PmMYB4 , a Transcriptional Activator from Pinus massoniana , Regulates Secondary Cell Wall Formation and Lignin Biosynthesis

: Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine ( Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4 , which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-ligniﬁed stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes ( PmPAL and PmCCoAOMT ) involved in SCW biosynthesis. In addition, PmMYB4 -OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees. PmMYB4 20-fold transgenic We also expression proﬁles of PAL4 and found no difference between the WT and PmMYB4 overexpressing transgenic lines These results suggest that overexpression of PmMYB4 in transgenic tobacco the expression of SCW-related genes.


Introduction
The formation of wood involves the development of mother cells of secondary xylem differentiated from the vascular cambium, elongation of cells, deposition of the SCW, and programmed cell death (PCD) [1]. Cell walls are the main component of wood, accounting for more than 90% of the dry weight of plants [2]. In plant cells, the cell wall consists mainly of the SCW [3]. During the formation of the SCW, the composition of the cell wall changes significantly, mainly because of the directional arrangement of cellulose, deposition of lignin, and changes in the composition of hemicellulose and proteins [4]. Similarly, the SCW thickness, chemical composition, and structure and the proportions of different cell types determine wood properties [3]. Lignin, cellulose, and hemicellulose are the main components of the SCW. The biosynthesis of these components is not only precisely regulated by structural genes but also strictly regulated by many transcription factors (TFs). Other studies of xylem cell wall biosynthesis in other plants have led to a regulation model that consists of four gene levels: (1) first-layer master switches (NAC genes; NAM, ATAF1/2, and CUC2 genes), (2) second-layer master switches (MYB (myeloblastosis) genes; v-myb avian myeloblastosis viral oncogene homologs), and (3) downstream regulators (TF genes) regulating (4) genes for SCWs [5][6][7][8][9][10].
A putative R2R3-MYB TF gene was obtained by BLAST searching in the Masson pine database(PRJNA655997) [24] using PgMYB4 (accession no. ABQ51220.1) as a query sequence. The ORF was amplified from the leaf cDNA of 2-year-old Masson pine by RT-PCR. The obtained sequence, named PmMYB4, encoded a deduced polypeptide of 490 amino acid residues with a calculated molecular weight of 54.2 KD and an isoelectric point of 5.43.
The N-terminus of PmMYB4 contained typical R2R3 imperfect repeats, which mediate binding to target DNA sequences and are highly conserved among R2R3-MYB proteins ( Figure S1). The comparison of the PmMYB4 amino acid sequence with those of other MYB proteins showed high identity with lignin-associated MYB proteins from Pinus taeda (PtMYB4, 61.02%) and Picea glauca (PgMYB4, 64.08%) (Figure 1 and Figure S1).
The N-terminus of PmMYB4 contained typical R2R3 imperfect repeats, which mediate binding to target DNA sequences and are highly conserved among R2R3-MYB proteins ( Figure S1). The comparison of the PmMYB4 amino acid sequence with those of other MYB proteins showed high identity with lignin-associated MYB proteins from Pinus taeda (PtMYB4, 61.02%) and Picea glauca (PgMYB4, 64.08%) (Figure 1 and Figure S1).  [6]; and PtrMYB174, PtrMYB175, PtrMYB090, PtrMYB161, PtrMYB088, PtrMYB059, and PtrMYB093 (MYBs as tertiary switches in the SCW pathway in Populus trichocarpa) are highlighted with blue boxes [6]. Group I represents MYBs as secondary switches in the SCW, and group II represents other MYBs. The GenBank accession numbers of these R2R3-MYB TFs are listed in the Materials and Methods.

