DoAP2/ERF89 activated the terpene synthase gene DoPAES in Dendrobium officinale and participated in the synthesis of β-patchoulene

Plant material

All of the D. officinale specimens and protocorm-like bodies (PLBs) used in this experiment were obtained from the lab at Anhui Agricultural University. D. officinale tissue culture seedlings were grown in an artificial climate box at 26 °C in a 12 h/12 h light cycle (Li et al., 2021). The induction methods used in this study for protocorms of Dendrobium officinale were based on those outlined by Lv et al. (2022). Tobacco was grown in a greenhouse at 25 °C in 16 h/8 h (light/dark) photoperiod conditions. Tobacco leaves that were 4–5 weeks old were used for the injection.

DNA isolation, RNA isolation, cDNA synthesis

A complete D. officinale plant was used for RNA and DNA extraction. The samples were immediately frozen in liquid nitrogen after collection and stored at −80 °C. The DNA extraction and RNA extraction kits used in the experiments were obtained from Chengdu Bafet Biotechnology Co, Chengdu, China.

Chemical profiling

The GC-MS method described by Li et al. (2021) was used to analyze the treated D. officinale PLBs. The volatile compounds of the PLBs were collected by headspace SPME and adsorbed by 75 µm CAR/PDMS fiber (Sigma-Aldrich, St. Louis, MO, USA) at 25 °C for 1 h. All captured volatile compounds were subsequently thermally desorbed and transferred to an Agilent 5975-6890N gas chromatography-mass spectrometry (GC-MS) instrument (Agilent Technologies, Santa Clara, CA, USA). The detected volatile compounds were identified and qualitatively analyzed using the National Institute of Standards and Technology (NIST) 2011 standard library (Xu et al., 2019a; Yahyaa et al., 2015).

Heterologous expression and enzyme activity assays

The pMAL-c2x vector containing the DoPAES gene was transformed into Escherichia coli BL21, which was cultured on the LB agar plate containing 100 mg/L ampicillin to achieve the hetero expression of DoPAES. The bacterial solution was shaken at 37 °C until OD₆₀₀ = 0.6, then the expression of bacterial cell fusion protein was induced by adding 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 16 °C. The bacterial solution was induced overnight at 16 °C and centrifuged at 12,000 r/min and 4 °C for 10 min. Part of the bacterial body weight was suspended in 1×PBS buffer, added into 5×SDS-PAGE loading buffer, mixed, and placed at 100 °C for 10 min to obtain an electrophoretic sample. The samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. The remaining bacteria were resuspended in 3 mL of pre-cooled buffer (20 mmol/L Tris pH 8.0, 10 mmol/L DTT, 5 mmol/L Na₂S₂O_5, and 10% glycerol) to obtain the crude enzyme solution and then sonicated (sonication power ratio 15%, crushing 8 s, interval 45 s, crushing 10 min). The crushed protein solution was centrifuged at 4 °C for 10 min at 12,000 r/min and the supernatant was collected to obtain the crude protein.

The recombinant protein was assayed in vitro by headspace extraction combined with GC-MS and 500 μL of target gene crude protein supernatant, 50 mmol/L Tris pH 7.5, 10 mmol/L MgCl₂, 10 μmol/L MnCl₂, and 1 μL FPP were added for a total volume of 1 mL. After incubating at 30 °C for 30 min, the sample was kept at room temperature for 1 h and then GC-MS detection was performed on the sample. The adsorbed compounds were analyzed using GC-MS according to the methods outlined by Li et al. (2021).

Identification of DoAP2/ERF genes

The AP2/ERF gene family sequences in A. thaliana were obtained from the Arabidopsis Information Resource (TAIR) website (https://www.arabidopsis.org/), and used to identify the AP2/ERF gene family members in D. officinale. Using the AP2/ERF sequences of A. thaliana as query sequences, a BLAST analysis was performed with the D. officinale genome using the Cluster Database at High Identity with Tolerance (CD-HIT; http://www.bioinformatics.org/cd-hit/) to remove redundant sequences when the thresholds were greater than 95%. The candidate sequences were further validated using PFAM (https://pfam.xfam.org/) to identify the AP2/ERF transcription factor family members in D. officinale. The physicochemical properties of each DoAP2/ERF protein were determined using the ProtParam online tool (https://web.expasy.org/protparam).

