Next Article in Journal
Epigenetics of Skeletal Muscle Atrophy
Previous Article in Journal
Fungicide-Loaded Liposomes for the Treatment of Fungal Diseases in Agriculture: An Assessment of Botrytis cinerea
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 15
10.3390/ijms25158361
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

Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses

by
Dengbao Wang
1,2,3,
Zimo Qiu
1,2,3,
Tao Xu
1,2,3,
Sheng Yao
1,2,3,
Meijing Chen
1,2,3,
Qianzi Li
1,2,3,
Romaric Hippolyte Agassin
1,2,3 and
Kongshu Ji
1,2,3,*
1
State Key Laboratory of Tree Genetics and Breeding, Nanjing Forestry University, Nanjing 210037, China
2
Key Open Laboratory of Forest Genetics and Gene Engineering of National Forestry & Grassland Administration, Nanjing 210037, China
3
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(15), 8361; https://doi.org/10.3390/ijms25158361
Submission received: 25 June 2024 / Revised: 26 July 2024 / Accepted: 29 July 2024 / Published: 31 July 2024
(This article belongs to the Special Issue Molecular Mechanisms of Zinc Finger Proteins in Biological Processes and Diseases)
Browse Figures
Review Reports Versions Notes

Abstract

:
Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif ‘QALGGH’ were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.

1. Introduction

Pinus massoniana, a widely distributed coniferous tree in southern China, is not only an economically important species for timber, wood pulp, and rosin but also plays a significant ecological role in the forest ecosystem [1]. The occurrence of seasonal soil drought in Southern China poses a significant natural constraint on the production and growth of Masson pine [2]. In addition, the prevalence of pine wood disease in China over the past four decades has caused a tremendous disaster for coniferous plants. Therefore, it is highly necessary to cultivate drought-resistant and disease-resistant Masson pine. A large number of transcription factors (TFs) were involved in the response to biotic and abiotic stresses [3,4,5,6,7,8,9,10,11,12,13]. Recent studies regarding comprehensive genome-wide sequencing, various transcriptome analyses, and the functional identification of numerous genes have demonstrated the involvement of various transcription-factor families in conferring abiotic stress tolerance in plants, including MYB [4,5], AP2/ERF [7], NAC [8], bHLH (basic helix–loop–helix) [10], bZIP (basic leucine zipper) [11], homeodomain [13], WRKY [14], HSF [15], MADS-box [16], and zinc finger proteins [17]. The large and diverse zinc finger protein family plays important roles in all aspects of plant growth and development [18]. The first zinc finger protein (TF IIIA) was initially observed in Xenopus laevis oocytes in 1985, and the term ‘zinc finger’ was coined to describe their distinctive structural resemblance to fingers [19]. Since then, zinc fingers with a variety of functions have been found in animals, plants, yeasts, and viruses [20].
Zinc finger proteins (ZFPs) play a critical role in transcriptional regulation, RNA binding, the regulation of apoptosis, and protein–protein interactions [21]. They can be classified as C2H2, C2C2, C2HC, C8, C2HC5, C3HC4, CCCH, C6, C4, and C4HC3 [22,23]. Among these types, C2H2-type zinc finger proteins are widely recognized as one of the most abundant and extensively investigated protein families in eukaryotes [8]. C2H2 ZFPs contain two cysteine (C) and two histidine (H) residues in each finger, and these residues bind a zinc ion that stabilizes the ZFP and specifically binds to a domain within the promoter of the gene it regulates [24]. In plants, numerous C2H2-ZFPs exhibit conserved structural features among themselves. Its sequence is characterized by C-X2-4-C-X12-H-X3-5-H (X represents any amino acid) [25]. In multiple-fingered proteins, the adjacent fingers are separated by a long spacer that is highly variable in length and sequence from each other [21]. Most fingers have a six-amino acid stretch, ‘QALGGH’, at a position corresponding to the N-terminal part of the recognition helix. They are called Q-type C2H2 ZFPs [26].
Currently, an increasing number of Q-type C2H2 ZFPs have been identified in various plants. Arabidopsis thaliana has been reported to possess a total of 176 C2H2 ZFPs [27], while 189, 109, 321, 54, and 47 C2H2 ZFPs have been identified in rice (Oryza sativa) [28], poplar (Populus trichocarpa) [29], soybean (Glycine max) [30], apple (Malus domestica Borkh.) [31] and wheat (Triticum aestivum) [32], respectively. Recent functional analysis has shown that C2H2-ZFPs are involved in regulating multiple growth-development processes and resisting biotic and abiotic stress in plants [12,20]. EPF1 was identified from Petunia as the first plant-specific ZFP that interacted with the promoter region [33]. Later, WZF1 was reported in wheat as a DNA-binding zinc finger protein that interacts with a cis element of histone genes [34]. C2H2-ZFP245 might function as a downstream component of CBF/DREB proteins by repressing the expression of some genes in response to cold or drought stress in O. sativa [35]. The overexpression of C2H2-SlZF3 in Solanum lycopersicum and A. thaliana resulted in the accumulation of AsA, thereby alleviating the oxidative damage caused by salt stress through the enhancement of the transgenic plants’ ROS-scavenging capacity [36]. In Glycine max, C2H2-GmSCOF-1 was induced by low temperature and abscisic acid (ABA) treatments but not by dehydration or high salinity [37]. These results suggest that C2H2-ZFPs act as transcriptional activators or repressors in different stress signal-transduction pathways to regulate the transcriptional levels of downstream genes.
However, the identification of C2H2-ZFPs and stress-related functional analysis in P. massoniana is still lacking. In recent years, the advancements in transcriptome and genome analysis have provided us with an opportunity to identify TF families in plants that play important roles in diverse stress responses. In this study, 57 PmC2H2-ZFPs were identified from three stress-related transcriptomes and characterized using bioinformatic analysis. Then, the expression profiles of Q-type PmC2H2 in different tissues and stresses were studied to better understand their roles in regulating P. massoniana stress response. The results of this study provide insights into the characterization of PmC2H2-ZFPs, as well as screening a potential regulatory gene in response to biotic and abiotic stresses in P. massoniana.

2. Results

2.1. Transcriptome-Wide Identification and Analysis of C2H2 ZFPs in P. massoniana

After excluding incomplete and highly homologous sequences (>97%), further confirmation of the conserved domain was obtained through prediction from SMART and CD-search. We identified 57 C2H2 ZFPs with a highly conserved C2H2 domain (e-value < 0.001) from three transcriptomes in response to biotic and abiotic stresses in P. massoniana (Table S1). We designated them as PmC2H2-1 to PmC2H2-57 (Table 1). These C2H2 ZFPs exhibited significant variations in length, ranging from 239 to 725 amino acids (Aa), with an average length of 477.34 aa. The molecular weight (MW) of PmC2H2s ranged from 27.41 kDa to 81.74 kDa, while the isoelectric point (pI) values ranged from 4.82 to 9.46. Subsequently, subcellular localization prediction results revealed that the majority of PmC2H2s were localized in the nucleus, except for one instance where PmC2H2-29 was found to be localized in the chloroplast.

