Abstract
The C2H2 zinc finger protein (C2H2-ZFP) gene family plays important roles in response to environmental stresses and several other biological processes in plants. Ginseng is a precious medicinal herb cultivated in Asia and North America. However, little is known about the C2H2-ZFP gene family and its functions in ginseng. Here, we identified 115 C2H2-ZFP genes from ginseng, defined as the PgZFP gene family. It was clustered into five groups and featured with eight conserved motifs, with each gene containing one to six of them. The family genes are categorized into 17 gene ontology subcategories and have numerous regulatory elements responsive to a variety of biological process, suggesting their functional differentiation. The 115 PgZFP genes were spliced into 228 transcripts at seed setting stage and varied dramatically in expression across tissues, developmental stages, and genotypes, but they form a co-expression network, suggesting their functional correlation. Furthermore, four genes, PgZFP31, PgZFP78-01, PgZFP38, and PgZFP39-01, were identified from the gene family that were actively involved in plant response to salt stress. These results provide new knowledge on origin, differentiation, evolution, and function of the PgZFP gene family and new gene resources for C2H2-ZFP gene research and application in ginseng and other plant species.
Similar content being viewed by others
Introduction
Ginseng, as a valuable medicinal herb, has widely been used for human health food, medicine, and cosmetics. Its main bioactive ingredients, ginsenosides, have been shown to prevent, inhibit, and treat several types of cancers1,2,3. Therefore, ginseng genome research has been pursued extensively in the recent years. Xu et al.4 reported the first draft of the ginseng genome. Kim et al.5 released the draft of the ginseng genome for another genotype, from which 59,352 genes were identified. Recently, Wang et al.6 reported a chromosome-sized assembly of the ginseng genome for the third genotype, from which 65,913 genes were identified. Wang et al.7 sequenced the transcriptome of a 4-year-old ginseng plant expressed in 14 tissues, assembled 248,992 transcript unigenes spliced from 130,557 gene models and quantified their expressions in the 14 tissues. These genomic and transcriptomic resources have provided useful tools for genome-wide identification and characterization of genes important for ginseng research and breeding.
The C2H2 zinc finger protein (ZFP) gene family has been shown to play important roles in plant response to abiotic and biotic stresses, plant growth and development, and hormone signal transduction. For instance, the over-expression of an Arabidopsis C2H2-ZFP gene, ZAT18, improved drought tolerance8. The rice bsr-d1 gene encoding a C2H2 transcription factor increased disease resistance and immunity against rice blast9. The tomato SlZFP2 gene participated in the regulation of ABA biosynthesis during fruit development, thereby inhibiting fruit development through the transcriptional inhibition of flowering regulator10. It has been reported that the C2H2-ZFP gene family actively takes part in plant response to salt stress, which is significant for crop production11. The expression of a tomato C2H2-ZFP gene, SlZF3, enhanced plant salt tolerance12. The expression of Zoysia japonica ZjZFN1 in Arabidopsis improved seed germination, plant adaptability to salt stress, and the percentage of green cotyledons under the salt stress condition13. The AZFs and STZ genes in Arabidopsis had transcriptional repression activities, thus responding to salt stress14. The OsZFP213 gene in rice was shown to interact with OsMAPK3, thus enhancing salt tolerance15. In soybean and Arabidopsis, GmZAT4 played an important role in PEG and NaCl stress tolerance and ABA response16.
Therefore, the genes of the C2H2-ZFP gene family have been genome-wide identified and characterized in serval plant species, including Arabidopsis that contains 176 C2H2-ZFP genes17, rice (189)18, poplar (109)19, Medicago truncatula (218)20, maize (211)21, and soybean (321)22. The C2H2-ZFP gene family is usually expressed as X2CX2–4CX12HX2–8H, where X represents any amino acid, and the number indicates the number of any amino acids23. It is characterized with its C2H2 zinc finger protein that forms coordination bonds with two pairs of cysteine and histidine residues and further forms a compact finger-like tetrahedral structure through β hairpins and α helices24. In addition, the gene family contains a few conserved domains, including DNA binding domain, transcription regulatory domain, and protein interaction domain23. The DNA binding domain is featured by a highly conserved QALGGH motif in which every amino acid is important for its DNA binding activity. Absence of the QALGGH motif in a C2H2-ZFP gene may reduce ABA sensitivity, thus stomatal size25, and influence inflorescence development26. Another unique feature of the gene family is length variation of the spacer between two adjacent zinc fingers of the C2H2 zinc finger protein, which may also affect DNA binding23. Moreover, some C2H2-ZFPs contain a highly conserved amino acid sequence (L/FDLNL/FxP), known the EAR motif, in carboxy terminal region27. The EAR motif is the smallest known transcription repression domain. When a single residue in the EAR motif changed, the transcription repression ability was greatly reduced or disappeared28. The three DLN amino acids of the hexa-peptide motif is indispensable for transcriptional inhibition and the presence of at least two DLN residues produced maximum transcriptional inhibition29.
Nevertheless, the C2H2-ZFP gene family has not been reported in ginseng. In the present study, we identified 228 C2H2-ZFP gene transcripts spliced from 115 C2H2-ZFP genes from Jilin ginseng, defined PgZFP genes, and characterized them in genetic diversity, evolution, expression, functional differentiation, and gene × gene interaction. Furthermore, we identified four PgZFP genes involved in ginseng response to salt stress, thus confirming the roles of the family in plant response to salt stress. Therefore, the findings of this study provide a comprehensive insight into the PgZFP gene family in ginseng, knowledge, and gene resources necessary for advanced research and breeding in ginseng and related species.
Materials and methods
Plant materials
Three sets of Jilin ginseng plant materials were used for this study (Supplemental Fig. S1A) These plant materials included 14 tissues of a 4-year-old cv. Damaya plant, roots of 5-, 12-, 18-, 25-year-old cv. Damaya plants, and 4-year-old plant roots of 42 cultivars (hereafter referred as to genotypes) coded from S1 to S42 (Supplemental Table S1). The seeds of these plant materials were all from our laboratory, which are available upon request. The 14 tissues of the 4-year-old cv. Damaya plant included fiber root, leg root, main root epiderm, main root cortex, arm root, rhizome, stem, leaf peduncle, leaflet pedicel, leaf blade, fruit peduncle, fruit pedicel, fruit, and seed7. The roots of 5-, 12-, 18-, 25-year-old plants all were collected from a cultivar, Damaya. The 42 genotypes were collected from the origin and diversity center of ginseng, Jilin Province, China where over 65% of the world ginseng was produced. These genotypes were representatives of the genetic diversity of Jilin ginseng.
Databases
To facilitate ginseng functional genomics research, we previously developed several databases of functional genes for Jilin ginseng. These databases included the gene sequence and expression database in the above 14 tissues of the 4-year-old plant (Database A)7, the gene sequence and expression database of the four different year-old plant roots (Database B)7, and the gene sequence and expression database of the 4-year-old plant roots of 42 genotypes (Database C)30. These databases were used for this study. Database A consists of 248,992 unique transcripts, with 59,092–113,456 unique transcripts per tissue. Database B contains 54,444–65,412 unique transcripts per year-old plant root. Database C is made of 48,211–79,134 unique transcripts per genotype. Moreover, three ginseng genome assemblies from three genotypes4,5,6 were also used for this study.
Identification of PgZFP gene transcripts
Database A was used to identify the C2H2-ZFP genes in ginseng. First, we retrieved the C2H2-ZFP Hidden Markov Model (HMM) (protein family: PF00096) from the Pfam database (Pfam 33.1; https://pfam.xfam.org/)31, used as queries to search ginseng Database A for C2H2-ZFP genes, and identified the putative C2H2-ZFP genes in ginseng. Then, the identified ginseng putative C2H2-ZFP genes were imported into iTAK (http://bioinfo.bti.cornell.edu/tool/itak) to further confirm the putative C2H2-ZFP genes by C2H2-ZFP conservative domain search32. The putative C2H2-ZFP genes that were confirmed to have the C2H2-ZFP domains were considered as ginseng C2H2-ZFP-encoding genes and defined as PgZFP genes (Supplemental Table S2). Finally, the ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) was used to identify the ORFs of the PgZFP genes. The relevant physiological and biochemical indicators of their putative protein sequences were predicted, including isoelectric point (PI), molecular weight (Da), grand average of hydropathicity, instability index, and aliphatic index, using Protparam (https://web.expasy.org/protparam/).
Distribution of the PgZFP gene family in the ginseng genome and its gene number variation within the ginseng species and synteny with Arabidopsis
We aligned the PgZFP genes identified above to three ginseng genome assemblies4,5,6 using Blastn to determine their distribution in the ginseng genome6 and the variation of the PgZFP gene family size among genotypes. The criteria of identity ≥ 99%, cover length ≥ 200 bp, and e-value ≤ 1.0E−100 were used for the alignment. The PgZFP genes were aligned to the Arabidopsis genome at criteria of identity ≥ 75%, cover length ≥ 80 bp, and e-value ≤ 1.0E−10, given that ginseng is distantly related with Arabidopsis (Supplemental Fig. S1B)33. The distribution of the gene family in the ginseng genome and its synteny with the Arabidopsis genome were constructed using the R-package Circlize34. The genes aligned to each genome assembly were compared to identify the pan- and core-transcriptomes of the gene family among the three ginseng genotypes.
Duplication, phylogeny, and evolution of the PgZFP gene family
To determine the origin, evolution, and phylogeny of the PgZFP gene family, we screened for the ORFs of the transcripts of all 115 PgZFP genes using the NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/), calculated the Ka/Ks ratio of the PgZFP genes recently duplicated, constructed their phylogenetic tree, and predicted their conserved domains. The transcripts with complete CDS were used for Ka/Ks calculation using the KaKs_Calculator35. The time of the gene duplication and divergence was estimated by T = Ks/2λ × 10−6, where Ks is synonymous nucleotide substitution rate and λ is the number of substitutions per synonymous site per generation, for which λ = 6.5 × 10−9 was used36,37,38.
The transcript that has the longest amino acid sequence for a PgZFP gene was used for constructing the phylogenetic tree of the gene family. The phylogenetic tree was constructed using the maximum-likelihood (ML) method of MEGA X39, with 1000 bootstrap replications. Forty-five C2H2-ZFPs from Arabidopsis were downloaded from Plant Transcription Factor Database (http://plntfdb.bio.uni-potsdam.de/v3.0/) and used as the outgroup and evolutionary reference.
MEME40 was used to predict the conserved domains in the PgZFP gene transcripts. The putative protein sequences of the 26 PgZFP genes that have the complete conservative domains were used as representatives for the conserved domain prediction of the genes. Motif length was set to 10–50 amino acids and the maximum number of motifs was set to 6, 8, and 10, respectively, while other parameters were set as default.
Functional divergence of the PgZFP genes
Studies has documented that the types of gene cis-regulatory elements, such as promoters, are related to the biological functionality of the genes, such as plant responses to hormones, abiotic and biotic stresses, and plant growth and development. We estimated the functional differentiation and divergence of the genes in the PgZFP gene family by gene ontology (GO) categorization and cis-regulatory element sequence analysis. The PgZFP gene transcripts were GO categorized using Blast2GO V5.041. The enrichment of the number of PgZFP gene transcripts categorized into each subcategory was tested by Chi-square test using the categorization of the transcripts expressed in the 4-year-old ginseng plant as the control7. The 1500-bp upstream sequences of the PgZFP genes were analyzed, the cis-regulatory elements of the genes were identified, and the types of the cis-regulatory elements were classified using PlantCARE database42.
Expression characterization and co-expression network of the PgZFP gene family and its hub genes
We characterized the PgZFP gene family by analysis of the expressions, heatmaps, and co-expression networks of its gene transcripts in 14 tissues of a 4-year-old plant, in 5-, 12-, 18-, and 25-year-old plant roots, and in the 4-year-old plant roots of 42 genotypes. The expressions of the PgZFP gene transcripts were extracted from Databases A, B, and C, respectively. The expression heatmaps of the genes were constructed using an R language package for heatmap construction and visualization. The co-expression networks of the genes were constructed using the BioLayout Express3D Version 3.2 software43. Furthermore, we tested the tendency that the PgZFP gene transcripts formed a co-expression network by Student’s t-test using 176 transcripts randomly selected from the 228 PgZFP gene transcripts identified in this study with the same numbers of transcripts randomly selected from Database A as a control. The test was carried out at a series of cutoff values from P ≤ 5.0E−02 to 1.0E−08, with 20 replicates per cutoff value. In addition, we identified the hub genes of the PgZFP gene family network using the following criteria: network cutoff P-value ≤ 0.001, connectivity ≥ 30, and the percentage of hub genes in the total number of genes in the network ≤ 5%. The connectivity of a gene in a network indicates not only the degree of its co-expression correlation with other genes, but also the number of genes with which it interacts in the network.
Response of ginseng to salt stress
To examine the potential function of the PgZFP gene family in response to salt stress, we stressed ginseng with the NaCl salt using its adventitious roots as experimental materials. The 1-cm adventitious roots were cultured and stressed on the B5 medium containing 0 mM, 20 mM, 40 mM, and 80 mM NaCl at 25 oC for 30 days. The lengths of the roots were measured, quickly frozen in liquid nitrogen, and stored at -80℃ for gene expression analysis.
Relative expressions of the PgZFP genes under salt stress
We first analyzed the PgZFP genes by aligning them to the salt-responsive genes identified in A. thaliana, STZ and AZF. The PgZFP genes that were best aligned to the STZ and AZF genes were then selected and subjected to comparative expression analysis using the ginseng adventitious roots stressed with and without salt by real-time quantitative PCR (qPCR) to test whether the genes of the PgZFP gene family respond to salt stress. The qPCR primers of the selected PgZFP genes were designed, based on their sequences (Supplemental Table S3). Total RNA was isolated from the adventitious roots stressed with the above different NaCl concentrations using the Trizol method. mRNA was purified from the total RNA, and first-strand cDNA was synthesized and used as qPCR templates. The CYP gene was used as the reference gene44. qPCR was conducted using the Applied Biosystems 7500 Real-Time System (ABI, USA) and the Ultra SYBR Mixture Kit (Low ROX) (ComWin, Beijing, China). The 2−ΔΔCT method was employed to determine the relative expressions of the PgZFP genes.
Research involving plants
Authors confirm that all methods were performed in accordance with the relevant guidelines and regulations. All plant materials used in this study were from the authors’ laboratory.
Results
Identification of the PgZFP gene transcripts and the biophysical and biochemical properties of their putative proteins
A total of 228 PgZFP gene transcripts were identified, with a sequence length from 202 to 5327 bp and an average length of 1419 bp (Supplemental Table S2). These transcripts were spliced from 115 PgZFP genes, with 1 to 20 transcripts per gene and an average of 2 transcripts per gene. The molecular weights of the putative proteins of the PgZFP gene transcripts varied from 3808 to 177,151 Daltons (Da), their isoelectric point (PIs) ranged from 4.55 to 12.13, the maximum grand average of their hydropathicities was 1.253, and the minimum grand average of their hydropathicities was − 1.512. Since only 16 of the PgZFP gene transcripts had a positive grand average of hydropathicity, we speculated that most genes of the PgZFP gene family code hydrophilic proteins. The instability indices of the PgZFP putative proteins were between 18.90 and 88.75, with most of them being greater than 40.00, suggesting that most PgZFP genes in the gene family code unstable proteins (Supplemental Table S4).
Distribution of the PgZFP gene family in the ginseng genome and its gene number variation within the ginseng species and synteny with Arabidopsis
Seventy-three of the 115 PgZFP genes identified in this study were mapped to all 24 chromosomes of the Chinese ginseng6, with each chromosome having 1–6 PgZFP genes. Seventeen of 73 PgZFP genes mapped to the Chinese ginseng genome6 were shown to be syntenic to 11 Arabidopsis orthologues in four of the five Arabidopsis chromosomes, Chromosomes 1, 3, 4, and 5, with each chromosome having 2–4 Arabidopsis orthologues (Fig. 1A). Of the 115 PgZFP genes identified in this study, 74 and 74 were aligned to the genomes of Chinese ginseng IR826 (Ref.4) and Korean ginseng ChP5, respectively. Comparative analysis showed that the pan-transcriptome of the gene family consisted of 149 PgZFP genes and the core-transcriptome of the gene family contained only 12 PgZFP genes, suggesting a wide variation of the gene family size among genotypes within P. ginseng.
The distribution of the PgZFP gene family in the ginseng genome and its family size variation among ginseng genotypes. (A) The positions of the PgZFP gene family in the Chinese ginseng genome6 and its synteny with the Arabidopsis genome. The PgZFP genes were mapped to all 24 ginseng chromosomes indicated by light blue bars and their Arabidopsis orthologues were mapped to four of the five Arabidopsis chromosomes indicated by yellow bars. (B) Venn diagram showing the pan- and core-transcriptomes of the PgZFP gene family. The numbers in the Venn diagram indicate the numbers of the PgZFP genes in the family that are genotype-specific, shared between the genotypes, and common among all three genotypes. The percentage of the genes was calculated by the numbers of genes divided by the total number of the PgZFP genes (221) aligned to the genome of each genotype times 100.
