Calcium Dynamics in Root Cells of Arabidopsis thaliana Visualized with Selective Plane Illumination Microscopy

Abstract

Selective Plane Illumination Microscopy (SPIM) is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET). Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca2+ signal percolation, predicted in previous studies, has been directly visualized.

Advances in Imaging Plant Cell Dynamics

“…Moreover, LSFM was combined recently with Förster resonance energy transfer (FRET) to address the respiration rates of root hairs using a FRET sensor for MgATP production (De Col et al, 2017). A similar FRET-based sensor was used to follow cytosolic calcium oscillations during root (Costa et al, 2013) and root hair growth.…”

Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma

“…eATP administration to root tip cells has been reported to trigger a cytosolic Ca 2+ transient in both aequorin expressing seedlings (Tanaka et al, 2010) and Cameleon (Tanaka et al, 2010;Krebs et al, 2012;Loro et al, 2012) with a consequent Ca 2+ accumulation in intracellular compartments including the mitochondria and the ER (Loro et al, 2012;Bonza et al, 2013;Wagner et al, 2015b). Specifically, in the root tip, eATP is initially perceived by the cells of the meristematic zone (lateral root cap cells), and afterward a Ca 2+ wave travels from this region to the elongation zone (Tanaka et al, 2010;Loro et al, 2012;Costa et al, 2013). We therefore analyzed plastidial Ca 2+ dynamics in response to 500 mM eATP in the root meristematic cells of seedlings.…”

Light-sheet microscopy: a tutorial

“…[167]. A standard ratiometric FRET effect is used to monitor the calcium dynamics of transgenic Arabidopsis plants, taking advantage of the LSFM planar sectioning.…”