Viewing stomata in action: Autonomous in planta imaging of individual stomatal movement

Stomata regulate plant gas exchange under changing environments, but observations of the dynamics of single stomata 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. Dark field imaging with green light was used to create 3D stacks from which 2D surface projection were constructed to resolve stomatal apertures within the field of view.

Stomatal dynamics of Chrysanthemum morifolium ( Chrysanthemum ) and Zea Mays (Maize) under dynamically changing light intensity were categorized, and a kinetic model was fitted to the data for quantitative comparison. In addition, we also resolved dynamics of the surface position of the leaf, related to dynamics of leaf thickness or bending.

Maize stomata oscillated frequently between open and closed states under constant growth light and these oscillating stomata responded faster to changes in light than non-oscillating stomata at the same aperture.

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.

In conclusion, our new system enables fine mapping of the heterogeneity of movement in neighboring stomata, providing new insights on the relations between stomatal dimensions, relative position and aperture changes under fluctuating light intensity.