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 KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 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 PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 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 KNAT7 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 KNAT7 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.

Understanding the Modus Operandi of Class II KNOX Transcription Factors in Secondary Cell Wall Biosynthesis

“…In a recent study performed in our laboratory by Ahlawat et al [17], transgenic poplar plants overexpressing PtKNAT7 and AtKNAT7 genes showed enhanced expression of the SCW genes CesA8, IRX9, PAL, and CCR, and reduced expression of the same genes in the poplar PtKNAT7 antisense plants. These results further suggested a positive regulatory role of KNAT7 in SCW formation in poplars.…”

“…Furthermore, a detailed investigation into the regulatory network and downstream targets of Class II KNOX TF proteins is required to understand the transcriptional regulation of SCW formation. These studies will help us to modify cell wall formation in transgenic plants and enhance saccharification, as we recently showed [14,17]. Our understanding of the molecular controls of the deposition of each call wall component will help us to design cell walls for improved biomass production and reduced recalcitrance to bioconversion to ethanol.…”

“…The suppression of KNAT7 function increased SCW formation in interfascicular fibers but resulted in reduced cell wall synthesis in xylary fibers with collapsed vessels, suggesting that it is a transcriptional suppressor [13,15]. Quite contrasting results were observed by other authors, who suggested that KNAT7 is a transcriptional activator [12,14,15,17,31,33]. Recent reports by Wang et al and Qin et al [15,16] reconciled these observations, suggesting that KNAT7 acts as a suppressor in interfascicular fibers but as an activator in vessels and xylary fibers.…”

“…Reduced expression of SCW genes, reduced lignin content, altered lignin composition (S/G ratio), and increased saccharification. [17] The successful complementation of Arabidopsis knat7 mutants with the overexpression of the cotton GhKNL1 gene [33] and poplar PtKNAT7 [13] rescued the defective irx phenotype of the knat7 mutants, suggesting the functional conservation of KNAT7 genes among Arabidopsis, cotton, and poplar. The overexpression of cotton GhKNL1 in Arabidopsis resulted in thinner interfascicular fibers and slightly thinner vessels walls without any change in the xylary fibers compared to control plants [33].…”

“…These TFs function by regulating the SCW biosynthetic genes that encode cellulose synthases (CesAs), xylan synthases, and lignin biosynthetic pathway enzymes. One of the Class II KNOTTED1-like homeodomain (KNOX) genes, KNAT7, has recently gained attention for its potential role in the transcriptional network regulating SCW biosynthesis [11][12][13][14][15][16][17]. This comprehensive review focuses on the recent developments in our understanding of the transcriptional networks involving Class II KNOX TFs in the regulation of SCW biosynthesis.…”