The WUSCHEL-Related Homeobox 4 (BpWOX4) Promotes Secondary Growth Through Lignin Biosynthesis Activation in Betula platyphylla

The WUSCHEL-Related Homeobox 4 (BpWOX4) Promotes Secondary Growth Through Lignin Biosynthesis Activation in Betula platyphylla | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The WUSCHEL-Related Homeobox 4 (BpWOX4) Promotes Secondary Growth Through Lignin Biosynthesis Activation in Betula platyphylla Haroon Rasheed, Dinghao Liu, Tingting Jin, Yijie Li, Chichi Winarsih, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7726648/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract Secondary growth is a key process in the growth and development of woody plants, influencing biomass accumulation and structural integrity. The WUSCHEL- related homeobox 4 ( BpWOX4 ) gene regulates vascular cambium activity in trees; however, its precise role in secondary growth in Betula platyphylla remains poorly understood. In this study, we functionally characterized BpWOX4 and its role in secondary growth through a combination of transcriptomic, physiological, and histological analyses. Transcriptome analysis among wild-type (WT), overexpression (O), and suppression (SE) lines revealed upregulation of lignin biosynthesis genes, such as PAL , 4CL , CCR , and CAD . Gene Ontology (GO) and KEGG enrichment analyses indicated activation of the phenylpropanoid and lignin biosynthesis pathways. Additionally, key transcription factors involved in secondary growth, including members of the MYB and NAC families, were significantly upregulated in overexpression lines. Furthermore, overexpression of BpWOX4 in B. platyphylla resulted in increased stem diameter and xylem thickness, as well as significantly higher lignin content. Together, these results provide new insights into the molecular mechanisms underlying secondary growth and identify BpWOX4 as a promising genetic target for enhancing biomass production and wood quality in forest trees. Secondary growth WOX Betula platyphylla Lignin Transcriptome analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Tree growth occurs through two different but interconnected processes, primary growth and secondary growth. Primary growth in plants refers to the increase in the length of the plant, driven by cell division in the apical meristem, whereas secondary growth refers to the proliferation of plant organs through cell division, cell wall biosynthesis, lignification, and programmed cell death(Zhou et al., 2023 ). These growth types manage the height, spread, and diameter of trees, shaping their form and functional adaptations across developmental stages. Cell division direction within vascular tissue is regulated by the interaction between the receptor kinase PXYv that expressed in meristematic cells, and its peptide ligand CLE41, produced by nearby phloem cells(Etchells & Turner, 2010 ). The cambium is a meristematic tissue and develops into a closed ring during secondary growth, forming the vascular cambium, and undergoes proliferation (active cell division). Secondary growth in plant stems results from the activity of the vascular cambium, a lateral meristem that produces secondary xylem (wood) toward the inside and secondary phloem toward the outside(Mellerowicz et al., 2001 ; Spicer & Groover, 2010 ). The secondary xylem not only conducts water up the stem, but it also develops a strong woody structure after substantial lignification and cell death, allowing the tree to grow taller(Morris et al., 2016 ). Lignin, a phenolic heteropolymer found in plants, exhibits varying structures and contents across different species, tissues, cell types, cell layers, and environments that serves significant biological functions. Lignin is a crucial component of secondary growth, directly affecting wood formation, vascular development, and structural integrity (Somerville et al., 2004 ). Due to its hydrophobic nature, lignin plays an essential role in water transport and serves as a major component of vascular tissue(Boyce et al., 2004 ; Coleman et al., 2008 ). Lignin is also deposited on the cell wall through the oxidative polymerization of lignin monomers(Smith et al., 2013 ). Following lignification, tubular elements and fibrous cells undergo programmed cell death. This process includes the disintegration of organelles, along with the degradation of the protoplast and part of the secondary cell wall that remains unlignified(Escamez & Tuominen, 2014 ). Studies have shown transcription factors, signaling pathways, and metabolic enzymes stimulate that lignin biosynthesis. The WOX gene family function as a key regulators of developmental processes in plants, including embryonic patterning, stem cell maintenance, and organogenesis(Rasheed et al., 2024 ). The WUSCHEL-related homeoboxes (WOX) gene family was first identified in Arabidopsis thaliana in 1996, with its role in shoot and floral development(Laux et al., 1996 ). According to evolutionary history, WOXs are divided into three clades: modern/WUS, intermediate clade, and ancient clade(Graaff et al., 2009 ). Previous study showed the WOX gene family has a function in structuring several early plant cell populations(Ji et al., 2010 ). Members of the WOX protein family are essential for the maintenance and proliferation of stem cells in cambium, the lateral meristem that responsible for generating all cellular components of wood(Galibina et al., 2023 ). Some WOX genes have been linked to lignin biosynthesis through their influence on gibberellin (GA) signaling and secondary cell wall formation(Y. Zhang et al., 2022 ). Overexpression of PtrWOX13A in poplar significantly enhanced the growth potential in transgenic lines and resulted in increased secondary cell wall thicknesses, longer fiber length, as well as lignin and hemicellulose contents(Y. Zhang et al., 2022 ). Overexpression of WOX4 in Populus resulted in increased cambial activity and led to a higher lignin deposition in xylem tissues(M. Zhang et al., 2023 ). Study in Arabidopsis revealed the loss of WOX4 did not affect xylem differentiation but impaired cambial cell proliferation(Suer et al., 2011 ). Another study demonstrated that the knockdown of GhWOX4 gene hindered secondary growth by reducing cambial width and division activity compared with control plants (Sajjad et al., 2021 ). White birch ( B. Platyphylla ) is a broad-leaved pioneer species in eastern Asia, where it contributes significantly to the stabilization and regeneration of forest ecosystem (Y. Wang et al., 2023 ). Due to its vast source of biomass, B. platyphylla is commonly used to make biofuel, building materials, pulp and paper, and other valuable chemicals(Zhao et al., 2019 ). It is a monoecious species with distinct male and female inflorescences, making it significantly different from model plant species like Arabidopsis and Antirrhinum (Endress, 1992 ). Our research investigates the function of BpWOX4 in B. platyphylla , identifies its regulatory mechanism and involvement in promoting secondary development and lignification in woody plant. This research gives new insights into the genetic regulation of vascular cambium activity and contributes to a better knowledge of secondary development in trees. 2. Materials and Methods 2.1Plants materials The clones of Betula platyphylla used in this study were propagated through in vitro tissue culture . The BpWOX4 gene was constructed from Betula platypylla cDNA using specific primers and then cloned into a vector via Agrobacterium -mediated transformation. Transgenic BpWOX4 -overexpression (OE), BpWOX4 -suppression (SE), and wild-type (WT) lines were subsequently cultivated in 25 cm × 25 cm pots and received standard irrigation and fertilization throughout their growth. All plants were grown under field conditions for two years in the Northeast Forestry University, Harbin, located in Heilongjiang Province, China. 2.2 RNA Sequencing Nine samples of B.platyphylla were selected for transcriptome analysis, comprising three biological replicates each from wild-type(WT1, WT2, WT3), overexpression (OE1, OE2, OE3), and suppression (SE1, SE2, SE3)lines. The shoot tissues were collected by scraping the stem after removing the bark and stored at -80℃ in an ultra-low temperature freezer. Subsequently, the samples were sent to Majorbio company (www.majorbio.com) for RNA extraction and transcriptome sequencing. Differential gene expression analysis was performed using the DESeq2 method to identify differentially expressed sequence ( DEGs across different groups. Additionally, pathway annotation analysis was conducts on the DEGs to elucidate their functional roles. All data analyses were carried out using the R language programming (cran.rstudio.com) and Python software. 2.3 RT-qPCR validation of DEGs RT-qPCR was performed to validate the transcript levels of selected genes. Total RNAs were extracted separately from the shoot of samples using the protocol provided with the E.Z.N.A Plant RNA Kit (www.omegabiotek.com.cn). A NanoDrop 2000 spectrophotometer was used to analyze RNA quantity and purity. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer. Only samples with an RNA Integrity Number (RIN) higher than 8 were kept at -80°C for further analysis. First-strand complementary DNA (cDNA) was synthesized from 7 μL of total RNA by using the Primescript TM RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) following the manufacturer’s protocol. QRT-PCR was performed on a 96-well Analytik Jena (aj) PCR instrument in accordance with the Lablead protocol (www.lablead.cn). Target gene The relative expression levels of target genes were determined using the2 - ΔΔCT method, with 18S rRNA and tubulin serving as reference genes. Primers with a temperature of 58 to 60°C and nucleotide lengths of 18 to 25 bp were designed using NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). 2.4 Lignin Content Determination Two-year-old B. platyphylla plants were selected for lignin determination. Stems were cut at a height of 3cm above ground using pruning shears, and the bark was removed from the sampled region. The sample were then oven-dried at 60°C for 72 hours. Lignin content was determined following the protocol provided by the Suzhou Grace Biotechnology CO., Ltd (www.geruisi-bio.com). Three biological replicates were used for each sample. Data analysis was performed using Python software. 