Cell-specific transcriptomics and knockout reveal aquaporin function in grass stomatal movements.

The high efficiency of grass stomatal movement is believed to be due to their unique four-celled structure, with two dumbbell-shaped guard cells (GCs) flanked by two subsidiary cells (SCs). The molecular interaction between these cells requires further investigation. To investigate genes, including those encoding aquaporins (AQPs), expressed in maize GCs and SCs, RNA sequencing was performed on microdissected GCs and SCs from epidermis harvested during day and night. A CRISPR-based tissue-specific knockout (KO) system was then used to target ZmPIPs belonging to the plasma membrane intrinsic protein1 (PIP1) subfamily in maize GCs or SCs. A general transcriptomic analysis first revealed that genes preferentially expressed in SCs compared with GCs during the day were primarily involved in lipid transport, localization, and metabolism, while genes more highly expressed in GCs were associated with photosynthesis, chloroplasts, and plastids. Interestingly, the expression of several AQP genes varied both spatially and temporally. The KO of ZmPIP1s in GCs resulted in greater stomatal opening than wild-type plants, particularly under mild water-deficit conditions. Overall, these findings reveal transcriptomic specificities between maize GCs and SCs, providing valuable insights into the regulation of rapid stomatal movements in grasses and highlight the role of ZmPIP1s in these processes.