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Abstract

Stomata are cellular pores on the leaf epidermis that regulate CO2 uptake for photosynthesis and limit water vapour loss. Stomata can open and close in response to different environmental triggers and the speed of stomatal movements defines how efficiently plants exchange gas and save water in fluctuating environments. Therefore, stomatal speed and water use efficiency (ratio between assimilated carbon and lost water through stomata) are important traits for plant productivity and tolerance to environmental stresses. Grasses such as wheat, and rice and the model species Brachypodium distachyon are the plant group displaying the fastest stomatal movements, a trait that is associated with their unique stomatal morphology. Grass stomata are formed by two dumbbell-shaped guard cells (GCs) flanked by two subsidiary cells (SCs). This morphology seems to result in optimal mechanics that facilitate the stomatal movements and the SCs appear to contribute with quick exchange of ions and water required for turgor-driven stomatal opening and closure. Still, the molecular mechanisms behind the efficient form and function of grass stomata remain highly unexplored. A comparison of the leaf transcriptome between wildtype B. distachyon (Bd21-3) and bdmute mutant plants, which lack SCs and show defective stomatal function, allowed identifying candidate genes potentially associated with grass stomata form and function. Therefore, I established a leaf-level gas exchange phenotyping platform to perform a reverse genetic screen to identify phenotypes associated with defective stomatal kinetics and water use efficiency. First, I characterized stomatal movements and gas exchange efficiency in wild-type B. distachyon with 120 individuals and verified that environmental fluctuations (e.g., light intensity, temperature) systemically affected leaf-level gas exchange measurements and stomatal anatomy, and observed significant correlations between stomatal anatomy and function. Secondly, the reverse genetic screen of 60 mutant lines yielded a few promising candidates from which I selected BdPRX76/BdPOX, a class III peroxidase for further characterization. bdpox mutants displayed lower intrinsic water- use efficiency and higher stomatal conductance levels due to increased stomatal size. Interestingly, bdpox mutants presented longer stomata but no differences in stomatal density. Therefore, this resulted in a unique disruption in the negative correlation between stomatal density and size. Contrary to stomata, prickle hair cells were reduced in bdpox mutants. Surprisingly, BdPOX was exclusively expressed in hair cells which suggested that BdPOX cell-autonomously promote prickle hair cell size and non-cell-autonomously constricts stomatal elongation. The analysis of cell wall autofluorescence and lignin stainings suggested that BdPOX acts in the lignification/hydroxycinnamates cross-linking at the hair cell base to support cell outgrowth. Finally, ectopic expression of BdPOX in the stomatal lineage restricted stomatal elongation by increasing phenolic content in the guard cells. Overall, these results suggest a coordination between stomata and prickle hair cell formation to optimize leaf functionality.