Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Plant lignocellulosic biomass, i.e., secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan, but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation, increases lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene co-expression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was not only regulated by the secondary cell wall hierarchical regulatory network, but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification, and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.