). Among the 91 PfPP2C proteins, 32.97% were predicted to be stable proteins, but the majority (67.03%) were predicted to be unstable. The theoretical instability index ranged from 42.32 to 94.39. The GRAVY values were all less than 0, ranging from −0.586 to −0.101, indicating that all 91 PP2C proteins were hydrophilic.

3.2. Prediction of the Subcellular Localization of PP2C Family Members of P. fortunei

Proteins are distributed throughout the cell to participate in physiological activities, and subcellular localization prediction can clearly show the respective protein prediction sites of PfPP2C gene family members (Table 1), thus inferring the functions of related genes. The predicted subcellular cellular localization results showed that the predicted sites of P. fortunei PP2C protein were in the nucleus, chloroplast, cytoplasm, mitochondria, and cell membrane (Figure 1). The protein prediction sites had 83.5% (76) of their members in the nucleus, followed by the chloroplasts (24) and the least number of members in the mitochondria (1). Members of the protein prediction site in the nucleus are distributed except for the K subfamily, and the distribution of members in the C, D, I, J, and L subfamilies reaches 100%. Members of protein prediction sites in chloroplasts are distributed in all but the D, I, J, and L subfamilies, with the K subfamily having a 100% distribution. Based on the results of the study, it can be inferred that most of the action sites of the PP2C gene family members of P. fortunei are in the nucleus and chloroplasts.

3.3. Chromosomal Localization of PP2C Genes in P. fortunei

The location of PP2C genes was mapped onto the chromosomes of P. fortunei (Figure 2). The PP2C genes were unevenly distributed on 19 chromosomes. Chromosome 11 had the highest number of PP2C genes (8), followed by chromosome Chr03 (7), and then Chr7, Chr18, Ch19, and Chr20 (6 on each). The lowest number of PP2C genes (1 gene) was on Chr12. The distribution of PP2C genes within the same chromosome was also uneven, with two or more genes forming gene clusters. The results of sequence and chromosomal localization analyses revealed that PfPP2C41 and PfPP2C42 encoded the same amino acid sequence but were located at different chromosomal positions. In general, there was no positive correlation between the length of a chromosome and the number of PP2C genes it contained. Most genes on the same chromosome did not belong to the same subclade in the evolutionary tree. These results suggested that different genes on the same chromosome may encode proteins with different functions.

3.4. Phylogenetic Analysis of Members of the PP2C Family in P. fortunei

Sequence analyses showed that the PP2C family genes in P. fortunei were poorly conserved at the N-terminal end, but strongly conserved at the C-terminal end, which contained common structural subdomains that are presumed to be functionally similar. To clarify the evolutionary relationships among PP2C family members in P. fortunei, 80 protein sequences of the A. thaliana PP2C family, 53 protein sequences of the P. trifoliata PP2C family, and 91 protein sequences of the P. fortunei PP2C family were used to construct a phylogenetic evolutionary tree using the neighbor-joining method with MEGA-X software. In the tree (Figure 3), the PP2C sequences from the three species were divided into 12 subfamilies, with those of Paulownia distributed among the 12 subfamilies. Subfamily E had the highest number of Paulownia PP2C genes (15 genes), followed by subfamily D (12 genes), while subfamily L had the lowest number of Paulownia PP2C genes (1 gene). The number of Paulownia PP2C genes in the other families ranged from 2 to 11. In general, all subfamilies A–L contained PP2C genes from A. thaliana, P. trifoliata, and P. fortunei. All three species showed similar distribution ratios of PP2C genes in the subfamilies, indicative of relatively consistent evolutionary relationships among A. thaliana, P. trifoliata, and P. fortunei.

3.5. Analysis of Conserved Structural Domains and Conserved Motifs of PP2C Family Members of P. fortunei

Analysis of the conserved structural domains revealed that all 91 PP2C protein sequences of P. fortunei contained conserved PP2C structural domains (Figure 4b). Among them, eight Paulownia PP2C proteins (PfPP2C16, PfPP2C45, PfPP2C52, PfPP2C69, PfPP2C74, PfPP2C81, PfPP2C83, and PfPP2C88) contained PP2C-2 structural domains; PfPP2C11 contained the most diverse conserved structural domains (PP2C, Pkinase, cNMP_binding, and PK_Tyr_Ser-Thr domains); and PfPP2C04 contained three conserved structural domains (PP2C, PK_Tyr_Ser-Thr, and Pkinase_fungal). The conserved structural domains of Yop-YscD_cpl and FHA were only present in PfPP2C48 and PfPP2C74, respectively.
We detected 20 conserved motifs in members of the PP2C family in P. fortune (Table 2), but the distribution of motifs differed significantly among subfamilies (Figure 4c). Among the 20 conserved motifs, motif 5 was only present in subfamilies C and D; motif 11 was only present in subfamily E; motif 20 was only present in subfamily H; motifs 9 and 17 were only present in subfamily D; and motif 19 was only present in subfamily C. This situation may be indicative of different functions of proteins in the different subfamilies. While each subfamily had unique motifs, subfamilies E, A, G, I, H, J, B, and F all contained the following motif structure: motifs 3, 16, 13, 10, 2, 6, 12, 4, 14, 1, 18, and 8; and the common motif structure in members of subfamilies C and D was motifs 3, 16, 7, 2, 6, 4, 14, 1, and 5. These findings indicated that many PfPP2C proteins share a high degree of similarity in the composition of their conserved motifs. PfPP2C27 and PfPP2C61 had a number of motifs missing compared with other members of the same subfamily, which may have resulted from sequence losses during tandem duplication of genes.

3.6. Structure of PP2C Genes in P. fortunei

To understand the structure of PfPP2C genes, their intron and exon composition was determined (Figure 5b). All the PfPP2C genes contained introns and exons in their sequences, with the number of exons ranging from 2 (in PfPP2C47, PfPP2C73, PfPP2C26, PfPP2C45, and PfPP2C70) to 20 (in PfPP2C23) and the number of introns ranging from 1 to 19. There were 15 exons 14 introns in PfPP2C11. In total, 36 PfPP2C genes (39.5%) contained four exons and 23 contained five exons. In total, 36 PfPP2C genes (39.5%) contained three introns and 22 contained four introns. These results indicated that the gene structure of PfPP2Cs is relatively well conserved. Apart from genes in subfamilies E and J, those in the other subfamilies contained similar numbers of exons and introns, with a difference of no more than three. For example, all twelve members of subfamily D had four exons and three introns except for PfPP2C27, which had five exons and four introns, and all four members of subfamily I had ten exons and nine introns. In addition, the exon distribution and sequence lengths of PfPP2C genes belonging to the same subfamily in the phylogenetic tree were not very different and somewhat conserved, suggesting that the genes within these subfamilies have similar functions.

3.7. Analysis of Cis-Acting Elements in Promoters of PP2C Genes in P. fortunei

We identified 20 cis-acting elements in the promoter regions of PP2C genes in P. fortunei (Figure 5c), including hormone-responsive elements, light-responsive elements, and stress-responsive elements. Among all the PfPP2C genes, 62.6% (57), 48.4% (44), 47.3% (43), 35.2% (32), and 33.0% (30) had methyl jasmonate (MeJA)-responsive, gibberellin (GA)-responsive, abscisic acid (ABA)-responsive, indole acetic acid (IAA)-responsive, and salicylic acid (SA)-responsive elements, respectively, in their promoter regions; moreover, 96.7% (88), 70.3% (64), 40.7% (37), and 56.0% (51) had response elements related to light regulation, anaerobic induction, meristem expression and abiotic stress/defense, respectively, in their promoter regions. A small number of PfPP2C gene promoters also contained specific response elements related to circadian rhythm regulation, cell cycle regulation, endosperm expression, wound response, zein metabolism, tissue growth, and development related to palisade mesophyll cell differentiation. These results suggested that members of the PP2C family of P. fortunei play important regulatory roles in responses to hormone induction, light regulation, and stress under adverse conditions.

3.8. Covariance Analysis of Members of the PP2C Family of P. fortunei

To explore the evolution of the PP2C family in P. fortunei, an intraspecific covariance analysis was conducted (Figure 6a). The results showed that 67 of the 91 PP2Cs in P. fortunei (74% of all PP2Cs in P. fortunei) were involved in 56 pairs of gene covariation events, suggesting that gene fragment duplication has played an important role in the evolution of the PP2C family in P. fortunei. The largest number of PFPP2C family members involved in covariance events was on chromosome Chr11 (six genes), followed by Chr3 and Chr19 (five genes each). To further elucidate the evolutionary relationships of PP2C genes between different species, a covariance analysis was performed on P. fortunei and A. thaliana (Figure 6b). We detected 98 pairs of gene covariation events between the two species, of which 54 AtPP2C genes (67.5% of all AtPP2Cs) and 67 PfPP2C genes (73.6% of all PfPP2Cs) were involved in gene covariation events. These results indicate a high degree of homology and similar evolutionary relationships between the PP2C family members of P. fortunei and A. thaliana, which have been highly conserved during evolution.

3.9. Expression Analysis of PP2C Family Members in P. fortunei

Analyses of RNAseq data from P. fortune infected (PFI) or uninfected (PF) with the mycoplasma that causes PaWB revealed that 80 of the 91 PfPP2C genes were expressed, and 11 were not (Figure 7a). The transcript levels of PP2C19, PfPP2C57, PfPP2C59, PfPP2C80, and PfPP2C81 significantly increased after the development of PaWB. A total of 10 PfPP2C genes showed decreased transcript levels after the development of PaWB in P. fortunei, among which PfPP2C02, PfPP2C12, and PfPP2C24 showed the most obvious decreases. The transcript levels of the remaining 66 PfPP2C genes, including PfPP2C38 and PfPP2C55, did not change significantly. After Rif treatment, 80 PfPP2C genes were expressed, while 11 PfPP2C genes were not (Figure 7b). As the Rif treatment time extended, the transcript levels of 5 PfPP2C genes, including PfPP2C02, PfPP2C12, and PfPP2C20, significantly continued increase, while the transcript levels of 19 PfPP2C genes, including PfPP2C19, PfPP2C52, PfPP2C69, and PfPP2C80, significantly continued to decrease. The transcript levels of 23 PfPP2C genes, including PfPP2C57 and PfPP2C68, showed no significant monotonous trend, and the transcript levels of the remaining 19 and 15 genes increased and then decreased and decreased and then increased, respectively. After MMS treatment, 78 PfPP2C genes had detectable transcript levels and 13 PfPP2C genes had no detectable expression (Figure 7c). Among them, eight PfPP2C genes, including PfPP2C02, PfPP2C12, and PfPP2C47, were up-regulated over time under MMS treatment. The transcript levels of PfPP2C19, PfPP2C52, PfPP2C69, PfPP2C80, and 14 other genes showed an overall decreasing trend, compared with their respective levels in the control. Another 10 genes showed an increasing and then decreasing transcript levels, and 25 genes showed a decreasing and then increasing transcript levels, while 21 genes did not show significant changes in transcript levels under MMS treatment
Summarizing the above results, PfPP2C19 and PfPP2C80 were up-regulated in P. fortunei affected by PaWB, but down-regulated by Rif and MMS treatments. PfPP2C02 and PfPP2C12 were down-regulated in in P. fortunei affected by PaWB and up-regulated by Rif and MMS treatments. These findings indicated that these genes play an important regulatory role in the development of PaWB. Further in-depth analyses of their roles will provide further insights into the molecular mechanism of PaWB.