PmMYB4 Is Primarily Expressed in Bark
We investigated the expression profiles of PmMYB4 in various tissues of Masson pine by semiquantitative RT-PCR and quantitative real-time PCR. The results showed that PmMYB4 was expressed in all tissues tested, but relatively high expression was found in bark, semi-lignified stems and new leaves ( Figure 2). The highest level of PmMYB4 expression was detected in bark, while the lowest was detected in roots and flowers. The PmMYB4 transcript levels in bark were approximately 120-fold higher than those in flowers, while the transcript levels in semi-lignified stems were 47-fold higher ( Figure 2). Although the expression of the PmMYB4 gene was not xylem-specific, it was mainly expressed in lignin-forming tissues, such as semi-lignified stems and bark, suggesting that PmMYB4 may play an important role in lignin biosynthesis during SCW formation. PmMYB4 is highlighted with a red box; PtrMYB21 and PtrMYB074 (MYBs as secondary switches in the SCW pathway in Populus trichocarpa) are highlighted with orange boxes [6]; and PtrMYB174, PtrMYB175, PtrMYB090, PtrMYB161, PtrMYB088, PtrMYB059, and PtrMYB093 (MYBs as tertiary switches in the SCW pathway in Populus trichocarpa) are highlighted with blue boxes [6]. Group I represents MYBs as secondary switches in the SCW, and group II represents other MYBs. The GenBank accession numbers of these R2R3-MYB TFs are listed in the Materials and Methods.

PmMYB4 Is Primarily Expressed in Bark
We investigated the expression profiles of PmMYB4 in various tissues of Masson pine by semiquantitative RT-PCR and quantitative real-time PCR. The results showed that PmMYB4 was expressed in all tissues tested, but relatively high expression was found in bark, semi-lignified stems and new leaves ( Figure 2). The highest level of PmMYB4 expression was detected in bark, while the lowest was detected in roots and flowers. The PmMYB4 transcript levels in bark were approximately 120-fold higher than those in flowers, while the transcript levels in semi-lignified stems were 47-fold higher ( Figure 2). Although the expression of the PmMYB4 gene was not xylem-specific, it was mainly expressed in lignin-forming tissues, such as semi-lignified stems and bark, suggesting that PmMYB4 may play an important role in lignin biosynthesis during SCW formation.

Transcriptional Activation of PmMYB4 and the Binding Ability of PmMYB4 with AC Elements
Yeast harboring pGBKT7-PmMYB4 grew well on SD/-4 medium and turned blue on

Figure 2.
Tissue-specific expression patterns were characterized by real-time quantitative PCR, in which expression levels were averaged from three biological repeats and three technical repeats. Flowers (F), roots (R), new leaves (NL), old leaves (OL), semi-lignified stems (SS), lignified stems (LS), and bark (BK).

Growth and Morphological Characteristics of PmMYB4 Transgenic Tobacco
The results of RT-PCR and qRT-PCR indicated that the PmMYB4 gene was integrated into the genomes of 6 independent transgenic plant lines and highly expressed in L1, L5, and L6 ( Figure 4A,B). To investigate whether the overexpression of PmMYB4 could impact plant growth, we monitored the individual growth of three transgenic plants(T1) from three lines and untransformed controls. Two months after planting in the soil, significant growth phenotype differences were observed between plants overexpressing PmMYB4 and the wild type (WT). Height measurements of the 3 lines clearly indicated that L1, L5, and L6 plants were significantly shorter than WT plants ( Figure 4C,D). Similarly, the transgenic lines had a significantly greater stem diameter (18.75%) than the WT ( Figure 4E). In addition, the overexpression lines produced fewer leaves (33.03%) than the WT ( Figure 4F). Generally, compared to the WT, lines overexpressing the MYB4 gene showed altered growth characteristics.
PmMYB4 and the wild type (WT). Height measurements of the 3 lines clearly indicated that L1, L5, and L6 plants were significantly shorter than WT plants ( Figure 4C,D). Similarly, the transgenic lines had a significantly greater stem diameter (18.75%) than the WT ( Figure 4E). In addition, the overexpression lines produced fewer leaves (33.03%) than the WT ( Figure 4F). Generally, compared to the WT, lines overexpressing the MYB4 gene showed altered growth characteristics.

Overexpression of PmMYB4 Positively Regulates SCW in Transgenic Tobacco
The three independent lines (L1, L5, and L6) had similar phenotypes, and L5, which showed the highest expression ( Figure 4B), was used for histochemical staining. In sections stained with phloroglucinol, which stains lignin, gray staining was more intense in the OE lines (Figure 5a,b). These results indicated that higher lignin deposition occurred in the OE lines than in the WT. We also found significantly increased lignin contents in the OE lines, as determined by chemical analysis (Table 1), which was consistent with the results of sections stained with phloroglucinol ( Figure 5a,b). The chemical analysis also

Overexpression of PmMYB4 Positively Regulates SCW in Transgenic Tobacco
The three independent lines (L1, L5, and L6) had similar phenotypes, and L5, which showed the highest expression ( Figure 4B), was used for histochemical staining. In sections stained with phloroglucinol, which stains lignin, gray staining was more intense in the OE lines (Figure 5a,b). These results indicated that higher lignin deposition occurred in the OE lines than in the WT. We also found significantly increased lignin contents in the OE lines, as determined by chemical analysis (Table 1), which was consistent with the results of sections stained with phloroglucinol ( Figure 5a,b). The chemical analysis also suggested that the cellulose content in the OE lines was higher than that in the WT. However, there were no differences in hemicellulose content among the WT and PmMYB4-overexpressing lines (Table 1). These results indicate that PmMYB4 has a positive effect on lignin and cellulose contents but no obvious effect on the hemicellulose content.
Increases in cell wall thickness result from increased deposition of xylose and cellu-lose [23]. To better understand the contribution of PmMYB4 overexpression to SCW biosynthesis, microscopy analyses were conducted to measure the thicknesses of the stems of WT and transgenic plants. Notably, SEM images showed that the thickness of the entire cell wall, including the SCW, was greater in the overexpression lines than in the WT (Figure 5c,d). These results showed that overexpression of PmMYB4 positively regulated SCW formation in transgenic tobacco.

Overexpression of PmMYB4 Affects the Expression of SCW Biosynthesis Genes in Transgenic Tobacco
To better understand the molecular mechanisms of PmMYB4-mediated SCW formation, especially the accumulation of lignin and cellulose, we used qRT-PCR analysis to analyze the expression profiles of SCW biosynthetic genes. The results of qRT-PCR analysis showed that all detected lignin biosynthetic genes (CCoAOMT1, CCoAOMT2, CCoA-OMT3, PAL1, CAD-LIKE, HCT, and 4CL) presented significant expression increases in the PmMYB4-overexpressing transgenic lines. The expression of CCoAOMT2, CCoAOMT3, and HCT was upregulated five-to 20-fold in the transgenic lines ( Figure 6). We also analyzed the expression profiles of PAL4 and found no difference between the WT and PmMYB4-overexpressing transgenic lines ( Figure 6). These results suggest that overexpression of PmMYB4 in transgenic tobacco affected the expression of SCW-related genes.  Increases in cell wall thickness result from increased deposition of xylose and cellulose [23]. To better understand the contribution of PmMYB4 overexpression to SCW biosynthesis, microscopy analyses were conducted to measure the thicknesses of the stems of WT and transgenic plants. Notably, SEM images showed that the thickness of the entire cell wall, including the SCW, was greater in the overexpression lines than in the WT (Figure 5c,d). These results showed that overexpression of PmMYB4 positively regulated SCW formation in transgenic tobacco.

Overexpression of PmMYB4 Affects the Expression of SCW Biosynthesis Genes in Transgenic Tobacco
To better understand the molecular mechanisms of PmMYB4-mediated SCW formation, especially the accumulation of lignin and cellulose, we used qRT-PCR analysis to analyze the expression profiles of SCW biosynthetic genes. The results of qRT-PCR analysis showed that all detected lignin biosynthetic genes (CCoAOMT1, CCoAOMT2, CCoAOMT3, PAL1, CAD-LIKE, HCT, and 4CL) presented significant expression increases in the PmMYB4overexpressing transgenic lines. The expression of CCoAOMT2, CCoAOMT3, and HCT was upregulated five-to 20-fold in the transgenic lines ( Figure 6). We also analyzed the expression profiles of PAL4 and found no difference between the WT and PmMYB4overexpressing transgenic lines ( Figure 6). These results suggest that overexpression of PmMYB4 in transgenic tobacco affected the expression of SCW-related genes. Error bars represent ± SD from three biological repeats and three technical repeats. The significance of differences between different lines was tested with the t-test. * p < 0.05 and ** p < 0.01. The GenBank accession numbers of these SCW-related genes are listed in the Materials and Methods.