2.5 Histochemical Staining of BpWOX4 Transgenic birch Stem segments were collected from six-month-old WT, OE, and RE birch lines and were manually sectioned. To detect the lignin deposition, transverse sections were stained with phloroglucinol-HCl, which produces a characteristic red coloration in lignified cell walls. The stained samples were examined under a light microscope, and anatomical features-including xylem width, number of cambial cell layers, and vascular tissue organization- were compared among the WT and both transgenic lines. 3. Results 3.1 RNA-seq Data Quality and Mapping Efficiency RNA sequencing produced between 42,538,698 and 56,062,972 raw reads per sample. After quality control, a high proportion of clean readings was maintained, ranging from 42,003,412 to 55,464,274, showing minimal loss during filtering. The subsequent alignment to the reference genome resulted in an efficient mapping rate across all samples, ranging from 91.44% to 92.09%, with an average mapping rate above 91%. This mapping efficiency demonstrates reliability and a high degree of sequence specificity to the reference genome. The GC content was consistent across all samples, ranging from 44.71% to 45.52%, indicating that the nucleotide makeup was uniform across experimental groups. The slight variations in GC content reflect natural biological differences rather than technical bias. Overall, the RNA-seq data quality was high, with adequate read depth, strong mapping efficiency, and stable GC content, ensuring requirement for downstream transcriptome analysis. 3.2 Analysis of Differentially Expressed Genes (DEGs) The DESeq2 package (version 4.5.0) in R was used to conduct differential expression analysis on all acquired reads, normalized as reads per kilobase of transcript per million mapped reads (RPKM)(Love et al., 2014 ). To identify differentially expressed genes (DEGs), pairwise comparisons were performed with criteria of log2 fold change (log2FC) > 1 or <-1 and a p-value (-log10padj). In the OE_ vs_ WT comparison, 4355 differently expressed genes (DEGs) were identified, with 2220 upregulated and 2135 downregulated. The SE_vs_WT comparison revealed a greater number of DEGs (6950), including 3468 upregulated and 3482 downregulated genes, indicating more comprehensive transcriptome changes in this group. Similarly, the OE_ vs_ SE analysis found 3989 DEGs, with 1935 genes upregulated and 2054 genes downregulated, indicating unique transcriptional profiles in these two groups. The distribution and overlap of DEGs were further assessed using Venn analysis. The analysis identified a set of common DEGs shared among comparisons, as well as unique subsets specific to OE or SE plants (Fig. 1 B). The existence of shared DEGs indicates a core transcriptional response to BpWOX4 disruption, whereas the unique subsets reveal regulatory alternations to each condition. To visualize the distribution of DEGs, volcano plots were constructed for each comparison. In the OE_vs_WT, the majority of genes clustered around the non-significant region, with significant upregulated and downregulated gene populations visible at higher Log2FC values (Fig. 1 C). The SE_VS_WT displayed a similar pattern but more significant DEGs, indicating the broader transcriptional impact of the suppressor element (Fig. 1 D). The OE_vs_SE revealed a balanced distribution of upregulated and downregulated genes, emphasizing the regulatory interaction between two conditions (Fig. 1 E). The volcano plots not only corroborated the DES analysis but also provided a clear visual representation of the magnitude and significance of gene expression changes. Beyond DEG counts, we analyzed global expression patterns using principal component analysis (PCA) and correlation heatmap. The PCA plot clearly separated OE, SE, and WT samples into distinct clusters, with OE and SE showing divergence from WT but partial overlap with each other (Fig. 1 F). This separation demonstrates that BpWOX4 expression status is a significant determinant of transcriptional variance across lines. The correlation heatmap further supported these findings, showing strong intra-group correlations(red) and weaker inter-group correlations (blue) (Fig. 1 G). This indicates consistent expression across biological replicates and robust transcriptional reprogramming between groups. In conclusion, our combined analysis of DEGs and volcano reveals the extensive transcriptional reprogramming in overexpression and suppression plants. The significant number of DEGs and their distribution in the volcano plot highlight the complexity of gene regulatory networks in response to genetic alterations. 3.3 GO and KEGG Enrichment Analysis To better understand the molecular processes regulating WOX gene activity in B. platyphylla , we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on WOX-overexpression (OE) and WOX-silenced (SE) transgenic lines compared with wild-type (WT). The analysis revealed that DEGs were significantly enriched in pathways related to secondary wall formation, hormone regulation, and stress responses (Fig. 2 ). The most significantly enriched KEGG pathway was phenylpropanoid biosynthesis (map00940). In addition, enrichment was shown in plant hormone signal transduction (map04075), plant–pathogen interaction (map04626), tryptophan metabolism (map00380, a precursor to auxin biosynthesis), and galactose metabolism (map00052). Gene Ontology (GO) enrichment showed that terms such as cell wall biogenesis, secondary metabolic process, phenylpropanoid catabolic process, and hormone-mediated signaling pathway were highly represented, indicating that BpWOX4 may regulate gene networks that are critical for secondary wall synthesis and hormonal signaling integration. 3.4 Differentially expressed TFs are regulated during secondary growth in B.platyphylla Transcription factors (TFs) regulate secondary growth, lignin biosynthesis, and ROS homeostasis in plants, and MYB, NAC, WRKY, and LIM are particularly important for this developmental processes (Khan et al., 2018 ; Q. Liu et al., 2018 ). A total of 779 transcription factor (TF) genes were identified in this study, among these, 80 MYB and 46 NAC transcription factors were significantly expressed. The differential expression of these TFs across our comparisons (OE vs. WT and SE vs. WT) highlights their dynamic role in modulating secondary growth and lignin-related pathways, which is a key finding of our study. 3.5 Lignin biosynthesis pathway is significantly enriched in the BpWOX4 plants Lignin pathway was found to be the most significant in the pathway analysis. Lignin maintains the structural integrity, strength, and hardness of the cell wall, supports water transportation, prevents cell wall penetration, and protects plants against pathogen invasion(Meng et al., 2021 ). In this study, the main synthetase genes in the phenylpropane biosynthesis pathway were identified by annotating and analyzing the sequence findings in the KEGG pathway. Our transcriptome analysis data revealed the important genes that are involved in the lignin biosynthesis pathway and we compared the lignin content between WT, OE, and SE lines. Our data shows that the lignin content of WT plants is greater than that of SE plants and less than OE plants. KEGG pathway analysis reveals that OE_ vs_ WT comparison posses a total of 32 genes expressed in this pathway, in which 13 are upregulated and 19 are down-regulated. A comparison of SE_ vs_ WT shows that a total of 55 genes are expressed in this pathway, of which 29 are upregulated and 26 are downregulated. A comparison of OE_ vs_ SE shows that a total of 35 genes are regulated in this pathway, of which 7 are upregulated and 29 are downregulated genes. Lignin is polymerized from three key monomers: ρ-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, also known as the H, G, and S monolignols (Eudes et al., 2012 ). These monolignols are produced by a sequence of up to ten enzymatic processes that sequentially deaminate phenylalanine, hydroxylate the phenyl ring at the 3, 4, and 5 positions, and reduce the acid end group of the propane side chain to alcohol (Shi et al., 2010 ). The three genes PAL, C4H, and 4CL are responsible for the first three reaction steps from phenylalanine to p-coumaroyl CoA, which are part of the common route of general phenylpropanoid biosynthesis, which includes lignin and flavonoids. A total of 122 differentially expressed genes (DEGs) were found to be directly involved in the biosynthesis of monolignols and their subsequent polymerization to form lignin among the WT, OE, and SE lines. The expression analysis of key genes in the phenylpropanoid pathway was found to reveal significant transcriptional changes across OE, SE, and WT lines. Phenylalanine ammonia-lyse (PAL), a critical enzyme that catalyzes the first step of the process, is highly upregulated in both OE compared to WT and SE relative to WT. Particularly BPChr03G03333.v1.1 and BPChr03G03311.v1.1had greater expression in SE compared to WT (log 2FC: 5.55 and 3.72 respectively) than in OE relative to WT (log 2FC: 1.94 and 1.73, respectively). This suggests that suppressing another pathway component may result in a compensatory reaction, such as increasing PAL expression in SE lines to maintain metabolic flow toward lignin production. Further downstream, BPChr05G17489.v1.1 encodes 4-Coumarate: CoA Ligase (4CL), which is slightly downregulated in SE compared to WT (log2FC: -1.24), while BPChr03G18648.v1.1 is downregulated in OE relative to SE (log2FC: -1.72). This indicates that despite enhanced PAL expression, a regulatory bottleneck at the 4CL step may limit the flow of phenylpropanoids. On the contrary, Hydroxycinnamoyl-CoA: Shikimate Hydroxycinnamoyl Transferase (HCT), an essential enzyme in monolignol biosynthesis, is significantly upregulated in OE compared to WT (log2FC: 5.28 and 4.74) for BPChr06G25266.v1.1 and BPChr06G25247.v1.1, respectively. This indicates a greater metabolic commitment to the production of lignin precursor. The expression of Caffeoyl-CoA O-Methyltransferase (CCoAMT) reveals contrasting trends, with BPChr08G03026.v1.1 highly downregulated in OE compared to WT (-3.69 log2FC), whereas BPChr09G12142.v1.1 is significantly upregulated in both OE compared to WT and SE compared to WT (lof2FC: 3.60 and 1.97, respectively). This demonstrates gene-specific regulatory differences, which may be driven by feedback control mechanisms or the unique function of various CCoAMT isoforms in monolignol modification. Similarly, Cinnamyl Alcohol Dehydrogenase (CAD), which catalyzes the last step in lignin monomer biosynthesis, showed upregulation in BPChr05G14782.v1.1 for both OE comparison to WT (1.64 lof2FC) and SE contrast to WT (2.62 log2FC), showing improved lignin biosynthesis potential in both conditions. In contrast, BPChr09G20203.v1.1 shows downregulation in both comparisons, suggesting an isoform-specific function in lignification. Interestingly, peroxidase genes (BPChr11G07155.v1.1), which help in lignin polymerization, are highly downregulated in OE comparison to WT (-3.20 loG2FC) and even more significantly downregulated in SE comparison to WT (-6.71 log2FC). This indicates that, while monolignol production may be increased, the final polymerization process may be inhibited, potentially resulting in altered lignin deposition and structural changes in the cell wall. The significant downregulations of peroxidases suggest a disturbance in lignin polymerization, which might impact cell wall stiffness and overall plant structural integrity. 