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

Plants defend against pathogens such as fungi by detecting an attack and initiating both structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx, calcium oscillations, and induces defense gene expression, yet the mechanisms by which these or other signals encode the external stressors or propagate signals plant-wide remain unclear. Here, we present a microfluidic system to examine intracellular calcium signals of the moss Physcomitrium patens upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent microscopy of juvenile moss cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon chitin removal. We developed an unbiased image segmentation algorithm to automatically locate regions with cell-specific oscillatory responses, using pixel-based k-means clustering, treating each time point as a separate dimension. Calcium dynamics were distinct across adjacent cells and distinguishable by cell type. Waves were dependent on time of day, adaptation time within the device, and stimulus timing. Cytosolic calcium waves, which rose and fell symmetrically within about 60 s, appeared spontaneously at night and with short adaptation time. Chitin increased wave frequency, amplitude, and duration, and repeated chitin pulses drove regular, plant-wide oscillations at a controlled frequency. This study complements prior investigations of whole plant and growth tip dynamics and provides new methods to comprehensively study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression.