Visualization of Cu²⁺ uptake and release in plant cells by fluorescence lifetime imaging microscopy

Abstract

A principal objective in life sciences is the visualization of biochemical processes. Fluorescence-based techniques are widely used to demonstrate transport of relevant substances across cellular membranes. In this paper we report a novel noninvasive, real-time fluorescence lifetime imaging microscopy method for visualizing uptake and release of divalent copper ions (Cu 2+ ) in vivo. For this purpose, we employed a green fluorescent protein (GFP) form able to change its fluorescence lifetime upon Cu 2+ binding. We demonstrate that this technique is selective for Cu 2+ . We show the reversible decrease of the fluorescence lifetime of GFP from 2.2 to 1.6 ns in Escherichia coli and from 1.8 to 1.3 ns in root cells of Arabidopsis after the addition of Cu 2+ . Cu 2+ uptake of epidermal tobacco cells leads to a drop of the GFP lifetime from 2.5 to 2.2 ns. In summary, the spatially resolved visualization of Cu 2+ distribution in vivo is demonstrated in prokaryote and eukaryote cells.

ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Responses

“…Positive clones were used for the verification of proper ZAT12-GFP and ZAT12DEAR-GFP fusion protein expression by transient transformation of N. benthamiana leaf epidermal cells. N. benthamiana infiltration was performed as described previously (Hötzer et al, 2012). After confirmation of the vector constructs and protein expression, transgenic plants were obtained by Agrobacterium tumefaciens-mediated floral dip transformation (Clough and Bent, 1998) of wild-type and zat12-3 plants.…”

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

“…Transformation of Nicotiana benthamiana leaf epidermis cells was performed as described previously (Hötzer et al, 2012).…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…A Cu 2+ sensor based on FRET between GFP as the donor and Cu 2+ has been reported (Hötzer et al, 2011) and employed for mapping Cu 2+ ion uptake and release in plant cells via FLIM (Hötzer et al, 2012). An Oregon Green-labelled apocarbonic anhydrase II Cu 2+ sensor has also recently been reported, where the Oregon Green fluorescence is quenched by FRET upon binding of Cu 2+ to the apocarbonic anhydrase II (McCranor et al, 2014).…”