Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis

Oxygen (O 2 ), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O 2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O 2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii . This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid. Significance Statement A high-throughput genetic screen was used to identify O 2 sensitive mutants of Chlamydomonas reinhardtii (Chlamydomonas throughout) that experience elevated oxidative stress in the light relative to WT cells. Identification of genes altered in these mutants offers opportunities to discover activities that a ) protect photosynthetic cells from oxidative damage, b ) participate in rapid assembly of photosynthetic complexes, which would limit accessibility of intermediates to O 2 , and/or c ) facilitate repair of damaged cellular complexes. A mutant from this screen disrupted for RMT2 , originally described as encoding a Rubisco methyltransferase, was defective for PSI biogenesis. Additionally, RMT2 appears to be enriched in/around the pyrenoid, a chloroplast localized compartment harboring much of the Chlamydomonas Rubisco, raising the possibility that this compartment plays a role in PSI biogenesis.