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Article

Systematic Analysis of the CCoAOMT Gene Family in Isatis indigotica and the Molecular Mechanism of CCoAOMT8-Mediated Flavonoid Synthesis Under Alkaline Stress Treatment

by
Bo Liu
1,†,
Lingyang Kong
1,†,
Junbai Ma
1,
Shan Jiang
1,
Lengleng Ma
1,
Jiao Xu
2,*,
Weichao Ren
1 and
Wei Ma
1,*
1
College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China
2
College of Jiamusi, Heilongjiang University of Chinese Medicine, Jiamusi 154000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2025, 14(11), 1518; https://doi.org/10.3390/biology14111518
Submission received: 17 September 2025 / Revised: 19 October 2025 / Accepted: 24 October 2025 / Published: 30 October 2025
(This article belongs to the Special Issue Research Progress on Salt Stress in Plants)
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Simple Summary

This study aims to identify and analyze the CCoAOMT gene family in Isatis indigotica at the whole-genome level and predict its impact on the growth and development of Isatis indigotica. Simultaneously, this study investigates the expression characteristics and response patterns of the CCoAOMT gene in I. indigotica under abiotic stress treatment and determines the content of flavonoids in Isatis indigotica roots. In addition, yeast one-hybrid experiments confirmed that IiWRKY48 and IiWRKY54 can activate the promoter of the CCoAOMT gene in I. indigotica, providing a key basis for further understanding the role of this gene in physiological processes and the secondary metabolism regulation of I. indigotica.

Abstract

Caffeoyl CoA O-methyltransferase (CCoAOMT) is one of the key regulatory enzymes in the lignin biosynthesis pathway. In addition, it participates in the modification of flavonoids, which significantly impacts plant growth, development, and antioxidant capacity, and it plays a crucial role in plant responses to stress and adversity. There is a current research gap in the CCoAOMT gene family of Isatis indigotica, particularly in terms of systematic identification and functional validation. Therefore, this study employed bioinformatics techniques to determine the composition of the CCoAOMT gene family in the Isatis indigotica genome. Eight members of the IiCCoAOMT gene family were identified, and their gene structures and motifs are relatively conserved. These members of the IiCCoAOMT family are located on three different chromosomes (3, 6, and 7) and exhibit significant tandem replication. According to phylogenetic research, IiCCoAOMT is divided into four distinct groups: Ia, Ib, Ic, and II. It is worth noting that the IiCCoAOMT genes in group Ia may be candidate genes involved in flavonoid biosynthesis and indirectly affect the content of flavonoid components., Subsequently, a yeast one-hybrid experiment verified that IiWRKY48 and IiWRKY54 could activate the CCoAOMT gene promoter in Isatis indigotica. These results provide a theoretical basis for understanding the function of CCoAOMT genes.

1. Introduction

Isatis indigotica is a biennial herbaceous plant of the Brassicaceae family [1]. It is a popular Chinese herbal medicine, and its medicinal parts are roots and leaves. The leaves of indigo, commonly known as Folium Isatidis [2], are widely used in the treatment of various diseases [3]. In traditional Chinese medicine, the basic theory attributes functions such as clearing heat and detoxifying, cooling the blood, and removing macules. The main components of indigo are lignin, indole alkaloids, and polysaccharide components [4]. These components have good clinical therapeutic effects in preventing and treating viral infections, immunodeficiency diseases, and cancers, such as influenza, mumps, pneumonia, allergic dermatitis, acute respiratory syndrome, and other diseases [5]. Plants face environmental fluctuations throughout their life cycles, including abiotic and biotic stress treatments. Therefore, plants develop various response mechanisms to adapt to these unfavorable conditions, and many genes are activated for expression. The expression level of stress treatment response genes may contribute to the survival of plants [6]. Typically, the control of gene expression in response to external elements is predominantly governed by families of enzymes, which are crucial adaptation mechanisms in plants [7].
The external environment has a significant influence on the normal growth and development of plants. External stress treatments such as drought, low temperatures, and saline–alkali stress treatment will cause different degrees of damage in plants [8], which may even lead to plant death [9]. Secondary metabolites in plants play a vital role in stress treatment reactions and are essential for their growth and development. The pathways of phenylpropane metabolism include lignin and flavonoids, which have different effects on stress treatment resistance. Lignin is one of the main substances that make up the plant’s cell wall. It can increase the hardness of the cell wall, play a better supporting role, and exhibit strong compressive resistance while also providing functions in disease resistance and stress treatment resistance [5]. Research has shown that high-nitrogen applications can lead to lignin deposition and reduced content [10]. As potent reactive oxygen species scavengers, plant flavonoids exhibit a strong antioxidant effect, responding effectively to various stress treatments and improving the tolerance and resistance of plants to both biotic and abiotic stress treatments. The latest research indicates that CCoAOMT is involved in lignin biosynthesis, as well as in erucic acid metabolism and isorhamnetin biosynthesis, through its role in methylating hydroxycinnamic acid [11] or flavonoid precursors [12]. The content of polymethoxyflavones in citrus fruits is significantly increased after being infected with Huanglongbing [13]. In addition, flavonoids play a crucial role in the growth and development of plants, as well as in the quality formation process of flowers and fruits. For example, flavonoid compounds are the main substances that affect flower color. They can induce the colonization of rhizobia and arbuscular mycorrhizal fungi in plant roots and improve the absorption of elements such as nitrogen and phosphorus by plants [14].
The year 1988 marked the discovery of CCoAOMT in carrot and parsley tissue culture cells, which was linked to their defensive mechanisms against fungal infections [15]. Caffeoyl-CoA O-methyltransferase (CCoAOMT) participates in the modification process of O-methylation. Unmodified flavonoids have poor lipid solubility and water solubility. Post-modification effects such as methylation, glycosylation, and hydroxylation can effectively improve the various biological activities of flavonoids and enrich the structure and function of flavonoids [8]. CCoAOMT participates in the methylation process of flavonoids [16]. CCoAOMT is dependent on Mg2+ and includes true CCoAOMT that specifically catalyzes caffeoyl-CoA and CCoAOMT, which have a preference for catalyzing caffeic acid esters and flavonoids [17]. For example, under drought stress treatment conditions, the expression level of the CCoAOMT gene in Paeonia ostii leaves increased significantly. When the PoCCoAOMT gene of Paeonia ostii was transferred into tobacco and subjected to drought treatment, it was found that tobacco plants with the PoCCoAOMT gene had stronger drought resistance than wild-type plants. [18]. In Arabidopsis, CCoAOMT1 plays a role in resisting drought stress treatment through the control of H2O2 buildup and the ABA and ROD signaling routes [19]. Eliminating the CCoAOMT enzyme gene in Arabidopsis led to a reduction in G lignin levels and a rise in S and H lignin amounts [20,21]. Additionally, the elimination of this gene results in an excessively sensitive state to salt stress treatment due to the prevention of root growth [22]. Tobacco plants with the caffeoyl-CoA-O-methyltransferase gene of the M14 line of sugar beet have strong resistance to salt stress treatment and drought stress treatment [23]. Throughout the growth and maturation of Lonicera macranthoides, the expression intensity of the LmCCoAOMT gene correlates with chlorogenic acid levels and plays a crucial role in chlorogenic acid synthesis [24]. The CCoAOMT gene also has a certain impact on resistance to bacterial wilt. The expression level of the CCoAOMT gene is higher in tomato varieties resistant to bacterial wilt and lower in susceptible varieties [25]. Genomic studies reveal the presence of CCoAOMT genes in Arabidopsis thaliana [20], rice [26], and poplar [27] (in the presented order). The CCoAOMT gene consists of eight preserved motifs labeled A to H [28].
High-quality genomic data on I. indigotica have been published [29]. Nonetheless, research into the evolutionary link and functional confirmation of the CCoAOMT family in I. indigotica remains scarce. This article determines the CCoAOMT gene family in the genome of I. indigotica through bioinformatics analysis, thus filling this gap. The qRT-PCR (quantitative real-time polymerase chain reaction) technique is used to detect gene expression in this family of leaves under alkali stress treatment at different time periods and to determine the content of chemical flavonoid components. The findings aid in comprehending CCoAOMT genes and offer fresh insights for additional studies on their traits.

2. Materials and Methods

2.1. Experimental Materials

The seeds used in the experiment were all from the Heilongjiang University of Traditional Chinese Medicine. Firstly, treat the samples with 70% (v/v) ethanol for 1 min; then, soak and disinfect the sample with a solution containing 0.5% (v/v) sodium hypochlorite for 10 min, and finally, rinse the sample 3–5 times with sterile distilled water. During the subsequent 24 h pretreatment period, the seeds were placed in a light-controlled incubator under strict growth conditions: temperature of 22 °C, relative humidity of 65%, light cycle of 16 h/8 h in the dark, and light intensity of 120 μ mol·m−2s−1. The seeds were sown in a flower pot made of vermiculite and nutrient soil at a ratio of 1:4 until 3–4 leaves grew, and they were transplanted into a pot with a diameter of 12 cm and further cultured for about 60 days. Select materials with good growth conditions and stable growth, and then, place them in a light-controlled incubator for 24 h to remove any damage. The initial pH value of the mixed nutrient soil was measured before the experiment (using a pH meter with a soil water ratio of 1:5) [30]. The measurement results showed that its pH value was 6.0 ± 0.2, which met the growth requirements of the experimental species. During the experiment, a 200 mmol NaHCO3 alkaline solution was poured every 24 h, with a pH value of 8.2 [31]. Meanwhile, the control group received an equal amount of sterile water for irrigation on the same day of treatment. Samples were taken at 0, 1, 3, and 5 days under the same cultivation and management conditions. During RT-PCR, flavonoid content, similarity evaluation, POD activity, and MDA content experiments, we selected three independent samples as biological replicates and set three technical replicates for each independent sample to ensure the accuracy and stability of the experiment. The newly collected samples were rapidly frozen using liquid nitrogen and stored in a refrigerator set to −80 °C.

