Next Article in Journal
Novel Mouse Cell Lines and In Vivo Models for Human High-Grade Neuroendocrine Lung Carcinoma, Small Cell Lung Carcinoma (SCLC), and Large Cell Neuroendocrine Carcinoma (LCNEC)
Previous Article in Journal
Genetic Architecture of Salt Tolerance in Cowpea (Vigna unguiculata (L.) Walp.) at Seedling Stage Using a Whole Genome Resequencing Approach
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 20
10.3390/ijms242015282
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.)

by
Yu Chen
1,2,†,
Haohan Zhao
1,†,
Yue Wang
1,
Xiaojun Qiu
1,
Gang Gao
1,
Aiguo Zhu
1,
Ping Chen
1,
Xiaofei Wang
1,
Kunmei Chen
1,
Jia Chen
1,
Peng Chen
2,* and
Jikang Chen
1,3,*
1
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410221, China
2
College of Agriculture, Guangxi University, Nanning 530004, China
3
National Breeding Center or Bast Fiber Crops, MARA, Changsha 410221, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(20), 15282; https://doi.org/10.3390/ijms242015282
Submission received: 13 September 2023 / Revised: 4 October 2023 / Accepted: 12 October 2023 / Published: 18 October 2023
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
The protein phosphatase 2C (PP2C), a key regulator of the ABA signaling pathway, plays important roles in plant growth and development, hormone signaling, and abiotic stress response. Although the PP2C gene family has been identified in many species, systematic analysis was still relatively lacking in ramie (Boehmeria nivea L.). In the present study, we identified 63 BnPP2C genes from the ramie genome, using bioinformatics analysis, and classified them into 12 subfamilies, and this classification was consistently supported by their gene structures and conserved motifs. In addition, we observed that the functional differentiation of the BnPP2C family of genes was restricted and that fragment replication played a major role in the amplification of the BnPP2C gene family. The promoter cis-regulatory elements of BnPP2C genes were mainly involved in light response regulation, phytohormone synthesis, transport and signaling, environmental stress response and plant growth and development regulation. We identified BnPP2C genes with tissue specificity, using ramie transcriptome data from different tissues, in rhizome leaves and bast fibers. The qRT-PCR results showed that the BnPP2C1, BnPP2C26 and BnPP2C27 genes had a strong response to drought, high salt and ABA, and there were a large number of stress-responsive elements in the promoter region of BnPP2C1 and BnPP2C26. The results suggested that BnPP2C1 and BnPP2C26 could be used as the candidate genes for drought and salt tolerance in ramie. These results provide a reference for further studies on the function of the PP2C gene and advance the development of the mechanism of ramie stress response, with a view to providing candidate genes for the molecular breeding of ramie for drought and salt tolerance.

1. Introduction

The type 2C protein phosphatases (PP2Cs) belong to a branch of phosphoprotein metal phosphatases (PPM), which are Mg2+ or Mn2+ dependent protein molecules and are widely found in plants and animals, especially in greater abundance in plants [1]. The PP2C is highly conserved during evolution and has a unique protein structure with an active catalytic region at the C-terminal or N-terminal end, and a structurally diverse extension region at the other end [2]. The structural diversity of the extension region allows for the diversification of PP2C genes and confers different biological functions to PP2C family genes [2]. In higher plants, PP2C plays an important role in plant growth and development, hormone signaling and abiotic stress responses [3,4].
The PP2C families have been reported in many plant species, such as Arabidopsis thaliana (80) [5], wheat (Triticum aestivum) (257) [6], cucumber (Cucumis sativus) (56) [7], walnut (Juglans regia) (44) [8], etc. Phylogenetic analysis classified the 80 AtPP2C genes in Arabidopsis into 13 subfamilies (A-L), while the other seven were not involved in the grouping. PP2C is a direct target of ABA receptor proteins (PYR/PYL/RCAR) in the ABA signaling pathway, and the interaction between PYR/PYL/RCAR proteins and PP2C activates downstream targets [9]. All of the nine members belonging to subgroup A of AtPP2Cs contain ABA-responsive elements in their promoter regions, among which ABA-insensitive 1 (ABI1) and ABA-insensitive 2 (ABI2) act by negatively regulating ABA signaling, and can also directly inhibit PP2C activity in vitro [10]. The B-branch members of PP2C genes mainly function in the mitogen-activated protein kinase (MAPK) signaling pathway, acting as negative regulators of plant hormone synthesis and defenses, and usually inactivating MPK4 and MPK6 for signal transduction regulation [11,12]. PP2C C-branch member and D-branch member genes regulate plant flowering development and salinity stress response, respectively [13,14]. It has been reported that many PP2C members are involved in the regulation of abiotic stresses such as high salt and drought in plants. For example, in Betula alba, overexpression of BpPP2C1 significantly enhanced the salt tolerance of plants [15]; PP2C49 negatively regulates plant salt tolerance by inhibiting the activity of Arabidopsis AtHKT1;1 [16]. ZmPP84, a member of the PP2C F-branch, negatively regulates plant drought tolerance by affecting maize stomatal closure and thereby inhibiting the ZmMEK1–ZmSIMK1 signaling pathway [17].
Ramie (Boehmeria nivea L.), a perennial fiber crop of the genus Boehmeria in the family Urticaceae, has been widely used as material for animal feed, the textile industry and the phytoremediation of heavy metal contaminated soil [18,19]. Ramie is one of the most important economic crops in Asia, and has been cultivated for over 4000 years [20]. Its production is governed by various abiotic and biotic stress conditions, such as drought, salt, and nutrient stresses. Drought stress is one of the major threats to ramie production, and under severe drought stress, ramie yield will be lost by more than 20% [20,21]. Several studies have reported that drought triggered the biosynthesis of ABA and induced leaf abscission and yield reduction in ramie [22]. Therefore, it is important to know the molecular mechanisms of ramie’s response to stresses. However, as one of the key regulators of the ABA signaling pathway, the BnPP2C gene family in ramie has not been studied previously.
Based on the genomic and transcriptomic data of ramie in our laboratory, this study was conducted to identify and analyze the gene family of BnPP2C using bioinformatics methods, and to analyze their expression patterns under stressful environments, with the aim of providing a reference basis for subsequent studies on the functions of BnPP2Cs, advancing the development of stress resistance mechanisms in ramie, thereby contributing to the genetic improvement of ramie for a resistant cultivar.

2. Results

2.1. Identification and Basic Information of PP2C Family Members in Ramie

In this study, a total of 63 PP2C family members from the whole genome database of ramie were identified, and were named BnPP2C1 to BnPP2C63, based on their distribution in chromosomes. Physicochemical property analysis showed that the protein sequences of the BnPP2C gene family members were highly variable, among which BnPP2C10 had the shortest amino acid sequence of 164 aa, and BnPP2C28 had the longest amino acid sequence of 1088 aa. The molecular weights of these BnPP2C proteins ranged from 17.95 kDa to 121.62 kDa, with PIs ranging from 4.66 to 9.63, including 14 basic proteins and 49 acidic proteins (Supplementary Table S1).
The predicted results of subcellular localization showed that 27 of the 63 BnPP2C members were localized in chloroplasts, 19 in the cytosol, and 12 in the nucleus; BnPP2C28 and BnPP2C38 were found in the endoplasmic reticulum, and BnPP2C56, BnPP2C55 and BnPP2C53 were found in the cytoskeleton, vascular membrane and mitochondrion, respectively (Supplementary Table S1).

2.2. Phylogenetic Tree Analysis

To investigate the phylogenetic relationship between ramie and Arabidopsis PP2C genes, a phylogenetic tree of 63 ramie BnPP2C and 80 Arabidopsis AtPP2C proteins were constructed using the neighbor joining method by MEGA11.0 software (Figure 1). The results showed that the 63 genes of the ramie PP2C family could be divided into 12 subfamilies, with subgroup A and subgroup E having the most BnPP2C members (8), followed by subgroup D and F1 (7), in the phylogenetic tree. Combining the data on the physicochemical properties of BnPP2C, it was found that the physicochemical properties of the proteins differed significantly among the subfamilies, while small differences were found within the same subfamily. Interestingly, none of the BnPP2C genes have been categorized into subgroup K, and no details have been reported on the function of PP2C subfamily K in other species.

2.3. BnPP2C Gene Structure Analysis and Conserved Motif Analysis

The conserved motifs, conserved structural domains, coding sequences (CDS) and the location of the untranslated region (UTR) of BnPP2C were analyzed (Figure 2). We integrated and visualized the evolutionary tree and motif prediction results of the BnPP2C family using TBtools software and used the MEME online website to predict the conserved motifs of the BnPP2C family members. The results revealed some differences in the type and number of conserved motifs contained in each protein sequence. In the same sub-family species, the BnPP2C members had similar conserved motif structures. The results suggested that most BnPP2C genes were evolutionarily conserved, and individual members might be functionally divergent.
The structural domain analysis of BnPP2C family genes revealed that all 63 BnPP2C family gene members had PP2C structural domains, among which five genes contained PP2C-C superfamily structural domains. In addition, BnPP2C59, BnPP2C28 and BnPP2C38 had PKc-like superfamily structural domains, which might play important roles in the signal transduction cascade reaction.
Different gene structures affected gene expression and protein function, and exon–intron structural changes played an important role in gene functional differentiation [12,23]. Based on the phylogenetic analysis of BnPP2C, it was found that members of the same subgroup had similar exon and intron structures, and there were significant differences in intron and exon structures among members of different subgroups; for example, BnPP2C49 had only one intron, but BnPP2C28 had 20 introns.

2.4. Chromosome Distribution and Gene Duplication of BnPP2Cs

After identification of the ramie PP2C gene family, 63 members of the BnPP2C family were mapped on the chromosomes by TBtools and renamed according to the chromosome number and the order of the position of each gene on the chromosome (BnPP2C1-BnPP2C63). The analysis showed that the 62 BnPP2C genes assembled were unevenly distributed on the 14 chromosomes of ramie (Figure 3), with most BnPP2Cs anchored in Chr1 (8). Only two BnPP2Cs in each were anchored in Chr5, Ch10 and Chr12. All of the BnPP2C genes located on Chr9 were distributed in the first half of the chromosome.
To further understand the amplification mechanism of BnPP2C, the collinearity of PP2C in the ramie genome was investigated (Figure 4; Supplementary Table S2). The results revealed that there were 10 co-linear gene pairs among members of the ramie PP2C gene family, with three pairs of tandem duplicated genes distributed on Chr2, Chr4 and Chr8, respectively. The remaining seven pairs of segmentally duplicated genes were distributed unevenly on the remaining chromosomes, except Chr4, Chr5, Chr9, Chr10 and Chr11, where BnPP2C36, BnPP2C11 and BnPP2C54 were mutually segmentally duplicated gene pairs. In addition, we performed collinearity analysis on ramie and Arabidopsis and detected 59 homologous gene pairs, among which no homologous genes with Arabidopsis were present on ramie Chr11 and the most PP2C homologous genes (11) were present on ramie Chr3 (Figure 5; Supplementary Table S2).
The Ka/Ks value determines whether the gene encoding the protein is under selection pressure, which has an important role in the evolutionary analysis of gene families (Ka/Ks > one for positive selection, Ka/Ks = one for neutral selection, and Ka/Ks < one for purifying selection) [24,25] (Supplementary Table S3). The analysis showed that, except for two gene pairs, BnPP2C7–BnPP2C62 and BnPP2C34–BnPP2C35, which were significantly differentiated and evolved over a long distance, the Ka/Ks values of all gene pairs were less than 1, indicating that the evolutionary process of these genes evolved mainly under purifying selection [26].

2.5. Cis-Acting Elements Analysis

To further clarify the potential function of the BnPP2C gene family, cis-acting elements in the upstream 2 kb region were analyzed (Figure 6; Supplementary Table S4). We realized that abundant responsive regulatory elements were found in the promoter region of BnPP2C, and a total of 50 cis elements were detected, which were mainly classified into four categories. The first category included the most responsive light-responsive elements (26), such as G-box, Box4, etc. The second category included the hormone-responsive elements (11), such as P-box, ABRE, etc. The third category included responsive stress response elements (5), such as MBS, ARE, etc. The fourth category included plant growth response elements (8), such as O2-site, circadian, etc.
Calculation of the number of elements revealed that the light-responsive element G-box and the hormone-responsive element ABRE had the largest number. The ARE element was the most distributed among the five stress elements, but the plant growth-responsive elements were present in very small numbers. Among them, G-box, Box4, ABRE and ARE elements were mainly distributed in BnPP2C family members. The hormones involved in the phytohormone response elements were MeJA, ABA, and GA, etc., among which ABRE had the largest number, followed by the CGTCA-motif and the TGACG-motif. The cis-elements involved in stress were less diverse but more numerous, mainly including cis-elements that are responsive to low temperature, hypoxia and drought stress, such as ARE elements. These results suggest that BnPP2Cs might play important regulatory roles in response to light regulation, hormone signaling and adversity stress.

2.6. Expression Profiling of PP2C Gene Family in Different Tissues of Ramie

In order to gain insight into the tissue-specific expression pattern of ramie, we used the transcriptome data to create a heat map of the relative expression of BnPP2C in roots, stems, leaves and bast fibers (Figure 7). The results showed that BnPP2Cs were differentially expressed in different tissues, among which BnPP2C51, BnPP2C31, BnPP2C35, BnPP2C48, BnPP2C44, BnPP2C43, BnPP2C60 and BnPP2C2 were highly expressed in bast fibers, while little or no expression was found in rootstocks and leaves, indicating that these genes might be accompanied in the synthesis of bast fibers. BnPP2C53, BnPP2C21, BnPP2C12, BnPP2C11, BnPP2C62, BnPP2C29, BnPP2C30, BnPP2C39, BnPP2C20, BnPP2C37 and BnPP2C45 were highly expressed in the stems, indicating that these genes may have a regulatory role in the growth and development of ramie stems. The results implied that BnPP2Cs might play multiple regulatory roles in growth and development.

