Targeted decoupling of <scp>FTO</scp> interaction with <scp>RCC1</scp> reprograms wood composition and confers <scp>UV</scp> ‐B tolerance in poplar

The human FTO, encoding the first identified reversible m6A RNA demethylase, has attracted considerable attention for its central roles in obesity and cancer pathogenesis (Fawcett & Barroso, 2010; Jia et al., 2011; Li et al., 2017; Su et al., 2018). Notably, plants lack an endogenous FTO homolog, raising intriguing questions about the effects and regulatory consequences of its introduction into plant systems. Previous studies demonstrated that heterologous expression of FTO in annual crops such as rice (Oryza sativa) and potato (Solanum tuberosum) substantially increased yield and biomass – by up to 50% – and improved key agronomic traits including root architecture, photosynthetic capacity, and drought resilience (Yu et al., 2021). These findings position FTO as a powerful candidate for transgenic crop improvement. However, whether FTO exerts similar functions in perennial woody species, which possess distinct life cycles and developmental constraints, remains entirely unexplored. This study addresses this critical knowledge gap by investigating the role of FTO in Populus, providing new insights into the conservation and divergence of epitranscriptomic regulatory mechanisms across plant lineages and offering innovative strategies for engineering biomass and stress adaptation in trees. To assess the impact of FTO on perennial woody plant growth and adaptation, we generated transgenic Populus (84K) lines constitutively overexpressing FTO (FTO-OE) (Fig. 1a,b). The successful overexpression of the FTO transgene was confirmed by western blot analysis, which detected elevated FTO protein levels in the transgenic plants (Supporting Information Fig. S1). Phenotypic analysis revealed no significant alteration in plant height and ground diameter between FTO-OE lines and wild-type (84K) controls in 4-month potted plant and 6-month field experiments (Fig 1a,b, S2). However, photosynthetic capacity was profoundly compromised in FTO-OE plants. The net photosynthetic rate (Pn) exhibited a marked 28.4% reduction (Fig. 1c). This impairment was associated with a significant 37.4% decrease in stomatal conductance (Gs) and a concomitant 3.5% reduction in intercellular CO2 concentration (Ci) (Fig. 1d,e), alongside diminished stomatal length (Figs 1f, S3). Collectively, these data indicate that stomatal limitation constitutes the primary mechanism underlying the FTO-OE-mediated suppression of Pn. Intriguingly, despite the substantial decrease in stomatal aperture typically associated with improved water conservation, both transpiration rate (Tr) and intrinsic water-use efficiency (WUEi) were significantly reduced in FTO-OE plants by 32.5 and 15.4%, respectively (Figs 1g, S4, S5). All these unexpected declines suggests that the impairment in photosynthetic carbon assimilation likely outweighs the potential benefit from reduced water loss (Figs 1g, S4, S5). Furthermore, a 12% reduction in Chl content (Fig. S6) implies potential inhibition of mesophyll photosynthetic function beyond stomatal constraints. The observed severe photosynthetic suppression without a concurrent reduction in plant height suggests potential compensatory adjustments in resource allocation. Our analysis also revealed that FTO overexpression significantly impaired secondary xylem development. Specifically, the width of the secondary xylem layer at the eighth internode in FTO-OE transgenic plants was reduced by 53% compared to wild-type (84K) controls (Figs 1h, S7), indicating a substantial inhibition of wood formation. Consistent with results in rice, potato, and soybean, FTO-OE Populus lines decreased m6A/A ratio from 0.83 to 0.74‰ (Fig. 1i) and increased fluorescence intensity per cell from 31.8 to 35.7. It is suggested that global m6A methylation significantly decreased and chromatin openness significantly increased in FTO-OE Populus lines (Figs 1j, S8). However, FTO overexpression in poplar resulted in impaired photosynthesis and unchanged growth trait, unlike the broader benefits observed in herbaceous crops. This constrained functionality implies the presence of unknown interacting factors within the poplar genome. Therefore, yeast two-hybrid (Y2H) were used to identify potential FTO-interacting proteins in poplar, which screened a total of 628 potential interacting factors. Among these genes, photosynthesis, generation of precursor metabolites and energy, translational elongation and translational termination were significantly enriched in biological process (Fig. S9). For molecular function, translation regulator activity, RNA binding and nucleic acid binding were significantly enriched (Fig. S9). Among these potential interacting factors is Regulator of Chromosome Condensation 1 (PtoRCC1) – a plant RCC1 family member implicated in abiotic stress responses (e.g. UV-B, salinity, oxidative stress) via regulation of protective genes (Zhang et al., 2025). Transcriptomic analyses indicated