Construction of phylogenetic trees

IQTREE was used for phylogenetic evolutionary tree construction and the output result files were inputted into the Interactive Tree Of Life (iTOL; https://itol.embl.de/) website for tree beautification.

Gene structure, conserved motif prediction, and GO analysis

Multiple predictions of conserved motifs in DoAP2/ERFs proteins were made. Motif elicitation (https://meme-suite.org/meme/) was expected to be maximized, and the maximum number of motifs was adjusted to 10. DoAP2/ERF gene exons and introns were analyzed using the GSDS 2.0 Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/) online tool to determine their location and quantity. DoAP2/ERF proteins were prepared using BLAST2GO’s non-redundant protein database software, and the DoAP2/ERF protein sequences were compared using NCBI’s protein blast tool, BLASTP. The GO annotation for each DoAP2/ERF was then retrieved from Map GO and classified using the WEGO (https://wego.genomics.cn/) online tool.

Heat map analysis

Transcriptome data of D. officinale (PRJNA348403) were used to obtain the gene expression data in eight tissues of D. officinale: column, sepal, white part of the root, green root tip, stem, leaf, lip, and buds. The transcriptional level of the DoAP2/ERF family of genes was analyzed using Tbtools Visualization. Based on the transcriptome data, a Pearson correlation coefficient calculation was performed on DoAP2/ERF family genes and DoPAES, and the calculated results were visually analyzed using the Cytoscape software.

Subcellular localization analysis

The DoPAES coding sequence was fused with the green fluorescent protein (GFP) and cloned into the pCAMBIA1305 vector. Using Arabidopsis protoplasts as an experimental material, a PEG-mediated transformation of Arabidopsis protoplasts was used to examine the subcellular localization of DoPAES. Fluorescence images of green fluorescent protein (GFP) and chlorophyll signal distribution in Arabidopsis protoplasts were observed under the IX81 Olympus confocal microscope at 488 and 640 nm. The control group was the pCAMBIA1305-GFP empty vector.

The coding sequences of DoAP2/ERFs were fused with a green fluorescent protein (GFP) and cloned into the pCAMBIA1305 vector, and the coding sequences of DoAP2/ERFs with a green fluorescent protein (GFP) were cloned into the pCAMBIA1305 vector and then transformed into Agrobacterium tumefaciens competent (GV3101). The methods used for injecting the suspension into tobacco leaves were based on methods used in previous studies (Ke et al., 2019). After one day in darkness followed by two days in normal light conditions, the injected leaves were observed under a confocal laser scanning microscope (LCSM).

Yeast one-hybrid assay

The transcriptional activity of DoAP2/ERFs was verified using the yeast-one-hybrid system, and the promoter of DoPAES was cloned into the pAbAi vector as bait (DoPAES-pAbAi). The full-length DoAP2/ERFs were fused into the pGAL4 activation vector (AD-pGADT7) to generate DoAP2/ERFs-pGADT7 as prey. They were then co-transformed into Y1H yeast cells and grown. The positive clones were screened on defective SD medium.

Dual-luciferase assay (Dual-LUC)

The DoPAES promoter was ligated into the pGreenII 0800-LUC reporter vector. These TFs were embedded into the pGreenII62-SK vector. The recombinant plasmid was transformed into Agrobacterium tumefaciens competent cells (GV3101) and then injected into the leaves of N. benthamiana and assessed for LUC activity using enzyme markers.

Transgenic expression and volatile terpenoid analysis

The RNA interference fragments of DoAP2/ERFs were connected with the pCAMBIA1305 vector and DoAP2/ERFs was also connected with the pCAMBIA1305 vector. The positive vector was transformed into competent cells of Agrobacterium tumefaciens strain GV3101 to prepare transiently transformed PLBs. PLBs were cultured in 25 °C liquid medium for three days in dark conditions and treated with Agrobacterium tumefaciens infection solution OD₆₀₀ = 0.6 for 10 min. PLBs were then cultured in co-medium at 25 °C in dark conditions for 3 days. Agrobacterium tumefaciens was then removed by washing with sterile distilled water and sterile water containing 0.3 mg/L Temetine. Finally, the PLBs were exposed to a selective medium containing 0.1 g/L hygromycin and 0.3 mg/L Temetine for light exposure for 3 days (Gnasekaran et al., 2014; Ma et al., 2020). GC-MS was used to detect the changes of terpenoids (Li et al., 2021). Ethyl-decanoate (200 μg/kg FW·h) was added to the samples as the internal standard. A quantitative analysis was performed according to the peak area of the internal standard, and three repeated analyses were performed for each sample. The contents of the volatiles were expressed as μg/kg FW·h.

