The cell wall, a key structural component of plant cells, was essential for regulating cell morphology, driving growth and development, and enabling cell expansion (Persson et al., 2007a). Cellulose, hemicellulose and pectin were the principal components of primary cell walls, while cellulose, hemicellulose and lignin were the principal components of secondary cell walls (Burton et al., 2010). It was evident that cellulose performed a pivotal function in processes such as cell growth, differentiation and signaling. In addition, hemicellulose had been observed to interact closely with cellulose microfibrils through hydrogen bonding (Welker et al., 2015).

Glycosyltransferases (GTs) are enzymes that catalyze the transfer of an activated sugar group to an acceptor substrate, which may consist of polysaccharides, peptides, lipids, or small molecules (Zabotina et al., 2021). Cellulose, hemicellulose, pectin, and lignin are the major polysaccharides in plants (Li et al., 2020). The cellulose synthase gene superfamily is a group of type-two glycosyltransferases (GT2), which is the largest family within GTs (Aspeborg et al., 2005). This superfamily could be further divided into two subfamilies: cellulose synthase (CesA) and nine cellulose synthase-like (Csl) subfamilies (CslA/B/C/D/E/F/G/H/J) (Yin et al., 2009, 2014). Cellulose was primarily synthesized by CesA (cellulose synthase), while some hemicelluloses were synthesized with the involvement of Csls (cellulose synthase-like proteins) (Yin et al., 2014). A new CslM subfamily had been identified in other flowering plants (Farrokhi et al., 2006; Little et al., 2018). Available studies had shown that the CslA gene was involved in the biosynthesis of the mannan and glucomannan backbone (Liepman et al., 2005; Goubet et al., 2009). The CslC genes, on the other hand, were responsible for the biosynthesis of the β-1,4-glucan backbone for xyloglucan synthesis (Cocuron et al., 2007). The Cs1D gene might be involved in the synthesis of cellulose or mannan in tip-growing cells (Park et al., 2011; Yin et al., 2011; Yang et al., 2020). In contrast, the CslF, CslH, and CslJ subfamilies catalyzed the biosynthesis of (1,3;1,4)-β-glucan, which were polymers commonly referred to as mixed ligated glucan (MLG) (Doblin et al., 2009; Vega-Sánchez et al., 2012; Little et al., 2019; Lou et al., 2022). However, the specific functions of the other four Csl subfamily members (CslB, CslE, CslG and CslM) remained to be elucidated.

The CesA/Csl gene families play a pivotal role in plant cell wall biosynthesis by mediating the synthesis of both cellulose and hemicellulose polysaccharides (McFarlane et al., 2014). In A. thaliana, AtCesA1, AtCesA3 and CesA6-like genes (AtCesA2/5/6/9) had been reported to be involved in primary wall cellulose synthesis, whereas the AtCesA4, AtCesA7 and AtCesA8 genes were responsible for secondary wall cellulose synthesis (Sod et al., 2023). Multiple CesA proteins assembled into the cellulose synthase complex CesA complexe (CSC), which was responsible for cellulose synthesis (Zhang et al., 2021). Csls, a member of the cellulose synthase superfamily, shared high sequence similarity with the CesA gene family.

CesA/Csl played a crucial role in plant growth and development (Polko and Kieber, 2019). For instance, the impairment of rice OsCSLD4 function resulted in a reduction of grain width and weight, whereas the overexpression of OsCSLD4 led to an increase in both grain width and weight (Zhao et al., 2022). The knockdown of HvCslF3 from barley through RNA interference resulted in a decrease in root growth rate, which was associated with a reduced elongation zone and a marked reduction in the root system (Lou et al., 2022). The PbrMADS52-PbrCSLD5 signaling pathway resulted in an elevated cellulose content within the cell wall of pear pollen tubes, consequently hindering the growth of the pollen tubes in Pyrus bretschneideri (Li et al., 2022). Likewise, the single mutants csld1 and csld4, as well as the double mutant csld1/csld4 in Arabidopsis, exhibited highly irregular pollen tube growth. This abnormality led to a substantial decrease in cellulose accumulation within the pollen tube wall and a pronounced disorganization of the pollen tube wall layers, ultimately impairing the genetic transmission of male gametophytes (Wang et al., 2011). Cellulose constitutes the primary component of secondary cell walls. In addition, CesA/Csls were also involved in stress response in plants. For instance, in rice, high temperature activates the kinase OsCDPK24/28, which phosphorylates OsHSFA4d at S146. This phosphorylation event subsequently leads to the differential regulation of HSP101 and CslF6 gene expression (Fang et al., 2025). Parallel studies examining gene expression changes in orchid stems under abiotic stress revealed distinct roles for Csl subfamilies: CslA appears to be a key subfamily in drought stress response across orchids of different life forms, whereas CslD may be particularly important for epiphytic and saprophytic orchids in adapting to freezing stress (Wang et al., 2022).