3.6 Validation of RNAseq data by qRT-PCR The qPCR validation results also confirmed that key genes involved in lignin biosynthesis, such as PAL, C4H, and COMT, were significantly upregulated in overexpression transgenic lines, supporting the hypothesis that BpWOX4 enhances lignification through transcriptional regulation of secondary cell wall biosynthesis pathways. Lignin is an important structural component of the secondary cell wall, providing mechanical support and resilience to biotic and abiotic stressors (Boerjan et al., 2003 ). Previous research has demonstrated that members of the WOX family can regulate lignin deposition in response to developmental and environmental signals, which supports our findings (J. Wang et al., 2013 ). 3.7 Histochemical Staining of BpWOX4 Transgenic Birch To study the effects of BpWOX4 on secondary growth, transverse sections of WT, OE, and SE plants were examined using histochemical staining and scanning electron microscopy (SEM) (Fig. 6 A-D). In OE plants, phloroglucinol-HCI staining shows greater lignin deposition than in WT plants, as well as a larger xylem area and more layers of cambial cells (Fig. 6 A-C).In contrast, SE plants showed much less lignin staining, thinner xylem regions, and fewer cambial cell layers, as well as uneven vascular development and disrupted secondary xylem structure. SEM analysis provided further evidence of cell wall alteration (Fig. 6 D). OE plants showed significantly thicker secondary cell walls in xylem fibers (1.12-fold greater than WT), whereas SE plants had weaker cell walls (0.9 times WT) and irregularly dispersed xylem cells. Quantitative measurement confirmed that BpWOX4 promotes secondary wall thickening, mostly through enhanced lignin deposition (Fig. 6 E). Phenotypic differences at the whole plant were also observed (Fig. 6 F), OE plants were significantly taller than WT. In contrast, SE plants were stunted, consistent with impaired vascular development and reduced secondary growth. Taken together, our findings show that BpWOX4 plays a critical role in promoting vascular cambium activity, secondary xylem differentiation, and lignin deposition. Overexpression of BpWOX4 promotes lignification and secondary wall thickening, whereas its suppression severely disrupts vascular organization and plant growth. 4. Discussion In tree species, different wood-related transcription factors, including NAC and MYB, have been demonstrated to play important roles in the coordinated control of secondary growth during wood formation(L. Yang et al., 2017 ). NAC transcription factors function as the master regulators by activating downstream transcription factors, including MYB, which in turn directly bind to promoters of lignin pathway genes and polymerization enzymes(Zhong et al., 2010 ). MYB transcription factors regulate nearly all stages of plant growth, development, and metabolism, and are primarily involved in controlling cell division, tissue differentiation, and the biosynthesis of both primary and secondary metabolites(Xiao et al., 2021 ). Specifically, NAC transcription factors indirectly influence key enzymes in lignin biosynthesis (PAL, C4H, 4CL, and COMT) through the activation of MYBs(J. Liu et al., 2015 ). A study in Arabidopsis showed that overexpression of EgNAC141 increased lignification, xylem development, and lignin content(Sun et al., 2021 ). The differential expression of these TFs across comparisons (OE vs. WT and SE vs. WT) highlights their dynamic role in modulating secondary growth and lignin-related gene networks. In this study, transcriptome analysis demonstrates that overexpression of BpWOX4 triggers significant changes in gene expression related to secondary growth. These results are in line with earlier research on the WOX gene family, which has been widely connected to cambial activity and vascular development in both herbaceous and woody species (Rasheed et al., 2024 ). GO and KEGG Enrichment Analysis showed that the overexpression of BpWOX4 significantly enriched many biological pathways that are directly related to secondary wall formation, hormone control, and stress responses. The most significantly enriched KEGG pathways were phenylpropanoid biosynthesis (map00940), which is most important for the synthesis of lignin, a crucial structural polymer deposited during secondary xylem growth(Zhao et al., 2022 ). This suggests that BpWOX4 may enhance lignin accumulation and cell wall reinforcement, which could explain the observed increase in secondary growth. The enrichment of plant hormone signal transduction (map04075) further supports the role of BpWOX4 function in mediating hormonal interaction, particularly auxin, cytokinin, and gibberellin, all of which regulate cambial activity and vascular differentiation(Virág et al., 2025 ). Additionally, the activation of tryptophan metabolism (map00380), a precursor to auxin biosynthesis, suggests that BpWOX4 may impact local formation of indole-3-acetic acid (IAA), hence supporting cambial cell proliferation through a hormone-regulated feedback mechanism(Jiang et al., 2022 ). Interestingly, the plant pathogen interaction pathway (map04626) was found to be enriched, suggesting that BpWOX4 may play a role in preparing the vascular tissue for defense against biotic stress, a critical function during wood production. Enrichment in galactose metabolism (map00052) indicates enhanced production or alteration of wall polysaccharides like hemicellulose and pectin, which help to maintain cell wall flexibility and structural integrity(Höftberger et al., 2022 ).Gene Ontology (GO) enrichment further supported these findings. Terms such as cell wall biogenesis, secondary metabolic process, phenylpropanoid catabolic process, and hormone-mediated signaling pathway were highly represented, indicating that BpWOX4 could be implicated in gene networks critical for secondary wall synthesis and hormonal signaling integration. The consistent enrichment of these functional categories in both OE and SE comparisons emphasizes that BpWOX4 plays an important role as a growth promoter of secondary metabolites by activating lignin biosynthesis. These findings suggest that BpWOX4 plays an important role in secondary development in B. platyphylla by regulating lignin biosynthesis, hormonal control, and stress-responsive metabolic changes. The enrichment of transcriptional regulators and cell wall-modifying enzymes reveals further mechanistic insight into how BpWOX4 promotes lignification and enhances woody biomass accumulation. In Arabidopsis thaliana, AtWOX4 has been demonstrated to sustain cambial stem cell activity, while in Populus, PtrWOX4 regulates vascular meristem proliferation and secondary growth(Hirakawa et al., 2010 ; Suer et al., 2011 ). Our research highlights the significance of BpWOX4 in xylem expansion and wood development, and further supports the conserved role of WOX genes in secondary growth control in tree species. Furthermore, the significant upregulation of lignin biosynthesis-related genes in BpWOX4 overexpressing lines implies its role in regulating the phenylpropanoid pathway, an essential metabolic pathway for the synthesis of lignin. Recent research has confirmed the biological activities of essential genes in lignin biosynthesis in several plants. For instance, phenylalanine ammonia-lyase (PAL) is the first key enzyme in lignin biosynthesis. In Brachypodium distachyon grass, knockdown of the BdPAL gene that encodes PAL enzyme resulted in a 43% reduction in lignin content and a 57% decrease in ferulate levels in stem cell walls, as well as delayed plant development and inhibited root growth(Cass et al., 2015 ). The 4-coumarate-CoA ligase (4CL) is the last step enzyme in the phenylpropanoid metabolic pathway, and suppression of the Os4CL3 gene expression in rice resulted in a significant decrease in lignin content and plant height(Gui et al., 2011 ). Furthermore, Trans-cinnamate 4-monooxygenase (C4H), a monooxygenase from the CYP73 subfamily of plant cytochrome P450 enzymes, also plays a role in lignin biosynthesis. In transgenic Nicotiana tabacum , reduced C4H activity led to a decrease in lignin content and altered lignin composition, specifically lowering the S/G unit ratio, without causing any abnormal plant growth(Sewalt et al., 1997 ). The phenylpropanoid biosynthesis pathway is essential for the secondary metabolite production, such as flavonoids and lignin. Our findings revealed a considerable increase in phenylpropanoid pathway activity in BpWOX4 overexpression lines, suggesting that this gene might influence broader metabolic processes other than lignin biosynthesis. These findings agree with earlier research, which suggests that transcription factors such as NAC and MYB can modulate wood composition and stress tolerance in forest species (Z. Wang et al., 2020 ; J. Yang et al., 2023 ). Multiple transcription factors, including MYB, NAC, WARKY, and LIM families, were found to be differentially expressed and are associated with the secondary growth and regulation of lignin biosynthesis. Overexpression of