Title : Integrated single-cell transcriptomics, cis-element profiling, and quantitative functional assays for mining maize bundle sheath cell-specific promoters
Abstract:
Maize (Zea mays L.) is a representative C4 crop in which carbon assimilation through the Calvin cycle primarily takes place in bundle sheath cells (BSCs). Identifying promoters capable of driving strong and specific gene expression in BSCs is of paramount importance for elucidating C4 photosynthetic mechanisms and advancing genetic engineering to improve crop yield. However, experimental validation and systematic activity comparison of promoters corresponding to BSC-enriched genes remain limited, hindering their application in C4 photosynthetic engineering. To address these challenges, this study established an integrated pipeline combining multi-source single-cell RNA sequencing (scRNA-seq) datasets with molecular, physiological, and biochemical approaches to systematically mine and functionally characterize maize BSC-associated promoters. First, 10 upstream promoter sequences (~2000 bp) of previously reported BSC marker genes (including PCK1, ME1, PRK2, SHBP1, RBCS1/2, RCA2, FBPA1/2, and MDH) were cloned. Quantitative dual-luciferase assays in maize protoplasts revealed that the PCK1 and SHBP1 promoters exhibited the highest transcriptional activities, while PRK2 showed relatively lower baseline activity. To clarify their cell-type expression specificities, stable transgenic maize plants carrying promoter-GUS reporter constructs were generated. Tissue section analysis demonstrated that the PRK2 promoter conferred strict BSC-specific expression, whereas the SHBP1 promoter exhibited non-specific activity across epidermal cells. To expand the repertoire of BSC candidate promoters, three published maize leaf scRNA-seq datasets were integrated, resulting in 81 conserved overlapping genes. Developmental expression profiling identified three candidate genes (Zm00001d041819, Zm00001d046786, and Zm00001d023376) with sustained high transcript abundance across leaf development stages. Dual-luciferase transient assays confirmed strong promoter activities for all three candidates, with Zm00001d041819 displaying the highest expression strength. Furthermore, by analyzing cis-regulatory motifs within known marker promoters, 15 core conserved motifs and 31 core transcription factor families were identified. A weighted regulatory scoring model incorporating core motif density and binding ratios was constructed, effectively screening 21 high-confidence BSC candidate promoters from the 81 overlapping genes. Finally, a cell-isolation workflow separating BSCs and mesophyll cells (MCs) was optimized, coupled with RT-qPCR to validate the cell-type-specific expression patterns of these candidate promoters. In summary, this study provides a quantitative activity ranking of classic BSC marker promoters and establishes an end-to-end framework of mining maize BSC-specific promoters integrating "scRNA-seq mining – cis-motif modeling – functional activity assay – cell isolation validation." These findings supply essential genetic tools for C4 photosynthetic engineering and Plant Synthetic Biology.
