Fig 1. Experimental workflow for the treatment, data collection, and quantitative evaluation of actin microfilament (MF) configurations in guard cells.

(A) Illustration of the workflow for image acquisition of Arabidopsis guard cell pairs from mock-, pathogen-, and PAMP-elicited samples. Sampling times are shown as Zeitgeber time, which refers to the experimental time. All experiments were conducted under 12 light/12 hour dark cycles; Zeitgeber time zero indicates the time at which the lights turn on. (B) Illustration of image collection for diurnal image sampling. (C) Guard cell images (n = 1793) collected by spinning disc confocal microscopy were processed and analyzed, and further classified based on MF configurations following immune elicitation (i.e., PAMP treatment, pathogen treatment). Using this approach, we evaluated stomatal aperture and three metric parameters for MF orientation, bundling and density, independently. Based on the three MF configuration parameters, images were classified into some MF configuration classes and time evolution of the class frequency was assessed.