MCA: Cell-Specific Promoters in Xylem Formation: A Transformative Tool for Tree Biology

Wood formation in trees is a fundamental biological process with profound implications for renewable products, carbon sequestration, and forest productivity. Despite decades of research, scientists lack precise molecular tools to study and manipulate individual cell types within wood-forming tissues, limiting progress in both basic tree biology and applied biotechnology. This project will develop a comprehensive toolkit of cell-type-specific promoters that turn on gene expression only in targeted cell populations within the wood-forming zone of poplar trees. By enabling researchers to precisely control where genes are active within a tree, this toolkit will accelerate discovery of how different wood cell types develop and function, while also supporting the engineering of trees with improved properties for biofuels, paper production, and carbon storage. The tools developed here will be freely shared with the broader plant science community, multiplying their impact across many research programs. Targeted manipulation of individual cell types addresses a long-standing bottleneck in tree biotechnology: the inability to modify wood properties such as lignin content in fiber cells or vessel wall composition without triggering unintended growth penalties or off-target effects throughout the plant. These capabilities are directly relevant to national priorities in advanced materials and manufacturing, offering new pathways to engineer trees as precision biological factories for next-generation bioproducts. This work directly advances NSF's mission to promote scientific progress and national prosperity by strengthening the foundational biotechnology needed to harness trees as sustainable resources. This project leverages recent breakthroughs in single-nucleus RNA sequencing to systematically identify, validate, and characterize promoters and cis-regulatory elements (CREs) with highly restricted, cell-type-specific expression patterns in the developing xylem of the genus Populus. Three interconnected projects will be pursued. First, existing and newly generated single-cell transcriptomic datasets will be mined to identify candidate genes with highly enriched expression in specific xylem cell populations, including cambial initials, fiber cells, vessel elements, and ray parenchyma, followed by computational prediction of minimal promoter regions conserved across multiple Populus species. Second, candidate promoters will be rapidly validated using Induced Somatic Sector Analysis (ISSA) combined with the RUBY reporter gene system, enabling visualization of cell-specific expression in biologically relevant woody tissue without full stable transformation; top-performing candidates will then be confirmed in stably transformed greenhouse-grown trees. Third, computationally predicted CREs will be used to design synthetic promoters with defined, modular regulatory architectures, testing whether cell-type specificity can be rationally engineered. The anticipated outcome is a validated, publicly available library of at least one well-characterized cell-type-specific promoter for 75% of the major xylem cell populations examined, along with a framework for synthetic promoter design in wood-forming tissues. This research directly advances biotechnology as a national priority by providing precision tools for tree improvement. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.