Drop-ANd-See: A Simple, Real-Time, and Noninvasive Technique for Assaying Plasmodesmal Permeability

Abstract

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.

Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose

“…The color threshold was adjusted to minimize autoflourescence. The parameters were as follows: hue 57–108, saturation 133–255, brightness 0–60, thresholding method default, threshold color white, color space HSP (the thresholding protocol is based upon DANS methods as described in Cui et al , 2015 ). Wilcoxon rank sum test was used; asterisks indicate P <0.05.…”

Plasmodesmata at a glance

“…Alternatively, membranepermeable dyes could be applied to intact plants in a non-invasive manner to measure plasmodesmal permeability at the tissue level. For example, the Drop-ANd-See (DANS) dye-loading assay utilizes the membrane-permeable non-fluorescent dye carboxyfluorescein diacetate, which enters epidermal cells once loaded onto an intact plant surface and is immediately converted into the membraneimpermeable fluorescent tracer carboxyfluorescein (Cui and Lee, 2016;Cui et al, 2015;Gui et al, 2014;Lee et al, 2011;Lim et al, 2016; Wang et al, 2013) (see poster). This method is an ideal choice when addressing questions that require real-time, non-invasive analyses.…”

A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

“…To evaluate the vitality of the transformed cells, the vital stain CFDA (50 mM in DMSO) was diluted immediately prior to use to a final working concentration of 1 mM in water. Stems were covered with a thin layer of CFDA by spreading small drops of a few µl each evenly over the Cuscuta stem and infection sites (adjusted from “Drop‐And‐See assay” [Cui et al, 2015]). After incubation for 10 minutes in the dark, the CFDA was removed by briefly rinsing with tap water, gently dried with paper, and viewed under a StereoLumar V12 stereo microscope using Zeiss filter set 38.…”