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Article

Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree

by
Qifeng Liu
1,†,
Bi Qin
2,3,†,
Dong Zhang
1,
Xiaoyu Liang
1,
Ye Yang
1,
Lifeng Wang
2,3,
Meng Wang
1,* and
Yu Zhang
1,*
1
Sanya Institute of Breeding and Multiplication, School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
2
Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture and Rural Affairs, State Key Laboratory Incubation Base for Cultivation & Physiology of Tropical Crops, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
3
Danzhou Investigation & Experiment Station of Tropical Crops, Ministry of Agriculture and Rural Affairs, Danzhou 571737, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(22), 16061; https://doi.org/10.3390/ijms242216061
Submission received: 29 September 2023 / Revised: 31 October 2023 / Accepted: 6 November 2023 / Published: 7 November 2023
(This article belongs to the Special Issue Abiotic Stress Tolerance in Plants: The Regulation of Reactive Oxygen Species and Plant Hormones)
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Abstract

:
Plant PP2C genes are crucial for various biological processes. To elucidate the potential functions of these genes in rubber tree (Hevea brasiliensis), we conducted a comprehensive analysis of these genes using bioinformatics methods. The 60 members of the PP2C family in rubber tree were identified and categorized into 13 subfamilies. The PP2C proteins were conserved across different plant species. The results revealed that the HbPP2C genes contained multiple elements responsive to phytohormones and stresses in their promoters, suggesting their involvement in these pathways. Expression analysis indicated that 40 HbPP2C genes exhibited the highest expression levels in branches and the lowest expression in latex. Additionally, the expression of A subfamily members significantly increased in response to abscisic acid, drought, and glyphosate treatments, whereas the expression of A, B, D, and F1 subfamily members notably increased under temperature stress conditions. Furthermore, the expression of A and F1 subfamily members was significantly upregulated upon powdery mildew infection, with the expression of the HbPP2C6 gene displaying a remarkable 33-fold increase. These findings suggest that different HbPP2C subgroups may have distinct roles in the regulation of phytohormones and the response to abiotic and biotic stresses in rubber tree. This study provides a valuable reference for further investigations into the functions of the HbPP2C gene family in rubber tree.

1. Introduction

PP2C (protein phosphatases type 2C) enzymes constitute a monomeric group of protein phosphatases with a specific action on serine/threonine residues, and they rely on the presence of Mg2+ or Mn2+ ions for their enzymatic activity. These enzymes are highly conserved throughout evolution and are found ubiquitously among various life forms, including archaea, bacteria, fungi, plants, and animals [1,2]. Notably, plants exhibit the highest abundance of PP2C-like proteins in comparison to other organisms [3]. The typical structure of most plant PP2C proteins comprises three distinctive motifs: a conserved catalytic structural domain located at the C-terminus, an extended region housing membrane-localized signaling sequences with diverse functions at the N-terminus, and a structural domain facilitating interactions akin to receptor kinases [4].
The PP2C gene family exerts a significant influence on plant growth and development due to its unique structure and function. These effects encompass a wide range of processes, such as plant root formation [5,6], leaf senescence [7,8], seed germination [9], plant innate immunity [10,11], and pollen germination [12,13]. Moreover, beyond its contributions to plant growth and development, the PP2C family plays a pivotal role in responding to both biotic and abiotic stresses, including fungal pathogens, extreme temperatures, drought, and salt stress [2,14,15,16,17,18]. Furthermore, the PP2C family is integral to hormone signaling pathways, including those involving abscisic acid [7,19,20], MeJA [21], and salicylic acid [21]. Consequently, genes within the PP2C family are pivotal in plant responses to adverse stress conditions and the regulation of various hormone pathways.
Despite extensive research on the PP2C gene family in other plant species, studies on this family in rubber tree are limited. Therefore, it is imperative to identify the PP2C gene family in rubber tree and analyze its expression under stress conditions. In our study, we performed a comprehensive analysis of 60 HbPP2C genes in rubber tree, encompassing physicochemical properties, phylogenetic relationships, gene structures, conserved domains and motifs, and promoter cis-acting elements. Additionally, we investigated the expression patterns of HbPP2C genes in six tissues of rubber tree, their response to abiotic stress, and their expression following infestation by powdery mildew. Our findings not only serve as a reference for delving deeper into the functions of the PP2C gene family, but also provide a foundational understanding of the potential molecular mechanisms of PP2C involvement in various hormonal regulatory pathways, responses to adversity stress, and immune regulation in rubber tree.

