Single-cell proteomics of Arabidopsis leaf mesophyll identifies drought stress-related proteins

The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and FAIMS-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing >80 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein-protein dynamics. Highlights This study describes the first application of scProteomics to study abiotic stress-induced proteome regulation in leaf mesophyll cells From a single Arabidopsis mesophyll protoplast, ∼2800 proteins on average were quantified with high precision using label free scProteomics scProteomics of water-deficit Arabidopsis leaves revealed known and novel proteins involved in drought response Single-protoplast proteomics revealed water deficit-induced changes in protein covariation networks independent of changes in abundance