2.2. Phylogenetic Analysis and Domain Analysis of PmC2H2s

A phylogenetic tree was constructed using the ML method, as shown in the left part of Figure 1, which facilitated the classification of these 57 PmC2H2 ZFPs into five distinct subgroups denoted as ‘I’ to ‘V’. Among them, group I represents the largest subgroup, comprising a total of 33 C2H2-ZFP members. The smallest group was Group III, which consisted of PmC2H2-8 and PmC2H2-43. Groups II, IV, and V contain 11, 8, and 3 members, respectively. According to the MEME program, for the identification of the conserved motifs of 57 C2H2 ZFPs (Figure 1), the amino acid length of the 10 motifs ranged from 8 to 50 (Table 2). We found that all members had at least one highly typical conserved C2H2 domain (motif 1). Motif 2 was distributed in all groups. In addition, motifs 5 and 7 were only found in group I. Motif 3 and motif 6 were found in six members in group III with an exception in group I (PmC2H2-12). In addition, we found that six members (PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33) contained the ‘QALGGH’ domain in group IV; they are also called Q-type C2H2 ZFPs. The occurrence of motif 4 was frequently observed in groups I and II. Motif 8 was distributed in all groups except Group V. Motif 9 occurs primarily in group I, with an exception in group II (PmC2H2-52). Motif 10 was distributed in group II and group V. In order to further determine the type and distribution of the C2H2 domains, a regular expression search showed that the most common C2H2 domain is C-X2-C-X12-H-X3-H, accounting for 49% of all C2H2, followed by C-X4-C-X12-H-X3-H and C-X4-C-X12-H-X4-H, accounting for 19% and 14%, respectively. However, the structure of C-X3-C-X12-H-X3-H and C-X3-C-X12-H-X5-H have not been found in Masson pine (Table S3). Considering that Q-type C2H2 is unique to plants and is widely involved in plant responses to both biotic and abiotic stresses, the functional studies of zinc finger proteins have predominantly focused on Q-type C2H2 members. However, there are no studies of Q-type C2H2 ZFPs that have been reported on Masson pine yet. Therefore, we selected these six Q-type members for further study (Table S2).

2.3. Subcellular Localization Analysis

Subcellular localization prediction indicated that most C2H2 TFs likely exert regulatory functions at the nucleus. In order to further comprehend the localization features of PmC2H2 ZFPs, PmC2H2-4 and PmC2H2-20 were selected for the experiment. A fluorescent signal was found after transient transformation in tobacco leaves. The GFP signal was distributed throughout the whole cell in the control; however, mGFP5 fused with PmC2H2-4 and PmC2H2-20 only showed fluorescence in the nucleus (Figure 2). The results indicated that PmC2H2-4 and PmC2H2-20 are nuclear localization proteins.

2.4. Expression Patterns of PmC2H2 ZFPs under Nematode and Drought Stress

Drought and nematodes are the two most important factors currently limiting the growth and development of the Masson pine. Therefore, two heatmaps were generated to analyze the expression levels of PmC2H2 ZFPs under nematode infestation and drought stress conditions, based on the transcriptome data (Figure 3). The transcriptome data represent gene-expression patterns in a specific temporal and spatial context, so only 34 and 22 expression patterns of PmC2H2 ZFPs were obtained from the RNA-seq transcriptomics data, respectively. After infection by nematodes, 18 members of the C2H2 family showed induced expression at different time points. The expression of 5 members was suppressed after nematode infection, while the expression levels of 11 members initially increased and then decreased over time. As the drought intensifies, the expression levels of 15 members tend to increase, while the expression levels of 7 members decrease.

2.5. Expression Patterns of Q-Type C2H2 ZFPs Genes in Different Tissues

The qRT-PCR analysis in Figure 4 showed the expression patterns of six Q-type PmC2H2s in eight different tissues: shoot apices (T); young needle leaves (YL); old needle leaves (OL); young stem (YS); old stem (OS); xylem (X); phloem (P); and root (R). It is evident from the results that PmC2H2-4, PmC2H2-5, and PmC2H2-20 exhibit high expression in needles. PmC2H2-16 and PmC2H2-20 expressed significantly in the root, while PmC2H2-33 expressed highly in the young stem. The expression levels of PmC2H2-4 and PmC2H2-5 in the phloem are extremely low, while PmC2H2-20 and PmC2H2-24 have the lowest expression in the xylem. In addition, PmC2H2-33 exhibited a low expression in the old stem. Notably, the expression of PmC2H2-16 in stems and the xylem was almost undetectable.

2.6. Expression Levels of Q-Type C2H2 ZFPs Genes under Abiotic Stresses

Figure 5 shows the expression patterns of six genes under different abiotic stresses. PmC2H2-20 and PmC2H2-24 were induced significantly by ABA treatment (Figure 5a), while other genes exhibited lower expression levels under ABA treatment. Under natural drought conditions, with the exception of PmC2H2-20 which showed suppressed expression on the 20th day of drought, other genes were induced at different stages of drought. PmC2H2-20 and PmC2H2-33 were suppressed significantly by ETH (Figure 5c), and PmC2H2-5 was not sensitive to ETH treatment. The expressions of PmC2H2-4, PmC2H2-16, and PmC2H2-24 increased significantly at first and then decreased after 6 h. The expression of PmC2H2-5 and PmC2H2-16 were suppressed under H2O2 treatment, while other genes were induced by H2O2 first, and then, their expression presented a downward trend (Figure 5d). The PmC2H2-20 gene exhibited no sensitivity to mechanical injury stress (Figure 5e), while the expression levels of the remaining five genes displayed an initial increase followed by a subsequent decrease over time, with peak expression observed at either 3 h or 6 h. The expressions of PmC2H2-16, PmC2H2-20, and PmC2H2-33 were inhibited by MeJA (Figure 5f), and the expression levels of PmC2H2-24 increased significantly after MeJA treatment. The expression of PmC2H2-20 was significantly inhibited under salt stress (Figure 5g), and the expression changes of PmC2H2-5 under salt stress were not obvious. The expression levels of PmC2H2-20 and PmC2H2-33 significantly decreased after PEG treatment, while the other four genes were induced to varying degrees (Figure 5h). After SA treatment, it was worth noting that the expression level of PmC2H2-20 significantly increased within 3 h and then recovered. The expression levels of PmC2H2-20 were found to be significantly suppressed in most treatments, except for ABA and SA treatment, suggesting that PmC2H2-20 may play a significant role in stress resistance in Masson pine.

