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A suppressor screen uncovers flu as a weak mutant and chloroplastic 1O2 triggers EX1-independent stress responses in Arabidopsis

ABSTRACT: • Singlet oxygen (1O2) is the main ROS produced in chloroplasts, particularly under environmental stress conditions. Studies using the Arabidopsis conditional flu (flu1-1) mutant have shown that chloroplastic 1O2 acts as a signal, activating retrograde signaling that eventually leads to photoinhibition or cell death. • Through a forward genetic screen for suppressors using EMS-mutagenized Arabidopsis flu1-1/ex1 mutants, we isolated two suppressor mutants (mut1 and mut2) that restored 1O2-induced stress responses. Using combined methods of map-based cloning and high-throughput sequencing, we mapped the causal mutations in both mut1 and mut2 to the FLU gene again. • We verified that the widely used flu1-1 is not the long-recognized null mutant, but rather a weak allele, encoding a variant that binds weakly to GluTR1 and leads to partial shutdown of tetrapyrrole biosynthesis in darkness. We further found that the FLU-knockout mutants produce more 1O2, activating transcriptional changes, enzymatic lipid peroxidation, and PCD even in the absence of EX1. Overexpression of SAFE1 suppressed 1O2-induced PCD in mut2 in a dosage-dependent manner. • Collectively, our findings redefine the nature of the widely used flu1-1 mutant and reveal that excessive 1O2 can overwhelm the inhibitory effect of endogenous SAFE1 and induce stress responses independently of EX1.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE292110 | GEO | 2026/05/02

REPOSITORIES: GEO

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Project description:Shortly after the release of singlet oxygen (1O2), 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. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment. Experiment Overall Design: Two individual biologica replicates, each containing material of five mature plants of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, respectively, were used for the microarray analysis. Plants were germinated on soil and kept under continuous light until the beginning of bolting and then transferred to the dark for 8 h. Dark-incubated mature plants were reilluminated for 30 min and subsequently harvested for RNA extraction. Experiment Overall Design: The EX1 (At4g33630) T-DNA insertion line SALK 002088 and EX2 (At1g27510) T-DNA insertion line SALK 012127 were obtained from the European Arabidopsis Stock Centre (NASC). Homozygous mutant lines were identified by PCR analysis by using T-DNA-, EX1- and EX2-specific primers. Both T-DNA-lines were crossed with a flu Col-0 line that had been obtained by 5 backcrosses of flu1-1 in Landsberg erecta with wild-type Columbia. The ex1/flu and ex2/flu mutant lines were crossed, and within the segregating F2 population triple mutants were identified by PCR-based genotyping. For the cultivation of mature plants, seeds of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, all in Col-0 ecotype, were sown on soil and plants were grown under continuous light (100 mol m 2 s 1).

Project description:We used the flu mutant of Arabidopsis and a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX) to address the interactions between different reactive oxygen species (ROS) signaling pathways. The conditional flu mutant of Arabidopsis accumulates excess protochlorophyllide in the dark within chloroplast membranes that upon illumination acts as a photosensitizer and generates singlet oxygen (1O2). Immediately after the release of singlet oxygen rapid changes in nuclear gene expression occur. Distinct sets of genes were activated that were different from those induced by other reactive oxygen species, superoxide or hydrogen peroxide (H2O2), suggesting that different types of active oxygen species activate distinct signaling pathways. It was not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX, line 14/2 PMID: 15165186). In the flu mutant overexpressing tAPX, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions. Experiment Overall Design: Arabidopsis thaliana rosette leaves were harvested after 2 h of reillumination following a 8h dark period for RNA extraction and hybridization on Affymetrix ATH1 microarrays. The entire experiment was performed six times, providing independent biological replicates. For each of the six experiments, all four lines, wild-type, thylakoidal ascorbate peroxidase overexpressor (over tAPX, line 14/2), flu mutant and flu plants overexpressing thylakoidal ascorbate peroxidase were grown for 3 weeks under continuous light at 90 mmol. m-2 . s-1, transferred to the dark for 8 h, and reilluminated for 120 min before the rosette leaves of at least 10 plants per line were harvested. Total RNAs from three separate biological experiments were pooled (= 1 biological rep.) for the preparation of cDNA and the subsequent synthesis of biotin-labeled complementary RNA as recommended by Affymetrix.

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A suppressor screen uncovers flu as a weak mutant and chloroplastic 1O2 triggers EX1-independent stress responses in Arabidopsis

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