The PmPAL and PmCCoAOMT Promoters Are Activated by PmMYB4
To confirm that PmMYB4 directly binds to the promoters of SCW biosynthesis genes, we conducted an analysis of cis-elements in the promoters of lignin biosynthetic genes (PAL and CCoAOMT genes). Then, a luciferase activity(LUC) assay was performed. A binary vector containing the CaMV 35S promoter and the CDS of PmMYB4 was used as the effector, while vectors containing the two individual promoter fragments and LUC reporter genes were used as reporters ( Figure 7B). Coexpression of effectors and reporters in the leaves of Nicotiana benthamiana significantly enhanced the relative LUC activity compared with that in mock-treated plants ( Figure 7E). The results suggest that PmMYB4 can bind to the promoters of lignin biosynthetic genes (PAL and CCoAOMT).
To further verify the above interactions identified in double-luciferase assays, the pBI121-NOGUS-PmMYB4 construct was used as an effector, and two promoters were fused with the minimal 35S promoter to drive the GUS reporter gene ( Figure 7B). GUS activity was detected in tobacco leaves following the co-transformation of lines harboring pBI121-NOGUS-PmMYB4 with the two promoters. By analyzing the gray value of tobacco leaves, we found that the leaves co-transformed with 35::PmMYB4 and the promoter have darker GUS staining ( Figure 7C,D). The results further showed that PmMYB4 could activate the promoters of PAL and CCoAOMT.
In addition, the presence of an SNBE NAC binding site indicates that PmMYB4 may be regulated by NAC as a secondary transcriptional switch in the SCW synthesis pathway of Masson pine ( Figure 7F). Error bars represent ± SD from three biological repeats and three technical repeats. The significance of differences between different lines was tested with the t-test. Asterisks represent significant differences between each OE line and WT (* p < 0.05, ** p < 0.01). The GenBank accession numbers of these SCW-related genes are listed in the Materials and Methods.

The PmPAL and PmCCoAOMT Promoters Are Activated by PmMYB4
To confirm that PmMYB4 directly binds to the promoters of SCW biosynthesis genes, we conducted an analysis of cis-elements in the promoters of lignin biosynthetic genes (PAL and CCoAOMT genes). Then, a luciferase activity (LUC) assay was performed. A binary vector containing the CaMV 35S promoter and the CDS of PmMYB4 was used as the effector, while vectors containing the two individual promoter fragments and LUC reporter genes were used as reporters ( Figure 7B). Coexpression of effectors and reporters in the leaves of Nicotiana benthamiana significantly enhanced the relative LUC activity compared with that in mock-treated plants ( Figure 7E). The results suggest that PmMYB4 can bind to the promoters of lignin biosynthetic genes (PAL and CCoAOMT).
To further verify the above interactions identified in double-luciferase assays, the pBI121-NOGUS-PmMYB4 construct was used as an effector, and two promoters were fused with the minimal 35S promoter to drive the GUS reporter gene ( Figure 7B). GUS activity was detected in tobacco leaves following the co-transformation of lines harboring pBI121-NOGUS-PmMYB4 with the two promoters. By analyzing the gray value of tobacco leaves, we found that the leaves co-transformed with 35::PmMYB4 and the promoter have darker GUS staining ( Figure 7C,D). The results further showed that PmMYB4 could activate the promoters of PAL and CCoAOMT.
In addition, the presence of an SNBE NAC binding site indicates that PmMYB4 may be regulated by NAC as a secondary transcriptional switch in the SCW synthesis pathway of Masson pine ( Figure 7F).

