Next Article in Journal
Defective Thyroglobulin: Cell Biology of Disease
Next Article in Special Issue
Drought-Stress Induced Physiological and Molecular Changes in Plants 2.0
Previous Article in Journal
Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells
Previous Article in Special Issue
Proteomic Analysis Revealed Different Molecular Mechanisms of Response to PEG Stress in Drought-Sensitive and Drought-Resistant Sorghums
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 21
10.3390/ijms232113603
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

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

Genome-Wide Identification of AP2/ERF Transcription Factor Family and Functional Analysis of DcAP2/ERF#96 Associated with Abiotic Stress in Dendrobium catenatum

by
Yuliang Han
†,
Maohong Cai
†,
Siqi Zhang
,
Jiawen Chai
,
Mingzhe Sun
,
Yingwei Wang
,
Qinyu Xie
,
Youheng Chen
,
Huizhong Wang
and
Tao Chen
*
Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, College of Life and Environmental Science, Hangzhou Normal University, Hangzhou 311121, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(21), 13603; https://doi.org/10.3390/ijms232113603
Submission received: 16 October 2022 / Revised: 29 October 2022 / Accepted: 3 November 2022 / Published: 6 November 2022
(This article belongs to the Special Issue Drought-Stress Induced Physiological and Molecular Changes in Plants 2.0)
Browse Figures
Versions Notes

Abstract

:
APETALA2/Ethylene Responsive Factor (AP2/ERF) family plays important roles in reproductive development, stress responses and hormone responses in plants. However, AP2/ERF family has not been systematically studied in Dendrobium catenatum. In this study, 120 AP2/ERF family members were identified for the first time in D. catenatum, which were divided into four groups (AP2, RAV, ERF and DREB subfamily) according to phylogenetic analysis. Gene structures and conserved motif analysis showed that each DcAP2/ERF family gene contained at least one AP2 domain, and the distribution of motifs varied among subfamilies. Cis-element analysis indicated that DcAP2/ERF genes contained abundant cis-elements related to hormone signaling and stress response. To further identify potential genes involved in drought stress, 12 genes were selected to detect their expression under drought treatment through qRT-PCR analysis and DcAP2/ERF#96, a nuclear localized ethylene-responsive transcription factor, showed a strong response to PEG treatment. Overexpression of DcAP2/ERF#96 in Arabidopsis showed sensitivity to ABA. Molecular, biochemical and genetic assays indicated that DcAP2ERF#96 interacts with DREB2A and directly inhibits the expression of P5CS1 in response to the ABA signal. Taken together, our study provided a molecular basis for the intensive study of DcAP2/ERF genes and revealed the biological function of DcAP2ERF#96 involved in the ABA signal.

1. Introduction

Transcription factors (TFs) are key regulators for plants to respond to various environmental stimuli and transmit stress signals. Many kinds of transcription factors, such as APETALA2/Ethylene Responsive Factor (AP2/ERF) [1], bHLH [2], MYB [3], Dof [4] and WRKY [5], have been proven to play vital roles in biotic and abiotic stress responses [6]. AP2/ERF widely exists in the plant kingdom and is one of the largest transcription factor families in plants [7], which was first discovered in Arabidopsis and soon found similar transcription factors in tobacco [8]. As the most representative domain of AP2/ERF family proteins, the AP2 domain consists of one α-helix and three β-sheets, with a range of 60–70 amino acid residues [9]. The AP2 domain can bind to some special cis-acting elements, such as dehydration responsive elements (DRE), C-repeat element (CRT) and GCC box, to regulate downstream gene expression [7,9,10]. This family is mainly divided into four subfamilies: AP2 (APETALA2), DREB (Dehydration Responsive Element-Binding), ERF (Ethylene Responsive Element Binding protein) and RAV (Related to ABI3/VP) [11]. The AP2 subfamily contains two tandem AP2 domains, which have been shown to be widely involved in floral organ development and regulation [12]. The RAV subfamily contains an AP2 domain and a B3 domain, which are thought to be involved in biotic and abiotic stresses, as well as its known involvement in flowering regulation [13]. The DREB and ERF subfamily both contain only one AP2 domain, but the 14th and 19th amino acids of their AP2 domains are different, resulting in a distinct affinity for different DNAs. DREB family proteins can specifically bind to DRE/CRT cis-acting elements, thereby regulating ABA, drought and low temperature responses, while the ERF subfamily proteins prefer to bind GCC-box related to ethylene response, disease resistance and abiotic stress [8].
Members of the AP2/ERF family have been identified and scaled in many species, including 146 in Arabidopsis thaliana [11], 163 in Oryza sativa [11], 134 in Fagopyum Tataricum [14], 364 in Salix matsudana [15], 163 in Zingiber officinale [16], 322 in Triticum aestivum [17], 189 in Panax ginseng [18] and 531 in Brassica campestris [19]. AP2/ERF transcription factor family has been confirmed to be involved in a variety of abiotic stress responses; for example, AtCBF1/2/3 control low temperature responses in Arabidopsis [20,21], ZmDREB2a participates in heat response in maize [22], and GmERF4/7 mediate salt stress response in soybean [23]. Overexpression of OsERF71 in rice has also been found to change the root structure of plants and enhance tolerance to drought stress [24]. However, to date, no studies have clarified the role of AP2/ERF family genes in the response to environmental stress of D. catenatum, so it is very important to identify DcAP2/ERF by genome-wide identification approach.
D. catenatum, as a typical orchid plant, is highly valued because its stem contains many medicinal ingredients [25]. In addition, due to harsh growth circumstances, often attached to rocks, tree trunks and cliffs (Figure S1), D. catenatum has strong resistance to various stresses [26]. Therefore, studying how D. catenatum resists and resolves stresses from nature may provide new inspiration for studying the mechanism of plants resisting stress. To date, only a few gene families have been identified in D. catenatum, which are involved in stress response, including MYB [15] and GRAS [27]. With the determination of the whole genome sequences of D. catenatum in recent years [28,29,30], it has become possible to systematically analyze the characteristics and functions of AP2/ERF family in D. catenatum.
In this study, we first performed genome-wide identification of DcAP2/ERF gene family using HMMER3.0 and a total of 120 DcAP2/ERF genes were identified. Second, all 120 genes were comprehensively analyzed by phylogenetic analysis, gene structure and domain visualization, and promoter motif prediction. Third, we presented the expression patterns of the AP2/ERF family in 9 different tissues and under low temperature stress. Finally, we detected the expression of 12 DcAP2/ERF genes under drought stress by qRT-PCR analysis and focused on analyzing the characteristics and functions of DcAP2ERF#96. Our research found that the heterologous expression of DcAP2ERF#96 causes at least 9 ABA downstream genes to be significantly inhibited, including P5CS1 and RD29A, which are usually used by researchers to measure the severity of abiotic stress faced by plants [31,32,33]. We also found the protein DREB2A, which interacts with DcAP2ERF#96 in D. catenatum. Together, our study reveals the vital role of AP2/ERF family in D. catenatum and provides several key candidate genes related to abiotic stress.

2. Results

2.1. Identification of AP2/ERF Genes in D. catenatum

To identify the DcAP2/ERF family members, a combined analysis of genome-wide and full-length transcriptome-wide was performed using HMM software and SMART online. After removing the duplicate transcripts, a total of 120 AP2/ERF genes were finally screened and identified in the D. catenatum genome, and the basic information of these genes was analyzed and summarized in Table 1, including their protein length, MW, PI and subcellular location. Among these genes, the largest protein is encoded by DcAP2ERF#47 with 733 aa, while the smallest protein is encoded by DcAP2ERF#2 with 117 aa. (Table 1). The molecular weight (MW) of proteins ranges from 13.24 to 81.46 kDa and the isoelectric point (PI) varies from 4.35 (DcAP2ERF#39) to 10.10 (DcAP2ERF#64). According to the analysis of the aliphatic index and grand average of hydropathicity (GRAVY), the DcAP2/ERF family proteins are generally hydrophilic. To analyze subcellular localization, all AP2/ERF proteins were predicted using PSORT. The results showed that most DcAP2/ERF proteins (78.3%) are located in the nucleus, which is consistent with the localization characteristics of the transcription factor family. In addition, 18.3% of proteins were predicted to be located in cytoplasm, and 3.3% of proteins were likely to be located in mitochondria (Table 1).

2.2. Phylogenetic Analysis of DcAP2/ERF Families

To study the genetic phylogeny of AP2/ERF family in Orchidaceae, phylogenetic trees were constructed using sequences from Arabidopsis thaliana (146 genes), D. catenatum (120 genes) and Phalaenopsis equestris (118 genes) (Figure 1A). As shown in Figure 1, all AP2/ERFs of the three species can be grouped into four subfamilies, namely, AP2, ERF, DREB and RAV, which suggests that the AP2/ERF family is evolutionarily conserved in the plant kingdom. From the view of family size, with the deepening of evolution, the number of genes in AP2/ERF family has been reduced from 139 in Arabidopsis thaliana to 118 in Phalaenopsis equestris and 120 in D. catenatum. The size of the RAV and DREB subfamily of D. catenatum and Phalaenopsis equestris was significantly smaller than that of Arabidopsis thaliana. The number of ERF subfamily members increased slightly compared with that of Arabidopsis thaliana, while the gene number and tree structures of the AP2 subfamily remained basically unchanged (Figure 1B). These changes showed that AP2/ERF family is conservative in the history of plant evolution, while making corresponding changes with different plant growth environments. Considering that orchids usually have stronger environmental adaptability than Arabidopsis thaliana, the adjustment of these gene group structures may help them cope with more complicated biotic and abiotic stresses.

