Validation of Fiber-Dominant Expressing Gene Promoters in Populus trichocarpa

Wood is an important raw material for industrial applications. Its fiber-specific genetic modification provides an effective strategy to alter wood characteristics in tree breeding. Here, we performed a cross-analysis of previously reported single-cell RNA sequencing and the AspWood database during wood formation to identify potential xylem fiber-dominant expressing genes in poplar. As a result, 32 candidate genes were obtained, and subsequently, we further examined the expression of these genes in fibers and/or vessels of stem secondary xylem using the laser capture microdissection technique and RT-qPCR. Analysis identified nine candidate genes, including PtrFLA12-2, PtrIRX12, PtrFLA12-6, PtrMYB52, PtrMYB103, PtrMAP70, PtrLRR-1, PtrKIFC2-3, and PtrNAC12. Next, we cloned the promoter regions of the nine candidate genes and created promoter::GUS transgenic poplars. Histochemical GUS staining was used to investigate the tissue expression activities of these gene promoters in transgenic poplars. In one month, transgenic plantlets grown in medium showed intensive GUS staining signals that were visible in the leaves and apical buds, suggesting substantial expression activities of these gene promoters in plantlets predominantly undergoing primary growth. In contrast, for three-month-old transgenic poplars in the greenhouse with predominantly developed secondary stem tissues, the promoters of seven of nine candidate genes, including PtrMYB103, PtrIRX12, and PtrMAP70, showed secondary xylem fiber-dominant GUS signals with considerable spatial specificity. Overall, this study presents xylem fiber-dominant promoters that are well-suited for specifically expressing genes of interest in wood fibers for forest tree breeding.