Discussion
The identification of the molecular switches that regulate secondary cell wall biogenesis during wood formation is essential for basic studies and also for the biotechnological manipulation of wood quality and quantity in woody plant species [25]. While the TFmediated transcriptional regulation of wood formation has been well studied in angiosperms [25][26][27], the understanding in conifers remains limited. Efforts to unravel the molecular regulation of coniferous secondary cell wall formation has implicated the R2R3-MYB family of TFs as transregulators of lignin biosynthesis and wood formation [28][29][30][31][32]. In this study, we elucidated a key TF, PmMYB4, associated with cell wall biogenesis in Masson pine and inferred an evolutionarily conserved regulatory mechanism of this TF on secondary cell wall formation. This study represents an important step towards understanding the coordinated transcriptional regulation that SCW formation in conifers.
Amino acid sequence alignment showed that the N-terminal region of PmMYB4 contains R2 and R3 domains that are conserved with their homologous genes but differ in the C-terminal region ( Figure S1). By subcellular localization and yeast one-hybrid assays, we determined that PmMYB4 is a transcriptional activator that targets the nucleus. Tissue expression pattern analysis showed that PmMYB4 was preferentially expressed in bark and semi-lignified stems. Interestingly, the expression level of MYB4 in young tissues was higher than that in mature tissues, suggesting that MYB4 may play an important role in plant growth and development.
One common strategy for functional characterization of a candidate gene is to downor up-regulate its expression by genetic transformation. The long generation time and long life span of conifers have been major obstacles to perform reverse genetic approaches

