Heterologous expression of Pinus massonianaVNS2 enhances secondary cell wall deposition in poplar

Heterologous expression of Pinus massonianaVNS2 enhances secondary cell wall deposition in poplar | 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 Heterologous expression of Pinus massonianaVNS2 enhances secondary cell wall deposition in poplar Yating Tian, Kang Wang, Hao Rong, Meng Xu, Zhouxian Ni, Li-an Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7931415/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract NAC-domain transcription factors (TFs) play central roles in regulating secondary cell wall (SCW) biosynthesis during wood formation. In this study, we identified PmVNS2 , a VND-subfamily NAC gene from Pinus massoniana , encoding a 353-amino acid protein with conserved NAC domains. PmVNS2 was preferentially expressed in developing xylem. Heterologous expression in Populus davidiana × P . bolleana significantly enhanced plant growth, increased the number of secondary xylem cell layers, and thickened SCWs (3.45 ± 0.27 μm vs. CK: 1.91 ± 0.18 μm). Transgenic lines showed upregulated expression of lignin biosynthetic genes ( CCR2 , C4H2 ), cellulose synthase genes ( CesA2B , CesA3A ), and MYB transcription factors ( MYB021 , MYB128 ), while xylan biosynthesis genes ( GT43B , GT43D ) were downregulated. These results demonstrate that PmVNS2 is a key regulator of SCW formation in conifers, offering a promising genetic target for wood quality improvement. Pinus massoniana NAC transcription factor Second cell wall Xylem differentiation Transgenic poplar Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message PmVNS2 promotes secondary cell wall formation and enhances xylem development in transgenic poplar. 1. Introduction Wood formation represents a complex biological process orchestrated through multi-tiered transcriptional regulatory networks that precisely coordinate cambial activity, secondary cell wall (SCW) biosynthesis, and programmed cell death (PCD) (Ye and Zhong 2015 ; Zhang, et al. 2024 ). This intricate mechanism involves the synergistic action of thousands of genes, and elucidating its molecular regulation provides a theoretical foundation for targeted genetic improvement of tree species. Notably, recent research has underscored the pivotal role of NAC-MYB transcriptional networks in regulating SCW formation across both angiosperms and gymnosperms (Akiyoshi 2020; Chen, et al. 2019 ; Hao, et al. 2024 ; Johnsson, et al. 2019 ; Nakano, et al. 2015 ; Wang, et al. 2020 ). The NAC-MYB network serves as the primary regulatory hub for xylem cell differentiation, with the VNS (VND-, NST/SND- and SMB-related proteins) subfamily of the NAC TF family emerging as a dominant player in SCW formation (Ge, et al. 2020 ; Hussey, et al. 2013 ; Nakano, et al. 2015 ; Yamaguchi, et al. 2008 ). In Arabidopsis , the VNS subfamily is further divided into three distinct groups: VND (vascular-related NAC domain, e.g., VND1 - VND7 ), NST (NAC secondary wall thickening promoting factors, e.g., NST1 , NST2 , and NST3 ), and SMB (Sombrero, SMB , BRN1 , and BRN2 )(Kubo, et al. 2005 ; Mitsuda, et al. 2007 ). Similarly, in Populus trichocarpa , 16 VNS proteins have been identified and classified into VND ( PtVNS01 - PtVNS08 ), NST ( PtVNS09 - PtVNS12 ), and SMB ( PtVNS13 - PtVNS16 ) subfamilies (Ohtani, et al. 2011 ; Takata, et al. 2019 ; Zhong, et al. 2010 ). MYB TFs, such as AtMYB46 and AtMYB83 , act as second-layer regulatory switches downstream of VNS proteins, further modulating SCW formation (Hussey, et al. 2013 ; Ko, et al. 2014 ; McCarthy, et al. 2009 ; Nakano, et al. 2015 ). The lignification process, integral to plant cell wall formation, involves intracellular monomer biosynthesis and polymerization, requiring upregulation of related enzyme genes by VNS and MYB proteins (Nakano, et al. 2015 ; Zhong and Ye 2012 ). The conservation of the SCW biosynthesis mechanism, involving VNS genes, has been observed across diverse vascular plant species (Hussey, et al. 2013 ; Valdivia, et al. 2013 ; Zhong, et al. 2010 ; Zhong, et al. 2011 ). For instance, while Physcomitrella patens lacks a typical SCW structure, poplar PpVNSs can regulate homologous MYB46 and MYB83 in P. patens (Bo Xu 2014). In Pinus taeda , the instantaneous overexpression of four VNS genes ( PtaVNS1 - 3 and PtaVNS5 ) induces ectopic SCW deposition and upregulates SCW biosynthesis-related genes (e.g., MYB4 , CesA , and XCP1 gene) in tobacco (Akiyoshi 2020). Additionally, the PpNAC1 gene from Pinus pinaster , a homolog of Arabidopsis NST3 and NST1 , plays a critical role in SCW biosynthesis. Its inhibition disrupts the radial pattern of stem vascular bundles and downregulates SCW biosynthesis-related genes, while its binding to the PpMYB4 promoter activates the expression of PpMYB8 , a downstream transcriptional regulator (Pascual, et al. 2018 ). In Picea glauca , PgNAC7 is a key regulatory factor for SCW biosynthesis in coniferous wood (Duval, et al. 2014 ; Lamara, et al. 2016 ). These findings suggest the conservation of biological functions and regulatory stability within the NAC TF family, further indicating that the NAC-MYB-based transcriptional regulatory network may have a common evolutionary origin across lower and higher vascular plants. Pinus massoniana Lamb (Pinaceae), celebrated for its rapid growth rate, straight trunk profile, and exceptional resilience to drought and in fertile soils, stands as a cornerstone species in the afforestation of barren hills, the establishment of large-diameter timber forests, and the sustainable production of industrial raw material across southern China (Quan and Ding 2017 ; Yu, et al. 2024 ; Zhang, et al. 2023 ). While prior studies have primarily focused on its gene functions in cellulose and lignin biosynthesis (Ha Van, et al. 2012 ; Ni, et al. 2020 ), the molecular regulatory mechanisms underlying SCW biosynthesis remain underexplored in this taxon. In this study, we identified a VND-subfamily gene in P. massoniana . Tissue-specific profiling revealed preferential PmVNS2 transcription in developing xylem. Heterologous overexpression of PmVNS2 in poplar enhanced the growth performance, secondary xylem cell layer proliferation, and SCW thickening. These findings provide valuable genetic resources for enhancing wood traits and lay the groundwork for future investigations into the molecular mechanisms of wood formation in conifer species. 2. Materials and methods 2.1. Plant materials All samples were collected from a full-sibling progeny test forest at the Zhangping Wuyi National Forestry Farm in Fujian Province, Southeast China. Tissues of the root, tender stem, developing xylem, megaspore, microspore, and cone (three biological duplications per tissue) were sampled for tissue-specific expression analysis of PmVNS2 . The transgenic material was P. davidiana × P. bolleana grown in Murashige and Skoog medium for 35–45 days. 2.2. DNA and RNA isolation, cDNA synthesis and qRT-PCR The DNA of P. massoniana and poplar was extracted using a Plant GenPrep DNA Kit (Zoman Biotech, Beijing, China). Total RNA was isolated from various tissues with the RNAprep Pure Total RNA Extraction Kit (Tiangeng Biotech, Beijing, China). RNA was reverse transcribed to cDNA for subsequent experiments using a Prime Script RT Master Mix Kit (TaKaRa, Dalian, China). qRT-PCR was conducted on an ABI ViiA 7 platform using gene-specific primers and FastStart Universal SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). PmDDI1 (selected as the reference gene by our research group) was used as the reference gene (Table S1 ), and three technical replicates were performed per sample per run. 2.3. Gene cloning and sequence analysis The gene sequence was obtained from our previous RNA-Seq analysis of developing xylem in P. massoniana (Ni, et al. 2020 ). To study the effect of the gene on SCW formation, we used cDNA from the xylem of P. massoniana as a template. The target fragments were amplified using Primer STAR Max DNA Polymerase (Takara, Dalian, China). The primers are listed in Table S1 . The open reading frames (ORFs) of the sequences were predicted using NCBI ORF Finder tool ( https://www.ncbi.nlm.nih.gov/orffinder/ ) (Wang, et al. 2020 ). The primary structure and physicochemical properties of the protein were analyzed using the online tools of ExPASy ProtParam ( https://web.expasy.org/protparam/ ) and ProtScale ( https://web.expasy.org/protscale/ ). The structural domain of the protein was predicted using the BlastP ( https://www.ncbi.nlm.nih.gov/cdd/ ). Multiple sequence alignment analysis was performed using Jalview 2.11.4.1. A phylogenetic tree was constructed using the maximum likelihood method (ML) in MEGA X with a bootstrap of 1000 (Kumar, et al. 2016 ). 2.4 Subcellular localization of PmVNS2 Using Nimble cloning technology, the target gene sequence was inserted into the pNC-Amp-GFP vector to generate the fusion expression construct 35S:: PmVNS2 -GFP for subcellular localization (Yan, et al. 2020 ). The isolation of P. massoniana protoplasts and PEG-mediated transformation were conducted according to methods previously described by Ye et al. (Ye, et al. 2022 ). The results were observed with an Axio Scope A1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). 2.5 Detection and phenotyping of transgenic plants The obtained target gene was connected with the binary plant expression vector pH35GS using the ClonExpress II One-Step Cloning Kit (Vazyme, Nanjing, China) to generate the 35S:: PmVNS2 overexpression vector. The 35S:: PmVNS2 recombinant plasmid was transferred into the leaves of P. davidiana × P. bolleana (Xu, et al. 2018 ). Transgenic plants were selected using kanamycin resistance screening, and DNA was extracted from the resistant plants for PCR verification. Using RNA from the stems of OE_ PmVNS2 lines as a template, qRT-PCR was performed to analyze their transcriptional expression levels, and Ef1α was used as a reference gene (Table S1 )(Xu et al., 2011). Phenotypic traits, including plant height, root number, and total root length, were measured in transgenic lines and CK lines grown on MS medium. Measurements were taken at four time points: 10 days, 20 days, 30 days, and 40 days after transplantation. For each transgenic line, five plants were selected, and their phenotypic data were recorded at each of these time points. Subsequently, all strains were transplanted to sterilized nutrient soil for growth, and plant height and ground diameter (1 cm above the roots) were measured at 60 days. The experiment did not span the winter season or any period inducing dormancy. 2.6. Paraffin section and scanning electron microscope analyses To compare the structural characteristics of SCWs between transgenic plants and CK plants, the following treatments were performed: Stem base segments approximately 2 mm in length were sectioned using the paraffin-embedding method (Xu et al., 2023 ) for histomorphological observation under an optical microscope. Following staining, the paraffin sections were imaged, and quantitative analysis, including measurement of secondary xylem cell layers and the pith-to-primary xylem area ratio, was performed using ImageJ software. For each sample, at least three randomly selected fields of view were analyzed: the "Cell Counter" plugin counted cell layers from the vascular cambium to the secondary xylem edge. At the same time, the "Freehand Selection" tool outlined the pith and primary xylem regions to measure their areas and calculate the ratio. Additionally, 5-mm stem segments were fixed in 4% glutaraldehyde, dehydrated through a graded ethanol series, and then processed using critical point drying. Ultrastructural observation and comparison of secondary cell walls were then conducted using a scanning electron microscope (SEM; Quanta 200, USA). From the SEM images, 10 randomly selected fields were analyzed, with at least 10 fiber cells measured per field using the instrument’s software to determine secondary cell wall thickness. The mean and standard deviation were recorded. All images were captured under consistent magnification and contrast settings to ensure data accuracy and comparability. (1) The bases of the stems were cut into 2-mm segments, which were processed by paraffin-embedded sectioning method (Xu, et al. 2023 ), and observed histomorphologically under a light microscope; (2) In addition, 5-mm stem segments were fixed in 4% glutaraldehyde and then dehydrated by gradient ethanol and critical point drying treatment, and SCWs were analyzed for ultrastructural comparison using a scanning electron microscope (Quanta 200, USA). 