Abstract
The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin β-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.
Highlights
In recent years, mounting environmental problems, such as global warming from fossil fuels, have increased the importance of sustainable and carbon-neutral bioresources
The transcription factor genes used in the present work were AtSND1, AtVND7, and AtMYB46, which are master regulators of secondary cell walls (SCWs) formation in A. thaliana (Kubo et al, 2005; Mitsuda et al, 2005, 2007; Zhong et al, 2006, 2007; Ko et al, 2009, 2014; Nakano et al, 2015; Ohtani and Demura, 2019; Kamon and Ohtani, 2021)
The key feature of this self-reinforced system is that the PtCesA18 promoter can be upregulated by AtSND1, AtVND7, and AtMYB46, since these transcription factors can recognize and bind their cis-regulatory elements, which are highly conserved among each ortholog (Zhong et al, 2010a; Ohtani et al, 2011; Kim et al, 2012; Zhong and Ye, 2012)
Summary
In recent years, mounting environmental problems, such as global warming from fossil fuels, have increased the importance of sustainable and carbon-neutral bioresources. One of the most important findings is the identification of key transcriptional factors of SCW formation; in vascular plants, a specific group of NAC (NAM/ATAF/CUC) family transcription factors, called VNS [VASCULAR-RELATED NAC-DOMAIN (VND), NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST)/SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND), and SOMBRERO (SMB)-related] proteins, were shown to function as master regulators of SCW formation, activating all of the events required for SCW formation in A. thaliana (Arabidopsis; Kubo et al, 2005; Mitsuda et al, 2005, 2007; Zhong et al, 2006, 2010b; Yamaguchi et al, 2008, 2011; Ohashi-Ito et al, 2010; Ohtani et al, 2011; Xu et al, 2014; Akiyoshi et al, 2020). Downstream of the VNS proteins, the MYB transcription factors, such as Arabidopsis MYB46 and MYB83, function to upregulate the expression of genes encoding SCW-related enzymes as secondary master regulators of xylem cell formation (Zhong et al, 2007; Ko et al, 2009, 2014; McCarthy et al, 2009, 2010; Nakano et al, 2010). The NAC–MYB-based transcriptional network of SCW formation is widely conserved among land plants (Zhong et al, 2010a; Xu et al, 2014; Nakano et al, 2015; Bowman et al, 2017; Ohtani et al, 2017a), suggesting that these NAC and MYB transcription factors would be effective targets for modifying the quantity and quality of lignocellulosic biomass
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