Blue‐light dependent reactive oxygen species formation by Arabidopsis cryptochrome may define a novel evolutionarily conserved signaling mechanism

Abstract

SummaryCryptochromes are widespread blue-light absorbing flavoproteins with important signaling roles. In plants they mediate de-etiolation, developmental and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cry1 activation by blue light also results in direct enzymatic conversion of molecular oxygen (O 2 ) to reactive oxygen species (ROS) and hydrogen peroxide (H 2 O 2 ) in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 can play a physiological role in vivo.ROS formation resulting from cry1 expression was measured by fluorescence assay in insect cell cultures and in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death was determined by colorimetric assay.We found that ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death.We conclude that ROS formation by cryptochromes may indeed be of physiological relevance and could represent a novel paradigm for cryptochrome signaling.

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

“…1K and L). Atcry2 nuclear localization has also been confirmed by nuclear 3D analysis as described previously 3 (data not shown). It is therefore evident that cry2 undergoes lightdependent translocation into the nucleus in this heterologous insect cell culture system, somewhat in contrast to what has been reported in plants.…”

“…In our original study 3 and in this accompanying manuscript, we have shown a potentially novel way in which cryptochromes may participate in signaling. Whereas there is convincing evidence that conformational changes induced by primary photochemical events lead to interaction with cry signaling partners, this does not exclude that formation of ROS may also play a role in cryptochrome signaling.…”

“…3 Living insect cells were treated with fluorescent substrate and then either kept in the dark or illuminated with blue light (Fig. 2).…”

“…Methods for AtCry2 expression, immunohistochemical staining, and fluorescence staining for ROS are described in our original paper. 3 Polyclonal antibody used for cry2 detection has been raised to the C-terminal domain as used previously. 6 …”

“…3 We show the accumulation of ROS using a fluorescence-based assay for the detection of H 2 O 2 in cell culture media (Amplex Ultra Red) and by direct visualization of ROS in living cells by immunofluorescence imaging techniques. Of specific interest, ROS were shown to accumulate in the nucleus in Atcry1 expressing cells, and therefore are in potential contact with transcriptional regulators and elements of the transcriptional machinery.…”