E. grandis, as one of the world’s three fast-growing tree species, has important economic, ecological and social values, and is widely used in paper, timber and other industrial fields. With the completion of genome sequencing of E. grandis (Myburg et al., 2014), a solid foundation for molecular biology research and genetic improvement of Eucalyptus has been provided. To date, the CesA/Csl gene family had been comprehensively characterized and studied in numerous plant species, including rice Oryza sativa (Ouyang et al., 2007), Sorghum bicolor (Paterson et al., 2009), Gossypium hirsutum (Li et al., 2017), Linum usitatissimum (Pydiura et al., 2015), Populus trichocarpa (Takata and Taniguchi, 2015) and Arabidopsis thaliana (Persson et al., 2007b). However, the CesA/Csl genes has not been identified in E. grandis. In view of the important roles of CesA and Csls genes in plant growth and development, the present study systematically identified the CesA and Csls family members in E. grandis, and analyzed their characteristics, including phylogenetic relationships, gene and protein structural characteristics, cis-acting elements and gene expression patterns. These results laid a theoretical foundation for in-depth study of the functions of CesA/Csls genes in cell wall synthesis during growth and development process in E. grandis.

Glycosyltransferases (GTs) were a class of enzymes that catalyze the attachment of sugar molecules to specific receptors via glycosidic bonds, and they were widely present in various organisms including plants, animals, and microorganisms (Lairson et al., 2008). In plants, these enzymes were particularly important as they directly participate in carbon allocation and energy storage by converting monosaccharides (such as glucose) produced through photosynthesis into disaccharides, oligosaccharides, and polysaccharides (Keegstra and Raikhel, 2001). In addition, glycosyltransferases drove the synthesis of plant cell wall polysaccharides (such as cellulose and hemicellulose) and were involved in the glycosylation modification of glycoproteins, glycolipids, and secondary metabolites (such as flavonoids and alkaloids) (Bowles et al., 2006). These functions played a crucial role in maintaining the structural integrity of plant cell walls, signal transduction, and stress resistance (Zhao et al., 2022). According to the classification of the Carbohydrate-Active enZymes Database (CAZy), plant glycosyltransferases had formed highly diversified gene families (such as the GT2 family), with members exhibiting significant differentiation in substrate specificity and function (Lombard et al., 2014).