3. Results 3.1. Gene cloning and sequence analysis of PmVNS2 Based on the previous transcriptome analysis, the phenylpropane biosynthesis pathway gene Cluster-15034.26926 was involved in wood formation (Ni, et al. 2020 ). Its ORF sequence was 1062 bp, and encoded 353 amino acids (Figure S1 a), which was named PmVNS2 because of its 98.78% similarity to P. taeda PtaVNS2 . The predicted molecular weight of the PmVNS2-encoded protein was 40.64 kDa, and the theoretical isoelectric point was 5.91, which was an acidic protein (Table S2 ). Sequence alignment revealed that the N-terminal of the PmVNS2 protein contains a typical NAC domain (A-E subdomains), and its C-terminal contains the LP-box and WQ-box motifs with transcriptional activation functions (Fig. 1 a, S1b). In addition, to explore the phylogenetic relationships, we constructed a phylogenetic tree using the maximum likelihood method based on the amino acid sequences of VNSs proteins from multiple species. The results showed that PmVNS2 clustered in the VND subgroup together with PtaVNS2 , OsSWN6 / 7 , and AtVND4 - 6 , and is closest in affinity to PtaVNS2 (Fig. 2 b). Therefore, we suggest that PmVNS2 belongs to the VND subfamily. 3.2. Expression patterns and Subcellular localization of PmVNS2 We investigated the expression profiles of PmVNS2 in different tissues of P. massoniana , including roots, microstrobilus, ovulate strobilus, immature stem, needles, cone, and developing xylem, using qRT-PCR (Fig. 2 a). Relative expression levels were normalized to root tissue. PmVNS2 exhibited predominant expression in developing xylem (6-fold higher than roots) and immature stem (3-fold higher than roots), suggesting its critical role in xylem formation. To further characterize its subcellular localization, we transiently transformed P. massoniana protoplasts with the 35S:: PmVNS2 -GFP fusion vector via PEG-mediated transfection. Fluorescence microscopy revealed exclusive nuclear localization of PmVNS2 -GFP, confirming its identity as a nuclear protein (Fig. 2 b). 3.3. Heterologous Transformation of PmVNS2 in Populus To verify the biological function of the PmVNS2 in the wood formation, we constructed an overexpression vector of 35S:: PmVNS2 to perform Agrobacterium -mediated leaf-disk plant transformations in P. davidiana × P. bolleana. Eight randomly selected lines with healthy growth performance were subjected to DNA detection. We found that the size of the amplified PCR fragments was consistent with our expectations, and no target bands were detected in CK (Figure S2 ). Transcript analysis demonstrated significant upregulation of PmVNS2 in transgenic plants, with the T7 line exhibiting a 597-fold increase relative to CK plants, followed by T1 (532-fold) and T6 (418-fold) lines (Fig. 3 ). The observed expression variability among eight independent lines likely stems from position effects caused by distinct T-DNA insertion sites. Based on these results, the T1, T6, and T7 lines were selected for subsequent functional analyses. 3.4. Overexpression of PmVNS2 promoted plant growth To explore the effect of overexpression of PmVNS2 on plant growth, preliminary observations were made on transgenic lines (T1, T6, and T7) and CK lines grown on MS medium for 40 days (Fig. 4 ). Transgenic lines showed significant growth advantages in plant height and root development, including root number and total root length (Fig. 4 a). From 0–20 days, transgenic lines showed slightly higher plant height (Fig. 4 b) and significant root growth advantage, with average root number and total root length reaching 2.00 and 2.52-fold of CK, respectively (Fig. 4 c, d). During 20–30 days, total root length was significantly increased in transgenic lines, especially in the T7 line (46.73 ± 1.62 cm vs. CK: 18.83 ± 2.84 cm) (Fig. 4 d). During 30–40 days, transgenic lines had 2.00-fold higher root number and 1.83-fold higher total root length than CK (Fig. 4 c, d), and the plant height of transgenic lines (an increase of 6.68 cm from 10–40 days) was significantly greater than that of CK lines (5.80 cm) (Fig. 4 b). After 60 days of growth in soil pots (Fig. 5 ), transgenic lines further exhibited enhanced phenotypic advantages: plant height (17.94 ± 0.97 cm vs. CK: 14.70 ± 0.92 cm) and ground diameter (2.61 ± 0.15 mm vs. CK: 1.99 ± 0.14 mm) were significantly greater than CK (Fig. 5 a-c). These findings indicate that PmVNS2 may orchestrate plant growth by coordinately enhancing both aboveground development and root system expansion. 3.5. PmVNS2 Overexpression Promotes SCW Deposition in Xylem Development Based on the enhanced radial growth observed in stems of PmVNS2 overexpressing lines in the above study (Fig. 5 a, c), to further elucidate the regulatory mechanism of this gene on SCW formation, we selected transgenic lines (T1, T7) and CK lines grown on MS medium for 40 days. Stem segments from the 10th-11th internodes were collected, and 2-mm-thick cross-sections were prepared. These sections underwent paraffin embedding, sectioning, and staining for histological observation and analysis. Microscopic observation revealed that the number of secondary xylem cell layers in transgenic lines increased approximately 1.5-fold compared to CK lines. Specifically, the T1 line averaged 18.1 ± 1.59 layers, the T7 line averaged 21.6 ± 1.35 layers, while the CK averaged 12.8 ± 1.47 layers (Fig. 6 a, b). Furthermore, the pith and primary xylem occupied relatively minor proportions of the stem cross-sectional area, with significant differences observed in their area ratios (T1: 3.15, T7: 2.87, CK: 3.52) (Fig. 6 a, c). This indicates a significantly increased proportion of xylem and a relatively reduced pith cavity in transgenic plants. Analysis of the stem cross-section ultrastructure using scanning electron microscopy (SEM) further demonstrated a highly significant difference in xylem cell wall thickness between transgenic lines (3.45 ± 0.27 µm) and CK lines (1.91 ± 0.18 µm) (Fig. 6 d, e). Collectively, these histomorphological and ultrastructural findings demonstrate that overexpression of PmVNS2 not only significantly promotes xylem cell differentiation and secondary wall deposition but also enhances the xylem construction process by regulating the spatial distribution between xylem and other tissues. To further dissect the molecular mechanism by which PmVNS2 promotes SCW deposition, we performed qRT-PCR analysis of the expression levels of SCW biosynthesis-related genes in the transgenic lines (Fig. 7 a). The results revealed that lignin biosynthesis genes (including CCR2 , C3H3 , C4H2 , 4CL5 , COMT2 , and HCT1 ) exhibited 2- to 5-fold upregulation in transgenic plants compared to CK plants, except for F5H2 . Meanwhile, the expression levels of cellulose biosynthesis enzyme genes CesA2B and CesA3A were also significantly increased, while xylan biosynthesis-related genes GT43B and GT43D showed down-regulation. These findings suggested that PmVNS2 may regulate the directional deposition of SCW by synergistically activating the lignin and cellulose biosynthesis pathways and inhibiting xylan production. Notably, NAC TFs serve as upstream regulatory hubs that initiate SCW formation by activating MYB TFs. Consistent with this mechanism, transgenic plants demonstrated significantly elevated expression levels of seven SCW-related MYB TFs ( MYB002 , MYB003 , MYB020 , MYB021 , MYB090 , MYB128 , and MYB158 ) compared to CK plants (Fig. 7 b). MYB128 displayed the most pronounced induction (> 8-fold), while other MYB genes showed 2-7-fold upregulation. This synergistic activation with the MYB regulatory network strongly implies that PmVNS2 might drive the ectopic deposition of SCW during wood formation by acting upstream of the NAC-MYB regulatory cascade. 4. Discussion Wood formation is controlled by a highly ordered transcriptional regulatory network involving VNS and MYB family transcription factors (Akiyoshi 2020; Hao, et al. 2024 ; Hussey, et al. 2013 ; Nakano, et al. 2015 ). Studies in model plants Arabidopsis and poplar have demonstrated that VNDs (VNS members) and NSTs function as master regulatory switches governing SCW formation through the initiation of gene expression cascades (Ohtani, et al. 2011 ; Takata, et al. 2019 ; Yamaguchi, et al. 2008 ; Zhong, et al. 2011 ; Zhong, et al. 2006 ; Zhong and Ye 2007 ). Through analysis of RNA-seq, we identified PmVNS2 , a VND subfamily member in P. massoniana . qRT-PCR revealed predominant expression of PmVNS2 in xylem tissues, followed by immature stems (Fig. 2 a), consistent with the characteristic spatial expression pattern of SCW biosynthesis-related genes that typically exhibit elevated expression in stem tissues, particularly mature stems (Li, et al. 2012 ; Yang 2015; Zhao, et al. 2014 ). To investigate the regulatory role of PmVNS2 in SCW development, we conducted functional characterization through heterologous overexpression in transgenic poplar systems. Wood formation depends on the continuous division and differentiation of vascular cambium cells, ultimately generating secondary xylem and phloem (Chen, et al. 2019 ; Ye and Zhong 2015 ). As core regulators for SCW biosynthesis, VNS family genes are functionally highly conserved across multiple species. For instance, overexpression of PtrWND2B / PtVNS10 (NST subfamily) and PtrWND6B / PtVNS08 (VND subfamily) in P. trichocarpa significantly induces SCW thickening in transgenic Arabidopsis (Ohtani, et al. 2011 ; Zhong, et al. 2011 ; Zhong, et al. 2010 ), while overexpression of PtoVNS11 in Populus tomentosa leads to abnormally compact cell walls in wood fibers and vessel cell walls (Yang 2015). Our study further demonstrates that overexpression of PmVNS2 , a VND subfamily member, not only increases the number of SCW cell layers in transgenic plants but also elevates cell wall thickness to 3.45 ± 0.27 µm, representing a 1.81-fold increase compared to CK plants (1.91 ± 0.18 µm) (Fig. 6 a, b). This observation aligns with the ectopic SCW deposition caused by heterologous expression of the P. taeda ortholog PtaVNS2 in tobacco (Akiyoshi 2020), corroborating the conserved regulatory role of the VND subfamily in SCW biosynthesis. Furthermore, the small-rosette and leaf curling phenotypes induced by PpNAC1 overexpression from P. pinaster in Arabidopsis mirror those reported for Arabidopsis SND1 / NST1 overexpression (Mitsuda, et al. 2005 ; Pascual, et al. 2018 ; Zhong, et al. 2006 ), suggesting functional convergence of VNS regulatory networks across divergent species. Although this study focuses on secondary xylem, ectopic expression of PmVNS2 may affect cell wall deposition in non-typical tissues, which warrants further investigation. Collectively, these findings demonstrate that VNS family genes, particularly the VND and NST subfamilies, play pivotal roles in wood formation by coordinating SCW thickening (Hussey, et al. 2013 ; Ohtani, et al. 2011 ). Genes involved in SCW biosynthesis typically exert bidirectional regulatory effects on plant growth through resource competition mechanisms. Previous studies have demonstrated that while overexpression of PtoLAC14 in P. tomentosa enhances cell wall thickening and lignification, it concurrently suppresses plant height growth (Qin, et al. 2020 ). Similarly, overexpression of PtrWND2B / 6B in P. trichocarpa results in shortened stems and reduced growth rates in transgenic plants (Zhong, et al. 2011 ). In contrast to these findings, our study reveals that PmVNS2 overexpression induces coordinated development of aboveground and belowground systems. During early developmental stages (0–40 days), transgenic plants exhibited pronounced growth advantages over CK plants: plant height increment increased by 15.2% (6.68 cm vs. CK 5.80 cm), while root number and total root length reached 2.00-fold and 1.83-fold of CK plants, respectively (Fig. 4 ). After 60 days of soil cultivation, transgenic plants displayed significantly greater stem diameter (2.61 ± 0.15 mm vs. CK: 1.99 ± 0.14 mm) and plant height compared to CK plants (Fig. 5 ). We propose that PmVNS2 may dynamically regulate the spatiotemporal pattern of SCW deposition, simultaneously enhancing cell wall mechanical strength and promoting holistic plant growth. This mechanism aligns with the effects of overexpressing the Arabidopsis homolog SND2 , which increases xylem thickness in poplar while enhancing plant height, stem diameter, leaf expansion, and root development (Han 2021 ). Our findings highlight the potential application value of PmVNS2 overexpression in coordinating plant growth with SCW deposition, though the growth characteristics of transgenic plants at maturity require further investigation. The two-layer cascade regulatory system composed of VNS-MYB TFs constitutes the master control network for SCW formation. As the top regulators of the cascade regulatory network, VNS TFs achieve multilevel regulation by coordinating the expression of downstream TFs, SCW biosynthesis-related genes, and PCD-related genes (Akiyoshi, et al. 2021 ; Nakano, et al. 2015 ; Taylor-Teeples, et al. 2015 ). In this study, we observed significant upregulation of MYB family genes, including MYB002 , MYB003 , MYB020 , MYB021 , MYB090 , MYB128 , and MYB158 in PmVNS2 overexpressing transgenic poplar. Notably, MYB002 / 021 and MYB003 / 020 were identified as secondary regulators in poplar, functionally complementing the SCW-deficient phenotypic defects in Arabidopsis MYB46 - MYB83 double mutants (McCarthy, et al. 2010 ; Pratyusha and Sarada 2022 ; Wegrzyn, et al. 2010 ; Zhong, et al. 2010 ). Moreover, the expression of the key lignin biosynthesis pathway genes, CCR2 , C4H2 and 4CL5 , was also significantly elevated. Previous studies have demonstrated that inhibition of these genes directly reduces lignin accumulation (Liu, et al. 2014 ; Ni, et al. 2020 ; Novaes, et al. 2010 ; Thévenin, et al. 2011 ; Zhang, et al. 2025 ). Additionally, PmVNS2 overexpression specifically activated the expression of the cellulose synthase genes ( CesA2B and CesA3A ), but inhibited the transcription of the xylan synthase genes ( GT43B & GT43D ). This suggests that PmVNS2 differentially regulates polysaccharide metabolic pathways to modulate cell wall composition, positively regulating lignin and cellulose biosynthesis, yet negatively regulating xylan synthesis. Collectively, our findings propose that PmVNS2 serves as a top regulator of SCW deposition during wood formation in conifers, with functional conservation mirroring the VNS regulatory networks reported in angiosperms. This discovery provides novel evidence supporting the evolutionary homology of SCW regulatory mechanisms between gymnosperms and angiosperms. 