2.3. Structure Analyses of the AP2/ERF Gene Family in D. catenatum

To further understand the structural characteristics of DcAP2/ERF family genes, the conserved motif, domain, intron and exon of DcAP2/ERFs were further analyzed (Figure 2). Using motif analysis, eight different motifs were predicted based on DcAP2/ERF protein sequences (Figure S2). We summarized the different motif patterns of each subfamily, and the results showed that motif 1, motif 3 and motif 4 exist in all four DcAP2/ERF subfamilies (Figure 2A). Compared with the ERF subfamily, the RAV subfamily lacks motif 2, while the DREB subfamily adds motif 7. Motif 7 only exists in the DREB subfamily and it is located outside the AP2 domain (Figure S3A). For the AP2 subfamily containing two AP2 domains, these proteins were mainly divided into two categories. Category I is composed of motif 3, motif 2, motif 1, motif 4 (first AP2 domain) and motif 5, motif 1, motif 4 (second AP2 domain). Category II is composed of motif 8, motif 1, motif 4 (first AP2 domain) and motif 3, motif 2, motif 6 (second AP2 domain). These data suggest that the double AP2 domain of the AP2 subfamily is not a simple duplication of the single AP2 domain, but has its own unique characteristics. For domain and structure analysis (Figure 2B,C), the characteristics of the gene members in each subfamily were consistent with previous reports [34]. It is worth noting that the intron structures of AP2 subfamily members are very complex, which is conducive to their flexible cutting and assembly so as to cope with different situations. The diversity of the AP2 domain model may be the result of gene evolution and selection. This partly explains why different subfamilies have preferences for different cis-acting elements [8].
Combined with the secondary structure of the AP2 domain, we further analyzed the motif of a typical AP2 domain (Figure S3B). Motif 3, motif 2 and the front half of motif 1 are distributed with one β-sheet, respectively. The back half of motif 1 and the first half of motif 4 form one α-helix. These secondary structures together form a complete AP2 domain. These findings are consistent with the previous description of the AP2 domain [35]. In addition, these results showed that although AP2/ERF family is complex at the motif level, it is still conserved in domain composition.

2.4. Cis-Acting Element Analysis of DcAP2/ERF Gene Promoters

To explore the putative functions of AP2/ERF family genes in D. catenatum, the 2000-bp upstream sequences of 120 genes were extracted to analyze the potential cis-elements. The predicted TF-binding motifs were classed into three categories: hormone-related elements, stress-related elements, and growth and development-related elements (Figure 3). Hormone response, such as auxin-responsiveness, ABA-responsiveness, GA-responsiveness, MeJA-responsiveness and SA-responsiveness. From the view of hormone response-related elements, a total of 113 genes have hormone response elements in the promoter region (Table S3), indicating that DcAP2/ERF family genes may be widely involved in hormone response pathways. Among these elements, the number of elements responding to MeJA and ABA was enriched, reaching 185 and 149, respectively, which indicated that DcAP2/ERF genes may respond to biotic and abiotic stress rapidly. Stress response-related elements were associated with anaerobic induction, drought induction, low temperature responsiveness and anoxic specific induction. In addition, the promoters of 68 genes in DcAP2/ERF family contain growth- and development-related response elements associated with meristem expression, zein metabolism, endoperm expression, circadian control, cell cycle regulation, flavonoid biosynthetic gene regulation, and others. Meristem expression elements and zein metabolism regulation elements accounted for the largest proportion, reaching 32 and 31, respectively (Figure 3).

2.5. GO Annotation Analysis of DcAP2/ERF Family Proteins

To further understand the molecular function of DcAP2/ERF proteins, GO annotations were conducted in this study. According to gene ontology, genes or gene products have three main characteristics: cell composition, molecular function and biological process. Their terminology labels can help us understand the protein functions [36]. Here, a total of 75 DcAP2/ERF proteins were assigned 20 GO terms, including 4 cellular component terms, 2 molecular function terms and 14 biological process terms (Figure S4, Table S2). Under the cellular component category, 32 proteins were identified as ‘cell’ (GO: 0005623), ‘cell part’ (GO: 0044464) and ’organelle’ (GO: 0043226). Under the molecular function category, 50 and 75 proteins were annotated for ‘binding’ (GO: 0005488) and ‘transcription regulator activity’ (GO:0140110), respectively. Under the biological process category, all annotated proteins are involved in ‘metabolic processes’ (GO: 0008152), ‘cellular processes’ (GO: 0009987) and ‘biological regulatory’ (GO: 0065007). In addition, there are 53 proteins involved in ‘response to stimulus’ (GO: 0050896). In summary, DcAP2/ERF proteins are a typical family of transcription factors with the potential ability to bind downstream DNA and regulate various physiological processes.

2.6. Expression Patterns of DcAP2/ERF Genes under Different Stresses

To study the expression pattern of DcAP2/ERF family genes in response to hormone and environmental stresses, the transcriptomic data of D. catenatum under MeJA and low-temperature treatments were analyzed. In chilling stress, a total of 29 genes showed significant changes under chilling treatment, of which 16 genes were significantly upregulated, especially DcAP2ERF#52, DcAP2ERF#7 and DcAP2ERF#98 (Table S4), and 13 genes were downregulated (Figure 4B), especially DcAP2ERF#84, DcAP2ERF#109 and DcAP2ERF#61. These data suggest that these genes may play important roles in the cold stress response and can be considered candidate genes for further study of cold stress biology.
For the MeJA treatment, the repetitions of three experimental groups and three control groups were clustered into two groups (Figure 5A). Under MeJA treatment, 9 genes were upregulated, and 11 genes were downregulated (Figure 5B). Among these genes, the expression of DcAP2ERF#44 and DcAP2ERF#71 was highly increased (about 18 times and 9 times, respectively) compared with the control (Table S5). This suggests that they may be strongly induced by JA signaling and perform important functions in D. catenatum.

2.7. Expression Patterns of DcAP2/ERF Genes in Different Organs

To study the tissue expression pattern and reveal the related function of DcAP2/ERF genes, we analyzed the transcriptome data of DcAP2/ERFs in different tissues (Figure S5). Among all 120 DcAP2/ERF genes, 14 DcAP2/ERF genes were constitutively expressed, 11 genes tended to be expressed in roots (including green root tip and white part of root), stem or leaves, and 34 genes tended to be expressed in flower organs (including sepal, labellum, gynostemium and pollinia). In addition, by the cluster analysis of tissues and organs, the expression pattern of flower organs was distinguished from roots, stems, leaves and their related tissues. The expression pattern of gynostemium is similar to that of sepal among these DcAP2/ERF genes, except DcAP2ERF#74, DcAP2ERF#64, DcAP2ERF#55, DcAP2ERF#50, DcAP2ERF#22 and DcAP2ERF#26. It is worth mentioning that the expression pattern of DcAP2/ERF genes in pollen is obviously different from those in other tissues. Some genes, such as DcAP2ERF#117, DcAP2ERF#24, DcAP2ERF#40, DcAP2ERF#41 and DcAP2ERF#59, are specifically expressed in pollen, indicating that these genes play important roles in pollen development.

2.8. Expression Pattern of DcAP2/ERFs under PEG6000 Treatment

Many ABA response elements in DcAP2/ERF family gene promoters and the excellent drought resistance of D. catenatum allow us to suspect that some DcAP2/ERF family genes are involved in the drought-tolerant response. Based on the expression of these genes in different transcriptomes (Figure 4, Figure 5 and Figure S5), we finally selected 12 genes of interest to test their expression levels under drought stress (Figure 6). After 20% PEG6000 treatment for 0 h, 2 h, 4 h and 8 h, respectively, leaves, stems and roots of D. catenatum were collected for RNA extraction and qRT-PCR analysis. As shown in Figure 6, most of the genes responding to drought are found in leaves, with 11 genes, while only 7 and 5 genes showed increased expression in stems and roots, respectively, under drought stress. Among these genes, four genes (DcAP2ERF#16, DcAP2ERF#50, DcAP2ERF#52, DcAP2ERF#78) showed obvious increased expression in all three tissues under drought stress. Interestingly, DcAP2ERF#1 and DcAP2ERF#86 only increased in leaves and stems, while they had no changes in roots, indicating that these genes may play different roles in D. catenatum in response to drought stresses. In addition, according to the differences in the expression levels and response times of these genes in the three tissues, we can infer that the three kinds of tissues showed different sensitivities in the face of drought stress.

2.9. DcAP2ERF#96 Encodes a Conserved AP2 Domain Transcription Factor Localized in the Nucleus

To further confirm the related genes involved in drought stress in the AP2/ERF family, we measured the biomass differences in several tissues of D. catenatum under drought stress and normal conditions (Figure 7). The results showed that the growth of the stem was significantly inhibited under three-month drought conditions (Figure 7A). In addition, stems, as the main medicinal parts of D. catenatum, had the most obvious changes in biomass under drought stress (Figure 7B). Based on this finding, we selected DcAP2ERF#96 as a potential gene for further study, which showed specifically decreased expression in the stem under PEG6000 treatment, as shown in Figure 6. Sequence analysis revealed that DcAP2ERF#96 is an AP2-like ethylene-responsive transcription factor. Subcellular localization in D. catenatum protoplasts and tobacco leaf cells showed that the DcAP2ERF#96 protein was located to the nucleus (Figure 8A,B). Previous studies have shown that AP2/ERF transcription factors can bind to GCC (AGCCGCC), DRE (GCCGAC), and CRT (ACCGAC) boxes to regulate downstream gene expression [6]. To confirm this, a Y1H assay was performed, and the results showed that DcAP2ERF#96 has a strong ability to bind to the CRT box. In addition, it also has weak binding activity to DRE and GCC box (Figure 8C), indicating that the binding motifs of DcAP2ERF#96 were conserved in D. catenatum. A transactivation activity assay showed that DcAP2ERF#96 had self-activating properties, and the self-activating regions existed in the N terminal (1–185 aa) and C-terminal (280–429 aa) of DcAP2ERF#96, while not the middle region (186–279 aa), which contain two AP2 domains (Figure 8D). These results confirm that DcAP2ERF#96 is a nuclear-localized transcription factor.