2.7. Analysis of Expression Patterns of BnPP2C Genes under Drought, Salt and ABA Treatments

The analysis of the promoter cis-acting element of BnPP2Cs suggested that this gene family might play an important role in response to abiotic stresses and hormonal regulation. Therefore, qRT-PCR was used to analyze the expression of 15 of these BnPP2C genes under ABA, PEG and salt treatments. The quality testing of RNA mainly includes concentration, purity and integrity testing, the results of which play an important role in the subsequent experiments. The concentration of RNA in this test was between 950 and 1200 ng/μL, and the OD260/OD280 were all around 2.0. Agarose electrophoresis found three bands, and the 28s and 18s bands were obvious, which indicated the integrity was better. The above results confirmed that the quality of the RNA was reliable and could be used for the subsequent experiments, and the results of its test are shown in the Appendix (Supplementary Table S6 and Supplementary Figure S1). Dissolution curve analysis is a method to verify primer specificity. We carried out the dissolution curve analysis and found that the dissolution curves of each primer were single peaks and that the Tm values were all greater than 80 °C, indicating that the primers were specific; the results are shown in Supplementary Figure S2. The 15 selected genes showed high homology with 15 of the cucumber PP2C family members, and the expression of these cucumber PP2C members were identified to be significantly up-regulated under drought, salt stress and ABA treatments. The treatment was conducted with 10% PEG, 100 mM NaCl and 100 μM ABA, as pre-experiments and references suggested [7,12]. The results showed that the expression of all of the 15 selected BnPP2C genes were down-regulated within 6 h of ABA treatment, but the expression of the BnPP2C1, 24, 26, 27, 29, 33, 43 and 48 genes gradually increased as the treatment time extended, with the highest expression level of BnPP2C41 at 24 h, which was about twice as high as that at 0 h. ABA suppressed the expression of BnPP2C48 genes at 24 h (Figure 8).
Under PEG-induced drought stress, the expression of all 14 BnPP2C1 genes were down-regulated at 6 h except for BnPP2C1, but the expression of BnPP2C7, 24, 26, 45, 46, 49 and 57 gradually increased with an increase in the stress time. Among them, the expression of BnPP2C1 gradually increased from the onset of stress, and the highest expression was at 24 h, which was about 7.5 times of that at 0 h. After 6 h of salt stress, only the expression of BnPP2C1 was up-regulated. After 12 h of salt stress, the expression of the 15 selected BnPP2C genes were all down-regulated compared to 0 h, with BnPP2C46 being the most significantly down-regulated. After 24 h of stress, only the expression of BnPP2C1, 17, 24, 26 and 27 genes were up-regulated compared with CK, with BnPP2C24 being the most significantly up-regulated, which was 1.7 times more than that at 0 h.
In summary, some genes showed similar expression patterns under PEG and ABA treatments, such as BnPP2C24 and BnPP2C27, with the lowest expression at 6 h, and gradually increasing thereafter. However, under salt stress treatment, BnPP2C24 and BnPP2C27 had the lowest expression at 12 h, indicating that BnPP2C genes have different expression patterns under different stress treatments, and the above genes might be involved in the response process of ramie to drought stress, salt stress and ABA treatment.

3. Discussion

3.1. Global Profile of the PP2C Gene Family of Ramie

The PP2C family is one of the most numerous and widely distributed gene families in plants, which plays important roles in coping with abiotic stresses such as drought and salinity, as well as in plant growth and development [27,28,29]. PP2C is known to be mainly involved in stress signaling, but most of the functions of PP2C remain undefined. Although the PP2C gene family has been extensively studied in other species, little research has been undertaken in ramie [30]. In this study, the genome-wide identification and screening of PP2C genes were carried out, followed by analyses of phylogenetics, gene structure, gene localization, covariate relationships, cis-acting elements, tissue expression, and the expression pattern under abiotic stresses. It laid an important foundation for understanding the potential molecular mechanism of ramie PP2C genes in the signal transduction of adversity and stress.
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 [31]. In the present study, 63 BnPP2C genes were identified and categorized into 12 subfamilies. Interestingly, none of the BnPP2C genes have been categorized into subgroup K, and no details have been reported on the function of PP2C subfamily K in other species [5]. The physicochemical properties of the members of the PP2C family of ramie differ among subfamilies, and most of them are acidic proteins. Protein localization predictions showed that the BnPP2C genes were mainly localized in chloroplasts and cytoplasm, which might be related to the anabolism of photosynthetic enzymes and to cell growth and development. Members of the same subgroup of the BnPP2C gene have similar exon–intron structures, and the proteins they encode have similar motif components [12,23]. In cucumber, the vast majority of CsPP2C genes were present in Motif 1 and 2, whereas in ramie the vast majority of BnPP2C genes were present in Motif 4, 7 and 9 structures. This pattern of motif arrangement may be closely related to the catalytic core structural domains of PP2C proteins, and further confirms that the PP2C gene family is highly conserved in evolution [32]. We also found the presence of PKc-like superfamily structural domains in BnPP2C28, 38 and 59, which may play a role in the signaling cascade response [33].
The phenomenon of gene duplication was one of the most important reasons for the diversification of gene functions, and segmental and tandem duplication were the main drivers of the evolution and expansion of family genes [34,35]. This might also be one of the reasons for the difference in the number of members of the PP2C gene family in ramie compared to other plants. The presence of three pairs of tandem-duplicated genes and seven pairs of segmental-duplicated genes among the ramie PP2C family suggests that segmental duplication plays a major role in the amplification of the BnPP2C gene family, which was consistent with the major amplification of the rice PP2C gene in Arabidopsis [5]. The results of Ka/Ks analysis showed that, except for two pairs of genes with significant sequence differentiation and a long evolutionary distance, the Ka/Ks values of the rest of the BnPP2C gene pairs were all less than 1, suggesting that the functional differentiation of ramie PP2C family genes was limited; similar results were found in woodland strawberry (Fragaria vesca), pineapple strawberry (Fragaria ananassa), cotton (Gossypium hirsutum) and tomato (Solanum lycopersicum) [12,36,37].