AmMYB308 and AmMYB330 in Antirrhinum majus suppressed the expression of genes involved in lignin biosynthesis, such as 4CL, C4H, and CAD, resulting in a reduction of lignin content by up to 17%(Tamagnone et al., 1998 ). The NAC transcription factor in Rosa roxburghii showed a strong negative correlation with several lignin biosynthesis-related genes, including 4CL, HCT, C3H, CCoAOMT, CCR, COMT, CAD, and POD(Li et al., 2022 ). In Nicotiana tabacum , NtLIM1 was found to specifically bind to the PALbox element and activate the expression of a β-glucuronidase reporter gene that is triggered by the promoter of the horseradish POD C2 (prxc2) gene(Kaothien et al., 2002 ). In conclusion, our transcriptome analysis identified 779 differentially expressed transcription factors that have different key roles in secondary growth and lignin biosynthesis. Numerous phytohormones, such as Indole-3-acetic acid (Steinwand et al., 2014 ), gibberellins (Singh et al., 2019 ), abscisic acid, and methyl jasmonic acid(W. Liu et al., 2021 ), have been identified as key regulatory molecules involved in the control of lignin biosynthesis in plants. Our results also showed that overexpression of BpWOX4 results in increased lignin content in Betula platyphylla . Further functional analysis of BpWOX4 could provide deeper insight into its interactions with other transcriptional regulators that control secondary metabolism in woody plants. The identification of BpWOX4 function in secondary development and lignin production brings up new possibilities for tree genetics and enhancement. Lignin modification has significant implications for the forestry and bioenergy sectors, where modified lignin composition can enhance wood quality and pulping efficiency. Furthermore, by using genetic engineering techniques like CRISPR/Cas, gene editing could improve lignin deposition without compromising plant structural integrity. Additionally, analyzing the interaction between BpWOX4 and phytohormonal signaling cascades could provide deeper insights into its regulatory mechanism. Declarations Author Contributions: H.R.: Experimental design, methodology, data analysis, and writing the original draft; X.W. and H.H.: Conceptualization, experimental design, funding acquisition, supervision, and manuscript editing; Y.L.: Genetically modified birch tree and conduct slicing and staining experiment; D.L., T.J., C.W., and B.H.: editing and improvement of the manuscript. The manuscript was read and approved by all authors. Funding: This work was supported by STI 2030-Major Projects (2023ZD0405603), the National Natural Science Foundation of China (31800556) Acknowledgment: We are thankful to Professor Jiang Jing and Liu Guifeng from the State Key Laboratory of Tree Genetics and Breeding for their guidance and assistance. Data availability: The original contributions presented in the study are included in the article. Ethical approval: Not applicable. Conflict of Interest: The authors declare no conflicts of interest that could have influenced the work reported in this paper . References Boerjan, W., Ralph, J., & Baucher, M. (2003). Lignin Biosynthesis. Annual Review of Plant Biology , 54 , 519–546. https://doi.org/10.1146/annurev.arplant.54.031902.134938 Boyce, C. K., Zwieniecki, M. A., Cody, G. D., Jacobsen, C., Wirick, S., Knoll, A. H., & Holbrook, N. M. (2004). Evolution of xylem lignification and hydrogel transport regulation. 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15:40:12","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114569,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/e68c6ddc80e9b4313cb8f959.png"},{"id":94471553,"identity":"0a3f471b-0a32-4218-86b5-a267d90eb802","added_by":"auto","created_at":"2025-10-27 15:38:04","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114661,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/e8ea749b01d9edaa1c34e7dd.png"},{"id":94471608,"identity":"9408b494-f9db-4932-839c-ae5d56c8e44b","added_by":"auto","created_at":"2025-10-27 15:38:37","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":228759,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/e1df78d2cfd7ec4141df6ab1.png"},{"id":94471833,"identity":"d3047ee3-bb70-44a5-8d81-83fa38d7df85","added_by":"auto","created_at":"2025-10-27 15:39:59","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153136,"visible":true,"origin":"","legend":"","description":"","filename":"PCTOD25007070structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/8b907d0010b3beb73d8419e2.xml"},{"id":94471652,"identity":"74735e0e-e01d-4a76-bb68-20eaf85501f2","added_by":"auto","created_at":"2025-10-27 15:39:07","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161323,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/2de0ebf703ae50016c4c2e52.html"},{"id":94471926,"identity":"4c6e0528-beb8-4fa7-ab31-12b301844fbd","added_by":"auto","created_at":"2025-10-27 15:40:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":804581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential Gene Expression Analysis (DEGs).\u003c/strong\u003e (A) Bar graph illustrates upregulated (red) and downregulated (blue) DEGs. (B) The Venn diagram shows common upregulated and downregulated DEGs. (C, D, E) Volcano plots of OE vs. WT, SE vs. WT, and OE vs. SE indicate log\u003csub\u003e2\u003c/sub\u003e fold change vs. -log Padj, with red (upregulated), blue (downregulated), and gray (non-significant) genes showing differential regulation. (F) The PCA plot shows WT (blue) vs OE (red) and SE (green), indicating divergence with partial similarity. (G) The correlation heatmap shows strong intra-group (red) and lower inter-group (blue) correlations, indicating changed regulatory networks.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/45c24fed20ed0b89cec7b4a2.jpeg"},{"id":94471894,"identity":"dd7394c0-d2c7-4e28-8a14-edc097d9ae98","added_by":"auto","created_at":"2025-10-27 15:40:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":661720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKEGG and GO Enrichment Analysis.\u003c/strong\u003e (A, B,C) GO enrichment analysis between OE_vs_WT, SE_vs_WT, and OE_vs_SE comparison, respectively (D, E,F). KEGG enrichment analysis for OE_vs_WT , SE_vs_WT, and OE_VS_SE. Dot size represents DEG count; color shows significance (Padjust). Enriched terms include phenylpropanoid biosynthesis, hormone signaling, and cell wall organization.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/68941da304cce8b88040f1bd.jpeg"},{"id":94471914,"identity":"c83aa55c-8a96-47b8-9691-76b5b2d5b852","added_by":"auto","created_at":"2025-10-27 15:40:19","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":316951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential Expression of Transcription Factors (TFs). \u003c/strong\u003e(a) Schematic illustration of the phenylpropanoid and lignin biosynthesis pathway showing transcription factors (TFs) associated with key enzymatic steps. Red arrows indicate positive transcriptional regulation, while green arrows indicate negative regulation. (B) Heatmap of Z-score normalized expression for NAC/MYB transcription factors across WT, OE, and SE, with red (upregulated) and blue (downregulated).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/cfdfd1d9a9fcaacdf564eaab.jpeg"},{"id":94471834,"identity":"ff5c0d47-d7d1-49a4-97c9-be0589b9fc72","added_by":"auto","created_at":"2025-10-27 15:39:59","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome-based identification of DEGs across the phenylpropanoid pathway: \u003c/strong\u003e(A)Cluster analysis identified key genes involved in monolignol biosynthesis with significant changes in expression. Red boxes indicate upregulated genes and green boxes indicate downregulated genes across different enzymatic steps, including PAL, 4CL, HCT, CoAMT, and CAD. The pathway highlights transcriptional regulation leading to altered biosynthesis of H-, G-, and S-monolignols. (B) Total gene count in the Phenylpropanoid pathway. (C) Heatmap of Z-score normalized expression for pathway genes WT, OE, and SE, with red (upregulated) indicating active lignin production. (D) Bar graph of lignin content (%) between OE, SE, and WT, with OE showing higher lignin content than WT and SE.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/480dd2a5dfa6884eed7999b7.jpeg"},{"id":94471904,"identity":"5ba8240e-5018-4d49-be07-637177eaebaf","added_by":"auto","created_at":"2025-10-27 15:40:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of RNA-seq data with RT-qPCR: \u003c/strong\u003eThe RNA-Seq and qRT-PCR fold changes.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/56d28f62e63ab50d4cd5fa2c.png"},{"id":94471792,"identity":"ab7ec716-839a-4a36-ad9d-b9caeec32654","added_by":"auto","created_at":"2025-10-27 15:39:50","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":404032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnatomical and morphological characterization of transgenic BpWOX4\u003c/strong\u003e: (A-C) Stem cross-section of WT and transgenic birch plants showing vascular tissue organization; (D) SEM images of xylem cells illustrating variation in secondary cell wall thickness; (E) Quantification of cell wall thickness, with OE plants significantly increased and SE plants reduced compared with WT (*p \u0026lt; 0.05); (F) Plant height analysis across separate lines.