Viewing Stomata in Action: Autonomous in Planta Imaging of Individual Stomatal Movement

Stomata regulate plant gas exchange under changing environments, but observations of single stomata dynamics in planta are sparse. We developed a compact microscope system that can measure the kinetics of tens of stomata in planta simultaneously, with sub-minute time resolution. Darkfield imaging with green light was used to create 3D stacks from which 2D surface projections were constructed to resolve stomatal apertures. Stomatal dynamics of Chrysanthemum morifolium (Chrysanthemum) and Zea mays (Maize) under changing light intensity were categorized, and a kinetic model was fitted to the data for quantitative comparison. Maize stomata transitioned frequently between open and closed states under constant growth light and these 'opening and closing' stomata, when closed, responded faster to a change to saturating light than steady-state closed stomata under the constant growth light. The faster opening response benefits CO₂ uptake under saturating light. The slow closure of Chrysanthemum stomata reduced water use efficiency (WUE). Over 50% showed delayed or partial closure, leading to unnecessarily large apertures after reduced light. Stomata with larger apertures had more lag and similar closure speeds compared to those with smaller apertures and lag, further reducing WUE. In contrast, maize stomata with larger apertures closed faster, with no lag.