Tuning Xylan Polymerisation Enhanced Fibre Digestibility Without Biomass Loss in Sheepgrass (Leymus chinensis)

ABSTRACT

Leymus chinensis (sheepgrass), a dominant perennial grass of the Eurasian Steppe, is a crucial source of carbohydrates and energy for ruminants. However, the lignocellulose recalcitrance severely limits its digestibility. Here, we targeted xylan, a major hemicellulose interacting with cellulose and lignin in cell wall. To improve digestibility, we knocked out two IRREGULAR XYLEM 14‐LIKE (LcIRX14L) genes, which encode key enzymes for xylan biosynthesis. The Lcirx14L knockout mutants exhibited reduced xylan molecular weight but produced similar amounts of biomass to wild‐type plants. Simon's staining and cellulase binding assays indicated enhanced cellulose accessibility in the Lcirx14L mutant. In addition, the Lcirx14L plant showed increased saccharification efficiency, dry matter and NDF digestibility, and production of volatile fatty acids by in vitro fermentation. This study thus provides novel insight for engineering forage grasses with both high yield and quality by modifying xylan polymerization.

Leymus chinensis (sheepgrass), a dominant perennial grass of the Eurasian Steppe, is a crucial source of carbohydrates and energy for ruminants. Although traditional breeding have improved its abiotic stresses tolerance and biomass, varieties renowned for superior forage quality remain scarce (Liu et al. 2019). As a key indicator of forage quality, neutral detergent fibre (NDF) digestibility directly affects animal feed intake and productivity (Oba and Allen 1999). However, the lignocellulose recalcitrance severely limits its digestibility (Wang et al. 2017). Here, we targeted xylan, a major hemicellulose interacting with cellulose and lignin in cell wall, achieving improved fibre digestibility without biomass loss in sheepgrass.

NDF primarily consists cellulose, hemicellulose, and lignin. The cellulose and xylan can be degraded into glucose and xylooligosaccharides, which are fermented into volatile fatty acids (VFAs) utilised as energy by ruminants (Mizrahi et al. 2021). We analysed the dynamics of NDF digestibility in Lc6‐5, a genotype with a well‐annotated genome and amenable to genetic manipulation (Li et al. 2023; Lin et al. 2023; Zhang, Tang, et al. 2024), across seven developmental stages from elongation to maturity. Both dry matter (DM) and NDF digestibility declined with maturity, verifying by in vitro saccharification assays (Figure S1). At the milky stage, sheepgrass biomass exhibits significantly lower NDF digestibility and sugar release than common oat ( Avena sativa ), a widely used forage grass (Figure S2). Correlation analysis indicated that the lignin content showed the most negative correlation with NDF digestibility (R = −0.8884, p = 7.55e⁻⁸), followed by xylan (R = −0.7890, p = 2.13e⁻⁵) and cellulose (R = −0.7243, p = 2.00e⁻⁴; Figure S3). Since lignin reduction often causes adverse pleiotropic effects, we focused on modifying xylan structure to reduce cell wall recalcitrance (Zhou et al. 2010).

IRREGULAR XYLEM 9/9‐LIKE (IRX9/9L) and IRX14/14L from glycosyltransferase 43 (GT43) family and IRX10/10L (GT47 family) participate in xylan backbone elongation (Zhang, Zhou, and Zhang 2024). The LcIRX genes were identified via homology to Arabidopsis and rice sequences (Figure 1a, Table S2). Two genes, Lc2Ns001383 and Lc2Xm054717 (designated as LcIRX14L), were selected for further study based on their high expression in various tissues (Figure 1a). Using CRISPR/Cas9, we generated a homozygous double mutant (cr‐1) lacking both LcIRX14L genes and a heterozygous mutant (cr‐2) with a mutation in one allele of Lc2Ns001383 (Figure 1b, Figure S4a). qRT‐PCR confirmed downregulation of Lc2Ns001383 in cr‐2 (Figure S4b). To evaluate the potential gene dosage effect of LcIRX14L, both cr‐1 and cr‐2 mutants were subjected to further phenotypic analysis. After 3 months of growth, the Lcirx14L mutants and WT did not differ significantly in plant height and dry weight (Student's t‐test, p > 0.05; Figure 1c–e), indicating that biomass production was not compromised.

FIGURE 1.