Gene duplication, phylogeny, and conserved motifs of the PgZFP gene family
We first estimated the duplication and selection mode of the PgZFP genes in the gene family. Analysis of 20 pairs of the PgZFP genes that have complete ORFs and have most recently duplicated revealed that these genes have been duplicated and diverged 18.0–40.7 million years ago (MYA). Approximately 55% of them have been subjected to purifying selection (Ka/Ks < 1.0 ± 0.1), 40% to neutral selection (Ka/Ks = 1.0 ± 0.1), and 5% to positive selection (Ka/Ks > 1.0 ± 0.1) (Supplemental Table S5).
To determine the origin and phylogeny of the PgZFP gene family in Jilin ginseng (Supplemental Fig. S1A), the putative proteins of the longest transcripts of all 115 PgZFP genes were used for the experiment (Supplemental Table S4). Forty-five representative A. thaliana (At) C2H2-ZFP genes selected from their phylogenetic tree (Supplemental Table S6) were used as outgroup (Supplemental Fig. S1B)33. The PgZFP genes were classified, with the AtC2H2-ZFP genes, into five clusters, defined I through V (Fig. 2). Cluster I included 26 PgZFP genes grouped with the members from the C1-2i, C1-3i, C1-4i, and C1-5i clusters of the AtC2H2-ZFP genes. Cluster II consisted of 25 PgZFP genes grouped with the members from the C2, C3, A3, and A4 subfamilies of the AtC2H2-ZFP genes. Cluster III was made of 46 PgZFP genes grouped with the members from the C1-1i, C1-2i, A1-a, A1-b, and A1-c clusters of the AtC2H2-ZFP genes. Cluster IV contained 11 PgZFP genes grouped with the members from the AtC2H2-ZFP A2 subfamily and B family. Cluster V was constituted of 7 PgZFP genes grouped with the only member from the AtC2H2-ZFP C2 subfamily17. These results suggested the ancient origin and diversity of the PgZFP gene family.
Phylogeny of the PgZFP gene family. The phylogenetic tree of the PgZFP gene family was constructed using all 115 PgZFP genes and the C2H2-ZFP genes of Arabidopsis thaliana (At) as outgroup. The subfamilies of the PgZFP gene family are indicated by I, II, III, IV, and V. The phylogenetic tree was constructed using the maximum-likelihood (ML) method with 1,000 bootstrap replications. The number nearby each cluster indicates the bootstrap confidence of the cluster in percentage (%).
Moreover, we analyzed the conserved motifs of the PgZFP gene family using the putative proteins of 26 representative PgZFP genes that have complete conserved domains within ORFs with criteria of motif length = 10–50 amino acids and a maximum motif number of 6, 8, or 10. When a maximum motif number of 6 was used, six conservative motifs were identified. When a maximum motif number of 8 was employed, eight conservative motifs were identified. When a maximum motif number of 10 was applied, ten conservative motifs were identified. Nevertheless, the six or eight motifs of the PgZFP genes identified with a maximum motif number of 6 or 8 were completely consistent with the first six or eight of the 10 motifs of the genes identified with a maximum motif number of 10. Figure 3A shows the eight conserved motifs of the PgZFP genes identified with the maximum motif number of 8, defined Motif 1 through Motif 8. Motif 1 was identified in 88.5% of the 26 PgZFP genes examined (Fig. 3B), indicating the evolutionary conservation of the PgZFP gene family. The QALGGH of Motifs 1 and 2 plays important roles in DNA binding8. The EXEXXAXCLXXL (L-box) of Motif 4 is a leucine rich region that is considered to play an important role in protein–protein interactions23. Motif 5, as EAR domain, includes the core DLNL sequence and has been proven to play an important role in transcriptional inhibition and abiotic stress response29,45.
The conserved motifs and their distribution in the PgZFP genes. (A) Conserved motifs of PgZFP proteins. (B) Conserved motif distribution in the PgZFP proteins. The phylogenetic tree of the PgZFP proteins were constructed using the maximum-likelihood (ML) method.
Functional divergence of the PgZFP genes
First, we categorized the PgZFP gene transcripts with gene ontology (GO). The 228 PgZFP gene transcripts were categorized into all three primary functional categories, MF (Molecular Function), BP (Biological Process), and CC (Cellular Component) (Fig. 4A). The MF category included 88 transcripts, of which 27 were MF-specific, 17 were categorized into both MF and CC categories, 13 were categorized into both MF and BP categories, and 31 were categorized into all three primary categories. The BP category included 49 transcripts, of which 3 were BP-specific, 2 were categorized into both BP and CC categories, and the remaining transcripts were categorized with MF, and with MF and CC as above. The CC category included 56 transcripts, of which 6 were CC-specific and the remaining transcripts were categorized with MF, with BP, and with MF and BP as above. The BP category was further categorized into eight subcategories at level 2, of which three were down-enriched in number of the PgZFP genes (P ≤ 0.05 or 0.01) (Fig. 4B). The MF category was categorized into three subcategories, catalytic activity, binding, and transcription regulator activity, of which the PgZFP genes involved in catalytic activity were down-enriched (P ≤ 0.01) and those involved in binding or transcription regulator activity were up-enriched (P ≤ 0.01). The CC category was categorized into six subcategories, of which two were down-enriched and two up-enriched in number of the PgZFP genes (P ≤ 0.05 or 0.01). These results suggested the functional differentiation, divergence, and specialty of the PgZFP genes.
(A) GO categorization of the PgZFP gene transcripts. (B) Enrichment of the PgZFP gene transcripts. BP, Biological Process; MF, Molecular Function; CC, Cellular Component.
Furthermore, we examined whether the GO categorization of the PgZFP gene family was consistent across tissues, developmental stages, and genotypes. The results showed that the PgZFP gene family was consistently categorized into the same 17 subcategories as above at level 2 across tissues, developmental stages, and genotypes (Supplemental Fig. S2). Nevertheless, the number of the PgZFP gene transcripts categorized into each of the 17 subcategories varied substantially across tissues, developmental stages, and genotypes.
