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Abstract

Stomata, tiny valves in the plant epidermis, regulate the gas exchange for photosynthesis and transpiration, thus playing an essential role in plants' water-use efficiency and stress tolerance. In most land plants, stomata are composed of two kidney-shaped guard cells (GCs) surrounding a central pore. However, novel stomatal morphologies have also been discovered in some species. For example, in grasses, two dumbbell-shaped GCs are flanked by two subsidiary cells (SCs) that contribute to faster stomatal movement and higher water-use efficiency of grasses. Stomata with SCs are also typical in most succulents. Succulents adopted an innovative photosynthetic lifestyle, where gas exchange occurs at night to avoid severe transpiration during the day to adapt to survival in arid conditions. The stomata in Kalanchoë laxiflora, an emerging model system in succulents, include two kidney-shaped GCs surrounded by three unequal-sized, circularly arranged SCs. Research showed that ions shuttle between GCs and SCs in the stomata of K. laxiflora, like grasses, suggesting that these SCs might be functionally relevant to stomatal movement. However, the development and function of these anisocytic SCs are unknown. In my study, I focused on the stomatal development in K. laxiflora. Firstly, I established protocols to establish K. laxiflora as a model system for stomatal research, including horticultural protocols, tissue culture-based genetic transformation protocols, staining protocols for microscopy imaging, and cloning protocols. Secondly, based on static imaging, I identified and analyzed each stage of stomatal development of the anisocytic, mesogenous stomatal complex in K. laxiflora. In addition, light microscope imaging and quantification in different pairs/sizes of wild type (WT) leaves showed that the stomata on the leaf epidermis gradually develop and differentiate into GCs as the leaves grow. Notably, the 6th pair of leaves are already “mature leaves” for stomatal development. Thirdly, CRISPR-CAS9-mediated gene editing, reporter lines, and overexpression lines suggested that the two orthologs of the key stomatal transcription factor AtMUTE, KlMUTEs, promote asymmetric divisions to form SCs in K. laxiflora. This is functionally opposite to AtMUTE, which terminates the asymmetric divisions and promotes cell fate transition and stomata differentiation in Arabidopsis thaliana. Furthermore, Bulk RNA-sequencing revealed a potential genetic program regulated by KlMUTE1 for promoting asymmetric divisions during stomatal development. Overall, this study revealed the development process of a novel stomatal morphotype and a genetic mechanism that regulates the formation of the anisocytic SCs in the succulent model K. laxiflora.