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Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling. The Arabidopsis thaliana EXECUTER1 (EX1) protein, a chloroplast-localized singlet oxygen (1O 2) sensor, undergoes tryptophan (Trp) 643 oxidation by 1O 2 , a chloroplast-derived and light-dependent reactive oxygen species. The indole side chain of Trp is vulnerable to 1O 2 , leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease. The perception of 1O 2 via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling. In this study, we discovered that the EX1-like protein EX2 also undergoes 1O 2 -dependent Trp530 oxidation and FtsH-dependent turnover, which attenuates 1O 2 signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation. Consistent with this finding, the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis, whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss–of- function mutants of EX2. Intriguingly, phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward 1O 2. Collectively, these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own 1O 2 -dependent oxidation, providing a new mechanistic insight into the regulation of EX1-mediated 1O 2 signaling. EXECUTER1 (EX1) and EX2 proteins have long been considered major players in mediating singlet oxygen (1O 2)-triggered chloroplast-to-nucleus retrograde signaling, but the specific mechanisms by which they regulate 1O 2 signaling have been unclear. This study demonstrates that the 1O 2 -driven oxidative modification of EX2 modulates EX1-mediated 1O 2 signaling by hindering the oxidation of EX1 that is essential for the initiation of 1O 2 signaling. [ABSTRACT FROM AUTHOR]