2.7. Transcriptional Activity Analysis

The transcriptional activity of Q-type C2H2 was analyzed. We successfully cloned PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 and constructed them into the PGBKT7 vector. The yeast strains that contained recombinant plasmids were cultured on the SD/-Trp medium first. Verified positive yeast colonies were collected onto the higher selective culture medium SD/-Trp/-His and SD/-Trp/-His/-Ala. The photographs of the yeast growth assay (Figure 6) showed that only PmC2H2-20 can grow on the severe selective medium (SD/-Trp/-His/-Ala) and activate the expression of reporter genes, resulting in the production of β-galactosidase, which turns substrate x-α-gal blue, whereas the remaining five genes did not exhibit any self-activation. This result suggested that the PmC2H2-20 can act as a transcriptional activator by binding to the promoter sequence of downstream reporter genes, thereby activating their transcription and helping P. massoniana resist stress.

3. Discussion

The C2H2-type zinc finger proteins are widely distributed among eukaryotes and play significant roles in diverse biological processes, including hormone signaling, DNA or RNA binding, and stress response [22,38,39,40]. Moreover, extensive exploration has been conducted to identify C2H2-type zinc finger proteins in various plant species. However, the lack of a reference genome for P. massoniana has created a knowledge gap in the comprehensive identification of C2H2 TF families in P. massoniana at the genomic level. Therefore, we can only temporarily fill this gap by using transcriptome data to identify stress-response TFs, thereby establishing a foundation for future research.
A total of 57 PmC2H2 ZFPs were identified from three transcriptomes and divided into five subgroups. Almost all PmC2H2 ZFPs were predicted to localize in the nucleus, supporting their role as a transcription factor in the nucleus. In addition, we confirmed the subcellular localization of PmC2H2-4 and PmC2H2-20 in tobacco leaves through instantaneous transformation, which is consistent with the prediction. In yeast and A. thaliana, C2H2 ZFPs were classified based on the quantity and configuration of ZF domains [41]. The C2H2 sequence is especially characterized by C-X2,4-C-X12-H-X4-5-H in P. massoniana. However, it lacks the forms of C-X3-C-X12-H-X3-H and C-X3-C-X12-H-X5-H compared to other plants. The majority type of conserved C2H2 domains exhibited in P. massoniana are C-X2-C-X12-H-X3-H.
In addition, previous researchers have demonstrated that the surrounding residues at the C-terminus of the ‘QALGGH’ motif play a pivotal role in facilitating specific DNA recognition [42]. The AGC(T) sequence was identified as the optimal binding site for the first zinc finger (ZF) of the Petunia ZPT2-2 protein, while the CAGT core was determined to be the preferred binding site for the second ZF [43]. This discovery confirms that flanking residues play a pivotal role in determining specificity for recognizing DNA target sequences and facilitating effective DNA binding [44]. We also identified six Q-type C2H2 ZFPs from Masson pine, all of which possess a ‘QALGGH’ motif. Current research has shown that Q-type C2H2 ZFPs are widely involved in plant biotic and abiotic stress responses. In addition, the expression of C2H2 ZFP is modulated by tissue-specific variations and abiotic stresses [45]. Consequently, we further investigated the expression patterns of six Q-type C2H2s under diverse stresses and tissues.
The role of C2H2 ZFPs as transcription factors in response to abiotic stresses has been extensively studied across various plant species [46]. The upregulation of a series of resistance genes can be induced by ABA, leading to their enhanced expression under conditions of drought and salt stress. This mechanism enhances the plant’s capacity to endure osmotic stress [47,48]. STZ/ZAT10 derived from A. thaliana can serve as a supplementary factor for yeast calcineurin mutants, enhancing salt tolerance in transgenic yeast [21,49,50]. The expression of LkZFP6 in Larix kaempferi can be induced by ABA treatment [51]. The qRT-PCR results revealed that PmC2H2-20 and PmC2H2-24 can be induced by ABA treatment, indicating that they may regulate Masson pine in response to abiotic stresses through the ABA-mediated signal pathway.
It is worth noting that PmC2H2-20 exhibits significant inhibition under nematode stress and treatments other than ABA and SA, indicating its involvement in the biotic and abiotic stress response, such as drought, H2O2, damage, MeJA, NaCl, and PEG. In addition, PmC2H2-24 can be induced by all treatments and nematode infection, suggesting its potential involvement in response to external stresses in P. massoniana. The stress-induced response pattern of the PmC2H2-4 closely resembles that of PmC2H2-24, with the exception that its expression level is repressed upon ABA treatment. The remaining three Q-type C2H2 ZFPs exhibited distinct expression patterns in response to multiple stresses, implying the involvement of Q-type C2H2 genes in response to abiotic stresses.
The tissue-specific expression of genes usually is preliminarily used to predict their corresponding functions [52]. Previous research reported that the expression profiles of plant C2H2-ZFPs vary in different tissues and abiotic stresses [12,45]. A tissue-specific expression analysis showed that PmC2H2-4, PmC2H2-5, and PmC2H2-20 may perform specific functions in needles. Whereas PmC2H2-16 and PmC2H2-24 function in the root, PmC2H2-33 functions in the young stem. A previous report showed that ZAT18 (AT3G53600) played a positive role in drought tolerance in Arabidopsis [53]. PmC2H2-16 and PmC2H2-24, which are induced by drought, may be correlated with root growth and drought tolerance in P. massoniana.
In this study, we found the transcriptional activation of PmC2H2-20 in the yeast system, indicating that PmC2H2-20 could activate the expression of downstream reporter genes. The C2H2-type zinc protein of A. thaliana has been demonstrated in relevant studies to serve as a pivotal regulator of ROS signaling, thereby functioning as an integrative factor for coordinating responses to diverse abiotic stresses [54].
Although the role of C2H2 ZFPs has been extensively studied in model plants, such as Arabidopsis and Oryza sativa, there is little research on their involvement in pinaceae’s tolerance to both biotic and abiotic stresses. Our study has successfully identified 57 C2H2 ZFPs from the transcriptomes of Masson pine for the first time and analyzed their expression patterns under two major stress conditions currently restricting the development of P. massoniana. Furthermore, we have thoroughly investigated the expression patterns of Q-type C2H2 members under various abiotic stresses and screened a potential candidate gene, PmC2H2-20, which may play a crucial role as an important regulatory factor in the stress response of P. massoniana. These findings provide insights for future functional studies on the C2H2 genes involved in stress resistance mechanisms in P. massoniana.

4. Materials and Methods

4.1. Identification of C2H2 Genes in P. massoniana

We obtained the Hidden Markov Model (HMM) profile of the C2H2 domain (PF00096) from the Pfam database (http://pfam.xfam.org/) (accessed on 2 August 2023). The HMM profile was used to search for C2H2 proteins from three P. massoniana transcriptomes, namely CO2 stress transcriptome (SRA accession: PRJNA561037) [55], drought stress transcriptome (SRA accession: PRJNA595650) [56], and P. massoniana, inoculated with the pine wood nematode transcriptome (SRA accession: PRJNA660087). A BLASTP search was performed against four transcriptomes using the Hidden Markov Model (HMM) profile. We selected sequences with the C2H2 domain (e-value < 0.001) and deleted repeated sequences with a similarity of more than 97%. Transcription Factor Prediction (http://planttfdb.gao-lab.org/prediction.php) (accessed on 2 August 2023) was used to predict putative C2H2 proteins. Then, we use SMART (https://smart.embl.de/) (accessed on 2 August 2023) and NCBI Conserved Domain Search (CD Search) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 2 August 2023) to check the conserved domain of PmC2H2 TFs. Molecular weights and isoelectric points (pI) of identified PmC2H2 proteins were calculated using the ExPaSy site6 (https://web.expasy.org/compute_pi/) (accessed on 2 August 2023).

4.2. Phylogenetic and Bioinformatics Analysis

The maximum likelihood (ML) method implemented in MEGA-X was employed to sample 1000 bootstraps from the phylogenetic tree [57]. For visualization purposes, the phylogenetic tree underwent editing using EvolView online software (v2) (https://www.evolgenius.info/evolview) (accessed on 3 August 2023). To identify potential conserved motifs, we utilized the Multiple Expectation Maximization for Motif Elicitation (MEME) program (https://meme-suite.org/meme/tools/meme) (accessed on 3 August 2023) and the number of motifs was set to 10. Subcellular localization prediction and analysis of PmC2H2 protein were conducted using CELLO (http://cello.life.nctu.edu.tw/) (accessed on 3 August 2023), WoLF PSORT (https://wolfpsort.hgc.jp/) (accessed on 3 August 2023), and Plant-mPLoc tools (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) (accessed on 3 August 2023).

4.3. Subcellular Localization Analysis

The open reading frame (ORF) regions of PmC2H2-4 and PmC2H2-20 without a stop codon were linked with the pCAMBIA-1302-mGFP5 vector (primers were listed in Table S4). After PCR verification, the positive Agrobacterium was transferred to an LB medium supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L), followed by incubation until the optical density (OD) value reached 0.6. Subsequently, it was co-cultivated with the P19 (RNA Silencing Inhibitor) Agrobacterium strain in a suspension containing 150 µM acetosyringone, 10 mM MgCl2, and 10 mM 2-(N-morpholino) ethanesulfonic acid (MES). The leaves of 4-week-old Nicotiana benthamiana were injected with the mixed solution. Afterwards, the infiltrated N. benthamiana plants were kept in darkness for 48 h. The LSM710 confocal microscope (Zeiss, Jena, Germany) was used to capture the GFP signal.

4.4. RNA-Seq Data Analysis

The RNA-seq data of Masson pine under drought stress (PRJNA595650) were collected at four different field capacities representing natural drought conditions: CK (normal water supply) (80 ± 5)%, LD (light drought stress mild) (65 ± 5)%, MD (moderate drought stress) (50 ± 5)%, and SD (severe drought stress) (35 ± 5)%. In the case of pine wood nematode treatment (PRJNA660087), needles were sampled from each lateral branch at various time points (0 d, 3 d, 10 d, 20 d, and 35 d) after inoculation. Fragments per kilobase of the exon model per million reads mapped (FPKM) values were calculated to estimate the abundance of C2H2 ZFP transcripts. Heat maps depicting partial gene expression patterns based on log2(FPKM + 1) values were generated using TBtools software (v2.034) [58], with analyses performed at the row scale.

4.5. Plant Materials and Abiotic Stress Treatments

Two-year-old, healthy, and uniformly sized seedlings of P. massoniana were selected for tissue-specific analysis and abiotic treatments. The drought treatment was performed as follows. Needles were collected five times over a period of 20 days, allowing for natural evaporation after watering at day 0. The samples were collected at five time points with the field capacity as follows: 0 d (67%); 3 d (63%); 7 d (58%); 12 d (46%); and 20 d (34%). The osmotic stress was induced by soaking the seedlings in the 15% polyethylene glycol (PEG6000) solution and 200 mM NaCl solution. The mechanical damage treatment method was performed by cutting the upper half of the needles. For plant hormones treatment, the selected seedlings were sprayed independently with 100 µM ABA (abscisic acid); 1 mM SA (salicylic acid); 10 mM H2O2 (hydrogen peroxide); 10 mM MeJA (methyl jasmonate); and 50 µM ETH (ethephon) solutions (50 mL) on the surface of needles. Afterward, needles were sampled at 0 h, 3 h, 6 h, 12 h, and 24 h after treatment. All treatments were conducted on three biological replicates.

4.6. RNA Extraction and qRT-PCR Analysis

The total RNA of P. massoniana was extracted following the protocol provided in the FastPure plant total RNA isolation kit (RC401, Vazyme Biotech, Nanjing, China). The RNA concentration and purity were measured with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA integrity was estimated by 1% agarose gel electrophoresis. First-strand cDNA was synthesized using the One-step gDNA Removal and cDNA Synthesis Kit (AT311, TransGen Biotech, Beijing, China). Primers for quantitative real-time reverse transcription PCR (qRT-PCR) were designed using Primer 5.0 (Table S4). SYBR Green reagents were used to detect the target sequence. Each PCR mixture (10 µL) contained 1 µL of diluted cDNA (20× dilution), 5 µL of SYBR Green Master Mix (11184ES03, Yeasen Biotech, Shanghai, China), 0.4 µL of each primer (10 µM), and 3.2 µL of ddH2O. The PCR program stages were (1) 95 °C for 2 min (preincubation); (2) 95 °C for 10 s, and (3) 60 °C for 30 s, repeated 40 times. The remaining steps use the instrument’s default settings. The PCR quality was estimated based on the melting curves. The alpha-tubulin (TUA) gene was used as a reference gene [59]. Three independent biological replicates and three technical replicates for each biological replicate were examined. Quantification was achieved using comparative cycle threshold (Ct) values, and gene-expression levels were calculated as 2(−∆∆Ct) [∆CT = CT Target − CT TUA. ∆∆Ct = ∆Ct Target − ∆Ct CK]. Duncan’s test was used to examine the significance between different columns in IBM SPSS Statistics (Version 25). The lowercase letters represent the significance between different columns (p < 0.05).

4.7. Transcriptional-Activation Activity Assay

The open reading frames of PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 were fused with the pGBKT7 vector (primer sequences were listed in Table S4). Subsequently, these recombinant plasmids were transformed into AH109 yeast strains (YC1010, Weidi Biotech, Shanghai, China), which were cultured on the SD/-Trp medium at 29 °C for 48 h. Verified positive yeast colonies were collected into 200ul ddH2O. Subsequently, a repetitive procedure was performed to withdraw 5 μL from the suspension and deposit it onto the culture medium SD/-Trp/-His/-Ade, which contained X-α-Gal as an indicator. The empty pGBKT7 was used as a negative control, and PmC3H20, previously confirmed to exhibit transcriptional self-activation, was employed as a positive control [17].