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/13f757dbf39fbed89280841a.jpeg"},{"id":103251391,"identity":"5f9d0f46-4d24-4f2e-ae0b-97cfcbd0fff8","added_by":"auto","created_at":"2026-02-23 16:08:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3842807,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/50922270-d380-47c4-8d7f-fff0225a4d5f.pdf"},{"id":94471924,"identity":"897ace87-6cc2-4237-8b7b-68aa0f863e45","added_by":"auto","created_at":"2025-10-27 15:40:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19805,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-7726648/v1/4293b3d7cb9a0dc7d5947e07.docx"}],"financialInterests":"","formattedTitle":"The WUSCHEL-Related Homeobox 4 (BpWOX4) Promotes Secondary Growth Through Lignin Biosynthesis Activation in Betula platyphylla","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTree growth occurs through two different but interconnected processes, primary growth and secondary growth. Primary growth in plants refers to the increase in the length of the plant, driven by cell division in the apical meristem, whereas secondary growth refers to the proliferation of plant organs through cell division, cell wall biosynthesis, lignification, and programmed cell death(Zhou et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These growth types manage the height, spread, and diameter of trees, shaping their form and functional adaptations across developmental stages. Cell division direction within vascular tissue is regulated by the interaction between the receptor kinase PXYv that expressed in meristematic cells, and its peptide ligand CLE41, produced by nearby phloem cells(Etchells \u0026amp; Turner, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The cambium is a meristematic tissue and develops into a closed ring during secondary growth, forming the vascular cambium, and undergoes proliferation (active cell division). Secondary growth in plant stems results from the activity of the vascular cambium, a lateral meristem that produces secondary xylem (wood) toward the inside and secondary phloem toward the outside(Mellerowicz et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Spicer \u0026amp; Groover, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The secondary xylem not only conducts water up the stem, but it also develops a strong woody structure after substantial lignification and cell death, allowing the tree to grow taller(Morris et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLignin, a phenolic heteropolymer found in plants, exhibits varying structures and contents across different species, tissues, cell types, cell layers, and environments that serves significant biological functions. Lignin is a crucial component of secondary growth, directly affecting wood formation, vascular development, and structural integrity (Somerville et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Due to its hydrophobic nature, lignin plays an essential role in water transport and serves as a major component of vascular tissue(Boyce et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Coleman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Lignin is also deposited on the cell wall through the oxidative polymerization of lignin monomers(Smith et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Following lignification, tubular elements and fibrous cells undergo programmed cell death. This process includes the disintegration of organelles, along with the degradation of the protoplast and part of the secondary cell wall that remains unlignified(Escamez \u0026amp; Tuominen, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Studies have shown transcription factors, signaling pathways, and metabolic enzymes stimulate that lignin biosynthesis. The WOX gene family function as a key regulators of developmental processes in plants, including embryonic patterning, stem cell maintenance, and organogenesis(Rasheed et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe WUSCHEL-related homeoboxes (WOX) gene family was first identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e in 1996, with its role in shoot and floral development(Laux et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). According to evolutionary history, WOXs are divided into three clades: modern/WUS, intermediate clade, and ancient clade(Graaff et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Previous study showed the WOX gene family has a function in structuring several early plant cell populations(Ji et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Members of the WOX protein family are essential for the maintenance and proliferation of stem cells in cambium, the lateral meristem that responsible for generating all cellular components of wood(Galibina et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some WOX genes have been linked to lignin biosynthesis through their influence on gibberellin (GA) signaling and secondary cell wall formation(Y. Zhang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overexpression of \u003cem\u003ePtrWOX13A\u003c/em\u003e in poplar significantly enhanced the growth potential in transgenic lines and resulted in increased secondary cell wall thicknesses, longer fiber length, as well as lignin and hemicellulose contents(Y. Zhang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Overexpression of \u003cem\u003eWOX4\u003c/em\u003e in \u003cem\u003ePopulus\u003c/em\u003e resulted in increased cambial activity and led to a higher lignin deposition in xylem tissues(M. Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Study in \u003cem\u003eArabidopsis\u003c/em\u003e revealed the loss of \u003cem\u003eWOX4\u003c/em\u003e did not affect xylem differentiation but impaired cambial cell proliferation(Suer et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Another study demonstrated that the knockdown of \u003cem\u003eGhWOX4\u003c/em\u003e gene hindered secondary growth by reducing cambial width and division activity compared with control plants (Sajjad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhite birch (\u003cem\u003eB. Platyphylla\u003c/em\u003e) is a broad-leaved pioneer species in eastern Asia, where it contributes significantly to the stabilization and regeneration of forest ecosystem (Y. Wang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to its vast source of biomass, \u003cem\u003eB. platyphylla\u003c/em\u003e is commonly used to make biofuel, building materials, pulp and paper, and other valuable chemicals(Zhao et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is a monoecious species with distinct male and female inflorescences, making it significantly different from model plant species like \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eAntirrhinum\u003c/em\u003e(Endress, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Our research investigates the function of \u003cem\u003eBpWOX4\u003c/em\u003e in \u003cem\u003eB. platyphylla\u003c/em\u003e, identifies its regulatory mechanism and involvement in promoting secondary development and lignification in woody plant. This research gives new insights into the genetic regulation of vascular cambium activity and contributes to a better knowledge of secondary development in trees.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1Plants materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clones of \u003cem\u003eBetula platyphylla\u003c/em\u003e used in this study were propagated through \u003cstrong\u003ein vitro tissue culture\u003c/strong\u003e. The \u003cem\u003eBpWOX4\u003c/em\u003e gene was constructed from \u003cem\u003eBetula platypylla\u003c/em\u003e cDNA using specific primers and then cloned into a vector via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Transgenic \u003cem\u003eBpWOX4\u003c/em\u003e-overexpression (OE), \u003cem\u003eBpWOX4\u003c/em\u003e-suppression (SE), and wild-type (WT) lines were subsequently cultivated in 25 cm \u0026times; 25 cm pots and received standard irrigation and fertilization throughout their growth. All plants were \u003cstrong\u003egrown under field conditions\u003c/strong\u003e for two years in the Northeast Forestry University, Harbin, located in Heilongjiang Province, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 RNA Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNine samples of \u003cem\u003eB.platyphylla\u003c/em\u003e were selected for transcriptome analysis, comprising three biological replicates each from wild-type(WT1, WT2, WT3), overexpression (OE1, OE2, OE3), and suppression (SE1, SE2, SE3)lines. The shoot tissues were collected by scraping the stem after removing the bark and stored at -80℃ in an ultra-low temperature freezer. Subsequently, the samples were sent to Majorbio company (www.majorbio.com) for RNA extraction and transcriptome sequencing. Differential gene expression analysis was performed using the DESeq2 method to identify differentially expressed sequence\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eDEGs across different groups. Additionally, pathway annotation analysis was conducts on the DEGs to elucidate their functional roles. All data analyses were carried out using the R language programming (cran.rstudio.com) and Python software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 RT-qPCR validation of DEGs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR was performed to validate the transcript levels of selected genes. Total RNAs were extracted separately from the shoot of samples using the protocol provided with the E.Z.N.A Plant RNA Kit (www.omegabiotek.com.cn). A NanoDrop 2000 spectrophotometer was used to analyze RNA quantity and purity. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer. Only samples with an RNA Integrity Number (RIN) higher than 8 were kept at -80\u0026deg;C for further analysis. First-strand complementary DNA (cDNA) was synthesized from 7 \u0026mu;L of total RNA by using the Primescript\u003csup\u003eTM\u003c/sup\u003e RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) following the manufacturer\u0026rsquo;s protocol. QRT-PCR was performed on a 96-well Analytik Jena (aj) PCR instrument in accordance with the Lablead protocol (www.lablead.cn). Target gene The relative expression levels of target genes were determined using the2\u003csup\u003e-\u003c/sup\u003e \u003csup\u003e\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method, with 18S rRNA and tubulin serving as reference genes. Primers with a temperature of 58 to 60\u0026deg;C and nucleotide lengths of 18 to 25 bp were designed using NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Lignin Content Determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-year-old \u003cem\u003eB. platyphylla\u003c/em\u003e plants were selected for lignin determination. Stems were cut at a height of 3cm above ground using pruning shears, and the bark was removed from the sampled region. The sample were then oven-dried at 60\u0026deg;C for 72 hours. Lignin content was determined following the protocol provided by the Suzhou Grace Biotechnology CO., Ltd (www.geruisi-bio.com). Three biological replicates were used for each sample. Data analysis was performed using Python software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Histochemical Staining of \u003cem\u003eBpWOX4\u003c/em\u003e Transgenic birch\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStem segments were collected from six-month-old WT, OE, and RE birch lines and were manually sectioned. To detect the lignin deposition, transverse sections were stained with phloroglucinol-HCl, which produces a characteristic red coloration in lignified cell walls. The stained samples were examined under a light microscope, and anatomical features-including xylem width, number of cambial cell layers, and vascular tissue organization- were compared among the WT and both transgenic lines.