2.2. Identification and Characterization of IiCCoAOMT Genes

We downloaded the Isatis indigotica genome file and referenced the isatis indigotica genome annotation information file from FIGHARE (https://figshare.com/, accessed on 17 July 2025). Then, the protein sequences of the CCoAOMT of Arabidopsis thaliana L. were downloaded from TAIR (https://www.arabidopsis.org/, accessed on 17 July 2025) [32] as reference sequences, and according to the SMART website (http://smart.embl-heidelberg.de/, accessed on 17 July 2025), the sequences obtained in the previous step were subjected to structure prediction. Sequences without the typical domain of the CCoAOMT protein were removed. Sequences with missing or incomplete domains were manually removed. When dealing with several transcripts of a single gene, the lengthiest transcript was chosen as the standard sequence. The remaining protein sequences were regarded as members of the IiCCoAOMT family. Using ExPASy (http://web.expasy.org/protparam/, accessed on 18 July 2025), the website predicts the physicochemical properties of the Isatis indigotica IiCCoAOMT protein [33]. Predicting the subcellular positioning of the IiCCoAOMT protein is possible via the Plant-mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 18 July 2025) [34]. The tertiary structure of the protein encoded by I. indigotica CCoAOMT is predicted, and protein homology tertiary structure modeling is performed using SWISS-MODEL (https://swissmodel.expasy.org/, accessed on 18 July 2025).

2.3. Analysis of the Gene Structure of I. indigotica CCoAOMT and the Conserved Motifs

Using the GFF annotation file of the Isatis indigotica genome, the intron–exon distribution of the Isatis indigotica CCoAOMT gene was obtained. Protein sequence alignment analysis was performed through the online software MEME Suite v5.5.2 (https://meme-suite.org/meme/, accessed on 18 July 2025) [35]. Set the significance threshold for E value to <1 × 10−5. The conserved motifs identified in the Isatis indigotica CCoAOMT protein were identified. The refined settings include the distribution of sites, assigning either 0 or 1 to each sequence; a motif count of 8; and preset values for additional parameters. TBtools (v2.136) software was used for multiple sequence alignment.

2.4. Phylogenetic Analysis of the Isatis indigotica CCoAOMT Gene

We performed multiple alignments of the selected CCoAOMT amino acid sequence using Clustal X v2.0 [36], and the phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA11 software [37] (https://megasoftware.net, accessed on 18 July 2025) with 1000 bootstrap replicates. Classify the members of the CCoAOMT gene family in I. indigotica based on the classification method and phylogenetic relationships of the CCoAOMT gene family.

2.5. Analysis of Gene Cis-Acting Elements

Extract the 2000 bp upstream sequence of the transcription start codon for all members of the CCoAOMT gene family and submit it to the online website PlantCARE [38] (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 18 July 2025). The website was used for predicting cis-acting components, and a self-written R-3.6.0 script was used for statistical analysis and the visualization of the prediction results.

2.6. Gene Chromosome Location and Collinearity Analysis

Obtain the gene structure annotations of the CCoAOMT gene from the database and retrieve chromosome positions from these annotations. TB-Tools 2.142 is used to display the accurate location of genes on each chromosome. Collinearity analysis was conducted on its genome using TBtools (v2.136), and it was analyzed with the genomes of Arabidopsis thaliana and grape, with visual analysis conducted.

2.7. RNA Extraction from Leaves at Different Treatment Times and Real-Time Fluorescence Quantitative PCR Analysis

The leaves of Isatis indigotica were washed clean with tap water.The SPAREasy Plant RNA Kit (AC0305) was used to extract total RNA from Isatis indigotica plants (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China). Subsequently, RNA quality was assessed using 1% agarose gel electrophoresis, followed by its reverse transcription into cDNA. Utilize a nanodrop spectrophotometer to ascertain the concentration of the acquired cDNA. Finally, use the extra kit to perform qRT-PCR experiments with cDNA as a template. The PCR procedure is as follows: 94 °C for 30 s; 45 cycles of 94 °C for 12 s; 58 °C for 30 s; and 72 °C for 45 s. Then, heat the sample at 79 °C for 1 s to read the plate. After the end of the last PCR cycle, increase the temperature from 55 °C to 99 °C at a rate of 0.5 °C/s to generate the melting curve of the sample. Use the EF1α gene as an internal reference gene [39]. Three biological replicates and three technical replicates were performed for each sample. The 2−ΔΔCt algorithm was employed to determine the comparative expression levels of each gene. Set three technical replicates for each experiment [40]. The above test steps are carried out according to the kit’s instructions. Use Primer (version 5.0) software to design qPCR primers. Beijing Ruiboxingke Biotechnology Co., Ltd. (Beijing, China) is the sole producer of all primers employed in this research.

2.8. Determination of Flavonoid Chemical Constituents in I. indigotica Tissue Samples

The experimental materials are Isatis indigotica leaves used at different time periods under the alkali stress treatment. The standard products are purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China), including luteolin (batch number WRQ-0000398), isoquercitrin (batch number DSTDY000603), and Folium Isatidis control herb (ycwp24062805). Methanol is of chromatographic grade, and the other reagents are of analytical grade. Water is ultrapure water. The following were used: Waters (Milford, MA, USA) XTERRA® MS C18 (250 mm × 4.6 mm, 5 μm); mobile phase: methanol (A) 0.1% formic acid aqueous solution (B); gradient elution: 0–75 min, 10% → 64% A; 75–80 min, 64% → 90% A; flow rate: 1.0 mL/min; detection wavelength: 254 nm; temperature: 35 °C; injection volume: 10 μL [41]. Three biological replicates and three technical replicates were analyzed for each sample.
Creating a Standard Solution: To craft a mixed reference solution, take an appropriate amount of luteolin-7-O-glucoside and isoquercitrin reference substances, weigh them precisely, dissolve them in a 70% methanol solution, and quantitatively dilute them to prepare a mixed reference solution containing the above reference substances at the desired concentration per 1 mL.
Preparation of the Test Solution: Weigh 0.5 g of Folium Isatidis medicinal material; cut it into pieces; place it in a stoppered conical flask; accurately add 10 mL of 70% methanol solution; seal it tightly, weigh it, and extract it by ultrasound (frequency: 50 kHz) for 45 min; weigh it again; make up the lost weight with 70% methanol solution; shake it well; filter it with a 0.45 μm organic microporous membrane; and take the subsequent filtrate to obtain it. Compare the fingerprint spectra of Isatis indigotica treated with alkali at different time periods, determine them according to the above chromatographic method, and evaluate the similarity using Isatis indigotica as the control medicinal material.

2.9. Yeast One-Hybrid Assay

Under abiotic stress treatment, WRKY transcription factors can specifically bind to the promoter W-box (TTGACC) element. The screening of W-box elements that bind to the IiCCoAOMT promoter and WRKY transcription factors was conducted through the plantCARE website. By reviewing the literature and constructing the WRKY gene family of Isatis indigotica, it was determined that WRKY48 and WRKY54 have regulatory co-functions. Therefore, yeast one-hybrid analysis was used to analyze the interactions between WRKY48 and 54 and the promoters of the IiCCoAOMMT3, IiC-CoAOMT4, and IiCCoAOMMT7 genes [42]. The promoters for the quartet of CCoAOMT enzyme genes were inserted into the pLacZi vector, using EcoR I and Sac I sites as baits. Cloning of the wrky transcription factor’s CDS region into the pGADT7 vector occurred using the EcoR I site as the target. The plasmids pLacZi-IiCCoAOMT3, pLacZi-IiCCoAOMT4, and pLacZi-IiCCoAOMT7, along with the blank vector pLacZi, were introduced into the EGY48 strain and grown in media with varying concentration levels to identify toxicity and self-activation. The vectors of WRK48, 54, and the recombinant vectors pLacZi-IiCCoAOMT3, pLacZi-IiCCoAOMT4, and pLacZi-IiCCoAOMT7 were transformed into EGY48 yeast. The recombinant plasmids of pLacZi and JG4-5, pLacZi and IiWRKY54-JG4-5, and JG4-5 and pLacZi-IiCCoAOMT7 were co-transformed into yeast strain EGY48 as negative controls.
The transformed yeast was dispersed on an SD/−Trp-Ura-Broth medium and cultured at 30 °C for 3 to 5 days. Positive colonies were picked and cultured in SD/−Trp-Ura-Broth liquid medium and then cultured on chromogenic medium at 30 °C for 1 to 3 days. Based on color development, it can be determined whether the reporter gene is activated. Three biological replicates and three technical replicates were performed for each sample.