Next, we analyzed the cis-regulatory elements of the PgZFP genes, such as promoter elements, because of their relationships with gene expression activities and potential biological functions. Since 73 of the 115 PgZFP genes were aligned to the Chinese ginseng genome6, the 1500-bp upstream sequences of the 73 PgZFP genes were searched for cis-regulatory elements. A total of 3709 cis-regulatory elements were identified for the 73 PgZFP genes and these elements were classified into 53 types, such as TATA-box, CAAT-box, SARE, ARE, AuxRE, and MBS. These elements are responsive to hormones, environmental stresses, and plant growth (Supplemental Fig. S3A). Of the 3709 cis-regulatory elements of the 73 PgZFP genes, 276 were responsive to hormones, including auxin, gibberellin, salicylic acid, abscisic acid, and MeJA (Supplemental Fig. S3B); 149 to environmental stresses, including defense, light, low-temperature, and drought (Supplemental Fig. S3C); and 82 to plant growth, such as seed, meristem expression, and endosperm expression (Supplemental Fig. S3D).
Expression characteristics of the PgZFP gene transcripts in different tissues, at different developmental stages, and across genotypes
We characterized the expressions of the PgZFP gene family spatially, temperately, and across genotypes collected across the origin and diversity center of Jilin ginseng in different aspects. Analysis of a random selection of transcripts from the PgZFP gene family showed that the expressions of different gene transcripts varied dramatically in a tissue, at a developmental stage, or in a genotype. Nevertheless, the expression of a gene transcript was relatively consistent across tissues, developmental stages, and genotypes, even though its expression also varied across tissues, developmental stages, and genotypes (Supplemental Fig. S4). Of the 228 transcripts of the PgZFP gene family identified in this study, only 54.4–71.9% expressed in a single tissue (Supplemental Fig. S5A) and 27.6–33.8% expressed at a single developmental stage of root (Supplemental Fig. S5B). Forty-six of them expressed at all four developmental stages of the roots and 13, 5, 4, and 14 expressed specifically at 5-, 12-, 18-, and 25-year-old roots, respectively (Supplemental Fig. S5C). Among the genotypes studied, 49.1–64.5% of the PgZFP gene transcripts expressed in the 4-year-old plant root of a genotype (Supplemental Fig. S5D). Transcript expression heatmap analysis showed that the expressions of a vast majority of the transcripts in the PgZFP gene family was independently regulated across tissues, across developmental stages, and across genotypes (Fig. 5). Only PgZFP94 and PgZFP80-05 were found to be co-regulated across the tissues analyzed (Fig. 5A); none of the genes analyzed was co-regulated across the developmental stages of roots (Fig. 5B); and PgZFP36-02 and PgZFP36-03 were co-regulated across genotypes (Fig. 5C).
Expression heatmaps of the PgZFP gene transcripts in 14 tissues (A), four different aged roots (B) and the 4-year-old roots of 42 genotypes (C).
Co-expression network of the PgZFP gene transcripts and its potential hub genes
The above phylogenetic analysis, GO categorization, and cis-regulatory element examination of the PgZFP gene family indicated that the gene family has substantially differentiated in sequence and functionality. The question is whether any relationship remains among the genes of the PgZFP gene family. Therefore, we conducted co-expression network analysis with PgZFP gene transcripts. The results showed that all 228 transcripts of the PgZFP gene family formed a single strong co-expression network (Fig. 6A). The network consisted of 228 nodes and 4745 edges that were clustered into eight clusters (Fig. 6B). In comparison, the network of the PgZFP gene family was much more robust than that of randomly selected ginseng unknown transcripts (Fig. 6C,D). Statistics showed that the PgZFP gene transcripts were more likely to form a co-expression network than the randomly selected ginseng unknown transcripts (Fig. 6E,F). These results suggested that although the gene family has substantially differentiated in sequence and functionality, the expression activities of its genes still maintain correlated, indicating their functional correlation. Further analysis revealed that eight of the 115 PgZFP genes, PgZFP79, PgZFP82, PgZFP114, PgZFP87, PgZFP01-02, PgZFP48, PgZFP63-04, and PgZFP30, likely played central roles in the network when P ≤ 0.001 was applied; therefore, these genes are likely the hub genes, with each gene having a connectivity of 30–43 (Supplemental Fig. S6). Moreover, we attempted to align these eight PgZFP genes to the Arabidopsis genome, but only PgZFP79 and its paralogous gene, PgZFP79P, were aligned to the At3G48430 (REF6) in the Arabidopsis genome (see Fig. 1). This gene was found to be a positive regulator of flowering in an FLC-dependent pathway in Arabidopsis46.
The co-expression network of the PgZFP gene transcripts expressed in the 4-year-old roots of 42 genotypes. (A) The co-expression network of the 228 PgZFP gene transcripts constructed at P ≤ 5.0E−02. It contains 228 gene transcript nodes and 4745 interaction edges. (B) The 12 clusters of the network. (C, D) Tendency of network formation at different P values: variation in numbers of nodes (C) and edges (D). (E,F) Statistics of the network formation tendency in numbers of edges (E) and nodes (F), with 20 replications. “**” indicates that the difference is significant at P ≤ 0.01; and “NS” indicates the difference is not significant at P ≤ 0.05. The randomly selected ginseng unknown gene transcripts were selected from Database A as controls.
Response of the PgZFP genes to salt stress in ginseng
Because the above cis-regulatory element analysis of the PgZFP gene family showed that the genes of the family are likely to be responsive to environmental stresses, we further studied the gene family in responses to environmental stresses, especially to salt stress. Sequence alignment analysis identified four PgZFP genes that were most well aligned to the Arabidopsis salt-responsive STZ and AZF genes, including PgZFP31, PgZFP78-01, PgZFP38, and PgZFP39-01. Of the four genes, PgZFP31 was aligned to the Chinese ginseng genome6 and subjected to cis-regulatory element analysis, showing its potential responsiveness to hormones, environmental stresses, and growth. These four PgZFP genes were all from Cluster III of the family tree. Ginseng adventitious roots were used for the experiment. Figure 7A,B shows that the ginseng adventitious roots were sensitive to salt stress (NaCl). When the concentration of salt increased to 40 mM, the growth of ginseng roots was significantly inhibited (Fig. 7A), indicated by shorter roots (P ≤ 0.05) (Fig. 7B). The relative expression levels of all four genes were up-regulated by salt (Fig. 7C). When the concentration of the salt approached 20 mM NaCl, the relative expressions of two of the four genes started to increase significantly. When the concentration of the salt approached 40 mM NaCl or higher, the relative expressions of all four genes increased significantly (P ≤ 0.05) or extremely significantly (P ≤ 0.01), suggesting that at least four genes in the PgZFP gene family are involved in plant response to salt stress.
The responses of the PgZFP gene family to salt stress. (A) Ginseng adventitious roots stressed with different concentrations of NaCl on the B5 medium for 30 days. (B) Variation of the lateral root length of the salt-stressed roots. (C) The relative expressions of the PgZFP genes selected from the gene family in the salt-stressed roots. The expressions of the genes in the roots stressed with salt were presented as the expression level relative to those of the genes in the roots without salt stress (0 mM NaCl) considered as “1”. The t-test was used to test the mean difference of the gene expressions between control and salt-stressed roots. “*”, significant at P ≤ 0.05; “**”, significant at P ≤ 0.01.
Discussion
The C2H2-ZFP gene family has been shown in several species, including Arabidopsis, rice, tomato, and soybean, to play important functions in plant responses to abiotic and biotic stresses, plant growth and development, and hormone signal transduction8,9,10,11,13,14,15,16. However, the gene family remains unknown in ginseng. This study has, for the first time, genome-wide identified and characterized the gene family in ginseng. A total of 228 C2H2-ZFP gene transcripts, alternatively spliced from 115 C2H2-ZFP genes, are identified and defined PgZFP genes. Therefore, the PgZFP gene family consists of at least 115 gene members. This size of the gene family in ginseng is comparable with those of the gene family in poplar (109)19 and tomato (99)47, but it is smaller than those of the gene family in Arabidopsis (176)17, rice (189)18, Medicago truncatula (218)20, maize (211)21, and soybean (321)22. This result indicates that the PgZFP gene family is a moderate gene family. Nevertheless, this number of the PgZFP genes was identified in the Chinese ginseng cv. Damaya. Pan-transcriptome analysis reveals the number of genes in the family varies substantially among genotypes of P. ginseng, with pan-transcriptome of 149 PgZFP genes and a core-transcriptome of only 12 genes, suggesting that ginseng has a dispensable transcriptome varying by at least 137 PgZFP genes.
The PgZFP gene family is distributed in all 24 chromosomes of the Chinese ginseng genome, but only 17 (23%) of its 73 mapped PgZFP genes are syntenic to 11 of the Arabidopsis C2H2-ZFP genes. Analysis of 46 of the 115 PgZFP genes that have complete CDS shows that 40 (87%) of them were duplicated in the period of 18–41 MYA, suggesting that gene duplication plays a major role in the gene family expansion. The Ka/Ks ratio analysis indicates that purifying and neutral selections drive the family evolution. The present PgZFP gene family is classified into five clusters along with the C2H2-ZFP genes from Arabidopsis, suggesting that the gene family is an ancient gene family that originated before splitting between ginseng and Arabidopsis. Each cluster of the PgZFP gene family has specific conserved motifs distinguishing from other clusters; nevertheless, most genes of the gene family contain the highly conserved QALGGH motif or its variant, such as R/KALGGH. A previous study showed that the change of any amino acid in QALGGH may affect its DNA binding ability, and the mutation of the Q amino acid greatly reduced its DNA binding ability48. The C2H2-ZFP genes containing both QALGGH and I/D/FLN motifs played important roles in plant response to biotic and abiotic stresses23.
The PgZFP gene family was categorized into 17 subcategories at Level 2 and revealed to have 53 types of cis-regulatory elements responsive to multiple biological processes, suggesting that during the evolutionary process, changes have substantially occurred in the structure and upstream regions of PgZFP genes and these changes have affected their functional diversity36,37,38. The functional diversity of the gene family is larger than those of PgbHLH49, PgNAC50, and PgGRAS51 gene families in ginseng that were categorized into 11, 8, and 15 subcategories, respectively. Of the 228 PgZFP gene transcripts, 134 (59%) have binding functions. In comparison, poplar has also most (106) of the C2H2-ZFP genes involved in binding19 and tomato has all annotated C2H2-ZFP genes involved in binding, including nuclear acid binding, organic cyclic compound binding, and heterocyclic compound binding47. This suggests that the PgZFP genes play a role in transcriptional regulation by binding to downstream target genes28,29,52.
The expression analyses of the PgZFP gene family have resulted in several interesting findings. First, most of the genes in the PgZFP gene family expressed at a relatively low level in a tissue, at a developmental stage, and in the root of a genotype. The PgZFP genes that actively expressed in one tissue, at one developmental stage, and in the root of one genotype also tended to actively express in other tissues, at other developmental stages, and in the roots of other genotypes. Second, it is apparent that the expression relationships of the genes in the family are not consistent with their phylogenetic relationships determined by amino acid sequence similarity, suggesting that the genes having similar sequences may not have similar expression patterns. Third, the expressions of the transcripts spliced from the same gene may be substantially different in a tissue, at a developmental stage, and in the root of a genotype. Finally, of the 115 genes in the PgZFP gene family the expressions of only a few are co-regulated, while the expressions of a vast majority are independently regulated. Nevertheless, the genes of the PgZFP gene family are more likely to form a co-expression interaction network, of which some play central roles in the network, indicating that the gene members of the gene family functionally remain correlated49.
Previous studies showed that the C2H2-ZFP gene family plays important roles in growth and development, and plant responses to hormones and biotic and abiotic stresses23. The cis-regulatory element analysis of the PgZFP genes in the present study provides another line of evidence on these roles of the genes in ginseng. Moreover, four genes of the PgZFP gene family, PgZFP31, PgZFP78-01, PgZFP38, and PgZFP39-01, have been identified that were involved in response to salt stress, suggesting that the PgZFP gene family indeed plays roles in plant responses to abiotic stresses, particularly to salt stress in ginseng. Interestingly, the four PgZFP genes are all from Cluster III of the gene family tree. Of the four salt-stress responsive PgZFP genes examined, PgZFP31 and PgZFP78-01 have similar zinc finger structures to STZ that is involved in plant response to salt stress in Arabidopsis14. Both PgZFP31 and PgZFP78-01 belong to C1-2i type zinc finger proteins, have a high similarity in the DLN motif, and contain the FDLNI/L motif. The similar result has been obtained for PgZFP38 and PgZFP39-01 in comparison with AZF1 that is also involved in plant response to salt stress in Arabidopsis14,53. These four PgZFP genes, therefore, provide gene resources for salt tolerance research and genetic improvement in ginseng.
Conclusion
The PgZFP gene family is an ancient gene family consisting of approximately 115 PgZFP genes distributed among all 24 chromosomes of the ginseng genome. It originated before splitting of ginseng from Arabidopsis and its genes have substantially diverged in amino acid sequences and functionality, since they duplicated 18–41 MYA. Nevertheless, conserved motifs exist among the putative proteins of the genes in the family. Different gene members in the gene family express differently in a tissue, at a developmental stage, and in a genotype and the expression of the same gene varies across tissues, developmental stages, and genotypes, which further indicates differentiation of their functionality. Nevertheless, the genes in the family tend to express correlatively, forming a co-expression network, suggesting their functional correlation. Biologically, the PgZFP gene family plays important roles in plant response to salt stress in ginseng, from which four PgZFP genes are identified to be involved in response to salt stress in ginseng.
Data availability
The data used for this study have been deposited at Sequence Read Archive (SRA) of National Center for Biotechnology Information (NCBI), BioProject PRJNA302556; and at Gene Expression Omnibus (GEO) of NCBI, SRP066368 and SRR13131364-SRR13131405. The plant materials are available from the corresponding authors, upon request.
References
Choi, J. S., Chun, K. S., Kundu, J. & Kundu, J. K. Biochemical basis of cancer chemoprevention and/or chemotherapy with ginsenosides. Int. J. Mol. Med. 32, 1227–1238 (2013).
Xu, F. Y. et al. The antitumor activity study of ginsenosides and metabolites in lung cancer cell. Am. J. Transl. Res. 8, 1708–1718 (2016).
Zheng, Y. et al. Antiproliferative effects of protopanaxadiol ginsenosides on human colorectal cancer cells. Biomed. Rep. 1, 555–558 (2013).
Xu, J. et al. Panax ginseng genome examination for ginsenoside biosynthesis. Gigaence 6, 1–15 (2017).
Kim, N. H. et al. Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol. J. 16, 1904–1917 (2018).
Wang, Z.-H. et al. Reshuffling of the ancestral core-eudicot genome shaped chromatin topology and epigenetic modification in Panax. Nat. Commun. 13, 1902 (2022).
Wang, K. et al. The spatial and temporal transcriptomic landscapes of ginseng, Panax ginseng C.A. Meyer. Sci. Rep. 5, 18283 (2015).
Yin, M. et al. The Arabidopsis Cys2/His2 zinc finger transcription factor ZAT18 is a positive regulator of plant tolerance to drought stress. J. Exp. Bot. 68, 2991–3005 (2017).
Li, W. et al. A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell 170, 114–126 (2017).
Lin, W. et al. The zinc finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato. Plant Physiol. 167, 931–949 (2015).
Zhu, J. K. Abiotic stress signaling and responses in plants. Cell 167, 313–324 (2016).
Li, Y. et al. The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol. J. 16, 1201–1213 (2018).
Teng, K. et al. Heterologous expression of a novel Zoysia japonica C2H2 zinc finger gene, ZjZFN1, improved salt tolerance in Arabidopsis. Front. Plant Sci. 9, 1159 (2018).
Sakamoto, H. et al. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol. 136, 2734–2746 (2004).
Zhang, Z. et al. A C2H2 zinc-finger protein OsZFP213 interacts with OsMAPK3 to enhance salt tolerance in rice. J. Plant Physiol. 229, 100–110 (2018).
Sun, Z. et al. Ectopic expression of GmZAT4, a putative C2H2-type zinc finger protein, enhances PEG and NaCl stress tolerances in Arabidopsis thaliana. 3 Biotech. 9, 1–10 (2019).
Englbrecht, C. C., Schoof, H. & Böhm, S. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genomics 5, 39 (2004).
Agarwal, P. et al. Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis. Plant Mol. Biol. 65, 467–485 (2007).
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 10, e0134753 (2015).
Jiao, Z. et al. Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula. BMC Plant Biol. 20, 401 (2020).
Wei, K., Pan, S. & Li, Y. Functional characterization of maize C2H2 zinc-finger gene family. Plant Mol. Biol. Rep. 34, 761–776 (2016).
Yuan, S. et al. Genome-wide identification and classification of soybean C2H2 zinc finger proteins and their expression analysis in legume-rhizobium symbiosis. Front. Microbiol. 9, 126 (2018).
Agnieszka, K. M. Involvement of plant C2H2-type zinc finger transcription factors in stress responses. Plant Sci. 185, 78–85 (2012).
Pabo, C. O., Peisach, E. & Grant, R. A. Design and selection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem. 70, 313–340 (2001).
Luo, X. et al. Over-expression of GsZFP1, an ABA-responsive C2H2-type zinc finger protein lacking a QALGGH motif, reduces ABA sensitivity and decreases stomata size. J. Plant Physiol. 169, 1192–1202 (2012).
Bao, Z., Zhang, N. & Hua, J. Endopolyploidization and flowering time are antagonistically regulated by checkpoint component MAD1 and immunity modulator MOS1. Nat. Commun. 5, 5628 (2014).
Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. & Ohme-Takagi, M. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression. Plant Cell 13, 1959–1968 (2001).
Hiratsu, K., Ohta, M., Matsui, K. & Ohme-Takagi, M. The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers. FEBS Lett. 514, 351–354 (2002).
Singh, P., Mathew, I. E., Verma, A., Tyagi, A. K. & Agarwal, P. Analysis of rice proteins with DLN repressor Motif/S. Int. J. Mol. Sci. 20, 1600 (2019).
Li, H. et al. Basic leucine zipper (bZIP) transcription factor genes and their responses to drought stress in ginseng, Panax ginseng C.A. Meyer. BMC Genomics 22, 316 (2021).
El-Gebali, S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, D427–D432 (2019).
Zheng, Y. et al. iTAK: A program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases. Mol. Plant 9, 1667–1670 (2016).
Lin, Y. et al. Evolution, functional differentiation, and co-expression of the RLK gene family revealed in Jilin ginseng, Panax ginseng C.A. Meyer. Mol. Genet. Genomics 293, 845 (2018).
Brors, B., Gu, L., Schlesner, M., Eils, R. & Gu, Z. Circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811–2812 (2014).
Zhang, Z. et al. KaKs_Calculator: Calculating Ka and Ks through model selection and model averaging. Genom. Proteom. Bioinform. 4, 259–263 (2006).
Faraji, S. et al. Investigation and computational analysis of the sulfotransferase (SOT) gene family in potato (Solanum tuberosum): Insights into sulfur adjustment for proper development and stimuli responses. Plants 10, 2597 (2021).
Abdullah, et al. The GASA gene family in Cacao (Theobroma cacao, Malvaceae): Genome wide identification and expression analysis. Agronomy 11, 1425 (2021).
Heidari, P., Faraji, S., Ahmadizadeh, M., Ahmar, S. & Mora-Polete, F. New insights into structure and function of TIFY genes in Zea mays and Solanum lycopersicum: A genome-wide comprehensive analysis. Front. Genet. 12, 657970 (2021).
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).
Bailey, T. L. et al. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).
Conesa, A. & Götz, S. Blast2GO: A comprehensive suite for functional analysis in plant genomics. Int. J. Plant Genomics 2008, 619832 (2008).
Lescot, M. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325–327 (2002).
Theocharidis, A., van Dongen, S., Enright, A. J. & Freeman, T. C. Network visualization and analysis of gene expression data using BioLayout Express3D. Nat. Protoc. 4, 1535–1550 (2009).
Li, L. et al. Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots. PLoS ONE 14, e0226168 (2019).
Huang, J. et al. A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance. Biochim. Biophys. Acta. 1769, 220–227 (2007).
Noh, B. et al. Divergent roles of a pair of homologous jumonji/zinc-finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16, 2601–2613 (2004).
Zhao, T. et al. Genome-wide analyses of the genetic screening of C2H2-type zinc finger transcription factors and abiotic and biotic stress responses in tomato (Solanum lycopersicum) based on RNA-seq data. Front. Genet. 11, 540 (2020).
Kubo, K. et al. Cys2/His2 zinc-finger protein family of petunia: Evolution and general mechanism of target-sequence recognition. Nucleic Acids Res. 26, 608–615 (1998).
Zhu, L. et al. The bHLH gene family and its response to saline stress in Jilin ginseng, Panax ginseng C.A. Meyer. Mol. Genet. Genomics 295, 877–890 (2020).
Liu, Q. et al. Identification, characterization and functional differentiation of the NAC gene family and its roles in response to cold stress in ginseng, Panax ginseng C.A. Meyer. PLoS ONE 15, e0234423 (2020).
Wang, N. et al. Transcriptome-wide identification, evolutionary analysis, and GA stress response of the GRAS gene family in Panax ginseng C.A. Meyer. Plants 9, 190 (2020).
Ciftci-Yilmaz, S. et al. 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. 282, 9260–9268 (2007).
Han, G. et al. C2H2 zinc finger proteins: Master regulators of abiotic stress responses in plants. Front. Plant Sci. 11, 115 (2020).
Acknowledgements
This work was supported by the Department of Science and Technology of Jilin Province (20210402043GH, 20200801063GH, 20190201264JC, 20190103104JH, 20180414077GH, and 20180101027JC), and the Development and Reform Commission of Jilin Province (2016C064 and 2018C047-3).
Author information
Authors and Affiliations
Contributions
M.P.Z. and Y.W. conceived and designed the study, and M.P.Z. revised the manuscript. Y.J., L.Y.L., Y.H., Y.F.W. and K.W. conducted data analysis, and L.Y.L. and M.Z. wrote the manuscript. Z.P. cultured the ginseng adventitious roots. L.Y.L., L.Z., L.L. and S.L. performed salt stress and conducted qRT-PCR. All the authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Jiang, Y., Liu, L., Pan, Z. et al. Genome-wide analysis of the C2H2 zinc finger protein gene family and its response to salt stress in ginseng, Panax ginseng Meyer. Sci Rep 12, 10165 (2022). https://doi.org/10.1038/s41598-022-14357-w
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-022-14357-w
- Springer Nature Limited