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. 2015;10(8):e1042647.
doi: 10.1080/15592324.2015.1042647.

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role

Nathalie Jourdan  1 , Carlos F Martino, Mohamed El-Esawi, Jacques Witczak, Pierre-Etienne Bouchet, Alain d'Harlingue, Margaret Ahmad
Affiliations

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role

Nathalie Jourdan et al. Plant Signal Behav. 2015.

Abstract

Cryptochromes are blue-light absorbing flavoproteins with many important signaling roles in plants, including in de-etiolation, development, and stress response. They interact with downstream signaling partners such as transcription factors and components of the proteasome, and thereby alter regulation of nuclear gene expression in a light dependent manner. In a prior study, it has also been shown that Arabidopsis cry1 activation by blue light results in direct enzymatic conversion of molecular oxygen (O2) to ROS (reactive oxygen species) in vivo leading to cell death in overexpressing lines. Here we extend these observations to show that Atcry2 is translocated from the cytosol to the nucleus in response to blue light illumination, resulting in nuclear accumulation of ROS in expressing insect cell cultures. These observations suggest that ROS formation may represent a novel means of signaling by Atcry2 distinct from, and perhaps complementary to, the currently known mechanism of light-mediated conformational change.

Keywords: ROS; cryptochrome; light signaling; nuclear localization; oxidative stress; photoreceptor.

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Figures

Figure 1.
Figure 1.
Subcellular localization of Atcry2 in insect cells by immunofluorescence staining and confocal microscopy.Sf21 cells stably expressing Atcry2 were fixed with paraformaldehyde, permeabilized with Triton X100 (A) to (F) or not (G) to (L), incubated with a rabbit polyclonal anti-Atcry2 antibody and an Alexa 488-conjugated anti-rabbit secondary antibody, DNA were stained with Dapi. Cells were observed with an inverted Leica TCS SP5 microscope. Images (A) to (F) show projection of optical sections, scale bar 10 μm. Images (G) to (L) show single confocal z-section that cross the nucleus, scale bar 10 μm.
Figure 2.
Figure 2.
Production and subcellular localization of ROS by Sf21 CRY2 exposed to blue light.Living Sf21 stably expressing CRY2 were exposed to dark or blue light, treated with DCFH-DA [5-(and-6)-chloromethyl-2′,7′-dichlorofluorecein diacetate] and viewed by an inverted Leica TCS SP5 microscope. Images show single confocal z section that cross the nucleus. Intense ROS staining can be seen inside the nucleus (N) whose membrane can be clearly observed by differential interference contrast (D. I. C.). Scale bars 10 μm. Methods for AtCry2 expression, immunohistochemical staining, and fluorescence staining for ROS are described in our original paper. Polyclonal antibody used for cry2 detection has been raised to the C-terminal domain as used previously.
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