Environmental stress often leads to an increased production of reactive oxygen species that are involved in plastid-to-nucleus retrograde signaling. Soon after the release of singlet oxygen ((1)O(2)) in chloroplasts of the flu mutant of Arabidopsis, reprogramming of nuclear gene expression reveals a rapid transfer of signals from the plastid to the nucleus. We have identified extraplastidic signaling constituents involved in (1)O(2)-initiated plastid-to-nucleus signaling and nuclear gene activation after mutagenizing a flu line expressing the luciferase reporter gene under the control of the promoter of a (1)O(2)-responsive AAA-ATPase gene (At3g28580) and isolating second-site mutations that lead to a constitutive up-regulation of the reporter gene or abrogate its (1)O(2)-dependent up-regulation. One of these mutants, caa39, turned out to be a weak mutant allele of the Topoisomerase VI (Topo VI) A-subunit gene with a single amino acid substitution. Transcript profile analysis of flu and flu caa39 mutants revealed that Topo VI is necessary for the full activation of AAA-ATPase and a set of (1)O(2)-responsive transcripts in response to (1)O(2). Topo VI binds to the promoter of the AAA-ATPase and other (1)O(2)-responsive genes, and hence could directly regulate their expression. Under photoinhibitory stress conditions, which enhance the production of (1)O(2) and H(2)O(2), Topo VI regulates (1)O(2)-responsive and H(2)O(2)-responsive genes in a distinct manner. These results suggest that Topo VI acts as an integrator of multiple signals generated by reactive oxygen species formed in plants under adverse environmental conditions.