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. 2022 Jul 22;6(7):e424. doi: 10.1002/pld3.424

Genome‐wide analysis of MYB family genes in Tripterygium wilfordii and their potential roles in terpenoid biosynthesis

Meng Xia 1, Lichan Tu 1,2, Yuan Liu 1, Zhouqian Jiang 1, Xiaoyi Wu 1,✉, Wei Gao 1,3, Luqi Huang 4
PMCID: PMC9307386  PMID: 35898558

Abstract

Terpenoids are a class of significant bioactive components in the woody vine of Tripterygium wilfordii. Previous studies have shown that MYB transcription factors play important roles in plant secondary metabolism, growth, and developmental processes. However, the MYB involved in terpenoid biosynthesis in Tripterygium wilfordii are unknown. To identify Tripterygium wilfordii MYB (TwMYB) genes that are involved in terpenoid biosynthesis, we conducted the genome‐wide analysis of the TwMYB gene family. A total of 207 TwMYBs were identified including 84 1R‐TwMYB, 117 R2R3‐TwMYB, four 3R‐TwMYB, and two 4R‐TwMYB genes. The most abundant R2R3‐TwMYBs together with their Arabidopsis homologs were categorized into 26 subgroups. Intraspecific collinearity analysis found that the 74.9% of the TwMYBs may be generated by segmental duplication events, and 36.7% of duplicated gene pairs were derived from the specific whole genome duplication (WGD) event in Tripterygium wilfordii. In addition, interspecies collinearity analysis found that 16 TwMYB genes formed homologous gene pairs with MYB genes in seven representative species, which indicated they may have a key role in evolution. Notably, we found that the TwMYB genes were differentially expressed in various tissues by expression pattern analysis. In order to further select the candidate genes related to terpenoid biosynthesis, the assay of Methyl jasmonate (MeJA) induction and analysis of phylogenetic tree was conducted. It was speculated that six candidate TwMYB genes (TwMYB33, TwMYB34, TwMYB45, TwMYB67, TwMYB102, and TwMYB103) are involved in regulating terpenoid biosynthesis. This study is the first systematic analysis of the TwMYB gene family and will lay a foundation for the functional characterization of TwMYB genes in the regulation of terpenoid biosynthesis.

Keywords: genome, MYB, terpenoid biosynthesis, Tripterygium wilfordii

1. INTRODUCTION

Tripterygium wilfordii Hook. f. is a woody vine of the Celastraceae and commonly used in traditional Chinese medicine for treating rheumatoid arthritis. Its major bioactive terpenoids, triptolide, and celastrol possess significant anti‐inflammatory, immunosuppressive, and antitumor activities that predominantly existed in the root (Noel et al., 2019). It is well‐known that isopentenyl pyrophosphate (IPP), as the key precursor for the biosynthesis of terpenoids, can be produced through the plastid‐located methylerythritol phosphate (MEP) pathway and cytosol‐located mevalonic acid (MVA) pathway. Though the two pathways are at different cellular localization, both conspire through exchange of IPP and regulatory interactions. At present, studies on terpenoids biosynthesis in Tripterygium wilfordii mainly concentrate on the gene discovery and functional study. Many pathway genes have been identified and characterized, such as MEP pathway genes, TwDXR (1‐deoxy‐D‐xylulose‐5‐phosphate reductoisomerase) and TwDXS (1‐deoxy‐D‐xylulose‐5‐phosphate synthase) (Tong et al., 2015); MVA pathway genes TwHMGR (3‐hydroxy‐3‐methylglutaryl CoA reductase) and TwHMGS (3‐hydroxy‐3‐methylglutaryl‐CoA synthase) (Liu et al., 2014); and downstream pathway genes, TwFPSs (farnesyl pyrophosphate synthase) (Zhao et al., 2015), TwSEs (squalene epoxidase) (Zhou et al., 2018), and TwCYP712Ks (Hansen et al., 2020). Although the terpenoids biosynthesis in Tripterygium wilfordii has attracted widespread attention, studies on the regulation mechanism in plants are limited.

Transcription factors act as key regulatory components of plant metabolites by switching the expression of functional genes. MYB proteins constitute one of the largest transcription factor families in plants and interact with various signaling networks to modulate plant growth processes and secondary metabolite biosynthesis. These proteins harbor highly conserved MYB domains at the N‐terminus, which usually bind to a specific DNA sequence (C/TAACG/TG). MYB domains consist of one to four imperfect repeats (R) in which each R possesses approximately 53 amino acids. On the basis of the R number, MYBs are classified into four groups, including 1R‐MYB (containing a single or a partial repeat), R2R3‐MYB (two repeats), 3R‐MYB (three repeats), and 4R‐MYB (four repeats). 1R‐MYBs play roles in flavonoid biosynthesis and trichome development by interacting with R2R3 MYB‐bHLH‐WD40 repeat complexes (MBW) (Zhang, Zhang, et al., 2019). 3R‐MYBs participate in cell cycle control, whereas 4R‐MYBs make up the smallest subgroup, and their functions are rarely known in plants (Liu et al., 2015). As the most abundant MYBs, R2R3‐MYBs are widely involved in various physiological activities and the regulation of primary and secondary metabolism. Terpenoids are isoprene‐based natural products with high chemical diversity in plants (Bergman et al., 2019). Some studies have focused on the effects of MYB on terpenoid biosynthesis. In terms of monoterpene, Yang et al. have found that FhMYB21L2 in Freesia hybrida can act directly on the FhTPS1 (terpenoid synthase) to promote it expression significantly, which ultimately leads to an increase in content of linalool (Yang et al., 2020). For diterpene, SmMYB36 in Salvia miltiorrhiza hairy roots has been identified to upregulate tanshinone contents through stimulation of the MEP pathway genes including SmDXS2, SmDXR, SmGGPPS (geranylgeranyl pyrophosphate synthase), and SmKSL1 (kaurene synthase like 1) (Ding et al., 2017). In the triterpene biosynthesis process, BpMYB21 and BpMYB61 perform different functions. BpMYB21 can upregulate the squalene epoxidase and cycloartenol synthase synthesis, whereas BpMYB61 can downregulate HMGR, squalene epoxidase, and β‐amyrin synthase (Yin et al., 2020). However, studies on the involvement of MYB family genes in the biosynthesis of terpenoids in Tripterygium wilfordii are limited.

The complete genomic sequence for plants can provide the opportunity to identify and analyze the features of the MYB gene family. Examples of these include Prunus persica (256 members) (Zhang, Ma, et al., 2018), Solanum tuberosum (233 members) (Li et al., 2019), Brachypodium distachyon (122 members) (Chen et al., 2017), and Physcomitrella patens (116 members) (Pu et al., 2020). It can be seen that the numbers of MYB genes identified in these plants differ greatly. In this study, we performed the comprehensive investigation and analysis of MYB genes in Tripterygium wilfordii for the first time, including the phylogenetic, motif composition, gene structure, cis‐acting elements, chromosomal localization, gene duplication event, synteny, and expression pattern. And then through the induction by Methyl jasmonate (MeJA) and the analysis of terpenoid related genes expression, it can be found that TwMYB33, TwMYB34, TwMYB45, TwMYB67, TwMYB102, and TwMYB103 are candidate MYB genes involved in terpenoids biosynthesis. Our results lay a foundation for the functional characterization and regulatory mechanisms of TwMYB genes in the terpenoid biosynthesis.

2. MATERIALS AND METHODS

2.1. Identification of MYB genes in Tripterygium wilfordii

Genome data of Tripterygium wilfordii were obtained from previous research works. We downloaded the protein sequences of previously reported full‐length Arabidopsis AtMYBs from the Arabidopsis Information Resource (TAIR) library (https://www.arabidopsis.org/). TwMYBs were identified from the Tripterygium wilfordii genome under the E‐value cutoff of 1e‐5 by BLASTP searches using AtMYBs as target sequences. A hidden Markov model (HMM) profile of the MYB DNA binding domain (PF00249) was downloaded from the Pfam database (http://pfam.xfam.org/) to identify TwMYBs. SMART (http://smart.embl-heidelberg.de/) and NCBI CDD online software (https://www.ncbi.nlm.nih.gov/Structure/) were used to predict the conserved domain of selected TwMYBs and eliminate the TwMYBs with incomplete conserved domains. In total, we identified 207 TwMYB genes. Theoretical isoelectric points (PI) and molecular weight (MW) values were computed using the ExPaSy online tool (https://web.expasy.org/protparam/), and subcellular localization values were predicted using the platform‐ProtComp 9.0 online tool (http://linux1.softberry.com/).

2.2. Intron‐exon structure, motif composition, cis‐acting elements, and phylogenetic analysis

The Gene Structure Display Service (http://gsds.gao-lab.org/) was used to predict the structure of introns and exons of TwMYB genes. The MEME v5.4.1 online tool (https://meme-suite.org/meme/tools/meme) was used to investigate conserved motifs. PlantCARE online software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict the cis‐acting elements within 2000 bp upstream of all TwMYB genes. The TBtools software (South China Agricultural University, Guangzhou, China) was used for gene structural visualization (Chen et al., 2020).

We subjected the protein sequences of Tripterygium wilfordii R2R3‐MYB genes together with the previously reported Arabidopsis R2R3‐MYB genes to perform multiple sequence alignments by the ClustalW method with the default settings. A neighbor‐joining (NJ) tree was then constructed based on the prior alignment result using the MEGA X software with the following parameters: JTT model, 1000 replicate bootstrap values, and pairwise deletion.

