Biology subjects

Jinkerson, R. E.

Publications and source records attributed to Jinkerson, R. E..

6 recordsLinked to original sources

bioRxiv2024

Biogenesis, engineering and function of membranes in the CO2-fixing pyrenoid

Summary ParagraphApproximately one-third of global CO2 assimilation is performed by the pyrenoid1, a liquid-like organelle found in most algae and some plants2. Specialized membranes are hypothesized to drive CO2 assimilation in the pyrenoid by delivering concentrated CO23,4, but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana. SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO2-limiting conditions, providing evidence for the function of membrane tubules in CO2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

molecular biology↗

bioRxiv2024

Cathepsin X is a conserved cell death protein involved in algal response to environmental stress

Phytoplankton play a crucial role in global primary production and can form vast blooms in aquatic ecosystems. Bloom demise and the rapid turnover of phytoplankton are suggested to involve programmed cell death (PCD) induced by diverse environmental stressors. However, fundamental knowledge of the PCD molecular components in algae and protists in general remains elusive. Previously, we revealed that early oxidation in the chloroplast predicted subsequent cell death or survival in isogenic subpopulations that emerged following H2O2 treatment in the diatom Phaeodactylum tricornutum. Here, we performed transcriptome analysis of sorted sensitive oxidized cells and resilient reduced cells, to discover genes linked to their contrasting fates. By cross-comparison with a large-scale mutant screen in the green alga Chlamydomonas reinhardtii, we identified functionally relevant conserved PCD gene candidates, including the cysteine protease cathepsin X/Z (CPX). CPX mutants in P. tricornutum CPX1 and C. reinhardtii CEP12 both exhibited profound resilience to oxidative stress, supporting a conserved function in algal PCD. P. tricornutum cpx1 mutants, generated using CRISPR-Cas9, also exhibited resilience to the toxic diatom-derived infochemical cyanogen bromide. Phylogenetic and predictive structural analyses show that CPX is highly conserved in eukaryotes, and algae of the green and red lineages exhibit strong structural similarity to human cathepsin CTSZ. CPX is expressed by diverse algae across the oceans and during toxic Pseudo-nitzschia blooms, supporting its ecological importance. Elucidating PCD components in algae sheds light on the evolutionary origin of PCD in unicellular organisms, and on the cellular strategies employed by the population to cope with stressful conditions.

microbiology↗

bioRxiv2023

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis

Oxygen (O2), 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 O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 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 StatementA high-throughput genetic screen was used to identify O2 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 O2, 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.

plant biology↗

bioRxiv2021

Identification of green lineage osmotic stress pathways

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the green alga Chlamydomonas reinhardtii to establish a foundational understanding of evolutionarily conserved osmotic-stress signaling pathways in the green lineage through transcriptomics, phosphoproteomics, and functional genomics approaches. Five genes acting across diverse cellular pathways were found to be essential for osmotic-stress tolerance in Chlamydomonas including cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs of these genes in the multicellular land plant Arabidopsis thaliana have conserved functional roles in stress tolerance and reveal a novel PROFILIN-dependent actin remodeling stage of acclimation that ensures cell survival and tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

plant biology↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2018

A genome-wide algal mutant library reveals a global view of genes required for eukaryotic photosynthesis

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms that complement the use of multicellular plants by enabling higher-throughput studies. Here, we generated a genome-wide, indexed library of mapped insertion mutants for the flagship unicellular alga Chlamydomonas reinhardtii (Chlamydomonas hereafter). The 62,389 mutants in the library, covering 83% of nuclear, protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We leveraged this feature to perform a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase CPL3, showed it is important for accumulation of multiple photosynthetic protein complexes. Strikingly, 21 of the 43 highest-confidence genes are novel, opening new opportunities for advances in our understanding of this biogeochemically fundamental process. This library is the first genome-wide mapped mutant resource in any unicellular photosynthetic organism, and will accelerate the characterization of thousands of genes in algae, plants and animals.

genomics↗