Discussion
The identification of the molecular switches that regulate secondary cell wall biogenesis during wood formation is essential for basic studies and also for the biotechnological manipulation of wood quality and quantity in woody plant species [25]. While the TF-mediated transcriptional regulation of wood formation has been well studied in angiosperms [25][26][27], the understanding in conifers remains limited. Efforts to unravel the molecular regulation of coniferous secondary cell wall formation has implicated the R2R3-MYB family of TFs as transregulators of lignin biosynthesis and wood formation [28][29][30][31][32]. In this study, we elucidated a key TF, PmMYB4, associated with cell wall biogenesis in Masson pine and inferred an evolutionarily conserved regulatory mechanism of this TF on secondary cell wall formation. This study represents an important step towards understanding the coordinated transcriptional regulation that SCW formation in conifers.
Amino acid sequence alignment showed that the N-terminal region of PmMYB4 contains R2 and R3 domains that are conserved with their homologous genes but differ in the C-terminal region ( Figure S1). By subcellular localization and yeast one-hybrid assays, we determined that PmMYB4 is a transcriptional activator that targets the nucleus. Tissue expression pattern analysis showed that PmMYB4 was preferentially expressed in bark and semi-lignified stems. Interestingly, the expression level of MYB4 in young tissues was higher than that in mature tissues, suggesting that MYB4 may play an important role in plant growth and development.
One common strategy for functional characterization of a candidate gene is to down-or up-regulate its expression by genetic transformation. The long generation time and long life span of conifers have been major obstacles to perform reverse genetic approaches in these woody plants [25]. Akiyoshi et al. (2019) avoided these difficulties by using heterologous expression of loblolly pine VNS genes in tobacco and Arabidopsis cells to infer TF functions regulating tracheid cell wall biosynthesis [33]. In this study, we produced transgenic tobaccos overexpressing the PmMYB4 gene derived from P. massoniana under the control of a constitutive 35S promoter via Agrobacterium-mediated transformation [34]. The transgenic plants were significantly shorter than the WT plants, but the stems of the transgenic plants were thicker, similar to AtMYB46 and EgMYB2 [15,35]. In Arabidopsis, up-regulation of AtMYB46 resulted in thickening of ectopic secondary wall observed in epidermis, cortex, and pith cells of stem cross-sections, and the severity of the ectopic secondary wall thickening phenotypes (e.g., dwarfing, leaf curling) was positively associated with the level of expression of the introduced MYB46 gene [9]. Goicoechea found that half the tobaccos transformed with the EgMYB2 construct (9/18) grew to only two-thirds of the size of the control plants. He believes that this phenomenon is related to the loss of top advantage [15]. Although the leaves showed signs of dysplasia, it was found that PmMYB4 overexpressors did not exhibit the curly leaf phenotype, which is typical of the MYB46 overexpressors( Figure 4C,F). Recently, it has been proposed that PpMyb4 and its orthologs, AtMYB46 and EgMyb2, could be nonspecific for regulating lignin biosynthesis because they are also involved in the regulation of cellulose and xylan biosynthesis [1]. To further verify whether PmMYB4 has similar functions to AtMYB46, section staining, scanning electron microscopy and lignin, cellulose, and hemicellulose content analyses were performed on the overexpression lines. Overexpression of PmMYB4 resulted in a thicker xylem and ectopic deposition of lignin (Figure 5a,b), which were consistent with the results of lignin content measurements (Table 1). Interestingly, the cellulose content in PmMYB4-overexpressing transgenic lines was also significantly increased, and the hemicellulose content did not change (Table 1). Previous studies have shown that AtMYB46 and EgMYB2 have a positive effect on hemicellulose content; however, Guo found that BplMYB46 has a negative effect on hemicellulose content [16]. Whether this could be a subfunctionalization remains to be further studied. In addition, several genes involved in the secondary wall biosynthesis were up-regulated in transgenic tobaccos. Key genes for enzymes of monolignol biosynthesis, such as CCoAOMT1, CCoAOMT2, CCoAOMT3, PAL1, PAL4, CAD-like, or HCT, were up-regulated in the transgenic plants.
It is interesting to note that the SMRE consensus sequences include three variants that are identical to the AC elements(AC-I, ACCTACC; AC-II, ACCAACC; and AC-III, ACCTAAC) that were previously known to be involved in the activation of lignin and other phenylpropanoid biosynthetic genes [35][36][37]. Since R2R3 MYB DNA-binding domains are highly conserved in their sequences [13], it is not surprising to find that the homologous genes of AtMYB46 bind to AC elements. Phylogenetic and expression analyses of PmMYB4 have suggested a role as a potential candidate ortholog of AtMYB46 genes regulating SCW formation (Figures 1, 6 and 7). The Y1H method showed that PmMYB4 can bind to AC-boxes(AC-I, AC-II, AC-III, and SMRE4). The promoter region of the SCW gene was predicted from the loblolly pine genome, and seven AC-boxes and two AC-boxes were found in the promoter regions of PAL and CCoAOMT, respectively ( Figure 7A). The promoter regions of PmPAL and PmCCoAOMT were cloned using Masson pine gDNA as the template with designed specific primers (Table S7). The results showed that the promoter regions of PmPAL and PmCCoAOMT contained AC-box motifs. To investigate whether PmMYB4 can activate the promoters of PmPAL and PmCCoAOMT, MYB and promoter constructs were generated in the 35S effector vector and LUC/GUS reporter vector, respectively, and were then transformed into 5-week-old N. benthamiana leaves. The results were consistent with the Y1H assay, and the LUC/GUS activity assay confirmed that PmMYB4 could activate the promoters of PmPAL and PmCCoAOMT. It is worth mentioning that the promoter of PmMYB4 also contains 3 SNBE motifs ( Figure 7F); however, whether PmMYB4 is a target gene of PmSWNs remains to be further studied. The results obtained in this work suggested that a transcriptional cascade similar to the AtMYB46 network defined in Arabidopsis [8,38] and poplar [39][40][41] is conserved in conifers.
The identification of PmMYB4 as a main regulator of this network involved in wood formation in P. massoniana is of great interest for fundamental studies in conifers but also for potential applications in tree biotechnology. To increase our knowledge about the transcriptional regulatory network operating in conifers, and given the complexity of the network demonstrated in other species, intensive research is necessary to fully clarify to what extent the transcriptional network could be conserved between gymnosperms and angiosperms.