GC-MS analysis of volatile terpenes from D. officinale PLBs

Volatile terpenes from D. officinale PLBs were detected by HS-SPME-GC-MS. Only eight sesquiterpenes were detected in the protocorms: β-Elemene (10%), β-Patchoulene (11%), Caryophyllene (31%), γ-Elemene (7%), α-Guaiene (4%), Humulene (6%), Longifolene (4%), and γ-Patchoulene (27%; Fig. 1). Patchoulene content was the highest in D. officinale PLBs, followed by Caryophyllene.

Identification and analysis of DoPAES in D. officinale

Based on the transcriptome data, the whole sequence of DoPAES was annotated. DoPAES had an overall length of 1,653 bp and could encode 550 amino acids. The DoPAES protein had a relative molecular weight of 64.9 kDa and an isoelectric point of 5.42. A phylogenetic tree analysis revealed that DoPAES is a member of the TPS-a subfamily and clusters with the plant TPS gene in charge of sesquiterpene synthesis (Fig. 2). Both DoPAES and tree members share the conserved domains of terpenoid synthases, including RXR, DDXXD, and NST/DTE (Fig. 3), which was assumed to be involved in the synthesis of β-patchoulene and was named DoPAES (GenBank: MT512059.1). The most closely-related gene, with 68.6% similarity, was the α-humulene synthase in Phalaenopsis equestris.

The tertiary spatial structure of the DoPAES protein was analyzed and predicted. The homology modeling of DoPAES was performed using 5-achiral aristolodene synthetase (4RNQ1.A) from tobacco. The homological similarities between DoPAES and the template protein sequence was 36.82%. The protein template was sesquiterpene synthetase, and the conformation of DoPAES was similar to the terpene synthetase of other species (Fig. S1).

To determine the subcellular localization of DoPAES, a vacant plasmid was fused with a GFP tag and transiently expressed in Arabidopsis protoplasts, and the results showed that the GFP-fused DoPAES exhibited cytoplasmic localization (Fig. 4).

Expression and enzyme activity analysis of the DoPAES protein

Volatile sesquiterpenes were mainly synthesized through the mevalonate (MVA) pathway. The successfully-constructed recombinant plasmid, pET32a-DoPAES, was introduced into BL21 expressing strains. The products were then detected by GC-MS after adding substrate FPP. The results showed, and in vitro enzyme activity confirmed, the catalytic activity of DoPAES and its role in sesquiterpene biosynthesis. The GC-MS analysis showed that the enzyme reaction products contained terpenoid β-patchoulene (Fig. 5). These results indicate that DoPAES can specifically catalyze the synthesis of β-patchoulene.

The volatile terpene components of D. officinale were previously detected and compared with the active products of DoPAES. Based on the retention time (RT) and ion peak of the target, β-patchoulene was found to be the main enzymatic product of DoPAES in D. officinale PLBs.

Identification of the D. officinale AP2/ERF gene family

The A. thaliana AP2/ERF amino acid sequence was used as a query to blast against the D. officinale genome to identify the members of the AP2/ERF family. As a result, 122 AP2/ERF candidate genes were screened out and 111 non-redundant sequences were identified and named DoAP2/ERF1-111. The 111 DoAP2/ERF genes were initially divided into three subfamilies based on the classification of structural domains (Fig. 6). The RAV subfamily (which contains one AP2 domain and one B3 domain), the ERF subfamily (which contains one AP2 domain), and the AP2 subfamily (which contains two AP2 domains). A phylogenetic analysis showed that the 111 DoAP2/ERFs could be divided into these subfamilies as follows: 14 in AP2, three in RAV, 92 in ERF, and two in the soloist subfamily, according to the priority classification rules of A. thaliana AP2/ERF TFs.

