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Abstract

Stomata, breathing pores located on leaf epidermal made up of paired guard cells surrounding a central pore, are identified in most aerial tissues of plants. They can regulate the entry of photosynthetic CO2 and loss of water vapor. Grasses show particularly intriguing differences in stomatal development programs compared to the dicots in multiple ways. A four-celled stomatal complex presents a unique morphology in grasses termed graminoid, a pair of center dumbbell-shaped guard cells (GCs) recruiting specialized support cells called subsidiary cells (SCs), which develop in parallel rows with specific cell identity. These SCs serve as supporters of GCs in the molecular and mechanical perspectives, to facilitate stomatal movement in response to environmental signals. Production of SCs involves subsidiary mother cells (SMCs) establishment, polarization, and SCs division. The molecular genetic regulation of stomatal development process has been extensively studied in the dicot Arabidopsis thaliana. Yet, this process in grasses still required more exploration, especially the formation of SCs.

In my thesis work, I have examined the comparative transcriptomics (wild-type (WT) vs. mutant (bdmute)), focusing mainly on novel genes involved in SC divisions. My studies are performed using the forage grass Brachypodium distachyon, a recently developed, genetically tractable model species related to wheat. First, I describe mutations in the candidate gene BdPOLAR from transcriptomics, and BdPAN1, whose known orthologue was described as a polarity regulator during SC formation in maize. Although both mutants display SC defects, quantification analysis suggests they work together to regulate SC divisions. Subsequently, I identify that BdPOLAR localizes in the SMCs periphery while mainly being absent at the sites of guard mother cells (GMCs), which is a novel, distal polarity domain, and BdPAN1 polarized at the GMC/SMC interface; thus, two polarity proteins build up an opposing, almost reciprocal polarity pattern. Further, comparative imaging and polarity index data suggest BdMUTE and BdPAN1 are required for the expression and polarization of BdPOLAR, respectively. Moreover, BdPOLAR and BdPAN1 display different functions to promote SC polarizations. BdPAN1 is responsible for pre-mitotic nuclear polarization, which resembles its orthologue in maize. Strikingly, BdPOLAR acquires novel functions that specify SMC division plane orientation and control SMC division capacity. In addition, leaf-level gas exchange measurement discovered that correct SC formation is required for sufficient graminoid stomatal functionality. Finally, I describe the polarity role of BdPOLAR homologue gene, BdPOLAR-LIKE, report that link between BdPOLAR and (Brassinosteroid) BR signaling/biosynthesis in Brachypodium, and discuss prospects for using time-lapse imaging to gain insight into SMC polarity.