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

Yogesh Kumar Ahlawat,Anne E Harman-Ware,Ajaya K Biswal,Crissa Doeppke,Akula Nookaraju,Chandrashekhar P Joshi

doi:10.3389/fpls.2021.762067

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.

Highlights

Plant cell walls serve as significant sinks for the irreversible sequestering of fixed atmospheric carbon (Pauly and Keegstra, 2008)
Investigations into the role of KNAT7, one of the members of the plant KNOX II transcription factors (TFs) family, began with the Arabidopsis microarray data mining studies where the KNAT7 gene was highly co-expressed with Cellulose synthase A (CESA) genes involved in the secondary cell wall (SCW) formation (Brown et al, 2005; Ehlting et al, 2005; Persson et al, 2005)
In another study by Pandey et al (2016), overexpression of NbKNAT7 in tobacco resulted in thickening of the SCW in the xylem while suppression of NbKNAT7 in tobacco VIGS and RNAi plants resulted in thinner xylem SCWs coupled with increased xylem area

Summary

Introduction

Plant cell walls serve as significant sinks for the irreversible sequestering of fixed atmospheric carbon (Pauly and Keegstra, 2008). Lignocellulosic biomass from secondary cell walls (SCW) is one of the most promising bioenergy feedstocks for producing second-generation bioethanol (Demura and Ye, 2010; Nookaraju et al, 2013; Zhong et al, 2019). Zhong et al (2008) suggested that NAC TFs are the top-tier master regulators directly activating the expression of several lower-level TFs, including MYBs and KNAT7. Secondtier regulators like MYB46 directly regulate the expression of third-tier targets like KNAT7, impacting the biosynthesis of SCW components, namely, cellulose, xylan, and lignin (Ko et al, 2012a; Rao and Dixon, 2018). Genetic manipulation of KNAT7 gene expression in poplars has not yet been reported, but such knowledge will assist in using transgenic woody biomass for bioethanol production in the future

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