2.3. Chromosomal localization, gene duplication, and synteny with other plants

The chromosomal locations of the TwMYB genes were obtained from our genome data and exhibited by TBtools software. We analyzed TwMYB gene duplication events and the syntenic relationship between the TwMYB genes and MYB genes from Populus trichocarpa, Manihot esculenta, Vitis vinifera, Arabidopsis thaliana, Brassica rapa, Musa acuminate, and Zea mays by a multiple collinear scanning toolkit (MCScanX). The NG (Nei‐Gojobori) method was used to estimate the synonymous (Ks) and non‐synonymous (Ka) substitution rates of each duplicated gene pair (Nei & T, 1986).

2.4. Expression pattern analysis of the TwMYB genes

The seven tissues of Tripterygium wilfordii, including root bark, root phloem, root xylem, leaf, flower, stem bark, and peeled stem, were harvested in August from Fujian Province (China) and used for transcriptome sequencing. Total RNA for each tissue was isolated using cetyltrimethylammonium bromide (CTAB) method (Chakraborty et al., 2020). RNA quality was examined by 1.0% agarose gel electrophoresis and spectrophotometry measuring 260/280 absorbance ratio. The high‐quality RNA was used in cDNA library construction by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and Illumina sequencing on Illumina HiseqXTen platform, generating 150 bp paired‐end reads. Raw RNA reads were filtered to clean reads by removing reads containing adapters and N (the proportion of N is greater than 10%) and low‐quality reads, as shown in Table S1. Clean reads of each tissue were mapped to the Tripterygium wilfordii genome by TopHat2 (Chen et al., 2020). The expression level (reads per kilobase per million mapped reads, RPKM value) for each gene was calculated by HTSeq44 using default parameters. All RPKM values of TwMYB genes were transformed into log2(RPKM + 1) and normalized ( X−μ/σ,μ=mean value,σ=standard deviation) to create the heatmap. Three biological replicate samples from each tissue were analyzed. The transcriptome sequence data have been deposited under NCBI BioProject number PRJNA542587.

2.5. MeJA treatment with Tripterygium wilfordii suspension cells

Tripterygium wilfordii suspension cells were cultured in Murashige & Skoog basal medium with .5 mg·L−1 2,4‐dichlorophenoxyacetic acid (2,4‐D), .5 mg·L−1 indole‐3‐butytric acid (IBA), .1 mg·L−1 kinetin (KT), and sucrose (pH = 5.8) at 25°C. We carried out induction after 12 days with 50‐μM MeJA and harvested at 0, 4, 12, 24, and 48 h post‐treatment. The total RNA for suspension cell was isolated using the Eastep® Super Total RNA Extraction Kit (Promega). Three biological replicate samples from suspension cells were analyzed. The RNA‐Seq sequencing of each suspension cell and public repository accession numbers are the same as in Section 2.4. Differentially expressed genes (DEGs) were identified in light of more than a two‐fold change in expression (log2 fold change > 1) and P < .05, as shown in Table S2.

3. RESULTS AND DISCUSSION

3.1. Identification of MYB genes in Tripterygium wilfordii

A total of 207 MYB genes were selected in Tripterygium wilfordii and named as TwMYB1 to TwMYB207 in the order of physical position on the chromosomes. According to the number of MYB DNA binding domains, 207 TwMYBs were divided into four groups including 84 1R‐MYB, 117 R2R3‐MYB, 4 3R‐MYB, and 2 4R‐MYB genes. The detailed information, including length of protein, pI, MW, and subcellular localization, were analyzed and listed in Table S3. Among the 207 identified TwMYB genes, TwMYB48 was found to be the smallest TwMYB protein with 74 aa, whereas TwMYB142 was found to be the largest TwMYB protein with 1,728 aa. The pI ranged from 4.27 to 10.04, whereas the MW ranged from 8,355.04 to 188,952.44 Da. Subcellular localization prediction revealed that 141 TwMYBs are located in the nucleus, two TwMYBs are located in the plasma membrane and endoplasmic reticulum, respectively, and 64 TwMYBs are located in the secreted zone. Many MYB genes in other plants were predicted to be located in non‐nuclear, such as Nicotiana tabacum L. and Solanum tuberosum L., and we speculated that these non‐nuclear localized MYB genes may bind to ligands to perform their functions, such as bHLH and WD40 repeat to form MBW complexes.

