Dissertation

Investigating the Biomechanics of Stomatal Maturation and Dynamics in Eudicots and Grasses

Harb, Leila Moussa Jaafar

2025

Overview

Stomata are microscopic pores on the leaf surface, flanked by two guard cells that regulate pore size in response to environmental stimuli such as light, CO₂ levels, temperature, and humidity. The regulation of pore size is essential for controlling water transpiration and enabling CO₂ diffusion into the plant for photosynthesis. Understanding stomatal dynamics is crucial for improving water-use efficiency, elucidating plant adaptation to changing environments, and identifying new strategies for crop improvement.In eudicots such as Arabidopsis thaliana , guard cells are formed through the activity of a series of transcription factors and associated cellular and signaling pathways. This is followed by the growth and maturation of the stomatal complex, a process that remains poorly understood in terms of geometry, cell wall dynamics, and mechanics. In Chapter 2, we used a combination of genetics, cell biology, microscopy, mechanical testing, and finite element modeling to investigate stomatal maturation in Arabidopsis from both geometric and mechanical perspectives. We found that stomatal complexes mature through a series of geometric milestones and that, during maturation, guard cells develop mechanical anisotropy and exhibit reduced turgor pressure.Stomata vary in shape and size across plant species. For instance, while Arabidopsis stomata are flanked by kidney-shaped guard cells, grass stomatal complexes are composed of dumbbell-shaped guard cells flanked by subsidiary cells. These subsidiary cells, which are morphologically distinct from other epidermal cells, are thought to aid in regulating pore size. Several hypotheses have been proposed to explain the interactions between guard and subsidiary cells in grass stomatal complexes; however, these models have yet to be rigorously validated. In Chapter 3, we investigated guard–subsidiary cell interactions in the grass species Brachypodium distachyon using targeted laser ablation, turgor pressure measurements, and two-dimensional computational simulations of stomatal cross-sections. Our findings revealed that, contrary to prevailing models, guard and subsidiary cell turgor pressures change in the same direction during stomatal opening and closing, rather than in an inversely proportional manner. Moreover, we identified the subsidiary cells as the primary drivers of stomatal function in Brachypodium, calling for a revision of current models of grass stomatal mechanics.Guard cell walls play a critical role in stomatal function by providing the stiffness necessary to allow anisotropic cell deformation in response to changes in turgor pressure. The cell wall is composed of carbohydrate polymers such as cellulose, hemicelluloses, and pectins. Grasses possess type II primary cell walls, which are typically low in pectin content. However, previous studies have highlighted the significance of homogalacturonan biosynthesis genes in monocots. In Chapter 4, we investigate stomatal dynamics in the gaut1 mutant of Brachypodium in comparison to the wild type. We find that gaut1 mutants exhibit variations in subsidiary cell size and pore area, highlighting the importance of homogalacturonan in regulating cell expansion. Additionally, we describe the development and testing of new protocols to stain the cell wall and visualize the full depth of grass stomatal complexes using multiple imaging techniques, including confocal microscopy, Airyscan microscopy, multiphoton microscopy, and micro-computed tomography, to support the development of an image segmentation pipeline to automatically measure the volumes and geometries of cells in stomatal complexes.In summary, these studies elucidate the biomechanical characteristics of stomatal maturation in the eudicot model Arabidopsis thaliana and critically assess current hypotheses regarding guard– subsidiary cell interactions and the functional importance of cell wall components in the grass model Brachypodium distachyon . Collectively, our findings advance the understanding of stomatal dynamics in both eudicots and grasses and offer new opportunities for enhancing stomatal traits to improve plant resilience and productivity.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biomechanics / Botany / Cell division / Cell growth / Cellular biology / Cellulose / Developmental biology / Flowers & plants / Grasses / Microscopy / Polymer chemistry / Polymers / Pore size / Transcription factors
• ISBN: 9798297669987