5. Conclusions Wood formation is governed by a hierarchical NAC-MYB regulatory network, in which NAC domain transcription factors function as apex regulatory components. This study demonstrates that PmVNS2 from P. massoniana exhibits predominant expression in xylem tissues. Heterologous overexpression of PmVNS2 activates downstream MYB TFs ( MYB020 / 021 ), lignin/cellulose biosynthesis genes ( CCR2 , C4H2 , CesA2B , and CesA3A ), while suppressing xylan synthesis genes ( GT43B / D ), synergistically promoting secondary xylem cell layer proliferation (+ 1.5-fold) and cell wall thickening (3.45 ± 0.27 µm vs. CK 1.91 ± 0.18 µm). This study demonstrates that VND orthologs in conifers regulate SCW deposition through the conserved NAC-MYB pathway, providing genetic resources for targeted improvement of wood properties in conifer species. Declarations Funding This research was funded by the National Natural Science Foundation of China (No. 32301552), the Guangxi Science and Technology Major Program (AA24263021), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Author contribution statement Y.T.T., Z.X.N., and L.A.X. conceived and designed the research. Y.T.T., K.W., and H.R. performed the experiments and contributed analysis tools. Y.T.T. and M.X. analyzed data. Y.T.T. wrote the manuscript. Z.X.N. and L.A.X. reviewed the manuscript. All authors read and approved the manuscript. Declaration of competing interest The authors declare that they have no conflict of interest. Data Availability Data will be made available on request. References Akiyoshi N, Ihara A, Matsumoto T, Takebayashi A, Hiroyama R, Kikuchi J, Demura T, Ohtani M (2021) Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties. Plant Cell Physiol 62:1963-1974. http://dx.doi.org/10.1093/pcp/pcab102 Akiyoshi NA, Nakano Y, Sano R, Kunigita Y, Ohtani M, Demura T (2020) Involvement of VNS NAC-domain transcription factors in tracheid formation in Pinus taeda. Tree Physiology 40:704-716. http://dx.doi.org/10.1093/treephys/tpz106 Bo Xu MO, Masatoshi Yamaguchi, Kiminori Toyooka, Mayumi Wakazaki (2014) Contribution of NAC Transcription Factors to Plant Adaptation to Land. Science 343:1505-1508. http://dx.doi.org/10.1126/science.1248417 Chen H, Wang JP, Liu HZ, Li HY, Lin YCJ, Shi R, Yang CM, Gao JH, Zhou CG, Li QZ, Sederoff RR, Li W, Chiang VL (2019) Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Populus trichocarpa. Plant Cell 31:602-626. http://dx.doi.org/10.1105/tpc.18.00620 Duval I, Lachance D, Giguere I, Bomal C, Morency MJ, Pelletier G, Boyle B, MacKay JJ, Seguin A (2014) Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development. J Exp Bot 65:2319-2333. http://dx.doi.org/10.1093/jxb/eru116 Ge S, Han X, Xu X, Shao Y, Zhu Q, Liu Y, Du J, Xu J, Zhang S (2020) WRKY15 Suppresses Tracheary Element Differentiation Upstream of VND7 During Xylem Formation. The Plant Cell 32:2307-2324. http://dx.doi.org/10.1105/tpc.19.00689 Ha Van H, Ho Van G, Ngo Van T, Zhang S, Wang Y (2012) Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees. Molecular Breeding 29:173-180. http://dx.doi.org/10.1007/s11032-010-9535-9 Han Z (2021) Functions of SND2-B2 Gene from Populus in Secondary Cell Wall Biosynthesis. Shandong Agricultural University, Shandong, China Hao Y, Lu F, Pyo SW, Kim MH, Ko JH, Yan X, Ralph J, Li Q (2024) PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar. Journal of Integrative Plant Biology 66:1658-1674. http://dx.doi.org/10.1111/jipb.13717 Hussey SG, Mizrachi E, Creux NM, Myburg AA (2013) Navigating the transcriptional roadmap regulating plant secondary cell wall deposition. Frontiers in Plant Science 4. http://dx.doi.org/10.3389/fpls.2013.00325 Johnsson C, Jin X, Xue WY, Dubreuil C, Lezhneva L, Fischer U (2019) The plant hormone auxin directs timing of xylem development by inhibition of secondary cell wall deposition through repression of secondary wall NAC-domain transcription factors. Physiologia Plantarum 165:673-689. http://dx.doi.org/10.1111/ppl.12766 Ko JH, Jeon HW, Kim WC, Kim JY, Han KH (2014) The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Annals of Botany 114:1099-1107. http://dx.doi.org/10.1093/aob/mcu126 Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes & Development 19:1855-1860. http://dx.doi.org/10.1101/gad.1331305 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. MOLECULAR BIOLOGY AND EVOLUTION 33:1870-1874. http://dx.doi.org/10.1093/molbev/msw054 Lamara M, Raherison E, Lenz P, Beaulieu J, Bousquet J, MacKay J (2016) Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce. The New phytologist 210:240-255. http://dx.doi.org/10.1111/nph.13762 Li Q, Lin Y-C, Sun Y-H, Song J, Chen H, Zhang X-H, Sederoff RR, Chiang VL (2012) Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa. Proceedings of the National Academy of Sciences 109:14699-14704. http://dx.doi.org/10.1073/pnas.1212977109 Liu Y, You S, Taylor-Teeples M, Li WL, Schuetz M, Brady SM, Douglas CJ (2014) BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 Interact and Regulate Secondary Cell Wall Formation via Repression of REVOLUTA. The Plant Cell 26:4843-4861. http://dx.doi.org/10.1105/tpc.114.128322 McCarthy RL, Zhong RQ, Fowler S, Lyskowski D, Piyasena H, Carleton K, Spicer C, Ye ZH (2010) The Poplar MYB Transcription Factors, PtrMYB3 and PtrMYB20, are Involved in the Regulation of Secondary Wall Biosynthesis. Plant and Cell Physiology 51:1084-1090. http://dx.doi.org/10.1093/pcp/pcq064 McCarthy RL, Zhong RQ, Ye ZH (2009) MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis. Plant and Cell Physiology 50:1950-1964. http://dx.doi.org/10.1093/pcp/pcp139 Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270-280. http://dx.doi.org/10.1105/tpc.106.047043 Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M (2005) The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17:2993-3006. http://dx.doi.org/10.1105/tpc.105.036004 Nakano Y, Yamaguchiz M, Endo H, Rejab NA, Ohtani M (2015) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Frontiers in Plant Science 6. http://dx.doi.org/10.3389/fpls.2015.00288 Ni ZX, Han X, Yang ZQ, Xu M, Feng YH, Chen YB, Xu LA (2020) Integrative analysis of wood biomass and developing xylem transcriptome provide insights into mechanisms of lignin biosynthesis in wood formation of Pinus massoniana. International Journal of Biological Macromolecules 163:1926-1937. http://dx.doi.org/10.1016/j.ijbiomac.2020.08.253 Novaes E, Kirst M, Chiang V, Winter-Sederoff H, Sederoff R (2010) Lignin and Biomass: A Negative Correlation for Wood Formation and Lignin Content in Trees. Plant Physiology 154:555-561. http://dx.doi.org/10.1104/pp.110.161281 Ohtani M, Nishikubo N, Xu B, Yamaguchi M, Mitsuda N, Goue N, Shi F, Ohme-Takagi M, Demura T (2011) A NAC domain protein family contributing to the regulation of wood formation in poplar. Plant Journal 67:499-512. http://dx.doi.org/10.1111/j.1365-313X.2011.04614.x Pascual MB, Llebres MT, Craven-Bartle B, Canas RA, Canovas FM, Avila C (2018) PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine. Plant Biotechnology Journal 16:1094-1104. http://dx.doi.org/10.1111/pbi.12854 Pratyusha DS, Sarada DVL (2022) MYB transcription factors—master regulators of phenylpropanoid biosynthesis and diverse developmental and stress responses. Plant Cell Reports 41:2245-2260. http://dx.doi.org/10.1007/s00299-022-02927-1 Qin SF, Fan CF, Li XH, Li Y, Hu J, Li CF, Luo KM (2020) LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar. BIOTECHNOLOGY FOR BIOFUELS 13. http://dx.doi.org/10.1186/s13068-020-01843-4 Quan W, Ding G (2017) Root tip structure and volatile organic compound responses to drought stress in Masson pine (Pinus massoniana Lamb.). Acta Physiologiae Plantarum 39. http://dx.doi.org/10.1007/s11738-017-2558-7 Takata N, Awano T, Nakata MT, Sano Y, Sakamoto S, Mitsuda N, Taniguchi T (2019) Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells. Tree Physiology 39:514-525. http://dx.doi.org/10.1093/treephys/tpz004 Taylor-Teeples M, Lin L, de Lucas M, Turco G, Toal TW, Gaudinier A, Young NF, Trabucco GM, Veling MT, Lamothe R, Handakumbura PP, Xiong G, Wang C, Corwin J, Tsoukalas A, Zhang L, Ware D, Pauly M, Kliebenstein DJ, Dehesh K, Tagkopoulos I, Breton G, Pruneda-Paz JL, Ahnert SE, Kay SA, Hazen SP, Brady SM (2015) An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517:571-U307. http://dx.doi.org/10.1038/nature14099 Thévenin J, Pollet B, Letarnec B, Saulnier L, Gissot L, Maia-Grondard A, Lapierre C, Jouanin L (2011) The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana. Molecular Plant 4:70-82. http://dx.doi.org/10.1093/mp/ssq045 Valdivia ER, Herrera MT, Gianzo C, Fidalgo J, Revilla G, Zarra I, Sampedro J (2013) Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot Brachypodium distachyon. Journal of Experimental Botany 64:1333-1343. http://dx.doi.org/10.1093/jxb/ers394 Wang CH, Zhao FQ, Liu JX, Liu HY (2020) Short communication: The essential role of N-glycosylation in the transport activity of bovine peptide transporter 2. Journal of Dairy Science 103:6679-6683. http://dx.doi.org/10.3168/jds.2019-16858 Wang Z, Mao Y, Guo Y, Gao J, Liu X, Li S, Lin Y-CJ, Chen H, Wang JP, Chiang VL, Li W (2020) MYB Transcription Factor161 Mediates Feedback Regulation of Secondary wall-associated NAC-Domain1 Family Genes for Wood Formation. Plant Physiology 184:1389-1406. http://dx.doi.org/10.1104/pp.20.01033 Wegrzyn JL, Eckert AJ, Choi M, Lee JM, Stanton BJ, Sykes R, Davis MF, Tsai CJ, Neale DB (2010) Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem. New Phytologist 188:515-532. http://dx.doi.org/10.1111/j.1469-8137.2010.03415.x Xu M, Chen C, Cai H, Wu L (2018) Overexpression of PeHKT1;1 Improves Salt Tolerance in Populus. Genes 9. http://dx.doi.org/10.3390/genes9100475 Xu W, Qi H, Shen T, Zhao M, Song Z, Ran N, Wang J, Xi M, Xu M (2023) Poplar coma morphogenesis and miRNA regulatory networks by combining ovary tissue sectioning and deep sequencing. iScience 26. http://dx.doi.org/10.1016/j.isci.2023.106496 Yamaguchi M, Kubo M, Fukuda H, Demura T (2008) VASCULAR-RELATED NAC-DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. Plant Journal 55:652-664. http://dx.doi.org/10.1111/j.1365-313X.2008.03533.x Yan P, Zeng YJ, Shen WT, Tuo DC, Li XY, Zhou P (2020) Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning. Frontiers in Bioengineering and Biotechnology 7. http://dx.doi.org/10.3389/fbioe.2019.00460 Yang LY, Hou YS, Zhao X, Lu WX, Li YL, Yang F, Tang SH, Luo KM (2015) Identification and characterization of a wood-associated NAC domain transcription factor PtoVNS11 from Populus tomentosa Carr. TREES-STRUCTURE AND FUNCTION 29:1091-1101. http://dx.doi.org/10.1007/s00468-015-1188-1 Ye YJ, Han X, Rong H, Qian RJ, Zheng J, Ni ZX, Xu LA (2022) Effect of the PmARF6 Gene from Masson Pine (Pinus massoniana) on the Development of Arabidopsis. GENES 13. http://dx.doi.org/10.3390/genes13030469 Ye ZH, Zhong RQ (2015) Molecular control of wood formation in trees. Journal of Experimental Botany 66:4119-4131. http://dx.doi.org/10.1093/jxb/erv081 Yu C, Lv J, Xu H (2024) Plant growth-promoting fungi and rhizobacteria control Fusarium damping-off in Mason pine seedlings by impacting rhizosphere microbes and altering plant physiological pathways. Plant and Soil. http://dx.doi.org/10.1007/s11104-024-06475-3 Zhang M, Zhu P, Agassin