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

The high efficiency of grass stomata is believed to be due to their structure and anatomy, with two dumbbell-shaped guard cells surrounded by two subsidiary cells (SCs) and an efficient functional molecular interaction between these cells. Important channels expressed in these cells are aquaporins (AQPs), which contribute to the dynamic of stomatal movement. To further investigate genes expressed in maize GCs and SCs, RNA sequencing was performed on microdissected GCs and SCs during day and night. A general transcriptomic analysis first revealed that genes upregulated in SCs compared to GCs during the day were primarily involved in lipid transport, lipid localization, and lipid metabolism, whereas 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. For example, the plasma membrane aquaporin ZmPIP1;5 and ZmPIP2;5 displayed a higher expression in GCs than in SCs during the day, whereas ZmPIP1;3 and ZmPIP2;1 exhibited an opposite pattern. To examine the specific roles of AQPs in these cells, a CRISPR-based tissue-specific knockout system was successfully used to silence ZmPIPs belonging to the PIP1 subfamily in either maize GCs or SCs. The targeted knockout of ZmPIP1s in these cells impaired stomatal movements in response to light, ABA, and CO2 compared with wild-type plants. 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. Overall design: Maize B104 seeds were germinated and transferred to potting soil, as previously described. The plants were grown in a phytotron under a 16/8 h light/dark cycle (25/22 °C) and a daytime light intensity of 200 mmol. m-2. s-1 at the top of the leaves. Humidity was maintained between 60% and 70% in the growth chamber. Epidermal strips were peeled from the third leaf of two-week-old maize seedlings. They were placed on microscope glass slides (plain microscope slides 76x26mm, 0.8 mm x 1.00 mm, AGL4242) and flash-frozen with liquid nitrogen. The samples were immediately stored at -80 °C until microdissection. The samples were collected at two times, i.e., day (two hours after the light onset) and night (two hours after the light offset). Four independent collections were carried out. On the day before the microdissection, the peeled epidermal strips were freeze-dried at -40 °C for 10 h under pressure of 10 Pa in a Labconco® Free-Zone Plus freeze-drier. At the end of the process, the temperature slightly increased to room temperature for 3 h. A few drops of absolute ethanol were placed on the strips to flatten them. The samples were stored in a desiccator under vacuum at room temperature until microdissection, to avoid rehydration. Guard cells (GC) and subsidiary cells (SC) were collected using a PALM MicroBeam system (Carl Zeiss MicroImaging GmbH). GCs or SCs were selected using Zeiss software and cut using a laser. After cutting, cells were pressure catapulted and collected immediately in a tube with an adhesive cap (Carl Zeiss™ 415190-9211-000). Approximately 1500 cells were collected in one tube, and two tubes were used to collect cells for each sample.