2. Results

2.1. Characterization and Analysis of PP2C Gene Family Members in Rubber Tree

A total of 60 HbPP2C genes were successfully identified in the rubber tree using the genome ‘Reyan733397’ as the reference, with Arabidopsis PP2C genes serving as the query sequences. Key features of the HbPP2C genes and their encoded proteins were predicted and are presented in Table 1. These characteristics included CDS (coding sequences) length, the number of amino acids, exons, molecular weights, and PI values. Among the HbPP2C genes, the CDS length ranged from 681 to 3285 base pairs, and the number of exons varied from 1 to 23. The number of amino acids of the HbPP2C proteins spanned from 226 to 1094, with PI values falling within the range of 4.62 to 9.76. Moreover, the molecular weights exhibited considerable diversity, ranging from 14,735.89 Da to 57,315.53 Da. The nomenclature for these HbPP2C genes was based on homologs found in A. thaliana. Subsequently, 40 HbPP2C proteins were selected for further investigation from subgroups A to L, as illustrated in Figure 1.
Table 1. Characteristics of the HbPP2C family of genes and the corresponding proteins in rubber tree.
Table 1. Characteristics of the HbPP2C family of genes and the corresponding proteins in rubber tree.
Gene NameGene IDLength of CDS (bp)Number of ExonsPredicted Protein
Siza (aa)MW (Da)PI
HbPP2C11106503511236441143,539.765.92
HbPP2C2110664056681422625,130.495.41
HbPP2C31106444061311343636,712.725.47
HbPP2C41106408242001566665,573.515.39
HbPP2C51106661311287742846,120.96.40
HbPP2C61106701751251441645,312.935.21
HbPP2C71106594181506450154,241.094.62
HbPP2C81106537661086636139,711.866.21
HbPP2C9110667550882529326,712.088.31
HbPP2C10110672253846528130,766.688.70
HbPP2C11110634536990832930,162.584.79
HbPP2C121106643541275842446,211.855.62
HbPP2C13110651406861128618,178.685.63
HbPP2C14110639512999433233,255.947.96
HbPP2C151106668081539551230,078.98.72
HbPP2C161106415331638454558,926.584.71
HbPP2C1711065240210321034339,528.755.39
HbPP2C181106387401515650456,592.875.49
HbPP2C191106519373285161094121,034.75.05
HbPP2C20110665418849428228,091.826.17
HbPP2C211106573791032934331,444.846.09
HbPP2C221106373161140337931,413.625.15
HbPP2C231106591641170238927,827.864.79
HbPP2C241106369871305343436,716.078.19
HbPP2C251106477241125437440,501.117.06
HbPP2C261106665759151230432,822.45.09
HbPP2C271106692361149438241,895.725.24
HbPP2C281106531721230240914,735.895.79
HbPP2C291106577422364478786,792.45.40
HbPP2C301106391381158438542,130.44.88
HbPP2C311106690481236541126,258.974.99
HbPP2C311106617012700389987,750.356.81
HbPP2C331106300511539551228,981.918.83
HbPP2C341106392971074335739,813.26.36
HbPP2C351106648331602453351,567.475.54
HbPP2C36110655005852428326,636.456.45
HbPP2C371106432251278342546,328.045.12
HbPP2C381106502391230340944,117.948.54
HbPP2C39110666277855328419,352.035.94
HbPP2C401106314531593453057,315.535.34
HbPP2C411106671581194439744,117.948.54
HbPP2C42110669425777425826,124.65.63
HbPP2C431106462011134537743,622.847.23
HbPP2C44110635324852828332,449.259.76
HbPP2C45110637488912230338,238.895.63
HbPP2C46110642221687422927,451.316.30
HbPP2C471106728641158438541,882.795.96
HbPP2C481106513681158438542,961.38.29
HbPP2C491106409181185539443,697.354.93
HbPP2C501106721781050434940,916.224.85
HbPP2C511106456153072231023110,551.675.44
HbPP2C52110668244948431534,981.154.67
HbPP2C531106469451647354859,173.584.68
HbPP2C541106468641107436840,969.808.44
HbPP2C551106558081548451555,811.26.24
HbPP2C5611064242916321154359,251.994.90
HbPP2C571106506671164538742,380.074.97
HbPP2C58110671158876829132,120.558.70
HbPP2C591106573548911029631,721.074.77
HbPP2C601106468951110336940,325.375.23
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Figure 1. The phylogenetic tree of PP2C family proteins among 60 HbPP2Cs, 78 OsPP2Cs, and 80 AtPP2Cs. The protein sequencess were aligned by ClustalW and the unrooted phylogenetic tree constructed by the neighbor-joining (NJ) method with 1000 bootstrap replicates. The letters A to L are 13 major subfamilies of PP2Cs. Different subfamilies are represented in different colors.
Figure 1. The phylogenetic tree of PP2C family proteins among 60 HbPP2Cs, 78 OsPP2Cs, and 80 AtPP2Cs. The protein sequencess were aligned by ClustalW and the unrooted phylogenetic tree constructed by the neighbor-joining (NJ) method with 1000 bootstrap replicates. The letters A to L are 13 major subfamilies of PP2Cs. Different subfamilies are represented in different colors.
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2.2. Phylogenetic Analysis of HbPP2Cs