4. Discussion

In plants, PP2Cs are an important class of protein phosphatases that regulate plant metabolism by catalyzing the dephosphorylation of phosphorylated proteins [30]. The number of PP2C family members varies widely among different plant species. For example, there are 80 PP2C genes in A. thaliana [11], 27 in V. vinifera [20], 86 in B. distachyum [22], 53 in P. trifoliata [24], 125 in P. heterocycla [25], 67 in Dendrobium catenatum [26], 90 in O. sativa [31], 122 in Vigna radiata [32], and 81 in Fagopyrum tataricum [33]. This suggests that the number of PP2C gene family members may be related to the genome size of the species, or may have changed during the course of evolution. In this study, we identified 91 PP2C family members in P. fortunei. The amino acid length, isoelectric point, and relative molecular weight varied widely among the putative PfPP2C proteins, and such variations may be related to their functional diversity. The chromosomal localization analyses revealed that the PP2C genes in P. fortunei are unevenly distributed on 19 chromosomes, with one to eight PP2C genes per chromosome. The distribution of PP2C genes was also uneven within the same chromosome, with two or more genes arranged in gene clusters. These patterns of chromosomal localization are similar to those of PP2C genes in P. trifoliata and V. radiata [24,32].
In the phylogenetic evolutionary analysis of PP2C genes in A. thaliana, P. trifoliata, and P. fortunei, the PP2C genes were divided into 12 subfamilies, each of which harbored P. fortunei PP2C genes. Consistent with this, the PP2C genes of A. thaliana and P. heterocycla are also distributed among 12 subfamilies [11,24]. Our results indicate that the PP2C genes of A. thaliana, P. trifoliata, and P. fortunei are similarly distributed among the 12 subfamilies, indicative of relatively consistent evolutionary relationships among these three species. Thus, the PP2C gene family has been conserved during evolution. We detected clear differences in the number and type of conserved structural domains among PfPP2C family members. Our results show that the cNMP_binding and Pkinase_fungal domains are conserved domains unique to PfPP2C11 and PfPP2C04, respectively. We also detected some variability in the distribution of conserved motifs among different subfamilies, and some similarities in the distribution of conserved motifs within each subfamily. Nearly 3/4 of PfPP2C proteins have the following motif structure: motifs 3, 16, 13, 10, 2, 6, 12, 4, 14, 1, 18, and 8. The distribution of conserved motifs in members of the A subclade of PPfPP2C is similar to that in members of the A subclade in A. thaliana. This high degree of affinity suggests that A subclade members in P. fortunei participate in the regulation of ABA signaling, similar to their counterparts in A. thaliana.
In terms of gene structure, PfPP2C genes have between 2 and 20 exons, similar to the members of the PP2C families in P. trifoliata and S. italica [24,34]. The type and number of cis-acting elements in the promoter region affect differential gene expression [35]. One of the most important roles of the PP2C gene family is in the regulation of ABA signaling [36]. In plants, subfamily A PP2Cs regulate early events in the ABA signaling pathway [37]. The involvement of PP2C proteins in this pathway varies among species, and among different organs and tissues of the same species [37]. For example, in A. thaliana, subfamily A PP2C proteins negatively regulate the ABA pathway, while AtPP2C-G1 positively regulates the ABA pathway and the response to high salt stress [38], and AtPP2C2 can significantly increase the response to ABA when overexpressed [39]. In this study, we detected cis-acting elements responsive to various phytohormones, such as MeJA, GA, ABA, IAA, and SA in the promoter regions of PfPP2C genes. In total, 7 of the 11 members of subfamily A contained ABA-responsive elements in their promoter regions, suggesting that subfamily A PfPP2Cs are involved in the regulation of the ABA signaling pathway. These results are consistent with those reported in studies on Zea mays and B. papyrifera [21,40]. Overall, 42 of the PfPP2C family members contain ABA-responsive elements in their promoter regions, and half of the PfPP2C genes have stress-responsive elements in their promoters. Therefore, we speculate that PfPP2Cs may regulate various physiological activities in plants via the ABA signaling pathway.
Previous studies have demonstrated that PP2Cs also participate in the disease resistance signaling pathway. When plants are attacked by fungi, bacteria, and viruses, various signaling molecules such as SA and JA are produced, and they trigger the expression of genes encoding components of the disease resistance response [30]. It has been suggested that PP2Cs may play a role in plant resistance to biological stresses, such as rust and powdery mildew [41]. In this study, we found that the expression of some PP2C genes in P. fortunei were significantly affected by PaWB and treatment with Rif and MMS. The expression of PfPP2C19 and PfPP2C80 increased during the formation of PaWB in P. fortunei, but decreased under Rif and MMS treatments, while PfPP2C02 and PfPP2C12 were down-regulated during the formation of PaWB, but up-regulated in response to Rif and MMS treatments. These findings suggest that these genes play an important regulatory role in the development of PaWB, but may have a complex regulatory network. Further research is needed to explore their roles in the development of PaWB.

