Cloning and Functional Analysis of PmMYB45, a Transcription Factor in Pinus massoniana

Abstract

MYB transcription factors (TFs) have been shown to be important regulators of plant growth and development, and the R2R3−MYB family plays an important role in the regulation of plant primary and secondary metabolism, growth, and development. In this study, the PmMYB45 gene sequence was successfully cloned from Pinus massoniana, with an open reading frame of 1476 bp, encoding 491 amino acids. The results of subcellular localization and transcriptional self-activation showed that the gene was localized in the nucleus and was self-activating. qPCR showed that the highest expression of PmMYB45 was found in stems. PmMYB45 can promote lignin synthesis when overexpressed in tobacco. These results suggest that PmMYB45 promotes lignin synthesis by regulating the lignin biosynthesis pathway. These findings improve our understanding of the mechanism of lignin biosynthesis in P. massoniana and provide a basis for molecular breeding functions. It also provides insights into the role of the MYB transcription factor family in lignin accumulation.

1. Introduction

Secondary metabolites (SMs) produced by plants are significant in various aspects, such as plant growth, metabolic control, signaling pathways, and protection against both biotic and abiotic stressors [1,2,3]. Lignin, as a widespread class of secondary metabolites in plants, is mainly involved in plant material transportation, defense against pests and pathogens, plant resistance to stunting, and response to various environmental stresses [4,5,6,7]. MYB transcription factors are one of the largest families of plant transcription factors, and R2R3-MYB transcription factors (TFs) are the most numerous among them, and they have a significant impact on the growth, development, and regulation of metabolism in plants [8]. MYB transcription factors play a significant role in the modulation of lignin secondary metabolism on a broad scale, the most representative being AtMYB46 as well as AtMYB83, which are the target genes of SND1, VND6, and VND7 [9]. AtMYB20, AtMYB43, and AtMYB42 activate lignin synthesis-related genes, and the silencing of these genes leads to a significant reduction in lignin synthesis and defective plant growth in Arabidopsis [10]. The loss of AtMYB75 function resulted in increased thickness of wood fibers in Arabidopsis inflorescence stems, some genes related to lignin biosynthesis were upregulated, and the S/G ratio of lignin monomer and total lignin content were also affected [11]. Involvement of MYB transcription factors in lignin biosynthesis has also been demonstrated in other species, such as the excessive expression of CmMYB8 results in the suppression of multiple genes responsible for lignin synthesis and subsequently reduces the lignin content in chrysanthemum plants [12]. VcMYB4a resulted in a reduction in lignin biosynthesis via the downregulation of blueberry calli 4CL, COMT, and CAD gene expression [13].

Pinus. massoniana, which has been considered the main wood resource for paper and artificial fiberboard, occupies a key position in the development of the forest tree [14]. To date, several genes related to secondary wall synthesis have been cloned and characterized from P. massoniana. PmMYB7 is highly expressed in the xylem and promotes lignin synthesis via interaction with PmCCoAOMT2 [15]. PmMYB4 binds to the AC-box motif to directly activate PAL and CCoAOMT promoters involved in the secondary cell wall (SCW) biosynthesis genes, and overexpression increases tobacco lignin deposition and increases SCW thickness [14]. Transgenic PmMYB6 upregulates several genes in the lignin biosynthesis pathway, resulting in increased lignin content in transgenic plants [16]. While specific transcription factors have been identified as playing a role in the control of secondary cell wall (SCW) biosynthesis, a complete understanding of this process remains elusive. Investigating additional transcription factors involved in the regulation of SCW biosynthesis in P. massoniana is of significant scientific importance for improving wood productivity and quality. It also plays a crucial role in developing new cultivars customized to meet human needs.

In this study, we identified the MYB TF PmMYB45 and mainly used bioinformatics-related methods to comprehensively analyze the coding protein properties, study the coding sequence features, and analyze the expression pattern of P. massoniana tissues, as well as the localization of the protein expression and the detection of transcriptional self-activation activity. We further overexpressed PmMYB45 in tobacco and analyzed its biological functions by means of cell biology and molecular biology.

