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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.

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