The CesA gene family was closely related to the biosynthesis of plant cellulose. During cell wall formation, the proteins encoded by CesA genes catalyzed the polymerization of glucose molecules to form cellulose microfibrils, which were crucial for maintaining the strength and structural stability of the cell wall (Zhang et al., 2016). In different plants, there were multiple members of the CesA gene family, and they exhibited functional differences (Kumar and Turner, 2015). For example, in A.thaliana, different CesA genes played specific roles in the synthesis of primary and secondary wall cellulose (Carroll and Specht, 2011). The Csl gene family was involved in the synthesis of various cell wall polysaccharides and was closely linked to the synthesis of substances such as hemicellulose (Richmond and Somerville, 2000; Doblin et al., 2010). The Csl gene family comprised numerous members, each with distinct functions, and exhibited specific expression patterns in different tissues and organs during plant growth and development (Hazen et al., 2002; Yin et al., 2009). In A.thaliana, 10 CesA genes and 31 cellulose synthase-like (Csl) genes had been identified, and based on phylogenetic analysis, they were further classified into one CesA family and six Csl families (CslA/B/C/D/E/G) (Persson et al., 2007b). Based on the phylogenetic analysis, we identified 21 EgCesA genes and 41 Csl genes, which were further classified into one CesA family and six Csl families. The CesA family can be divided into two subclasses, with EgCesA1/3/6 and EgCesA4/7/8 serving as representative members. EgCesA1/3/6 is likely participated in the synthesis of primary wall fibers during cell elongation, with EgCesA1a/3b/6b/6i having higher expression levels in rapidly growing tissues such as the shoot apex, young leaves, and root tips. In contrast, EgCesA4/7/8 plays a role in cellulose synthesis during the thickening of secondary wall at the lignification stage, with increased expression levels observed in mature xylem and fiber cells ( Figures 1 , 6 ). These is consistent with that in Arabidopsis and poplar (Taylor et al., 2003; Desprez et al., 2007; Abbas et al., 2020), suggesting their conservation in these plant species. CslD genes (CslD1, CslD6a and CslD3) in A. thaliana were involved in the synthesis of mannose-containing polysaccharides (Liepman et al., 2005; Yin et al., 2011). The CslD genes were localized on the Golgi membrane, where they catalyzed the synthesis of 1,4-β-D-glucomannan by substrate GDP-glucose and GDP-mannose (Huang et al., 2018; Yang et al., 2020). In the CslD subfamily shown in Figure 6 , EgCslD2 is highly expressed in roots and also highly expressed in stem apex, suggesting that CslD proteins may be involved in cellulose synthesis during root hair and shoot growth. Eg CslD4 shows low expression levels in all tissues; Eg CslD6a and Eg CslD6b show higher expression levels in roots and stem apex, with lower levels in other regions. The diverse expression patterns of these group members suggest that subfunctionalization of genes may have occurred during their evolutionary process.

A phylogenetic tree analysis was conducted on the CesA/Csls gene families of E. grandis, P. trichocarpa, A. thaliana, and P. patens. P. patens is a bryophyte that serves as an outgroup for calibrating phylogenetic time nodes, facilitating the identification of the evolutionary origin of E. grandis. A. thaliana, a model organism for eudicots, diverged early in evolutionary history, and its annotated genome facilitates the examination of gene family alterations in E. grandis. P. trichocarpa, a woody eudicot that diverged approximately 100 million years ago, serves as a reference for investigating wood formation and metabolic processes in E. grandis. Although both species exhibit rapid growth, they differ in their lignin composition. Analyzing genes such as CesA provides insights into the evolutionary trajectory of lignin biosynthesis. Clustering analysis revealed that EgCesA3c clustered in the same branch with AtCesA3 from A. thaliana, which were involved in primary cell wall cellulose synthesis (Desprez et al., 2007). This suggested that these E. grandis CesA genes may be involved in the synthesis of primary cell wall cellulose. EgCesA4, EgCesA7, and EgCesA8 were phylogenetically closer to AtCesA4, AtCesA7, and AtCesA8 from A.thaliana, respectively, which were involved in secondary cell wall cellulose synthesis (Arioli et al., 1998). There were 10 CesA genes in A. thaliana, whereas 21 CesA genes were identified in E. grandis, and although there was a significant increase in number, it was still three genes that were involved in secondary cell wall synthesis (EgCesA4, EgCesA7, and EgCesA8), and they were significantly expressed in the xylem in the subsequent tissue expression analysis. Therefore, it was inferred that these genes played a core role in secondary cell wall cellulose synthesis. P. patens genes were mainly distributed within the CesA, CslA, CslC and CslD families and exhibited a clustered distribution. In contrast, the tree species E. grandis and P. trichocarpa were more evenly distributed across the phylogenetic tree. This might be attributed to the fact that P. patens was a lower plant with a relatively low level of differentiation and slow species evolution. In contrast, P. trichocarpa and E. grandis have undergone more pronounced differentiation during evolution, better adapting to environmental changes, which reflected the strong stress resistance and stability of these species.

