SUMMARYThe conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ( 1 O 2 ) in plastids during a darkto-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate 1 O 2 -mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of

Shortly after the release of singlet oxygen ((1)O(2)) 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 (1)O(2)-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 (1)O(2) 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 (1)O(2) was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the (1)O(2)-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 (1)O(2)-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.

Recent findings indicate that perturbations of the mitochondrial electron transport chain (METC) can cause extended longevity in evolutionarily diverse organisms. To uncover the molecular basis of how altered METC increases lifespan in C. elegans, we performed an RNAi screen and revealed that three predicted transcription factors are specifically required for the extended longevity of mitochondrial mutants. In particular, we demonstrated that the nuclear homeobox protein CEH-23 uniquely mediates the longevity but not the slow development, reduced brood size, or resistance to oxidative stress associated with mitochondrial mutations. Furthermore, we showed that ceh-23 expression levels are responsive to altered METC, and enforced overexpression of ceh-23 is sufficient to extend lifespan in wild-type background. Our data point to mitochondria-to-nucleus communications to be key for longevity determination and highlight CEH-23 as a novel longevity factor capable of responding to mitochondrial perturbations. These findings provide a new paradigm for how mitochondria impact aging and age-dependent diseases.

Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the “aging” Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses.

Plastid development in seedlings of Arabidopsis thaliana is affected by the transfer of 1 O2-mediated retrograde signals from the plastid to the nucleus and changes in nuclear gene expression during late embryogenesis. The potential impact of these mechanisms on plastid differentiation is maintained throughout seed dormancy and becomes effective only after seed germination. Inactivation of the 2 nuclear-encoded plastid proteins EXECUTER1 and EXECUTER2 blocks 1 O2-mediated retrograde signaling before the onset of dormancy and impairs normal plastid formation in germinating seeds. This long-term effect of 1 O2 retrograde signaling depends on the recruitment of abscisic acid (ABA) during seedling development. Unexpectedly, ABA acts as a positive regulator of plastid formation in etiolated and light-grown seedlings.

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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.

SUMMARY Retrograde plastid-to-nucleus signaling tightly controls and coordinates nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activities. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens have in common that they target plastid-specific but not extra-plastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the caa33 (constitutive activator of AAA-ATPase) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division thereby perturbing plastid homeostasis. This results in pre-conditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the plant’s capacity to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the plant’s susceptibility to stress and emphasizes the need to consider the possible impact of pre-conditioning on retrograde plastid-to-nucleus signaling.