A comprehensive analysis of the phylogenetic relationships among the domains in HbPP2C proteins, OsPP2C proteins, and AtPP2C proteins was conducted. The PP2C proteins of three species were categorized into 13 groups, denoted as subgroups A to L and the S subgroup. The HbPP2C family consisted of varying numbers of members in each subgroup, with 9, 3, 4, 7, 5, 4, 3, 5, 3, 2, 2, 3, and 2 members in subgroups A, B, C, D, E, F1, F2, G, H, I, J, K, and L, respectively (Figure 1). Notably, subgroups A, D, and F contained five or more members. The phylogenetic tree analysis revealed a parallel evolutionary relationship between PP2C in rubber tree, Oryza sativa, and Arabidopsis. The distribution of PP2C among subgroups in rubber tree closely resembled that in Arabidopsis and O. sativa.

2.3. Conserved Domain and Motifs of HbPP2C Proteins in Rubber Tree

To further understand the function of HbPP2Cs, an analysis was conducted on the structure of 60 HbPP2C proteins. These proteins exhibited a common PP2Cc domain, but the positioning of this domain within the HbPP2C protein varied significantly (Figure 2). Notably, HbPP2C19 possessed the most abundant conserved structural domains, including PP2Cc, two CAP_EDs, and PKc_like domains. On the other hand, the FHA structural domain was exclusively present in HbPP2C17. In addition to identifying the HbPP2C domains, MEME website (https://meme-suite.org/meme/tools/meme, accessed on 19 May 2023) was employed to identify and map 11 conserved motifs. The majority of amino acid sequences were organized in the order of “motif8-motif1-motif5-motif4-motif2-motif6-motif3-motif7-motif10-motif9-motif11”. However, during the evolution of the PP2Cs, certain motifs appear to have been lost. All HbPP2C sequences lacked several motifs. For instance, HbPP2C26 and HbPP2C55 possessed only three conserved motifs. Most HbPP2C proteins that clustered within the same subfamily shared three or more conserved domains or motifs (Figure 2 and Table S2). The genes within the same subgroup contained the majority of motif-encoding sequences, suggesting that these family members may have similar functions.

2.4. Structure of PP2C Genes in Rubber Tree

To comprehend the organization of HbPP2C genes, we generated diagrams illustrating the composition of their introns and exons (Figure 3). With the exception of HbPP1C13, which lacked introns, the HbPP2C gene sequences consisted of multiple introns and exons. The number of exons varied from 1 to 23, while the number of introns ranged from 1 to 22. Notably, of the 60 HbPP2C genes, 42 possessed between 3 and 5 exons. Among these, 23 genes contained 4 exons and 3 introns. These findings suggest a high degree of conservation in the gene structures of HbPP2Cs. Apart from the K, L, and J subfamily genes, the other subfamily genes displayed similar counts of exons and introns, with differences of no more than three. For instance, all members of subfamily B featured four exons, while all three members of subfamily F2 exhibited eight exons and seven introns. Moreover, HbPP2C genes belonging to the same subfamily in the phylogenetic tree showed comparable exon distributions and sequence lengths, indicating a certain level of conservation among genes within these subfamilies and suggesting that these genes may have similar functions.

2.5. Cis-Acting Element Analysis of the Promoters of the HbPP2C Genes

The promoters of all 60 HbPP2C genes were subjected to cis-acting element analysis. The results indicated that there were more than 38 distinct cis-elements (Figure 3 and Table S1). These elements included five types related to plant hormones: gibberellin (P-box and GARE motif), salicylic acid (TCA element), abscisic acid (ABRE), methyl jasmonate (TGACG motif and CGTCA motif), and auxin (TGA element). This suggests that the HbPP2C gene family may play a role in regulating responses to phytohormones. Each promoter sequence included one to five types of plant hormone-related elements. Specifically, 42 genes had abscisic acid-responsive elements, 15 genes had auxin-responsive elements, 32 genes had gibberellin-responsive elements, 35 genes had methyl jasmonate-responsive elements, and 13 genes had salicylic acid-responsive elements (Figure 3). This indicates that a single gene may contain multiple instances of the same hormone-related element, with the highest frequency observed in the abscisic acid response element. Furthermore, these promoters also contained other important cis-elements related to responses to biotic and abiotic stresses. Examples include elements responsive to low-temperature adversity (CCGAAA), elements related to light- and drought-induced MYB binding sites (MBS), elements related to defense and stress responsiveness (TC-rich repeats), elements involved in light responsiveness (G-box), and elements related to meristem expression (CAT-box).