2.10. DcAP2ERF#96 Interacted with DcDREB2A and Negatively Regulate ABA Signaling in Arabidopsis

To further investigate the function of DcAP2ERF#96 in response to drought, the two overexpression lines of Arabidopsis thaliana #3 and #6 with the highest gene expression levels were selected for further study. Compared with wild-type (Col-0), the leaf area and root length of the two overexpression lines were significantly reduced in 1/2 MS with 10 μM ABA, suggesting that DcAP2ERF#96 had an ABA-sensitive phenotype (Figure 9A–C). To further explore whether DcAP2ERF#96 participates in the ABA signaling pathway in response to drought, the expression of 9 genes related to the ABA signaling pathway was selected and detected under ABA treatment (Figure 9D). Results showed that all 9 genes except AtADH1 were significantly upregulated after 10 μM ABA treatment in Col-0. However, the relative expression levels of these genes were significantly reduced in DcAP2ERF#96 overexpression lines compared with Col-0, which confirmed our hypothesis that DcAP2ERF#96 acts as a transcriptional repressor to negatively regulate ABA signaling under drought stress in plants. Cis-elements analysis of these 9 gene promoters showed that three gene promoters contain DRE, CRT and GCC box: The AtP5CS1 promoter contains one GCC box; the AtRD29A promoter contains three CRT boxes and one DRE box; and the AtRAB18 promoter contains one DRE element. All the elements are located within the range of 500-bp upstream sequences of genes. (Figure 10A) Y1H results showed that DcAP2ERF#96 can directly bind to the promoter of P5CS1 and RD29A in yeast, but there is no indication that it can combine with RAB18 promoter (Figure 10B). At the same time, our Y2H results confirmed that DcAP2ERF#96 interacts with DREB2A-1 (LOC110112950) and DREB2A-2 (LOC110113533), which have been reported to regulate the expression of RD29A in Arabidopsis [37] (Figure 10C). These results suggested that DcAP2ERF#96 negatively regulates the expression of P5CS1 and RD29A in Arabidopsis thaliana, thereby affecting its response to the ABA signal (Figure 10D).

3. Discussion

D. catenatum, a precious Chinese herbal medicine of Orchidaceae, has great medicinal and economic value [27]. AP2/ERF family genes are widely involved in various physiological activities and biotic/abiotic stress responses of plants. However, the role of AP2/ERF family genes in D. catenatum remains uncertain. In this study, 120 DcAP2/ERF genes were detected, and their phylogenetic relationship, domain composition, gene structure, cis-acting elements and gene ontology were analyzed. Furthermore, the tissue expression profile and different responses of DcAP2/ERF family genes under cold environment, MeJA treatment and simulated drought were also explored. Based on these results, we discussed the potential functions of some genes in D. catenatum.
In phylogenetic analysis, we found obvious structural similarities of phylogenetic trees and adjustment of subfamily proportion among Arabidopsis thaliana, D. catenatum and Phalaenopsis equestris (Figure 1), which indicated that orchidaceae had a conserved evolutionary process. We also found that the number of introns in the AP2 subfamily was quite greater than those in other subfamilies (Figure 2). Previous studies have shown that introns may be gradually lost in the process of evolution [38]. According to this theory, we speculate that the AP2 subfamily may be one of the origins of the evolution of other subfamilies. Other studies have shown that the AP2 domain in plant genes may be derived from bacterial or viral HNH endonucleases, and various subfamilies have gradually been generated during the long evolutionary process [39]. In general, the evolutionary relationship between the various AP2/ERF subfamilies still remains to be explored.
Transcriptional regulation plays a leading role in the regulation of gene expression, which is mainly controlled by its cis-acting element on the gene promoter [40,41]. We analyzed the cis-acting elements in all DcAP2/ERF family promoter regions (Figure 3) and described their functions by gene ontology (Figure S4). The results showed that the cis-acting elements responding to MeJA and ABA accounted for the largest proportion, which was similar to the promoter characteristics of AP2/ERF family in other plants like wheat [17] and ginger [16]. At the same time, gene ontology also analyzed DcAP2/ERF proteins that have typical features of the general transcription factor families and have the ability to respond to stimuli and signal transduction.
Drought is a common abiotic stress that can cause serious damage to the growth and development of plants [42]. Drought stress will directly lead to cell dehydration, which produces an ABA signal and causes a series of tolerance responses in plants, including promoting stomatal closure of guard cells and regulating the expression of related genes [43]. Previous studies have suggested that some AP2/ERF family genes are closely related to drought stress and ABA [44,45]. We selected 12 genes of interest of DcAP2/ERF family, and tested their expression through PEG6000 treatment simulating an arid environment (Figure 6). The results confirmed our hypothesis that the expression of most selected genes increased under simulated drought conditions. As the most strongly upregulated gene, the homologous gene of DcAP2ERF#1 in Arabidopsis has been confirmed to control the synthesis of epidermal wax [46], and the increase of epidermal wax in D. catenatum leaves may be beneficial in reducing water loss and surviving longer under drought stress. In the stem, the changes in the expression levels of all tested genes were significantly smaller than those in leaves and roots, which may be due to the fact that stems are rich in polysaccharides, polyphenols and other substances and have strong resistance to drought stress. After comparing the biomass of different tissues of D. catenatum under drought conditions, the stem is considered to play a key role in the process of D. catenatum resisting drought stress (Figure 7). At the same time, we noticed a decrease in the expression of DcAP2ERF#96 in stems under simulated drought conditions (Figure 6). Molecular biology and transgenic techniques including subcellular localization, Y1H, Y2H and transgenic experiments confirmed that DcAP2ERF#96 functions as a transcriptional repressor to regulate the ABA signaling pathway and interacts with DREB2A in D. catenatum. The downregulation of its expression in stems may be one of the self-rescue responses of D. catenatum under drought stress, which is conducive to the transmission of ABA signals in the whole plant. (Figure 8, Figure 9 and Figure 10). In addition, the expression of some genes downstream of ABA, such as AtABI1, AtABI5 and AtADH1, were also significantly reduced (Figure 9D), which may be caused by DcAP2ERF#96 indirect regulation, but this speculation remains to be further tested. In general, this study systematically analyzed the DcAP2/ERF family of D. catenatum and preliminarily studied the role of DcAP2ERF#96 in the ABA signaling pathway. Further functional verification should be carried out to understand the role of DcAP2/ERF family genes under different conditions.

4. Materials and Methods

4.1. Identification of AP2/ERF Genes in D. Catenatum

To perform genome-wide identification of the DcAP2/ERF gene family, the whole D. catenatum genome and its annotation information were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 1 November 2022). The Hidden Markov model (HMM) of the AP2 domain (pfam: PF00847) and B3 domain (pfam: PF02362) were downloaded from the Pfam database (http://www.sanger.ac.uk/Software/Pfam/, accessed on 1 November 2022). The potential DcAP2/ERF genes were searched by HMMER3.0 (e-value < e−5). All screened sequences were verified by the SMART database (http://smart.embl-heidelberg.de/, accessed on 1 November 2022) [47]. After removing duplicate transcripts, a total of 120 AP2/ERF family genes were found in D. catenatum and named as DcAP2ERF#1 to DcAP2ERF#120. Basic information, such as the isoelectric point and molecular weight of genes, was analyzed by ExPASy (https://www.expasy.org/, accessed on 1 November 2022). The subcellular localization of genes was predicted using PSORT (https://www.genscript.com/psort.html, accessed on 1 November 2022).
Using the same method, we identified 118 AP2/ERF family genes from Phalaenopsis equestris. The 146 AP2/ERF genes of Arabidopsis thaliana were downloaded from PlantTFDB (http://planttfdb.gao-lab.org/index.php, accessed on 1 November 2022) [48].

4.2. Phylogenetic Tree Analysis of the DcAP2/ERF Protein

The phylogenetic tree was constructed by MEGA7 using the neighbor-joining method with a bootstrap test (1000 replicates). Multiple sequence alignments of all proteins were completed by MUSCLE before phylogenetic analysis [49]. For the graphic display, the final phylogenetic trees were modified using ITOL (https://itol.embl.de/, accessed on 1 November 2022).

4.3. Gene Structure, Conserved Domain, Motif and Promoter Analyses of AP2/ERF Family Members

The gene structure information was directly generated by TBtools according to the gene annotation file. The conserved domain information of AP2/ERF family proteins was calculated by Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 1 November 2022) [50]. The conserved motif of the full length of AP2/ERF proteins was analyzed using the online MEME website (https://meme-suite.org/meme/tools/meme, accessed on 1 November 2022) [51] by the zero or one occurrence per sequence (zoops) strategy and set the number of searchable motifs to 8. Other parameters are website default settings 4.4. Cis-acting element analyses of DcAP2/ERF family genes
The promoter sequences (2000-bp upstream of gene) were obtained from the D. catenatum genome. All cis-acting elements were calculated by PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 November 2022) [52].

4.4. Plant Materials and Methods of Stress Treatment

‘Honggan ruanjiao’ a well-known D. catenatum variety, was used in this study. In order to detect the expression pattern of DcAP2/ERF family under drought conditions, 6-month-old seedlings were treated with 20% PEG6000 to mimic drought conditions. After treatment for 0, 2, 4 and 8 h, respectively, three samples (root, stem and leaf) were harvested and frozen in liquid nitrogen for RNA extraction.
In addition, DcAP2ERF#96 was transformed into Arabidopsis thaliana to obtain overexpression lines. All overexpression lines were selected by hygromycin and the relative expression level of these lines was detected by qRT-PCR (Figure S6).

4.5. RNA Extraction and Quantitative/Real-Time-PCR (qRT-PCR) Analysis

The total RNA of plant materials was extracted using Trizol reagent (Coolaber, Beijing, China). One microgram of total RNA was used to synthesize cDNA with HiScript II Q Select RT SuperMix for qRT-PCR (Vazyme, Nanjing, China). qRT-PCR was performed on a CFX384 real-time system (BIO-RAD, Hercules, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The experiments were repeated three times independently. The actin2 gene was used as an internal control. The primers used in this assay are listed in Table S1.

4.6. Expression Pattern Analysis of DcAP2/ERF Genes

To reveal the expression patterns of all AP2/ERF family genes in D. catenatum, the raw RNA-seq data were downloaded from the NCBI Sequence Read Archive (SRA) database. The transcriptome data of root (SRX2938667), stem (SRR4431600), leaf (SRR4431601), white part of root (SRR4431598), Green root tip (SRR4431599), sepal (SRR4431597), labellum (SRR4431602), pollinia (SRR5722145) and gynostemium (SRR4431596) were obtained for tissue expression analyses [29]. The raw RNA-seq data of leaves under 20 °C conditions (SRR3210630, SRR3210635 and SRR3210636) and 0 °C conditions for 20 h (SRR3210613, SRR3210621 and SRR3210626) were downloaded for low temperature stress analyses [53]. By clustering, six groups of transcriptomic data were grouped into two categories according to temperature, indicating that the experimental data were reliable and had significant differences between the control and chilling treatments (Figure 4A). The raw RNA-seq data of 1mM MeJA (SRR14635790, SRR14635791, SRR14635792) treatment for 4 h and control (SRR14635793, SRR14635796, SRR14635797) were downloaded for hormone treatment analyses [54]. All raw data were converted by the Kallisto method and normalized by transcripts per million (TPM). Differential expression analysis and volcano plot drawing were visualized using Tbtools.

4.7. Gene Ontology Annotation Analysis

Gene Ontology (GO) analysis of DcAP2/ERF family proteins was done by eggNOG-mapper (http://eggnog-mapper.embl.de/, accessed on 1 November 2022) and visualized by the WEGO website (https://wego.genomics.cn/, accessed on 1 November 2022). The GO annotation results are listed in Table S2.

4.8. Subcellular Localization of DcAP2ERF#96

The full-length coding sequences of DcAP2ERF#96 were amplified and cloned into the pEarleyGate 101 vector to produce the DcAP2ERF#96-GFP fusion construct. To observe the localization in protoplasts of D. catenatum, young leaves of six-month-old D. catenatum were used for protoplast preparation and transformation. 10 μg plasmid was transformed into 100 μL protoplast suspension, and fluorescence was observed after 12 h [55]. To observe the localization in tobacco, four-week-old N. benthamiana was used for gene transient expression. The activated GV3101 Agrobacterium containing the constructed plasmid was incubated with infection solution (10 mM MES, 10 mM MgCl2 and 200 µM AS) for 2 h and then injected into the young leaves of tobacco (N. benthamiana). Fluorescence was observed at 488 nm and 560 nm using a confocal microscope after 2 days.

4.9. Yeast One-Hybrid (Y1H) and Yeast Two-Hybrid (Y2H) Assays

For the Y1H assay, the full-length CDS of DcAP2ERF#96 was cloned into the pB42AD vector. Three tandem repeats of the motif sequences (DRE, CRT, GCC) were cloned into the pLacZi2μ vector. The combined plasmids were co-transformed into yeast strain EGY48 and selected on the SD/-Ura/-Trp agar medium. Colonies were then plated onto SD/-Ura/-Trp agar medium containing raffinose, galactose and 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) for blue color development. For the Y2H assay, the full-length CDS and three truncated fragments of DcAP2ERF#96 were cloned into the pGBKT7 vector. The constructed plasmids were transformed into AH109 yeast competent with empty pGADT7 using the Yeastmaker Yeast Transformation System (Clontech, Shanghai, China). Transformation cells were plated on SD/-Trp/-Leu medium to screen positive clones. After 4 days, positive clones were transferred to SD/-Ade/-His/-Leu/-Trp dropout medium to detect self-activation. All relevant primer sequences are listed in Table S1.

4.10. Acquisition and Handling of Arabidopsis Transgenic Lines

The full-length CDS of DcAP2ERF#96 was cloned into the pGWB512 vector containing the 35S promoter. Arabidopsis thaliana inflorescences were infected by the floral dip method and the selection of transgenic positive lines by MS medium containing hygromycin. The two lines with the highest expression level were identified and selected by qRT-PCR, and their seeds were placed on MS medium for germination to a root length of 1 cm, then transferred to 1.2% MS medium containing 10 μM ABA, and cultured for 5 days before observation phenotype.

5. Conclusions

In this study, 120 AP2/ERF genes were identified in D. catenatum for the first time, and their characteristics were further analyzed. These results contribute to our understanding of the classification and evolution trend of AP2/ERF family of orchids. At the same time, according to transcriptome data, we further analyzed the expression pattern of DcAP2/ERF family genes and selected 12 genes of interest from them to test their responses to drought stress. In addition, we preliminarily studied the characteristics of transcription factor DcAP2ERF#96 and found its interacting proteins and promoters according to its inhibition effect on the ABA signaling pathway. However, the specific molecular mechanism of DcAP2ERF#96 in response to abiotic stress in D. catenatum remains to be further studied.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms232113603/s1.

