The cell wall is a unique external protective structure in plant cells, providing mechanical support, maintaining normal metabolic functions, and facilitating responses to environmental stress. It is essential for plant growth and development (Gigli-Bisceglia et al., 2020) As such, the cell wall is closely connected to physiological processes such as absorption, transpiration, transport, and secretion. Plant cell walls are typically comprised of the middle lamella, primary wall, and secondary wall, with primary components including cellulose, hemicellulose, lignin, and pectin (Kubicek et al., 2014).

Pectin, cellulose, and hemicellulose make up the majority of the primary wall. Cellulose is interconnected by hydrogen bonds, providing structural strength and support to the cell wall. Xylan, the predominant hemicellulose in plant cell walls, consists of a β- (1→4)-linked D-xylose backbone with L-arabinose and 4-O-methyl-glucuronic acid side chains (Zabotina, 2012). It mediates interactions between the pectin/lignin matrix and cellulose microfibrils, affecting wall flexibility biomass recalcitrance (Kang et al., 2019; Zhong et al., 2019; Yu et al., 2022). The three types of pectin-homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II)-are structurally complex polysaccharides found in plant cell walls made up of α-D-galacturonic acid residues connected by 1,4-bonds. The most prevalent sugar residue in all plant primary walls is galacturonic acid (GalA) (Ridley et al., 2001). In plant cell walls, HG, a polymer of α-1,4-linked D-galacturonic acid, makes up more than 60% of the pectin (Caffall and Mohnen, 2009). Pectin regulates the cell wall’s porosity, contributing to its plasticity and permeability (Mohnen, 2008). These carbohydrates play critical roles in the composition and metabolism of plant cell walls.

Glycosyltransferases (GTs) are enzymes that transfer sugar moieties from active donor molecules to specific acceptor molecules, thereby catalyzing the creation of glycosidic connections. The manufacture of disaccharides, oligosaccharides, and polysaccharides is facilitated by these enzymes (Sinnott, 1990; Lairson et al., 2008). Glycosyltransferases were categorized into 138 families as of March 2025 based on sequence similarity and substrate recognition (http://www.cazy.org/GlycosylTransferases.html). Among these, the GT8 family has a vital part in plant cell wall production and abiotic stress response (Wang et al., 2024). Evolutionary analysis has classified GT8 into seven subfamilies: Galacturonic acid transferases (GAUT), Galacturonic acid transferase-like transferases (GATL), GATL-related (GATR), Galactinol synthases (GolS), and Plant glycogen starch synthesis initiation protein A (PGSIP-A), PGSIP-B, and PGSIP-C (Yin et al., 2010; Kong et al., 2019). The production of pectin and xylan is the main function of GAUT, GATL, and GATR, which also aid in cell wall remodeling (Sterling et al., 2006). PGSIP proteins are involved in initiating starch biosynthesis (Chatterjee et al., 2005), while GolS is a key enzyme in the synthesis of cotton oligosaccharides, playing a role in plant abiotic stress responses (Taji et al., 2002). In Arabidopsis thaliana, the GT8 gene family comprises 41 proteins categorized into four major subfamilies: GAUT, GATL, GolS, and PGSIP (Yin et al., 2010). Among these, QUASIMODO1 (QUA1) and GALACTURONOSYL TRANSFERASE1 (GAUT1) have been shown to be involved in pectin biosynthesis (Bouton et al., 2002). In particular, QUA1 is expressed in vascular tissues and influences the activity of β-1→4-D-xylan synthase and α-1→4-D-galacturonic acid transferase, which are involved in the synthesis of pectin and hemicellulose (Orfila et al., 2005). GAUT1 encodes a galacturonic acid polysaccharide glycosyltransferase directly involved in pectin synthesis, and alterations in its function significantly affect plant cell wall composition and function (Caffall and Mohnen, 2009). AtGATL1/PARVUS was initially thought to play a role in pectin synthesis (Bouton et al., 2002), However, further studies revealed that the PARVUS gene, which is essential for xylan biosynthesis, is expressed in cells undergoing secondary wall thickening. Its loss of function leads to reduced mechanical strength of the plant cell wall (Lee et al., 2007). AtGAUT12/IRX8 (IRREGULAR XYLEM 8) is also crucial for xylan synthesis. Both PARVUS and IRX8 contribute to the synthesis of tetramer-saccharides at xylan’s reducing end (Brown et al., 2007; Peña et al., 2007), and A. thaliana irx8 mutants exhibit reduced xylan and lignin content, along with partial reductions in pectin and cellulose (Persson et al., 2007). Additionally, GUX1and GUX2 are involved in xylan side chain synthesis, particularly by adding GlcA to the xylan backbone (Rennie et al., 2012). In Populus deltoides, PdGATL1.1 and PdGATL1.2 are the closest orthologs of the A. thaliana gene PARVUS and function in xylan biosynthesis (Suo et al., 2022). RNAi-mediated knockdown of GAUT12.1 in Populus reduced xylan and pectin content during wood formation, along with decreased biomass recalcitrance. Given the conserved role of GAUT12 in A. thaliana, these results suggest that GAUT12.1 similarly plays a key role in pectin and xylan biosynthesis in Populus (Biswal et al., 2015; Biswal et al., 2018). These genes are vital for plant cell wall construction and mechanical support. For a deeper comprehension of the dynamic process of woody plant cell wall formation, it is imperative to investigate the functional partitioning and evolution of the GT8 gene family.Eucalyptus (Eucalyptus robusta Smith), a species from the Myrtaceae family, is known for rapid growth, strong adaptability, high economic value and diverse uses. It is one of the three major fast-growing tree species in artificial afforestation, alongside poplar and pine (Kirch et al., 2011). With a short growth cycle, rapid regeneration, and strong soil nutrient dependency, Eucalyptus is a key species in southern artificial forests and a critical species for timber reserves (Melesse and Zewotir, 2017). The GT8 glycosyltransferase family has been studied in various species. GT8 family member numbers vary across species: 41 in A. thaliana (Yin et al., 2010), 40 in Oryza sativa (Kong et al., 2019), 40 in Solanum lycopersicum and 56 in Malus domestica members (Wang et al., 2024). However, research on the GT8 family in E. grandis remains limited. This study identifies the GT8 gene family in E. grandis based on bioinformatics analysis. We analyzed various features including physicochemical properties, phylogenetic trees, gene structures, conserved motifs, chromosome locations, and synteny relationships using multiple bioinformatics tools: TBtools-II (v1.120), MEGA11 (v13.0), and Jalview (v2.11.2.6), along with other online platforms. These analyses provide a foundation for further exploring the functional roles of GT8 genes in E. grandis.

The GT8 gene family is widely present in plant genomes, and studies have demonstrated that it is essential for plant cell wall biosynthesis and reaction to abiotic stress. E. grandis, characterized by its rapid growth and high economic value, is a key species for wood reserves. Therefore, studying the functional differentiation and evolution of the GT8 gene family in E. grandis is vital for the formation and utilization of its wood. Currently, the GT8 family has been reported in various species, but research on the GT8 family in woody plants such as E. grandis is relatively scarce. Using the GT8 genes of A. thaliana as a reference, we discovered 52 GT8 genes in E. grandis. Analysis of their physicochemical properties showed that the EgGT8 genes are unevenly distributed across 11 chromosomes, with most family members being unstable. Subcellular localization predictions displayed that EgGT8 proteins are extensively dispersed in the Golgi apparatus and endoplasmic reticulum, and the bulk of these genes (94.23%) encode hydrophilic proteins. The E. grandis GT8 family’s protein sequences and domains are generally very conserved, indicating that these genes are important for plant physiology, warranting further functional exploration.

