Enhancement of Secondary Cell Wall Formation in Poplar Xylem Using a Self-Reinforced System of Secondary Cell Wall-Related Transcription Factors

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.

Wood of trees: Cellular structure, molecular formation, and genetic engineering

“…Heterologous overexpression of SWN1 from rice under the control of the NST3 promoter resulted in a 57% increase in the physical strength of the stem of transgenic Populus (Sakamoto et al, 2016). Overexpression of Arabidopsis SND1 in Populus resulted in enhanced SCW thickening (Nakano et al, 2022). The upregulation of the alternative-spliced PtrWND1B transcript in Populus resulted in a considerable increase in xylem fiber cell wall thickness (Zhao et al, 2014).…”

“…Several MYB transcription factors that regulate SCW formation have been studied in order to modify the composition of wood SCWs. Overexpression of AtMYB46 , which is directly downstream of SND1 in the regulatory network for SCW biosynthesis, resulted in a lower lignin S/G ratio and acetylated hemicellulose in Populus (Nakano et al, 2022). MYB115 overexpression decreased lignin quantity and increased the S/G ratio, which improved saccharification and bioethanol yield (Fan et al, 2022).…”

The Regulation of Xylem Development by Transcription Factors and Their Upstream MicroRNAs

“…Previous studies have shown that, like MYB genes, NAC transcription factors are mainly involved in the regulation of plant stress responses [ 57 ]. Later, it was also found that NAC transcription factors play an important regulatory role in xylem development [ 58 , 59 ].…”

Tetraploidization promotes radial stem growth in poplars

“…The method for NMR footprinting analysis was essentially described by Hori et al (2020), Akiyoshi et al (2021), and Nakano et al (2022). Briefly, stem samples were freeze-dried and debarked and then ground in an automill (TK-AM7, Tokken) for 10 min.…”

“…To further estimate the cell wall properties, we measured enzymatic saccharification efficiency (Nakano et al 2022;Ohtani et al 2017;Okubo-Kurihara et al 2016;Yang et al 2006). Cellulose and hemicellulose in powder samples of whole shoots, which were grown in an agar medium for four weeks, were saccharified to quantify the amount of released glucose after 0-, 5-, or 24-h enzymatic incubation.…”