2.6. Expression Profiles of HbPP2Cs in Different Tissues

To analyze the expression of HbPP2C genes under different stress treatments and to explore the potential functional roles of various subfamilies, 40 HbPP2C genes were randomly selected for experimentation in this study. As depicted in Figure 4, HbPP2Cs exhibited high expression levels in branches, followed by flowers, leaves, bark, and roots, with the lowest expression observed in latex. Specifically, 29 of these genes demonstrated high expression in branches. Among these genes, subfamilies A, B, C, and D exhibited notable expression in branches. Additionally, subfamilies B, C, F1, and J displayed high expression specifically in branches and flowers. Furthermore, HbPP2C46 and HbPP2C23 exhibited high expression in branches, leaves, flowers, and bark. These results suggest that the HbPP2C genes may play essential roles in these tissues (Figure 4).

2.7. Expression of HbPP2C Genes under Abiotic Stress Conditions

To further investigate the role of HbPP2Cs in response to abiotic stress, we examined the expression of HbPP2C genes in rubber tree following treatments with ABA, drought, glyphosate, high temperature, and low temperature. After ABA treatment, 36 HbPP2C genes were upregulated in rubber tree leaves (Figure 5). Notably, members of subfamily A, specifically HbPP2C6 and HbPP2C53, showed significant upregulation, with up to 31- and 25-fold increases observed at 10 and 24 h, respectively. Following drought treatment, the expression levels of 20 HbPP2C genes increased notably from 6 to 9 days after treatment (Figure 6). Among them, the expression levels of HbPP2C2, HbPP2C25, HbPP2C40, HbPP2C29, HbPP2C46, HbPP2C23, HbPP2C6, and HbPP2C44 were strongly upregulated. The C subfamily member HbPP2C29 exhibited the most substantial upregulation, reaching up to 33-fold. After glyphosate treatment, the expression of 29 HbPP2C genes remained unchanged (Figure 7). However, during 6 and 24 h of treatment, the expression of HbPP2C54, HbPP2C24, and HbPP2C59 was significantly upregulated by up to 60-fold or more. In response to the low-temperature treatment of rubber tree, the expression trends of genes from subclades A, B, C, and D exhibited an initial upregulation, followed by a decrease and then another upregulation (Figure 8). HbPP2C3 and HbPP2C24 maintained high expression levels during 3–12 h of treatment, with upregulation reaching approximately 100-fold. The expression of A, B, and F1 subfamily members was upregulated from 6 to 12 h after high-temperature treatment (Figure 9). Notably, HbPP2C9 and HbPP2C10 were upregulated 32 times after 12 h of treatment.
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Figure 5. Expression of 40 HbPP2Cs in rubber tree subjected to ABA treatment.
Figure 5. Expression of 40 HbPP2Cs in rubber tree subjected to ABA treatment.
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Figure 6. Expression analysis of 40 HbPP2Cs in rubber tree under drought treatment.
Figure 6. Expression analysis of 40 HbPP2Cs in rubber tree under drought treatment.
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Figure 7. Expression patterns of 40 HbPP2Cs in rubber tree in response to glyphosate treatment.
Figure 7. Expression patterns of 40 HbPP2Cs in rubber tree in response to glyphosate treatment.
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Figure 8. Expression analysis of 40 HbPP2Cs in rubber tree under low-temperature treatment.
Figure 8. Expression analysis of 40 HbPP2Cs in rubber tree under low-temperature treatment.
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Figure 9. Expression patterns of 40 HbPP2Cs in rubber tree under high-temperature treatment.
Figure 9. Expression patterns of 40 HbPP2Cs in rubber tree under high-temperature treatment.
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2.8. Expression of the HbPP2C Gene under Biotic Stress

