Genome-wide survey and expression analysis of the OMT gene family in Stephania japonica

Plant materials

The S. japonica plants were cultivated and harvested in Wuhan, China and the ages of S. japonica plants are 1 year. The stems, leaves, roots, and shoots of S. japonica were collected for transcriptome sequencing and quantitative real-time polymerase chain reaction experiment.

Identification and sequence analysis of OMT gene family members in S. japonica

The genome assemblies and annotations were obtained from the National Center for Biotechnology Information (NCBI) GenBank accession number PRJNA888087 (Leng et al., 2024). The OMT genes sequences of the S. japonica were acquired from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with the accession numbers of OR240135 (SjCOMT) and OR240192 (SjCCoAOMT). In order to obtain the protein sequence of the candidate OMT gene, the amino acid sequences of 24 OMT members in Arabidopsis thaliana (https://www.arabidopsis.org/) were used as the reference for Blastp search (e-value < 1e⁻⁵).

The methyltransferase domains, including COMT (PPF00891) and CCoAOMT (PF01596), were obtained from Pfam (http://pfam.xfam.org) and were used as the query in Hidden Markov Model (HMM) searches using TBtools with the cutoff at 0.05. Further, all putative proteins were sent to NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for blast-p. Proteins with non-significant similarity were sent to to Softberry (http://linux1.softberry.com/) for correction and proteins without target domain were manually removed. Finally, the remaining candidate genes containing the complete OMT domain were regarded as OMT genes in S. japonica and renamed according to their relative location on chromosomes.

ExPASy (https://web.expasy.org/compute_pi/) was used to acquire the physical and chemical properties of OMT protein, such as isoelectric points (PI) and molecular weight (MW). WoLF PSORT (https://wolfpsort.hgc.jp/) was utilized to predict subcellular localization of OMT protein (Horton et al., 2007). Finally, the chromosomal location image of OMT genes was visualized using TBtools (Chen et al., 2020).

Multiple sequence alignment and phylogenetic analysis

The OMT protein sequences of A. thaliana and Vitis vinifera were obtained from the EnsemblPlants database (http://plants.ensembl.org/index.html) and reference (Lu et al., 2022), respectively. Multiple sequence alignments (MSA) of these protein sequences were performed using the MEGA11. With 100 bootstrap replications, the phylogenetic tree of COMT and CCoAOMT in S. japonica was constructed using the MEGA11 and Maximum-likelihood (ML) methods.

Investigation of gene structure and conserved motif

The MEME online tool (https://meme-suite.org/meme/tools/meme) was employed to identify the conserved motifs of OMT proteins in S. japonica, with the following parameters: maximum number of motifs = 6; optimum motif length= 6–50 residues (Bailey et al., 2006). By analyzing the S. japonica genome sequences, the exon-intron structure of the OMT members was investigated, and TBtools was used to visualize the gene structure.

RNA extraction and qRT-PCR analysis

RT-qPCR was performed to confirm the expression levels of selected OMT genes (SjCOMT11, SjCOMT13, SjCOMT21, SjCOMT37, SjCOMT33, SjCOMT15, and SjCOMT45). The Plant Total RNA Isolation Kit (FOREGENE, Chengdu, China) was used to extract total RNA from different samples following the instruction. The cDNA was synthesized from total RNA using the RT Easy TM II (FOREGENE, Chengdu, China). Table S1 contained the primer sequences for qRT-PCR analysis.

The ChamQ Universal SYBR qPCR Master Mix (FOREGENE, Chengdu, China) was used to conduct qRT-PCR reactions. The total amplification method was 10 μL, which consist of 2 × SYBR Master Mix (5 μL), ddH₂O (3.8 μL), forward and reverse primer (0.4 μL, 10 mM), cDNA (0.4 μL, 2.5 ng/μL). The amplification process consisted of the pre-denaturation at 95 °C for 180 S, denaturation at 95 °C for 10 S, and renaturation at 55 °C for 30 S. A total of 40 cycles were executed. The melting curve analysis was used to verify the specificity of the amplicon for each primer pair. The comparative Ct (2^−ΔΔCt) method (Livak & Schmittgen, 2001) was used to calculate transcripts amounts. Normalization was performed using GAPDH as the internal reference (Jin et al., 2019). Three biological replicates per sample were used for the analysis. ANOVA analysis was performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant.

Expression analysis of OMTs in different tissues

The expression profiles of SjCCoAOMTs and SjCOMTs in five tissues (root, leaf, flowers, bud and stem) were analyzed to verify expression levels using the RNA-seq data. The log2 (FPKM+1) values were used for normalization and the expression heat map was generated by TBtools.

Analysis of cis-acting elements of OMTs

The 1,400 bp upstream sequences of OMT genes in S. japonica genome were extracted as promoter sequences for prediction. The cis-acting elements related to plant growth and development, hormone response and biotic stress, abiotic stresses and TF binding sites were identified using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cis-acting elements results were further visualized by TBtools.

Identification and classification of OMTs in S. japonica

According to the sequence comparison, six CCoAOMT genes and fifty-two COMT genes were identified from the genome data of S. japonica after a series of screens (including BLASTP searching, Pfam analyses and eliminating short sequences). These OMT genes were named SjCCoAOMT1 to SjCCoAOMT6 and SjCOMT1 to SjCOMT52. The characteristics of SjCCoAOMTs and SjCOMTs were further analyzed and summarized in Table S2, including molecular weight (MW), cDNA sequence length, chromosome location, deduced protein sequence length, isoelectric point (pI), and subcellular localization. The length of encoded protein sequences in the SjCCoAOMT genes showed significant variation, ranging from 684 (SjCCoAOMT2) to 852 bp (SjCCoAOMT1) and 227 to 283 amino acids. As for the SjCOMT genes, the length of encoded protein sequences ranged from 256 to 393 amino acids, with the cDNA length ranging from 771 (SjCOMT25) to 1,182 bp (SjCOMT31). The MW of SjCCoAOMT proteins ranged from 25,522.7 (SjCCoAOMT2) to 31,330.55 Da (SjCCoAOMT1), and SjCOMT proteins ranged from 28,019.58 (SjCOMT25) to 43,948.65 Da (SjCOMT48). Most of the pI of SjCCoAOMT had values from 4.85 (SjCCoAOMT4) to 6.00 (SjCCoAOMT2), and only one alkaline protein exhibited a pI value higher than 8, namely SjCCoAOMT1 (8.96). As for SjCOMT, all pI ranged from 4.88 (SjCOMT33) to 6.63 (SjCOMT31). The subcellular localization predictions revealed that only SjCCoAOMT1 was situated in the chloroplast, and other SjCCoAOMTs were located in the cytoplasm region. As for SjCOMTs, SjCOMT4, SjCOMT10, SjCOMT17, SjCOMT31, SjCOMT36, and SjCOMT37 were located in the chloroplast; SjCOMT39 was located in the endoplasmic reticulum; SjCOMT13, SjCOMT23, SjCOMT28, SjCOMT30, SjCOMT40, and SjCOMT43 were located in the cytoskeleton. The remaining SjCOMTs were all located in the cytoplasm region.

Phylogenetic, orthologues analysis and chromosome distribution of OMTs in S. japonica

The physical localization map (Fig. 1) showed the distribution of SjCCoAOMT genes and SjCOMT genes on 11 chromosomes of S. japonica. It was obvious that the distribution of OMT genes was not homogeneous. Notably, four SjCCoAOMTs were concentrated on chromosome 6, while SjCCoAOMT1 and SjCCoAOMT6 were distributed separately on chromosome 2 and chromosome 9, respectively. SjCOMT genes were widely distributed on all chromosomes. Among them, 46% of SjCOMT genes were concentrated on chromosome 1 and chromosome 2, whereas chromosome 6 and chromosome 9 each had only one SjCOMT gene. Several duplicate gene pairs of SjCOMT genes could be found in chromosomes 1, 2, 3, 4 and 7.

In order to gain a comprehensive understanding of the evolutionary relationship of SjCCoAOMT genes and SjCOMT genes, the phylogenetic tree was created using the alignment of the full-length protein sequences of V. vinifera (10 CCoAOMT and 37 COMT), A. thaliana (seven CCoAOMT and 17 COMT), and S. japonica (six CCoAOMT and 52 COMT). It was found that OMT members were divided into two major subfamilies, CCoAOMT and COMT, based on their sequence similarity and topology structures (Fig. 2). Based on the sequence similarity and topology structures of the phylogenetic tree, OMT proteins were classed into two major subfamilies, One subfamily contained CCoAOMT members from the three species, while the other subfamily contained COMT members from the three species (Fig. 2). This finding suggested that the OMT genes remained highly conserved throughout plant evolution, spanning various species.

Orthologous OMT genes in A.thaliana, S. japonica and V. vinifera were identified through orthofinder. Finally, 26 of orthologous genes from A.thaliana and S. japonica, 27 from A.thaliana and V. vinifera, and 34 from S. japonica and V. vinifera were identified. The COMT genes had 5, 10 and 11 of orthologous genes in A.thaliana, S. japonica and V. vinifera. The CCoAOMT genes had seven, six and nine pairs of orthologous genes in A.thaliana, S. japonica and V. vinifera.

Gene structure and protein motif analysis of OMTs in S. japonica

Phylogenetic, MEME online tool and gene structure analysis were used to further illustrate the structural diversity of the SjCCoAOMT and SjCOMT in S. japonica, respectively (Fig. 3A). Six conserved motifs (motif 1–motif 6) were predicted, which exhibited specificity to individual groups (Fig. 3B). Different OMT groups exhibited varying compositions of structural motifs, whereas the same group contained similar motifs. The MEME results revealed that 85% of SjCOMT proteins contained six motifs, while SjCOMT proteins including SjCOMT2, SjCOMT4, SjCOMT5, SjCOMT19, SjCOMT21, SjCOMT25, SjCOMT37, and SjCOMT46 have four or five motifs. Notably, there were two motif 1 existed in SjCOMT19. Motif 1, motif 2, motif 4, and motif 5 were distributed in SjCCoAOMT1 to SjCCoAOMT6, which indicated that these motifs might play an essential role in the function regulation. While motif 3 and motif 6 were only absent in the SjCCoAOMT1.

OMT genes that were closely associated within the same phylogenetic cluster exhibited identical exon-intron arrangements, and those members belonging to the same group were more similar in their number and length of exons and introns (Fig. 3C). The number of exons in SjCCoAOMT2, SjCCoAOMT3, SjCCoAOMT4, SjCCoAOMT5, and SjCCoAOMT6 were mostly five or six, only SjCCoAOMT1 possessed eight exons. While the number of exons in SjCOMTs varied from two to five. The size of the exons and the length of the intron varied widely. These findings demonstrated that SjCCoAOMT and SjCOMT possess different structural patterns based on their features.

Expression profiles of OMTs in S. japonica

Investigating the expression of SjCCoAOMT and SjCOMT in S. japonica is essential to elucidate their potential function. We examined the expression patterns of SjCCoAOMTs and SjCOMTs in the root, bud, flower, leaf, and stem based on RNA-seq data which have been deposited under the National Center for Biotechnology Information (NCBI) GenBank accession number PRJNA888087 (Fig. 4). According to the clustering analysis, there was significant variability in the heat map of SjCCoAOMT and SjCOMT members in five tissues. SjCCoAOMT6 was highly expressed in five tissues, but SjCCoAOMT1, 3 and 4 were barely expressed in all tissues. In buds, only SjCCoAOMT5 and 2 were moderately expressed. Interestingly. As for SjCOMT members, there were 12 members (SjCOMT13, SjCOMT15, SjCOMT21, SjCOMT33, SjCOMT45, SjCOMT14, SjCOMT29, SjCOMT18, SjCOMT11, SjCOMT37, SjCOMT44, SjCOMT31) highly expressed in root. SjCOMT15, SjCOMT45, SjCOMT14, SjCOMT29, SjCOMT18, SjCOMT11, SjCOMT37, SjCOMT44, and SjCOMT49 were significantly expressed in the stem. Furthermore, 22 SjCOMT members were barely expressed in the root, bud, flower, leaf, and stem, such as SjCOMT46, SjCOMT28 and SjCOMT34. While SjCOMT45, SjCOMT14, SjCOMT29, SjCOMT18 and SjCOMT44 exhibited significant expression in five tissues. SjCOMT22, SjCOMT12 and SjCOMT39 were only highly expressed in bud and leaf.

Gene expression analysis of OMTs in S. japonica

Due to the high content of cepharanthine in the root of S. japonica, seven OMT members specifically expressed in the root were selected for qRT-PCR validation according to their expression in the transcriptome analysis (Table S3, Fig. 5). SjCOMT11 and SjCOMT13 showed specific expression levels mainly in root. SjCOMT21, SjCOMT33 and SjCOMT37 were significantly expressed in the root and slightly expressed in the stem, bud and leaf. SjCOMT15 and SjCOMT45 were not only significantly expressed in root, but also expressed highly in leaf. The findings were generally in line with the transcriptome information in the root.

Analysis of cis-acting elements of OMTs in S. japonica

The promoter, a key structure affecting the binding affinity of RNA polymerase, can influence the level of gene expression (Liu et al., 2019). Analyzing the cis-acting elements in the promoter sequence was an important way to understand the regulatory functions of OMT genes. Thus, the cis-acting elements in the promoters (1,400 bp upstream from the transcription start sites) of OMT members in S. japonica were further analyzed (Fig. 6). The cis-acting elements could be categorized into four primary groups based on their function: plant growth and development, phytohormone responsive, abiotic and biotic stress and TF binding sites. Among them, light-responsive elements, including ATCT-motif, Box4, CAG-motif, chs-CMA2a, GA-motif, GATA-motif, G-box, GC-motif, GTGGC-motif, I-box, LAMP-element, GT1-motif, Sp1, TCCC-motif, TCT-motif and ATC-motif, were the most abundant elements and distributed in most members, which was consistent with the distribution among the cis-acting elements in blueberry (Liu et al., 2021). These findings suggest these identified motifs might be important for light mediated regulation in S. japonica. Many cis-acting elements related to phytohormone responsive, such as CGTCA-motif and TGACG-motif related to MeJA-responsiveness, ABRE related to abscisic acid responsiveness elements, were present in large numbers in OMT genes. Furthermore, ARE, a cis-acting element necessary for anaerobic induction, was also found. CCAAT-box and MBS were the binding sites of the MYBHv1 and MYB transcription factors. Each CCoAOMT possessed 8–12 cis-acting elements. As for COMT, the number of cis-acting elements in each gene ranged from 5 (SjCOMT20, SjCOMT37) to 23 (SjCOMT24). In CCoAOMT, light-responsive elements were mainly distributed in SjCCoAOMT2 and SjCCoAOMT5, and MeJA-responsiveness was abundant in SjCCoAOMT1 and SjCCoAOMT6. Among them, SjCOMT15 contained the most abundant light-responsive elements (9), while only SjCOMT38 did not have light-responsive elements. In summary, OMT genes were involved in the growth, development and resistance to stress in S. japonica.