RH, Yao S, Wang D, Huang Z, Zhang C, Hao Q, Ji K (2023) Identification, Classification and Characterization of bZIP Transcription Factor Family Members in Pinus massoniana Lamb. Forests 14. http://dx.doi.org/10.3390/f14010155 Zhang S, Cao L, Chen D, Chang R, Cao J, Zhang Q, Qin Y, Liu G, Xu Z (2025) PtrVINV2 is dispensable for cellulose synthesis but essential for salt tolerance in Populus trichocarpa Torr. and Gray. Plant Biotechnology Journal. http://dx.doi.org/10.1111/pbi.70022 Zhang Y, Chen S, Xu L, Chu S, Yan X, Lin L, Wen J, Zheng B, Chen S, Li Q (2024) Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar. The Plant Cell 36:1806-1828. http://dx.doi.org/10.1093/plcell/koae040 Zhao Y, Sun J, Xu P, Zhang R, Li L (2014) Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species. Plant Physiology 164:765-776. http://dx.doi.org/10.1104/pp.113.231134 Zhong R, Lee C, Ye ZH (2010) Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Mol Plant 3:1087-1103. http://dx.doi.org/10.1093/mp/ssq062 Zhong R, McCarthy RL, Lee C, Ye Z-H (2011) Dissection of the Transcriptional Program Regulating Secondary Wall Biosynthesis during Wood Formation in Poplar. Plant Physiology 157:1452-1468. http://dx.doi.org/10.1104/pp.111.181354 Zhong R, Ye ZH (2012) MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol 53:368-380. http://dx.doi.org/10.1093/pcp/pcr185 Zhong RQ, Demura T, Ye ZH (2006) SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18:3158-3170. http://dx.doi.org/10.1105/tpc.106.047399 Zhong RQ, Lee CH, Ye ZH (2010) Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors. Plant Physiology 152:1044-1055. http://dx.doi.org/10.1104/pp.109.148270 Zhong RQ, Ye ZH (2007) Regulation of cell wall biosynthesis. Current Opinion in Plant Biology 10:564-572. http://dx.doi.org/10.1016/j.pbi.2007.09.001 Supplementary Files AminoacidsequenceoftheVNSprotein.xlsx SupplementalFigure.docx SupplementalTable1.docx SupplementalTable2.docx Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 30 Nov, 2025 Reviewers agreed at journal 04 Nov, 2025 Reviewers invited by journal 04 Nov, 2025 Editor assigned by journal 29 Oct, 2025 First submitted to journal 29 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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16:54:52","extension":"xml","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148454,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25012720structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/b368bff6edb863927489b5df.xml"},{"id":96241551,"identity":"fbe75894-a7a0-4215-97e5-3bd2da795340","added_by":"auto","created_at":"2025-11-19 07:10:57","extension":"html","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165694,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/30d74dd7e8179f42f95c5da8.html"},{"id":95857044,"identity":"46b81591-56bd-4567-9062-4ddc6350b6d2","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":438387,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment and phylogenetic analysis of VNS proteins. (a) Sequence alignment of \u003cem\u003ePmVNS2\u003c/em\u003e with \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003eAtVND4\u003c/em\u003e, \u003cem\u003ePopulus\u003c/em\u003e \u003cem\u003ePtrWND4A\u003c/em\u003e, and \u003cem\u003eP. taeda\u003c/em\u003e \u003cem\u003ePtaVNS2\u003c/em\u003e. (b) The maximum likelihood phylogenetic tree of \u003cem\u003ePmVNS2\u003c/em\u003e and VNS orthologs from diverse taxa was resolved into three major clades (color-coded). The PmVNS2 protein is highlighted with a red dot. The complete list of gene IDs and their amino acid sequences for this cluster is provided in the Supplementary File (Amino acid sequence of the VNS protein).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/ca8a2ee3e0c05dd2bc1af2f3.png"},{"id":95857050,"identity":"ed70d7b0-195f-4ebd-b06f-4c2ccd0b6641","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165861,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003ePmVNS2\u003c/em\u003e in \u003cem\u003eP. massoniana\u003c/em\u003e. (a) Expression levels of \u003cem\u003ePmVNS2\u003c/em\u003ein seven tissues of \u003cem\u003eP. massoniana\u003c/em\u003e: roots (R), microstrobilus (M), ovulate strobilus (O), immature stem (S), needle (L), cone (C), and developing xylem (X). Date presented as mean ± SD (n = 3). Different lowercase letters indicate statistical differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (b) Subcellular localization of PmVNS2 in \u003cem\u003eP. massoniana\u003c/em\u003e protoplasts. eGFP channels: location of fusion gene; Auto channels: autofluorescence of protoplasts; Bright channels: cellular state. Merge1: combination of eGFP and Auto, Merge2: integration of eGFP, Auto, and Bright. Bars = 10 μm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/6dfe1fea768d290945d6a1db.png"},{"id":96240295,"identity":"d10cd750-14c1-4833-88c7-1aee140313f9","added_by":"auto","created_at":"2025-11-19 07:08:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32921,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003ePmVNS2\u003c/em\u003e in transgenic and CK lines. qRT-PCR analysis was performed using cDNA derived from stems of plants grown on MS medium for 40 days. Date presented as mean ± SD (n = 3). Different colors represent the CK lines and different \u003cem\u003ePmVNS2\u003c/em\u003e transgenic lines.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/6a142d6b232c53090ea129ba.png"},{"id":95857049,"identity":"1d673b5f-c42b-47b0-92dc-7b5ecb8ae9da","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":461198,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic results of \u003cem\u003ePmVNS2\u003c/em\u003etransgenic lines and CK lines. (a) Phenotypic observations of the CK and transgenic T1, T6, and T7 lines at 10, 20, 30, and 40 days after microcutting regeneration. Scale bars: = 1 cm. (b) Statistical results of plant height of CK and three transgenic T1, T6, and T7 lines. (c) Results of statistical analysis of root number and total root length (d) of CK and three transgenic T1, T6, and T7 lines. Different lowercase letters indicate statistical differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/b9b4d4aed724e75e4c6c6151.png"},{"id":96240880,"identity":"1a0cf25a-113b-4ff2-b54a-5926772df8ac","added_by":"auto","created_at":"2025-11-19 07:09:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":330661,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypes of \u003cem\u003ePmVNS2\u003c/em\u003e transgenic lines and CK grown for 60 days in soil pots. (a) Phenotypic results of transgenic T1, T6, and T7 lines grown on MS medium for 40 days and CK lines transplanted in soil for 60 days. (b) Statistical analysis of plant height and ground diameter (1 cm above the roots) of CK and three transgenic T1, T6, and T7 lines (c). Different lowercase letters indicate statistical differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/bb05875c65880fe7696f0de2.png"},{"id":96240740,"identity":"74421a9e-4734-49a5-ba65-ef3a8655f5ea","added_by":"auto","created_at":"2025-11-19 07:09:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":391949,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characterization of stems in transgenic lines. (a) Cross-section observation of basal stems from CK, T1, and T7 lines regenerated via microcuttings (40 days). Xy: xylem (indicated), Xf: xylem fiber cells, Xv: xylem vessels. Scale bars: 50 μm, 100 μm. (b, c) Quantification of xylem cell layer numbers and pith/primary xylem area ratios in CK, T1, and T7 lines. (d) SEM images of basal stem cross-sections from CK, T1, and T7 lines. Scale bar: 10 μm. (e) Secondary cell wall thickness in CK, T1, and T7 lines. Data are presented as mean ± SD (n = 3). Statistical significance (*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) was evaluated via Student’s t-test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/33d90af41794f31e8be79521.png"},{"id":95857045,"identity":"169bc63a-04b0-487a-92ab-6ad77e2aff34","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52236,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional activation of the SCW biosynthesis pathway. (a) qRT-PCR analysis of stems of \u003cem\u003ePmVNS2\u003c/em\u003e overexpressing plants regenerated from microcuttings for 40 days. Genes analyzed include lignin biosynthesis pathway genes (\u003cem\u003ePAL4\u003c/em\u003e, \u003cem\u003eC4H2\u003c/em\u003e,\u003cem\u003e HCT1\u003c/em\u003e, \u003cem\u003eC3H3\u003c/em\u003e, \u003cem\u003eCCOAOMT1\u003c/em\u003e, \u003cem\u003eCCR2\u003c/em\u003e, \u003cem\u003eCOMT2\u003c/em\u003e, \u003cem\u003eCAD1\u003c/em\u003e, \u003cem\u003eF5H2\u003c/em\u003e), cellulose biosynthesis genes (\u003cem\u003eCesA2B\u003c/em\u003e, \u003cem\u003eCesA3A\u003c/em\u003e), and xylan biosynthesis genes (\u003cem\u003eGT43B\u003c/em\u003e,\u003cem\u003e GT43D\u003c/em\u003e). (b) Members of the MYB TF family, which regulates SCW deposition, were broadly activated in \u003cem\u003ePmVNS2\u003c/em\u003eoverexpression plants. All primers are shown in Table S1. Data are presented as mean ± SD (n = 3). Statistical significance (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) was evaluated via Student’s t-test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/a11ea6357d921013aa2e040f.png"},{"id":101151872,"identity":"fba5e9e2-9680-480c-bfea-9c1a95603967","added_by":"auto","created_at":"2026-01-26 16:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2689351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/587badcd-99a7-4bdb-ab5b-99b7d1e20afd.pdf"},{"id":95857052,"identity":"f36ec695-2bd8-4d4d-b068-7877bd4def74","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":12383,"visible":true,"origin":"","legend":"","description":"","filename":"AminoacidsequenceoftheVNSprotein.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/6546e81c3519a910f15329ba.xlsx"},{"id":95857054,"identity":"5a8d5e8b-0610-4409-9c88-88dfe325a7cf","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":306691,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/1021385b4e1b2cc1db20ba6f.docx"},{"id":95857058,"identity":"e4e236af-68dc-45ee-a49f-4fc52ba48768","added_by":"auto","created_at":"2025-11-13 16:54:52","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":18718,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/fdadd9994ccf3c713ef41ccf.docx"},{"id":96240467,"identity":"356041ed-47ef-4819-9b6f-b6f45e437fb2","added_by":"auto","created_at":"2025-11-19 07:08:56","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":19400,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7931415/v1/79429942f33a08bec56ce507.docx"}],"financialInterests":"","formattedTitle":"Heterologous expression of Pinus massonianaVNS2 enhances secondary cell wall deposition in poplar","fulltext":[{"header":"Key message","content":"\u003cp\u003e\u003cem\u003ePmVNS2\u003c/em\u003e promotes secondary cell wall formation and enhances xylem development in transgenic poplar.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eWood formation represents a complex biological process orchestrated through multi-tiered transcriptional regulatory networks that precisely coordinate cambial activity, secondary cell wall (SCW) biosynthesis, and programmed cell death (PCD) (Ye and Zhong \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This intricate mechanism involves the synergistic action of thousands of genes, and elucidating its molecular regulation provides a theoretical foundation for targeted genetic improvement of tree species. Notably, recent research has underscored the pivotal role of NAC-MYB transcriptional networks in regulating SCW formation across both angiosperms and gymnosperms (Akiyoshi 2020; Chen, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hao, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Johnsson, et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe NAC-MYB network serves as the primary regulatory hub for xylem cell differentiation, with the VNS (VND-, NST/SND- and SMB-related proteins) subfamily of the NAC TF family emerging as a dominant player in SCW formation (Ge, et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hussey, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yamaguchi, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, the VNS subfamily is further divided into three distinct groups: VND (vascular-related NAC domain, e.g., \u003cem\u003eVND1\u003c/em\u003e-\u003cem\u003eVND7\u003c/em\u003e), NST (NAC secondary wall thickening promoting factors, e.g., \u003cem\u003eNST1\u003c/em\u003e, \u003cem\u003eNST2\u003c/em\u003e, and \u003cem\u003eNST3\u003c/em\u003e), and SMB (Sombrero, \u003cem\u003eSMB\u003c/em\u003e, \u003cem\u003eBRN1\u003c/em\u003e, and \u003cem\u003eBRN2\u003c/em\u003e)(Kubo, et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mitsuda, et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Similarly, in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, 