In response to biotic stress, we analyzed HbPP2C gene expression in rubber tree leaves from 0 to 24 h after powdery mildew infection. The results revealed differential expression patterns of HbPP2Cs following powdery mildew infection. The expression levels of all HbPP2C genes increased to a certain extent after 3 h of powdery mildew infection (Figure 10). Notably, genes from the A and F1 subfamilies, such as HbPP2C6, HbPP2C54, and HbPP2C20, exhibited significant upregulation in expression at 3, 6 and 12 h post-infection. The upregulation could exceed 10-fold, with HbPP2C6 showing a remarkable upregulation of 33-fold, 21-fold, and 15-fold at 3, 6, and 12 months of infestation, respectively.
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Figure 10. Expression analysis of 40 HbPP2Cs in rubber tree under powdery mildew infection.
Figure 10. Expression analysis of 40 HbPP2Cs in rubber tree under powdery mildew infection.
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3. Discussion

3.1. Detailed Characterization and Evolution of HbPP2Cs in Rubber Tree

Extensive research has been conducted on PP2C genes in model and other species, highlighting their significance. In Arabidopsis, a total of 80 PP2C genes have been identified, while in O. sativa, 78 PP2C genes have been characterized [22]. Furthermore, 209, 131, and 56 PP2C genes have been identified in Brassica juncea [13], Brassica rapa [23], and cucumber [3], respectively, while 62 PP2C genes have been found in strawberry [24]. These findings illustrate the variation in the number of PP2C family genes among various species. In this study, 60 HbPP2C genes were identified in rubber tree for the first time using bioinformatics methods, and the expression of 40 representative HbPP2Cs from different subpopulations was analyzed. To analyze the evolution of the HbPP2C family in rubber tree, an evolutionary tree was constructed, and it was discovered that the HbPP2C family exhibited similarity to that of A. thaliana and O. sativa, categorized into 13 subgroups [22] (Figure 1). The number of genes in each subfamily ranged from 2 to 9. Phylogenetic analysis showed that each subfamily of Arabidopsis, O. sativa, and rubber tree contained PP2C proteins. This indicates that certain PP2C proteins emerged prior to the divergence of monocotyledons and dicotyledons. Therefore, it can be inferred that the structure and function of HbPP2Cs are conserved during rubber tree evolution. Additionally, we noted that there were 13 K subfamily members in Brachypodium distachyon [25], while both rubber tree and Arabidopsis had only three K subfamily members. This suggests significant variability in the distribution of individual subfamily members across different species.
The diversity in exon–intron number and positional patterns often plays a significant role in the evolutionary history of gene families. In this study, we conducted an analysis of the gene structures of HbPP2C based on their phylogenetic relationships. The exon–intron structures showed that most members within the same subfamily shared similar exon and intron numbers but exhibited substantial differences in exon and intron lengths. Most homologous genes have highly similar amino acid sequences and exon–intron compositions. Despite the expression diversification of homologous gene pairs, their functions were preserved. It was also found that there are great disparities in the number and distribution of exons among different subfamily members. For example, the L subfamily had exon numbers ranging from 3 to 16, while the H subfamily ranged from 5 to 8 (Table 1, Figure 3). Despite the genes in these two subfamilies having close genetic relationships, there was a significant disparity in their exon–intron arrangements. All HbPP2C genes without introns were members of subfamily G. Genes lacking introns amplified rapidly, suggesting that in the HbPP2C gene family, amplification in subfamily G may be linked to gene duplication. This demonstrates that expression diversification may happen within a brief timeframe, whereas changes in functional diversity require a long period of evolution.
The catalytic structural domain of PP2C phosphatase has been shown to have eleven conserved motifs, four of which play a crucial role in maintaining Mg2+/Mn2+ homeostasis [4,26]. Our results demonstrated that HbPP2Cs within the same subfamily shared similar conserved domains and motifs. However, differences were observed between members of different subfamilies, suggesting that HbPP2Cs within the same taxonomic group share a close evolutionary relationship (Figure 3). Additionally, motif 1, motif 6, and motif 7 either significantly increased or decreased in number and shifted in position. Conversely, motif 9, motif 10, and motif 11 were lost during the evolutionary process of some PP2C proteins. For instance, members of subfamily B lost motif 9 and motif 11. These observations may be related to natural selection driving different genetic variations in rubber plants in response to various environmental challenges. This highlights the role of natural selection in shaping the genetic diversity of rubber plants in the face of adversity.