3.2. The Roles of the BnPP2C Gene Family in Response to Abiotic Stresses

Currently, drought and salt stress have become the key factors limiting the productivity of ramie, and the excavation of key drought and salt tolerance genes is important for the advancement of ramie breeding programs [38,39]. It was shown that some PP2C genes in both cucumber and wheat showed a tendency to be up-regulated in expression after treatment with drought, salt stress, or ABA, which was consistent with the results of this study on ramie [7]. In walnut, JrPP2C17 showed a strong response to drought, high salt and ABA [8]. GlPP2C1 silencing increased the content of Ganoderma lingzhi polysaccharides (GL-PS) through Slt2, thereby enhancing cell wall resistance [40]. Some genes may have opposite roles in different plants. Compared with the wild type, the enhanced drought tolerance of the maize Zmpp2c26 mutant is mainly regulated by the dephosphorylation of ZmMAPK3 and ZmMAPK7 by ZmPP2C26. However, overexpression of ZmPP2C26L and ZmPP2C26S significantly reduced drought tolerance in Arabidopsis and rice [41]. The qRT-PCR results in this study showed that some BnPP2C genes showed similar expression patterns under PEG and NaCl treatments, such as BnPP2C1, BnPP2C24, BnPP2C26 and BnPP2C27 genes, presenting a U-type curve. Interestingly, the expression of BnPP2C24 decreased and did not change significantly under ABA treatment for 24 h, suggesting that the BnPP2C24 gene might be a negative regulator of the ABA-mediated signaling pathway. However, BnPP2C1, BnPP2C 26 and BnPP2C27 genes may positively regulate the ABA signaling pathway in response to adversity stress, thereby enhancing ramie resilience; similar results were observed in cucumber [7].
Cis-acting elements are important regulators of plant hormone responses and resistance to various adversities [42]. Cis-acting elements were detected to be responsive to various phytohormones, such as MeJA, GA, ABA, IAA and SA, in the promoter regions of PfPP2C genes in Paulownia fortunei [31]. The multi-roles of PP2C genes were also reported in A. thaliana [43], Saccharum spontaneum [3] and other species. The promoter regions of BnPP2Cs were enriched with light-responsive elements and hormone-responsive elements, such as G-box, Box4, P-box, ABRE, etc., which suggested that BnPP2Cs might play important roles in responding to photoperiodic and hormone-induced processes [44]. Among them, there were also multiple stress-responding elements in the promoter regions of BnPP2C1 and BnPP2C26, and these results supported the fact that both BnPP2C1 and BnPP2C26 may enhance ramie resistance by participating in the ABA signaling pathway.

3.3. Candidate Genes for Improving Drought and Salt Tolerance

Designing crops that are more productive, coupled with being more environmentally resilient, is a major goal of breeding scientists. Understanding the synergies between crop yield and stress response is critical to developing new varieties [45,46]. Abiotic stresses can lead to ramie root failure, slow growth, leaf loss and other undesirable phenomena, and finally impact fiber quality and yield [47,48]. As the global climate and environment degenerate, multi-stress conditions, especially drought coupled with salt stress, have become more and more frequent in agricultural production, especially affecting food crop [49], fiber crop [50] and horticulture crop [51].
In this study, we found a large number of stress-responsive elements in the promoter regions of BnPP2C1 and BnPP2C26, which were positively regulated in the ABA signaling pathway and abiotic stress response mechanisms. In summary, BnPP2C1 and BnPP2C26 can be used as candidate genes to improve salt and drought tolerance in ramie, but further studies are needed to reveal their specific functional mechanisms in ramie growth and resistance to adversity stress.

4. Materials and Methods

4.1. Identification of BnPP2C Family Members and Analysis of Physicochemical Properties

The BnPP2C family members were identified from the whole-genome data of ramie in our laboratory. The hidden Markov model (HMM) mapping of the PP2C structural domain (PF00481) was first obtained through the Pfam database (http://pfam.xfam.org/, accessed on 5 April 2023) and we downloaded the PP2C protein sequence of Arabidopsis from the TAIR website (The Arabidopsis Information Resource, https://www.Arabidopsis.org/, accessed on 5 April 2023). The target genes were searched using HMMER3.0 software with a threshold E-value ≤ 1 × 10−5 [52,53]. The protein sequences of the candidate BnPP2C genes were submitted to CD-search (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 7 April 2023) and SMART (http://smart.embl-heidelberg.de/, accessed on 7 April 2023) for protein structural domain analysis, removing redundancies and retaining protein sequences containing PP2C structural domains [54].
The physicochemical properties of BnPP2C proteins, including amino acid number, molecular weight and theoretical pI, were predicted by ExPASy ProtParam (https://web.expasy.org/protparam/, accessed on 10 April 2023) [55]. The subcellular localization of BnPP2C proteins was predicted by the Wolf PSORT website (https://wolfpsort.hgc.jp/, accessed on 10 April 2023) [56].

4.2. Phylogenetic Tree Construction of the BnPP2C Gene Family

The 63 BnPP2C and 80 AtPP2C protein sequences were imported into MEGA11 software together; the ClustalW method was used for multiple sequence alignment, and the neighbor joining method was used to construct a phylogenetic tree (the bootstrap value was set to 1000 and other parameters were set as default) [57]. The ramie PP2C proteins were subdivided into different subfamilies, according to the classification of AtPP2Cs. The phylogenetic tree was embellished and visualized using the online software iTOL (https://itol.embl.de/, accessed on 12 April 2023) [58].

4.3. Analysis of BnPP2C Gene Structure, Conserved Motifs and Conserved Structural Domains

The conserved patterns (CMs) of BnPP2C proteins were predicted on the MEME Suite 5.4.1 online tool (https://meme-suite.org/meme/tools/meme/, accessed on 14 April 2023) with the conserved pattern parameter set to 10 and other parameters set as default. The Gene Structure View function in TBtools was used to generate an overall map of the conserved patterns, conserved structural domains and gene structures of BnPP2Cs [59,60].

4.4. Chromosomal Localization and Covariance Analysis of BnPP2C Gene

The chromosome length information and PP2C gene position information in the chromosome were extracted from the ramie genome annotation file using TBtools software, and the family members are named BnPP2C1 to BnPP2C63 based on their location on the chromosome [61].
Gene duplication between ramie PP2C family genes within and between species (ramie and Arabidopsis) was analyzed using MCScanX and visualized using Advanced Circos. Using the KaKs_Calculator 2.0, we computed the synonymous (Ks) and non-synonymous (Ka) substitutions of the BnPP2C gene pairs [62,63].

4.5. Analysis of Cis-Acting Elements of the BnPP2C Gene Family

The first 2000 bp of upstream sequences of the start codon of the 63 identified ramie PP2C genes were extracted using TBtools for Cis-acting prediction through the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 April 2023) and their potential related functions, and the number of each acting element was counted by Excel 2019 [64].

4.6. Tissue-Specific Expression Analysis of the BnPP2C Gene Family

The specific expression of BnPP2C gene in different tissues (root, stem, leaf and bast) was investigated using the ramie transcriptome database “Zhongzhu No. 1” in our laboratory, and the expression heat map of BnPP2C gene in four different tissues was drawn using TBtools [61].

4.7. Plant Materials and Growth Conditions

Ramie seeds were rinsed well, soaked in pure water for 10 min, and evenly beaten on the surface of nutrient soil with a pipette gun, then covered with about 1cm of soil, watered and placed in a greenhouse (humidity (60 ± 5%), temperature (30 ± 2 °C)). When the ramie seedlings reached four true leaves, we selected seedlings of roughly uniform growth and transplanted them into the hydroponics apparatus for half a month, during which the nutrient solution was added. We set up three treatment groups (T1: 100 mM NaCl; T2: 100 μM ABA; T3: 10% PEG) and one control group, with six biological replicates for each treatment, and the stress conditions were selected with reference to the expression pattern analysis of the PP2C family members in cucumber. Ramie leaves after 0, 6, 12 and 24 h of different treatments were taken, wrapped in tinfoil, placed in liquid nitrogen and stored in a −80 °C refrigerator.

4.8. RNA Sample Extraction and qPCR Analysis

We performed qPCR analysis using samples from the above three treatments and four time periods. Firstly, gene-specific primers were designed through the Primer-BLAST online website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 29 April 2023). The SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.) and Evo M-MLV RT Premix for qPCR Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.) were used for total RNA extraction and reverse transcription. RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and RNA integrity was analyzed using 1% agarose gel electrophoresis. The 18s gene was used as an internal control (accession number: EU747115) and qRT-PCR was performed using a 2× SYBR Mixture (Biomiga(Beijing, China) Co., Ltd.) fluorescence quantification kit. The reaction system was as follows: 2× SYBR Green Pro Taq HS Premix: 5 μL, cDNA after 2000-fold dilution: 2 μL, forward and reverse primers: 0.2 μL, and ddH2O: 2.6 μL. The reaction procedure was as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 56 °C for 20 s, extension at 72 °C for 15 s, 40 cycles. Three technical replicates were set for each reaction. The relative expression of the BnPP2Cs was calculated using the 2-ΔΔCt method and plotted using GraphPad Prism v8.0 software [65,66]. The primer sequences are shown in Supplementary Table S5.

4.9. Statistics and Analysis of Data

Excel 2019 was used for statistics and analysis of data, and bar charts were created using Prism software.

5. Conclusions

A comprehensive analysis of the PP2C family genes in ramie was carried out in this study. We identified 63 BnPP2C genes using bioinformatics methods and classified them into 12 subfamilies based on the taxonomy of Arabidopsis thaliana; their conserved sequence features, gene structure and motif composition consistently supported the classifications. The physicochemical properties varied among subfamilies, with the majority being acidic proteins. The subcellular localization predictions revealed that the ramie PP2C genes were mainly localized in the chloroplast and cytoplasm. Synteny analysis revealed that fragment replication played a major role in the amplification of the BnPP2C gene family. The analysis of promoter cis-acting elements showed that members of BnPP2C respond positively to plant growth, hormone signaling and stress stimuli. Based on transcriptomic data, BnPP2Cs were found to have significant tissue expression specificity. By analyzing the expression pattern of BnPP2Cs, we identified some key genes associated with drought and salt stress. In conclusion, these results provide a useful reference for further investigation of the BnPP2C gene functions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242015282/s1.