16 VNS proteins have been identified and classified into VND (\u003cem\u003ePtVNS01\u003c/em\u003e-\u003cem\u003ePtVNS08\u003c/em\u003e), NST (\u003cem\u003ePtVNS09\u003c/em\u003e-\u003cem\u003ePtVNS12\u003c/em\u003e), and SMB (\u003cem\u003ePtVNS13\u003c/em\u003e-\u003cem\u003ePtVNS16\u003c/em\u003e) subfamilies (Ohtani, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Takata, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). MYB TFs, such as \u003cem\u003eAtMYB46\u003c/em\u003e and \u003cem\u003eAtMYB83\u003c/em\u003e, act as second-layer regulatory switches downstream of VNS proteins, further modulating SCW formation (Hussey, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ko, et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; McCarthy, et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The lignification process, integral to plant cell wall formation, involves intracellular monomer biosynthesis and polymerization, requiring upregulation of related enzyme genes by VNS and MYB proteins (Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhong and Ye \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe conservation of the SCW biosynthesis mechanism, involving VNS genes, has been observed across diverse vascular plant species (Hussey, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Valdivia, et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For instance, while \u003cem\u003ePhyscomitrella patens\u003c/em\u003e lacks a typical SCW structure, poplar \u003cem\u003ePpVNSs\u003c/em\u003e can regulate homologous \u003cem\u003eMYB46\u003c/em\u003e and \u003cem\u003eMYB83\u003c/em\u003e in \u003cem\u003eP. patens\u003c/em\u003e (Bo Xu 2014). In \u003cem\u003ePinus taeda\u003c/em\u003e, the instantaneous overexpression of four \u003cem\u003eVNS\u003c/em\u003e genes (\u003cem\u003ePtaVNS1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e and \u003cem\u003ePtaVNS5\u003c/em\u003e) induces ectopic SCW deposition and upregulates SCW biosynthesis-related genes (e.g., \u003cem\u003eMYB4\u003c/em\u003e, \u003cem\u003eCesA\u003c/em\u003e, and \u003cem\u003eXCP1\u003c/em\u003e gene) in tobacco (Akiyoshi 2020). Additionally, the \u003cem\u003ePpNAC1\u003c/em\u003e gene from \u003cem\u003ePinus pinaster\u003c/em\u003e, a homolog of \u003cem\u003eArabidopsis NST3\u003c/em\u003e and \u003cem\u003eNST1\u003c/em\u003e, plays a critical role in SCW biosynthesis. Its inhibition disrupts the radial pattern of stem vascular bundles and downregulates SCW biosynthesis-related genes, while its binding to the \u003cem\u003ePpMYB4\u003c/em\u003e promoter activates the expression of \u003cem\u003ePpMYB8\u003c/em\u003e, a downstream transcriptional regulator (Pascual, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In \u003cem\u003ePicea glauca\u003c/em\u003e, \u003cem\u003ePgNAC7\u003c/em\u003e is a key regulatory factor for SCW biosynthesis in coniferous wood (Duval, et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lamara, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These findings suggest the conservation of biological functions and regulatory stability within the NAC TF family, further indicating that the NAC-MYB-based transcriptional regulatory network may have a common evolutionary origin across lower and higher vascular plants.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePinus massoniana\u003c/em\u003e Lamb (Pinaceae), celebrated for its rapid growth rate, straight trunk profile, and exceptional resilience to drought and in fertile soils, stands as a cornerstone species in the afforestation of barren hills, the establishment of large-diameter timber forests, and the sustainable production of industrial raw material across southern China (Quan and Ding \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yu, et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While prior studies have primarily focused on its gene functions in cellulose and lignin biosynthesis (Ha Van, et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ni, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the molecular regulatory mechanisms underlying SCW biosynthesis remain underexplored in this taxon. In this study, we identified a VND-subfamily gene in \u003cem\u003eP. massoniana\u003c/em\u003e. Tissue-specific profiling revealed preferential \u003cem\u003ePmVNS2\u003c/em\u003e transcription in developing xylem. Heterologous overexpression of \u003cem\u003ePmVNS2\u003c/em\u003e in poplar enhanced the growth performance, secondary xylem cell layer proliferation, and SCW thickening. These findings provide valuable genetic resources for enhancing wood traits and lay the groundwork for future investigations into the molecular mechanisms of wood formation in conifer species.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant materials\u003c/h2\u003e\u003cp\u003eAll samples were collected from a full-sibling progeny test forest at the Zhangping Wuyi National Forestry Farm in Fujian Province, Southeast China. Tissues of the root, tender stem, developing xylem, megaspore, microspore, and cone (three biological duplications per tissue) were sampled for tissue-specific expression analysis of \u003cem\u003ePmVNS2\u003c/em\u003e. The transgenic material was \u003cem\u003eP. davidiana\u003c/em\u003e \u0026times; \u003cem\u003eP. bolleana\u003c/em\u003e grown in Murashige and Skoog medium for 35\u0026ndash;45 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. DNA and RNA isolation, cDNA synthesis and qRT-PCR\u003c/h2\u003e\u003cp\u003eThe DNA of \u003cem\u003eP. massoniana\u003c/em\u003e and poplar was extracted using a Plant GenPrep DNA Kit (Zoman Biotech, Beijing, China). Total RNA was isolated from various tissues with the RNAprep Pure Total RNA Extraction Kit (Tiangeng Biotech, Beijing, China). RNA was reverse transcribed to cDNA for subsequent experiments using a Prime Script RT Master Mix Kit (TaKaRa, Dalian, China). qRT-PCR was conducted on an ABI ViiA 7 platform using gene-specific primers and FastStart Universal SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). \u003cem\u003ePmDDI1\u003c/em\u003e (selected as the reference gene by our research group) was used as the reference gene (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and three technical replicates were performed per sample per run.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Gene cloning and sequence analysis\u003c/h2\u003e\u003cp\u003eThe gene sequence was obtained from our previous RNA-Seq analysis of developing xylem in \u003cem\u003eP. massoniana\u003c/em\u003e (Ni, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To study the effect of the gene on SCW formation, we used cDNA from the xylem of \u003cem\u003eP. massoniana\u003c/em\u003e as a template. The target fragments were amplified using Primer STAR Max DNA Polymerase (Takara, Dalian, China). The primers are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe open reading frames (ORFs) of the sequences were predicted using NCBI ORF Finder tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Wang, et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The primary structure and physicochemical properties of the protein were analyzed using the online tools of ExPASy ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ProtScale (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protscale/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protscale/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The structural domain of the protein was predicted using the BlastP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignment analysis was performed using Jalview 2.11.4.1. A phylogenetic tree was constructed using the maximum likelihood method (ML) in MEGA X with a bootstrap of 1000 (Kumar, et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Subcellular localization of PmVNS2\u003c/h2\u003e\u003cp\u003eUsing Nimble cloning technology, the target gene sequence was inserted into the pNC-Amp-GFP vector to generate the fusion expression construct 35S::\u003cem\u003ePmVNS2\u003c/em\u003e-GFP for subcellular localization (Yan, et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The isolation of \u003cem\u003eP. massoniana\u003c/em\u003e protoplasts and PEG-mediated transformation were conducted according to methods previously described by Ye et al. (Ye, et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The results were observed with an Axio Scope A1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Detection and phenotyping of transgenic plants\u003c/h2\u003e\u003cp\u003eThe obtained target gene was connected with the binary plant expression vector pH35GS using the ClonExpress II One-Step Cloning Kit (Vazyme, Nanjing, China) to generate the 35S::\u003cem\u003ePmVNS2\u003c/em\u003e overexpression vector. The 35S::\u003cem\u003ePmVNS2\u003c/em\u003e recombinant plasmid was transferred into the leaves of \u003cem\u003eP. davidiana\u003c/em\u003e \u0026times; \u003cem\u003eP. bolleana\u003c/em\u003e (Xu, et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Transgenic plants were selected using kanamycin resistance screening, and DNA was extracted from the resistant plants for PCR verification. Using RNA from the stems of OE_\u003cem\u003ePmVNS2\u003c/em\u003e lines as a template, qRT-PCR was performed to analyze their transcriptional expression levels, and \u003cem\u003eEf1α\u003c/em\u003e was used as a reference gene (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)(Xu et al., 2011).\u003c/p\u003e\u003cp\u003ePhenotypic traits, including plant height, root number, and total root length, were measured in transgenic lines and CK lines grown on MS medium. Measurements were taken at four time points: 10 days, 20 days, 30 days, and 40 days after transplantation. For each transgenic line, five plants were selected, and their phenotypic data were recorded at each of these time points. Subsequently, all strains were transplanted to sterilized nutrient soil for growth, and plant height and ground diameter (1 cm above the roots) were measured at 60 days. The experiment did not span the winter season or any period inducing dormancy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Paraffin section and scanning electron microscope analyses\u003c/h2\u003e\u003cp\u003eTo compare the structural characteristics of SCWs between transgenic plants and CK plants, the following treatments were performed: Stem base segments approximately 2 mm in length were sectioned using the paraffin-embedding method (Xu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) for histomorphological observation under an optical microscope. Following staining, the paraffin sections were imaged, and quantitative analysis, including measurement of secondary xylem cell layers and the pith-to-primary xylem area ratio, was performed using ImageJ software. For each sample, at least three randomly selected fields of view were analyzed: the \"Cell Counter\" plugin counted cell layers from the vascular cambium to the secondary xylem edge. At the same time, the \"Freehand Selection\" tool outlined the pith and primary xylem regions to measure their areas and calculate the ratio. Additionally, 5-mm stem segments were fixed in 4% glutaraldehyde, dehydrated through a graded ethanol series, and then processed using critical point drying. Ultrastructural observation and comparison of secondary cell walls were then conducted using a scanning electron microscope (SEM; Quanta 200, USA). From the SEM images, 10 randomly selected fields were analyzed, with at least 10 fiber cells measured per field using the instrument\u0026rsquo;s software to determine secondary cell wall thickness. The mean and standard deviation were recorded. All images were captured under consistent magnification and contrast settings to ensure data accuracy and comparability.