3.2. HbPP2C Genes Are Specifically Expressed in Branches

Previous studies showed that PP2C genes are differentially expressed in different tissues and can act in different tissues [27]. For example, Yang’s results showed that MtPP2C genes showed different expression patterns in eight tissues of Medicago truncatula, including roots, stems, leaves, vegetative buds, flowers, pods, and nodules. Among them, MtPP2C2, MtPP2C4, and MtPP2C15 were highly expressed in eight tissues, while MtPP2C34, MtPP2C35, MtPP2C36, and other genes had low expression levels in all eight tissues. Some MtPP2C genes showed significantly different tissue-specific expression patterns in the eight tissues, e.g., MtPP2C32 was significantly more highly expressed in roots than in other tissues [15]. In the present study, it was observed that the HbPP2C genes were most highly expressed in branches, followed by flowers, leaves, bark, and roots, and least expressed in latex. Except for HbPP2C gene subfamilies J and F1, all other subfamily members showed high expression in branches, demonstrating that HbPP2Cs may play an instrumental role in the growth and development of rubber tree. Furthermore, it should be noted that the expression patterns of HbPP2C genes within the same subfamily differ in different tissues. In fact, even within different tissues, the expression patterns of the same gene exhibit variations. This suggests the presence of functional diversity among them.

3.3. HbPP2C Genes Play Important Roles in the ABA Pathway and Drought Stress Response

Drought, as one of the most severe abiotic stresses, poses a significant threat to plant growth and crop yield [28]. Plants have evolved a series of complex signaling pathways to resist drought stress, and ABA plays a key role in this process [29,30]. The ABA signaling process is the earliest reported signaling pathway involved in PP2Cs and is also the most widely investigated [31]. In this study, we found that many were related to various phytohormone-associated elements and stress-responsive elements in the promoter sequence of HbPP2Cs. This suggests that the number and type of cis-acting elements in the promoter region of the HbPP2C gene may be related to its response to stress. Previous studies have shown that wheat [32], barley [17], maize [16,33,34], O. sativa, and Arabidopsis PP2C genes play important roles in plant responses to abiotic stresses, especially in ABA signaling [12,35,36,37]. In cucumber plants, the expression levels of CsPP3C11, 17–23, 45, 54, 55, and 2 were significantly upregulated under drought and salt treatments [3]. In Brachypodium distachyon [25], six genes of subfamily A were highly induced by exogenous ABA treatments, and similarly, BdPP2C of subfamily A was also upregulated to different degrees by drought, among which BdPP2C36, BdPP2C37, and BdPP2C44 consistently exhibited upregulation and sustained high levels in response to all stress treatments. In addition, BdPP2C70 of subfamily D, BdPP2C13 of subfamily F, and BdPP2C32 of subfamily G also showed strong expression levels under ABA treatments [25], indicating that some members of the other subfamilies may be involved and act as regulators in the ABA signaling pathway. Previous research results have shown that both glyphosate and drought can induce the regulation of ABA in plants [38]. Consistent with the results of this study, glyphosate, ABA, and drought highly induced the expression of most members of the HbPP2C subfamily A, B and, C genes in rubber tree at different time intervals, with a significant increase in the expression of genes. Thus, genes of subfamily A, B, and C HbPP2Cs may play an instrumental role in the ABA signaling pathway and drought stress response in rubber tree.

3.4. HbPP2C Genes Regulate the Temperature Stress Response in Rubber Tree

High- and low-temperature stress can induce PP2C expression in a variety of plants, such as A. thaliana [22], maize [39], and cotton [5]. Plants overexpressing subfamily B members of the maize ZmPP2C family in tobacco had higher germination rates and antioxidant enzyme activity under low-temperature stress and enhanced tolerance to cold stress, suggesting that ZmPP2C2 may be a positive regulator of plant resistance to low-temperature stress [39]. Additionally, FGT2 is a member of subfamily D of the Arabidopsis PP2C family and is involved in the regulation of high-temperature stress memory by interacting with the phospholipase PLDα2 [40]. In the present study, after low-temperature treatment, each family member of HbPP2Cs (A, B, C, D, F1, J) showed significant upregulation at 3 h and 12 h (Figure 8). The expression levels of each HbPP2C family member (A, B, D, F1) were significantly increased at 12 h after high-temperature treatment (Figure 9). Hence, HbPP2Cs participate in the response of plants to temperature stress, and the functions of the members of subfamilies A, B, C, D, F1, and J deserve further exploration.

