Dual and spatially resolved drought responses in the Arabidopsis leaf mesophyll revealed by single-cell transcriptomics.

Drought stress imposes severe challenges on agriculture by impacting crop performance. Understanding drought responses in plants at a cellular level is a crucial first step toward engineering improved drought resilience. However, the molecular responses to drought are complex as they depend on multiple factors, including the severity of drought, the profiled organ, its developmental stage or even the cell types therein. Thus, deciphering the transcriptional responses to drought is especially challenging. In this study, we investigated tissue-specific responses to mild drought (MD) in young Arabidopsis thaliana (Arabidopsis) leaves using single-cell RNA sequencing (scRNA-seq). To preserve transcriptional integrity during cell isolation, we inhibited RNA synthesis using the transcription inhibitor actinomycin D, and demonstrated the benefits of transcriptome fixation for studying mild stress responses at a single-cell level. We present a curated and validated single-cell atlas, comprising 50 797 high-quality cells from almost all known cell types present in the leaf. All cell type annotations were validated with a new library of reporter lines. The curated data are available to the broad community in an intuitive tool and a browsable single-cell atlas (http://www.single-cell.be/plant/leaf-drought). We show that the mesophyll contains two spatially separated cell populations with distinct responses to drought: one enriched in canonical abscisic acid-related drought-responsive genes, and another one enriched in genes involved in iron starvation responses. Our study thus reveals a dual adaptive mechanism of the leaf mesophyll in response to MD and provides a valuable resource for future research on stress responses.