\u003c/p\u003e\u003cp\u003e(1) The bases of the stems were cut into 2-mm segments, which were processed by paraffin-embedded sectioning method (Xu, et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and observed histomorphologically under a light microscope; (2) In addition, 5-mm stem segments were fixed in 4% glutaraldehyde and then dehydrated by gradient ethanol and critical point drying treatment, and SCWs were analyzed for ultrastructural comparison using a scanning electron microscope (Quanta 200, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Gene cloning and sequence analysis of PmVNS2\u003c/h2\u003e\u003cp\u003eBased on the previous transcriptome analysis, the phenylpropane biosynthesis pathway gene \u003cem\u003eCluster-15034.26926\u003c/em\u003e was involved in wood formation (Ni, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its ORF sequence was 1062 bp, and encoded 353 amino acids (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), which was named \u003cem\u003ePmVNS2\u003c/em\u003e because of its 98.78% similarity to \u003cem\u003eP. taeda PtaVNS2\u003c/em\u003e. The predicted molecular weight of the PmVNS2-encoded protein was 40.64 kDa, and the theoretical isoelectric point was 5.91, which was an acidic protein (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Sequence alignment revealed that the N-terminal of the PmVNS2 protein contains a typical NAC domain (A-E subdomains), and its C-terminal contains the LP-box and WQ-box motifs with transcriptional activation functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, S1b). In addition, to explore the phylogenetic relationships, we constructed a phylogenetic tree using the maximum likelihood method based on the amino acid sequences of VNSs proteins from multiple species. The results showed that \u003cem\u003ePmVNS2\u003c/em\u003e clustered in the VND subgroup together with \u003cem\u003ePtaVNS2\u003c/em\u003e, \u003cem\u003eOsSWN6\u003c/em\u003e/\u003cem\u003e7\u003c/em\u003e, and \u003cem\u003eAtVND4\u003c/em\u003e-\u003cem\u003e6\u003c/em\u003e, and is closest in affinity to \u003cem\u003ePtaVNS2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Therefore, we suggest that \u003cem\u003ePmVNS2\u003c/em\u003e belongs to the VND subfamily.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Expression patterns and Subcellular localization of PmVNS2\u003c/h2\u003e\u003cp\u003eWe investigated the expression profiles of \u003cem\u003ePmVNS2\u003c/em\u003e in different tissues of \u003cem\u003eP. massoniana\u003c/em\u003e, including roots, microstrobilus, ovulate strobilus, immature stem, needles, cone, and developing xylem, using qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Relative expression levels were normalized to root tissue. \u003cem\u003ePmVNS2\u003c/em\u003e exhibited predominant expression in developing xylem (6-fold higher than roots) and immature stem (3-fold higher than roots), suggesting its critical role in xylem formation. To further characterize its subcellular localization, we transiently transformed \u003cem\u003eP. massoniana\u003c/em\u003e protoplasts with the 35S::\u003cem\u003ePmVNS2\u003c/em\u003e-GFP fusion vector via PEG-mediated transfection. Fluorescence microscopy revealed exclusive nuclear localization of \u003cem\u003ePmVNS2\u003c/em\u003e-GFP, confirming its identity as a nuclear protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Heterologous Transformation of PmVNS2 in Populus\u003c/h2\u003e\u003cp\u003eTo verify the biological function of the \u003cem\u003ePmVNS2\u003c/em\u003e in the wood formation, we constructed an overexpression vector of 35S::\u003cem\u003ePmVNS2\u003c/em\u003e to perform \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated leaf-disk plant transformations in \u003cem\u003eP. davidiana\u003c/em\u003e \u0026times; \u003cem\u003eP. bolleana.\u003c/em\u003e Eight randomly selected lines with healthy growth performance were subjected to DNA detection. We found that the size of the amplified PCR fragments was consistent with our expectations, and no target bands were detected in CK (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Transcript analysis demonstrated significant upregulation of \u003cem\u003ePmVNS2\u003c/em\u003e in transgenic plants, with the T7 line exhibiting a 597-fold increase relative to CK plants, followed by T1 (532-fold) and T6 (418-fold) lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The observed expression variability among eight independent lines likely stems from position effects caused by distinct T-DNA insertion sites. Based on these results, the T1, T6, and T7 lines were selected for subsequent functional analyses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Overexpression of PmVNS2 promoted plant growth\u003c/h2\u003e\u003cp\u003eTo explore the effect of overexpression of \u003cem\u003ePmVNS2\u003c/em\u003e on plant growth, preliminary observations were made on transgenic lines (T1, T6, and T7) and CK lines grown on MS medium for 40 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Transgenic lines showed significant growth advantages in plant height and root development, including root number and total root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). From 0\u0026ndash;20 days, transgenic lines showed slightly higher plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and significant root growth advantage, with average root number and total root length reaching 2.00 and 2.52-fold of CK, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). During 20\u0026ndash;30 days, total root length was significantly increased in transgenic lines, especially in the T7 line (46.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62 cm vs. CK: 18.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). During 30\u0026ndash;40 days, transgenic lines had 2.00-fold higher root number and 1.83-fold higher total root length than CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d), and the plant height of transgenic lines (an increase of 6.68 cm from 10\u0026ndash;40 days) was significantly greater than that of CK lines (5.80 cm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 60 days of growth in soil pots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), transgenic lines further exhibited enhanced phenotypic advantages: plant height (17.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97 cm vs. CK: 14.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 cm) and ground diameter (2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mm vs. CK: 1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mm) were significantly greater than CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). These findings indicate that \u003cem\u003ePmVNS2\u003c/em\u003e may orchestrate plant growth by coordinately enhancing both aboveground development and root system expansion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5. PmVNS2 Overexpression Promotes SCW Deposition in Xylem Development\u003c/h2\u003e\u003cp\u003eBased on the enhanced radial growth observed in stems of \u003cem\u003ePmVNS2\u003c/em\u003e overexpressing lines in the above study (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, c), to further elucidate the regulatory mechanism of this gene on SCW formation, we selected transgenic lines (T1, T7) and CK lines grown on MS medium for 40 days. Stem segments from the 10th-11th internodes were collected, and 2-mm-thick cross-sections were prepared. These sections underwent paraffin embedding, sectioning, and staining for histological observation and analysis. Microscopic observation revealed that the number of secondary xylem cell layers in transgenic lines increased approximately 1.5-fold compared to CK lines. Specifically, the T1 line averaged 18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59 layers, the T7 line averaged 21.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 layers, while the CK averaged 12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47 layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Furthermore, the pith and primary xylem occupied relatively minor proportions of the stem cross-sectional area, with significant differences observed in their area ratios (T1: 3.15, T7: 2.87, CK: 3.52) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, c). This indicates a significantly increased proportion of xylem and a relatively reduced pith cavity in transgenic plants. Analysis of the stem cross-section ultrastructure using scanning electron microscopy (SEM) further demonstrated a highly significant difference in xylem cell wall thickness between transgenic lines (3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u0026micro;m) and CK lines (1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). Collectively, these histomorphological and ultrastructural findings demonstrate that overexpression of \u003cem\u003ePmVNS2\u003c/em\u003e not only significantly promotes xylem cell differentiation and secondary wall deposition but also enhances the xylem construction process by regulating the spatial distribution between xylem and other tissues.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further dissect the molecular mechanism by which \u003cem\u003ePmVNS2\u003c/em\u003e promotes SCW deposition, we performed qRT-PCR analysis of the expression levels of SCW biosynthesis-related genes in the transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The results revealed that lignin biosynthesis genes (including \u003cem\u003eCCR2\u003c/em\u003e, \u003cem\u003eC3H3\u003c/em\u003e, \u003cem\u003eC4H2\u003c/em\u003e, \u003cem\u003e4CL5\u003c/em\u003e, \u003cem\u003eCOMT2\u003c/em\u003e, and \u003cem\u003eHCT1\u003c/em\u003e) exhibited 2- to 5-fold upregulation in transgenic plants compared to CK plants, except for \u003cem\u003eF5H2\u003c/em\u003e. Meanwhile, the expression levels of cellulose biosynthesis enzyme genes \u003cem\u003eCesA2B\u003c/em\u003e and \u003cem\u003eCesA3A\u003c/em\u003e were also significantly increased, while xylan biosynthesis-related genes \u003cem\u003eGT43B\u003c/em\u003e and \u003cem\u003eGT43D\u003c/em\u003e showed down-regulation. These findings suggested that \u003cem\u003ePmVNS2\u003c/em\u003e may regulate the directional deposition of SCW by synergistically activating the lignin and cellulose biosynthesis pathways and inhibiting xylan production. Notably, NAC TFs serve as upstream regulatory hubs that initiate SCW formation by activating MYB TFs. Consistent with this mechanism, transgenic plants demonstrated significantly elevated expression levels of seven SCW-related MYB TFs (\u003cem\u003eMYB002\u003c/em\u003e, \u003cem\u003eMYB003\u003c/em\u003e, \u003cem\u003eMYB020\u003c/em\u003e, \u003cem\u003eMYB021\u003c/em\u003e, \u003cem\u003eMYB090\u003c/em\u003e, \u003cem\u003eMYB128\u003c/em\u003e, and \u003cem\u003eMYB158\u003c/em\u003e) compared to CK plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). \u003cem\u003eMYB128\u003c/em\u003e displayed the most pronounced induction (\u0026gt;\u0026thinsp;8-fold), while other \u003cem\u003eMYB\u003c/em\u003e genes showed 2-7-fold upregulation. This synergistic activation with the MYB regulatory network strongly implies that \u003cem\u003ePmVNS2\u003c/em\u003e might drive the ectopic deposition of SCW during wood formation by acting upstream of the NAC-MYB regulatory cascade.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWood formation is controlled by a highly ordered transcriptional regulatory network involving VNS and MYB family transcription factors (Akiyoshi 2020; Hao, et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hussey, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Studies in model plants \u003cem\u003eArabidopsis\u003c/em\u003e and poplar have demonstrated that VNDs (VNS members) and NSTs function as master regulatory switches governing SCW formation through the initiation of gene expression cascades (Ohtani, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Takata, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yamaguchi, et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhong and Ye \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Through analysis of RNA-seq, we identified \u003cem\u003ePmVNS2\u003c/em\u003e, a VND subfamily member in \u003cem\u003eP. massoniana\u003c/em\u003e. qRT-PCR revealed predominant expression of \u003cem\u003ePmVNS2\u003c/em\u003e in xylem tissues, followed by immature stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), consistent with the characteristic spatial expression pattern of SCW biosynthesis-related genes that typically exhibit elevated expression in stem tissues, particularly mature stems (Li, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yang 2015; Zhao, et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To investigate the regulatory role of \u003cem\u003ePmVNS2\u003c/em\u003e in SCW development, we conducted functional characterization through heterologous overexpression in transgenic poplar systems.