Ten GT8 genes (EgGUX08, EgGUX12, EgGUX15, EgGUX16, EgGUX17, EgGUX18, EgGUX19, EgGUX20, EgGUX21, and EgGUX22) form a tightly linked cluster on E. grandis chromosome 2 ( Figure 2 ), showing high structural similarity-a hallmark of tandem duplication. These group II genes share conserved motifs and domains, suggesting retained biochemical functions and potential involvement in common pathways. Analysis of E. grandis GT8 genes identified only four collinear gene pairs: EgGATL4B and EgGolS2, EgGUX13 and EgGUX14, EgGATL2 and EgGATL1B, EgGUX05 and EgGolS1 ( Figure 3A ). These genes represent non-contiguously distributed repetitive sequences in the genome, with phylogenetic evidence supporting their origin through dispersed duplication events. Syntenic gene pairs exhibit conserved motifs and similar gene architectures ( Figure 1 ), indicating their potential functional conservation via dosage effects. In contrast, non-syntenic gene pairs demonstrate substantial divergence in gene structures, suggesting evolutionary functional differentiation. Such divergent paralogs likely mediate distinct biological processes contributing to growth and development in E. grandis.

Comparative genomic analysis revealed that E. grandis exhibits significantly stronger collinearity in GT8 genes with P. alba than with A. thaliana ( Figures 3B, C ). This phenomenon can be attributed to two primary factors: First, both E. grandis and P. alba belong to woody clade, while A. thaliana is classified to herbaceous clade, indicating closer phylogenetic relationship between the former two species. Second, as woody plants, Eucalyptus and Populus require more conserved GT8 gene functions for secondary cell wall formation, resulting in greater gene diversification.

E. grandis exhibits greater GT8 gene family expansion (11 chromosome pairs) compared to A. thaliana (5 pairs) ( Figure 7B ), likely due to whole-genome duplication. This expansion facilitated functional diversification of GT8 genes, enhancing environmental adaptation. Duplicated genes may evolve specialized functions through sequence divergence and expression changes, forming adaptive networks. Future studies could employ CRISPR and comparative genomics to investigate GT8 functional mechanisms.

Previous work has demonstrated that IRX8, a member of the GT8 gene family, contributes to the production of the tetrasaccharide that contains galacturonic acid at the reducing end of xylan (Brown et al., 2007). The protein encoded by GAUT1 functions as a galacturonosyltransferase, directly participating in pectin biosynthesis (Caffall and Mohnen, 2009). In the phylogenetic tree constructed for GT8 genes across E. grandis, A. thaliana, S. moellendorffii, P. patens, and P. alba, these genes cluster within Group III. This suggests that EgGAUT1, EgGAUT12, and other Group III members may share functional similarities with IRX8 and GAUT1, potentially contributing to both pectin and xylan biosynthesis. Additionally, EgGUX02 and EgGUX04 exhibit close phylogenetic relationships with AtGUX1 and AtGUX2, indicating their possible involvement in xylan side-chain modifications by incorporating glucuronic acid (GlcA) into the xylan backbone (Rennie et al., 2012; Lyczakowski et al., 2021). Similarly, EgGolS4 and EgGolS5 cluster with AtGolS2 and AtGolS4, suggesting their potential roles as essential enzymes in the production of raffinose family oligosaccharides (Taji et al., 2002). Furthermore, EgGATL8 and EgGATL4A are closely related to AtGATL4 and AtGATL9, suggesting their possible role in the formation of the tetrasaccharide that contains galacturonic acid at the reducing end of xylan (Peña et al., 2007). These results offer insightful information about the functional diversification of GT8 genes, yet further experimental validation and functional characterization are required in future studies.

Expression data from E. grandis seedlings under various abiotic stresses and plant hormone treatments underscore the importance of MeJA. MeJA is a significant plant hormone, and previous studies have demonstrated that MeJA is crucial for maintaining ion homeostasis (Mir et al., 2018) and in cell stress responses (Piotrowska et al., 2009; Bali et al., 2019; Shri et al., 2019). In MeJA-treated E. grandis seedlings, the expression of GT8 genes was generally upregulated. The cis-element analysis showed that all EgGT8 family members contain hormone response elements, including those responsive to MeJA, strongly indicating a strong bond between the EgGT8 gene family and MeJA, which could be further analyzed in subsequent studies.

In situations including salt stress, phosphorus deficiency, boric acid deficiency, SA treatment, and MeJA treatment, EgGUX10 and EgGUX14 displayed increased levels of expression, according to heatmap analysis of RNA-Seq data. This suggests that these two genes may participate in hormone signal transduction pathways. As key nodes in the signaling chain, they may respond to hormone signals and regulate the expression of downstream genes, thus coordinating the plant’s response to different environmental signals. Moreover, EgGUX10 and EgGUX14 might function synergistically under environmental stress, controlling plant development and growth as well as adversity adaptation.

Under both boric acid deficiency and salicylic acid (SA) treatment, the expression level of the EgGATL1A gene was significantly upregulated, suggesting its involvement in both biotic and abiotic stress responses ( Figure 7 ). Phylogenetic analysis reveals that EgGATL1A clusters with AtGATL1, which have known to mediate glucuronoxylan biosynthesis during secondary cell wall thickening, in the same evolutionary clade, demonstrating high sequence and functional similarity (Lee et al., 2007). We therefore propose that EgGATL6 similarly participates in glucuronoxylan biosynthesis.

Tissue-specific expression analysis of the GT8 gene family in E. grandis reveals that several EgGT8 members are highly expressed in xylem and phloem, while others are predominantly expressed in leaves. Overall, EgGT8 genes exhibit highest expression levels in xylem, mature leaves, and lateral branches. This expression pattern aligns well with the enrichment of MYB-binding cis-elements in the promoter regions of GT8 genes, suggesting that these genes may be regulated by MYB transcription factors and participate in secondary cell wall modification. Based on the structural features of the GT8 gene family, we propose a hypothesis that the MYB-GT8 module enhances mechanical strength of the cell wall by glycosylating lignin precursors. This modification helps E. grandis respond to mechanical stress, maintain leaf morphology and structure, and regulate the synthesis and reinforcement of cell walls in lateral branches, thereby ensuring normal photosynthesis and promoting lateral branch development. This proposed mechanism is consistent with functional studies of PtrGT8D in Populus (Li et al., 2011), although tandemly duplicated genes in the E. grandis GT8 family may have further diverged to acquire subfunctions in regulating different substrates.

To evaluate the natural selection pressures acting on the E. grandis GT8 gene family, we calculated the ratio of non-synonymous (Ka) to synonymous (Ks) substitutions (u) for the EgGT8 gene pairs ( Supplementary Table 1 ). When the Ka/Ks ratio is < 1, = 1, or > 1, it indicates purifying, neutral, and positive selection, respectively. The data suggest that the majority of these gene pairs are under purifying selection, with only a few exhibiting signs of neutral selection. This likely indicates that these genes have been primarily subjected to conserved selection throughout evolution to maintain their functional integrity.

We analyzed 52 members of the GT8 gene family in E. grandis from the perspectives of phylogeny, gene structure, and proteomics. These genes were classified into four subfamilies, and a series of conserved protein structures were identified. We analyzed the phylogenetic relationships between different species. Expression profile analysis of related genes under different tissue types and treatments indicated that members of the GT8 gene family play important roles in plant resistance to environmental stress, a process associated with their involvement in multiple cell wall biosynthesis pathways. Based on the distribution of key GT8 family members from published literature and expression profile data, we identified potential key genes that may function within the E. grandis GT8 family. This will assist in future functional studies and provide valuable insights for breeders in improving wood quality and cultivating superior varieties.