5. Conclusions

In this study, we identified a total of 57 C2H2 ZFPs in P. massoniana and performed a bioinformatics analysis. These members were classified into five subgroups and six Q-type C2H2 ZFPs were selected for further investigation to explore their expression patterns under various stress conditions and in different tissues. Subcellular localization prediction results revealed nuclear localization for all genes except PmC2H2-29, while experimental validation confirmed the nuclear localization of PmC2H2-4 and PmC2H2-20. Two heatmaps showed that PmC2H2s could respond to pine wood nematodes stress and drought, respectively. However, we noticed that the expression levels of PmC2H2-20 are down-regulated, except for its induction under ABA stress. Furthermore, the transcriptional activation activity of PmC2H2-20 was detected, indicating its role as a potential transcriptional activator involved in stress-response mechanisms. This study establishes a theoretical basis for further investigation into C2H2 TFs associated with pine wood nematode and drought resistance in P. massoniana, while also presenting PmC2H2-20 as a potential candidate gene that exhibits significant responsiveness to stress.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25158361/s1.

Author Contributions

Conceptualization, D.W. and Z.Q.; methodology, Z.Q., S.Y. and T.X.; formal analysis, Z.Q., D.W. and M.C.; resources, Q.L. and D.W.; writing—original draft preparation, D.W. and Z.Q.; writing—review and editing, Z.Q., D.W., R.H.A. and K.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key R&D Program of China (2022YFD2200202), and the project was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Xiang, W.; Liu, S.; Deng, X.; Shen, A.; Lei, X.; Tian, D.; Zhao, M.; Peng, C. General allometric equations and biomass allocation 469 of Pinus massoniana trees on a regional scale in southern China. Ecol. Res. 2011, 26, 697–711. [Google Scholar] [CrossRef]
  2. Du, M.; Ding, G.; Cai, Q. The Transcriptomic Responses of Pinus massoniana to Drought Stress. Forests 2018, 9, 326. [Google Scholar] [CrossRef]
  3. Erpen, L.; Devi, H.S.; Grosser, J.W.; Dutt, M. Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants. Plant Cell Tissue Organ Cult. PCTOC 2017, 132, 1–25. [Google Scholar] [CrossRef]
  4. Roy, S. Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome. Plant Signal Behav. 2016, 11, e1117723. [Google Scholar] [CrossRef]
  5. Wang, X.; Niu, Y.; Zheng, Y. Multiple Functions of MYB Transcription Factors in Abiotic Stress Responses. Int. J. Mol. Sci. 2021, 22, 6125. [Google Scholar] [CrossRef] [PubMed]
  6. Zhang, Y.; Xia, P. The DREB transcription factor, a biomacromolecule, responds to abiotic stress by regulating the expression of stress-related genes. Int. J. Mol. Sci. 2023, 243, 125231. [Google Scholar] [CrossRef]
  7. Ma, Z.; Hu, L.; Jiang, W. Understanding AP2/ERF transcription factor responses and tolerance to various abiotic stresses in plants: A comprehensive review. Int. J. Mol. Sci. 2024, 25, 893. [Google Scholar] [CrossRef]
  8. Wang, Z.; Dane, F. NAC (NAM/ATAF/CUC) transcription factors in different stresses and their signaling pathway. Acta Physiol. Plant. 2013, 35, 1397–1408. [Google Scholar] [CrossRef]
  9. Li, W.; Pang, S.; Lu, Z.; Jin, B. Function and mechanism of WRKY transcription factors in abiotic stress responses of plants. Plants 2020, 9, 1515. [Google Scholar] [CrossRef]
  10. Radani, Y.; Li, R.; Korboe, H.M.; Ma, H.; Yang, L. Transcriptional and post-translational regulation of plant bHLH transcription factors during the response to environmental stresses. Plants 2023, 12, 2113. [Google Scholar] [CrossRef]
  11. Liu, H.; Tang, X.; Zhang, N.; Li, S.; Si, H. Role of bZIP transcription factors in plant salt stress. Int. J. Mol. Sci. 2023, 24, 7893. [Google Scholar] [CrossRef]
  12. Liu, Y.; Khan, A.R.; Gan, Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int. J. Mol. Sci. 2022, 23, 2730. [Google Scholar] [CrossRef] [PubMed]
  13. Gong, S.; Ding, Y.; Hu, S.; Ding, L.; Chen, Z.; Zhu, C. The role of HD-Zip class I transcription factors in plant response to abiotic stresses. Physiol. Plant. 2019, 167, 516–525. [Google Scholar] [CrossRef] [PubMed]
  14. Fan, F.; Wang, Q.; Li, H.; Ding, G.; Wen, X. Transcriptome-Wide identification and expression profiles of masson pine wrky transcription factors in response to low phosphorus stress. Plant Mol. Biol. Rep. 2021, 39, 1–9. [Google Scholar] [CrossRef]
  15. Von Koskull-Döring, P.; Scharf, K.D.; Nover, L. The diversity of plant heat stress transcription factors. Trends Plant Sci. 2007, 12, 452–457. [Google Scholar] [CrossRef] [PubMed]
  16. Zhang, X.; Li, T.; Chen, H.; Tang, H.; Mu, Y.; Gou, L.; Haibib, A.; Lan, X.; Ma, J. The wheat (Triticum aestiveum L.) MADS-box transcription factor TaMADS32 plays a role in response to abiotic stresses. Biotechnol. Biotechnol. Equip. 2022, 36, 451–461. [Google Scholar] [CrossRef]
  17. Wang, D.; Yao, S.; Agassin, R.H.; Zhang, M.; Lou, X.; Huang, Z.; Zhang, J.; Ji, K. Transcriptome-Wide identification of CCCH-type zinc finger proteins family in Pinus massoniana and RR-TZF proteins in stress response. Genes 2022, 13, 1639. [Google Scholar] [CrossRef]
  18. Han, G.; Qiao, Z.; Li, Y.; Wang, C.; Wang, B. The roles of CCCH zinc-finger proteins in plant abiotic stress tolerance. Int. J. Mol. Sci. 2021, 22, 8327. [Google Scholar] [CrossRef]
  19. Miller, J.; McLachlan, A.D.; Klug, A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985, 4, 1609–1614. [Google Scholar] [CrossRef]
  20. Han, G.; Lu, C.; Guo, J.; Qiao, Z.; Sui, N.; Qiu, N.; Wang, B. C2H2 zinc finger proteins: Master regulators of abiotic stress responses in plants. Front. Plant Sci. 2020, 11, 115. [Google Scholar] [CrossRef]
  21. Sakamoto, H.; Maruyama, K.; Sakuma, Y.; Meshi, T.; Iwabuchi, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol. 2004, 136, 2734–2746. [Google Scholar] [CrossRef] [PubMed]
