Microfluidic Interrogation of Chitin-Induced Calcium Oscillations in the Moss Physcomitrium patens

Plants defend against pathogens such as fungi by initiating coordinated structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx and calcium oscillations that drive defense gene expression, yet the mechanisms by which these signals encode stressor intensity and propagate systematically remain unclear. Here, we present a microfluidic system to characterize intracellular calcium dynamics in protonemal colonies of the moss <i>Physcomitrium patens</i> (Hedw.) upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent imaging of cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon stimulus removal. We implemented an unbiased image segmentation algorithm using pixel-based <i>k</i>-means clustering to automatically locate regions with specific oscillatory signatures. Calcium dynamics were distinct across adjacent cells, distinguishable by cell type, and significantly modulated by circadian rhythm, adaptation time within the device, and stimulus timing. Cytosolic calcium oscillations, which rose and fell symmetrically within about 60 s, occurred spontaneously during the subjective night and following short adaptation periods. Chitin elicited strong oscillations with increased frequency, amplitude, and duration, and repeated pulses entrained regular, colony-wide oscillations at the stimulation interval. This study complements prior investigations of whole plant and growth tip dynamics and provides a quantitative framework to study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression.