Integration of Stress-Related and Reactive Oxygen Species-Mediated Signals by Topoisomerase VI in Arabidopsis Thaliana
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Integration of stress-related and reactive oxygen species-mediated signals by Topoisomerase VI in Arabidopsis thaliana Klára Simková a, Fanny Moreaub,c,d,1, Piotr Pawlaka,1,2, Cécile Vrietb,c,d,1, Aiswarya Baruaha,e, Cristina Alexandrea, Lars Henniga,f, Klaus Apela,e,3, and Christophe Laloia,b,c,d,3 aDepartment of Biology, Zurich-Basel Plant Science Center, Eidgenössiche Technische Hochschule Zurich, CH-8092 Zurich, Switzerland; bLaboratoire de Génétique et Biophysique des Plantes, Aix-Marseille University, Marseille F-13009, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche Biologie Végétale et de Microbiologie Environmentale, Marseille F-13009, France; dCommissariat à l’Energie Atomique, Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Marseille F-13009, France; eBoyce Thompson Institute for Plant Research, Ithaca, NY 14853; and fDepartment of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, SE-75007 Uppsala, Sweden Edited by Frank Van Breusegem, Vlaams Instituut voor Biotechnologie-Universiteit Gent, Ghent, Belgium, and accepted by the Editorial Board August 20, 2012 (received for review February 6, 2012) 1 Environmental stress often leads to an increased production of Other consequences of increased O2 generation inside plastids reactive oxygen species that are involved in plastid-to-nucleus include a drastic reduction in the growth rate of mature plants and 1 1 retrograde signaling. Soon after the release of singlet oxygen ( O2) the bleaching and death of seedlings (10). All these O2-mediated in chloroplasts of the flu mutant of Arabidopsis, reprogramming of responses are genetically regulated by the two plastid proteins, nuclear gene expression reveals a rapid transfer of signals from the EXECUTER1 and EXEXUTER2, required for the translocation 1 plastid to the nucleus. We have identified extraplastidic signaling of O2-derived signals from the plastid to the nucleus (12, 13). 1 1 O signaling does not seem to operate via an isolated signaling constituents involved in O2-initiated plastid-to-nucleus signaling 2 and nuclear gene activation after mutagenizing a flu line express- pathway but rather as part of a complex signaling network that ing the luciferase reporter gene under the control of the promoter integrates various extra- and intracellular cues (14). We pre- of a 1O -responsive AAA-ATPase gene (At3g28580) and isolating viously used a genetic approach to penetrate this complexity. A 2 fl 1 second-site mutations that lead to a constitutive up-regulation of transgenic u line expressing an O2-responsive reporter gene was 1 mutagenized, and we isolated second-site mutations that either the reporter gene or abrogate its O2-dependent up-regulation. One of these mutants, caa39, turned out to be a weak mutant allele led to a constitutive up-regulation of the reporter gene or abro- gated its 1O -dependent up-regulation (14). The reporter gene of the Topoisomerase VI (Topo VI) A-subunit gene with a single 2 consisted of the luciferase ORF and the promoter of the 1O - amino acid substitution. Transcript profile analysis of flu and flu 2 responsive AAA-ATPase nuclear gene of Arabidopsis (8, 10). Here caa39 mutants revealed that Topo VI is necessary for the full acti- fi 1 we report on the identi cation and characterization of mutant vation of AAA-ATPase and a set of O2-responsive transcripts in 1 1 caa39, whose response to O2 is impaired. We found that caa39 is response to O2. Topo VI binds to the promoter of the AAA-ATPase 1 a weak mutant allele of the Topoisomerase VI (Topo VI) A- and other O2-responsive genes, and hence could directly regulate subunit (AtTOP6A) gene with a single amino acid substitution. their expression. Under photoinhibitory stress conditions, which 1 1 Under photoinhibitory stress conditions, AtTOP6A is indispens- enhance the production of O2 and H2O2, Topo VI regulates O2- 1 able for the selective activation of several O2-responsive nuclear responsive and H O -responsive genes in a distinct manner. These 2 2 genes and at the same time may act as a repressor of H2O2-re- results suggest that Topo VI acts as an integrator of multiple signals sponsive genes. This dual activity assigns a key role to Topo VI as generated by reactive oxygen species formed in plants under an integrator of multiple ROS signals that are released by plants adverse environmental conditions. in response to adverse environmental conditions. oxidative stress | light stress | cell death Results Isolation and Characterization of the caa39 Mutant. The Arabidopsis + lants are often exposed to environmental changes that ad- flu AAA:LUC line, which expresses the luciferase (LUC) re- 1 Pversely affect their growth and