  22. Kielbowicz-Matuk, A. Involvement of plant C2H-type zinc finger transcription factors in stress responses. Plant Sci. 2012, 185, 78–85. [Google Scholar] [CrossRef] [PubMed]
  23. Faraji, S.; Rasouli, S.H.; Kazemitabar, S.K. Genome-wide exploration of C2H2 zinc finger family in durum wheat (Triticum turgidum ssp Durum): Insights into the roles in biological processes especially stress response. Biometals 2018, 31, 1019–1042. [Google Scholar] [CrossRef] [PubMed]
  24. Wang, L.; Sadeghnezhad, E.; Nick, P. Upstream of gene expression: What is the role of microtubules in cold signalling? J. Exp. Bot. 2020, 71, 36–48. [Google Scholar] [CrossRef] [PubMed]
  25. Wolfe, S.A.; Nekludova, L.; Pabo, C.O. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 183–212. [Google Scholar] [CrossRef] [PubMed]
  26. Ciftci-Yilmaz, S.; Mittler, R. The zinc finger network of plants. Cell. Mol. Life Sci. 2008, 65, 1150–1160. [Google Scholar] [CrossRef] [PubMed]
  27. Englbrecht, C.C.; Schoof, H.; Bohm, S. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genom. 2004, 5, 39. [Google Scholar] [CrossRef] [PubMed]
  28. Agarwal, P.; Arora, R.; Ray, S.; Singh, A.K.; Singh, V.P.; Takatsuji, H.; Kapoor, S.; Tyagi, A.K. Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis. Plant Mol. Biol. 2007, 65, 467–485. [Google Scholar] [CrossRef] [PubMed]
  29. Liu, Q.; Wang, Z.; Xu, X.; Zhang, H.; Li, C. Genome-Wide analysis of C2H2 zinc-finger family transcription factors and their responses to abiotic stresses in poplar (Populus trichocarpa). PLoS ONE 2015, 10, e0134753. [Google Scholar] [CrossRef]
  30. Yuan, S.; Li, X.; Li, R.; Wang, L.; Zhang, C.; Chen, L.; Hao, Q.; Zhang, X.; Chen, H.; Shan, Z.; et al. Genome-Wide identification and classification of soybean C2H2 zinc finger proteins and their expression analysis in legume-rhizobium symbiosis. Front. Microbiol. 2018, 9, 126. [Google Scholar] [CrossRef]
  31. Liu, K.; Hou, Q.; Yu, R.; Deng, H.; Shen, L.; Wang, Q.; Wen, X. Genome-wide analysis of C2H2 zinc finger family and their response to abiotic stresses in apple. Gene 2024, 904, 148164. [Google Scholar] [CrossRef] [PubMed]
  32. Cheuk, A.; Houde, M. Genome wide identification of C1-2i zinc finger proteins and their response to abiotic stress in hexaploidy wheat. Mol. Genet. Genom. 2016, 291, 873–890. [Google Scholar] [CrossRef] [PubMed]
  33. Takatsuji, H.; Mori, M.; Benfey, P.N.; Ren, L.; Chua, N.H. Characterization of a zinc finger DNA-binding protein expressed specifically in Petunia petals and seedlings. EMBO J. 1992, 11, 241–249. [Google Scholar] [CrossRef] [PubMed]
  34. Sakamoto, A.; Minami, M.; Huh, G.H.; Iwabuchi, M. The putative zinc-finger protein WZF1 interacts with a cis-acting element of wheat histone genes. Eur. J. Biochem. 1993, 217, 1049–1056. [Google Scholar] [CrossRef]
  35. Huang, J.; Wang, J.F.; Wang, Q.H.; Zhang, H.S. Identification of a rice zinc finger protein whose expression is transiently induced by drought, cold but not by salinity and abscisic acid. DNA Seq. 2005, 16, 130–136. [Google Scholar] [CrossRef] [PubMed]
  36. Li, Y.; Chu, Z.; Luo, J.; Zhou, Y.; Cai, Y.; Lu, Y.; Xia, J.; Kuang, H.; Ye, Z.; Ouyang, B. The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol. J. 2018, 16, 1201–1213. [Google Scholar] [CrossRef] [PubMed]
  37. Kim, J.C.; Lee, S.H.; Cheong, Y.H.; Yoo, C.M.; Lee, S.I.; Chun, H.J.; Yun, D.J.; Hong, J.C.; Lee, S.Y.; Lim, C.O.; et al. A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants. Plant J. 2001, 25, 247–259. [Google Scholar] [CrossRef] [PubMed]
  38. Liu, Y.; Liu, D.; Hu, R.; Hua, C.; Ali, I.; Zhang, A.; Liu, B.; Wu, M.; Huang, L.; Gan, Y. AtGIS, a C2H2 zinc-finger transcription factor from Arabidopsis regulates glandular trichome development through GA signaling in tobacco. Biochem. Biophys. Res. Commun. 2017, 483, 209–215. [Google Scholar] [CrossRef] [PubMed]
  39. Muthamilarasan, M.; Bonthala, V.S.; Mishra, A.K.; Khandelwal, R.; Khan, Y.; Roy, R.; Prasad, M. C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses. Funct. Integr. Genom. 2014, 14, 531–543. [Google Scholar] [CrossRef]
  40. Lyu, T.; Liu, W.; Hu, Z.; Xiang, X.; Liu, T.; Xiong, X.; Cao, J. Molecular characterization and expression analysis reveal the roles of Cys2/His2 zinc-finger transcription factors during flower development of Brassica rapa subsp. chinensis. Plant Mol. Biol. 2020, 102, 123–141. [Google Scholar] [CrossRef]
  41. Bohm, S.; Frishman, D.; Mewes, H.W. Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins. Nucleic Acids Res. 1997, 25, 2464–2469. [Google Scholar] [CrossRef]
  42. Kubo, K.I.; Sakamoto, A.; Kobayashi, A.; Rybka, Z.; Kanno, Y.; Nakagawa, H.; Takatsuji, H. Cys2/His2 zinc-finger protein family of petunia: Evolution and general mechanism of target-sequence recognition. Nucleic Acids Res. 1998, 26, 608–615. [Google Scholar] [CrossRef]
  43. Yoshioka, K.; Fukushima, S.; Yamazaki, T.; Yoshida, M.; Takatsuji, H. The plant zinc finger protein ZPT2-2 has a unique mode of DNA interaction. J. Biol. Chem. 2001, 276, 35802–35807. [Google Scholar] [CrossRef] [PubMed]
  44. Takatsuji, H.; Matsumoto, T. Target-sequence recognition by separate-type Cys2/His2 zinc finger proteins in plants. J. Biol.Chem. 1996, 271, 23368–23373. [Google Scholar] [CrossRef]
  45. Ding, Q.; Zhao, H.; Zhu, P.; Jiang, X.; Nie, F.; Li, G. Genome-wide identification and expression analyses of C2H2 zinc finger transcription factors in Pleurotus ostreatus. PeerJ 2022, 10, e12654. [Google Scholar] [CrossRef] [PubMed]
  46. Shi, H.; Chan, Z. The cysteine2/histidine2-type transcription factor zinc finger of Arabidopsis thaliana 6-activated c-repeat-binding factor pathway is essential for melatonin-mediated freezing stress resistance in Arabidopsis. J. Pineal Res. 2014, 57, 185–191. [Google Scholar] [CrossRef]