3.2. Phylogenetic tree analysis of MYB genes in Tripterygium wilfordii and Arabidopsis thaliana

In plants, the R2R3‐MYB subfamily is widely studied and possesses clear functions. Arabidopsis is considered to be a model plant and its MYBs (AtMYBs) are well studied. Thus, we performed a combined phylogenetic analysis of 117 R2R3‐TwMYB and 104 R2R3‐AtMYB to predict functions of TwMYB genes, as shown in Figure 1. R2R3‐MYBs are divided into 25 subgroups in Arabidopsis based on the conservation of the DNA‐binding domain and motifs in the C‐terminal domains, which have been named S1 to S25 (Dubos et al., 2010). Our results showed that all R2R3‐MYB genes in Tripterygium wilfordii and Arabidopsis thaliana could fall into 26 subgroups, where 22 subgroups were highly consistent with the previous reports and other groups were named from S26 to S29. Among these subgroups, 21 out of 26 subgroups were present in both Tripterygium wilfordii and Arabidopsis. Orthologous genes usually share similar functions and are clustered in the same subgroups. For instance, subgroup 5 is well‐known to play crucial roles in the positive regulation of proanthocyanidin biosynthesis so that the TwMYB in this group might also play similar roles (Wang et al., 2019). There were three species‐specific R2R3‐MYB subgroups (S27, S28, and S29) in Tripterygium wilfordii. Two species‐specific subgroups (S6 and S24) only contain AtMYBs. Subgroup 6 (AtMYB75, AtMYB90, AtMYB113, and AtMYB114) and Subgroup 24 (AtMYB53, AtMYB92, and AtMYB93) have been shown to be involved in the regulation of anthocyanin and suberin biosynthesis, respectively (Legay et al., 2015; Munoz‐Gomez et al., 2021; To et al., 2020). These species‐specific genes suggest that these proteins might have specialized roles that have been either lost or gained after divergence from their last common ancestor. Some AtMYBs such as AtMYB35 and AtMYB80 were not included in a definite subgroup in Arabidopsis thaliana due to low statistical support. However, we found both AtMYB35 and AtMYB80 could cluster with TwMYB53 and TwMYB164 in S26, which suggests this subgroup expanded, and their functional roles differentiated in plants.

FIGURE 1.

FIGURE 1

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Phylogenetic analysis of R2R3‐MYB members in Tripterygium wilfordii and Arabidopsis thaliana . The phylogenetic tree was generated with 1,000 bootstrap replicates using the neighbor‐joining (NJ) method. The R2R3‐MYB genes are classified into Subgroup 3 to Subgroup 7 and Subgroup 9 to Subgroup 29 with different colors. The TwMYBs are labeled with squares, whereas the AtMYBs are labeled with dots.

3.3. Gene structure, motif composition, and promoter cis‐acting element analysis

To get comprehensive information of the TwMYBs, we performed gene structure, motif composition, and cis‐acting element analysis, as shown in Figure S1. Structural analysis with the intent to provide valuable information concerning evolution was performed. The gene structures of 207 TwMYB genes showed that they all contained MYB DNA binding domain, and the number of introns varied from 0 to 23, listed in Table S4. The majority of TwMYB genes had two introns (48.8%) or one intron (18.8%). Some TwMYB genes had a large number of introns, including TwMYB52 (23 introns), TwMYB121 (20 introns), TwMYB107 (18 introns), TwMYB76 (15 introns), and TwMYB73 (14 introns), which were all from the 1R‐MYB family. Furthermore, 13 TwMYB genes were found to be intron‐less. Exons were lost and gained during gene evolution, perhaps leading to functional diversity within the TwMYB family.

We identify a total of 10 distinct motifs and named them motif 1–10, as shown in Figure S2. Among them, motif 3 formed R1 MYB domain; motif 3, motif 7, and motif 1 together formed R2 MYB domain; and motif 4 and motif 2 together formed R3 MYB domain. The MYB domains were usually located close to the N‐terminal region, and the N‐terminal region was therefore quite conserved. We analyzed the motif compositions of 207 TwMYB and found that the TwMYB within a subgroup shared similar motif compositions, listed in Table S5. The results of motif composition are consistent with those of the above phylogenetic tree. Interestingly, Motif 5 and motif 9 were only found in Subgroup S27. And motif 10 was only found in some 1R‐MYBs including TwMYB24, TwMYB25, TwMYB46, TwMYB74, TwMYB78, TwMYB81, TwMYB113, TwMYB157, TwMYB172, and TwMYB177. These unique motifs may contribute to functional divergence.

To study the expression regulation of the TwMYB genes, putative cis‐elements within promoter regions were predicted, listed in Table S6. The results showed that TwMYB genes included three main types of cis‐acting elements. One type was responsiveness elements of phytohormones (abscisic acid, auxin, gibberellin, MeJA, and salicylic acid). Most of TwMYBs would respond to abscisic acid, which is a key endogenous messenger interacting with MYB genes to regulate plant physiology and metabolite metabolism (Golldack et al., 2014). The second type was environmental stress responsiveness element including low temperature responsiveness element, and defense and stress responsiveness element, which suggested that these TwMYBs may participate in the responses to various stress conditions. The third type was light responsiveness element. Except TwMYB188, other TwMYBs all contained this element. TwMYB gene promoters also contained circadian control elements and MYB binding sites that are involved in drought, flavonoid biosynthetic genes regulation, and light responsiveness.

3.4. Chromosomal distribution and duplication events

It is found that 207 TwMYB genes are unevenly distributed on 23 chromosomes, as shown in Figure 2. Chromosome 07 contained the most TwMYBs (16 genes), whereas chromosome 05 and chromosome 09 harbored the least TwMYBs (4 genes). Few genes were evenly distributed on a chromosome, instead most clustered on certain fragments of the chromosome (Li et al., 2016). As defined in an earlier study where nearby genes located within 200 kb were defined as a cluster, we thusly identify a total of 63 TwMYB genes to form 29 clusters on different chromosomes. However, there were no tandemly duplicated TwMYB genes.

FIGURE 2.

FIGURE 2

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Distribution of TwMYB genes on Tripterygium wilfordii chromosomes. Chromosome numbers are shown on left side of the bar. TwMYB genes are labeled to the right of the chromosomes. The TwMYB genes marked with green box indicates the TwMYB gene clusters which the distance between nearby genes within 200 kb.

Gene duplication events drive the evolution of gene families such as WGD, whole‐genome triplication event (WGT), and rearrangements (Clark and Donoghue, 2018). Thus, we performed duplication event analysis of the TwMYB genes using MCScanX and found 128 pairs of segmental‐duplicated gene pairs in total on Tripterygium wilfordii chromosomes, as shown in Figure 3. About 74.9% of the TwMYB genes (155 TwMYBs) might be generated by duplication events. Thus, we deduced that duplication events might be one of the major drivers of the TwMYB gene evolution. Tripterygium wilfordii underwent a core eudicot γ triplication event (116–128 MYA) and specific WGT event (Ks = .27 ± .077, 20.7 ± 5.94 MYA) (Feng et al., 2020; Tu et al., 2020). Generally, Ks value is used to estimate the divergence time of the duplication events according to T = Ks/2r (r, represents a substitution rate of 6.5 × 10−9 mutations per site per year for eudicots). The Ks values of the TwMYB duplicated gene pairs ranged from .126 to 6.85, as listed in Table S4. Fifteen duplicated gene pairs possessed higher Ks values (2.0–6.8), which indicates that they might have evolved from a more ancient duplication event. About 36.7% of duplicated gene pairs were distributed on 14–26 MYA, which derived from the specific WGT event in Tripterygium wilfordii. In conclusion, TwMYB was an ancient gene family and expanded with a specific WGT event. Moreover, the type of the most TwMYB gene duplications was triplication‐type, which means there are three segmental duplication genes. These results also support that the specific WGT event played critical roles in the expansion of the TwMYB gene family.

FIGURE 3.

FIGURE 3

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Segmental duplication events and inter‐chromosomal relationships between TwMYB genes. Gray lines indicate all synteny blocks in the Tripterygium wilfordii genome. The red lines indicate three segmental duplication genes. The green lines indicate two segmental duplication genes. The blue lines indicate multiple segmental duplication genes. The chromosome number is indicated on the inside of each chromosome.

The Ka/Ks ratio refers to the number of nonsynonymous substitutions (Ka) per nonsynonymous site to the number of synonymous substitutions (Ks) per synonymous site. Ka and Ks have to happen inevitably during plant evolution. Ka is assumed to cause changes in the sequence and potentially conformation and function of the protein, whereas Ks does not generate any change of the amino acid due to degeneracy properties in genetic codes. Based on neutral theory, values of Ka and Ks should logically be equal. However, when the changes of gene caused by Ka are more adaptive for the environment, the Ka/Ks ratio would be greater than 1, which indicates the corresponding gene underwent positive selection. On the contrary, the gene would undergo purifying selection and the Ka/Ks ratio <1 when the changes of gene caused by Ka are negative for plant adaptability. The Ka/Ks ratios were calculated in order to estimate selection pressure in duplicated genes and listed in Table S7. Notably, TwMYB6 and 37, TwMYB8 and 105, TwMYB34 and 69, TwMYB51 and 176, TwMYB47 and 185, TwMYB70 and 99, TwMYB71 and 105, TwMYB86 and 131, TwMYB86 and 176, and TwMYB88 and 183 had no Ks value (NaN) indicating that segmental duplication caused mutation at the nucleic acid level but not at the amino acid level. The Ka/Ks ratios of the other TwMYB duplications were all less than 1, suggesting that the TwMYB duplicated genes may have undergone purifying selective pressure during evolution. Purifying selection is the process of removing deleterious mutations. The duplicated gene pair of TwMYB48 and TwMYB87 possessed the smaller Ks and the largest Ka/Ks values, which indicates that these two genes might have experienced more purified selection.

3.5. Syntenic analysis

To further explore evolution of the TwMYBs, we constructed a synteny analysis between TwMYB and MYB genes in seven other plant species, including close dicot species (Populus trichocarpa and Manihot esculenta), distant dicot species (Vitis vinifera, Arabidopsis thaliana, and Brassica rapa), and monocots (Musa acuminata and Zea mays), as shown in Figure S3. The TwMYB genes were homologous to genes in other plants, and syntenic relationship was observed with Manihot esculenta (174 gene pairs dispersed on all chromosomes), Populus trichocarpa (177 gene pairs dispersed on all chromosomes), Vitis vinifera (166 gene pairs dispersed on all chromosomes), Brassica rapa (130 gene pairs dispersed on all chromosomes), Arabidopsis thaliana (138 gene pairs dispersed on all chromosomes), Zea mays (46 orthologous gene pairs dispersed on all chromosomes), and Musa acuminata (37 gene pairs dispersed on all chromosomes except chromosome 3 and chromosome 4).

There are 16 TwMYB genes that can form homologous gene pairs with MYB genes from any one of the seven species mentioned above, as shown in Figure 4. It suggests that these genes may have existed before the divergence of these plant species. Among them, multiple homologous gene pairs of TwMYB1, TwMYB2, TwMYB14, TwMYB28, TwMYB29, TwMYB60, TwMYB61, TwMYB119, TwMYB110, TwMYB128, TwMYB185, and TwMYB193 were found in different plants, which indicated that they may play a key role in evolution, the number of these homologous gene pairs as listed in Table 1. Interestingly, 137 TwMYB genes can form homologous gene pairs with one of five dicot species, while cannot form homologous gene pairs with two monocots. It might imply that these pairs may have appeared after the divergence of dicot and monocot species. The detailed information of syntenic gene pairs is provided in Table S8.

FIGURE 4.

FIGURE 4

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The MYB genes formed syntenic pairs between Tripterygium wilfordii and seven other representative species. The yellow horizontal bars indicate the total number of MYB syntenic pairs between different species and Tripterygium wilfordii. The different species correspond to the dots. The vertically connected filled dark dots represent shared MYB syntenic pairs between these species and the top vertical bars show the number of shared syntenic pairs. Populus trichocarpa , Manihot esculenta , Vitis vinifera , Arabidopsis thaliana , and Brassica rapa were dicot species, and Musa acuminata and Zea mays were monocots.

TABLE 1.

The number of mutual MYB homologous gene pairs in seven species

Gene ID Arabidopsis thaliana Brassica rapa Manihot esculenta Populus trichocarpa Vitis vinifera Musa acuminata Zea mays
TwMYB1 4 5 3 4 2 3 2
TwMYB2 2 2 3 3 2 1 1
TwMYB14 3 2 4 5 2 1 1
TwMYB28 5 3 4 5 3 4 1
TwMYB29 3 3 2 2 2 1 1
TwMYB61 3 1 4 5 3 3 2
TwMYB60 2 2 4 5 2 3 1
TwMYB119 2 2 4 5 3 1 1
TwMYB110 4 3 4 5 2 1 1
TwMYB128 4 3 4 4 2 3 1
TwMYB185 3 3 4 3 3 2 2
TwMYB193 3 2 4 5 3 4 2
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3.6. Expression patterns of TwMYB genes in different plant tissues

Functional redundancy is often common in the MYB family (Millar & Gubler, 2005). Thus, it is necessary to study TwMYB gene expression patterns in various organs to provide important clues for gene function. Expression profiles of the 207 TwMYB genes were analyzed using the publicly available RNA seq data including root bark, root phloem, root xylem, leaf, flower, stem bark, and peeled stem of Tripterygium wilfordii, as shown in Figure 5. Among the 207 TwMYB genes, the transcripts for 203 genes were found in at least one of seven tissues, and they were largely varied (RPKM values higher than 0); 161 TwMYB genes were expressed in all seven tissues, suggesting that they may participate in plant growth and development and perform their functions in multiple aspects. Zhang et al. used the convention that when the expression level of a gene in one tissue was at least twofold higher than in other organs and RPKM values greater than 1, it could be considered that this gene was predominantly expressed in this tissue (Zhang, Xu, et al., 2018). According to this principle, we found that TwMYBs have found to possess tissue‐specific expression patterns, and the results are listed in Table 2. Interestingly, we found that TwMYB45 and TwMYB116 predominantly expressed in root bark and were clustered in the same subgroup, S14. In this subgroup, we can also find AtMYB36 and AtMYB87 that have definite functions. AtMYB36 has been shown to regulate the transition from proliferation to differentiation in the primary root and proliferation/differentiation transition in the lateral root meristem, and AtMYB87 has been shown to function as a regulator of root growth by regulating cell wall organization and remodeling (Fernandez‐Marcos et al., 2017; Fujiwara et al., 2014). Thus, we speculated TwMYB45 and TwMYB116 may participate in root growth and development, and their root bark‐specific expression pattern might support this speculation. Similarly, TwMYB4, TwMYB15, and AtMYB18 clustered in subgroup16, and AtMYB18 has been demonstrated to be related to root growth (Yang et al., 2009). So, we speculated the TwMYB4 and TwMYB15 that specifically expressed in root xylem might also be involved in root growth. Finally, we got four candidate genes to regulate root growth and development, including TwMYB45, TwMYB116, TwMYB4, and TwMYB15.

FIGURE 5.

FIGURE 5

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Heatmaps of the differentially expressed TwMYB genes in various organs and root tissues. Gene transcript abundance values were represented in diverse colors, with red indicating higher expression level and blue indicating lower expression level, as shown in the bar at the top of figure.

TABLE 2.

The expression pattern of TwMYBs in various tissues

Tissue TwMYB
Root bark TwMYB27, TwMYB45, TwMYB116
Root phloem TwMYB101
Root xylem TwMYB4, TwMYB15
Leaf TwMYB1, TwMYB18, TwMYB24, TwMYB46, TwMYB103, TwMYB122
Flower TwMYB19, TwMYB21, TwMYB93, TwMYB141, TwMYB152, TwMYB158, TwMYB186
Stem bark TwMYB30, TwMYB185
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3.7. Identification of TwMYB related to terpenoid biosynthesis

MeJA can upregulate the contents of terpenoids in Tripterygium wilfordii by inducing terpenoid synthesis pathway gene expression (Guan et al., 2017; Huo et al., 2021; Liu et al., 2014; Zhang, Su, et al., 2019; Zhao et al., 2015). To speculate the TwMYBs putatively involved in terpenoid biosynthesis, the variation trends of some DEGs of Tripterygium wilfordii suspension cells during MeJA treatment are illustrated, as shown in Figure 6. TwAACT, TwDXS, TwDXR, TwHMGS, TwTPS, and TwFPS were upregulated in response to MeJA within 12 h of treatment, and their expression subsequently decreased. Similar to terpenoid biosynthesis genes mentioned above, TwMYB33, TwMYB34, TwMYB45, and TwMYB67 were also upregulated within 12 h and subsequently decreased. TwHMGR and TwSE were upregulated within 4 h and downregulated at 12–24 h. We found that TwMYB102 and TwMYB103 had similar pattern with TwHMGR and TwSE as well. It deduced that these TwMYBs might be candidate genes related to terpenoid biosynthesis and might regulate the accumulation of terpenoids.

FIGURE 6.

FIGURE 6

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Expression patterns of TwMYB and terpene biosynthetic genes in response to MeJA. Relative expression levels of the genes in suspension cells upon 0 (negative control), 4, 12, 24, and 48 h of MeJA treatment are shown. TwHMGR, TwAACT, TwDXS, TwDXR, TwTPS, TwHMGS, TwFPS, and TwSE were tested as positive controls. Standard deviation bars were obtained from three measurements.

To further investigate whether the candidate TwMYBs might be involved in terpenoid biosynthesis, we analyze the phylogenetic relationships between candidate TwMYBs and the MYBs that have been verified to directly regulate the genes in terpenoid biosynthetic pathway. On the basis of the results of MeJA induction, we found candidate TwMYBs might affect the upstream genes of terpenoid biosynthesis. It is reported that FhMYB21L2, SmMYB36, and BpMYB21 can regulate the upstream genes and all belong to R2R3‐MYBs (Ding et al., 2017; Yang et al., 2020; Yin et al., 2020). Thus, we chose TwMYB33, TwMYB34, TwMYB45, TwMYB67, and TwMYB102 for phylogenetic analysis, as shown in Figure 7. The TwMYB33, TwMYB34, and TwMYB45 proteins clustered with SmMYB36. SmMYB36 has been demonstrated to be the DXR and DXS regulator. Thus, we deduced that TwMYB33, TwMYB34, and TwMYB45 may have the similar function. We also found that the TwMYB67 and TwMYB102 proteins clustered with FhMYB21L2 and BpMYB21, respectively. It illustrates that they may function as TPS and SE regulator like FhMYB21L2 and BpMYB21. The phylogeny analysis supports the results of the MeJA treatment assay. Among them, TwMYB45 was predominantly expressed in the root bark, which is consistent with the fact that representative diterpenoids, triptolide, also accumulated in the root bark. Furthermore, we also calculate the Pearson correlation coefficient between TwMYB genes and terpenoid biosynthesis pathway genes based on gene expression patterns in seven different tissues, as shown in Table S9. The result showed that the expression patterns of TwMYB45, TwDXR, and TwDXS were similar and the Pearson correlation coefficients between them were .776 and .93. Thus, we concluded that TwMYB45 may influence the expression of TwDXR and TwDXS to regulate the accumulation of triptolide. There are no reports on the regulation of terpenoid biosynthesis by 1R‐MYB. We did not perform phylogeny analysis on TwMYB103, which belongs to 1R‐MYB.

FIGURE 7.

FIGURE 7

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Phylogenetic analysis of TwMYB33, TwMYB34, TwMYB45, TwMYB67, and TwMYB102 and FhMYB21L2, SmMYB36, and BpMYB21 that regulate terpenoid biosynthesis

4. CONCLUSION

A total of 207 TwMYB genes were identified from Tripterygium wilfordii genome. In order to perform a comprehensive analysis of TwMYB genes character, evolution, and potential functions, we systematically analyzed the phylogenetic tree, cis‐acting element, gene structure, motif composition, chromosome localization, colinearity, expression patterns in different tissues, and response to MeJA. The number of R2R3‐TwMYB genes is the most, and they can be divided into 26 subgroups with R2R3‐AtMYBs. The specific WGT event greatly contributes to the TwMYB gene family expansion. In the treatment of MeJA, it is found that some TwMYBs have the same expression pattern with upstream genes in terpenoid biosynthetic pathway. Based on all results above, six candidate genes including TwMYB33, TwMYB34, TwMYB45, TwMYB67, TwMYB102, and TwMYB103 are very likely to be involved in terpenoids biosynthesis, and their exact function should be studied in the future. Our research lays a comprehensive foundation for the study of MYB transcription factors in Tripterygium wilfordii and helps us to interpret the potential functions of candidate TwMYB genes involved in terpenoid biosynthesis.

CONFLICT OF INTEREST

The authors declare no competing interests.

AUTHOR CONTRIBUTIONS

Meng Xia performed the research, analysis, and interpretation of data for the work and wrote the manuscript (original draft). Lichan Tu performed the experiments of RNA‐seq. Yuan Liu did the contributions to the conception of the work and data curation. Zhouqian Jiang did the contributions to the conception of the work and revised the work. Xiaoyi Wu performed the supervision, writing—review and editing, final approval of the version, and project administration. Wei Gao performed the conceptualization, methodology, and supervision. Luqi Huang was responsible for the agreement to be accountable for all aspects of the work.

Supporting information

Figure S1. Gene structures, motif composition, and cis‐acting elements analysis of the TwMYB genes.

Figure S2. The putative conserved motifs in TwMYB proteins.

Figure S3. Synteny analysis of MYB genes between Tripterygium wilfordii and seven other representative species.

Click here for additional data file. (1.7MB, pdf)

Table S1. The quality data of transcriptome sequence.

Table S2. Differentially expressed genes of TwMYBs and terpenoid biosynthetic pathway genes with induction by MeJA.

Table S3. Details of the identified TwMYB genes.

Table S4. The number of intron‐exon of identified TwMYB genes.

Table S5. Motif of the identified R2R3‐TwMYB genes.

Table S6. Cis‐elements of the TwMYB genes.

Table S7. Details information of the segmental gene pairs.

Table S8. Orthologous relationships between Tripterygium wilfordii and other seven plant species.

Table S9. Pearson correlation coefficient between TwMYBs and terpenoid biosynthesis pathway genes.

Click here for additional data file. (138.3KB, xlsx)

ACKNOWLEDGMENT

This work was supported by National Natural Science Foundation of China (No. 81803650).

Xia, M. , Tu, L. , Liu, Y. , Jiang, Z. , Wu, X. , Gao, W. , & Huang, L. (2022). Genome‐wide analysis of MYB family genes in Tripterygium wilfordii and their potential roles in terpenoid biosynthesis. Plant Direct, 6(7), e424. 10.1002/pld3.424

Xiaoyi Wu, Wei Gao, and Luqi Huang contributed equally to this work and share correspondence authorship.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Gene structures, motif composition, and cis‐acting elements analysis of the TwMYB genes.

Figure S2. The putative conserved motifs in TwMYB proteins.

Figure S3. Synteny analysis of MYB genes between Tripterygium wilfordii and seven other representative species.

Click here for additional data file. (1.7MB, pdf)

Table S1. The quality data of transcriptome sequence.

Table S2. Differentially expressed genes of TwMYBs and terpenoid biosynthetic pathway genes with induction by MeJA.

Table S3. Details of the identified TwMYB genes.

Table S4. The number of intron‐exon of identified TwMYB genes.

Table S5. Motif of the identified R2R3‐TwMYB genes.

Table S6. Cis‐elements of the TwMYB genes.

Table S7. Details information of the segmental gene pairs.

Table S8. Orthologous relationships between Tripterygium wilfordii and other seven plant species.

Table S9. Pearson correlation coefficient between TwMYBs and terpenoid biosynthesis pathway genes.

Click here for additional data file. (138.3KB, xlsx)

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