The physicochemical characteristics of the DoAP2/ERF proteins were also examined. The results showed DoAP2/ERF10 was responsible for encoding the largest number of amino acid termini (661), whereas DoAP2/ERF17 displayed the highest molecular weight value, reaching 71.34. DoAP2/ERF17 was also identified as the heaviest of the 111 transcription factors because of its overall atom count of 9,736. The theory pI of the DoAP2/ERFs ranged from 4.35 (DoAP2/ERF98) to 10.1 (DoAP2/ERF96). All of the DoAP2/ERF proteins had negative values for the grand average of hydropathicity (GRAVY), indicating that they are all hydrophilic proteins. All of the instability indices were high, indicating that these proteins are unstable (Table S1). These findings suggest that DoAP2/ERF proteins might have distinct functions in various cell sites.

Phylogenetic analysis of the AP2/ERF gene family of D. officinale

A phylogenetic tree was built using 111 DoAP2/ERF and 147 A. thaliana AP2/ERF transcription factor families. Based on the results, the ERF subgroup, with the greatest number of family members, was further divided into 10 different branches, denoted I through X. The AP2 subgroup consisted of 14 D. officinale and 18 A. thaliana members; the RAV subgroup contained three D. officinale and six RAV subgroup members of A. thaliana. The results also showed that the majority of the superfamilies and subfamilies in the evolutionary tree were produced by A. thaliana and D. officinale AP2/ERF family member genes, showing that AP2/ERF family member genes may be homologous and may have evolved from the same ancestor.

Conserved motif analysis of the AP2/ERF gene family of D. officinale

The MEME online tool was used to characterize the potentially conserved motifs of the 111 DoAP2/ERF sequences from the amino acid sequences of DoAP2/ERF members (Fig. 7). Ten conserved motifs were identified, numbered 1 through 10 (Fig. S3). The majority of each group’s members had similar motifs. Although each subfamily of transcription factors had a distinct length, they all had conserved motifs that were largely the same. For instance, motif 1 was found in all 111 DoAP2/ERF family genes, proving that it was a crucially conserved pattern in AP2/ERF genes. The protein sequences of transcription factors contained several conserved motifs, which may function as potential DNA-binding sites to help regulate gene expression.

Gene structure analysis of the D. officinale AP2/ERF gene family

The locations of exons and introns of the 111 DoAP2/ERF sequences were analyzed using the GSDS online tool to further explore the function of the D. officinale AP2/ERF gene family (Fig. 8). The phylogenetic topological categorization of gene families was also supported by gene structure research. The number of introns in AP2/ERF family genes varied from one to nine in earlier reports on A. thaliana and Syntrichia caninervis (Jofuku et al., 1994; Li et al., 2017a). The exon and intron architectures of DoAP2/ERF genes were similar within the same subclass. The results showed that the majority of AP2 subfamily members had numerous exon and intron distributions. Although there were differences in the number, the positions of the introns and the exon regions were relatively conserved. The intronic sections were large and the exonic regions were shorter. Similar results were observed in ERF subfamily members, with the exception that each member of groups X and VII had two exonic regions, one at either end and one in the middle, respectively. Most ERF subfamily members only had one exonic area and no intronic regions outside of this region. The AP2 subfamily’s exon distribution was comparable for the soloist and had several introns. All AP2/ERF family members had the same conserved region at their N-terminus, which may be crucial to their functions.

GO analysis of the AP2/ERF gene family of D. officinale

The 111 DoAP2/ERF proteins from D. officinale underwent GO enrichment analysis. A total of 24 GO elements were enhanced when the P-value was less than 0.05. Blast2GO software was used to annotate the 111 D. officinale AP2/ERF proteins in 24 GO categories, visualize the GO terms and predict their participation in various biological processes, and WEGO was used to classify the GO terms (Fig. S4). Based on similarities in amino acid sequences, DoAP2ERF protein sequences were divided into three main groups: cellular component (CC), molecular function (MF) and biological process (BP), with 18 proteins belonging to the “biological process” category, four proteins grouped into “cellular component,” and two proteins classified as “molecular function.” The three most enriched items in the functional category of cellular components were cells, cell parts, and organelles. Transcription regulator activity in the biological process category of molecular function had the largest enrichment degree. Metabolic activities, cellular processes, biological control, and regulation of biological processes were the four entries with the highest enrichment. The GO analysis showed that the DoAP2/ERF transcription factor family was involved in most of the life processes of plant cells.

