The chloroplast translocon subunit TOC33 relays singlet oxygen-induced chloroplast-to-nucleus retrograde signaling in Arabidopsis

Abstract

Chloroplasts engaged in oxygenic photosynthesis frequently overproduce reactive oxygen species (ROS) under stress conditions, with singlet oxygen (1O2) being particularly harmful due to its high reactivity and short lifespan. The nuclear-encoded chloroplast protein EXECUTER1 (EX1) senses elevated 1O2 levels through Trp643 oxidation and undergoes proteolysis, a process essential for activating 1O2-induced EX1-mediated chloroplast-to-nucleus retrograde signaling (1O2-EX1 signaling). However, the link between EX1 proteolysis and subsequent nuclear transcriptome changes remains unclear. In this study, we isolated SOF1 (suppressor of flu 1) through a forward genetic screen using EMS-mutagenized flu mutant seeds of Arabidopsis thaliana harboring Flag-fused EX1 driven by its native promoter (referred to as fluEX1). Like flu, fluEX1 plants conditionally produce 1O2 in chloroplasts upon a dark-to-light shift. In the fluEX1 sof1, all 1O2-induced stress responses are largely suppressed, despite 1O2 levels being similar to those in the fluEX1. SOF1 encodes the chloroplast outer envelope-anchored preprotein import receptor TOC33. While TOC33 loss does not affect EX1 import, abundance, localization, and 1O2-induced proteolysis in the chloroplast, it blocks 1O2-induced chloroplast-to-nucleus retrograde signaling. TOC33 interacts with the UVR domain of EX1 (EX1-UVR) in the chloroplast envelope, facilitating 1O2-induced decrease of the chloroplast EX1-UVR and increase of the nucleus EX1-UVR. Moreover, ectopic expression of EX1-UVR outside of the chloroplast bypasses the restrictive barrier imposed by the chloroplast envelope, activating 1O2 signaling and inducing stress responses. Our findings demonstrate that SOF1/TOC33 mediates 1O2-EX1 signaling from the chloroplast to the nucleus and that the EX1-UVR domain can substitute for full-length EX1 in this signaling pathway.