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 RNA-seq Data Quality and Mapping Efficiency\u003c/h2\u003e\u003cp\u003eRNA sequencing produced between 42,538,698 and 56,062,972 raw reads per sample. After quality control, a high proportion of clean readings was maintained, ranging from 42,003,412 to 55,464,274, showing minimal loss during filtering. The subsequent alignment to the reference genome resulted in an efficient mapping rate across all samples, ranging from 91.44% to 92.09%, with an average mapping rate above 91%. This mapping efficiency demonstrates reliability and a high degree of sequence specificity to the reference genome. The GC content was consistent across all samples, ranging from 44.71% to 45.52%, indicating that the nucleotide makeup was uniform across experimental groups. The slight variations in GC content reflect natural biological differences rather than technical bias. Overall, the RNA-seq data quality was high, with adequate read depth, strong mapping efficiency, and stable GC content, ensuring requirement for downstream transcriptome analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Analysis of Differentially Expressed Genes (DEGs)\u003c/h2\u003e\u003cp\u003eThe DESeq2 package (version 4.5.0) in R was used to conduct differential expression analysis on all acquired reads, normalized as reads per kilobase of transcript per million mapped reads (RPKM)(Love et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To identify differentially expressed genes (DEGs), pairwise comparisons were performed with criteria of log2 fold change (log2FC)\u0026thinsp;\u0026gt;\u0026thinsp;1 or \u0026lt;-1 and a p-value (-log10padj). In the OE_ vs_ WT comparison, 4355 differently expressed genes (DEGs) were identified, with 2220 upregulated and 2135 downregulated. The SE_vs_WT comparison revealed a greater number of DEGs (6950), including 3468 upregulated and 3482 downregulated genes, indicating more comprehensive transcriptome changes in this group. Similarly, the OE_ vs_ SE analysis found 3989 DEGs, with 1935 genes upregulated and 2054 genes downregulated, indicating unique transcriptional profiles in these two groups. The distribution and overlap of DEGs were further assessed using Venn analysis. The analysis identified a set of common DEGs shared among comparisons, as well as unique subsets specific to OE or SE plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The existence of shared DEGs indicates a core transcriptional response to BpWOX4 disruption, whereas the unique subsets reveal regulatory alternations to each condition.\u003c/p\u003e\u003cp\u003eTo visualize the distribution of DEGs, volcano plots were constructed for each comparison. In the OE_vs_WT, the majority of genes clustered around the non-significant region, with significant upregulated and downregulated gene populations visible at higher Log2FC values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The SE_VS_WT displayed a similar pattern but more significant DEGs, indicating the broader transcriptional impact of the suppressor element (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The OE_vs_SE revealed a balanced distribution of upregulated and downregulated genes, emphasizing the regulatory interaction between two conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The volcano plots not only corroborated the DES analysis but also provided a clear visual representation of the magnitude and significance of gene expression changes.\u003c/p\u003e\u003cp\u003eBeyond DEG counts, we analyzed global expression patterns using principal component analysis (PCA) and correlation heatmap. The PCA plot clearly separated OE, SE, and WT samples into distinct clusters, with OE and SE showing divergence from WT but partial overlap with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This separation demonstrates that \u003cem\u003eBpWOX4\u003c/em\u003e expression status is a significant determinant of transcriptional variance across lines. The correlation heatmap further supported these findings, showing strong intra-group correlations(red) and weaker inter-group correlations (blue) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). This indicates consistent expression across biological replicates and robust transcriptional reprogramming between groups.\u003c/p\u003e\u003cp\u003eIn conclusion, our combined analysis of DEGs and volcano reveals the extensive transcriptional reprogramming in overexpression and suppression plants. The significant number of DEGs and their distribution in the volcano plot highlight the complexity of gene regulatory networks in response to genetic alterations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 GO and KEGG Enrichment Analysis\u003c/h2\u003e\u003cp\u003eTo better understand the molecular processes regulating \u003cem\u003eWOX\u003c/em\u003e gene activity in \u003cem\u003eB. platyphylla\u003c/em\u003e, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on WOX-overexpression (OE) and WOX-silenced (SE) transgenic lines compared with wild-type (WT). The analysis revealed that DEGs were significantly enriched in pathways related to secondary wall formation, hormone regulation, and stress responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The most significantly enriched KEGG pathway was phenylpropanoid biosynthesis (map00940). In addition, enrichment was shown in plant hormone signal transduction (map04075), plant\u0026ndash;pathogen interaction (map04626), tryptophan metabolism (map00380, a precursor to auxin biosynthesis), and galactose metabolism (map00052).\u003c/p\u003e\u003cp\u003eGene Ontology (GO) enrichment showed that terms such as cell wall biogenesis, secondary metabolic process, phenylpropanoid catabolic process, and hormone-mediated signaling pathway were highly represented, indicating that \u003cem\u003eBpWOX4\u003c/em\u003e may regulate gene networks that are critical for secondary wall synthesis and hormonal signaling integration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Differentially expressed TFs are regulated during secondary growth in \u003cem\u003eB.platyphylla\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTranscription factors (TFs) regulate secondary growth, lignin biosynthesis, and ROS homeostasis in plants, and MYB, NAC, WRKY, and LIM are particularly important for this developmental processes (Khan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Q. Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A total of 779 transcription factor (TF) genes were identified in this study, among these, 80 MYB and 46 NAC transcription factors were significantly expressed. The differential expression of these TFs across our comparisons (OE vs. WT and SE vs. WT) highlights their dynamic role in modulating secondary growth and lignin-related pathways, which is a key finding of our study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Lignin biosynthesis pathway is significantly enriched in the \u003cem\u003eBpWOX4\u003c/em\u003e plants\u003c/h2\u003e\u003cp\u003eLignin pathway was found to be the most significant in the pathway analysis. Lignin maintains the structural integrity, strength, and hardness of the cell wall, supports water transportation, prevents cell wall penetration, and protects plants against pathogen invasion(Meng et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, the main synthetase genes in the phenylpropane biosynthesis pathway were identified by annotating and analyzing the sequence findings in the KEGG pathway. Our transcriptome analysis data revealed the important genes that are involved in the lignin biosynthesis pathway and we compared the lignin content between WT, OE, and SE lines. Our data shows that the lignin content of WT plants is greater than that of SE plants and less than OE plants. KEGG pathway analysis reveals that OE_ vs_ WT comparison posses a total of 32 genes expressed in this pathway, in which 13 are upregulated and 19 are down-regulated. A comparison of SE_ vs_ WT shows that a total of 55 genes are expressed in this pathway, of which 29 are upregulated and 26 are downregulated. A comparison of OE_ vs_ SE shows that a total of 35 genes are regulated in this pathway, of which 7 are upregulated and 29 are downregulated genes.\u003c/p\u003e\u003cp\u003eLignin is polymerized from three key monomers: ρ-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, also known as the H, G, and S monolignols (Eudes et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These monolignols are produced by a sequence of up to ten enzymatic processes that sequentially deaminate phenylalanine, hydroxylate the phenyl ring at the 3, 4, and 5 positions, and reduce the acid end group of the propane side chain to alcohol (Shi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The three genes PAL, C4H, and 4CL are responsible for the first three reaction steps from phenylalanine to p-coumaroyl CoA, which are part of the common route of general phenylpropanoid biosynthesis, which includes lignin and flavonoids.\u003c/p\u003e\u003cp\u003eA total of 122 differentially expressed genes (DEGs) were found to be directly involved in the biosynthesis of monolignols and their subsequent polymerization to form lignin among the WT, OE, and SE lines. The expression analysis of key genes in the phenylpropanoid pathway was found to reveal significant transcriptional changes across OE, SE, and WT lines. Phenylalanine ammonia-lyse (PAL), a critical enzyme that catalyzes the first step of the process, is highly upregulated in both OE compared to WT and SE relative to WT. Particularly BPChr03G03333.v1.1 and BPChr03G03311.v1.1had greater expression in SE compared to WT (log 2FC: 5.55 and 3.72 respectively) than in OE relative to WT (log 2FC: 1.94 and 1.73, respectively). This suggests that suppressing another pathway component may result in a compensatory reaction, such as increasing PAL expression in SE lines to maintain metabolic flow toward lignin production.