Plant Materials and Stress Treatments
Eight-year-old Masson pine trees were propagated at Nanjing Forestry University (NJFU) in Jiangsu Province, China. Seven tissues, including flowers, roots, new leaves, old leaves, semi-lignified stems, lignified stems, and bark, were directly frozen in liquid nitrogen and stored at −80 • C until RNA extraction. Total RNA was isolated from each sample using an RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (Tiangen Biotech, Beijing, China) following the manufacturer's instructions.
Nicotiana benthamiana seedlings were planted in an artificial illumination incubator under appropriate conditions (22 • C with a 16-h light and 8-h dark photoperiod). Seeds were sterilized with 10% NaClO for 15 min and then sown in 1/2 MS medium. After 2 days of vernalization (4 • C dark), we placed the samples in an incubator for one week. At nine days after sowing (DAS), we transplanted the seedlings into a substrate (mixture of 60% turfy soil, 30% pearlite, and 10% vermiculite).

Cloning of the Full-Length PmMYB4 Coding Sequence (CDS) and Promoter
PmMYB4 was cloned from a cDNA library constructed from RNA isolated from needle tissue using the Prime Script 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). A pair of primers was designed according to the full-length coding region of the MYB4 gene sequence of P. taeda (GenBank: DQ399059.1). The PCR products were cloned into the pEASYT1 (Transgen, Beijing, China) vector, transformed into E. coli DH5α, and then sequenced. All primers used in these assays are listed in Supplementary Table S1. The molecular weight and isoelectric point of the PmMYB4 protein were determined using tools from the ExPASy website.
We extracted genomic DNA from 2-year-old Masson pine (DP320, Tiangen Biotech, Beijing, China) and isolated the 1781-bp promoter using three pairs of primers. The specific methods described in the Takara Genome Walking Kit instructions were followed. Based on the sequencing results, we designed a full-length specific primer ProPmMYB4-F/R for the PmMYB4 promoter to amplify the product of the full-length promoter. The online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 15 March 2021) was used to predict and analyze the cis-acting promoter elements. The primers are listed in Supplementary Table S1.

Sequence and Phylogenetic Analysis
The amino acid sequences of R2R3-MYB TFs were obtained from NCBI GenBank (https://www.ncbi.nlm.nih.gov) (accessed on 2 March 2021). Then, the sequences were used to construct a phylogenetic tree via the neighbor-joining (NJ) method using a Poisson model in MEGAX software (Park, PA, USA) [25] (https://www.megasoftware.net/) (accessed on 13 March 2021), and multiple sequence alignment was carried out with DNA-MAN software [42]. The GenBank accession numbers of these R2R3-MYB TFs are listed in Section 4.12.

Transcriptional Activation Analysis of PmMYB4
The protein-coding region of MYB4 was inserted into the pGBKT7 (Clontech, Shiga, Japan) vector (Table S2), and the construct was transformed into the yeast strain Saccharomyces cerevisiae AH109, together with pGADK7 by the LiAc/DNA/PEG method, Forests 2021, 12, 1618 11 of 15 as described previously [43]. The transformed yeast cells were plated on SD/-T for the selection of positive clones and were then transferred to dropout SD4 medium lacking tryptophan (Trp), leucine (Leu), histidine (His), and adenine (Ade) for the transactivation assay. The empty pGBKT7 vector was used as the negative control.

Agrobacterium-Mediated Transformation of Tobacco
The binary vector plasmid pBI121 harboring the desired PmMYB4 gene (Table S4), in which PmMYB4 was under the control of the CaMV 35S constitutive promoter, was introduced into Agrobacterium tumefaciens strain EHA105 by the freeze-thaw method [45]. Tobacco leaf discs were inoculated with an infective suspension (OD600 = 0.6) of regenerated A. tumefaciens with gentle shaking at 200 rpm for 10 min. Then, the leaf discs were dried using sterile paper towels and co-cultivated on MS medium containing 0.4 mg/L N-6-benzyladenine (6-BA), 0.1 mg/L 1-naphthylcetic acid (NAA), 0.01 mg/L thidiazuron (TDZ), 6 g/L agar, 30 g/L sucrose, and 200 mM acetosyringone (AS) at pH 5.8 and incubated in the dark at 28 • C for 2 days. Subsequently, the leaf discs were transferred to MS medium supplemented with 0.4 mg/L 6-BA, 0.1 mg/L NAA, 6 g/L agar, 30 g/L sucrose, 400 mg/L cefotaxime, and 50 mg/L kanamycin at pH 5.8 under 16/8 h light/dark conditions at 25 ± 1 • C in a phytotron to screen for putative transformant explants. Thereafter, the selected shoots were transferred first to half-strength MS rooting medium and then to soil and were propagated for complementary experiments. All transgenic and WT plants were acclimated and grown in a greenhouse at 18-23 • C under 60% humidity with 18 h of light and 6 h of dark daily at NJFU.

Plant Height and Biomass Measurements
The height from the basal stem to the tallest part of the plant and the stem diameter 5 cm above the soil were measured in 2-month-old tobacco plants from each transgenic and WT line. In addition, the number of leaves of each plant was recorded for comparison. Three lines are those that we chose, and each line contains six technical replicates.

Microscopy and Histochemistry
The stems (5th internode) of 2-month-old transgenic plants and WT plants were used for scanning electron microscopy (SEM) according to the protocol previously described by Yu et al. (2011) [46]. The image analysis software IMAGEJ (https://imagej.nih.gov/ij/) (accessed on 9 July 2021) was employed to quantify the morphological parameters of xylem cells (mm) and wall thickness.
Stems of 2-month soil-grown OE and WT PmMYB4 tobacco plants were fixed in dyeing solution (25 mg phloroglucinol powder, 25 mL of 100% methanol, and 25 mL 37% glacial acetic acid), and sections were obtained via the freehand slicing method. The stem sections were stained with phloroglucinol-HCl and examined by light microscopy [9]. At least three technical replicates were performed in these experiments.

Measurement of Lignin, Cellulose, and Hemicellulose Components in Transgenic Tobacco
Sections were obtained via the freehand slicing method. The stem sections were stained with phloroglucinol-HCl and examined by light microscopy [47]. The acetyl bromide (AcBr) method was used to estimate the total lignin content [48]. Cellulose and hemicellulose contents were measured by the Van Soest method [49]. The percent contents of cellulose, lignin and hemicellulose determined in the three biological replicates, and six technical replicate experiments were then averaged.

Transient Expression and LUC/GUS Activity Assay
The full-length CDS of PmMYB4 was amplified by PCR with gene-specific primers (Table S6) and then ligated into the plant binary vector pGreenII 62-SK driven by the CaMV 35S promoter. This construct was used as an effector in the LUC activity assay. Promoter fragments (PAL 1528 bp and CCoAOMT 646 bp) were independently cloned (Table S1) and ligated into the pGreen-0800-35 mini vector to produce various LUC reporters, and these constructs were used as effectors. All of these vectors were individually transformed into the A. tumefaciens strain GV3101. The effectors and reporters were infiltrated into the leaves of 2-week-old tobacco (Nicotiana benthamiana) plants by using the agroinfiltration method [50]. After 48 h of incubation, LUC activity was measured with a GloMax ® 20/20 luminometer.
The PAL and CCoAOMT promoters were fused with the 35S CaMV minimal promoter to drive the expression of the GUS gene in the pBI121 vector using the primers listed in Table S7. The full CDS of PmMYB4 was cloned into pBI121-NO-GUS under the control of the 35S promoter (35S:PmMYB4) as the effector. The reporters and effector were simultaneously infiltrated into the leaf epidermal cells of N. benthamiana as described above. After incubation at 28 • C for 2 days in the dark, blade samples with a diameter of 1 cm were collected with a hole punch and decolorized with 75% ethanol. Finally, GUS staining was performed under a stereomicroscope (SZX16, OLYMPUS, Tokyo, Japan) and a stereofluorescence microscope (M205FA, Leica, Wetzlar, Germany). The gray value of GUS was analyzed by IMAGEJ.

Statistical Analysis
All experimental data were obtained from at least three replicates, and statistical analysis was performed with Student's t-test. In all experiments, significant differences in the data were evaluated by one-way ANOVA. * p < 0.05, and ** p < 0.01.

GenBank Accession Numbers of Genes Used in This Study
The GenBank accession numbers of genes used in this study are as follows: PgMYB1