Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases

Background Xylan, a major component of the secondary cell wall in dicots, is often recalcitrant due to its acetylation and interaction with cellulose and lignin. To fine-tune the xylan structure and improve the processing of lignocellulosic biomass, overexpression of cell wall-degrading microbial enzymes is a viable option that can enhance cell wall properties and immunity. Here, we expressed a glycosyl hydrolase (GH10) xylanase (XYL) from Aspergillus nidulans and a carbohydrate esterase (CE)1 acetyl xylan esterase (AXE) from Aspergillus niger , or both enzymes simultaneously (AXE/XYL), under the control of the constitutive Cauliflower mosaic virus 35S promoter (35S) and the woody-tissue-specific Populus trichocarpa GT43B promoter (WP) in Arabidopsis thaliana and studied their effects on the cell wall, saccharification properties, and biotic resistance. Results Transgenic WP:AXE/XYL lines exhibited an irregular xylem phenotype and compromised deposition of cell wall components, which correlated with downregulation of the responsible genes as revealed by RNA sequencing analysis. In contrast, 35S:AXE/XYL plants did not show any deformities, possibly because XYL expression was lower than in WP:AXE/XYL or due to variation in transgene expression. Biochemical analyses revealed reduced acetyl content in 35S:AXE and 35S:XYL/AXE lines, while total xylan content was increased in 35S:XYL, 35S:AXE, and 35S:XYL/AXE expressing plants. Notably, cellulose, xylan digestibility, and ethanol production were highest in the 35S:XYL/AXE line, surpassing the parental lines. Moreover, the 35S:XYL/AXE lines showed enhanced resistance to Pseudomonas syringae . Comparative RNA sequencing of xylobiose-treated and 35S:AXE/XYL plants revealed altered expression of defense-related genes. This was supported by elicitor assays, which demonstrated that xylo-oligosaccharides present in the 35S:XYL/AXE lines can promote the activation of immune marker genes along with increased accumulation of reactive oxygen species. Conclusions In summary, simultaneous hydrolysis of the xylan backbone and its acetylation represents a promising approach to boost both lignocellulosic biomass quality and plant immunity.