2. Materials and Methods

2.1. Plant Material

In this study, we used P. massoniana seedlings, which were grown from seeds in the Seed Orchard of Baisha State Forest Farm in Shanghang County, Fujian Province, and cultivated in pots in our laboratory; the seeds of Arabidopsis and tobacco (Nicotiana x sanderae was used for the transgene, Nicotiana benthamiana was used for subcellular localization) used in this experiment were stored at 4 °C in our laboratory for a long time and were grown in a greenhouse at 24 °C under a 16/8 h light/dark cycle and supplemental light (4500lx). The genetic transformation system of P. massoniana was not successfully established. We selected Arabidopsis and tobacco for the functional validation of Masson pine genes, which serve as plant biological indicators.

2.2. Cloning of Genes and Promoters

RNA was isolated from the tissues of a 2-year-old P. massoniana seedling using the FastPure Universal Plant TotalRNA Isolation Kit (Vazmy Biotechnology, Nanjing, China). cDNA was extracted by reverse transcription of the first strand cDNA from the total RNA using a One-step gDNA Removal and cDNA Synthesis SuperMix kit (Yeasen Biotechnology, Shanghai, China). Specific primers were designed using Primer Premier 5.0 software (Table S1); the ORF sequence of PmMYB45 was cloned from P. massoniana by PCR using a 50 μL PCR reaction system. The polymerase chain reaction (PCR) was conducted with the following specified conditions: pre−denaturation at 98 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 1 min, annealing at 58 °C for 30 s, and extension at 72 °C for 40 s, holding at 72 °C for 5 min. The ORF fragment was attached to the Blunt Vector using the pClone007 Blunt Simple Vector Kit (Tsingke Biotechnology, Nanjing, China). Using the Pinus taeda genome database, we established a local database of PmMYB45 and compared it with it, the nucleic acid sequence of about 2000 bp upstream of the gene fragment with the highest similarity was selected as the reference sequence, specific primers were designed, the gDNA of P. massoniana leaves was used as the PCR template, the PCR reaction procedure was consistent with ORF cloning after it was sent to Tsingke Biotech (Nanjing, China) for sequencing, and finally, it was pieced together using the online software SeqMan1.0.

2.3. Bioinformatics Analysis of the Coding Region of the PmMYB45 Gene

The online software was utilized to predict the hydrophobicity, signal peptide, transmembrane region, secondary structure, and tertiary structure of the PmMYB45 protein such as Expasy−ProtScale3.0 (https://web.expasy.org/protscale, accessed on 28 February 2024), SignalP4.1 server (https://services.healthtech.dtu.dk/service.php?SignalP, accessed on 28 February 2024), TMHMM2.0 Server (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0, accessed on 28 February 2024), SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=%20npsa_sopma.html, accessed on 28 February 2024), SWISS−MODEL (https://swissmodel.expasy.org/, accessed on 28 February 2024). The amino acid sequence of PmMYB45 and other homologous genes were sequenced using the online software Clustalw2.1 (https://cdnai.gameax.cn/#/home, accessed on 1 March 2024). The MYB protein sequences of other species were downloaded from the NCBI database, and the MEGA5.1 software was used to perform multiple sequence comparisons of protein sequences and construct phylogenetic evolutionary trees. The cis-acting elements of the promoter were analyzed and predicted using the online analysis website PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 February 2024).

2.4. RT-qPCR

RNA was extracted from various tissues and subsequently converted into complementary cDNA. Different tissues, including Roots (R), Young stems (YS), Old stems (OS), Xylem (X), young leaves (YN), old leaves (ON), and Phloem (P). Horsetail pine PmTUA (KM496535.1) was used as an internal reference gene in P. massoniana [17]. The specific primers for each gene used in the RT−qPCR test can be found in Table S1. The setup was referred to the Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix instructions, and RT−qPCR was performed using the TP700 real-time PCR machine (Takara, Dalian, China); at the end of the reaction, relative expression was calculated using the 2^−ΔΔCt method [18].

2.5. Subcellular Localization of PmMYB45 Protein

The ORF fragment of PmMYB45 was inserted into the PJIT166−eGFP vector containing the 35S promoter. The PJIT166−eGFP vector carrying GFP alone was used as a control. 35S∷PmMYB45−eGFP and control vector were transformed into Agrobacterium tumefaciens (GV3103), and tobacco leaves (grown for 3–4 weeks) were infested with Agrobacterium-mediated transient transformation. After dark incubation for 12 h, they were grown in an incubator with a photoperiod of 16/8 for 24−36 h. The expression of fusion vectors was detected at the end of the photoperiod by applying the nucleus-specific dye DAPI staining, followed by examination using laser confocal microscopy (UV excitation wavelength of 488 nm).