3.5. HbPP2Cs Play an Influential Role in the Response to Biological Adversity Stresses

In addition to abiotic stresses, biotic stresses such as insect pests and pathogenic bacteria also threaten plant growth. Plants have developed a series of mechanisms to resist these stresses, among which PP2Cs also have a positive regulatory function. Research has indicated that the expression of tomato PP2C genes is triggered by the fungus Cladosporium fulvum [41] and the oomycete Phytophthora parasitica [9,42]. Among the walnut (Juglans regia) PP2C family genes, JrPP2C36 expression was significantly upregulated after inoculation with Colletotrichum gloeosporioide [19]. In this study, the expression levels of rubber tree HbPP2C genes were determined after inoculation with powdery mildew bacteria, and the expression of A (HbPP2C6, HbPP2C56, HbPP2C54, HbPP2C37) and F1 (HbPP2C20, HbPP2C58) subfamily members was significantly upregulated by up to 33-fold (Figure 10). Overall, the HbPP2C genes may also exert an active role when rubber tree is exposed to biotic stresses.

4. Materials and Methods

4.1. Plant Material

Seedlings and tissue samples of the rubber tree cultivar ‘Reyan 73397’ at various growth stages were sourced from the experimental base of the Rubber Research Institute, Chinese Academy of Tropical Agriculture Sciences (19°51′51 N; 109°55′63 E). Samples of 20 g roots, 20 g branches, 20 g leaves, 10 g flowers, 5 g bark and 500 mL latex of three 18-year-old healthy rubber trees were collected and used for expression analysis in different tissues. At the same time, healthy seedlings with 1 canopy of new leaves and consistent height were selected and planted in flower pots using peat soil. The seedlings were cultured with Hoagland nutrient solution every two weeks for gene expression pattern analysis.

4.2. Plant Treatment

Multiple groups of rubber seedlings were subjected to specific treatments, with each group consisting of three plants for experimentation. Various groups of rubber seedlings underwent specific treatments, with three plants included in each experimental group. For the hormone and chemical treatment of leaves, one group of rubber seedlings was exposed to a solution containing 200 μmol L−1 glyphosate and 200 μmol L−1 ABA. In contrast, another group of rubber seedlings received a spray of distilled water. Leaves were collected at various time points, including 0, 0.5, 2, 6, 12, 24, and 48 h post-treatment. In the drought treatment experiment, two groups of rubber seedlings were initially saturated with water in an incubator for 10 days. Subsequently, one group continued to receive regular water, serving as the control, while the other group’s water supply was discontinued. Leaves from both groups were collected at different time points: 0, 1, 3, 6, and 9 days after the cessation of watering. For the high- and low-temperature treatment experiments, rubber seedlings were placed in an incubator set at 40 °C/16 °C for the experimental group, while the control group was maintained at 25 °C. Leaves were collected from both groups at varying time intervals: 0, 3, 6, and 12 h post-treatment. In the powdery mildew infestation experiment, one group was inoculated with the Oidium heveae isolate HO-73, while the control group remained untreated. These rubber seedlings were then nurtured at 20 °C under conditions of 70–90% relative humidity, with a 12 h light/dark cycle. Leaves from both groups were collected at different time points: 0, 0.5, 2, 6, 12, 24, and 48 h post-treatment. Each experiment was conducted three times, and all collected samples were promptly frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

4.3. Gene Sequence Acquisition

The PP2C sequences of Arabidopsis thaliana and O. sativa were acquired from the TAIR database (https://www.arabidopsis.org/, accessed on 19 May 2023) and O. sativa Genome Annotation Project (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml, accessed on 19 May 2023). The A. thaliana PP2C sequences served as templates for searching for HbPP2C sequences within the rubber tree genome. Sequences retrieved via BLAST were carefully curated to remove any duplicate entries. Subsequently, the open reading frames of the identified genes were searched using the NCBI ORF finder website (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 19 May 2023). Then, we employed the NCBI Conserved Domain Search to predict the amino acid sequences of these candidate PP2C genes.

4.4. HbPP2C Structural and Functional Analyses

Information regarding the PP2C genes, involving exon numbers and coding sequences (CDS), was sourced from the NCBI website and subsequently verified using FGENESH (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind, accessed on 22 May 2023). The theoretical isoelectric point (PI) and molecular weight of the PP2C genes were calculated using the ProtParam website (https://web.ExPASy.org/protparam/, accessed on 25 May 2023). For further analysis, the PP2C gene promoter sequences (2 kb upstream of the gene initiation codon) were subjected to cis-element analysis utilizing the Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 29 May 2023) and then visualized using TBtools. Furthermore, the amino acid sequences of all 60 PP2C genes were used for motif prediction with the MEME Suite website (https://meme-suite.org/meme/, accessed on 22 May 2023). The results were acquired in MEME.xml format and subsequently processed for visualization using TBtools software (Version 2.003).