Knocking out LcIRX14L enhances fibre digestibility without biomass loss. (a) Phylogenetic analysis and expression profiles of IRX9/10/14 homologues. Two LcIRX14L genes are labelled in green. Scale bar, log₂(FPKM + 1). N/A, not available. (b) Sequences of LcIRX14L in WT and mutants. (c) Phenotypes of WT and Lcirx14L mutants. Scale bars, 5 cm. (d, e) Plant height (d), and dry weight (e) of WT and Lcirx14L mutants after 3 months of growth. (f) SEC profiles of xylans from WT and Lcirx14L using UPLC‐FLR. (g) Degree of polymerisation (DP) of xylan. (h) Adsorption kinetics curves of DO15 dye in WT and Lcirx14L mutants. (i) Distribution coefficients of the dye in WT and Lcirx14L mutants. (j) Cellulase binding assays using AF488‐labelled cellulases and DR23 in internode cross‐sections. Scale bars, 50 μm. (k) Quantification of fluorescence intensities in (j). (l) Glucose release efficiency of WT and Lcirx14L mutants. (m–o) NDF content (m), DM digestibility (n), and NDF digestibility (o) of WT and Lcirx14L plants. (p) VFA production following 48 h digestion by rumen fluid. All data were analysed using Student's t‐test. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant.

Xylose content, the most abundant monomer of xylan, was measured across different tissues via GC–MS. The cr‐1 mutant showed significantly reduced xylose in leaf and sheath, indicating impaired xylan accumulation (Figure S5a). UPLC and linkage analysis revealed a clear shift in the xylan elution profile and a lower degree of polymerisation (DP) in cr‐1, indicating that cr‐1 produces smaller xylan polymers and shorter xylan backbones than WT (Figure 1f,g). Intriguingly, these residual xylan displayed increased density of side‐chain decorations (2,4‐Xyl, 3,4‐Xyl, 2,3,4‐Xyl), indicating altered xylan structure (Figure S5b). The cr‐2 exhibited a comparable xylan structure to that of the WT.

We hypothesised the altered xylan structure may improve cell wall digestibility. Simon's staining assays demonstrated enhanced cellulose accessibility in the cr‐1 mutant, validated by in situ binding assays with AF488‐labelled cellulases (Figure 1h–k). These changes led to superior saccharification, with 1.8–3.0‐fold higher cellulose hydrolysis and 2.5–3.5‐fold higher xylan hydrolysis in cr‐1, while maintaining comparable cellulose content to WT (Figure 1l, Figure S6). Furthermore, the cr‐1 exhibited significantly lower NDF content and approximately 6 percentage points higher digestibility for both DM and NDF in rumen fermentation (Figure 1m–o). VFA concentrations were significantly increased in fermented cr‐1 samples compared with WT (Figure 1p, Figure S7). Therefore, LcIRX14L mutation enhances NDF digestibility and VFA production, thereby improving the nutritional value for ruminants.

In summary, we generated a sheepgrass mutant with enhanced fibre digestibility and comparable biomass by disrupting LcIRX14L via CRISPR/Cas9 (Figure S8). Unlike lignin reduction strategies, fine‐tuning xylan structure can improve digestibility without biomass loss. Although cr‐1 produces a shorter xylan backbone and denser sidechains, the absence of irregular xylem defects suggests that the residual xylan structure retains sufficient integrity for vascular development. This altered xylan likely modifies interactions among cellulose, xylan, and lignin, enhancing cell wall accessibility for degradation. Further investigation into the mechanisms underpinning the increase of sidechain modification may provide insights into the regulation of xylan biosynthesis. The high conservation of IRX14L across major Poaceae species indicates that fine‐tuning xylan structure is a promising strategy for improving the quality of forage and bioenergy grasses (Figure S9).

Author Contributions

X.C., B.Z., and S.Z. conceived the project and designed the research. M.Z., Z.L., L.C., S.T., Z.L., T.L., and F.C. performed the experiments and analysed the data. J.W., L.M., D.Z., X.Z., and D.P. provided sheepgrass materials. D.B., Z.S., X.S., and Y.Z. provided technical support and conceptual advice. M.Z., X.S., S.Z., B.Z., and X.C. wrote the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Appendix S1: pbi70651‐sup‐0001‐Supinfo.pdf.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.