  47. Dong, T.; Park, Y.; Hwang, I. Abscisic acid: Biosynthesis, inactivation, homoeostasis and signalling. Essays Biochem. 2015, 58, 29–48. [Google Scholar]
  48. Ullah, A.; Manghwar, H.; Shaban, M.; Khan, A.H.; Akbar, A.; Ali, U.; Ali, E.; Fahad, S. Phytohormones enhanced drought tolerance in plants: A coping strategy. Environ. Sci. Pollut. R. 2018, 25, 33103–33118. [Google Scholar] [CrossRef]
  49. Lippuner, V.; Cyert, M.S.; Gasser, C.S. Two classes of plant cDNA clones differentially complement yeast calcineurin mutants and increase salt tolerance of wild-type yeast. J. Biol. Chem. 1996, 271, 12859–12866. [Google Scholar] [CrossRef]
  50. Sakamoto, H.; Araki, T.; Meshi, T.; Iwabuchi, M. Expression of a subset of the Arabidopsis Cys2/His2-type zinc-finger protein gene family under water stress. Gene 2000, 248, 23–32. [Google Scholar] [CrossRef]
  51. Shao, L.; Li, L.; Huang, X.; Fu, Y.; Yang, D.; Li, C.; Yang, J. Identification of C2H2 zinc finger genes through genome-wide association study and functional analyses of LkZFPs in response to stresses in Larix kaempferi. BMC Plant Biol. 2023, 23, 298. [Google Scholar] [CrossRef] [PubMed]
  52. Hu, X.; Zhu, L.; Zhang, Y.; Xu, L.; Li, N.; Zhang, X.; Pan, Y. Genome-wide identification of C2H2 zinc-finger genes and their expression patterns under heat stress in tomato (Solanum lycopersicum L.). PeerJ 2019, 7, e7929. [Google Scholar] [CrossRef]
  53. Yin, M.; Wang, Y.; Zhang, L.; Li, J.; Quan, W.; Yang, L.; Wang, Q.; Chan, Z. The Arabidopsis Cys2/His2 zinc fnger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress. J. Exp. Bot. 2017, 68, 2991–3005. [Google Scholar] [CrossRef]
  54. Ciftci-Yilmaz, S.; Morsy, M.R.; Song, L.; Coutu, A.; Krizek, B.A.; Lewis, M.W.; Warren, D.; Cushman, J.; Connolly, E.L.; Mittler, R. The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress. J. Biol. Chem. 2007, 282, 9260–9268. [Google Scholar] [CrossRef]
  55. Wu, F.; Sun, X.; Zou, B.; Zhu, P.; Ji, K. Transcriptional analysis of Masson pine (Pinus massoniana) under high CO2 stress. Genes 2019, 10, 804. [Google Scholar] [CrossRef]
  56. Zhang, J.; Wang, D.; Chen, P.; Zhang, C.; Yao, S.; Hao, Q.; Agassin, R.H.; Ji, K. The transcriptomic analysis of the response of Pinus massoniana to drought stress and a functional study on the ERF1 transcription factor. Int. J. Mol. Sci. 2023, 24, 11103. [Google Scholar] [CrossRef]
  57. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  58. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; Xia, R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  59. Zhu, P.H.; Ma, Y.Y.; Zhu, L.Z.; Chen, Y.; Ji, K.S. Selection of suitable reference genes in Pinus massoniana Lamb. under different abiotic stresses for qPCR normalization. Forests 2019, 10, 632. [Google Scholar] [CrossRef]
Ijms 25 08361 g001
Figure 1. Phylogenetic analysis and motif distribution of PmC2H2 ZFPs. Branches with different colors represented different subgroups. The subgroup was named from “I” to “V”.
Figure 1. Phylogenetic analysis and motif distribution of PmC2H2 ZFPs. Branches with different colors represented different subgroups. The subgroup was named from “I” to “V”.
Ijms 25 08361 g001
Ijms 25 08361 g002
Figure 2. Subcellular localization analysis of PmC2H2-4 and PmC2H2-20 proteins in N. benthamiana leaves. The scale in the images is 20 μm. pCAMBIA-1302-mGFP5 was the control. DAPI—4′,6-diamidino-2-phenylindole, a blue fluorescent dye that shows DNA location. Chloroplast—chloroplast auto-fluorescence, displays the location of chloroplasts. GFP—green fluorescence protein, displays the location of the target protein. Bright—bright field. Merged—merged picture of four overlapped channels.
Figure 2. Subcellular localization analysis of PmC2H2-4 and PmC2H2-20 proteins in N. benthamiana leaves. The scale in the images is 20 μm. pCAMBIA-1302-mGFP5 was the control. DAPI—4′,6-diamidino-2-phenylindole, a blue fluorescent dye that shows DNA location. Chloroplast—chloroplast auto-fluorescence, displays the location of chloroplasts. GFP—green fluorescence protein, displays the location of the target protein. Bright—bright field. Merged—merged picture of four overlapped channels.
Ijms 25 08361 g002
Ijms 25 08361 g003
Figure 3. Transcriptional profiles of C2H2 ZFPs in P. massoniana under nematode stress and drought stress. (a) Different days after inoculation with pine wood nematodes: 0 (CK), 3, 10, 20, and 35 d. (b) Different soil water content represents different drought levels: CK (normal water supply) (80 ± 5)%, LD (light drought stress mild) (65 ± 5)%, MD (moderate drought stress) (50 ± 5)%, and SD (severe drought stress) (35 ± 5)%. A heatmap was generated using log2 (FPKM + 1) values, then normalized by row scale. The color scale represents relative expression levels.
Figure 3. Transcriptional profiles of C2H2 ZFPs in P. massoniana under nematode stress and drought stress. (a) Different days after inoculation with pine wood nematodes: 0 (CK), 3, 10, 20, and 35 d. (b) Different soil water content represents different drought levels: CK (normal water supply) (80 ± 5)%, LD (light drought stress mild) (65 ± 5)%, MD (moderate drought stress) (50 ± 5)%, and SD (severe drought stress) (35 ± 5)%. A heatmap was generated using log2 (FPKM + 1) values, then normalized by row scale. The color scale represents relative expression levels.
Ijms 25 08361 g003
Ijms 25 08361 g004aIjms 25 08361 g004b
Figure 4. The relative expression levels of six Q-type PmC2H2s in different tissues: shoot apices (T), young needle leaf (YL), old needle leaf (OL), young stem (YS), old stem (OS), xylem (X), phloem (P), and root (R). The relative expression level in ‘T’ was set as “1”. The same lowercase letters between different columns indicate no significant difference. The highest column is marked with ‘a’, then ‘b’, and so on. Completely different lowercase letters between different columns indicate a significant difference, p < 0.05. More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters.