5. Conclusions

At present, research on P. fortunei and its molecular biology lags behind that on other plants, and much less is known about the function of its PP2C proteins than about those of model plants, such as A. thaliana, O. sativa, and Glycine max. In this study, we analyzed P. fortunei PP2C family members to determine the chromosomal distribution, evolutionary relationships, and gene structure, and the conserved structural domains and conserved motifs in their encoded proteins. We also determined their transcript profiles before and after the development of PaWB, and identified four genes closely related to PaWB development (PfPP2C02, PfPP2C12, PfPP2C19, and PfPP2C80) among the 91 PP2C genes in P. fortunei. The results of this study provide a reference for future studies on the structure, function, and regulatory roles of the PP2C gene family in Paulownia, and provide clues about the PP2C proteins that may participate in the formation of PaWB.

Author Contributions

G.F. conceived and designed the experiments; Z.Z. and P.Z. performed the experiments and wrote the paper; M.D. and Y.C. contributed reagents and analyzed the data. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Academic Scientist Fund for Zhongyuan Scholars of Henan Province (grant 2018 [99]) and the National Key Research and Development Program (Grant No. 2016YFD0600106).

Data Availability Statement

Not applicable.

Acknowledgments

We thank Jennifer Smith for editing the English text of a draft of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Prediction of subcellular localization.
Figure 1. Prediction of subcellular localization.
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Figure 2. Chromosome mapping of PP2C family members in P. fortunei.
Figure 2. Chromosome mapping of PP2C family members in P. fortunei.
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Figure 3. Phylogenetic tree of the PP2C gene family in A. thaliana (At), P. trifoliata (Pt), and P. fortunei (Pf).
Figure 3. Phylogenetic tree of the PP2C gene family in A. thaliana (At), P. trifoliata (Pt), and P. fortunei (Pf).
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Figure 4. Subfamily grouping (a), conserved structural domain analysis (b) and conserved motif analysis (c) of PP2C gene family of P. fortunei.
Figure 4. Subfamily grouping (a), conserved structural domain analysis (b) and conserved motif analysis (c) of PP2C gene family of P. fortunei.
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Figure 5. Subfamily grouping (a), analysis of the gene structure (b) and cis-element (c) of the PP2C gene family in P. fortunei.
Figure 5. Subfamily grouping (a), analysis of the gene structure (b) and cis-element (c) of the PP2C gene family in P. fortunei.
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Figure 6. Collinearity analysis of PP2C gene families in different species: (a) Collinearity analysis of the PP2C gene family in P. fortunei; (b) Collinearity analysis of PP2C gene families in P. fortune and A. thaliana.
Figure 6. Collinearity analysis of PP2C gene families in different species: (a) Collinearity analysis of the PP2C gene family in P. fortunei; (b) Collinearity analysis of PP2C gene families in P. fortune and A. thaliana.
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Figure 7. Expression analysis of the PP2C gene of P. fortunei during the development of witches’ broom: (a) I is the P. fortunei seedling, II is the PaWB seedling; (b) III is the PaWB seedling, IV, V, VI, and VII are the PaWB seedlings treated with Rif (30 mg/L) for 5, 10, 15, and 30 days, respectively; (c) VIII is the PaWB seedling, IX, X, XI, and XII are the PaWB seedlings treated with MMS (20 mg/L) for 5, 10, 15, and 30 days, respectively.
Figure 7. Expression analysis of the PP2C gene of P. fortunei during the development of witches’ broom: (a) I is the P. fortunei seedling, II is the PaWB seedling; (b) III is the PaWB seedling, IV, V, VI, and VII are the PaWB seedlings treated with Rif (30 mg/L) for 5, 10, 15, and 30 days, respectively; (c) VIII is the PaWB seedling, IX, X, XI, and XII are the PaWB seedlings treated with MMS (20 mg/L) for 5, 10, 15, and 30 days, respectively.
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Table
Table 1. Physicochemical properties of PP2C family members in P. Fortunei.
Table 1. Physicochemical properties of PP2C family members in P. Fortunei.
Gene NameGene IDNumber of
Amino Acids		Molecular
Weight		Theoretical PIInstability IndexAliphatic IndexGRAVYPredicted Location(s)
PfPP2C01Pfo01g001610.129432,296.668.2440.8583.54−0.408Nucleus
PfPP2C02Pfo01g002750.147352,519.655.2248.9891.46−0.268Nucleus
PfPP2C03Pfo01g006380.139243,064.514.8140.5081.84−0.283Nucleus
PfPP2C04Pfo01g009870.165572,156.195.5831.2890.37−0.137Nucleus
PfPP2C05Pfo02g010590.136940,963.876.8932.0386.40−0.213Chloroplast/Nucleus
PfPP2C06Pfo02g010660.142646,080.745.8737.4793.31−0.133Chloroplast
PfPP2C07Pfo02g014240.127930,485.437.1242.2483.23−0.368Nucleus
PfPP2C08Pfo02g016010.138643,014.958.4848.7987.10−0.317Nucleus
PfPP2C09Pfo02g019750.139744,228.388.6645.2287.63−0.258Nucleus
PfPP2C10Pfo03g000530.137942,820.766.4449.3189.74−0.339Nucleus
PfPP2C11Pfo03g006870.11081119,853.485.0339.8789.16−0.207Cell membrane/Nucleus
PfPP2C12Pfo03g008490.155360,968.455.3353.0493.24−0.203Nucleus
PfPP2C13Pfo03g009450.129432,627.047.6747.4379.25−0.473Nucleus
PfPP2C14Pfo03g013130.137742,129.129.5146.1590.69−0.359Nucleus
PfPP2C15Pfo03g013680.163170,004.715.5038.1179.41−0.380Chloroplast/Nucleus
PfPP2C16Pfo03g015100.143348,321.815.2437.0982.66−0.379Chloroplast/Mitochondrion
PfPP2C17Pfo04g000480.139744,173.328.7244.8487.88−0.247Nucleus
PfPP2C18Pfo04g003840.128030,717.546.7635.4080.79−0.444Nucleus
PfPP2C19Pfo04g006590.142946,293.987.4939.1287.48−0.190Chloroplast
PfPP2C20Pfo04g006660.137241,415.266.4233.5685.43−0.252Nucleus
PfPP2C21Pfo05g000400.134938,472.544.7354.6991.35−0.101Nucleus
PfPP2C22Pfo05g003700.139743,555.965.2544.4183.73−0.188Nucleus
PfPP2C23Pfo05g003720.11107124,807.735.5646.7382.18−0.366Nucleus
PfPP2C24Pfo05g010690.140544,340.195.2564.3278.49−0.347Nucleus
PfPP2C25Pfo05g011250.166974,601.595.1542.6778.73−0.456Chloroplast/Nucleus
PfPP2C26Pfo06g004460.127030,030.466.7651.4389.59−0.260Nucleus
PfPP2C27Pfo06g004710.119622,216.768.9253.2994.39−0.143Nucleus
PfPP2C28Pfo07g001190.144948,913.547.1645.8476.88−0.407Nucleus
PfPP2C29Pfo07g005140.142245,245.838.3428.4888.06−0.159Nucleus
PfPP2C30Pfo07g009030.180188,560.945.2746.3774.59−0.481Chloroplast
PfPP2C31Pfo07g014460.129331,684.044.9336.3879.52−0.355Nucleus
PfPP2C32Pfo07g014670.135339,022.995.2034.3575.47−0.392Nucleus
PfPP2C33Pfo07g015080.128230,638.325.5049.4875.46−0.321Nucleus
PfPP2C34Pfo08g002420.134838,000.295.6949.5988.76−0.228Nucleus
PfPP2C35Pfo08g010120.134337,581.195.1739.8970.52−0.551Nucleus
PfPP2C36Pfo08g013880.155560,319.534.9740.9491.98−0.166Nucleus
PfPP2C37Pfo09g009830.152657,753.165.2641.7579.94−0.358Nucleus
PfPP2C38Pfo09g014660.128431,332.796.8440.2690.63−0.348Nucleus
PfPP2C39Pfo09g017020.128331,287.576.1432.4488.55−0.332Chloroplast/Cytoplasm
PfPP2C40Pfo10g007720.135838,635.146.2754.4984.41−0.159Chloroplast/Nucleus
PfPP2C41Pfo10g009060.142546,090.326.8359.0972.68−0.363Nucleus
PfPP2C42Pfo10g009070.142546,090.326.8359.0972.68−0.363Nucleus
PfPP2C43Pfo10g012720.139444,063.266.5543.7191.75−0.293Nucleus
PfPP2C44Pfo10g013980.149154,041.385.2341.9672.06−0.542Chloroplast/Nucleus
PfPP2C45Pfo11g000240.137942,063.305.0863.5383.43−0.275Nucleus
PfPP2C46Pfo11g001200.150554,945.554.7545.4588.22−0.202Nucleus
PfPP2C47Pfo11g001760.134938,194.484.6034.9080.37−0.296Nucleus
PfPP2C48Pfo11g002190.160165,992.206.3451.3691.70−0.155Chloroplast
PfPP2C49Pfo11g008040.138842,005.645.3256.6883.69−0.206Chloroplast/Cytoplasm
PfPP2C50Pfo11g011060.143847,647.395.2237.0383.70−0.217Nucleus
PfPP2C51Pfo11g013940.131334,056.084.9346.4181.02−0.337Nucleus
PfPP2C52Pfo11g015860.140344,718.896.5444.2990.77−0.345Nucleus
PfPP2C53Pfo12g008400.173181,194.225.6239.6377.36−0.479Chloroplast/Nucleus
PfPP2C54Pfo14g000920.155760,286.224.8639.8289.57−0.218Nucleus
PfPP2C55Pfo14g006170.126528,851.948.5934.9893.89−0.114Nucleus
PfPP2C56Pfo14g009710.134837,801.875.3151.3789.60−0.255Chloroplast/Nucleus
PfPP2C57Pfo15g011470.146150,501.815.8741.4082.47−0.206Chloroplast/Nucleus
PfPP2C58Pfo15g012450.139042,239.895.1153.7182.54−0.201Chloroplast
PfPP2C59Pfo16g002080.147152,221.335.2752.5669.92−0.493Nucleus
PfPP2C60Pfo16g008470.152657,751.385.0538.3180.68−0.319Chloroplast/Nucleus
PfPP2C61Pfo16g013780.112013,508.304.6836.3889.33−0.458Chloroplast/Cytoplasm
PfPP2C62Pfo17g001200.148953,137.058.8534.2787.36−0.150Chloroplast
PfPP2C63Pfo17g006640.143947,981.436.6544.2476.79−0.424Chloroplast
PfPP2C64Pfo18g001250.154859,083.534.6140.5088.03−0.126Chloroplast
PfPP2C65Pfo18g001790.134637,658.616.5540.9985.69−0.249Nucleus
PfPP2C66Pfo18g003260.138742,305.595.1061.4082.17−0.318Nucleus
PfPP2C67Pfo18g003900.136039,673.585.0035.3973.44−0.414Nucleus
PfPP2C68Pfo18g005750.146149,990.828.6257.4874.49−0.279Chloroplast
PfPP2C69Pfo18g006540.137340,537.836.4959.3272.92−0.463Nucleus
PfPP2C70Pfo19g000370.139743,812.264.9261.7384.08−0.222Nucleus
PfPP2C71Pfo19g001430.137541,296.747.5535.7480.13−0.324Nucleus
PfPP2C72Pfo19g002000.150655,328.184.9449.3187.11−0.198Nucleus