Author Contributions

Y.H., M.C., H.W. and T.C. initiated and designed the research, Y.H., S.Z., J.C., M.S. and Y.C. performed the experiments, J.C., Y.W. and Q.X. analyzed the data, Y.H. wrote the paper. M.C. revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Zhejiang Provincial Key Research & Development Project Grants (2018C02030), the Starting Research Fund from Hangzhou Normal University (2019QDL015) and the Zhejiang Provincial Natural Science Foundation of China (Grant NO. LQ22C130001).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the other datasets supporting the conclusions of this article are included within the article and its additional files.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gutterson, N.; Reuber, T.L. Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr. Opin. Plant Biol. 2004, 7, 465–471. [Google Scholar] [CrossRef] [PubMed]
  2. Toledo-Ortiz, G.; Huq, E.; Quail, P.H. The Arabidopsis Basic/Helix-Loop-Helix Transcription Factor Family. Plant Cell 2003, 15, 1749–1770. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Dubos, C.; Stracke, R.; Grotewold, E.; Weisshaar, B.; Martin, C.; Lepiniec, L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010, 15, 573–581. [Google Scholar] [CrossRef]
  4. Yang, X.; Tuskan, G.A.; Cheng, Z.-M. Divergence of the Dof Gene Families in Poplar, Arabidopsis, and Rice Suggests Multiple Modes of Gene Evolution after Duplication. Plant Physiol. 2006, 142, 820–830. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Networks of WRKY Transcription Factors in Defense Signaling. Available online: networks-of-wrky-transcription-factors-in-defense-signaling-5d5wsqb4ip.pdf (accessed on 1 November 2022).
  6. Feng, K.; Hou, X.-L.; Xing, G.-M.; Liu, J.-X.; Duan, A.-Q.; Xu, Z.-S.; Li, M.-Y.; Zhuang, J.; Xiong, A.-S. Advances in AP2/ERF super-family transcription factors in plant. Crit. Rev. Biotechnol. 2020, 40, 750–776. [Google Scholar] [CrossRef]
  7. Okamuro, J.K.; Caster, B.; Villarroel, R.; Van Montagu, M.; Jofuku, K.D. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. USA 1997, 94, 7076–7081. [Google Scholar] [CrossRef] [Green Version]
  8. Ohme-Takagi, M.; Shinshi, H. Ethylene-lnducible DNA Binding Proteins That lnteract with an Ethylene-Responsive Element. Plant Cell 1995, 7, 173–182. [Google Scholar]
  9. Allen, M.D.; Yamasaki, K.; Ohme-Takagi, M.; Tateno, M.; Suzuki, M. A novel mode of DNA recognition by a β-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J. 1998, 17, 5484–5496. [Google Scholar] [CrossRef] [Green Version]
  10. Riechmann, J.L.; Meyerowitz, E. The AP2/EREBP Family of Plant Transcription Factors. Biol Chem. 1998, 379, 633–646. [Google Scholar]
  11. Nakano, T.; Suzuki, K.; Fujimura, T.; Shinshi, H. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice. Plant Physiol. 2006, 140, 411–432. [Google Scholar] [CrossRef] [Green Version]
  12. François, L.; Verdenaud, M.; Fu, X.; Ruleman, D.; Dubois, A.; Vandenbussche, M.; Bendahmane, A.; Raymond, O.; Just, J.; Bendahmane, M. A miR172 target-deficient AP2-like gene correlates with the double flower phenotype in roses. Sci. Rep. 2018, 8, 12912. [Google Scholar] [CrossRef] [Green Version]
  13. Matías-Hernández, L.; Aguilar-Jaramillo, A.E.; Marín-González, E.; Suárez-López, P.; Pelaz, S. RAV genes: Regulation of floral induction and beyond. Ann. Bot. 2014, 114, 1459–1470. [Google Scholar] [CrossRef]
  14. Liu, Q.; Kasuga, M.; Sakuma, Y.; Abe, H.; Miura, S.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Two Transcription Factors, DREB1 and DREB2, with an EREBP/AP2 DNA Binding Domain Separate Two Cellular Signal Transduction Pathways in Drought- and Low- Temperature-Responsive Gene Expression, Respectively, in Arabidopsis. Plant Cell 1998, 10, 1391–1406. [Google Scholar] [CrossRef] [Green Version]
  15. Zhang, T.; Cui, Z.; Li, Y.; Kang, Y.; Song, X.; Wang, J.; Zhou, Y. Genome-Wide Identification and Expression Analysis of MYB Transcription Factor Superfamily in Dendrobium catenatum. Front. Genet. 2021, 12, 714696. [Google Scholar] [CrossRef]
  16. Xing, H.; Jiang, Y.; Zou, Y.; Long, X.; Wu, X.; Ren, Y.; Li, Y.; Li, H.-L. Genome-wide investigation of the AP2/ERF gene family in ginger: Evolution and expression profiling during development and abiotic stresses. BMC Plant Biol. 2021, 21, 561. [Google Scholar] [CrossRef]
  17. Riaz, M.W.; Lu, J.; Shah, L.; Yang, L.; Chen, C.; Mei, X.D.; Xue, L.; Manzoor, M.A.; Abdullah, M.; Rehman, S.; et al. Expansion and Molecular Characterization of AP2/ERF Gene Family in Wheat (Triticum aestivum L.). Front. Genet. 2021, 12, 632155. [Google Scholar] [CrossRef]
  18. Chen, J.; Zhou, Y.; Zhang, Q.; Liu, Q.; Li, L.; Sun, C.; Wang, K.; Wang, Y.; Zhao, M.; Li, H.; et al. Structural variation, functional differentiation and expression characteristics of the AP2/ERF gene family and its response to cold stress and methyl jasmonate in Panax ginseng C.A. Meyer. PLoS ONE 2020, 15, e0226055. [Google Scholar] [CrossRef] [Green Version]
  19. Ghorbani, R.; Zakipour, Z.; Alemzadeh, A.; Razi, H. Genome-wide analysis of AP2/ERF transcription factors family in Brassica napus. Physiol. Mol. Biol. Plants 2020, 26, 1463–1476. [Google Scholar] [CrossRef]
  20. Stockinger, E.J.; Gilmour, S.J.; Thomashow, M.F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA 1997, 94, 1035–1040. [Google Scholar] [CrossRef] [Green Version]
  21. Gilmour, S.J.; Zarka, D.G.; Stockinger, E.J.; Salazar, M.P.; Houghton, J.M.; Thomashow, M.F. Low temperature regulation of theArabidopsisCBF family of AP2 transcriptional activators as an early step in cold-inducedCORgene expression. Plant J. 1998, 16, 433–442. [Google Scholar] [CrossRef]
  22. Qin, F.; Kakimoto, M.; Sakuma, Y.; Maruyama, K.; Osakabe, Y.; Tran, L.-S.P.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L: ZmDREB2A in drought and heat stress response. Plant J. 2007, 50, 54–69. [Google Scholar] [CrossRef] [PubMed]
  23. Zhang, G.; Chen, M.; Chen, X.; Xu, Z.; Li, L.; Guo, J.; Ma, Y. Isolation and characterization of a novel EAR-motif-containing gene GmERF4 from soybean (Glycine max L.). Mol. Biol. Rep. 2010, 37, 809–818. [Google Scholar] [CrossRef] [PubMed]
  24. Lee, D.-K.; Jung, H.; Jang, G.; Jeong, J.S.; Kim, Y.S.; Ha, S.-H.; Do Choi, Y.; Kim, J.-K. Overexpression of the OsERF71 Transcription Factor Alters Rice Root Structure and Drought Resistance. Plant Physiol. 2016, 172, 575–588. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Zheng, K.; Cai, Y.; Chen, W.; Gao, Y.; Jin, J.; Wang, H.; Feng, S.; Lu, J. Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo. Front. Plant Sci. 2021, 12, 669458. [Google Scholar] [CrossRef] [PubMed]
  26. Wan, X.; Zou, L.-H.; Zheng, B.-Q.; Tian, Y.-Q.; Wang, Y. Transcriptomic profiling for prolonged drought in Dendrobium catenatum. Sci. Data 2018, 5, 180233. [Google Scholar] [CrossRef] [Green Version]
  27. Zeng, X.; Ling, H.; Chen, X.; Guo, S. Genome-wide identification, phylogeny and function analysis of GRAS gene family in Dendrobium catenatum (Orchidaceae). Gene 2019, 705, 5–15. [Google Scholar] [CrossRef]
  28. Zhang, G.-Q.; Xu, Q.; Bian, C.; Tsai, W.-C.; Yeh, C.-M.; Liu, K.-W.; Yoshida, K.; Zhang, L.-S.; Chang, S.-B.; Chen, F.; et al. The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution. Sci. Rep. 2016, 6, 19029. [Google Scholar] [CrossRef] [Green Version]
  29. Zhang, G.-Q.; Liu, K.-W.; Li, Z.; Lohaus, R.; Hsiao, Y.-Y.; Niu, S.-C.; Wang, J.-Y.; Lin, Y.-C.; Xu, Q.; Chen, L.-J.; et al. The Apostasia genome and the evolution of orchids. Nature 2017, 549, 379–383. [Google Scholar] [CrossRef] [Green Version]
  30. Niu, Z.; Zhu, F.; Fan, Y.; Li, C.; Zhang, B.; Zhu, S.; Hou, Z.; Wang, M.; Yang, J.; Xue, Q.; et al. The chromosome-level reference genome assembly for Dendrobium officinale and its utility of functional genomics research and molecular breeding study. Acta Pharm. Sin. B 2021, 11, 2080–2092. [Google Scholar] [CrossRef]
  31. Shrestha, A.; Fendel, A.; Nguyen, T.H.; Adebabay, A.; Kullik, A.S.; Benndorf, J.; Leon, J.; Naz, A.A. Natural diversity uncovers P5CS1 regulation and its role in drought stress tolerance and yield sustainability in barley. Plant Cell Environ. 2022, pce.14445. [Google Scholar] [CrossRef]
  32. Kasuga, M.; Liu, Q.; Miura, S.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol. 1999, 17, 287–291. [Google Scholar] [CrossRef]
  33. Du, L.; Huang, X.; Ding, L.; Wang, Z.; Tang, D.; Chen, B.; Ao, L.; Liu, Y.; Kang, Z.; Mao, H. TaERF87 and TaAKS1 synergistically regulate TaP5CS1/TaP5CR1—mediated proline biosynthesis to enhance drought tolerance in wheat. New Phytol. 2022, nph.18549. [Google Scholar] [CrossRef]
  34. Sheng, L.; Ma, C.; Chen, Y.; Gao, H.; Wang, J. Genome-Wide Screening of AP2 Transcription Factors Involving in Fruit Color and Aroma Regulation of Cultivated Strawberry. Genes 2021, 12, 530. [Google Scholar] [CrossRef]
  35. Wessler, S.R. Homing into the origin of the AP2 DNA binding domain. Trends Plant Sci. 2005, 10, 54–56. [Google Scholar] [CrossRef]
  36. Goyal, N.; Bhatia, G.; Sharma, S.; Garewal, N.; Upadhyay, A.; Upadhyay, S.K.; Singh, K. Genome-wide characterization revealed role of NBS-LRR genes during powdery mildew infection in Vitis vinifera. Genomics 2020, 112, 312–322. [Google Scholar] [CrossRef]
  37. Sakuma, Y.; Maruyama, K.; Osakabe, Y.; Qin, F.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Functional Analysis of an Arabidopsis Transcription Factor, DREB2A, Involved in Drought-Responsive Gene Expression. Plant Cell 2006, 18, 1292–1309. [Google Scholar] [CrossRef] [Green Version]
  38. Roy, S.W.; Penny, D. A Very High Fraction of Unique Intron Positions in the Intron-Rich Diatom Thalassiosira pseudonana Indicates Widespread Intron Gain. Mol. Biol. Evol. 2007, 24, 1447–1457. [Google Scholar] [CrossRef] [Green Version]
  39. Magnani, E.; Sjölander, K.; Hake, S. From Endonucleases to Transcription Factors: Evolution of the AP2 DNA Binding Domain in Plants. Plant Cell 2004, 16, 2265–2277. [Google Scholar] [CrossRef] [Green Version]
  40. Hernandez-Garcia, C.M.; Finer, J.J. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014, 217–218, 109–119. [Google Scholar] [CrossRef] [Green Version]
  41. Zou, C.; Sun, K.; Mackaluso, J.D.; Seddon, A.E.; Jin, R.; Thomashow, M.F.; Shiu, S.-H. Cis-regulatory code of stress-responsive transcription in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 2011, 108, 14992–14997. [Google Scholar] [CrossRef] [Green Version]
  42. Agurla, S.; Gahir, S.; Munemasa, S.; Murata, Y.; Raghavendra, A.S. Mechanism of Stomatal Closure in Plants Exposed to Drought and Cold Stress. In Survival Strategies in Extreme Cold and Desiccation; Iwaya-Inoue, M., Sakurai, M., Uemura, M., Eds.; Advances in Experimental Medicine and Biology; Springer Singapore: Singapore, 2018; Volume 1081, pp. 215–232. ISBN 9789811312434. [Google Scholar]
  43. Nakashima, K.; Yamaguchi-Shinozaki, K. ABA signaling in stress-response and seed development. Plant Cell Rep. 2013, 32, 959–970. [Google Scholar] [CrossRef] [PubMed]
  44. Ahn, H.; Jung, I.; Shin, S.-J.; Park, J.; Rhee, S.; Kim, J.-K.; Jung, W.; Kwon, H.-B.; Kim, S. Transcriptional Network Analysis Reveals Drought Resistance Mechanisms of AP2/ERF Transgenic Rice. Front. Plant Sci. 2017, 8, 1044. [Google Scholar] [CrossRef] [PubMed]
  45. Zhao, Q.; Hu, R.; Liu, D.; Liu, X.; Wang, J.; Xiang, X.; Li, Y. The AP2 transcription factor NtERF172 confers drought resistance by modifying NtCAT. Plant Biotechnol. J. 2020, 18, 2444–2455. [Google Scholar] [CrossRef] [PubMed]
  46. Park, C.S.; Go, Y.S.; Suh, M.C. Cuticular wax biosynthesis is positively regulated by WRINKLED4, an AP2/ERF-type transcription factor, in Arabidopsis stems. Plant J. 2016, 88, 257–270. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Letunic, I.; Bork, P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018, 46, D493–D496. [Google Scholar] [CrossRef]
  48. Jin, J.; Tian, F.; Yang, D.-C.; Meng, Y.-Q.; Kong, L.; Luo, J.; Gao, G. PlantTFDB 4.0: Toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res. 2017, 45, D1040–D1045. [Google Scholar] [CrossRef] [Green Version]
  49. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [Green Version]
  50. Lu, S.; Wang, J.; Chitsaz, F.; Derbyshire, M.K.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Marchler, G.H.; Song, J.S.; et al. CDD/SPARCLE: The conserved domain database in 2020. Nucleic Acids Res. 2020, 48, D265–D268. [Google Scholar] [CrossRef] [Green Version]
  51. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef] [Green Version]
  52. Lescot, M. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  53. Wu, Z.-G.; Jiang, W.; Chen, S.-L.; Mantri, N.; Tao, Z.-M.; Jiang, C.-X. Insights from the Cold Transcriptome and Metabolome of Dendrobium officinale: Global Reprogramming of Metabolic and Gene Regulation Networks during Cold Acclimation. Front. Plant Sci. 2016, 7, 1653. [Google Scholar] [CrossRef] [Green Version]
  54. Li, C.; Shen, Q.; Cai, X.; Lai, D.; Wu, L.; Han, Z.; Zhao, T.; Chen, D.; Si, J. JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen. BMC Plant Biol. 2021, 21, 360. [Google Scholar] [CrossRef]
  55. Chai, D.; Lee, S.M.; Ng, J.H.; Yu, H. l-Methionine sulfoximine as a novel selection agent for genetic transformation of orchids. J. Biotechnol. 2007, 131, 466–472. [Google Scholar] [CrossRef]
Ijms 23 13603 g001 550
Figure 1. Phylogenetic analysis of AP2/ERF in Arabidopsis thaliana, D. catenatum and Phalaenopsis equestris. (A) Phylogenetic trees of three species were constructed by the neighbor-joining method with 1000 bootstrap replications. Each gene cluster was labeled with distinguishable colors. (B) Gene quantity and structure of different subfamilies of AP2/ERF family in three species.
Figure 1. Phylogenetic analysis of AP2/ERF in Arabidopsis thaliana, D. catenatum and Phalaenopsis equestris. (A) Phylogenetic trees of three species were constructed by the neighbor-joining method with 1000 bootstrap replications. Each gene cluster was labeled with distinguishable colors. (B) Gene quantity and structure of different subfamilies of AP2/ERF family in three species.
Ijms 23 13603 g001
Ijms 23 13603 g002 550
Figure 2. Phylogenetic relationships, conserved motifs, protein domain and gene structure in DcAP2/ERF genes. (A) Conserved motif analysis of DcAP2/ERFs. The four subfamilies of genes were colored on the left side, the yellow color represented the ERF subfamily, the green color represented the DREB subfamily, the pink color represented the RAV subfamily, and the blue color represented the AP2 subfamily. (B) Conserved domain analysis of DcAP2/ERFs. (C) Gene structure analysis of DcAP2/ERFs. The black lines in each gene represent the introns.
Figure 2. Phylogenetic relationships, conserved motifs, protein domain and gene structure in DcAP2/ERF genes. (A) Conserved motif analysis of DcAP2/ERFs. The four subfamilies of genes were colored on the left side, the yellow color represented the ERF subfamily, the green color represented the DREB subfamily, the pink color represented the RAV subfamily, and the blue color represented the AP2 subfamily. (B) Conserved domain analysis of DcAP2/ERFs. (C) Gene structure analysis of DcAP2/ERFs. The black lines in each gene represent the introns.
Ijms 23 13603 g002
Ijms 23 13603 g003 550
Figure 3. Classification and analysis of cis-acting elements in the promoter regions of DcAP2/ERF genes. The 2 kb region upstream of the genes was analyzed using the PlantCARE website. Graphs in different colors represent different classes of cis-acting elements. The numbers indicate the amount of each cis-acting element.
Figure 3. Classification and analysis of cis-acting elements in the promoter regions of DcAP2/ERF genes. The 2 kb region upstream of the genes was analyzed using the PlantCARE website. Graphs in different colors represent different classes of cis-acting elements. The numbers indicate the amount of each cis-acting element.
Ijms 23 13603 g003
Ijms 23 13603 g004 550
Figure 4. Expression of DcAP2/ERF genes in response to cold stress. (A) Heat map showing the expression pattern DcAP2/ERF genes in leaves under cold stress for 20 h. The red, green, yellow and blue arcs in the outer ring of the heat map represent the range of ERF, DREB, RAV and AP2 subfamily, respectively. (B) The volcano plot showing the upregulation and downregulation of genes under low-temperature treatment.
Figure 4. Expression of DcAP2/ERF genes in response to cold stress. (A) Heat map showing the expression pattern DcAP2/ERF genes in leaves under cold stress for 20 h. The red, green, yellow and blue arcs in the outer ring of the heat map represent the range of ERF, DREB, RAV and AP2 subfamily, respectively. (B) The volcano plot showing the upregulation and downregulation of genes under low-temperature treatment.
Ijms 23 13603 g004
Ijms 23 13603 g005 550
Figure 5. Expression of DcAP2/ERF genes in response to MeJA. (A) Heat map showing expression pattern DcAP2/ERF genes under 1 mM MeJA. The red, green, yellow and blue arcs in the outer ring of the heat map represent the range of ERF, DREB, RAV and AP2 subfamily, respectively. (B) The volcano plot showing the upregulation and downregulation of genes under 1 mM MeJA treatment.
Figure 5. Expression of DcAP2/ERF genes in response to MeJA. (A) Heat map showing expression pattern DcAP2/ERF genes under 1 mM MeJA. The red, green, yellow and blue arcs in the outer ring of the heat map represent the range of ERF, DREB, RAV and AP2 subfamily, respectively. (B) The volcano plot showing the upregulation and downregulation of genes under 1 mM MeJA treatment.
Ijms 23 13603 g005
Ijms 23 13603 g006 550
Figure 6. Expression patterns of 12 selected genes in leaves, stems and roots under 20% PEG6000 treatment. The orange color represented leaves, the green color represented stems, and the yellow color represented roots. DcACTIN was used as an internal control. Values are presented as means ± SD (n = 3). (* p < 0.05, ** p < 0.01, Student’s t-test).
Figure 6. Expression patterns of 12 selected genes in leaves, stems and roots under 20% PEG6000 treatment. The orange color represented leaves, the green color represented stems, and the yellow color represented roots. DcACTIN was used as an internal control. Values are presented as means ± SD (n = 3). (* p < 0.05, ** p < 0.01, Student’s t-test).
Ijms 23 13603 g006
Ijms 23 13603 g007 550
Figure 7. The growth status of D. catenatum stems is strongly affected by drought stress. (A) The growth status of D. catenatum under normal and extreme drought conditions. (B) Statistics on biomass loss under different degrees of drought stress.
Figure 7. The growth status of D. catenatum stems is strongly affected by drought stress. (A) The growth status of D. catenatum under normal and extreme drought conditions. (B) Statistics on biomass loss under different degrees of drought stress.
Ijms 23 13603 g007
Ijms 23 13603 g008 550
Figure 8. DcAP2ERF#96 exhibits significant transcription factor characteristics. (A,B) Subcellular localization of DcAP2ERF#96 protein in D. catenatum protoplasts (A) and in the leaf epidermal cells of N. benthamiana (B). D53-mCherry was used as a nuclear marker. Bars = 20 μm. The fluorescence signals were detected by confocal microscopy. (C) Yeast one-hybrid verification of the binding of DcAP2ERF#96 protein to three tandem repeats of CRT box, DRE box and GCC box. (D) Yeast two-hybrid validation of DcAP2ERF#96 protein self-activation regions. The transformed yeast cells were plated on DDO (SD/-Trp/-Leu) and QDO (SD/-Trp/-Leu/-His/-Ade).
Figure 8. DcAP2ERF#96 exhibits significant transcription factor characteristics. (A,B) Subcellular localization of DcAP2ERF#96 protein in D. catenatum protoplasts (A) and in the leaf epidermal cells of N. benthamiana (B). D53-mCherry was used as a nuclear marker. Bars = 20 μm. The fluorescence signals were detected by confocal microscopy. (C) Yeast one-hybrid verification of the binding of DcAP2ERF#96 protein to three tandem repeats of CRT box, DRE box and GCC box. (D) Yeast two-hybrid validation of DcAP2ERF#96 protein self-activation regions. The transformed yeast cells were plated on DDO (SD/-Trp/-Leu) and QDO (SD/-Trp/-Leu/-His/-Ade).
Ijms 23 13603 g008
Ijms 23 13603 g009 550
Figure 9. DcAP2ERF#96 overexpression lines showed sensitivity to ABA. (A) 10-d-old seedlings were transferred to vertical plates containing 1/2 MS + 10 μM ABA and its control (1/2 MS). Phenotypes were observed and recorded one week later. Bars = 1 cm. (B,C) Leaf area (B) and root length (C) statistics of Col-0 and DcAP2ERF#96 overexpression lines under 10 μM ABA treatment (n = 9). (D) Relative expression levels of ABA signaling pathway-related genes in Col-0 and overexpression lines after 10 μM ABA treatment for one week. AtACTIN2 was used as an internal control. Values are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, Student’s t-test.
Figure 9. DcAP2ERF#96 overexpression lines showed sensitivity to ABA. (A) 10-d-old seedlings were transferred to vertical plates containing 1/2 MS + 10 μM ABA and its control (1/2 MS). Phenotypes were observed and recorded one week later. Bars = 1 cm. (B,C) Leaf area (B) and root length (C) statistics of Col-0 and DcAP2ERF#96 overexpression lines under 10 μM ABA treatment (n = 9). (D) Relative expression levels of ABA signaling pathway-related genes in Col-0 and overexpression lines after 10 μM ABA treatment for one week. AtACTIN2 was used as an internal control. Values are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, Student’s t-test.
Ijms 23 13603 g009
Ijms 23 13603 g010 550
Figure 10. DcAP2ERF#96 interacts with DcDREB2A and inhibits P5CS1 and RD29A in Arabidopsis thaliana. (A) Distribution of DRE, CRT and GCC motifs in ABA-related gene P5CS1, RD29A and RAB18 promoters. (B) Yeast one hybrid verification of the binding of DcAP2ERF#96 to P5CS1, RD29A and RAB18 promoters. The pB42AD-DcAP2ERF#96 and pLacZi2μ with different promoters were co-transformed into the EGY48 yeast strain. The transformed yeast cells were plated on DDO (SD/-Ura/-Trp) with X-Gal. (C) Yeast two hybrids verified the interaction of DcAP2ERF#96 with DREB2A-1 and DREB2A-2. The transformed yeast cells were plated on DDO (SD/-Trp/-Leu) and QDO (SD/-Trp/-Leu/-His/-Ade). (D) Regulatory model for the regulation of the ABA signal by DcAP2ERF#96 in Arabidopsis thaliana and D. catenatum. Overexpression of DcAP2ERF#96 inhibits the expression of ABA downstream genes such as RD29A and P5CS1 by binding to their promoters, thereby affecting plant sensitivity to ABA signaling. DcAP2ERF#96 can interact with DREB2A protein in D. catenatum, whose homologous gene in Arabidopsis has been reported to positively regulate the expression of RD29A.
Figure 10. DcAP2ERF#96 interacts with DcDREB2A and inhibits P5CS1 and RD29A in Arabidopsis thaliana. (A) Distribution of DRE, CRT and GCC motifs in ABA-related gene P5CS1, RD29A and RAB18 promoters. (B) Yeast one hybrid verification of the binding of DcAP2ERF#96 to P5CS1, RD29A and RAB18 promoters. The pB42AD-DcAP2ERF#96 and pLacZi2μ with different promoters were co-transformed into the EGY48 yeast strain. The transformed yeast cells were plated on DDO (SD/-Ura/-Trp) with X-Gal. (C) Yeast two hybrids verified the interaction of DcAP2ERF#96 with DREB2A-1 and DREB2A-2. The transformed yeast cells were plated on DDO (SD/-Trp/-Leu) and QDO (SD/-Trp/-Leu/-His/-Ade). (D) Regulatory model for the regulation of the ABA signal by DcAP2ERF#96 in Arabidopsis thaliana and D. catenatum. Overexpression of DcAP2ERF#96 inhibits the expression of ABA downstream genes such as RD29A and P5CS1 by binding to their promoters, thereby affecting plant sensitivity to ABA signaling. DcAP2ERF#96 can interact with DREB2A protein in D. catenatum, whose homologous gene in Arabidopsis has been reported to positively regulate the expression of RD29A.
Ijms 23 13603 g010
Table
Table 1. Characteristics of DcAP2/ERF family proteins.
Table 1. Characteristics of DcAP2/ERF family proteins.
IDLOCRenameLength
(aa)		Mw
(Da)		PIAliphatic IndexGRAVYLocation
XM_020816501.1LOC110092113DcAP2ERF#139343,675.569.2759.49−0.678mitochondria
XM_020816597.2LOC110092178DcAP2ERF#211713,244.555.361.88−0.913nucleus
XM_020817122.2LOC110092532DcAP2ERF#320522,653.277.9267.61−0.666nucleus
XM_020817685.2LOC110092963DcAP2ERF#434438,910.424.8962.21−0.691nucleus
XM_020817704.2LOC110092982DcAP2ERF#519921,704.649.3671.16−0.545nucleus
XM_020817821.2LOC110093058DcAP2ERF#614215,996.035.3167.39−0.497mitochondria
XM_020818820.2LOC110093822DcAP2ERF#727330,323.275.1277.95−0.467cytoplasm
XM_020818851.2LOC110093837DcAP2ERF#827330,499.495.2871.5−0.49cytoplasm
XM_020818859.2LOC110093843DcAP2ERF#924026,225.99.6975.71−0.383nucleus
XM_020818985.2LOC110093926DcAP2ERF#1037840,998.346.3456.9−0.635nucleus
XM_020819386.2LOC110094210DcAP2ERF#1143147,967.916.3562.48−0.686nucleus
XM_020820818.2LOC110095317DcAP2ERF#1248752,737.336.8159.16−0.591nucleus
XM_020820987.1LOC110095445DcAP2ERF#1325828,269.687.7866.67−0.537nucleus
XM_020821323.2LOC110095684DcAP2ERF#1415517,183.339.9865.03−0.815nucleus
XM_020821563.2LOC110095855DcAP2ERF#1513414,806.869.7164.18−0.525cytoplasm
XM_020821729.2LOC110095987DcAP2ERF#1628531,434.65.4481.47−0.432nucleus
XM_020822089.2LOC110096240DcAP2ERF#1721924,140.19.1459.77−0.591cytoplasm
XM_020822222.2LOC110096335DcAP2ERF#1823124,602.719.268.18−0.349nucleus
XM_020822428.2LOC110096456DcAP2ERF#1927529,678.796.4660.51−0.476nucleus
XM_020823171.2LOC110096991DcAP2ERF#2014716,174.86564.35−0.612nucleus
XM_020823245.2LOC110097036DcAP2ERF#2121022,952.78.4167.57−0.503nucleus
XM_020823401.2LOC110097153DcAP2ERF#2247853,136.396.5266.95−0.597nucleus
XM_020823475.2LOC110097214DcAP2ERF#2318120,350.635.3661.16−0.646nucleus
XM_020823706.2LOC110097366DcAP2ERF#2416618,041.336.4360.6−0.581cytoplasm
XM_020824867.2LOC110098141DcAP2ERF#2520121,854.615.8369.1−0.4nucleus
XM_020825001.2LOC110098242DcAP2ERF#2634739,167.065.6456.57−0.847nucleus
XM_020825318.2LOC110098477DcAP2ERF#2732335,258.228.3258.08−0.645nucleus
XM_020825410.2LOC110098547DcAP2ERF#2819320,380.734.4970−0.454nucleus
XM_020825656.2LOC110098714DcAP2ERF#2917419,198.489.8258.51−0.936nucleus
XM_020825700.2LOC110098779DcAP2ERF#3021523,420.826.4456.88−0.686nucleus
XM_020826383.2LOC110099287DcAP2ERF#3123326,407.919.2666.95−0.771nucleus
XM_020826509.2LOC110099386DcAP2ERF#3223926,222.629.2174.44−0.535nucleus
XM_020826512.1LOC110099389DcAP2ERF#3325328,355.926.3575.89−0.687nucleus
XM_020826786.2LOC110099586DcAP2ERF#3425528,810.66.2265.73−0.436cytoplasm
XM_020827298.2LOC110099956DcAP2ERF#3517019,093.625.6986.06−0.573cytoplasm
XM_020827868.2LOC110100389DcAP2ERF#3622924,589.958.9176.77−0.172nucleus
XM_020827870.1LOC110100391DcAP2ERF#3716418,123.614.8686.34−0.033cytoplasm
XM_020827871.2LOC110100392DcAP2ERF#3816418,035.274.5985.67−0.157cytoplasm
XM_020827872.1LOC110100393DcAP2ERF#3917819,751.314.3583.99−0.19cytoplasm
XM_020827873.1LOC110100394DcAP2ERF#4012213,445.69.2796.890.025cytoplasm
XM_020827909.2LOC110100414DcAP2ERF#4117919,288.484.5169.44−0.491nucleus
XM_020828052.2LOC110100522DcAP2ERF#4216518,544.948.1165.88−0.66cytoplasm
XM_020828120.2LOC110100557DcAP2ERF#4326830,011.645.267.8−0.532nucleus
XM_020828122.2LOC110100558DcAP2ERF#4428931,823.95.474.05−0.407cytoplasm
XM_020828709.2LOC110100983DcAP2ERF#4515516,800.784.8369.48−0.427nucleus
XM_020828734.2LOC110100999DcAP2ERF#4621023,031.596.4463.76−0.604nucleus
XM_020829170.2LOC110101314DcAP2ERF#4773381,463.466.8264.6−0.577nucleus
XM_020829570.2LOC110101604DcAP2ERF#4817418,520.726.8266.32−0.37nucleus
XM_020829885.2LOC110101821DcAP2ERF#4924427,139.949.2379.22−0.437nucleus
XM_020830439.2LOC110102213DcAP2ERF#5033836,717.669.3969.85−0.426nucleus
XM_020831356.2LOC110102864DcAP2ERF#5119121,091.729.1260.94−0.654nucleus
XM_020831553.2LOC110103007DcAP2ERF#5233336,879.235.8371.02−0.514nucleus
XM_020831585.2LOC110103039DcAP2ERF#5320522,548.566.9263.85−0.415nucleus
XM_020831635.2LOC110103078DcAP2ERF#5435039,719.188.6362.74−0.763nucleus
XM_020831820.2LOC110103210DcAP2ERF#5531934,146.415.2355.49−0.573nucleus
XM_020832156.2LOC110103436DcAP2ERF#5623326,398.059.5961.55−0.839nucleus
XM_020832246.1LOC110103500DcAP2ERF#5721623,871.264.6563.7−0.469mitochondria
XM_020832253.2LOC110103508DcAP2ERF#5818820,706.095.4455.74−0.648nucleus
XM_020832331.2LOC110103559DcAP2ERF#5933337,914.716.2352.82−0.984nucleus
XM_020832378.2LOC110103593DcAP2ERF#6018219,679.185.3584.95−0.175nucleus
XM_020834156.2LOC110104876DcAP2ERF#6122624,995.955.1776.42−0.376nucleus
XM_020834164.2LOC110104880DcAP2ERF#6213815,094.935.4476.45−0.46cytoplasm
XM_020834254.2LOC110104943DcAP2ERF#6331735,188.215.5666.81−0.568nucleus
XM_020834863.1LOC110105385DcAP2ERF#6414616,196.6710.166.99−0.646nucleus
XM_020835088.2LOC110105539DcAP2ERF#6535139,673.699.7558.38−0.915nucleus
XM_020835287.2LOC110105686DcAP2ERF#6623726,183.237.0669.58−0.675nucleus
XM_020835818.2LOC110106077DcAP2ERF#6738842,370.139.2557.24−0.649nucleus
XM_020836334.2LOC110106428DcAP2ERF#6819321,736.56.1966.27−0.59cytoplasm
XM_020836335.2LOC110106429DcAP2ERF#6925928,586.498.9681.85−0.388nucleus
XM_020837398.2LOC110107204DcAP2ERF#7062869,816.296.1759.54−0.671nucleus
XM_020837735.2LOC110107464DcAP2ERF#7138841,489.167.8155.67−0.557nucleus
XM_020838142.2LOC110107770DcAP2ERF#7226628,625.148.8973.46−0.421nucleus
XM_020838192.2LOC110107803DcAP2ERF#7325127,642.365.6771.95−0.498nucleus
XM_020838224.2LOC110107825DcAP2ERF#7418820,344.919.354.68−0.677cytoplasm
XM_020838231.2LOC110107829DcAP2ERF#7548153,471.977.6471.81−0.516nucleus
XM_020838234.2LOC110107830DcAP2ERF#7647652,887.257.6471.13−0.535nucleus
XM_020838388.2LOC110107938DcAP2ERF#7729532,730.655.5767.83−0.523nucleus
XM_020838721.2LOC110108187DcAP2ERF#7837742,334.33560.34−0.746nucleus
XM_020838765.2LOC110108219DcAP2ERF#7937641,253.266.2859.26−0.623nucleus
XM_020838815.2LOC110108252DcAP2ERF#8020521,740.449.3467.27−0.423nucleus
XM_020838948.2LOC110108340DcAP2ERF#8125026,683.778.5664.16−0.449nucleus
XM_020839309.2LOC110108607DcAP2ERF#8217719,603.157.8159.77−0.459cytoplasm
XM_020839427.2LOC110108677DcAP2ERF#8338141,657.985.1358.71−0.519nucleus
XM_020840002.2LOC110109092DcAP2ERF#8414315,701.749.5162.94−0.524nucleus
XM_020840101.2LOC110109163DcAP2ERF#8514616,192.6810.166.99−0.652nucleus
XM_020840120.2LOC110109181DcAP2ERF#8625626,949.369.269.1−0.32nucleus
XM_020840182.2LOC110109222DcAP2ERF#8737240,701.754.6859.87−0.761nucleus
XM_020840524.2LOC110109455DcAP2ERF#8826328,948.725.1248.37−0.681nucleus
XM_020840628.2LOC110109529DcAP2ERF#8932335,656.474.8562.29−0.542nucleus
XM_020840999.2LOC110109796DcAP2ERF#9019421,956.896.3172.99−0.591nucleus
XM_020841375.2LOC110110072DcAP2ERF#9132635,893.829.6478.34−0.444nucleus
XM_020841449.2LOC110110123DcAP2ERF#9219021,631.678.9865.32−0.522nucleus
XM_020841821.2LOC110110379DcAP2ERF#9330734,245.395.8570.26−0.536nucleus
XM_020842046.2LOC110110526DcAP2ERF#9437741,376.995.8762.65−0.665nucleus
XM_020842532.1LOC110110883DcAP2ERF#9524927,664.279.4670.92−0.62nucleus
XM_020843531.2LOC110111594DcAP2ERF#9642847,796.645.6459.53−0.581cytoplasm
XM_020844032.2LOC110111961DcAP2ERF#9739343,606.049.362.39−0.647nucleus
XM_020844150.2LOC110112064DcAP2ERF#9821323,769.37554.23−0.665cytoplasm
XM_020844655.2LOC110112434DcAP2ERF#9918019,766.056.6361.28−0.763nucleus
XM_020844798.2LOC110112545DcAP2ERF#10048253,271.166.4768.05−0.595nucleus
XM_020844875.2LOC110112594DcAP2ERF#10131134,278.275.2970.96−0.433nucleus
XM_020844987.2LOC110112684DcAP2ERF#10264069,342.866.0361.78−0.474nucleus
XM_020845292.2LOC110112910DcAP2ERF#10319821,803.575.7169.49−0.397nucleus
XM_020845879.2LOC110113332DcAP2ERF#10416017,799.229.7862.19−0.819nucleus
XM_020846166.2LOC110113533DcAP2ERF#10527130,721.335.2656.24−0.851nucleus
XM_020846170.2LOC110113537DcAP2ERF#10638442,198.394.7354.24−0.736nucleus
XM_020846319.2LOC110113656DcAP2ERF#10735039,815.184.8955.54−0.903nucleus
XM_020847119.2LOC110114282DcAP2ERF#10819821,677.036.8459.29−0.67nucleus
XM_020847550.2LOC110114618DcAP2ERF#10926228,562.386.7876.45−0.426cytoplasm
XM_020847791.2LOC110114791DcAP2ERF#11026829,590.175.956.16−0.538nucleus
XM_020848468.2LOC110115279DcAP2ERF#11113814,972.247.8684.93−0.132nucleus
XM_020848863.2LOC110115597DcAP2ERF#11227130,293.535.1957.68−0.744nucleus
XM_020849558.2LOC110116094DcAP2ERF#11320521,591.067.757.85−0.299mitochondria
XM_020850163.2LOC110116551DcAP2ERF#11425128,723.569.2467.21−0.648cytoplasm
XM_028691742.1LOC110112950DcAP2ERF#11527931,067.056.1669.21−0.6nucleus
XM_028692461.1LOC110096264DcAP2ERF#11632936,673.855.2964.62−0.631cytoplasm
XM_028694856.1LOC110099516DcAP2ERF#11718220,153.729.3564.45−0.713nucleus
XM_028696782.1LOC110098115DcAP2ERF#11820121,811.555.8368.11−0.396nucleus
XM_028698356.1LOC110103792DcAP2ERF#11962867,8775.860.25−0.61nucleus
XM_028699460.1LOC110115745DcAP2ERF#12024826,592.459.0151.57−0.605nucleus
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Han, Y.; Cai, M.; Zhang, S.; Chai, J.; Sun, M.; Wang, Y.; Xie, Q.; Chen, Y.; Wang, H.; Chen, T. Genome-Wide Identification of AP2/ERF Transcription Factor Family and Functional Analysis of DcAP2/ERF#96 Associated with Abiotic Stress in Dendrobium catenatum. Int. J. Mol. Sci. 2022, 23, 13603. https://doi.org/10.3390/ijms232113603

AMA Style

Han Y, Cai M, Zhang S, Chai J, Sun M, Wang Y, Xie Q, Chen Y, Wang H, Chen T. Genome-Wide Identification of AP2/ERF Transcription Factor Family and Functional Analysis of DcAP2/ERF#96 Associated with Abiotic Stress in Dendrobium catenatum. International Journal of Molecular Sciences. 2022; 23(21):13603. https://doi.org/10.3390/ijms232113603

Chicago/Turabian Style

Han, Yuliang, Maohong Cai, Siqi Zhang, Jiawen Chai, Mingzhe Sun, Yingwei Wang, Qinyu Xie, Youheng Chen, Huizhong Wang, and Tao Chen. 2022. "Genome-Wide Identification of AP2/ERF Transcription Factor Family and Functional Analysis of DcAP2/ERF#96 Associated with Abiotic Stress in Dendrobium catenatum" International Journal of Molecular Sciences 23, no. 21: 13603. https://doi.org/10.3390/ijms232113603

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