2.10. Determination of Antioxidant Content in I. indigotica

The malondialdehyde content assay kit(BN0049-W96) and POD (BN0048-W96) enzyme activity assay kit (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China) were used to determine the malondialdehyde content in the tissue samples of Isatis indigotica. Weigh approximately 0.1 g of tissue (or 0.5 g for adequately moist samples), introduce 1 mL of the extraction mixture, blend in an ice bath, centrifuge at 4 °C × 12,000 rpm for 10 min, collect the supernatant, and store it on ice for analysis. Combine the active solution with the sample as needed, transfer 200 μL of the supernatant into a 96-well plate, and measure absorbance A at wavelengths of 532 nm and 600 nm, ΔA = A532-A600. Calculate the malondialdehyde content using the following formula: MDA (malondialdehyde) content (nmol/g fresh weight) = 32.3 × ΔA ÷ W, where W is the sample mass, g. Weigh about 0.1 g of tissue (0.25 g for samples with sufficient moisture), add 1 mL of extraction solution, and perform ice bath homogenization. Place the supernatant in a centrifuge at 4 °C and 12,000 rpm for 10 min; then, chill it on ice for analysis. Mix the test solutions, reagent 1, reagent 2, and reagent 3, in a 96-well plate as required. Immediately read the absorbance value A1 at 470 nm, and after 1 min, read A2, ΔA A2-A1. The following was used: POD (Δ OD470/min/g fresh weight) = 100 × ΔA ÷ W, where W denotes sample mass, g. POD (peroxidase) enzyme activity is defined as increasing the absorbance value at 470 nm by 1 unit U per gram of tissue per minute in the reaction system. The above experiment involves three biological replicates and three technical replicates for each sample.

2.11. Statistical Analysis

The biological experiment was conducted with three biological and technical replicates, and statistical evaluation was performed using one-way analysis of variance (Tukey test). Measurement information is presented in the form of the mean plus or minus the standard deviation. Use p < 0.05 as the benchmark for statistical correlation and p < 0.01 as the benchmark for significance comparison.

3. Results

3.1. Identification and Physicochemical Property Analysis

Eight predicted IiCCoAOMT genes were identified from the Isatis indigotica genome (Supplementary Table S1). The IiCCoAOMT genes contain two conserved domains: UDP-glucuronosyl and UDP (Glucosyl transferase and 0-methyltransferase (PF01596.17)) [43]. Some genes also have only one domain: O-methyltransferase. Furthermore, an analysis was conducted on the dimensions of the protein, the isoelectric point, its molecular mass, and the subcellular positioning of IiCCoAOMT (Table 1). The findings revealed that the briefest segment, the IiCCoAOMT5 protein, comprises 147 amino acid units. The number of amino acid residues in the remaining seven sequences is around 200. The lengths of the IiCCoAOMT gene sequences vary greatly. Among them, the IiCCoAOMT2 gene sequence is the longest (834), and the gene sequence lengths are between 400 and 850 bp. The theoretical isoelectric points of proteins range between 4.96 (IiCCoAOMT3) and 9.37 (IiCCoAOMT5). It has been determined that there are both acidic and alkaline proteins. The prediction results of the subcellular localization show that IiCCoAOMT2, IiCCoAOMT3, IiCCoAOMT4, and IiCCoAOMT6 are located in the chloroplast, IiCCoAOMT5 is located in the nucleus, IiCCoAOMT7 is located in the peroxisome, and IiCCoAOMT8 is situated in the cytoplasm. The basic details of all members of the IiCCoAOMT family are summarized in Table 1. Using the SWISS-MODEL tool for the homology modeling of IiCCoAOMT, a three-dimensional model is constructed (Figure 1). It can be seen that IiCCoAOMT is composed of two subunits, contains abundant α-helices and random coils, and has two SAM binding regions. As shown in the figure, the three-dimensional model of IiCCoAOMT2 differs significantly from that of other IiCCoAOMT genes. A genetic mutation has likely occurred.

3.2. Analysis of the Gene Structure of I. indigotica CCoAOMT and the Conserved Motifs and Sequence Alignment of Its Encoded Protein

To further study the similarity and diversity of motifs in different IiCCoAOMT proteins, MEME software is used for protein domain prediction (Figure 2 and Supplementary Figure S1). The results show that the protein structure of IiCCoAOMT is relatively conserved. A total of eight relatively conserved motifs (named Motif1~8, respectively) were identified. Among them, Motif1, Motif6, and Motif7 are the most conserved and are common motifs in each group (Figure 2A). In addition to the common motifs, each group of motifs also has certain specificities. Motif8 is unique to the IiCCoAOMT7 and IiCCoAOMT8 genes. Such motifs may be related to the diversity of functions of the IiCCoAOMT gene. The variation between exons and introns plays a crucial role in the evolution of gene families, offering compelling proof for analyzing evolutionary paths [44,45]. For a better understanding of IiCCoAOMT’s gene architecture, we acquired the genomic and CDS sequence details of each gene based on Isatis indigotica’s genomic data, followed by the creation of an exon–intron structure diagram. (Figure 2B). The results show that the number of exons and introns per IiCCOAOMT family member was relatively conserved. Most IiCCoAOMT genes contain five exons and four introns. This indicates that four introns are the main structural form of the IiCCoAOMT gene. Multiple sequence alignment was performed on eight IiCCoAOMT proteins to study their conserved protein domains with respect to existence and location (Supplementary Figure S2). The IiCCoAOMT gene contains two conserved domains: UDP-glucuronosyl and UDP (Glucosyl transferase and 0-methyltransferase (PF01596.17)). Some genes also have only one domain, O-methyltransferase, which is similar to the results of other plant species.

3.3. Phylogenetic Analysis of IiCCoAOMT Family Members

To determine the evolutionary relationship between IiCCoAOMT and CCoAOMT of other model plants, a phylogenetic tree was constructed using 37 CCoAOMTs from four plant species (17 from Dendrocalamus farinosus [15], 7 from Glycine max, 6 from rice, and 7 from Arabidopsis) (Supplementary Table S2). The results show that the eight IiCCoAOMT genes are divided into four subfamilies (Figure 3). Clustered CCoAOMT protein sequences within a single branch tend to exhibit more functional resemblance. The I. indigotica CCoAOMT family proteins are divided into three subgroups: Subgroup Ia contains two IiCCoAOMT proteins, namely, IiCCoAOMT3 and IiCCoAOMT4. Subgroup Ib contains four IiCCoAOMT proteins, namely, IiCCoAOMT1, IiCCoAOMT5, IiCCoAOMT6, and IiCCoAOMT7. Subgroup II contains two IiCCoAOMT proteins, namely, IiCCoAOMT2 and IiCCoAOMT8. The majority of members grouped under the same subgroup exhibit comparable motif configurations, suggesting that proteins derived from the same sub-branch might serve similar roles. Research revealed that both I. indigotica and the prototype plant Arabidopsis are members of the Brassicaceae family. In each subfamily, the IiCCoAOMT gene is closest to the AtCCoAOMT gene. For example, these include AtCCoAOMT3 and IiCCoAOMT8 and IiCCoAOMT4 and AtCCoAOMT1, indicating that the evolutionary relationship between the members of the IiCCoAOMT family and the members of the AtCCoAOMT family is the closest. Consequently, the gene functions might be analogous between the IiCCoAOMT gene family members and those in the AtCCoAOMT gene family.

3.4. Cis-Acting Element Analysis of IiCCoAOMT Gene Promoter

To explore the mechanism of action of the IiCCoAOMT gene in stress treatment response and development, the PlantCARE online tool was used to analyze the 2000 bp upstream promoter sequence of the IiCCoAOMT gene and its cis-acting elements (Figure 4). A total of 42 core promoter elements were identified and classified into six categories according to their functions: plant hormone response, light response element, abiotic stress treatment response, plant growth hormone, and core. A great number of cis-acting elements were found upstream of the eight IiCCoAOMT genes, revealing that light signals regulate IiCCoAOMT. Furthermore, a multitude of plant hormone reaction components exist, including ABRE and TGACG-Motif, alongside binding locations for transcription factors like MYB and W-box and stress treatment response elements such as STRE, WUN motif, and LTR. In addition, the W-box is exclusive to IiCCoAOMT1, IiCCoAOMT3, IiCCoAOMT6, and IiCCoAOMT7, suggesting that the WRKY transcription factor may specifically bind to these four members of the IiCCoAOMT gene family. IiCCoAOMT3, IiCCoAOMT4, IiCCoAOMT5, IiCCoAOMT6, and IiCCoAOMT7 contain methyl jasmonate (MeJA) response elements (CGTCA motif and TGACG motif). Among them, IiCCoAOMT4 contains the most MeJA response elements. These findings suggest that multiple hormones control IiCCOAOMT. It is worth noting that the upstream regions of the IiCCoAOMT1, IiCCoAOMT2, IiCCoAOMT3, IiCCoAOMT4, IiCCoAOMT5, IiCCoAOMT6, and IiCCoAOMT7 genes all contain MYB binding sites involved in the regulation of flavonoid biosynthesis.

3.5. Chromosome Localization and Homology Analysis of IiCCoAOMT Genes

A gene’s chromosomal position is shaped by its evolutionary history. Therefore, our study shows that the members of the IiCCoAOMT gene family are randomly distributed on three chromosomes (Figure 5). According to the chromosomal distribution of CCoAOMT, it is named IiCCoAOMT1-IiCCoAOMT8. Among them, there are four genes on chromosome 7, and there are two genes on chromosomes 3 and 6 (Figure 5B). From this, it can be seen that the CCoAOMT gene is irregularly distributed on the chromosome [46,47]. To elucidate the evolutionary link among IiCCoAOMT genes, we examined their interspecific collinearity. However, through analysis, we only found one homologous pair (IiCCoAOMT1, IiCCoAOMT5, IiCCoAOMT6, and IiCCoAOMT7) (Figure 5A). The duplicate gene pairs in IiCCoAOMT may have similar functions, indicating that these chromosomal fragments may not be differentiated entirely during evolution and may have redundancy in function [48]. To further understand the evolutionary relationship of IiCCoAOMT, we constructed an interspecific collinearity analysis diagram of three species: I. indigotica, grape, and Arabidopsis. According to the analysis, there are only four collinear relationships between I. indigotica and I. indigotica alone, and there are a total of four collinear relationships among the three species of I. indigotica, grape, and Arabidopsis (Figure 5C). From the above analysis results, the homology between I. indigotica and Arabidopsis is slightly higher.

3.6. Analysis of IiCCoAOMT Gene Expression Patterns Under Different Time Periods of Alkali Treatment

The cultivation of I. indigotica on a large scale in Northeast China is significantly influenced by its ability to withstand cold and its salinity–alkalinity balance. It is reported that cold tolerance and salinity–alkalinity can affect the content of flavonoids and, at the same time, improve the stress treatment recovery ability of I. indigotica. The expression level of genes under alkali treatments at different time points in plants can reflect their functions and possible regulatory pathways to a certain extent, and this is closely related to the growth and development of plants. This study found that when I. indigotica was treated with alkali for 0d, 1d, 3d, and 5d, the leaves of I. indigotica gradually wilted as time went by, while the normal control group did not show leaf wilting, forming a sharp contrast with the treatment group (Figure 6).
To further verify the function of the IiCCoAOMT gene and analyze the impact of alkali stress treatment on the IiCCoAOMT gene, through qRT-PCR technology, we verified that as the treatment time prolongs, the change range of the IiCCoAOMT1 gene is relatively small, and the overall state is gradually decreasing. The genes IiCCoAOMT2, IiCCoAOMT5, and IiCCoAOMT8 first increased and then decreased; the genes IiCCoAOMT3 and IiCCoAOMT4 first increased and then decreased; the genes IiCCoAOMT6 and IiCCoAOMT7 showed a gradually increasing trend (Supplementary Table S3). In summary, the qRT-PCR verification results reveal that the IiCCoAOMT genes show different expression patterns in response to alkali stress treatment, indicating that these genes are specific in expression (Figure 7).

3.7. Analysis of Flavonoid Metabolite Content of IiCCoAOMT Gene

The content of luteolin, isoquercetin, and flavonoids in I. indigotica treated with alkali stress treatment at different time periods was detected. The results showed that the content of isoquercetin and luteolin changed at different treatment times. The content of quercetin first increased and then decreased, showing an overall downward trend. It reached its peak on the first day, which was twice the content of the blank group. The range of content variation of luteolin is relatively small, with almost no change in content, showing a first decreasing and then increasing trend, with a subsequent decrease (Figure 8). Comparing Figure 6 with Figure 7 reveals that the content change in isoquercetin is consistent with the expression trend of the IiCCoAOMT8 gene. The IiCCoAOMT8 gene may regulate the expression of the flavonoid metabolite isoquercetin. Nonetheless, the variation in luteolin’s content aligns with the expression pattern of IiCCoAOMT1. The IiCCoAOMT1 gene may directly or indirectly regulate the expression of luteolin.
The similarity evaluation software for traditional Chinese medicine chromatographic fingerprint analysis was used in our analysis (Figure 9). The characteristic response curve and fingerprint spectrum are shown in the figure. Two chromatographic peaks were identified using the standard medicinal herb Daqingye as a control. By comparing with the control sample, the characteristic peaks were identified. It was found that peak 1 was hesperidin, and peak 2 was isoquercetin (Supplementary Figure S3 and Supplementary Table S4). The similarity results revealed high homology across different treatment time periods, with only slight differences in content, indicating that abiotic stress treatment impacts gene expression.

3.8. The Effect of Alkaline Stress Treatment on POD Activity and MDA Content in I. indigotica Tissue Samples

The CCoAOMT gene is associated with the synthesis of multiple methoxy flavones (PMFs), which, in turn, are related to antioxidant indicators. The activity of antioxidant enzymes, such as POD, is an essential indicator for evaluating the antioxidant level of plants. POD can clear excess reactive oxygen species and prevent oxidative damage. As shown in Figure 10, an extension of alkaline treatment times is associated with higher POD enzyme activity, which, in turn, strengthens the plant’s stress treatment resistance, indicating a significant relationship. MDA is a secondary product of membrane damage. When plants are subjected to stress treatment, MDA accumulates significantly at various time periods, and its content increases considerably at the cellular level. The above results indicate that the degree of membrane lipid peroxidation is high in 1d and 3d treatments, with a significant relationship, suggesting that growth conditions may influence it.

3.9. IiWrky48 and IiWrky54 Can Bind to the Promoter CCoAOMT Gene

Yeast one-hybrid is a typical method for detecting the interaction between proteins and DNA. To determine whether the promoter of the target gene can bind to IiWRKY48 and IiWRKY54, a literature review revealed that the Arabidopsis WRKY gene (AT2G30250.1) regulates enzyme genes. Therefore, all WRKY genes of Isatis indigotica were co-constructed with it. As shown in Supplementary Figure S4, IiWRKY48 and Arabidopsis WRKY are on the same branch, and yeast one-hybrid experiments were conducted. The IiWRKY48-JG4-5 and IiWRKY54-JG4-5 constructs, which contain two promoters, and the pLacZi-IiCCoAOMT4 construct were co-transformed into the yeast strain EGY48, as shown in Figure 11. The yeast that was co-transformed with IiWRKY48-JG4-5, IiWRKY54-JG4-5, and pLacZi-IiCCoAOMT4 grew well, indicating that IiWRKY48-JG4-5 and IiWRKY54-JG4-5 can directly interact with the promoter of the pLacZi-IiCCoAOMT4 gene, and the colonies turned blue. However, the IiWRKY48-JG4-5 domain and IiWRKY54-JG4-5 cannot interact with other pLacZi-IiCCoAOMT domains. This result indicates that the IiWRKY48-JG4-5 and IiWRKY54-JG4-5 domains can directly interact with the domain of pLacZi-IiCCoAOMT4 and regulate its transcription.

4. Discussion

CCoAOMT is a class of S-adenosyl-L-methionine methyltransferases. The gene is crucial for plants’ physiological growth and their reaction to external stressors. The dried leaves (Isatis indigotica Fort. folium) and roots (Isatidis radix) of I. indigotica are traditional Chinese medicines, which have curative effects such as clearing heat and detoxification, cooling the blood, and resolving carbuncles. I. indigotica can also be used as an industrial dye. Therefore, I. indigotica is a medicinal and economic plant widely planted all over the world. Studying the regulatory mechanism of the growth and development of I. indigotica will help solve possible problems in I. indigotica breeding and improve the overall quality of I. indigotica.

4.1. Expansion of CCoAOMT Gene Family

The IiCCoAOMT gene family has not been identified yet, making the function of the IiCCoAOMT gene largely unclear. In this study, through bioinformatics techniques, a total of eight IiCCoAOMT genes were identified and classified into four subfamilies; notably, subgroup Ic contains no genes. The number and subgroup classification of these members are similar to those of other species. For example, there are CCoAOMT genes in Marchantia paleacea [49] and Populus [47], and they are all divided into four subfamilies. These results all indicate that the CCoAOMT gene family is conserved during the long evolutionary process. However, there is no information about CCoAOMT in Isatis indigotica [50,51]. Therefore, utilizing the I. indigotica genome document, we pinpointed the IiCCoAOMT gene family and examined the expression trends of an individual gene under alkali stress treatment.

4.2. Bioinformatics Analysis of the IiCCoAOMT Gene Family

In addition, CCoAOMT is vital in the production of lignin, flavonoids, and phenylpropanes. The conformation of a protein is determined by the gene’s structure, which, in turn, dictates its function, resulting in genes with analogous protein structures often performing analogous functions (Figure 2). According to the study and analysis, all IiCCoAOMT have one to eight structural motifs, which may be key to maintaining the functional preservation of the gene family. Except for IiCCoAOMT2, motif 5 exists in all other IiCCoAOMTs. Given the vital role of motif sequences at both ends in enzyme function and substrate attachment, the lack of motif five could result in the distinct functionality of IiCCoAOMT2. The protein structures of IiCCoAOMT1, IiCCoAOMT6, and IiCCoAOMT7 are the same, and the exon–intron gene distribution is also consistent. IiCCoAOMT3, IiCCoAOMT4, and IiCCoAOMT5 have specific institutional motifs, which may lead to the diversification of IiCCoAOMT gene functions. Gene duplication is considered to be the main factor leading to the expansion of gene families and the diversity of gene functions. The chromosomal examination of eight IiCCoAOMT gene locations revealed a dense distribution of three IiCCoAOMT genes on chromosome 7, indicative of proximal gene duplication, akin to Arabidopsis thaliana and poplar [52], and potentially linked to gene evolution. The remaining IiCCoAOMT genes are scattered on chromosomes 3 and 6 (Figure 5). Within poplar trees, the CCoAOMT genes are uniformly dispersed across chromosomes 1, 8, 9, and 10 [46].
Cis-regulatory components within the gene promoter area are crucial in controlling patterns of gene expression [53]. Research indicates that the Mybplant motif, located in the promoter region of genes responsible for phenylpropane biosynthesis, controls lignin production by attaching to the P-box element [54]. In our study, both IiCCoAOMT4 and IiCCoAOMT5 contain P-box elements. These genes are thought to participate in lignin synthesis. In addition, cis-acting elements related to ABA, GA, MeJA, and light responses were also found in the promoter of the IiCCoAOMT gene. Treatment with MeJA swiftly enhances the expression of lignin synthase genes, such as CCoAOMT, and fosters the synthesis of lignin [55,56]. Furthermore, ABRE and W-box have been verified as key cis-acting components in the promoter triggered by alkali stress treatment [42]. In most of the eight genes of IiCCoAOMTs, there are ABA response elements (ABREs). The W-box’s cis-acting element binding site under salt stress treatment suggests their possible involvement in I. indigotica’s reaction to alkali stress treatment. Consequently, we analyzed the CCoAOMT gene’s expression under salt stress treatment in I. indigotica and examined the potential regulatory mechanism.
Based on phylogenetic tree analysis, it has been shown that AtCCoAOMT6 is involved in the biosynthesis of phenylpropanoid polyamine polymers in Arabidopsis [57]. Research has confirmed AtCoAOMT7’s involvement in the creation of phenylpropane and flavonoids, showing a marked inclination towards the methylation of both flavonoids and dihydroflavonoids [58]. Therefore, we speculate that IiCCoAOMT1, IiCCoAOMT5, IiCCoAOMT6, and IiCCoAOMT7 are on the same branch as AtCCoAOMT7 and may be involved in the biosynthesis of flavonoids. According to qRT-PCR, IiCCoAOMT1 and IiCCoAOMT8 are consistent with the expression trends of luteolin and isoquercetin, respectively. The CCoAOMT gene is also associated with the synthesis of multiple methoxyflavones (PMFs), which in turn are related to antioxidant indicators. The CCoAOMT gene plays a crucial role in plants, particularly in dicotyledonous plants, where it is involved in the synthesis of multiple methoxyflavones (PMFs). Multimethoxyflavones are a class of compounds with various biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, which are of great significance to human health. It is speculated that the CCoAOMT gene regulates the production of multimethoxyflavonoids and has effects in antioxidant, anticancer, anti-obesity, and anti-inflammatory aspects. Consistent with the changes in IiCCoAOMT3, IiCCoAOMT6, and IiCCoAOMT7 in qRT-PCR results, the trend in MDA oxidation index contents is consistent with that of IiCCoAOMT2 contents. These genes may play a role in antioxidant activity.

4.3. Response of the IiCCoAOMT Family to Alkaline Treatment

When plants are subjected to alkaline stress treatment, the expression and activity of this gene undergo significant changes, becoming an essential part of plant defense mechanisms and maintaining internal balance. A high pH environment can damage the membrane structure and function of roots and interfere with ion absorption and balance, particularly leading to the fixation and loss of essential elements such as iron and phosphorus and thereby causing a series of physiological metabolic disorders. To cope with this stress treatment, plants activate complex signaling networks, among which the response of the CCoAOMT gene is particularly critical. It can rely on S-adenosylmethionine to catalyze the generation of various secondary metabolites, such as flavonoids, alkaloids, and phytohormones. Research has shown that O-methylation modification can enhance the affinity of flavonoids for lipids and proteins, expand the distribution range of flavonoids in cells, and be beneficial for improving the adaptability of plants to stress treatment during growth and development [59,60].
In our study, we detected the expression levels of the CCoAOMT gene in Isatis indigotica under alkaline stress treatment at the same concentration but different times: IiCCoAOMT3, IiCCoAOMT4, IiCCoAOMT6, and IiCCoAOMT7. The overall expression of these genes showed an upward trend, indicating that alkaline treatment increased the expression of these genes.
This study observed that the expression patterns of IiCCoAOMT3 and IiCCoAOMT8 were highly correlated with the accumulation trends of isoquercetin and luteolin, strongly suggesting their key roles in flavonoid B-ring methylation modification. This correspondence may stem from precise transcriptional regulation, where upstream transcription factors (WRKY) are activated under specific stress or developmental signals, thereby synergistically regulating the expression of IiCCoAOMT genes and the entire flavonoid pathway’s structural genes. At the same time, post-transcriptional regulation or post-translational modifications of enzyme activity may also fine-tune its final function. In addition, as a key node enzyme in the phenylpropane metabolism network, CCoAOMT simultaneously serves the synthesis of lignin monomers and some flavonoids. For example, many CCOAOMT genes in VvCCoAOMT4 and other plant species [61] are involved in the synthesis of flavonoids, particularly anthocyanins. CcWRKY activates the transcription of MYB by binding to its promoter W-box, increasing the content of capsaicin and demonstrating its critical role in capsaicin synthesis [62]. Other research has shown that inhibiting CcWRKY25 significantly decreases the transcription of PAL and CBG genes, which are crucial in the production of phenylpropane [63]. Therefore, upregulation of IiCCoAOMT3/8 expression may trigger competitive allocation of common substrates (such as caffeoyl CoA) between lignin and flavonoid synthesis pathways. The significant increase in flavonoid content in this study may indicate that under specific physiological conditions, the enzyme is preferentially allocated to the flavonoid branch pathway, or its enzymatic properties are more inclined to catalyze flavonoid methylation. To fundamentally verify the physiological function of the IiCCoAOMT gene and further explore the above mechanisms, the core research direction will be the use of CRISPR/Cas9 technology to create functional deletion mutants or construct overexpression transgenic plants in the future. In the mutant, we expect to observe a significant reduction in methylated flavonoids and the accumulation of intermediate products such as luteolin. In overexpression or heterologous expression systems, we expect to see an increase in the content of target flavonoids.

5. Conclusions

In this study, we accurately located eight IiCCoAOMT genes in I. indigotica. We extensively investigated the genetic structure, domains, and preserved motifs of CCoAOMT to reveal its evolutionary connections across different plants. In addition, our study showed changes in gene expression patterns during exposure to alkaline stress treatment at various time intervals. Through yeast one-hybrid experiments, the IiCCoAOMT gene is likely involved in regulating the biosynthesis of flavonoids. Due to resource and time constraints, we mainly use Y1H for large-scale screening and qualitative identification, which is a limitation of our research. We plan to explore the details of these interactions further through quantitative enzyme activity measurement and mutation analyses of key sites in subsequent research. Despite this limitation, our Y1H data can support our main conclusion. In summary, this study provides a new perspective on the prospective characteristics of the IiCCoAOMT gene. In addition, this lays the foundation for a more comprehensive analysis of the stress response process of the CCoAOMT gene in I. indigotica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology14111518/s1, Figure S1: motif sequence of the CCoAOMT gene family in I. indigotica. Figure S2: Multiple sequence alignment results of IiCCoAOMT proteins. Figure S3: UPLC chromatogram A: standard sample diagram; B. C, D, E, F: Sample images with different processing times. Figure S4: Arabidopsis WRKY gene and Isatis indigotica WRKY gene co construct phylogenetic tree. Table S1: CCoAOMT gene sequence of Isatis indigotica. Table S2: Amino acid sequences of CCoAOMT in 4 species. Table S3: QRT PCR Primers. Table S4: Fingerprint similarity matching result.

Author Contributions

Conceptualization, methodology, formal analysis, investigation, and writing—original draft, B.L. Supervision, investigation, and writing—review and editing, L.K. Investigation, validation, and software, J.M. Investigation and validation, S.J. Data curation, investigation, and validation, L.M. Methodology, validation, and supervision, J.X. Investigation, methodology, supervision, and visualization, W.R. Conceptualization, supervision, project administration, methodology, writing—review and editing, and funding acquisition, W.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the following projects: National Key Research and development Project, research and demonstration of collection, screening and breeding technology of ginseng and other genuine medicinal materials, Project No. 2021YFD1600901; Heilongjiang Province “double first-class” discipline collaborative innovation achievement project: Quality optimization and deep processing of characteristic Chinese medicinal materials in the lower cold region of great health industry, Project No. LJGXCG2023-058; and Heilongjiang Touyan Innovation Team Program (grant number: [2019] No. 5).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed in this study are included in the main text and its Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Manzoor, M.A.; Manzoor, M.M.; Li, G.; Abdullah, M.; Han, W.; Wenlong, H.; Shakoor, A.; Riaz, M.W.; Rehman, S.; Cai, Y. Genome-wide identification and characterization of bZIP transcription factors and their expression profile under abiotic stress treatmentes in Chinese pear (Pyrus bretschneideri). BMC Plant Biol. 2021, 21, 413. [Google Scholar] [CrossRef]
  2. Huazhen, Q.; Bo, S.; Shiyang, L. Comparing Research in Antiviral Action of Rhizoma et Radix Baphicacanthis cusiae and Radix isatidis against Influenza A Virus. Chin. Arch. Tradit. Chin. Med. 2009, 27, 168–169. [Google Scholar] [CrossRef]
  3. Chang, S.-J.; Chang, Y.-C.; Lu, K.-Z.; Tsou, Y.-Y.; Lin, C.-W. Antiviral Activity of Isatis indigotica Extract and Its Derived Indirubin against Japanese Encephalitis Virus. Evid.-Based Complement. Altern. Med. 2012, 2012, 925830. [Google Scholar] [CrossRef]
  4. Zhou, W.; Zhang, X.-Y. Research Progress of Chinese Herbal Medicine Radix isatidis (Banlangen). Am. J. Chin. Med. 2013, 41, 743–764. [Google Scholar] [CrossRef] [PubMed]
  5. Wang, Q.H.; Du, T.T.; Zhang, Z.H.; Ji, M.; Hu, H.Y.; Chen, X.G. Research progress in pharmacological action and mechanism of chlorogenic acid, Yaoxue Xuebao. J. Pharm. 2020, 55, 2273–2280. [Google Scholar] [CrossRef]
  6. Akula, R.; Ravishankar, G.A. Influence of abiotic stress treatment signals on secondary metabolites in plants. Plant Signal. Behav. 2014, 6, 1720–1731. [Google Scholar] [CrossRef]
  7. Wang, Z.; Zhang, Z.; Wang, P.; Qin, C.; He, L.; Kong, L.; Ren, W.; Liu, X.; Ma, W. Genome-wide identification of the NAC transcription factors family and regulation of metabolites under salt stress treatment in Isatis indigotica. Int. J. Biol. Macromol. 2023, 240, 124436. [Google Scholar] [CrossRef] [PubMed]
  8. Sun, S.N.; Wen, L.Y.; Liu, Z.W.; Li, X.X.; Gao, J.P.; Geng, R.M.; Jiang, C.H.; Sun, M.M.; Pu, W.X.; Yang, A.G. Identification and Functional Analysis of tobacco NtCCoAOMT Gene Family. Mol. Plant Breed. 2025, 1–11. Available online: https://link.cnki.net/urlid/46.1068.S.20230522.1128.006 (accessed on 18 July 2025).
  9. Golldack, D.; Li, C.; Mohan, H.; Probst, N. Tolerance to drought and salt stress treatment in plants: Unraveling the signaling networks. Front. Plant Sci. 2014, 5, 151. [Google Scholar] [CrossRef] [PubMed]
  10. Pitre, F.E.; Pollet, B.; Lafarguette, F.; Cooke, J.E.; MacKay, J.J.; Lapierre, C. Effects of increased nitrogen supply on the lignification of poplar wood. J. Agric. Food Chem. 2007, 55, 10306–10314. [Google Scholar] [CrossRef]
  11. Do, C.-T.; Pollet, B.; Thévenin, J.; Sibout, R.; Denoue, D.; Barrière, Y.; Lapierre, C.; Jouanin, L. Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta 2007, 226, 1117–1129. [Google Scholar] [CrossRef] [PubMed]
  12. Day, A.; Dehorter, B.; Neutelings, G.; Czeszak, X.; Chabbert, B.; Belingheri, L.; David, H. Caffeoyl-coenzyme A 3-O-methyltransferase enzyme activity, protein and transcript accumulation in flax (Linum usitatissimum) stem during development. Physiol. Plant. 2001, 113, 275–284. [Google Scholar] [CrossRef] [PubMed]
  13. Shi, Y.; Liu, Y.; Chen, J.J.; Zhang, H.P.; Zeng, J.W.; Huang, H.J.; Tian, J.; Peng, S.; Xu, J. The effect of Huanglong disease pathogen infection on flavonoids and volatile compounds in tea branches and citrus fruits. J. Huazhong Agric. Univ. 2020, 39, 24–33. [Google Scholar] [CrossRef]
  14. Zhang, C.L.; Chen, P.; Zhong, Y.M.; Zhou, C.Y.; Liang, G.H.; Shen, D.H.; Wu, Q.Y. Cloning and Expression of Flavonoid O-methyltransferase Gene in Ginkgo biloba, Yuanyi Xuebao. J. Hortic. 2012, 39, 355–362. [Google Scholar] [CrossRef]
  15. Wang, Y.; Wang, T.; Qi, S.; Zhao, J.; Kong, J.; Xue, Z.; Sun, W.; Zeng, W. Genome-wide identification, expression profiling, and protein interaction analysis of the CCoAOMT gene family in the tea plant (Camellia sinensis). BMC Genom. 2024, 25, 238. [Google Scholar] [CrossRef]
  16. Kim, B.-G.; Sung, S.H.; Chong, Y.; Lim, Y.; Ahn, J.-H. Plant Flavonoid O-Methyltransferases: Substrate Specificity and Application. J. Plant Biol. 2010, 53, 321–329. [Google Scholar] [CrossRef]
  17. Liu, X.M. Cloning and Functional Validation of Flavonoids O-Methyltransferase Genes in Ponkan. Master’s Thesis, Southwest University, Chongqing, China, 2021. [Google Scholar] [CrossRef]
  18. Zhang, X.Y. Study on the Response of Fengdan to Drought Stress Treatment and the Function of CCoAOMT Gene. Master’s Thesis, Yangzhou University, Yangzhou, China, 2020. [Google Scholar] [CrossRef]
  19. Chun, H.J.; Lim, L.H.; Cheong, M.S.; Baek, D.; Park, M.S.; Cho, H.M.; Lee, S.H.; Jin, B.J.; No, D.H.; Cha, Y.J.; et al. Arabidopsis CCoAOMT1 Plays a Role in Drought Stress treatment Response via ROS- and ABA-Dependent Manners. Plants 2021, 10, 831. [Google Scholar] [CrossRef]
  20. Kai, K.; Mizutani, M.; Kawamura, N.; Yamamoto, R.; Tamai, M.; Yamaguchi, H.; Sakata, K.; Shimizu, B. Scopoletin is biosynthesized viaortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J. 2008, 55, 989–999. [Google Scholar] [CrossRef]
  21. Fellenberg, C.; van Ohlen, M.; Handrick, V.; Vogt, T. The role of CCoAOMT1 and COMT1 in Arabidopsis anthers. Planta 2012, 236, 51–61. [Google Scholar] [CrossRef]
  22. Chun, H.J.; Baek, D.; Cho, H.M.; Lee, S.H.; Jin, B.J.; Yun, D.-J.; Hong, Y.-S.; Kim, M.C. Lignin biosynthesis genes play critical roles in the adaptation of Arabidopsis plants to high-salt stress treatment. Plant Signal. Behav. 2019, 14, 1625697. [Google Scholar] [CrossRef]
  23. Gong, S.L. Study on the Function of Caffeoyl Coenzyme A-O-Methyltransferase Gene in Sugarbeet M14 Strain. Master’s Thesis, Heilongjiang University, Harbin, China, 2013. [Google Scholar]
  24. Huang, Y.Z.; Qiao, Z.Q.; Liu, S.S.; Chen, K.X.; Huang, X.Z.; He, G. Cloning and Functional Analysis of LmCCoAOMT Gene from Loniceramacranthoides Hand-Mazz. Genom. Appl. Biol. 2021, 40, 325–333. [Google Scholar]
  25. Miao, L.X. Cloning of AFLP Molecular Markers and Related Genes for Tomato Resistance to Bacterial Wilt Disease. Ph.D. Thesis, Zhejiang University, Hangzhou, China, 2008. [Google Scholar]
  26. Yokoyama, R.; Nishitani, K. Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis. Plant Cell Physiol. 2004, 45, 1111–1121. [Google Scholar] [CrossRef] [PubMed]
  27. Shi, R.; Sun, Y.-H.; Li, Q.; Heber, S.; Sederoff, R.; Chiang, V.L. Towards a Systems Approach for Lignin Biosynthesis in Populus trichocarpa: Transcript Abundance and Specificity of the Monolignol Biosynthetic Genes. Plant Cell Physiol. 2010, 51, 144–163. [Google Scholar] [CrossRef]
  28. Joshi, C.P.; Chiang, V.L. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol. Biol. 1998, 37, 663–674. [Google Scholar] [CrossRef]
  29. Kang, M.; Wu, H.; Yang, Q.; Huang, L.; Hu, Q.; Ma, T.; Li, Z.; Liu, J. A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine. Hortic. Res. 2020, 7, 18. [Google Scholar] [CrossRef]
  30. Ma, L.; Kong, L.; Jiang, S.; Ma, J.; He, L.; Wu, J.; Zhang, X.; Wu, W.; Ma, W.; Ren, W. Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress. Int. J. Mol. Sci. 2025, 26, 8131. [Google Scholar] [CrossRef]
  31. Jiang, M.; Wang, Z.; Ren, W.; Yan, S.; Xing, N.; Zhang, Z.; Li, H.; Ma, W. Identification of the bZIP gene family and regulation of metabolites under salt stress treatment in Isatis indigotica. Front. Plant Sci. 2022, 13, 1011616. [Google Scholar] [CrossRef]
  32. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  33. Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.C.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 1999, 112, 531–552. [Google Scholar] [PubMed]
  34. Newbigin, E.; Chou, K.-C.; Shen, H.-B. Plant-mPLoc: A Top-Down Strategy to Augment the Power for Predicting Plant Protein Subcellular Localization. PLoS ONE 2010, 5, e11335. [Google Scholar] [CrossRef]
  35. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef] [PubMed]
  36. Larkin, M.A.; Blackshields, G.; Brown, N.P.; Chenna, R.; McGettigan, P.A.; McWilliam, H.; Valentin, F.; Wallace, I.M.; Wilm, A.; Lopez, R.; et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948. [Google Scholar] [CrossRef]
  37. Tamura, K.; Stecher, G.; Kumar, S.; Battistuzzi, F.U. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef] [PubMed]
  38. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. 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]
  39. Qu, R.; Miao, Y.; Cui, Y.; Cao, Y.; Zhou, Y.; Tang, X.; Yang, J.; Wang, F. Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune. BMC Mol. Biol. 2019, 20, 9. [Google Scholar] [CrossRef]
  40. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  41. Luo, W.Y.; Duan, H.X.; Liu, X.P.; Chen, X.; Zhang, Y.; Li, Z.Y. Simultaneous determination of six anti-tumor active ingredients in Da Qing Ye by HPLC multiple evaluation method. Chin. Pharm. 2018, 29, 2635–2639. [Google Scholar]
  42. Xiao, Y.; Feng, J.X.; Li, Q.; Zhou, Y.Y.; Bu, Q.T.; Zhou, J.H.; Tan, H.X.; Yang, Y.B.; Zhang, L.; Chen, W.S. IiWRKY34 positively regulates yield, lignan biosynthesis and stress tolerance in Isatis indigotica. Acta Pharm. Sin. B 2020, 10, 2417–2432. [Google Scholar] [CrossRef]
  43. Ma, Q.; Yan, Q.; Zhang, Z.S.; Wu, F.; Zhang, J.Y. Identification, Evolution, and Expression Analysis of CCoAOMT Gene Family in Alfalfa. J. Grassl. Ind. 2021, 30, 144–156. [Google Scholar]
  44. Shiu, S.-H.; Bleecker, A.B. Expansion of the Receptor-Like Kinase/Pelle Gene Family and Receptor-Like Proteins in Arabidopsis. Plant Physiol. 2003, 132, 530–543. [Google Scholar] [CrossRef]
  45. Wang, L.; Zhu, W.; Fang, L.; Sun, X.; Su, L.; Liang, Z.; Wang, N.; Londo, J.P.; Li, S.; Xin, H. Genome-wide identification of WRKY family genes and their response to cold stress treatment in Vitis vinifera. BMC Plant Biol. 2014, 14, 103. [Google Scholar] [CrossRef]
  46. Akhter, S.; Sami, A.A.; Toma, T.I.; Jahan, B.; Islam, T. Caffeoyl-CoA 3-O-methyltransferase gene family in jute: Genome-wide identification, evolutionary progression and transcript profiling under different quandaries. Front. Plant Sci. 2022, 13, 1035383. [Google Scholar] [CrossRef] [PubMed]
  47. Zhao, H.; Qu, C.; Zuo, Z.; Cao, L.; Zhang, S.; Xu, X.; Xu, Z.; Liu, G. Genome Identification and Expression Profiles in Response to Nitrogen Treatment Analysis of the Class I CCoAOMT Gene Family in Populus. Biochem. Genet. 2021, 60, 656–675. [Google Scholar] [CrossRef]
  48. Lynch, M.; Conery, J.S. The Evolutionary Fate and Consequences of Duplicate Genes. Science 2000, 290, 1151–1155. [Google Scholar] [CrossRef]
  49. Xu, R.-X.; Ni, R.; Gao, S.; Fu, J.; Xiong, R.-L.; Zhu, T.-T.; Lou, H.-X.; Cheng, A.-X. Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea. Plant Sci. 2022, 314, 111102. [Google Scholar] [CrossRef]
  50. Zhang, L.; Li, Q.; Chen, J.-F.; Chen, W.-S. Computational identification and systematic classification of novel GRAS genes in Isatis indigotica. Chin. J. Nat. Med. 2016, 14, 161–176. [Google Scholar] [CrossRef] [PubMed]
  51. Xiao, L.; Ren, J.-Z.; Li, Q.; Yang, B.; Liu, Z.-J.; Chen, R.-B.; Zhang, L. Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica. J. Integr. Med. 2023, 21, 77–88. [Google Scholar] [CrossRef] [PubMed]
  52. Barakat, A.; Choi, A.; Yassin, N.B.M.; Park, J.S.; Sun, Z.; Carlson, J.E. Comparative genomics and evolutionary analyses of the O-methyltransferase gene family in Populus. Gene 2011, 479, 37–46. [Google Scholar] [CrossRef]
  53. 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] [PubMed]
  54. Tamagnone, L.; Merida, A.; Parr, A.; Mackay, S.; Culianez-Macia, F.A.; Roberts, K.; Martin, C. The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell. 1998, 10, 135–154. [Google Scholar] [CrossRef]
  55. Lin, S.-J.; Yang, Y.-Z.; Teng, R.-M.; Liu, H.; Li, H.; Zhuang, J. Identification and expression analysis of caffeoyl-coenzyme A O-methyltransferase family genes related to lignin biosynthesis in tea plant (Camellia sinensis). Protoplasma 2020, 258, 115–127. [Google Scholar] [CrossRef]
  56. Sehr, E.M.; Agusti, J.; Lehner, R.; Farmer, E.E.; Schwarz, M.; Greb, T. Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation. Plant J. 2010, 63, 811–822. [Google Scholar] [CrossRef]
  57. Yang, K.; Li, L.; Lou, Y.; Zhu, C.; Li, X.; Gao, Z. A regulatory network driving shoot lignification in rapidly growing bamboo. Plant Physiol. 2021, 187, 900–916. [Google Scholar] [CrossRef]
  58. Liu, X.; Zhao, C.; Gong, Q.; Wang, Y.; Cao, J.; Li, X.; Grierson, D.; Sun, C.; Costa, F. Characterization of a caffeoyl-CoA O-methyltransferase-like enzyme involved in biosynthesis of polymethoxylated flavones in Citrus reticulata. J. Exp. Bot. 2020, 71, 3066–3079. [Google Scholar] [CrossRef] [PubMed]
  59. Koirala, N.; Thuan, N.H.; Ghimire, G.P.; Thang, D.V.; Sohng, J.K. Methylation of flavonoids: Chemical structures, bioactivities, progress and perspectives for biotechnological production. Enzyme Microb. Technol. 2016, 86, 103–116. [Google Scholar] [CrossRef] [PubMed]
  60. Cheng, Y.; Zhou, Y.; Yang, Y.; Chi, Y.J.; Zhou, J.; Chen, J.Y.; Wang, F.; Fan, B.; Shi, K.; Zhou, Y.H.; et al. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors. Plant Physiol. 2012, 159, 810–825. [Google Scholar] [CrossRef] [PubMed]
  61. Widiez, T.; Hartman, T.G.; Dudai, N.; Yan, Q.; Lawton, M.; Havkin-Frenkel, D.; Belanger, F.C. Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases. Plant Mol. Biol. 2011, 76, 475–488. [Google Scholar] [CrossRef]
  62. Zhu, Z.; Sun, B.; Cai, W.; Zhou, X.; Mao, Y.; Chen, C.; Wei, J.; Cao, B.; Chen, C.; Chen, G.; et al. Natural variations in the MYB transcription factor MYB31 determine the evolution of extremely pungent peppers. New Phytol. 2019, 223, 922–938. [Google Scholar] [CrossRef]
  63. Zhang, L.; Wu, D.; Zhang, W.; Shu, H.; Sun, P.; Huang, C.; Deng, Q.; Wang, Z.; Cheng, S. Genome-Wide Identification of WRKY Gene Family and Functional Characterization of CcWRKY25 in Capsicum chinense. Int. J. Mol. Sci. 2023, 24, 11389. [Google Scholar] [CrossRef]
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Figure 1. Tertiary structure analysis of IiCCoAOMT proteins.
Figure 1. Tertiary structure analysis of IiCCoAOMT proteins.
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Figure 2. Structure of IiCCoAOMT gene and conserved motif analysis of its encoded protein. (A) IiCCoAOMT conservative domain analysis; (B) exon–intron structure analysis.
Figure 2. Structure of IiCCoAOMT gene and conserved motif analysis of its encoded protein. (A) IiCCoAOMT conservative domain analysis; (B) exon–intron structure analysis.
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Figure 3. CCoAOMTs phylogenetic tree of I. indigotica, Arabidopsis. thaliana, Dendrocalamus farinosus, Oryza sativa, and Glycine max. In total, 45 CCoAOMT proteins were constructed using the neighbor-joining (NJ) method. The CCoAOMT protein is divided into Ia, Ib, Ic, and II. The proteins of I. indigotica are labeled with red, and various colors and shapes distinguish different varieties of CCoAOMTs.
Figure 3. CCoAOMTs phylogenetic tree of I. indigotica, Arabidopsis. thaliana, Dendrocalamus farinosus, Oryza sativa, and Glycine max. In total, 45 CCoAOMT proteins were constructed using the neighbor-joining (NJ) method. The CCoAOMT protein is divided into Ia, Ib, Ic, and II. The proteins of I. indigotica are labeled with red, and various colors and shapes distinguish different varieties of CCoAOMTs.
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Figure 4. Analysis of cis-acting elements of the IiCCoAOMT gene family. Different colors in the IiCCoAOMTs promoter region indicate cis elements with various functions.
Figure 4. Analysis of cis-acting elements of the IiCCoAOMT gene family. Different colors in the IiCCoAOMTs promoter region indicate cis elements with various functions.
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Figure 5. Chromosome localization and collinearity analysis of the IiCCoAOMT gene. (A) Intraspecific collinearity analysis of the IiCCoAOMTs gene. The gray background shows all the same linear segments in the genome of I. indigotica. The red line shows the identical linear IiCCoAOMT gene pairs. (B) The number of chromosomes of the IiCCoAOMTs gene is shown on the left side of each chromosome. The respective genes are marked on the right side of the chromosome. (C) Interspecific collinearity of IiCCoAOMT. Duplicate IiCCoAOMT gene pairs are linked with red lines.
Figure 5. Chromosome localization and collinearity analysis of the IiCCoAOMT gene. (A) Intraspecific collinearity analysis of the IiCCoAOMTs gene. The gray background shows all the same linear segments in the genome of I. indigotica. The red line shows the identical linear IiCCoAOMT gene pairs. (B) The number of chromosomes of the IiCCoAOMTs gene is shown on the left side of each chromosome. The respective genes are marked on the right side of the chromosome. (C) Interspecific collinearity of IiCCoAOMT. Duplicate IiCCoAOMT gene pairs are linked with red lines.
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Figure 6. Phenotype of I. indigotica plants at different time periods under alkaline treatment. The phenotype of Isatis indigotica. Use a 200 mmol NaHCO3 alkaline solution with a pH value of 8.2 for treatment. With an increase in processing time, the plant height of I. indica decreased, the leaves wilted, and the growth rate slowed down.
Figure 6. Phenotype of I. indigotica plants at different time periods under alkaline treatment. The phenotype of Isatis indigotica. Use a 200 mmol NaHCO3 alkaline solution with a pH value of 8.2 for treatment. With an increase in processing time, the plant height of I. indica decreased, the leaves wilted, and the growth rate slowed down.
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Figure 7. Quantitative RT-PCR analyses of IiCCoAOMT genes. Testing through significance analysis method. Quantitative RT-PCR results were normalized for IiCCoAOMT homeostasis. The data is the average of three replicates (mean ± SD, n = 3). The significance of chemical content in leaves of I. indigotica under alkaline stress treatment was studied using one-way analysis of variance and Tukey’s multiple-comparison test. (* p < 0.05; *** p < 0.001; **** p < 0.0001).
Figure 7. Quantitative RT-PCR analyses of IiCCoAOMT genes. Testing through significance analysis method. Quantitative RT-PCR results were normalized for IiCCoAOMT homeostasis. The data is the average of three replicates (mean ± SD, n = 3). The significance of chemical content in leaves of I. indigotica under alkaline stress treatment was studied using one-way analysis of variance and Tukey’s multiple-comparison test. (* p < 0.05; *** p < 0.001; **** p < 0.0001).
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Figure 8. Analysis of changes in flavonoid content. (A) Luteolin content. (B) Isocercetin content. The data is the average of three replicates (mean ± SD, n = 3). The significance of chemical content in leaves of I. indigotica under alkaline stress treatment was studied using one-way analysis of variance and Tukey’s multiple-comparison test. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Figure 8. Analysis of changes in flavonoid content. (A) Luteolin content. (B) Isocercetin content. The data is the average of three replicates (mean ± SD, n = 3). The significance of chemical content in leaves of I. indigotica under alkaline stress treatment was studied using one-way analysis of variance and Tukey’s multiple-comparison test. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
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Figure 9. UPLC fingerprint spectrum of the Isatis indigotica sample. Use the ultrasonic extraction method to extract the sample, and transfer the extraction solution to a liquid phase bottle for measurement. (A) The peak spectrum of I. indigotica as a reference, and (B) analysis of the fingerprint spectra and reference drugs of I. indigotica at different stages.
Figure 9. UPLC fingerprint spectrum of the Isatis indigotica sample. Use the ultrasonic extraction method to extract the sample, and transfer the extraction solution to a liquid phase bottle for measurement. (A) The peak spectrum of I. indigotica as a reference, and (B) analysis of the fingerprint spectra and reference drugs of I. indigotica at different stages.
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Figure 10. (A) POD activity; (B) MDA content. Data are means (mean ± SD, n = 3) of three replicates. Significance of SOD enzyme activity was analyzed using one-way ANOVA followed by Tukey’s multiple-comparisons test. (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 10. (A) POD activity; (B) MDA content. Data are means (mean ± SD, n = 3) of three replicates. Significance of SOD enzyme activity was analyzed using one-way ANOVA followed by Tukey’s multiple-comparisons test. (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Figure 11. Yeast one-hybrid screening of target genes that bind to IiWRKY48 and IiWRKY54. When pLacZi IiCCoAOMT3 co-transformed with pJG4-5-IiWRKY48 and pJG4-5-IiWRKY54, the colony appeared blue, indicating that transcription factors IiWRKY48 and IiWRKY54 interact with the IiCCoAOMT3 gene. (A) shows the interaction between bait protein and prey protein in yeast one hybrid experiments. (B) shows a schematic diagram of the IiCCoAOMT and IiWRKY combination interaction.
Figure 11. Yeast one-hybrid screening of target genes that bind to IiWRKY48 and IiWRKY54. When pLacZi IiCCoAOMT3 co-transformed with pJG4-5-IiWRKY48 and pJG4-5-IiWRKY54, the colony appeared blue, indicating that transcription factors IiWRKY48 and IiWRKY54 interact with the IiCCoAOMT3 gene. (A) shows the interaction between bait protein and prey protein in yeast one hybrid experiments. (B) shows a schematic diagram of the IiCCoAOMT and IiWRKY combination interaction.
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Table 1. The information of IiCCoAOMT members.
Table 1. The information of IiCCoAOMT members.
Gene NameAccession NumberChromosomeNumber of Amino AcidsProtein Length (aa)Molecular Weight (KDa)Isoelectric PointSubcellular
Localization Predicted
IiCCoAOMT1Iin23419.t1Chr377425728.945.21Specific location not predicted
IiCCoAOMT2Iin27867.t1Chr383427730.778.84Chloroplast. Cytoplasm
IiCCoAOMT3Iin25371.t1Chr669923226.194.96Chloroplast
IiCCoAOMT4Iin25370.t1Chr669923226.315.17Chloroplast
IiCCoAOMT5Iin11784.t1Chr744414716.679.37Nucleus
IiCCoAOMT6Iin10582.t1Chr770523426.454.97Chloroplast
IiCCoAOMT7Iin22645.t1Chr771723826.70 5.27Peroxisome
IiCCoAOMT8Iin25369.t1Chr780126630.245.48Cytoplasm
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Liu, B.; Kong, L.; Ma, J.; Jiang, S.; Ma, L.; Xu, J.; Ren, W.; Ma, W. Systematic Analysis of the CCoAOMT Gene Family in Isatis indigotica and the Molecular Mechanism of CCoAOMT8-Mediated Flavonoid Synthesis Under Alkaline Stress Treatment. Biology 2025, 14, 1518. https://doi.org/10.3390/biology14111518

AMA Style

Liu B, Kong L, Ma J, Jiang S, Ma L, Xu J, Ren W, Ma W. Systematic Analysis of the CCoAOMT Gene Family in Isatis indigotica and the Molecular Mechanism of CCoAOMT8-Mediated Flavonoid Synthesis Under Alkaline Stress Treatment. Biology. 2025; 14(11):1518. https://doi.org/10.3390/biology14111518

Chicago/Turabian Style

Liu, Bo, Lingyang Kong, Junbai Ma, Shan Jiang, Lengleng Ma, Jiao Xu, Weichao Ren, and Wei Ma. 2025. "Systematic Analysis of the CCoAOMT Gene Family in Isatis indigotica and the Molecular Mechanism of CCoAOMT8-Mediated Flavonoid Synthesis Under Alkaline Stress Treatment" Biology 14, no. 11: 1518. https://doi.org/10.3390/biology14111518

APA Style

Liu, B., Kong, L., Ma, J., Jiang, S., Ma, L., Xu, J., Ren, W., & Ma, W. (2025). Systematic Analysis of the CCoAOMT Gene Family in Isatis indigotica and the Molecular Mechanism of CCoAOMT8-Mediated Flavonoid Synthesis Under Alkaline Stress Treatment. Biology, 14(11), 1518. https://doi.org/10.3390/biology14111518

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