PtoRCC1 expression is elevated in mature xylem and modestly downregulated under salt stress (Fig. S10). This gene was selected as a candidate for this study based on its potential interaction with FTO, which may subsequently modulate xylem development and environmental adaptation. Subsequent protein pull-down and firefly luciferase complementation assays yielded consistent results with the Y2H analysis, confirming a physical interaction between FTO and PtoRCC1. (Figs 2a, S11). Structural analysis confirmed the RCC1 domain adopts a conserved seven-bladed β-propeller fold across eukaryotes (Fig. S12) (Renault et al., 1998). Given this high conservation, we propose that FTO-RCC1 interaction likely represents a general mechanistic feature in plant lineage. To elucidate differences in FTO-RCC1 interactions across species, we performed molecular docking analysis. Results revealed species-specific interaction patterns in algae, mosses, ferns, gymnosperms, angiosperms and mammals (Table S1). We predicted the interaction between FTO and RCC proteins in humans and mice. The results showed that FTO interacted with RCC in both mammals, but the interaction sites were different from those in plants (Table S1). Therefore, we speculate that although the RCC protein itself is highly conserved in sequence and structure, the functional modules composed of FTO-RCC may serve very different biological goals in animals and plants. Critical differences emerged at the interaction interface: in Populus spp. (Populus tomentosa, Populus alba, Populus trichocarpa) and the gymnosperm Pinus taeda, RCC1-Glu341/Glu268 amino acid residue formed a salt bridge with FTO-Arg10. RCC1-Leu347/Gly348/Gln299 amino acid residue hydrogen bonds with FTO-Asn447 (Fig. 2b). Strikingly, these interactions were absent in rice and potato – species where human FTO expression enhances biomass and drought resistance (Table S1). It is implied that the absence of key amino acid residue interactions may liberate FTO from inhibitory partners in annual crops, thereby unlocking its potential for genetic improvement. Structural analysis of FTO-altered (Arg10/Asn447 are altered to Phe or Leu) confirmed preserved tertiary structure (Fig. S13) but completely abrogated binding to RCC1 in two members of Populus sect. Populus (P. tomentosa, P. alba) (Table S2). This demonstrates that FTO-Arg10(R10)/Asn447(N477) are essential for mediating RCC1 interactions in poplar. This provides a structural basis for the observed limitations in genetic improvement of woody plants via FTO introduction, contrasting with benefits seen in herbaceous crops lacking these critical interactions. This suggests that plants dependent on FTO-R10/N477-mediated binding may be unsuitable candidates for FTO-based engineering strategies. To further validate the role of this site, we generated mutations in the interaction domain based on molecular structure analysis and overexpressed the mutated FTO gene (mFTO, in which Arg10 was replaced by Leu and Asn447 was replaced by Phe) in poplar (Fig. S14). Y2H analysis showed that mFTO decoupled with PtoRCC1 (Fig. 2c). Subcellular localization analysis revealed that mFTO remains localized to the nucleus (Fig. S15). mFTO-OE Populus lines decreased m6A/A ratio from 0.83 to 0.61‰ and increased fluorescence intensity per cell from 52.9 to 61.5 (Figs S16, S17). It is suggested that mFTO-OE confer global m6A demethylation and chromatin openness more than FTO-OE in poplar. mFTO-OE lines exhibited significantly induced height and ground diameter in field experiments (Figs 2d, S18). In terms of wood components, overexpression of mFTO significantly increased hemicellulose content in transgenic poplar, with elevated levels of d-galactose polymer (Fold Change = 1.37, P < 0.001) and d-xylose polymer (Fold Change = 1.20, P < 0.01), resulting in a 23% rise in total hemicellulose (P < 0.05) (Fig. 2e,f; Tables S3, S4). The microscopic observation results of fiber morphology showed that there was no significant difference in the cell wall thickness of mFTO-OE fibers, but the fiber length increased significantly (Fig. S19). By contrast, lignin components and holocellulose remained unchanged (Fig. 2g; Table S3). These modifications in cell wall composition, specifically enhanced hemicellulose accumulation without altering lignin, suggest improved recalcitrance properties for biomass processing. Overexpression of mFTO in poplar significantly alters free sugar accumulation, as evidenced by pronounced increases in mannose, arabinose, galactose, glucose-6-phosphate, maltose, and fructose (Fold Change: 1.26–1.77; P < 0.05). Sucrose levels remained unchanged (Table S4). These findings suggest that mFTO enhanced accumulation of key sugars may improve carbon availability, supporting growth and stress responses in poplar. Further characterization of lignin structure via 2D-HSQC NMR revealed a significant shift in monomeric composition: the relative abundance of syringyl (S) lignin decreased by 7.71%, whereas guaiacyl (G) lignin content was stable. This resulted in a marked decrease