Identification and analysis of genes involved in tetrapyrrole metabolism and plastid-derived ROS-mediated signaling in Chlamydomonas reinhardtii
Final Report Abstract
Despite many decades of intensive research on the control of tetrapyrrole biosynthesis and TBS-mediated retrograde signaling in plants and green algae, many unknowns exist about the nature of contributing factors and the mode of action of these two pathways. In principle, the transcriptional and posttranslational regulation for the pathway of TBS in light-exposed organisms ensures low steady-state levels of photoreactive TBS intermediates. Any accumulation of these tetrapyrrole intermediates would induce a plastid-derived signaling pathway, which is generated by singlet oxygen (1O2). The aim of the project application is to identify new genes for components and regulatory factors in the model organism Chlamydomonas reinhardtii and to characterize their function in the TBS pathway and in plastid-derived retrograde signaling, which is triggered by 1O2 generated by protoporphyrin IX accumulation. Apart from an old mutant collection originally generated by Profs Christoph Beck and Kris Niyogi, as a new resource for mutant screens, two different new collections of insertional mutants were independently generated in the course of this ongoing project: mutants (i) screened for pale green, but mainly Chl-free mutant phenotype during growth in darkness, and (ii) produced in a protoporphyrin IX-accumulating and chlorophyll deficient strain background, which showed a defect in the 1O2 signaling pathway. The report describes the outcome of the work plan, which includes the mutant-specific experimental approaches for the analysis of the mutants which are affected either in retrograde signaling or the TBS pathway. Methods of genetics, biochemistry, physiology, and molecular biology were used to unravel the complexity of the genome structure of the analysed mutants or to elucidate the complexity of intracellular signaling and metabolic networks, which interfere with the 1O2-induced retrograde signaling pathways. Transcriptome data were analyzed to correlate data from metabolomics approaches to determine the effects on gene expression and metabolite levels for the respective mutants. While the analysis of the insertional mutant bleR2809-279 accumulating MgProtoME in darkness was hampered by the 4-month lockdown period at the Humboldt-Universität zu Berlin, the evaluation of the sequencing approach of the most promising yellow-in-the-dark bleR1208-32 mutant did not reveal an obvious and immediately eye-catching mutant gene, responsible for the mutant phenotype. The screen for the 1O2 signaling mutants revealed several promising candidates, among them, the identified mutated genes, which code for TREHALOSE 6-PHOSPHATE PHOSPHATASE (TPS2) or RIBOSOMAL PROTEIN S6-KINASE (S6K). Both genes and their gene products connect 1O2 signaling pathways to either the sugar signaling pathway or to translational response and other signaling cascades. The analyses of these two mutants indicated a complex network of signal components that indicate a cross-talk of the 1O2 signaling other signal pathways and metabolic processes Last but not least, characterization of the available non-identified CF mutants, which were initially obtained by UV-mutagenesis, revealed several allelic mutants of genes for enzymes in the Mg porphyrin pathway of TBS. The detection of a series of green revertants of the CF215 mutant and its derivative strains was more striking. CF215 was initially characterized to contain a putative mutation in the CHLD gene. However, resequencing of the original mutant and their brown and green derivatives revealed a novel mutant gene encoding the cytochrome P450 CYP97, which was suggested to be responsible for the green revertant phenotype and modified distribution of protoporphyrin for heme and chlorophyll synthesis.
Publications
- The function of PROTOPORPHYRINOGEN IX OXIDASE in Chl biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii. Communications Biology 2:159 (2019)
Brzezowski P, Ksas B, Havaux M, Grimm B, Chazaux M, Peltier G, Johnson X, Alric J
(See online at https://doi.org/10.1038/s42003-019-0395-5) - (2021).Trehalose-6-phosphate links singlet oxygen-induced signalling with metabolic signalling in Chlamydomonas reinhardtii
Al Youssef, W., Feil, R., Saint-Sorny, M., Johnson, X., Lunn, J.E., Grimm, B., and Brzezowski, P.
(See online at https://doi.org/10.1101/2021.11.03.467076)
