Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars

Abstract

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous <i>KNAT7</i> gene expression on SCW formation in transgenic poplars, we prepared poplar <i>KNAT7</i> (<i>PtKNAT7</i>) overexpression (<i>PtKNAT7</i>-OE) and antisense suppression (<i>PtKNAT7</i>-AS) vector constructs for the generation of transgenic poplar lines <i>via Agrobacterium</i>-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed <i>Arabidopsis KNAT7</i> (<i>AtKNAT7-OE</i>) in transgenic poplars. In all these constructs, the expression of <i>KNAT7</i> transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar <i>PtKNAT7-OE</i> and <i>AtKNAT7-OE</i> lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. <i>PtKNAT7-AS</i> lines, however, showed reduced expression of many SCW biosynthesis genes downstream of <i>KNAT7</i> accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three <i>KNAT7</i> knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of <i>KNAT7</i> expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.