in the S : G ratio to 1.24 (Fig. 2h). Concurrently, the level of p-coumaric acid (pCA) esterification (pCA2,6) onto lignin increased by 10.6% (Fig. 2h). These results demonstrate that mFTO overexpression disrupts normal secondary growth, alters cell wall composition and lignin structure. Therefore, mFTO overexpression significantly elevated hemicellulose and soluble sugar levels of mFTO-OE poplar might exhibit strong potential as a dedicated bioenergy feedstock optimized for sugar release. To further elucidate the function of mFTO-OE in environmental adaptation, mFTO-OE and wild-type (84K) Populus plants were subjected to salt (200 mM NaCl) and UV-B stress. mFTO-OE lines exhibited pronounced hypersensitivity to salinity, displaying severe leaf wilting under salt stress (Fig. 2i). SOD activity and POD activity were not induced in mFTO-OE lines; the CAT activity decreased, and more H2O2 was accumulated, resulting in significantly elevated superoxide anion (O2−) accumulation vs 84 K (Figs S20, S21), demonstrating mFTO negatively regulated poplar salt tolerance. Conversely, under UV-B radiation, mFTO-OE plants maintained normal growth (Fig. 2j). SOD and POD activities were significantly induced in mFTO-OE lines; the activity of CAT increases while the content of H2O2 decreases, accelerating the removal of O2− (Figs S21, S22), indicating mFTO positively regulates UV-B stress resistance. This phenotype reveals that mFTO-OE enhanced UV-B resilience suggests potential for niche expansion into high-altitude, high-radiation environments, thereby influencing montane ecological resource partitioning. This study uncovers a novel mechanism whereby engineered mFTO modulates secondary cell wall composition in poplar by selectively decoupling its interaction with the chromosome condensation regulator RCC1 via residue-specific mutations. This manipulation alters hemicellulose accumulation and enhances stress-responsive traits, revealing a previously unknown regulatory module in xylem development and environmental adaptation. We further demonstrate an innovative protein engineering strategy that optimizes exogenous FTO function by rationally redesigning key interaction sites, thereby overcoming endogenous regulatory constraints. This approach provides a precise and potent breeding tool that surpasses conventional transgenic methods, offering new possibilities for the genetic improvement of perennial trees. This study highlights the unique effects of introduced human FTO in poplar trees, which prompts us to think more broadly about the core functional differences of FTO among animal and plant systems. In animals, especially mammals, FTO is mainly located in the cell nucleus and functions as an m6A demethylase. Its primary substrate is the m6A modification within mRNA. By influencing mRNA splicing, stability, and translation, it plays a key role in energy homeostasis, adipogenesis, immune regulation, and other processes. In sharp contrast, our findings in poplar and preliminary evidence in other plants such as rice and potato suggest that human FTO may have unique regulatory patterns in plants in addition to targeting and regulating m6A methylated transcripts. Although it also affects growth and stress response in poplar trees, its core mechanism may not be the classical m6A demethylation pathway defined around animal FTO. These fundamental functional differences pose-specific challenges for the future use of FTO for plant improvement. First, there are the challenges of multi-effectiveness and precise regulation. Constitutive overexpression may lead to trade-off effects, such as altering stress sensitivity or resource allocation, thereby offsetting growth dominance. As our research indicates, human FTO is not a universal positive regulatory factor in plants. Therefore, to obtain an ideal phenotype, a precise, tissue-specific, or inducible expression system may be required, rather than a simple global overexpression (Bai et al., 2024). Another major obstacle in the application is the incomplete understanding of the regulatory pathways of human FTO in plants. Before truly considering its biotechnological applications, it is necessary to identify its true substrates more comprehensively (Su et al., 2018), interacting proteins, and downstream targets in plants to avoid unexpected consequences for plant development and metabolism. To generate FTO-OE and mFTO overexpression the FTO and mFTO were downstream of the in the and introduced into plants. We a total of potential FTO-OE transgenic and mFTO-OE transgenic poplar We a preliminary molecular of these and western blot and selected based on protein expression levels for analysis. To the conservation of the RCC1 protein across animals and a analysis was RCC1 protein from and Populus were from the protein sequence was the in to of key within functional a was the based on the analysis indicated that the was the for this To assess the of the were with at The resulting the of RCC1 proteins among animals and plants. 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