4.5. Sequence Alignment and Phylogenetic Analysis

A total of 80 Arabidopsis PP2C protein sequences and 78 O. sativa PP2C protein sequences were obtained from the TAIR database and O. sativa Genome Annotation Project. To analyze the obtained PP2C amino acid sequences, Multiple Sequence Alignment (MUSCLE) within Molecular Evolutionary Genetic Analysis (MEGA X) software (Version 10.0.5) was employed using default parameters. Subsequently, a phylogenetic analysis was conducted using the neighbor-joining (NJ) method, and the parameters were validated through 1000 bootstrap replicates. Additionally, a phylogenetic tree was constructed based on these sequences.

4.6. Expression Analysis of HbPP2Cs Using qRT–PCR

RNA extraction from all rubber tree samples followed the protocols provided by the TIANGEN Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Takara, Tokyo, Japan). Subsequently, the obtained 1 µg RNA samples were subjected to reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, Beijing, China), and the resulting cDNA was utilized for qRT–PCR experiments. The primers for qRT–PCR were designed using Primer 5 (Version 5.00), with HbActin (GenBank accession: HO004792) serving as the internal control gene (Table S3). The qRT-PCRs were conducted using the SYBR Premix Ex Taq II Kit (Takara, Japan) in a total volume of 20 µL. Each treatment consisted of three biological replicates per sample and three technical replicates. Expression patterns were analyzed employing the 2−∆∆CT method. Statistical analysis was carried out using one-way ANOVA, and multiple comparison analyses were performed using Tukey’s test at a significance level of p < 0.05. The obtained results were visualized using heatmaps generated with TBtools.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242216061/s1.

Author Contributions

Conceived and designed the experiments: M.W., B.Q., Y.Y. and Y.Z.; performed the experiments: D.Z. and Q.L.; analyzed the data: X.L. and L.W.; contributed reagents/materials/analysis tools: X.L., B.Q. and M.W.; wrote the manuscript: Q.L., L.W. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (32160370), as well as the Modern Agro-industry Technology Research System (CARS-33-BC1) and Hainan University Collaborative Innovation Center Project (XTCX2022NYC28).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this article and are available upon reasonable request to the corresponding author.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 2. Conserved structural domains and motifs of the HbPP2C protein. The Conserved structural domains and motifs of the HbPP2C proteins were analyzed using the NCBI Conserved Domain Search and the MEME web server. Colored blocks represent conserved domains or motifs. The letters express different subfamilies of HbPP2Cs.
Figure 2. Conserved structural domains and motifs of the HbPP2C protein. The Conserved structural domains and motifs of the HbPP2C proteins were analyzed using the NCBI Conserved Domain Search and the MEME web server. Colored blocks represent conserved domains or motifs. The letters express different subfamilies of HbPP2Cs.
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Figure 3. Gene structure and promoter cis-acting elements of HbPP2Cs in rubber tree. The letters express different subfamilies of HbPP2Cs.
Figure 3. Gene structure and promoter cis-acting elements of HbPP2Cs in rubber tree. The letters express different subfamilies of HbPP2Cs.
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Figure 4. Expression profiles of 40 HbPP2Cs in different tissues in rubber tree. The color bar represents the gene expression level, with red indicating high expression level, white indicating no expression, and brown indicating low expression level of transcript abundance. The letters express different subfamilies of HbPP2Cs (the same in Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10).
Figure 4. Expression profiles of 40 HbPP2Cs in different tissues in rubber tree. The color bar represents the gene expression level, with red indicating high expression level, white indicating no expression, and brown indicating low expression level of transcript abundance. The letters express different subfamilies of HbPP2Cs (the same in Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10).
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Liu, Q.; Qin, B.; Zhang, D.; Liang, X.; Yang, Y.; Wang, L.; Wang, M.; Zhang, Y. Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree. Int. J. Mol. Sci. 2023, 24, 16061. https://doi.org/10.3390/ijms242216061

AMA Style

Liu Q, Qin B, Zhang D, Liang X, Yang Y, Wang L, Wang M, Zhang Y. Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree. International Journal of Molecular Sciences. 2023; 24(22):16061. https://doi.org/10.3390/ijms242216061

Chicago/Turabian Style

Liu, Qifeng, Bi Qin, Dong Zhang, Xiaoyu Liang, Ye Yang, Lifeng Wang, Meng Wang, and Yu Zhang. 2023. "Identification and Characterization of the HbPP2C Gene Family and Its Expression in Response to Biotic and Abiotic Stresses in Rubber Tree" International Journal of Molecular Sciences 24, no. 22: 16061. https://doi.org/10.3390/ijms242216061

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