Deep single-cell proteomics reveals cellular heterogeneity in the cold response of Arabidopsis leaves.

Mass spectrometry (MS)-based proteomic analysis at single-cell resolution in complex plant tissues is challenging. Here, we present a highly sensitive single-cell proteomics (SCP) workflow that integrates key steps including single-cell sorting, sample pretreatment, MS detection, and data processing, with specific optimizations tailored for plant cells. Using this customized workflow, we achieved great identification depth, quantifying approximately 4,200 protein groups from a single mesophyll cell. In the proof-of-principle application in the cold response research of Arabidopsis leaves, we achieved accurate cell typing/clustering based on proteomic information at the single-cell level in plants. Moreover, we revealed cell-type-specific differences in cold response and quantified the dynamic changes of cold-responsive proteins at single-cell resolution. Among 100 randomly sorted epidermal cells, two major cell types (guard cell and pavement cell) were identified. The observed discordance at single-cell level between proteome and transcriptome in mesophyll cells further underscores the importance of plant SCP technologies. Our deep plant SCP workflow paves the way for future applications across diverse tissues/organs and enables comprehensive insights into plant biology.