A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

Abstract

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

Glucosylhydroxyceramides Modulate Secretion Machinery of a Subset of Plasmodesmata Proteins and a Change in the Callose Accumulation

“…HPTS loading assays and aniline blue staining ref. (Han et al, 2014; Kumar et al, 2016) were used to assess the permeability of the PD. GFP fluorescence intensity and callose signal intensity were measured using ImageJ software.…”

The 28 Ser Amino Acid of Cucumber Mosaic Virus Movement Protein Has a Role in Symptom Formation and Plasmodesmata Localization

“…Callose at PD was stained with aniline blue (2:3 ratio of 0.1% aniline blue and 1 M glycine pH: 9.5) by infiltration into leaves 10 min before visualization [ 35 ]. For plasmolysis, leaf sections were incubated in 10% NaCl and the plasmolyzed cells were examined by confocal microscopy.…”

α1-COP delivers sphingolipid modifiers and controls plasmodesmal callose deposition in Arabidopsis

“…To measure symplasmic connectivity using the HPTS (8-Hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt, SIGMA-ALDRICH) dye movement assay, a symplasmic dye tracer, was loaded on the top of sharply trimmed etiolated three-day-old Arabidopsis hypocotyls as shown in previous publications (Han et al, 2014; Kumar et al, 2016). A cover slip was placed between each cut hypocotyl surface and the MS agar.…”

“…For dye loading, individual agar blocks containing HPTS (5 mg/mL) were placed on the cut hypocotyl surface. After a 5 min loading period, the seedlings were washed in water for 15 min, and then fluorescent probe movements were observed by confocal microscopy (Kumar et al, 2016).…”

“…CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 22, 2021. ; https://doi.org/10.1101/2021.03.22.436362 doi: bioRxiv preprint tracer, was loaded on the top of sharply trimmed etiolated three-day-old Arabidopsis hypocotyls as shown in previous publications (Han et al, 2014;Kumar et al, 2016). A cover slip was placed between each cut hypocotyl surface and the MS agar.…”