development and may ulti- porter gene under the control of the O2-responsive AAA- mately result in the death of the plant. Most of these stress ATPase promoter (14), was mutagenized and screened for conditions disrupt the metabolic balance of cells and increase the second-site mutants that either constitutively up-regulate the 1 production of reactive oxygen species (ROS) (1). ROS may be O2-responsive reporter gene or have lost the ability to respond toxic and cause oxidative damage, or they may act as signaling molecules and activate the plant’s defenses against environ- mental stress (2, 3). The specificity of these responses is largely Author contributions: K.S., K.A., and C.L. designed research; K.S., F.M., P.P., C.V., A.B., C.A., determined by the chemical identity of the ROS (4, 5). Research and C.L. performed research; K.S., F.M., P.P., C.V., L.H., K.A., and C.L. analyzed data; and K.S., in the past has primarily been concerned with studying the bi- L.H., K.A., and C.L. wrote the paper. F.M., P.P. and C.V. contributed equally to this work. •– fl ological activities of superoxide anion (O2 ) and hydrogen per- The authors declare no con ict of interest. 1 oxide (H O ) (6, 7), whereas the analysis of singlet oxygen ( O ) This article is a PNAS Direct Submission. F.V.B. is a guest editor invited by the 2 2 • 2 and hydroxyl radical ( OH) has been impeded by the lack of Editorial Board. suitable experimental systems and detection techniques. Only Data deposition: The microarray data have been deposited in ArrayExpress, www.ebi.ac. recently, by using the conditional flu mutant of Arabidopsis uk/arrayexpress, (ID no. E-TABM-1076). 1 1 thaliana, has O2 been shown to be involved in plastid-to-nucleus F.M., P.P., and C.V. contributed equally to this work. 1 retrograde signaling. Immediately after the onset of O2 genera- 2Present address: Department of Genetics and Animal Breeding, Poznan University of Life tion in plastids of Arabidopsis, changes in nuclear gene expression Sciences, 60-637 Poznan, Poland. 1 reveal a rapid transfer of O2-derived signals from the plastid to 3To whom correspondence may be addressed. E-mail: [email protected] or christophe. 1 the nucleus (8–10). Several O -responsive genes are different from [email protected]. •− 2 •− those activated by O2 or H2O2, suggesting that O2 /H2O2- and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1 O2-dependent signaling occurs via distinct pathways (8, 10, 11). 1073/pnas.1202041109/-/DCSupplemental. 16360–16365 | PNAS | October 2, 2012 | vol. 109 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1202041109 Downloaded by guest on September 27, 2021 1 + to O2. Six flu AAA:LUC caa mutants were isolated that in 10-d- AAA-ATPase and BAP1, whereas the H2O2-responsive marker old seedlings constitutively activate both the reporter gene and gene FER1 was not or barely affected) despite the fact that caa39 the endogenous AAA-ATPase gene, and hence carry trans-acting growth phenotype is much less severe than in the AtTOP6A knock- mutations (14). In caa mutants with mutations in genes that out mutants or the other Topo VI mutants (Fig. 2). In contrast, 1 genetically form part of the O2-signaling pathway and that also impairment of the SPO11 meiotic recombination endonuclease 1 contain the flu mutation, generation of O2 by a dark-to-light (D/ function in spo11-1-3 and spo11-2 mutants resulted in very dif- L) shift should not further enhance the constitutive expression of ferent expression profiles (Fig. 2). These results support the no- 1 1 O2-responsive genes. Based on this criterion, only one of the six tion that AtTOP6A modulates O2-responsive gene expression 1 caa mutants, caa39, could be directly linked to O2 signaling (14– while being part of the Topo VI complex. 16). Until 5 d old, caa39 mutant seedlings grown under contin- 1 uous light were phenotypically similar to wild-type seedlings (Fig. AtTOP6A Regulates O2-Triggered Stress Responses. To confirm that 1 1A). Afterward they developed chlorotic spots and spontaneous AtTOP6A represents a genuine O2 signaling component, we 1 cell death, and their growth slowed relative to wild-type (Fig. 1 A further analyzed the flu caa39 mutant under O2-producing and E). conditions. As cotyledons of 10-d-old flu seedlings grown under continuous light are no longer able to accumulate significant CAA39 Encodes the A-Subunit of Arabidopsis Topo VI. The visible amounts of the photosensitizer protochlorophyllide (Pchlide) in morphological alterations and constitutive expression of the the dark and, hence, do not release 1O during reillumination, + 2 AAA:LUC reporter gene cosegregated as a single recessive this analysis was performed in 5-d-old seedlings. The expression + Mendelian trait when crossed with the parental flu AAA:LUC of LUC and the endogenous AAA-ATPase was only moderately line (14).