\u003c/p\u003e\u003cp\u003eFurther downstream, BPChr05G17489.v1.1 encodes 4-Coumarate: CoA Ligase (4CL), which is slightly downregulated in SE compared to WT (log2FC: -1.24), while BPChr03G18648.v1.1 is downregulated in OE relative to SE (log2FC: -1.72). This indicates that despite enhanced PAL expression, a regulatory bottleneck at the 4CL step may limit the flow of phenylpropanoids. On the contrary, Hydroxycinnamoyl-CoA: Shikimate Hydroxycinnamoyl Transferase (HCT), an essential enzyme in monolignol biosynthesis, is significantly upregulated in OE compared to WT (log2FC: 5.28 and 4.74) for BPChr06G25266.v1.1 and BPChr06G25247.v1.1, respectively. This indicates a greater metabolic commitment to the production of lignin precursor. The expression of Caffeoyl-CoA O-Methyltransferase (CCoAMT) reveals contrasting trends, with BPChr08G03026.v1.1 highly downregulated in OE compared to WT (-3.69 log2FC), whereas BPChr09G12142.v1.1 is significantly upregulated in both OE compared to WT and SE compared to WT (lof2FC: 3.60 and 1.97, respectively). This demonstrates gene-specific regulatory differences, which may be driven by feedback control mechanisms or the unique function of various CCoAMT isoforms in monolignol modification.\u003c/p\u003e\u003cp\u003eSimilarly, Cinnamyl Alcohol Dehydrogenase (CAD), which catalyzes the last step in lignin monomer biosynthesis, showed upregulation in BPChr05G14782.v1.1 for both OE comparison to WT (1.64 lof2FC) and SE contrast to WT (2.62 log2FC), showing improved lignin biosynthesis potential in both conditions. In contrast, BPChr09G20203.v1.1 shows downregulation in both comparisons, suggesting an isoform-specific function in lignification. Interestingly, peroxidase genes (BPChr11G07155.v1.1), which help in lignin polymerization, are highly downregulated in OE comparison to WT (-3.20 loG2FC) and even more significantly downregulated in SE comparison to WT (-6.71 log2FC). This indicates that, while monolignol production may be increased, the final polymerization process may be inhibited, potentially resulting in altered lignin deposition and structural changes in the cell wall. The significant downregulations of peroxidases suggest a disturbance in lignin polymerization, which might impact cell wall stiffness and overall plant structural integrity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Validation of RNAseq data by qRT-PCR\u003c/h2\u003e\u003cp\u003eThe qPCR validation results also confirmed that key genes involved in lignin biosynthesis, such as PAL, C4H, and COMT, were significantly upregulated in overexpression transgenic lines, supporting the hypothesis that \u003cem\u003eBpWOX4\u003c/em\u003e enhances lignification through transcriptional regulation of secondary cell wall biosynthesis pathways. Lignin is an important structural component of the secondary cell wall, providing mechanical support and resilience to biotic and abiotic stressors (Boerjan et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Previous research has demonstrated that members of the WOX family can regulate lignin deposition in response to developmental and environmental signals, which supports our findings (J. Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Histochemical Staining of \u003cem\u003eBpWOX4\u003c/em\u003e Transgenic Birch\u003c/h2\u003e\u003cp\u003eTo study the effects of BpWOX4 on secondary growth, transverse sections of WT, OE, and SE plants were examined using histochemical staining and scanning electron microscopy (SEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). In OE plants, phloroglucinol-HCI staining shows greater lignin deposition than in WT plants, as well as a larger xylem area and more layers of cambial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).In contrast, SE plants showed much less lignin staining, thinner xylem regions, and fewer cambial cell layers, as well as uneven vascular development and disrupted secondary xylem structure. SEM analysis provided further evidence of cell wall alteration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). OE plants showed significantly thicker secondary cell walls in xylem fibers (1.12-fold greater than WT), whereas SE plants had weaker cell walls (0.9 times WT) and irregularly dispersed xylem cells. Quantitative measurement confirmed that \u003cem\u003eBpWOX4\u003c/em\u003e promotes secondary wall thickening, mostly through enhanced lignin deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Phenotypic differences at the whole plant were also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), OE plants were significantly taller than WT. In contrast, SE plants were stunted, consistent with impaired vascular development and reduced secondary growth.\u003c/p\u003e\u003cp\u003eTaken together, our findings show that BpWOX4 plays a critical role in promoting vascular cambium activity, secondary xylem differentiation, and lignin deposition. Overexpression of BpWOX4 promotes lignification and secondary wall thickening, whereas its suppression severely disrupts vascular organization and plant growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn tree species, different wood-related transcription factors, including NAC and MYB, have been demonstrated to play important roles in the coordinated control of secondary growth during wood formation(L. Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). NAC transcription factors function as the master regulators by activating downstream transcription factors, including MYB, which in turn directly bind to promoters of lignin pathway genes and polymerization enzymes(Zhong et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMYB transcription factors regulate nearly all stages of plant growth, development, and metabolism, and are primarily involved in controlling cell division, tissue differentiation, and the biosynthesis of both primary and secondary metabolites(Xiao et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specifically, NAC transcription factors indirectly influence key enzymes in lignin biosynthesis (PAL, C4H, 4CL, and COMT) through the activation of MYBs(J. Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). A study in \u003cem\u003eArabidopsis\u003c/em\u003e showed that overexpression of \u003cem\u003eEgNAC141\u003c/em\u003e increased lignification, xylem development, and lignin content(Sun et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The differential expression of these TFs across comparisons (OE vs. WT and SE vs. WT) highlights their dynamic role in modulating secondary growth and lignin-related gene networks. In this study, transcriptome analysis demonstrates that overexpression of \u003cem\u003eBpWOX4\u003c/em\u003e triggers significant changes in gene expression related to secondary growth. These results are in line with earlier research on the \u003cem\u003eWOX\u003c/em\u003e gene family, which has been widely connected to cambial activity and vascular development in both herbaceous and woody species (Rasheed et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGO and KEGG Enrichment Analysis showed that the overexpression of \u003cem\u003eBpWOX4\u003c/em\u003e significantly enriched many biological pathways that are directly related to secondary wall formation, hormone control, and stress responses. The most significantly enriched KEGG pathways were phenylpropanoid biosynthesis (map00940), which is most important for the synthesis of lignin, a crucial structural polymer deposited during secondary xylem growth(Zhao et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This suggests that \u003cem\u003eBpWOX4\u003c/em\u003e may enhance lignin accumulation and cell wall reinforcement, which could explain the observed increase in secondary growth. The enrichment of plant hormone signal transduction (map04075) further supports the role of \u003cem\u003eBpWOX4\u003c/em\u003e function in mediating hormonal interaction, particularly auxin, cytokinin, and gibberellin, all of which regulate cambial activity and vascular differentiation(Vir\u0026aacute;g et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, the activation of tryptophan metabolism (map00380), a precursor to auxin biosynthesis, suggests that \u003cem\u003eBpWOX4\u003c/em\u003e may impact local formation of indole-3-acetic acid (IAA), hence supporting cambial cell proliferation through a hormone-regulated feedback mechanism(Jiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Interestingly, the plant pathogen interaction pathway (map04626) was found to be enriched, suggesting that \u003cem\u003eBpWOX4\u003c/em\u003e may play a role in preparing the vascular tissue for defense against biotic stress, a critical function during wood production. Enrichment in galactose metabolism (map00052) indicates enhanced production or alteration of wall polysaccharides like hemicellulose and pectin, which help to maintain cell wall flexibility and structural integrity(H\u0026ouml;ftberger et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).Gene Ontology (GO) enrichment further supported these findings. Terms such as cell wall biogenesis, secondary metabolic process, phenylpropanoid catabolic process, and hormone-mediated signaling pathway were highly represented, indicating that \u003cem\u003eBpWOX4\u003c/em\u003e could be implicated in gene networks critical for secondary wall synthesis and hormonal signaling integration. The consistent enrichment of these functional categories in both OE and SE comparisons emphasizes that \u003cem\u003eBpWOX4\u003c/em\u003e plays an important role as a growth promoter of secondary metabolites by activating lignin biosynthesis. These findings suggest that \u003cem\u003eBpWOX4\u003c/em\u003e plays an important role in secondary development in \u003cem\u003eB. platyphylla\u003c/em\u003e by regulating lignin biosynthesis, hormonal control, and stress-responsive metabolic changes. The enrichment of transcriptional regulators and cell wall-modifying enzymes reveals further mechanistic insight into how \u003cem\u003eBpWOX4\u003c/em\u003e promotes lignification and enhances woody biomass accumulation.