2.6. Transcriptional Activation Activity Assay

pGBKT7−PmMYB45 fusion vector was constructed by homologous recombination. The pGBKT7−PmMYB45 plasmid and pGADT7 plasmid were co-transformed into AH109 yeast-competent cells as an experimental group. pGBKT7−p53 and pGADT7−largeT co-transfer AH109 were positive control, and pGBKT7−laminC with pGADT7−largeT were negative control and coated to the corresponding defective type screening plates. After PCR examination, positive bacteria were inoculated in 5 mL of SD/-Trp liquid medium, shaken overnight, and spot-coated onto SD/-Trp/-Leu, SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade/X-α-Gal plates.

2.7. GUS Staining

This paper describes a slightly improved method of GUS staining with reference to that reported by Chu [19]. First, A. thaliana was immersed in a 1/2MS hypertonic solution containing a 25% sucrose mass fraction for 15 min at 25 °C. Subsequently, A. thaliana was infected with Agrobacterium-mediated transient transformation and incubated at 120 r/min for 2.5 h at 25 °C. This was followed by placing it in co-culture medium (1/2MS + 120 μmol/L AS + 1.5 mg/L KT + 0.5 mg/L NAA + 3% sucrose, pH = 5.4) for 72 h in the dark at (23 ± 2) °C. Finally, A. thaliana was immersed in GUS staining solution (Leagene Biotechnology, Beijing, China), protected from light overnight at 37 °C, and then destained with a solution of V (ethanol):V (acetic acid) = 3:1. After completion of decolorization, a microscope was used to take pictures.

2.8. Genetically Modified Tobacco

According to the ORF sequence of PmMYB45 and the PBI121 vector sequence, a pair of specific primers was designed using Primer Premier 5 software for PCR amplification, and then the PCR products were connected to the plant binary vector PBI121. We transformed the resulting vector 35S:PmMYB45 into A. tumefaciens using the freezing-thawing transformation method. As described previously, tobacco transformation was mediated by A. tumefaciens [20]. First, leaf discs were infected with A. tumefaciens cultures containing 35S::PmMYB45 for 8−10 min, placed in medium (MS + 0.2 mg/L NAA + 2 mg/L 6-BA), and incubated in dark conditions for 3 d. Second, leaves were transferred to medium (MS + 0.2 mg/L NAA + 2 mg/L 6−BA + 250 mg/L Cef and 50 mg/L Kan) for screening. Until after small green shoots had grown. They were grown for another 15 days, and then the green shoots were cut and transferred to containing 100 mg/L Cef and 25 mg/L Kan for rooting screening of the resistant plants. Positive transgenic tobacco was identified for further study using PCR and qRT−PCR (S1).

2.9. Chemical Staining of Xylem

Phloroglucinol is a commonly used chemical reagent that can be utilized to detect lignin. When phloroglucinol reacts with lignin, it will appear red or purplish-red, enabling the determination of lignin content based on the depth of the stain. Sections were prepared from stem segments of the stem base and sixth leaf petioles of transgenic and wild-type tobacco. The samples were stained with phloroglucinol and examined under a microscope.

2.10. Determination of Lignin Monomer Content

Tobacco stem segments were taken and put into a mortar grinding. About 0.1 g of the sample was weighed, 2 mL of pre-cooled ultra-pure water was added, and the sample was soaked in a constant temperature water bath at 90 °C for 30 min. Then, the filtrate was taken, the pH was adjusted to 2.0, and it was extract with ethyl acetate three times. A nitrogen-blowing instrument was taken and used for blow drying; the sample was dissolved with a mobile phase and was filtered with a pinhead filter to be measured.

2.11. Scanning Electron Microscope

Stems of 2-month-old wild-type and transgenic tobacco were promptly immersed in FAA tissue fixative for 24 h. Subsequently, the stems were dried and sectioned. Transverse sections of stems were photographed for observation using a scanning electron microscope (FEI Quanta 200, Hillsboro, WA, USA).

