Large-scale single-cell profiling of stem cells identifies redundant regulators of shoot development and yield trait variation

Plant shoot stem cells generate organs essential for food, feed, and biofuels. However, plant single-cell analyses struggled to capture these rare cells or to detect stem cell regulators like CLAVATA3 and WUSCHEL. Here, we dissected stem cell-enriched shoot tissues from maize and Arabidopsis for single-cell RNA sequencing (scRNA-seq), and we optimized protocols to recover thousands of CLAVATA3- and WUSCHEL-expressing cells. A cross-species comparison identified deeply conserved stem cell regulators. We also profiled maize stem cell mutants, validated candidate regulators with spatial transcriptomics, and confirmed their roles in shoot development. These include a family of RNA-binding protein genes and two families of sugar kinase genes. Finally, we show how coupling large-scale single-cell profiling with allelic variation analysis in diverse maize germplasm serves as a valuable resource for identifying stem cell regulators significantly associated with grain yield components. Our discoveries advance the study of shoot stem cells and open avenues for rational crop engineering.