The unique morphology of grass stomata enables rapid responses to environmental changes. Deciphering the basis for these responses is critical for improving food security. We have developed a planta platform of single nucleus RNA-sequencing by combined fluorescence-activated nuclei flow sorting, and used it to identify cell types in mature and developing stomata from 33,098 nuclei of the maize epidermis. Guard and subsidiary cells displayed differential expression of genes, besides those encoding transporters, involved in the abscisic acid, CO2, Ca2+, starch metabolism, and blue light signaling pathways, implicating coordinated signal integration in speedy stomatal responses, and of genes affecting cell wall plasticity, implying a more sophisticated relationship between guard and subsidiary cells in stomatal development and dumbbell-shaped guard cell formation. The trajectory of stomatal development identified in young tissues, and by comparison to the bulk RNA-seq data of the MUTE defective mutant in stomatal development, confirmed known features and shed light on key participants in stomatal development. Our study provides a valuable, comprehensive, and fundamental foundation for further insights into grass stomatal function.

Background The leaf epidermis functions to prevent the loss of water and reduce gas exchange. As an interface between the plant and its external environment, it helps prevent damage, making it an attractive system for studying cell fate and development. In monocotyledons, the leaf epidermis grows from the basal meristem that contains protodermal cells. Leaf protoderm zone is covered by the leaf sheath or coleoptile in maize and wheat, preventing traditional exogenous phytohormone application methods, such as directly spraying on the leaf surface or indirectly via culture media, from reaching the protoderm areas directly. The lack of a suitable application method limits research on the effect of phytohormone on the development of grass epidermis. Results Here, we describe a direct and straightforward method to apply exogenous phytohormones to the leaf protoderms of maize and wheat. We used the auxin analogs 2,4-D and cytokinin analogs 6-BA to test the system. After 2,4-D treatment, the asymmetrical division events and initial stomata development were decreased, and the subsidiary cells were induced in maize, the number of GMC (guard mother cell), SMC (subsidiary mother cell) and young stomata were increased in wheat, and the size of the epidermal cells increased after 6-BA treatment in maize. Thus, the method is suitable for the application of phytohormone to the grass leaf protodermal areas. Conclusions The method to apply hormones to the mesocotyls of maize and wheat seedlings is simple and direct. Only a small amount of externally applied substances are needed to complete the procedure in this method. The entire experimental process lasts for ten days generally, and it is easy to evaluate the phytohormones’ effect on the epidermis development.

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Background: The maize leaf epidermis is function as protection against water loss and gas exchange, contacting the environment and avoiding the damage, which is an attractive system for studying the process of cell fate and development. In monocots, leaves epidermis grown from basal meristem, which contains protodermal cells. The leaf protoderm zone was covered by the leaf sheath or coleoptile in maize, the classic exogenously phytohormone application method, such as spraying on leaf surface or adding in the culture media can’t apply the phytohormone to the protoderm areas directly, which restricts the research about phytohormone effect epidermal development.Results: Here we described a simple and direct method for exogenously application of phytohormone to maize leaf protoderm. We use the auxin analogs 2,4-D to test the system, and the asymmetrical division events which initial stomata development were decreased and the subsidiary cells were induced in advance after 2,4-D treatment. This result was the same as other similar studies’ results, indicated that the method is suitable for been used for application phytohormone to the maize leaf protodermal areas.Conclusions: The method, applied hormones on the mesocotyls of the maize seedlings, is simple and direct. Only a small amount of externally applied substances is required to complete this experiment through this method. The entire experiment process just last 10 days generally and it is easy to survey the phytohormone's effect on the epidermis development.