Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS) which oxidize DNA, proteins and lipids. Reactive oxygen species can also act as signals to induce acclimation through chloroplast degradation, cell death and nuclear gene expression. To better understand the mechanisms behind ROS signaling from chloroplasts, we have used the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates the ROS singlet oxygen (1 O 2) leading to chloroplast degradation and eventually cell death. Here we have mapped mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in post-transcriptional gene expression. These mutants exhibited broadly reduced chloroplast gene expression, impaired chloroplast development and reduced chloroplast stress signaling. Levels of 1 O 2 , however, could be uncoupled from chloroplast degradation, suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild-type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. While broad inhibition of plastid transcription with rifampicin was also able to suppress cell death in fc2 mutants, specific reductions in plastid gene expression using other mutations was not always sufficient. Together these results suggest that plastid gene expression, or the expression of specific plastid genes by PPR30 and mTERF0, is a necessary prerequisite for chloroplasts to activate the 1 O 2 signaling pathways to induce chloroplast quality control pathways and/or cell death.

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways, but the mechanisms are largely unknown.Here we characterize one fc2 suppressor mutation and map it to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis.The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling.Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that provide nucleotides for chloroplast function, thereby allowing stress signaling to occur.

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to control chloroplast quality control, cell death, and gene expression. The mechanisms behind these pathways remain largely unknown. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways. Here we mapped one fc2 suppressor mutation to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for cytoplasmic de novo CTP (and dCTP) synthesis. The ctps2 mutation blocks singlet oxygen signals by specifically reducing plastid (not mitochondrial) transcripts and DNA content. These phenotypes are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting that ctps2 blocks signaling by limiting nucleotides for plastid genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed that CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for plastid DNA and chloroplast signaling. Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by CTPS enzymes that may have evolved specialized functions in providing nucleotides to specific subcellular compartments.

Running head: Plastid gene expression and cellular degradationSignificance summary: Reactive oxygen species accumulate in the chloroplast (photosynthetic plastids) and signal for stress acclimation by inducing chloroplast degradation, cell death, and changes in nuclear gene expression. We have identified two chloroplast-localized proteins involved in gene regulation that are required to transmit these signals, suggesting that proper plastid gene expression and chloroplast development is necessary to activate chloroplast controlled cellular degradation and nuclear gene expression pathways. Summary:Chloroplasts constantly experience photo-oxidative stress while performing photosynthesis. This is particularly true under abiotic stresses that lead to the accumulation of reactive oxygen species (ROS). While ROS leads to the oxidation of DNA, proteins, and lipids, it can also act as a signal to induce acclimation through chloroplast degradation, cell death, and nuclear gene expression. Although the mechanisms behind ROS signaling from chloroplasts remain mostly unknown, several genetic systems have been devised in the model plant Arabidopsis to understand their signaling properties. One system uses the plastid ferrochelatase two (fc2) mutant that conditionally accumulates the ROS singlet oxygen ( 1 O2) leading to chloroplast degradation and eventually cell death. Here we have mapped three mutations that suppress chloroplast degradation in the fc2 mutant and demonstrate that they affect two independent loci (PPR30 and mTERF9) encoding chloroplast proteins predicted to be involved in posttranscriptional gene expression. Mutations in either gene were shown to lead to broadly reduced chloroplast gene expression, impaired chloroplast development, and reduced chloroplast stress signaling. In these mutants, however, 1 O2 levels were uncoupled to chloroplast degradation suggesting that PPR30 and mTERF9 are involved in ROS signaling pathways. In the wild type background, ppr30 and mTERF9 mutants were also observed to be less susceptible to cell death induced by excess light stress. Together these results suggest that plastid gene expression (or the expression of specific plastid genes) is a necessary prerequisite for chloroplasts to activate 1 O2 signaling pathways to induce chloroplast degradation and/or cell death.