\u003c/p\u003e\u003cp\u003eWood formation depends on the continuous division and differentiation of vascular cambium cells, ultimately generating secondary xylem and phloem (Chen, et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ye and Zhong \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). As core regulators for SCW biosynthesis, VNS family genes are functionally highly conserved across multiple species. For instance, overexpression of \u003cem\u003ePtrWND2B\u003c/em\u003e/\u003cem\u003ePtVNS10\u003c/em\u003e (NST subfamily) and \u003cem\u003ePtrWND6B\u003c/em\u003e/\u003cem\u003ePtVNS08\u003c/em\u003e (VND subfamily) in \u003cem\u003eP. trichocarpa\u003c/em\u003e significantly induces SCW thickening in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Ohtani, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), while overexpression of \u003cem\u003ePtoVNS11\u003c/em\u003e in \u003cem\u003ePopulus tomentosa\u003c/em\u003e leads to abnormally compact cell walls in wood fibers and vessel cell walls (Yang 2015). Our study further demonstrates that overexpression of \u003cem\u003ePmVNS2\u003c/em\u003e, a VND subfamily member, not only increases the number of SCW cell layers in transgenic plants but also elevates cell wall thickness to 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u0026micro;m, representing a 1.81-fold increase compared to CK plants (1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;m) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). This observation aligns with the ectopic SCW deposition caused by heterologous expression of the \u003cem\u003eP. taeda\u003c/em\u003e ortholog \u003cem\u003ePtaVNS2\u003c/em\u003e in tobacco (Akiyoshi 2020), corroborating the conserved regulatory role of the VND subfamily in SCW biosynthesis. Furthermore, the small-rosette and leaf curling phenotypes induced by \u003cem\u003ePpNAC1\u003c/em\u003e overexpression from \u003cem\u003eP. pinaster\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e mirror those reported for \u003cem\u003eArabidopsis SND1\u003c/em\u003e/\u003cem\u003eNST1\u003c/em\u003e overexpression (Mitsuda, et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Pascual, et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), suggesting functional convergence of VNS regulatory networks across divergent species. Although this study focuses on secondary xylem, ectopic expression of \u003cem\u003ePmVNS2\u003c/em\u003e may affect cell wall deposition in non-typical tissues, which warrants further investigation. Collectively, these findings demonstrate that VNS family genes, particularly the VND and NST subfamilies, play pivotal roles in wood formation by coordinating SCW thickening (Hussey, et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ohtani, et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGenes involved in SCW biosynthesis typically exert bidirectional regulatory effects on plant growth through resource competition mechanisms. Previous studies have demonstrated that while overexpression of \u003cem\u003ePtoLAC14\u003c/em\u003e in \u003cem\u003eP. tomentosa\u003c/em\u003e enhances cell wall thickening and lignification, it concurrently suppresses plant height growth (Qin, et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, overexpression of \u003cem\u003ePtrWND2B\u003c/em\u003e/\u003cem\u003e6B\u003c/em\u003e in \u003cem\u003eP. trichocarpa\u003c/em\u003e results in shortened stems and reduced growth rates in transgenic plants (Zhong, et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In contrast to these findings, our study reveals that \u003cem\u003ePmVNS2\u003c/em\u003e overexpression induces coordinated development of aboveground and belowground systems. During early developmental stages (0\u0026ndash;40 days), transgenic plants exhibited pronounced growth advantages over CK plants: plant height increment increased by 15.2% (6.68 cm vs. CK 5.80 cm), while root number and total root length reached 2.00-fold and 1.83-fold of CK plants, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). After 60 days of soil cultivation, transgenic plants displayed significantly greater stem diameter (2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mm vs. CK: 1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 mm) and plant height compared to CK plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We propose that \u003cem\u003ePmVNS2\u003c/em\u003e may dynamically regulate the spatiotemporal pattern of SCW deposition, simultaneously enhancing cell wall mechanical strength and promoting holistic plant growth. This mechanism aligns with the effects of overexpressing the \u003cem\u003eArabidopsis\u003c/em\u003e homolog \u003cem\u003eSND2\u003c/em\u003e, which increases xylem thickness in poplar while enhancing plant height, stem diameter, leaf expansion, and root development (Han \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our findings highlight the potential application value of \u003cem\u003ePmVNS2\u003c/em\u003e overexpression in coordinating plant growth with SCW deposition, though the growth characteristics of transgenic plants at maturity require further investigation.\u003c/p\u003e\u003cp\u003eThe two-layer cascade regulatory system composed of VNS-MYB TFs constitutes the master control network for SCW formation. As the top regulators of the cascade regulatory network, VNS TFs achieve multilevel regulation by coordinating the expression of downstream TFs, SCW biosynthesis-related genes, and PCD-related genes (Akiyoshi, et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nakano, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Taylor-Teeples, et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, we observed significant upregulation of MYB family genes, including \u003cem\u003eMYB002\u003c/em\u003e, \u003cem\u003eMYB003\u003c/em\u003e, \u003cem\u003eMYB020\u003c/em\u003e, \u003cem\u003eMYB021\u003c/em\u003e, \u003cem\u003eMYB090\u003c/em\u003e, \u003cem\u003eMYB128\u003c/em\u003e, and \u003cem\u003eMYB158\u003c/em\u003e in \u003cem\u003ePmVNS2\u003c/em\u003e overexpressing transgenic poplar. Notably, \u003cem\u003eMYB002\u003c/em\u003e/\u003cem\u003e021\u003c/em\u003e and \u003cem\u003eMYB003\u003c/em\u003e/\u003cem\u003e020\u003c/em\u003e were identified as secondary regulators in poplar, functionally complementing the SCW-deficient phenotypic defects in \u003cem\u003eArabidopsis MYB46\u003c/em\u003e-\u003cem\u003eMYB83\u003c/em\u003e double mutants (McCarthy, et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pratyusha and Sarada \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wegrzyn, et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhong, et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Moreover, the expression of the key lignin biosynthesis pathway genes, \u003cem\u003eCCR2\u003c/em\u003e, \u003cem\u003eC4H2\u003c/em\u003e and \u003cem\u003e4CL5\u003c/em\u003e, was also significantly elevated. Previous studies have demonstrated that inhibition of these genes directly reduces lignin accumulation (Liu, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ni, et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Novaes, et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Th\u0026eacute;venin, et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhang, et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, \u003cem\u003ePmVNS2\u003c/em\u003e overexpression specifically activated the expression of the cellulose synthase genes (\u003cem\u003eCesA2B\u003c/em\u003e and \u003cem\u003eCesA3A\u003c/em\u003e), but inhibited the transcription of the xylan synthase genes (\u003cem\u003eGT43B\u003c/em\u003e \u0026amp; \u003cem\u003eGT43D\u003c/em\u003e). This suggests that \u003cem\u003ePmVNS2\u003c/em\u003e differentially regulates polysaccharide metabolic pathways to modulate cell wall composition, positively regulating lignin and cellulose biosynthesis, yet negatively regulating xylan synthesis. Collectively, our findings propose that \u003cem\u003ePmVNS2\u003c/em\u003e serves as a top regulator of SCW deposition during wood formation in conifers, with functional conservation mirroring the VNS regulatory networks reported in angiosperms. This discovery provides novel evidence supporting the evolutionary homology of SCW regulatory mechanisms between gymnosperms and angiosperms.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWood formation is governed by a hierarchical NAC-MYB regulatory network, in which NAC domain transcription factors function as apex regulatory components. This study demonstrates that \u003cem\u003ePmVNS2\u003c/em\u003e from \u003cem\u003eP. massoniana\u003c/em\u003e exhibits predominant expression in xylem tissues. Heterologous overexpression of \u003cem\u003ePmVNS2\u003c/em\u003e activates downstream MYB TFs (\u003cem\u003eMYB020\u003c/em\u003e/\u003cem\u003e021\u003c/em\u003e), lignin/cellulose biosynthesis genes (\u003cem\u003eCCR2\u003c/em\u003e, \u003cem\u003eC4H2\u003c/em\u003e, \u003cem\u003eCesA2B\u003c/em\u003e, and \u003cem\u003eCesA3A\u003c/em\u003e), while suppressing xylan synthesis genes (\u003cem\u003eGT43B\u003c/em\u003e/\u003cem\u003eD\u003c/em\u003e), synergistically promoting secondary xylem cell layer proliferation (+\u0026thinsp;1.5-fold) and cell wall thickening (3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u0026micro;m vs. CK 1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;m). This study demonstrates that VND orthologs in conifers regulate SCW deposition through the conserved NAC-MYB pathway, providing genetic resources for targeted improvement of wood properties in conifer species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No. 32301552), the Guangxi Science and Technology Major Program (AA24263021), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.T.T.,\u0026nbsp;Z.X.N., and\u0026nbsp;L.A.X. conceived and designed the research. Y.T.T., K.W., and\u0026nbsp;H.R.\u0026nbsp;performed the experiments and contributed analysis tools.\u0026nbsp;Y.T.T. and M.X. analyzed data. Y.T.T. wrote the manuscript. Z.X.N. and L.A.X.\u0026nbsp;reviewed the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkiyoshi N, Ihara A, Matsumoto T, Takebayashi A, Hiroyama R, Kikuchi J, Demura T, Ohtani M (2021) Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties. Plant Cell Physiol 62:1963-1974. http://dx.doi.org/10.1093/pcp/pcab102\u003c/li\u003e\n\u003cli\u003eAkiyoshi NA, Nakano Y, Sano R, Kunigita Y, Ohtani M, Demura T (2020) Involvement of VNS NAC-domain transcription factors in tracheid formation in Pinus taeda. Tree Physiology 40:704-716. http://dx.doi.org/10.1093/treephys/tpz106\u003c/li\u003e\n\u003cli\u003eBo Xu MO, Masatoshi Yamaguchi, Kiminori Toyooka, Mayumi Wakazaki (2014) Contribution of NAC Transcription Factors to Plant Adaptation to Land. Science 343:1505-1508. http://dx.doi.org/10.1126/science.1248417\u003c/li\u003e\n\u003cli\u003eChen H, Wang JP, Liu HZ, Li HY, Lin YCJ, Shi R, Yang CM, Gao JH, Zhou CG, Li QZ, Sederoff RR, Li W, Chiang VL (2019) Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Populus trichocarpa. Plant Cell 31:602-626. http://dx.doi.org/10.1105/tpc.18.00620\u003c/li\u003e\n\u003cli\u003eDuval I, Lachance D, Giguere I, Bomal C, Morency MJ, Pelletier G, Boyle B, MacKay JJ, Seguin A (2014) Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development. J Exp Bot 65:2319-2333. http://dx.doi.org/10.1093/jxb/eru116\u003c/li\u003e\n\u003cli\u003eGe S, Han X, Xu X, Shao Y, Zhu Q, Liu Y, Du J, Xu J, Zhang S (2020) WRKY15 Suppresses Tracheary Element Differentiation Upstream of VND7 During Xylem Formation. The Plant Cell 32:2307-2324. http://dx.doi.org/10.1105/tpc.19.00689\u003c/li\u003e\n\u003cli\u003eHa Van H, Ho Van G, Ngo Van T, Zhang S, Wang Y (2012) Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees. Molecular Breeding 29:173-180. http://dx.doi.org/10.1007/s11032-010-9535-9\u003c/li\u003e\n\u003cli\u003eHan Z (2021) Functions of SND2-B2 Gene from Populus in Secondary Cell Wall Biosynthesis. Shandong Agricultural University, Shandong, China\u003c/li\u003e\n\u003cli\u003eHao Y, Lu F, Pyo SW, Kim MH, Ko JH, Yan X, Ralph J, Li Q (2024) PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar. Journal of Integrative Plant Biology 66:1658-1674. http://dx.doi.org/10.1111/jipb.13717\u003c/li\u003e\n\u003cli\u003eHussey SG, Mizrachi E, Creux NM, Myburg AA (2013) Navigating the transcriptional roadmap regulating plant secondary cell wall deposition. Frontiers in Plant Science 4. http://dx.doi.org/10.3389/fpls.2013.00325\u003c/li\u003e\n\u003cli\u003eJohnsson C, Jin X, Xue WY, Dubreuil C, Lezhneva L, Fischer U (2019) The plant hormone auxin directs timing of xylem development by inhibition of secondary cell wall deposition through repression of secondary wall NAC-domain transcription factors. Physiologia Plantarum 165:673-689. http://dx.doi.org/10.1111/ppl.12766\u003c/li\u003e\n\u003cli\u003eKo JH, Jeon HW, Kim WC, Kim JY, Han KH (2014) The MYB46/MYB83-mediated transcriptional regulatory programme is a gatekeeper of secondary wall biosynthesis. Annals of Botany 114:1099-1107. http://dx.doi.org/10.1093/aob/mcu126\u003c/li\u003e\n\u003cli\u003eKubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes \u0026amp; Development 19:1855-1860. http://dx.doi.org/10.1101/gad.1331305\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. MOLECULAR BIOLOGY AND EVOLUTION 33:1870-1874. http://dx.doi.org/10.1093/molbev/msw054\u003c/li\u003e\n\u003cli\u003eLamara M, Raherison E, Lenz P, Beaulieu J, Bousquet J, MacKay J (2016) Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce. The New phytologist 210:240-255. http://dx.doi.org/10.1111/nph.13762\u003c/li\u003e\n\u003cli\u003eLi Q, Lin Y-C, Sun Y-H, Song J, Chen H, Zhang X-H, Sederoff RR, Chiang VL (2012) Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa. Proceedings of the National Academy of Sciences 109:14699-14704. http://dx.doi.org/10.1073/pnas.1212977109\u003c/li\u003e\n\u003cli\u003eLiu Y, You S, Taylor-Teeples M, Li WL, Schuetz M, Brady SM, Douglas CJ (2014) BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 Interact and Regulate Secondary Cell Wall Formation via Repression of REVOLUTA. The Plant Cell 26:4843-4861. http://dx.doi.org/10.1105/tpc.114.128322\u003c/li\u003e\n\u003cli\u003eMcCarthy RL, Zhong RQ, Fowler S, Lyskowski D, Piyasena H, Carleton K, Spicer C, Ye ZH (2010) The Poplar MYB Transcription Factors, PtrMYB3 and PtrMYB20, are Involved in the Regulation of Secondary Wall Biosynthesis. Plant and Cell Physiology 51:1084-1090. http://dx.doi.org/10.1093/pcp/pcq064\u003c/li\u003e\n\u003cli\u003eMcCarthy RL, Zhong RQ, Ye ZH (2009) MYB83 Is a Direct Target of SND1 and Acts Redundantly with MYB46 in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis. Plant and Cell Physiology 50:1950-1964. http://dx.doi.org/10.1093/pcp/pcp139\u003c/li\u003e\n\u003cli\u003eMitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270-280. http://dx.doi.org/10.1105/tpc.106.047043\u003c/li\u003e\n\u003cli\u003eMitsuda N, Seki M, Shinozaki K, Ohme-Takagi M (2005) The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17:2993-3006. http://dx.doi.org/10.1105/tpc.105.036004\u003c/li\u003e\n\u003cli\u003eNakano Y, Yamaguchiz M, Endo H, Rejab NA, Ohtani M (2015) NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Frontiers in Plant Science 6. http://dx.doi.org/10.3389/fpls.2015.00288\u003c/li\u003e\n\u003cli\u003eNi ZX, Han X, Yang ZQ, Xu M, Feng YH, Chen YB, Xu LA (2020) Integrative analysis of wood biomass and developing xylem transcriptome provide insights into mechanisms of lignin biosynthesis in wood formation of Pinus massoniana. International Journal of Biological Macromolecules 163:1926-1937. http://dx.doi.org/10.1016/j.ijbiomac.2020.08.253\u003c/li\u003e\n\u003cli\u003eNovaes E, Kirst M, Chiang V, Winter-Sederoff H, Sederoff R (2010) Lignin and Biomass: A Negative Correlation for Wood Formation and Lignin Content in Trees. Plant Physiology 154:555-561. http://dx.doi.org/10.1104/pp.110.161281\u003c/li\u003e\n\u003cli\u003eOhtani M, Nishikubo N, Xu B, Yamaguchi M, Mitsuda N, Goue N, Shi F, Ohme-Takagi M, Demura T (2011) A NAC domain protein family contributing to the regulation of wood formation in poplar. Plant Journal 67:499-512. http://dx.doi.org/10.1111/j.1365-313X.2011.04614.x\u003c/li\u003e\n\u003cli\u003ePascual MB, Llebres MT, Craven-Bartle B, Canas RA, Canovas FM, Avila C (2018) PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine. Plant Biotechnology Journal 16:1094-1104. http://dx.doi.org/10.1111/pbi.12854\u003c/li\u003e\n\u003cli\u003ePratyusha DS, Sarada DVL (2022) MYB transcription factors\u0026mdash;master regulators of phenylpropanoid biosynthesis and diverse developmental and stress responses. Plant Cell Reports 41:2245-2260. http://dx.doi.org/10.1007/s00299-022-02927-1\u003c/li\u003e\n\u003cli\u003eQin SF, Fan CF, Li XH, Li Y, Hu J, Li CF, Luo KM (2020) LACCASE14 is required for the deposition of guaiacyl lignin and affects cell wall digestibility in poplar. BIOTECHNOLOGY FOR BIOFUELS 13. http://dx.doi.org/10.1186/s13068-020-01843-4\u003c/li\u003e\n\u003cli\u003eQuan W, Ding G (2017) Root tip structure and volatile organic compound responses to drought stress in Masson pine (Pinus massoniana Lamb.). Acta Physiologiae Plantarum 39. http://dx.doi.org/10.1007/s11738-017-2558-7\u003c/li\u003e\n\u003cli\u003eTakata N, Awano T, Nakata MT, Sano Y, Sakamoto S, Mitsuda N, Taniguchi T (2019) Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells. Tree Physiology 39:514-525. http://dx.doi.org/10.1093/treephys/tpz004\u003c/li\u003e\n\u003cli\u003eTaylor-Teeples M, Lin L, de Lucas M, Turco G, Toal TW, Gaudinier A, Young NF, Trabucco GM, Veling MT, Lamothe R, Handakumbura PP, Xiong G, Wang C, Corwin J, Tsoukalas A, Zhang L, Ware D, Pauly M, Kliebenstein DJ, Dehesh K, Tagkopoulos I, Breton G, Pruneda-Paz JL, Ahnert SE, Kay SA, Hazen SP, Brady SM (2015) An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517:571-U307. http://dx.doi.org/10.1038/nature14099\u003c/li\u003e\n\u003cli\u003eTh\u0026eacute;venin J, Pollet B, Letarnec B, Saulnier L, Gissot L, Maia-Grondard A, Lapierre C, Jouanin L (2011) The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana. Molecular Plant 4:70-82. http://dx.doi.org/10.1093/mp/ssq045\u003c/li\u003e\n\u003cli\u003eValdivia ER, Herrera MT, Gianzo C, Fidalgo J, Revilla G, Zarra I, Sampedro J (2013) Regulation of secondary wall synthesis and cell death by NAC transcription factors in the monocot Brachypodium distachyon. Journal of Experimental Botany 64:1333-1343. http://dx.doi.org/10.1093/jxb/ers394\u003c/li\u003e\n\u003cli\u003eWang CH, Zhao FQ, Liu JX, Liu HY (2020) Short communication: The essential role of N-glycosylation in the transport activity of bovine peptide transporter 2. Journal of Dairy Science 103:6679-6683. http://dx.doi.org/10.3168/jds.2019-16858\u003c/li\u003e\n\u003cli\u003eWang Z, Mao Y, Guo Y, Gao J, Liu X, Li S, Lin Y-CJ, Chen H, Wang JP, Chiang VL, Li W (2020) MYB Transcription Factor161 Mediates Feedback Regulation of Secondary wall-associated NAC-Domain1 Family Genes for Wood Formation. Plant Physiology 184:1389-1406. http://dx.doi.org/10.1104/pp.20.01033\u003c/li\u003e\n\u003cli\u003eWegrzyn JL, Eckert AJ, Choi M, Lee JM, Stanton BJ, Sykes R, Davis MF, Tsai CJ, Neale DB (2010) Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem. New Phytologist 188:515-532. http://dx.doi.org/10.1111/j.1469-8137.2010.03415.x\u003c/li\u003e\n\u003cli\u003eXu M, Chen C, Cai H, Wu L (2018) Overexpression of PeHKT1;1 Improves Salt Tolerance in Populus. Genes 9. http://dx.doi.org/10.3390/genes9100475\u003c/li\u003e\n\u003cli\u003eXu W, Qi H, Shen T, Zhao M, Song Z, Ran N, Wang J, Xi M, Xu M (2023) Poplar coma morphogenesis and miRNA regulatory networks by combining ovary tissue sectioning and deep sequencing. iScience 26. http://dx.doi.org/10.1016/j.isci.2023.106496\u003c/li\u003e\n\u003cli\u003eYamaguchi M, Kubo M, Fukuda H, Demura T (2008) VASCULAR-RELATED NAC-DOMAIN7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots. Plant Journal 55:652-664. http://dx.doi.org/10.1111/j.1365-313X.2008.03533.x\u003c/li\u003e\n\u003cli\u003eYan P, Zeng YJ, Shen WT, Tuo DC, Li XY, Zhou P (2020) Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning. Frontiers in Bioengineering and Biotechnology 7. http://dx.doi.org/10.3389/fbioe.2019.00460\u003c/li\u003e\n\u003cli\u003eYang LY, Hou YS, Zhao X, Lu WX, Li YL, Yang F, Tang SH, Luo KM (2015) Identification and characterization of a wood-associated NAC domain transcription factor PtoVNS11 from Populus tomentosa Carr. TREES-STRUCTURE AND FUNCTION 29:1091-1101. http://dx.doi.org/10.1007/s00468-015-1188-1\u003c/li\u003e\n\u003cli\u003eYe YJ, Han X, Rong H, Qian RJ, Zheng J, Ni ZX, Xu LA (2022) Effect of the PmARF6 Gene from Masson Pine (Pinus massoniana) on the Development of Arabidopsis. GENES 13. http://dx.doi.org/10.3390/genes13030469\u003c/li\u003e\n\u003cli\u003eYe ZH, Zhong RQ (2015) Molecular control of wood formation in trees. Journal of Experimental Botany 66:4119-4131. http://dx.doi.org/10.1093/jxb/erv081\u003c/li\u003e\n\u003cli\u003eYu C, Lv J, Xu H (2024) Plant growth-promoting fungi and rhizobacteria control Fusarium damping-off in Mason pine seedlings by impacting rhizosphere microbes and altering plant physiological pathways. Plant and Soil. http://dx.doi.org/10.1007/s11104-024-06475-3\u003c/li\u003e\n\u003cli\u003eZhang M, Zhu P, Agassin RH, Yao S, Wang D, Huang Z, Zhang C, Hao Q, Ji K (2023) Identification, Classification and Characterization of bZIP Transcription Factor Family Members in Pinus massoniana Lamb. Forests 14. http://dx.doi.org/10.3390/f14010155\u003c/li\u003e\n\u003cli\u003eZhang S, Cao L, Chen D, Chang R, Cao J, Zhang Q, Qin Y, Liu G, Xu Z (2025) PtrVINV2 is dispensable for cellulose synthesis but essential for salt tolerance in Populus trichocarpa Torr. and Gray. Plant Biotechnology Journal. http://dx.doi.org/10.1111/pbi.70022\u003c/li\u003e\n\u003cli\u003eZhang Y, Chen S, Xu L, Chu S, Yan X, Lin L, Wen J, Zheng B, Chen S, Li Q (2024) Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar. The Plant Cell 36:1806-1828. http://dx.doi.org/10.1093/plcell/koae040\u003c/li\u003e\n\u003cli\u003eZhao Y, Sun J, Xu P, Zhang R, Li L (2014) Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species. Plant Physiology 164:765-776. http://dx.doi.org/10.1104/pp.113.231134\u003c/li\u003e\n\u003cli\u003eZhong R, Lee C, Ye ZH (2010) Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Mol Plant 3:1087-1103. http://dx.doi.org/10.1093/mp/ssq062\u003c/li\u003e\n\u003cli\u003eZhong R, McCarthy RL, Lee C, Ye Z-H (2011) Dissection of the Transcriptional Program Regulating Secondary Wall Biosynthesis during Wood Formation in Poplar. Plant Physiology 157:1452-1468. http://dx.doi.org/10.1104/pp.111.181354\u003c/li\u003e\n\u003cli\u003eZhong R, Ye ZH (2012) MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes. Plant Cell Physiol 53:368-380. http://dx.doi.org/10.1093/pcp/pcr185\u003c/li\u003e\n\u003cli\u003eZhong RQ, Demura T, Ye ZH (2006) SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18:3158-3170. http://dx.doi.org/10.1105/tpc.106.047399\u003c/li\u003e\n\u003cli\u003eZhong RQ, Lee CH, Ye ZH (2010) Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors. Plant Physiology 152:1044-1055. http://dx.doi.org/10.1104/pp.109.148270\u003c/li\u003e\n\u003cli\u003eZhong RQ, Ye ZH (2007) Regulation of cell wall biosynthesis. Current Opinion in Plant Biology 10:564-572. http://dx.doi.org/10.1016/j.pbi.2007.09.001\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-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pinus massoniana, NAC transcription factor, Second cell wall, Xylem differentiation, Transgenic poplar","lastPublishedDoi":"10.21203/rs.3.rs-7931415/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7931415/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNAC-domain transcription factors (TFs) play central roles in regulating secondary cell wall (SCW) biosynthesis during wood formation. In this study, we identified\u003cem\u003ePmVNS2\u003c/em\u003e, a VND-subfamily NAC gene from \u003cem\u003ePinus massoniana\u003c/em\u003e, encoding a 353-amino acid protein with conserved NAC domains. \u003cem\u003ePmVNS2\u003c/em\u003e was preferentially expressed in developing xylem. Heterologous expression in \u003cem\u003ePopulus davidiana\u003c/em\u003e × \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ebolleana\u003c/em\u003e significantly enhanced plant growth, increased the number of secondary xylem cell layers, and thickened SCWs (3.45 ± 0.27 μm vs. CK: 1.91 ± 0.18 μm). Transgenic lines showed upregulated expression of lignin biosynthetic genes (\u003cem\u003eCCR2\u003c/em\u003e, \u003cem\u003eC4H2\u003c/em\u003e), cellulose synthase genes (\u003cem\u003eCesA2B\u003c/em\u003e, \u003cem\u003eCesA3A\u003c/em\u003e), and MYB transcription factors (\u003cem\u003eMYB021\u003c/em\u003e, \u003cem\u003eMYB128\u003c/em\u003e), while xylan biosynthesis genes (\u003cem\u003eGT43B\u003c/em\u003e, \u003cem\u003eGT43D\u003c/em\u003e) were downregulated. These results demonstrate that \u003cem\u003ePmVNS2\u003c/em\u003e is a key regulator of SCW formation in conifers, offering a promising genetic target for wood quality improvement.\u003c/p\u003e","manuscriptTitle":"Heterologous expression of Pinus massonianaVNS2 enhances secondary cell wall deposition in poplar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 16:54:47","doi":"10.21203/rs.3.rs-7931415/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-12-01T03:18:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-04T10:57:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-04T07:11:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-29T16:29:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-10-29T04:39:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"64e6c919-0833-4a05-b463-cd185c879bb0","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:03:24+00:00","versionOfRecord":{"articleIdentity":"rs-7931415","link":"https://doi.org/10.1007/s00299-026-03717-9","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-01-20 15:58:27","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-11-13 16:54:47","video":"","vorDoi":"10.1007/s00299-026-03717-9","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03717-9","workflowStages":[]},"version":"v1","identity":"rs-7931415","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7931415","identity":"rs-7931415","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}