Author Contributions

Conceptualization, Y.C. and X.Q.; methodology, Y.C., H.Z. and X.Q.; software, Y.C. and X.Q.; validation, G.G., P.C. (Ping Chen) and H.Z.; formal analysis, Y.C.; investigation, Y.C. and Y.W.; resources, G.G., K.C., J.C. (Jia Chen) and X.W.; data curation, Y.C.; writing—original draft preparation, Y.C., H.Z. and J.C. (Jikang Chen); writing—review and editing, G.G. and P.C. (Peng Chen); visualization, Y.C.; supervision, J.C. (Jikang Chen) and G.G.; project administration, J.C. (Jikang Chen) and G.G.; funding acquisition, J.C. (Jikang Chen) and A.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Youth Talent Program of Hunan Province (2022RC1013), the China Agriculture Research System for Bast and Leaf Fiber Crops (CARS-16), the Foreign Expert Project (G2022051017L) and the Agriculture Science and Technology Innovation Project (ASTIP-IBFC-04).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kamada, R.; Kudoh, F.; Ito, S.; Tani, I.; Janairo, J.I.B.; Omichinski, J.G.; Sakaguchi, K. Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors. Pharmacol. Ther. 2020, 215, 107622. [Google Scholar] [CrossRef] [PubMed]
  2. Schweighofer, A.; Hirt, H.; Meskiene, I. Plant PP2C phosphatases: Emerging functions in stress signaling. Trends Plant Sci. 2004, 9, 236–243. [Google Scholar] [CrossRef] [PubMed]
  3. Huang, X.; Liang, Y.; Zhang, R.; Zhang, B.; Song, X.; Liu, J.; Lu, M.; Qin, Z.; Li, D.; Li, S.; et al. Genome-wide identification of the PP2C gene family and analyses with their expression profiling in response to cold stress in wild sugarcane. Plants 2023, 12, 2418. [Google Scholar] [CrossRef] [PubMed]
  4. Yang, Q.; Liu, K.; Niu, X.; Wang, Q.; Wan, Y.; Yang, F.; Li, G.; Wang, Y.; Wang, R. Genome-wide identification of PP2C genes and their expression profiling in response to drought and cold stresses in Medicago truncatula. Sci. Rep. 2018, 8, 12841. [Google Scholar] [CrossRef]
  5. Xue, T.; Wang, D.; Zhang, S.; Ehlting, J.; Ni, F.; Jakab, S.; Zheng, C.; Zhong, Y. Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis. BMC Genom. 2008, 9, 550. [Google Scholar] [CrossRef] [PubMed]
  6. Yu, X.; Han, J.; Wang, E.; Xiao, J.; Hu, R.; Yang, G.; He, G. Genome-wide identification and homoeologous expression analysis of PP2C genes in wheat (Triticum aestivum L.). Front. Genet. 2019, 10, 561. [Google Scholar] [CrossRef]
  7. Zhang, G.; Zhang, Z.; Luo, S.; Li, X.; Lyu, J.; Liu, Z.; Wan, Z.; Yu, J. Genome-wide identification and expression analysis of the cucumber PP2C gene family. BMC Genom. 2022, 23, 563. [Google Scholar] [CrossRef]
  8. Sisi, C.; Jieru, D.; Peidong, C.; Zhaolong, Z.; Yihang, W.; Shuwen, C.; Yan, T.; Tianyu, W.; Guiyan, Y. Transcriptome-wide identification of walnut PP2C family genes in response to external stimulus. BMC Genom. 2022, 23, 640. [Google Scholar] [CrossRef]
  9. Lim, C.W.; Baek, W.; Jung, J.; Kim, J.H.; Lee, S.C. Function of ABA in stomatal defense against biotic and drought stresses. Int. J. Mol. Sci. 2015, 16, 15251–15270. [Google Scholar] [CrossRef]
  10. Wu, Z.; Luo, L.; Wan, Y.; Liu, F. Genome-wide characterization of the PP2C gene family in peanut (Arachis hypogaea L.) and the identification of candidate genes involved in salinity-stress response. Front. Plant Sci. 2023, 14, 1093913. [Google Scholar] [CrossRef]
  11. Meskiene, I.; Baudouin, E.; Schweighofer, A.; Liwosz, A.; Jonak, C.; Rodriguez, P.L.; Jelinek, H.; Hirt, H. Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase. J. Biol. Chem. 2003, 278, 18945–18952. [Google Scholar] [CrossRef]
  12. Guo, L.; Lu, S.; Liu, T.; Nai, G.; Ren, J.; Gou, H.; Chen, B.; Mao, J. Genome-wide identification and abiotic stress response analysis of PP2C gene family in woodland and pineapple strawberries. Int. J. Mol. Sci. 2023, 24, 4049. [Google Scholar] [CrossRef] [PubMed]
  13. Haider, M.S.; Kurjogi, M.M.; Khalil-Ur-Rehman, M.; Fiaz, M.; Pervaiz, T.; Jiu, S.; Haifeng, J.; Chen, W.; Fang, J. Grapevine immune signaling network in response to drought stress as revealed by transcriptomic analysis. Plant Physiol. Biochem. 2017, 121, 187–195. [Google Scholar] [CrossRef] [PubMed]
  14. Galbiati, M.; Simoni, L.; Pavesi, G.; Cominelli, E.; Francia, P.; Vavasseur, A.; Nelson, T.; Bevan, M.; Tonelli, C. Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells. Plant J. Cell Mol. Biol. 2008, 53, 750–762. [Google Scholar] [CrossRef]
  15. Xing, B.; Gu, C.; Zhang, T.; Zhang, Q.; Yu, Q.; Jiang, J.; Liu, G. Functional Study of BpPP2C1 revealed its role in salt stress in Betula platyphylla. Front. Plant Sci. 2021, 11, 617635. [Google Scholar] [CrossRef]
  16. Chu, M.; Chen, P.; Meng, S.; Xu, P.; Lan, W. The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1. J. Integr. Plant Biol. 2021, 63, 528–542. [Google Scholar] [CrossRef]
  17. Guo, Y.; Shi, Y.; Wang, Y.; Liu, F.; Li, Z.; Qi, J.; Wang, Y.; Zhang, J.; Yang, S.; Wang, Y.; et al. The clade F PP2C phosphatase ZmPP84 negatively regulates drought tolerance by repressing stomatal closure in maize. New Phytol. 2023, 237, 1728–1744. [Google Scholar] [CrossRef]
  18. Chen, K.; Mou, P.; Zhu, A.; Chen, P.; Chen, J.; Gao, G.; Wang, X.; Feng, X.; Yu, C. A comparative study of different methods for the determination of cadmium in various tissues of ramie (Boehmeria nivea L.). Environ. Monit. Assess. 2023, 195, 1009. [Google Scholar] [CrossRef]
  19. Ma, Y.S.; Jie, H.D.; Zhao, L.; Lv, X.Y.; Liu, X.C.; Tang, Y.Y.; Zhang, Y.; He, P.L.; Xing, H.C.; Jie, Y.C. Identification of the Xyloglucan endotransglycosylase/hydrolase (XTH) gene family members expressed in Boehmeria nivea in response to cadmium stress. Int. J. Mol. Sci. 2022, 23, 16104. [Google Scholar] [CrossRef] [PubMed]
  20. Rehman, M.; Fahad, S.; Saleem, M.; Hafeez, M.; Rahman, M.; Liu, F.; Deng, G. Red light optimized physiological traits and enhanced the growth of ramie (Boehmeria nivea L.). Photosynthetica 2020, 040, 58922–58931. [Google Scholar] [CrossRef]
  21. Rasheed, A.; Jie, Y.; Nawaz, M.; Jie, H.; Ma, Y.; Shah, A.N.; Hassan, M.U.; Gillani, S.F.A.; Batool, M.; Aslam, M.T.; et al. Improving drought stress tolerance in ramie (Boehmeria nivea L.) using molecular techniques. Front. Plant Sci. 2022, 13, 911610. [Google Scholar] [CrossRef] [PubMed]
  22. Tan, L.; Yun, C.; Chen, P.; Wang, Y.; Zhu, J.; Chen, J.; Lu, L.; Xiong, H. Effects of exogenous salicylic acid on physiological characteristics and yield of ramie under drought stress and re-watering. J. Nucl. Agric. Sci. 2016, 30, 388–395. [Google Scholar]
  23. Long, M.; Betrán, E.; Thornton, K.; Wang, W. The origin of new genes: Glimpses from the young and old. Nat. Rev. Genet. 2003, 4, 865–875. [Google Scholar] [CrossRef]
  24. Lynch, M.; Conery, J.S. The evolutionary fate and consequences of duplicate genes. Science 2000, 290, 1151–1155. [Google Scholar] [CrossRef] [PubMed]
  25. Li, J.; Zhang, Z.; Vang, S.; Yu, J.; Wong, G.K.; Wang, J. Correlation between Ka/Ks and Ks is related to substitution model and evolutionary lineage. J. Mol. Evol. 2009, 68, 414–423. [Google Scholar] [CrossRef]
