Drop-ANd-See: a simple, real-time, and noninvasive technique for assaying plasmodesmal permeability.

Gating of plasmodesmata (PD) is a highly dynamic cellular process spatiotemporally controlled by various physiological, developmental, and environmental conditions. Here, we describe a quantitative approach named Drop-ANd-See (DANS), which allows for a real-time, in situ assessment of plasmodesmal permeability in an array of comparative studies. The power of the DANS assay lies in its simplicity: a membrane-permeable, non-fluorescent dye is loaded onto the adaxial epidermis of an intact leaf; the absorbed dye is cleaved by cellular esterases and become fluorescent yet membrane-impermeable; this symplasmic form then diffuses via PD through the mesophyll and into the abaxial epidermis, where the extent of fluorescent dye spreading can be imaged and quantified by confocal microscopy as a measure of cell-to-cell permeability. By employing this DANS assay, rapid changes in PD permeability upon chemical, biological, or environmental treatments can be easily analyzed. Furthermore, PD permeability as a phenotype or a trait of interest can be evaluated using various genetic backgrounds or mutants. We provide hereby an easy-to-follow visual guide of the DANS assay using Arabidopsis plants as an example along with a description of the step-by-step protocol.