\u003c/p\u003e\u003cp\u003eIn Arabidopsis thaliana, \u003cem\u003eAtWOX4\u003c/em\u003e has been demonstrated to sustain cambial stem cell activity, while in Populus, \u003cem\u003ePtrWOX4\u003c/em\u003e regulates vascular meristem proliferation and secondary growth(Hirakawa et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Suer et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Our research highlights the significance of \u003cem\u003eBpWOX4\u003c/em\u003e in xylem expansion and wood development, and further supports the conserved role of WOX genes in secondary growth control in tree species. Furthermore, the significant upregulation of lignin biosynthesis-related genes in \u003cem\u003eBpWOX4\u003c/em\u003e overexpressing lines implies its role in regulating the phenylpropanoid pathway, an essential metabolic pathway for the synthesis of lignin. Recent research has confirmed the biological activities of essential genes in lignin biosynthesis in several plants. For instance, phenylalanine ammonia-lyase (PAL) is the first key enzyme in lignin biosynthesis. In \u003cem\u003eBrachypodium distachyon\u003c/em\u003e grass, knockdown of the \u003cem\u003eBdPAL\u003c/em\u003e gene that encodes PAL enzyme resulted in a 43% reduction in lignin content and a 57% decrease in ferulate levels in stem cell walls, as well as delayed plant development and inhibited root growth(Cass et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The 4-coumarate-CoA ligase (4CL) is the last step enzyme in the phenylpropanoid metabolic pathway, and suppression of the \u003cem\u003eOs4CL3\u003c/em\u003e gene expression in rice resulted in a significant decrease in lignin content and plant height(Gui et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Furthermore, Trans-cinnamate 4-monooxygenase (C4H), a monooxygenase from the CYP73 subfamily of plant cytochrome P450 enzymes, also plays a role in lignin biosynthesis. In transgenic \u003cem\u003eNicotiana tabacum\u003c/em\u003e, reduced C4H activity led to a decrease in lignin content and altered lignin composition, specifically lowering the S/G unit ratio, without causing any abnormal plant growth(Sewalt et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe phenylpropanoid biosynthesis pathway is essential for the secondary metabolite production, such as flavonoids and lignin. Our findings revealed a considerable increase in phenylpropanoid pathway activity in \u003cem\u003eBpWOX4\u003c/em\u003e overexpression lines, suggesting that this gene might influence broader metabolic processes other than lignin biosynthesis. These findings agree with earlier research, which suggests that transcription factors such as \u003cem\u003eNAC\u003c/em\u003e and \u003cem\u003eMYB\u003c/em\u003e can modulate wood composition and stress tolerance in forest species (Z. Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; J. Yang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Multiple transcription factors, including MYB, NAC, WARKY, and LIM families, were found to be differentially expressed and are associated with the secondary growth and regulation of lignin biosynthesis. Overexpression of \u003cem\u003eAmMYB308\u003c/em\u003e and \u003cem\u003eAmMYB330\u003c/em\u003e in \u003cem\u003eAntirrhinum majus\u003c/em\u003e suppressed the expression of genes involved in lignin biosynthesis, such as 4CL, C4H, and CAD, resulting in a reduction of lignin content by up to 17%(Tamagnone et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The NAC transcription factor in \u003cem\u003eRosa roxburghii\u003c/em\u003e showed a strong negative correlation with several lignin biosynthesis-related genes, including 4CL, HCT, C3H, CCoAOMT, CCR, COMT, CAD, and POD(Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In \u003cem\u003eNicotiana tabacum\u003c/em\u003e, \u003cem\u003eNtLIM1\u003c/em\u003e was found to specifically bind to the PALbox element and activate the expression of a β-glucuronidase reporter gene that is triggered by the promoter of the horseradish POD C2 (prxc2) gene(Kaothien et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, our transcriptome analysis identified 779 differentially expressed transcription factors that have different key roles in secondary growth and lignin biosynthesis. Numerous phytohormones, such as Indole-3-acetic acid (Steinwand et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), gibberellins (Singh et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), abscisic acid, and methyl jasmonic acid(W. Liu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), have been identified as key regulatory molecules involved in the control of lignin biosynthesis in plants. Our results also showed that overexpression of \u003cem\u003eBpWOX4\u003c/em\u003e results in increased lignin content in \u003cem\u003eBetula platyphylla\u003c/em\u003e. Further functional analysis of \u003cem\u003eBpWOX4\u003c/em\u003e could provide deeper insight into its interactions with other transcriptional regulators that control secondary metabolism in woody plants.\u003c/p\u003e\u003cp\u003eThe identification of \u003cem\u003eBpWOX4\u003c/em\u003e function in secondary development and lignin production brings up new possibilities for tree genetics and enhancement. Lignin modification has significant implications for the forestry and bioenergy sectors, where modified lignin composition can enhance wood quality and pulping efficiency. Furthermore, by using genetic engineering techniques like CRISPR/Cas, gene editing could improve lignin deposition without compromising plant structural integrity. Additionally, analyzing the interaction between \u003cem\u003eBpWOX4\u003c/em\u003e and phytohormonal signaling cascades could provide deeper insights into its regulatory mechanism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eH.R.: Experimental design, methodology, data analysis, and writing the original draft; X.W. and H.H.: Conceptualization, experimental design, funding acquisition, supervision, and manuscript editing; Y.L.: Genetically modified birch tree and conduct slicing and staining experiment; D.L., T.J., C.W., and B.H.: editing and improvement of the manuscript. The manuscript was read and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was supported by STI 2030-Major Projects (2023ZD0405603), the National Natural Science Foundation of China (31800556)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe are thankful to Professor Jiang Jing and Liu Guifeng from the State Key Laboratory of Tree Genetics and Breeding for their guidance and assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The original contributions presented in the study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval: Not applicable.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest that could have influenced the work reported in this paper\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoerjan, W., Ralph, J., \u0026amp; Baucher, M. 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Full-Length Transcriptome Analysis of the Secondary-Growth-Related Genes of Pinus massoniana Lamb. with Different Diameter Growth Rates. \u003cem\u003eForests\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(4). https://doi.org/10.3390/f14040811\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Secondary growth, WOX, Betula platyphylla, Lignin, Transcriptome analysis","lastPublishedDoi":"10.21203/rs.3.rs-7726648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7726648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSecondary growth is a key process in the growth and development of woody plants, influencing biomass accumulation and structural integrity. The WUSCHEL- related homeobox 4 (\u003cem\u003eBpWOX4\u003c/em\u003e) gene regulates vascular cambium activity in trees; however, its precise role in secondary growth in \u003cem\u003eBetula platyphylla\u003c/em\u003e remains poorly understood. In this study, we functionally characterized \u003cem\u003eBpWOX4\u003c/em\u003e and its role in secondary growth through a combination of transcriptomic, physiological, and histological analyses. Transcriptome analysis among wild-type (WT), overexpression (O), and suppression (SE) lines revealed upregulation of lignin biosynthesis genes, such as \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003e4CL\u003c/em\u003e, \u003cem\u003eCCR\u003c/em\u003e, and \u003cem\u003eCAD\u003c/em\u003e. Gene Ontology (GO) and KEGG enrichment analyses indicated activation of the phenylpropanoid and lignin biosynthesis pathways. Additionally, key transcription factors involved in secondary growth, including members of the MYB and NAC families, were significantly upregulated in overexpression lines. Furthermore, overexpression of \u003cem\u003eBpWOX4\u003c/em\u003e in \u003cem\u003eB. platyphylla\u003c/em\u003e resulted in increased stem diameter and xylem thickness, as well as significantly higher lignin content. Together, these results provide new insights into the molecular mechanisms underlying secondary growth and identify \u003cem\u003eBpWOX4\u003c/em\u003e as a promising genetic target for enhancing biomass production and wood quality in forest trees.\u003c/p\u003e","manuscriptTitle":"The WUSCHEL-Related Homeobox 4 (BpWOX4) Promotes Secondary Growth Through Lignin Biosynthesis Activation in Betula platyphylla","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 14:19:06","doi":"10.21203/rs.3.rs-7726648/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-16T02:57:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-13T09:32:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T05:40:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-09-29T22:07:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1a6e3ba7-b87a-4c6b-b8b7-e52fea6f0d94","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:05:15+00:00","versionOfRecord":{"articleIdentity":"rs-7726648","link":"https://doi.org/10.1007/s11240-025-03334-6","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-02-19 15:59:38","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-10-27 14:19:06","video":"","vorDoi":"10.1007/s11240-025-03334-6","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03334-6","workflowStages":[]},"version":"v1","identity":"rs-7726648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7726648","identity":"rs-7726648","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}