The expression heatmap analysis based on RNA-Seq data revealed that out of the gene set examined, 61 genes exhibited expression activity, with EgCslD1 being silent and showing no detectable expression signal. Among the expressed genes, EgCesA4, EgCesA7, and EgCesA8 demonstrated significant tissue-specific and developmental stage-specific expression patterns. These three genes were highly expressed in the xylem tissue of 6-month-old plants, with higher expression than those in other tissues and developmental stages. Moreover, EgCesA4, EgCesA7, and EgCesA8 also showed high expression levels in the 3rd, 5th, 7th, 9th, and 11th internodes of 6-month-old plants, as well as in the xylem tissue of 6-year-old plants. This expression pattern further confirms that EgCesA4, EgCesA7, and EgCesA8 were involved in the process of secondary wall thickening, specifically the cellulose biosynthetic pathway during the lignification stage, playing a crucial regulatory role in the formation of plant secondary cell walls and the lignification process. Cellulose synthase constitutes a rose knot-like cellulose synthase complex (CSC) at the plasma membrane, and two types of CSCs are involved in coordinated cellulose biosynthesis in the secondary cell wall of Populus, forming a multilayered structure that differentiates the xylem cells of Populus that undergo secondary cell wall thickening (Xi et al., 2017). In the present study, the high expression of EgCesA4, EgCesA7, and EgCesA8 in xylem in E. grandis might also constitute a complex involved in secondary cell wall synthesis and thickening. As is typical of woody plants, CSC and other complexes play important roles in cellulose structural characterization and multilayer wall structure formation during cell wall thickening in P. trichocarpa and E. grandis. Protein interaction network analysis is beneficial for predicting the composition of the cellulose synthase complex and elucidating its biological functions. Therefore, further investigation of the protein interaction network is necessary to characterize cellulose synthase complexes (CSCs) and their roles in cell wall synthesis.

When comparing the three-dimensional (3D) structures of proteins from different subfamilies, significant differences were observed. The results demonstrated high structural conservation within subfamily members, exhibiting only marginal variations ( Figure 8 ). These structural variations were primarily attributed to the varying lengths of α-helices, β-turns, and γ-irregular coils among the proteins. The differences in these secondary structural elements resulted in altered folding angles of the proteins in space, and these changes in spatial conformation were highly likely to constitute the structural basis for the distinct functions of these proteins. Understanding the relationships between structure and function in cellulose synthases (CesAs) has been significantly enhanced by the partial elucidation of the structure of the CesA8 protein derived from poplar (PDB Entry-6WLB) using cryo-electron microscopy (Purushotham et al., 2020). PttCesA8 protein is a ortholog of Arabidopsis AtCesA8 and cotton GhCesA1, all of which belong to the cross-species CesA8 clade that facilitates the synthesis of cellulose in secondary walls (Zou et al., 2018). We compare the structure of EgCESA8 with PttCESA8 and AtCESA8, and found high similarity in these proteins, supporting the accuracy of the analysis ( Supplementary Figure S2 ). The multiple alignment sequence were further conducted, suggesting their high conservation in the plant-conserved region (PCR), the class-specific region (CSR) and the conserved aspartic acids (D) and the QXXRW motif for donor and acceptor binding ( Supplementary Figure S1 ). However, the members of CslA and CslC lack the PCR and CSR regions (Daras et al., 2021), resulting in a low-level similarity with CesA in structure ( Figure 8 ). The rice fc17 mutant of the CesA4 protein, which is the ortholog of Arabidopsis CesA8 participated in secondary cell wall synthesis, contains an amino acid substitution (F426S) in the PCR region. The fc17 mutation influenced plant growth and resulted in increased lodging resistance, and also exhibited increased biomass saccharification efficiency and a decrease in cellulose content, accompanied by a compensatory rise in hemicellulose and lignin levels (Li et al., 2018). FxVTxK motif, which lies near the catalytic domain, exist within a substrate gating loop. Various mutation in this region showed change of the cellulose production (Daras et al., 2021). Therefore, these observations suggest that the PCR and FxVTxK motifs could serve as potential targets for cell wall modification in biotechnological applications. The FxVTxK motif was also investigated in EgCESA8, PttCESA8 and AtCesA8 ( Supplementary Figure S1 ), and showing the conservation in this region. Additional functional studies on the FxVTxK motif in EgCESA8 are essential for understanding its role in cellulose synthesis. Through the analysis of the CSC complex structure formed by the proteins encoded by the three genes EgCesA4, EgCesA7, and EgCesA8, it was found that there was a cavity structure within the complex. It was speculated that this cavity structure could specifically bind to the substrate uridine diphosphate glucose (UDP-glucose). Knockout mutants of secondary cell wall CesA, including irx5-4 (cesa4), irx3-7 (cesa7), and irx1-7 (cesa8) with T-DNA insertions, display a range of phenotypic characteristics such as dark green leaves and inflorescence stems, collapsed xylem vessels, decreased plant height and cellulose content (Kumar et al., 2017). This complex CSC model provided more intuitive and compelling structural biological evidence for the involvement of EgCesA4, EgCesA7 and EgCesA8 in the cellulose biosynthetic pathway during the lignification stage, further elucidating the mechanism of action of these three genes in this biological process at the molecular level.

Cis-regulatory elements analysis provides crucial information for transcriptional regulation of CslC/CslA gene ( Supplementary Table S2 ). The majority of these gene promoters included response elements for hormones and abiotic stress ( Figure 5 ). Specifically, EgCesA1a and EgCslA2 exhibited consistently high expression levels across various treatments ( Figure 7 ), which aligns with the presence of numerous cis-acting elements associated with hormone and stress responses ( Figure 5 ). In addition, the high expression of these genes may also result from the combined effects of cross-talk among multiple signaling pathways (Wang et al., 2020), protein multifunctionality (Liu et al., 2017), and species-specific adaptive evolution (Wei et al., 2025), reflecting a strategy of “module reuse” by which plants cope with complex environmental stresses within a limited genome. Further functional investigation of these EgCesA/Csls by overexpression or knockout of these genes may help to elucidate their function in response to different stress conditions. Among the cis-acting elements identified in EgCesA/Csls genes promoters, MYB transcription factors related elements were widely distributed and presented in over half of the identified EgCesA/Csls ( Figure 5 ). It was well-known the MYB transcription factors were highly expressed and widely involved in various stress responses, including drought and light responses, thereby regulating plant stress resistance, growth and development, and metabolic processes. In addition, MYB transcription factors could bind to the AC-box to regulate lignin biosynthesis genes (Xiao et al., 2021). Then they could directly promote secondary cell wall biosynthesis by regulating lignin, hemicellulose and cellulose pathway genes. Therefore, Data analysis from the heatmap showed that many members of this family were significantly expressed in the xylem, with lower expression in other tissues such as leaves, further highlighting their impact on cell wall formation. It has been found that two BBR-BPC transcription factors BPC1/BPC2 in Arabidopsis positively regulate plant β-1,4-galactan (a major component of pectin) accumulation (Yan et al., 2021). The binding sites of BBR-BPC were the most prevalent in the EgCesA/Csls promoters ( Supplementary Figure S2 ), suggesting their potential roles in cell wall synthesis.

In this study, we identified 62 EgCesA/Csls genes containing cellulose synthase domains and classified them into seven subfamilies (CesA, CslA, CslC, CslB, CslD, CslE, and CslG). Furthermore, expression profile analysis showed that CesA/Csls genes exhibited different expression patterns in various tissues of E. grandis. Specifically, EgCesA4, EgCesA7, and EgCesA8 were likely to be primarily involved in cellulose synthesis during the secondary wall thickening (lignification) stage according to their high expression in xylem. This finding lays a robust foundation for further investigation into the CesA/Csls-mediated molecular mechanisms underlying the growth and development process of E. grandis. Additionally, it offers a promising theoretical foundation and genetic resources for the enhancement of wood quality in E. grandis, as well as for improving biomass utilization efficiency through genetic engineering strategies.