  26. Prince, V.E.; Pickett, F.B. Splitting pairs: The diverging fates of duplicated genes. Nat. Rev. Genet. 2002, 3, 827–837. [Google Scholar] [CrossRef] [PubMed]
  27. Chen, Y.; Zhang, J.B.; Wei, N.; Liu, Z.H.; Li, Y.; Zheng, Y.; Li, X.B. A type-2C protein phosphatase (GhDRP1) participates in cotton (Gossypium hirsutum) response to drought stress. Plant Mol. Biol. 2021, 107, 499–517. [Google Scholar] [CrossRef]
  28. Zhang, B.; Chen, N.; Peng, X.; Shen, S. Identification of the PP2C gene family in paper mulberry (Broussonetia papyrifera) and its roles in the regulation mechanism of the response to cold stress. Biotechnol. Lett. 2021, 43, 1089–1102. [Google Scholar] [CrossRef] [PubMed]
  29. Khan, N.; Ke, H.; Hu, C.M.; Naseri, E.; Haider, M.S.; Ayaz, A.; Amjad Khan, W.; Wang, J.; Hou, X. Genome-wide identification, evolution, and transcriptional profiling of PP2C gene family in Brassica rapa. BioMed Res. Int. 2019, 2019, 2965035. [Google Scholar] [CrossRef]
  30. Chen, C.; Yu, Y.; Ding, X.; Liu, B.; Duanmu, H.; Zhu, D.; Sun, X.; Cao, L.; Zaib-Un-Nisa; Li, Q.; et al. Genome-wide analysis and expression profiling of PP2C clade D under saline and alkali stresses in wild soybean and Arabidopsis. Protoplasma 2018, 255, 643–654. [Google Scholar] [CrossRef]
  31. 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. [Google Scholar] [CrossRef]
  32. Shi, Y. Serine/threonine phosphatases: Mechanism through structure. Cell 2009, 139, 468–484. [Google Scholar] [CrossRef]
  33. Zhang, M.; Zhang, S. Mitogen-activated protein kinase cascades in plant signaling. J. Integr. Plant Biol. 2022, 64, 301–341. [Google Scholar] [CrossRef] [PubMed]
  34. Zhang, L.; Ma, C.; Kang, X.; Pei, Z.Q.; Bai, X.; Wang, J.; Zheng, S.; Zhang, T.G. Identification and expression analysis of MAPK cascade gene family in foxtail millet (Setaria italica). Plant Signal. Behav. 2023, 18, 2246228. [Google Scholar] [CrossRef]
  35. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef] [PubMed]
  36. Shazadee, H.; Khan, N.; Wang, J.; Wang, C.; Zeng, J.; Huang, Z.; Wang, X. Identification and expression profiling of protein phosphatases (PP2C) gene family in Gossypium hirsutum L. Int. J. Mol. Sci. 2019, 20, 1395. [Google Scholar] [CrossRef]
  37. Qiu, J.; Ni, L.; Xia, X.; Chen, S.; Zhang, Y.; Lang, M.; Li, M.; Liu, B.; Pan, Y.; Li, J.; et al. Genome-wide analysis of the protein phosphatase 2C genes in tomato. Genes. 2022, 13, 604. [Google Scholar] [CrossRef]
  38. Wu, S.; Xue, S.; Iqbal, Y.; Xing, H.; Jie, Y. Seasonal nutrient cycling and enrichment of nutrient-related soil microbes aid in the adaptation of ramie (Boehmeria nivea L.) to nutrient-deficient conditions. Front. Plant Sci. 2021, 12, 644904. [Google Scholar] [CrossRef]
  39. Huang, C.; Wei, G.; Jie, Y.; Wang, L.; Zhou, H.; Ran, C.; Huang, Z.; Jia, H.; Anjum, S.A. Effects of concentrations of sodium chloride on photosynthesis, antioxidative enzymes, growth and fiber yield of hybrid ramie. Plant Physiol. Biochem. 2014, 76, 86–93. [Google Scholar] [CrossRef]
  40. Wang, Z.; Chen, J.H.; Wang, L.S.; Ding, J.; Zhao, M.W.; Liu, R. GlPP2C1 silencing increases the content of Ganoderma lingzhi Polysaccharide (GL-PS) and enhances Slt2 phosphorylation. J. Fungi 2022, 8, 949. [Google Scholar] [CrossRef]
  41. Lu, F.; Li, W.; Peng, Y.; Cao, Y.; Qu, J.; Sun, F.; Yang, Q.; Lu, Y.; Zhang, X.; Zheng, L.; et al. ZmPP2C26 alternative splicing variants negatively regulate drought tolerance in maize. Front. Plant Sci. 2022, 13, 851531. [Google Scholar] [CrossRef] [PubMed]
  42. Nishimura, N.; Sarkeshik, A.; Nito, K.; Park, S.Y.; Wang, A.; Carvalho, P.C.; Lee, S.; Caddell, D.F.; Cutler, S.R.; Chory, J.; et al. PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. Plant J. Cell Mol. Biol. 2010, 61, 290–299. [Google Scholar] [CrossRef] [PubMed]
  43. Liu, X.; Zhu, Y.; Zhai, H.; Cai, H.; Ji, W.; Luo, X.; Bai, X. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner. Biochem. Biophys. Res. Commun. 2012, 422, 710–715. [Google Scholar] [CrossRef]
  44. Ezer, D.; Shepherd, S.J.K.; Brestovitsky, A.; Dickinson, P.; Cortijo, S.; Charoensawan, V.; Box, M.S.; Biswas, S.; Jaeger, K.E.; Wigge, P.A. The G-Box transcriptional regulatory code in Arabidopsis. Plant Physiol. 2017, 175, 628–640. [Google Scholar] [CrossRef]
  45. Zhang, H.; Zhao, Y.; Zhu, J.K. Thriving under stress: How plants balance growth and the stress response. Dev. Cell 2020, 55, 529–543. [Google Scholar] [CrossRef] [PubMed]
  46. Qiu, X.; Zhao, H.; Abubakar, A.S.; Shao, D.; Chen, J.; Chen, P.; Yu, C.; Wang, X.; Chen, K.; Zhu, A. Genome-wide analysis of AP2/ERF gene superfamily in ramie (Boehmeria nivea L.) revealed their synergistic roles in regulating abiotic stress resistance and ramet development. Int. J. Mol. Sci. 2022, 23, 15117. [Google Scholar] [CrossRef]
  47. Chen, K.; Chen, P.; Qiu, X.; Chen, J.; Gao, G.; Wang, X.; Zhu, A.; Yu, C. Regulating role of abscisic acid on cadmium enrichment in ramie (Boehmeria nivea L.). Sci. Rep. 2021, 11, 22045. [Google Scholar] [CrossRef]
  48. An, X.; Chen, J.; Zhang, J.; Liao, Y.; Dai, L.; Wang, B.; Liu, L.; Peng, D. Transcriptome profiling and identification of transcription factors in ramie (Boehmeria nivea L. Gaud) in response to PEG treatment, using Illumina paired-end sequencing technology. Int. J. Mol. Sci. 2015, 16, 3493–3511. [Google Scholar] [CrossRef]
  49. Zhang, H.; Song, J.; Dong, F.; Li, Y.; Ge, S.; Wei, B.; Liu, Y. Multiple roles of wheat ferritin genes during stress treatment and TaFER5D-1 as a positive regulator in response to drought and salt tolerance. Plant Physiol. Biochem. 2023, 202, 107921. [Google Scholar] [CrossRef]
  50. Wang, Y.; Yu, Y.; Wan, H.; Ni, Z. Sea-island cotton (Gossypium barbadense L.) GbTCP5 improves plant adaptation to drought and salt stress by directly activating GbERD7, GbUBC19, and GbGOLS2 expression. Ind. Crops Prod. 2023, 203, 117209. [Google Scholar] [CrossRef]
  51. Li, F.; Chen, G.; Xie, Q.; Zhou, S.; Hu, Z. Down-regulation of SlGT-26 gene confers dwarf plants and enhances drought and salt stress resistance in tomato. Plant Physiol. Biochem. 2023, 203, 108053. [Google Scholar] [CrossRef]
  52. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, 412–419. [Google Scholar] [CrossRef] [PubMed]
  53. Reiser, L.; Subramaniam, S.; Zhang, P.; Berardini, T. Using the Arabidopsis Information Resource (TAIR) to find information about Arabidopsis genes. Curr. Protoc. 2022, 2, 574. [Google Scholar] [CrossRef] [PubMed]
  54. Letunic, I.; Khedkar, S.; Bork, P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Res. 2021, 49, 458–460. [Google Scholar] [CrossRef] [PubMed]
  55. Artimo, P.; Jonnalagedda, M.; Arnold, K.; Baratin, D.; Csardi, G.; de Castro, E.; Duvaud, S.; Flegel, V.; Fortier, A.; Gasteiger, E.; et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012, 40, 597–603. [Google Scholar] [CrossRef] [PubMed]
  56. Yu, C.S.; Cheng, C.W.; Su, W.C.; Chang, K.C.; Huang, S.W.; Hwang, J.K.; Lu, C.H. CELLO2GO: A web server for protein subcellular localization prediction with functional gene ontology annotation. PLoS ONE 2014, 9, 99368. [Google Scholar] [CrossRef]
  57. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  58. Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: An online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49, 293–296. [Google Scholar] [CrossRef]
  59. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics 2015, 31, 1296–1297. [Google Scholar] [CrossRef]
  60. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, 202–208. [Google Scholar] [CrossRef]
  61. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  62. Su, J.; Song, S.; Wang, Y.; Zeng, Y.; Dong, T.; Ge, X.; Duan, H. Genome-wide identification and expression analysis of DREB family genes in cotton. BMC Plant Biol. 2023, 23, 169. [Google Scholar] [CrossRef]
  63. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef]
  64. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  65. Khan, N.; Hu, C.M.; Khan, W.A.; Wang, W.; Ke, H.; Huijie, D.; Zhishuo, Z.; Hou, X. Genome-wide Identification, Classification, and Expression Pattern of Homeobox Gene Family in Brassica rapa under Various Stresses. Sci. Rep. 2018, 8, 16265. [Google Scholar] [CrossRef]
  66. Xu, J.; Xu, X.; Tian, L.; Wang, G.; Zhang, X.; Wang, X.; Guo, W. Discovery and identification of candidate genes from the chitinase gene family for Verticillium dahliae resistance in cotton. Sci. Rep. 2016, 6, 29022. [Google Scholar] [CrossRef] [PubMed]
Ijms 24 15282 g001
Figure 1. The phylogenetic tree of BnPP2C proteins and AtPP2C proteins. The BnPP2Cs are labeled with red circles and AtPP2Cs with blue stars. Different colors represent different subfamilies.
Figure 1. The phylogenetic tree of BnPP2C proteins and AtPP2C proteins. The BnPP2Cs are labeled with red circles and AtPP2Cs with blue stars. Different colors represent different subfamilies.
Ijms 24 15282 g001
Ijms 24 15282 g002
Figure 2. The phylogenetic tree, conserved motifs, conserved domain, and gene structure of the PP2C gene family in ramie. (a) The neighbor joining tree with 1000 bootstrap replicates of all BnPP2C proteins. (b) The composition of conserved motifs in PP2C proteins of ramie. (c) Conserved structure of BnPP2C identified by the NCBI Batch CD-search tool. (d) The gene structure of BnPP2C genes. The green blocks are CDS, yellow blocks are UTR.
Figure 2. The phylogenetic tree, conserved motifs, conserved domain, and gene structure of the PP2C gene family in ramie. (a) The neighbor joining tree with 1000 bootstrap replicates of all BnPP2C proteins. (b) The composition of conserved motifs in PP2C proteins of ramie. (c) Conserved structure of BnPP2C identified by the NCBI Batch CD-search tool. (d) The gene structure of BnPP2C genes. The green blocks are CDS, yellow blocks are UTR.
Ijms 24 15282 g002
Ijms 24 15282 g003
Figure 3. Chromosome distribution of the PP2C gene family in ramie. The left scale indicates the chromosome length (Mb).
Figure 3. Chromosome distribution of the PP2C gene family in ramie. The left scale indicates the chromosome length (Mb).
Ijms 24 15282 g003
Ijms 24 15282 g004
Figure 4. The collinearity analysis of BnPP2Cs. The gray lines indicate all co-linear gene pairs in ramie, and red lines indicate co-linear gene pairs in BnPP2Cs. The color on the chromosome represents the gene density, with red indicating the highest density and blue the lowest density.
Figure 4. The collinearity analysis of BnPP2Cs. The gray lines indicate all co-linear gene pairs in ramie, and red lines indicate co-linear gene pairs in BnPP2Cs. The color on the chromosome represents the gene density, with red indicating the highest density and blue the lowest density.
Ijms 24 15282 g004
Ijms 24 15282 g005
Figure 5. The collinearity analysis of BnPP2Cs and AtPP2Cs. The gray lines indicate all co-linear gene pairs between ramie and Arabidopsis, and the green lines indicate co-linearity of ramie PP2Cs with Arabidopsis.
Figure 5. The collinearity analysis of BnPP2Cs and AtPP2Cs. The gray lines indicate all co-linear gene pairs between ramie and Arabidopsis, and the green lines indicate co-linearity of ramie PP2Cs with Arabidopsis.
Ijms 24 15282 g005
Ijms 24 15282 g006
Figure 6. Cis-regulatory element analysis of the BnPP2C genes. Note: The different colors on the left represent the different elements (hormone response elements, light response elements, stress response elements and plant growth related elements) and the distribution of the 50 acting elements in the 63 BnPP2C genes is shown in the heat map on the right.
Figure 6. Cis-regulatory element analysis of the BnPP2C genes. Note: The different colors on the left represent the different elements (hormone response elements, light response elements, stress response elements and plant growth related elements) and the distribution of the 50 acting elements in the 63 BnPP2C genes is shown in the heat map on the right.
Ijms 24 15282 g006
Ijms 24 15282 g007
Figure 7. Expression profiles of 63 BnPP2C genes based on RNA-seq data: red and blue boxes indicate high and low expression levels of BnPP2Cs, respectively.
Figure 7. Expression profiles of 63 BnPP2C genes based on RNA-seq data: red and blue boxes indicate high and low expression levels of BnPP2Cs, respectively.
Ijms 24 15282 g007
Ijms 24 15282 g008
Figure 8. Quantitative real-time PCR analysis of BnPP2C genes in response to 10% PEG (a), 100 mM NaCl (b) and 100 μM ABA (c). Note: The relative gene expression was calculated using the 2−∆∆Ct method, and its value is the average of three biological replicates.
Figure 8. Quantitative real-time PCR analysis of BnPP2C genes in response to 10% PEG (a), 100 mM NaCl (b) and 100 μM ABA (c). Note: The relative gene expression was calculated using the 2−∆∆Ct method, and its value is the average of three biological replicates.
Ijms 24 15282 g008
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Chen, Y.; Zhao, H.; Wang, Y.; Qiu, X.; Gao, G.; Zhu, A.; Chen, P.; Wang, X.; Chen, K.; Chen, J.; et al. Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.). Int. J. Mol. Sci. 2023, 24, 15282. https://doi.org/10.3390/ijms242015282

AMA Style

Chen Y, Zhao H, Wang Y, Qiu X, Gao G, Zhu A, Chen P, Wang X, Chen K, Chen J, et al. Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.). International Journal of Molecular Sciences. 2023; 24(20):15282. https://doi.org/10.3390/ijms242015282

Chicago/Turabian Style

Chen, Yu, Haohan Zhao, Yue Wang, Xiaojun Qiu, Gang Gao, Aiguo Zhu, Ping Chen, Xiaofei Wang, Kunmei Chen, Jia Chen, and et al. 2023. "Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.)" International Journal of Molecular Sciences 24, no. 20: 15282. https://doi.org/10.3390/ijms242015282

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop