Stomatal Anatomy and Function Across Drought Tolerant and Drought Sensitive Grasses
Previous work comparing E. nindensis and the desiccation sensitive plant E. curvula showed that E. nindensis adult leaves are desiccation tolerant, but not the older, juvenile leaves Vander Willigen et al. 2001, 2003).We will compare the developmental programs of juvenile and adult leaves in each of the species, including quantitative and qualitative analyses of leaf anatomical features. Qualitative comparisons include presence/absence of cell types such as hair cells, bulliform cells, and cork cells. We will examine the structure of apical hydathodes, which are a potential avenue for water loss/dehydration. Hand sections will be used to determine vascular anatomy. Quantitative measurements may include stomatal features such as density and size/area, vein density, bulliform cell area, xylem vessel number and area, and bundle sheath cell size. Images will be quantified using ImageJ. We will compare adult leaves before and after dehydration, and adult leaves in recovered vs never watered plants, to determine if tolerant and resistant grasses alter the developmental program of newly emerging leaves after desiccation.
Fully expanded leaf samples will be examined using scanning electron microscopy and confocal microscopy. Confocal and histochemical staining will be used to examine the cell walls and cuticles, including general cell wall stains (Propidium iodide, Direct Red 23 and/or calcofluor), cuticle stains (Fluorol Yellow 088 and Nile Red) and lignin (Basic Fuchsin).
We will use gas exchange, and direct observations of stomata to address stomatal behaviors during different developmental stages, during dry-down, and during rehydration (if applicable). We hypothesize that gas exchange measurements may not accurately reflect stomatal behavior due to a decrease in available soil water, and that stomatal behavior is an important factor in the regulated dehydration of desiccation tolerant grasses. To address this, we will directly examine stomata using microscopic methods (SEM, methacrylate impressions, and/or confocal microscopy), in conjunction with gas exchange measurements. We will also sample leaves to measure abaxial and adaxial stomatal aperture at the same time, to determine the correlation (or lack thereof) between aperture and transpiration. We will also examine chlorophyll degradation in stomata vs mesophyll cells using confocal microscopy.
We have developed an unpublished method to segment and characterize the anatomical features of maize stomata. We will adapt our current Mask-RCNN segmentation model trained on maize confocal images to recognize stomata from other grasses, including tef, finger millet and related desiccation tolerant species. Using this model, we will perform high-throughput analyses of stomata of desiccation tolerant and sensitive plants.