Figure 4. The relative expression levels of six Q-type PmC2H2s in different tissues: shoot apices (T), young needle leaf (YL), old needle leaf (OL), young stem (YS), old stem (OS), xylem (X), phloem (P), and root (R). The relative expression level in ‘T’ was set as “1”. The same lowercase letters between different columns indicate no significant difference. The highest column is marked with ‘a’, then ‘b’, and so on. Completely different lowercase letters between different columns indicate a significant difference, p < 0.05. More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters.
Ijms 25 08361 g004aIjms 25 08361 g004b
Ijms 25 08361 g005aIjms 25 08361 g005bIjms 25 08361 g005c
Figure 5. The figure above shows the expression levels of five PmC2H2 genes under different abiotic treatments, namely (a) ABA, (b) drought, (c) ETH, (d) H2O2, (e) mechanical damage, (f) MeJA, (g) NaCl, (h) PEG, and (i) SA. The absence of any significant difference is indicated by the presence of identical lowercase letters across different columns. Conversely, the presence of completely distinct lowercase letters between different columns signifies a statistically significant difference (p < 0.05). In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters. The relative expression at 0 h is normalized to “1”.
Figure 5. The figure above shows the expression levels of five PmC2H2 genes under different abiotic treatments, namely (a) ABA, (b) drought, (c) ETH, (d) H2O2, (e) mechanical damage, (f) MeJA, (g) NaCl, (h) PEG, and (i) SA. The absence of any significant difference is indicated by the presence of identical lowercase letters across different columns. Conversely, the presence of completely distinct lowercase letters between different columns signifies a statistically significant difference (p < 0.05). In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters. The relative expression at 0 h is normalized to “1”.
Ijms 25 08361 g005aIjms 25 08361 g005bIjms 25 08361 g005c
Ijms 25 08361 g006
Figure 6. Transcriptional activation assay of six PmC2H2 genes. Empty pGBKT7 vector was used as a negative control and pGBKT7-PmC3H20 was used as a positive control.
Figure 6. Transcriptional activation assay of six PmC2H2 genes. Empty pGBKT7 vector was used as a negative control and pGBKT7-PmC3H20 was used as a positive control.
Ijms 25 08361 g006
Table 1. The characteristics of 57 C2H2 proteins identified and their subcellular localization prediction.
Table 1. The characteristics of 57 C2H2 proteins identified and their subcellular localization prediction.
Gene IDAaMw (kDa)pISubcellular Localization
PmC2H2-146153.285.68Nucleus
PmC2H2-268876.548.58Nucleus
PmC2H2-369672.089.15Nucleus
PmC2H2-455961.935.47Nucleus
PmC2H2-553258.455136.23Nucleus
PmC2H2-661766.99.1Nucleus
PmC2H2-739544.676.59Nucleus
PmC2H2-861766.875.59Nucleus
PmC2H2-952257.315.49Nucleus
PmC2H2-1035037.358.77Nucleus
PmC2H2-1159865.218.94Nucleus
PmC2H2-1244448.939.22Nucleus
PmC2H2-1338543.035.93Nucleus
PmC2H2-1465168.656.53Nucleus
PmC2H2-1572576.179.19Nucleus
PmC2H2-1655861.935.81Nucleus
PmC2H2-1741146.685.43Nucleus
PmC2H2-1872576.679.31Nucleus
PmC2H2-1923927.418.01Nucleus
PmC2H2-2035939.95.85Nucleus
PmC2H2-2140045.445.47Nucleus
PmC2H2-2237743.589.46Nucleus
PmC2H2-2342446.516.65Nucleus
PmC2H2-2438743.525.89Nucleus
PmC2H2-2535137.694.82Nucleus
PmC2H2-2645950.477.01Nucleus
PmC2H2-2743447.898.84Nucleus
PmC2H2-2871981.745.28Nucleus
PmC2H2-2928230.888.93Chloroplast
PmC2H2-3029832.778.06Nucleus
PmC2H2-3126230.157.21Nucleus
PmC2H2-3241445.998.84Nucleus
PmC2H2-3361969.25.88Nucleus
PmC2H2-3441546.886.68Nucleus
PmC2H2-3514916.29.42Nucleus
PmC2H2-3615617.889.2Nucleus
PmC2H2-3761667.235.63Nucleus
PmC2H2-3820622.948.53Nucleus
PmC2H2-3919620.939.1Nucleus
PmC2H2-4020222.129.51Nucleus
PmC2H2-4115516.999.33Nucleus
PmC2H2-4232634.6517.74Nucleus
PmC2H2-4366569.545.63Nucleus
PmC2H2-4418119.999.71Nucleus
PmC2H2-4514016.179.87Nucleus
PmC2H2-4629832.78.41Nucleus
PmC2H2-4722825.17.1Nucleus
PmC2H2-4852159.886.36Nucleus
PmC2H2-4952057.438.18Nucleus
PmC2H2-5021924.318.79Nucleus
PmC2H2-5146853.088.61Nucleus
PmC2H2-5236740.878.21Nucleus
PmC2H2-5354860.014.96Nucleus
PmC2H2-5456661.775.5Nucleus
PmC2H2-5513716.099.5Nucleus
PmC2H2-5620523.66.38Nucleus
PmC2H2-5717719.219.1Nucleus
Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.
Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.
MotifLengthMotif ConsensusMotif Logo
121CEICGKGFSRPQNLQQHMRTHIjms 25 08361 i001
215GCGKRFSVVSDLKRHIjms 25 08361 i002
350WKLRQRTTKEIRKRVYICPEPTCVHHDPSRALGDLTGIKKHFCRKHGEKKIjms 25 08361 i003
432SKTCGTREYRCDCGTLFSRRDSFITHRAFCDAIjms 25 08361 i004
541YSCPFEGCRRNKBHPKFKPLKSIRSLRNHYKRSHCPKMYTCIjms 25 08361 i005
637QQNTTIKRKRNLPGTPDPDAEVIALSPKTLMATNRFVIjms 25 08361 i006
750YSSASESVDLPNSRTLPRPSALVGGSMPPAPQSMMGQFSSKVSSSSQKKHIjms 25 08361 i007
821CSICGRSFTSKQALKGHIRVHIjms 25 08361 i008
98TGEKPFVCIjms 25 08361 i009
1029QQQQHTSTPQMSATALLQKAAQMGATASNIjms 25 08361 i010
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

Wang, D.; Qiu, Z.; Xu, T.; Yao, S.; Chen, M.; Li, Q.; Agassin, R.H.; Ji, K. Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses. Int. J. Mol. Sci. 2024, 25, 8361. https://doi.org/10.3390/ijms25158361

AMA Style

Wang D, Qiu Z, Xu T, Yao S, Chen M, Li Q, Agassin RH, Ji K. Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses. International Journal of Molecular Sciences. 2024; 25(15):8361. https://doi.org/10.3390/ijms25158361

Chicago/Turabian Style

Wang, Dengbao, Zimo Qiu, Tao Xu, Sheng Yao, Meijing Chen, Qianzi Li, Romaric Hippolyte Agassin, and Kongshu Ji. 2024. "Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses" International Journal of Molecular Sciences 25, no. 15: 8361. https://doi.org/10.3390/ijms25158361

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