PfPP2C73Pfo19g002890.137842,036.044.8335.0179.89−0.337Nucleus
PfPP2C74Pfo19g003480.160066,319.146.2047.5388.42−0.215Chloroplast
PfPP2C75Pfo19g005950.123326,049.777.0552.3172.79−0.389Nucleus
PfPP2C76Pfo20g000210.153859,481.426.9945.8778.79−0.353Chloroplast
PfPP2C77Pfo20g001120.139443,765.706.1239.8190.10−0.275Nucleus
PfPP2C78Pfo20g004520.142746,141.065.5363.4371.66−0.367Nucleus
PfPP2C79Pfo20g005770.136339,132.587.0244.4480.55−0.199Cell membrane/Nucleus
PfPP2C80Pfo20g008850.138742,878.695.1145.0267.05−0.586Nucleus
PfPP2C81Pfo20g009100.138942,573.537.5544.6992.06−0.119Nucleus
PfPP2C82Pfoxxg008780.138542,539.379.1143.3289.82−0.331Nucleus
PfPP2C83Pfoxxg011050.143248,285.765.7838.3577.64−0.458Nucleus
PfPP2C84Pfoxxg015210.138042,806.715.9349.3492.63−0.236Nucleus
PfPP2C85Pfoxxg021750.163169,947.475.5640.2279.10−0.394Nucleus
PfPP2C86Pfoxxg021830.143348,153.285.5440.1573.93−0.310Nucleus
PfPP2C87Pfoxxg022160.129331,764.124.8736.5978.19−0.369Nucleus
PfPP2C88Pfoxxg025250.143248,177.565.5834.2377.87−0.419Nucleus
PfPP2C89Pfoxxg026240.163270,046.615.6339.9679.13−0.392Nucleus
PfPP2C90Pfoxxg026500.138542,563.439.1142.3242.32−0.318Nucleus
PfPP2C91Pfoxxg028980.138042,792.685.9349.5692.37−0.237Nucleus
Table
Table 2. Conserved motif information of the PP2C gene family in P. fortunei.
Table 2. Conserved motif information of the PP2C gene family in P. fortunei.
MotifLengthAmino Acid Sequence Information
motif 129LTPEDEFLILASDGLWDVLSNZEAVDJVR
motif 216DLYVANVGDSRAVLCR
motif 315TFFGVFDGHGGPGAA
motif 415GGLAVSRAIGDRYLK
motif 550NPRGGPARRLVKAALFRAAKKREMRYSELKKIDQGVRRHYHDDITVIVIF
motif 615AIQLTVDHKPNREDE
motif 741DVJKKAFSATEEEFLSLVDRQWMIKPZJASVGSCCLVGVIC
motif 815RGSKDBITVIVVDFK
motif 941GRVDGLLWYKDLGHHVNGEFSMAVVQANNLLEDQSQLESGP
motif 1011SGTTAVTALVI
motif 1141TPGRVFLNGSSKYASLFTQQGKKGVNQDAMIVWENFGGQED
motif 1211RERIEAAGGRV
motif 1315KKAJKKAFLKTDKEL
motif 1411PYLIAEPEVTV
motif 1518GRRREMEDAVAAIPDLCG
motif 1615FVKDNLFENVLKELK
motif 1721RSLHPDDSQIVVLKHKVWRVK
motif 1815PDPEAAAKRLVEEAL
motif 1929SLGSQNLQWAQGKAGEDRVHVVVSEEHGW
motif 2041NEKIEKPTVK YGQAAQSKKGEDYFLIKTDCQRVPGBPSTSF
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MDPI and ACS Style

Zhao, Z.; Zhang, P.; Deng, M.; Cao, Y.; Fan, G. Genome-Wide Identification and Expression Analyses of the PP2C Gene Family in Paulownia fortunei. Forests 2023, 14, 207. https://doi.org/10.3390/f14020207

AMA Style

Zhao Z, Zhang P, Deng M, Cao Y, Fan G. Genome-Wide Identification and Expression Analyses of the PP2C Gene Family in Paulownia fortunei. Forests. 2023; 14(2):207. https://doi.org/10.3390/f14020207

Chicago/Turabian Style

Zhao, Zhenli, Peiyuan Zhang, Minjie Deng, Yabing Cao, and Guoqiang Fan. 2023. "Genome-Wide Identification and Expression Analyses of the PP2C Gene Family in Paulownia fortunei" Forests 14, no. 2: 207. https://doi.org/10.3390/f14020207

APA Style

Zhao, Z., Zhang, P., Deng, M., Cao, Y., & Fan, G. (2023). Genome-Wide Identification and Expression Analyses of the PP2C Gene Family in Paulownia fortunei. Forests, 14(2), 207. https://doi.org/10.3390/f14020207

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