Heat map analysis of the AP2/ERF gene family of D. officinale

Transcriptome sequencing (RNA-Seq) data from eight different tissues of D. officinale were downloaded from the National Center for Biotechnology Information (NCBI) using the BioProject accession number PRJNA348403. The expression patterns of DoAP2/ERF genes are shown in Fig. S5. The majority of the family genes, including DoAP2/ERF101, DoAP2/ERF10, and DoAP2/ERF65, had very low expression levels in these eight tissues, with some having no expression or expressing in just select tissues. A small number of genes, including DoAP2/ERF07, DoAP2/ERF26, DoAP2/ERF27, DoAP2/ERF37, and DoAP2/ERF29, were substantially expressed in different tissues. Overall, the majority of the AP2/ERF family of genes were expressed in the flower tissue.

The expression patterns of DoAP2/ERF and DoPAES

In plants, the AP2/ERF transcription factor family is crucial. A gene co-expression analysis was carried out based on transcriptome data to investigate the spatiotemporal regulation of DoAP2/ERF and terpenoid synthases DoPAES. The highly-correlated DoAP2/ERF89 and DoAP2/ERF47 were chosen based on the findings of the correlation screening (Fig. 9), as they may help control the expression of DoPAES.

Identification and characterization of DoAP2/ERFs

DoAP2/ERF89’s full-length cDNA was 801 bp long, encoding 266 amino acid sequences, with a molecular weight of 28.62 kDa. DoAP2/ERF47’s full-length cDNA was 981 bp long, encoding 326 amino acid sequences, with a molecular weight of 35.89 kDa. In accordance with the prior evolutionary tree, the protein sequence analysis revealed that DoAP2/ERF47 contained AP2 and B3 binding domains and belonged to the RAV subfamily, whereas DoAP2/ERF89 contained an AP2-binding domain and belonged to the ERF subfamily.

Subcellular localization analysis

DoAP2/ERF89 and DoAP2/ERF47 were cloned into the pCAMBIA-1305 vector and transformed into tobacco plants using Agrobacterium tumefaciens strain GV3101. The results showed that DoAP2/ERF89-GFP and DoAP2/ERF47-GFP were only found in the nucleus and displayed blue after fusion in the same cell, whereas the GFP fluorescence of the empty vector was distributed throughout tobacco leaf cells (Fig. 10). These results demonstrated the localization of the DoAP2/ERF89 and DoAP2/ERF47 proteins in the nucleus.

Analysis of the interaction between DoAP2/ERF and DoPAES

In order to determine whether DoAP2/ERF89 and DoAP2/ERF47 were involved in the regulation process of DoPAES, the promoter of cloned DoPAES was linked to the pAbAi vector and verified by yeast one-hybrid assay (Y1H). The results showed that the bait strain co-expressing DoAP2/ERF89, DoAP2/ERF47, and proDoPAES grew well in SD/-Leu medium containing the antibiotic Aureobasidin A (Fig. 11), indicating that DoAP2/ERF89 and DoAP2/ERF47 could bind to the promoter of DoPAES.

The previous results showed that the expression trend of DoAP2/ERF47 and DoAP2/ERF89 were consistent with DoPAES in D. officinale, suggesting that these TFs may regulate the expression of DoPAES. Dual-LUC analysis further confirmed that these TFs regulated the expression of DoPAES. The DoPAES promoter was placed into the pGreenII0800-LUC vector, while these transcription factors were embedded into the pGreenII62-SK vector. The results revealed that DoAP2/ERF89 considerably activated the DoPAES promoter compared to the control group, although DoAP2/ERF47 activation was less pronounced (Fig. 12).

DoAP2/ERF89 promotes the formation of terpenoids from D. officinale

Volatile terpene concentrations in the control group (CK), DoAP2/ERFs-OE, and DoAP2/ERFs-RNAi were determined through the transient expression of candidate genes in D. officinale PLBs. The results showed that overexpression of DoAP2/ERF89 increased the content of β-patchoulene compared with CK. The amount of β-patchoulene in PLBs was decreased when DoAP2/ERF89 was silenced (Fig. 13). DoAP2/ERF47 also had an impact on terpenoid synthesis, but not on the synthesis of β-patchoulene. These findings indicate that the synthesis of β-patchoulene in D. officinale is regulated by DoAP2/ERF89.