Shortly after the release of singlet oxygen (¹O₂) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a ¹O₂-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by ¹O₂ but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether ¹O₂ was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the ¹O₂-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that ¹O₂-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development.