Chloroplast quality control pathways are dependent on plastid DNA synthesis and nucleotides provided by cytidine triphosphate synthase two.

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.

Roles for the chloroplast-localized pentatricopeptide repeat protein 30 and the 'mitochondrial' transcription termination factor 9 in chloroplast quality control.

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 O2 ) 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 O2 , 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 O2 signaling pathways to induce chloroplast quality control pathways and/or cell death.