2.12. Statistical Analysis

The experimental data were collected from three separate biological replicates, and all graphs were created using Microsoft Excel. Statistical analysis was then conducted using GraphPad Prism 8 software. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

3. Results

3.1. Cloning and Structural Analysis of PmMYB45

According to the 57 MYB proteins of P. massoniana identified in the previous phase by our research group, PmMYB45, located in the S37 subgroup, may participate in lignin biosynthesis [21]. Using PCR technology, the PmMYB45 gene was cloned from P. massoniana. The gene’s open reading frame (ORF) was 1317ps long and encoded 438 amino acids (Supplementary Table S1 and Figure S1). The SMART online program predicted that the protein contains two common SANT structural domains and belongs to the MYB family (Figure 1A). Additionally, the NCBI website’s prediction of conserved structural domains revealed that PmMYB45 included the MYB DNA structural domain, along with the PLAN03091, REB1, and SANT structural domains (Figure 1B).

The 1321 bp promoter sequence upstream of PmMYB45 was cloned and analyzed using PlantCARE. In addition to core promoter elements and light-responsive elements such as the TATA box and CAAT box, the PmMYB45 promoter also contains elements for phytohormone-related responses, such as abscisic cis-acting element and salicylic cis-acting element (Figure 2). According to the findings presented, it is postulated that the expression of the PmMYB45 promoter could potentially be influenced by both photoperiodic and phytohormone signaling pathways.

3.2. Bioinformatics Analysis of the PmMYB45

The protein’s primary structure was predicted using the online software Expasy-ProtScale, as shown, and it can be observed that the score of PmMYB45 is mainly in the negative region, indicating that it is a protein with hydrophilicity (Figure 3A). The secondary structure of the protein was analyzed using SOPMA online prediction. The results showed that the secondary structure of the PmMYB45 protein consisted of random coil, extended strand, and Beta turn. Among them, the random coil accounted for 60.05%; the Alpha helix accounted for 24.66% of the sequence; the extended strand accounted for 9.59% of the sequence; and the Beta turn was 5.71% (Figure 3B). The tertiary structure of the protein was predicted online using the homology modeling method via the SWISS-MODEL website. The main structures constituting it were identified as random coil and random coil, which is consistent with the secondary structure prediction (Figure 3C). The signal peptide and transmembrane structures of PmMYB45 were analyzed using SignalP1.6 and TMHMM Sever v.2.0 online programs. PmMYB45 did not contain signaling peptides and transmembrane structures, indicating that it is a nonsecretory protein (Figure 3D,E).

In this paper, a phylogenetic tree was constructed using MEGA6.0 software. The PmMYB45 protein sequences were used to construct the phylogenetic tree along with MYB protein sequences from other species. The analysis revealed that PmMYB45 shares a high degree of homology with other plant MYBs known to be involved in secondary wall formation (Figure 4A, Supplementary Table S2). The protein sequence closely resembling PmMYB45 was identified by querying the National Center for Biotechnology Information (NCBI) database using the BLASTN. The results showed that all five proteins have R2 and R3 structural domains, indicating that they were all R2R3−MYB TFs (Figure 4B).

3.3. Analysis of the Tissue-Specific Expression Pattern of PmMYB45

The qRT−PCR technique was used to evaluate the expression levels of PmMYB45 in different tissues of P. massoniana, with the PmTUA gene used as a reference. High levels of PmMYB45 expression were detected in old and young stems, and the lowest levels were detected in roots. The expression of PmMYB45 in P. massoniana was tissue-specific (Figure 5A). To further investigate the spatial and temporal expression patterns, the 35S∷PmMYB45−GUS vector containing the 1.3 kb promoter region upstream of PmMYB45 was introduced into Arabidopsis. Histochemical analysis of GUS expression after the transformation of the plants showed that PmMYB45 promoter-driven staining was expressed predominantly in stems as well as the veins of mature leaves. This restricted expression pattern suggests that the PmMYB45 gene demonstrates expression in tissues that are undergoing the process of secondary cell wall thickening (Figure 5B).

3.4. Subcellular Localization and Transcriptional Activation Analysis

To verify whether the protein encoded by the PmMYB45 gene can localize in the nucleus of plant cells and thus bind to the promoters of downstream genes to achieve its transcriptional regulatory role, a 35S::PmMYB45−eGFP fusion gene was generated and transiently transformed in tobacco by A. rhizogenes. The findings indicated that the expression of PmMYB45 was localized specifically within the nucleus, while the green fluorescent protein (GFP) activity produced by the control 35S::eGFP transgene was observed to be dispersed throughout the entire cell. (Figure 6A). To determine the transcriptional activity of PmMYB45, its ORF was fused to PGBKT7 and transferred into the yeast strain AH109. Yeast transformants carrying the PGBKT7::PmMYB45 fusion gene were able to grow on selective media and induce the expression of the α-galactosidase reporter gene (Figure 6B). Suggesting that PmMYB45 has transcriptional activity, consistent with PmMYB45 functioning as a transcription factor.

3.5. Overexpression of PmMYB45 in Tobacco Promotes Lignin Accumulation

To preliminarily investigate the function of PmMYB45, it was first overexpressed in tobacco. Several transgenic plants were identified and screened by DNA and RNA levels (Supplemental Figure S1). The expression level of PmMYB45 was detected by qRT−PCR. Two lines (five replicates per line) with good growth status were randomly selected for further analysis. The results showed that it was successfully expressed in the transgenic lines, with expression levels 179 and 27-fold higher than that of the wild type (Supplemental Table S1, Figure 7B). There were some morphological differences between the transgenic plants and the wild-type control. Phenotypic observations revealed that the transgenic plants exhibited a slightly declined growth, and all of them were smaller in plant height compared to the wild type but had increased stem thickness (Figure 7C,D). These findings suggest that the overexpression of PmMYB45 may play a role in the secondary growth of tobacco.

To evaluate the influence of transgenic plants on lignin biosynthesis, the stems and petioles of PmMYB45 transgenic plants were sectioned and subjected to phloroglucinol staining to analyze the distribution of lignin. The results showed that the number of lignin cell layers in the petiole of overexpression plants was significantly increased (Figure 8A), and the thickness of lignin in the stems of PmMYB45 transgenic plants was increased in transgenic plants compared with that in the wild type (Figure 8B), which indicated that PmMYB45 could promote the synthesis of lignin in the transgenic plants. The xylem cell wall thickness of the transverse section was observed by scanning electron microscopy (SEM). It was found that the cell wall thickness of transgenic tobacco increased significantly compared with that of WT (Figure 8C,D).

3.6. The Lignin Monomers in Stems of Wild Type and Transgenic Tobacco

Since PmMYB45 transgenic plants exhibit higher lignin content than wild−type plants, we conducted an assay to determine if the change in lignin content leads to a change in lignin monomer content. We analyzed the lignin monomer content in the stems of both WT and transgenic plants. Our results are shown as follows: G lignin content increased, S lignin also increased, and the G/S ratio changed significantly (

Table 1. Contents of lignin monomers in tobacco stems.

	WT	L2	L4
G Lignin (ng/g)	63.28 ± 0.13	113.72 ± 4.18 ****	283.12 ± 7.28 ****
H Lignin (ng/g)	11.42 ± 0.12	20.99 ± 1 *	118.35 ± 4.39 ****
S Lignin (ng/g)	66.92 ± 1.39	64.77 ± 0.73	91.48 ± 1.11 ****
S/G	1.06 ± 0.02	0.58 ± 0.01 ****	0.32 ± 0.01 ****

The values shown in the table are mean ± SE (n = 3). Different numbers of “*” indicate significant differences (* p < 0.05, **** p < 0.0001). Data are shown as mean ± SE, with three biological replicates.

).

3.7. Effect of PmMYB45 Overexpression on Key Enzyme Genes for Secondary Cell Wall Formation in Tobacco

To further demonstrate that PmMYB45 regulates lignin biosynthesis, we utilized RT-qPCR to analyze the relative expression of enzyme genes that are specifically involved in lignin monomer biosynthesis. The results showed that 4CL5, PAL1, CAD, and HCT were significantly upregulated in all transgenic tobacco compared with wild-type plants (Figure 9). These data suggest that PmMYB45 is indeed involved in regulating lignin biosynthesis.

4. Discussion

Cell walls are the main component of wood biomass, and studying the process of cell wall formation can help humans obtain new species that fulfill their needs. Numerous MYB TFs involved in the regulation of secondary wall biosynthesis have been extensively and intensively studied in the herb A. thaliana and the woody plant Populus tremula [22]. In contrast, there is limited research on the identification of transcription factors responsible for controlling the process of secondary wall biosynthesis in P. massoniana. In order to comprehend the molecular regulation mechanism of secondary wall biosynthesis in P. massoniana, we validated the PmMYB45 TF associated with SCW synthesis based on the identified R2R2-MYB TFs. We employed transgenesis to carry out a functional study with a novel perspective for investigating related metabolite contents.

In analyzing its tissue expression and promoter analysis, we also noted that the PmMYB45 gene is also abundantly expressed in tobacco-lignified tissues, such as stems as well as the veins of mature leaves (Figure 5B), where a number of structural genes and transcription factors involved in lignin biosynthesis are also specifically expressed, such as PtoMYB158, PtoMYB189, and PtoMYB152 [23,24]. This suggests that PmMYB45 may be involved in the regulation of lignin biosynthesis. The sites of expression of the TFs overlap with those of lignin synthesis, and the two may be related. Previous studies have shown that PmMYB45 belongs to subfamily 37, along with PtrMYB009, PtrMYB123, PtrMYB153, PtrMYB115, PtrMYB201, PtrMYB006, PtrMYB126, PtrMYB050, PtrMYB051, PtrMYB060, and PtrMYB061. Branch and members of subfamily 37 have been reported to play key roles in lignin biosynthesis [25,26]. In this branch, PtrMYB6 inhibits secondary cell wall development in poplar and A. thaliana [27]. PtrMYB061 is homologous to AtMYB46 and activates cellulose and hemicellulose synthesis [28]. Overexpression of PmMYB45 in tobacco resulted in changes in plant growth traits, with a significant decrease in plant height, an increase in basal stem diameter, and an increase in the thickness of xylem ductal cell wall, which is consistent with the results of PtMYB8 overexpression in spruce(Picea asperata), which resulted in slow growth of the plant, dwarfing, and an increase in the accumulation of lignin [29]. Overexpression of PmMYB45 resulted in a significant upregulation of genes related to the lignin synthesis pathway (4CL, CAD, HCT, PAL1) in tobacco, suggesting that PmMYB45 can influence multiple target genes to regulate their expression. It was shown that overexpression and upregulation of secondary wall biosynthesis genes resulted in the accumulation of transcripts, stronger xylem lignification, and thicker secondary walls in plants [30]. The PtoMYB170 promoter was able to activate the expression of the CCOAOMT1 and CCR2 genes. Knockdown of PtoMYB170 resulted in the weakening of lignin deposition, as well as the loosening and collapsing of the xylem structure. This indirectly indicates that a target gene can be regulated by multiple TFs simultaneously, leading to varying expression levels of the same gene in different plants [31]. In addition, the non-specific binding to the gene promoter caused by MYB overexpression may be indirectly affected by ectopic MYB overexpression [29]. Furthermore, the non-specific attachment to the gene promoter induced by the overexpression of MYB could potentially be indirectly influenced by the ectopic overexpression of MYB. Therefore, there are many reasons for the upregulation of downstream/target genes in PmMYB45 overexpressing plants.

In summary, PmMYB45 functions as a transcriptional activator that stimulates the expression of crucial enzyme genes in the lignin biosynthesis pathway, thereby enhancing lignin biosynthesis. However, the mechanism of how to realize the regulatory function is not clear. Delving deeper into its function and exploring its potential interaction with other TFs to create a network map of secondary wall formation in coniferous species would be a promising area for further research.

5. Conclusions

In this study, we isolated the MYB transcription factor PmMYB45 and functionally characterized it. Our results indicate that PmMYB45 is a transcriptional activator localized in the nucleus. Overexpression in tobacco promotes the expression of lignin biosynthesis genes, resulting in an increase in xylem thickness in transgenic plants. PmMYB45 is involved in the regulation of lignin biosynthesis during the formation of the secondary cell wall. This factor may be a specific regulator. To prove this, the application of antisense techniques can be considered in future experiments to more accurately determine the function of the gene.