Biology subjects

Grossman, A. R.

Publications and source records attributed to Grossman, A. R..

16 recordsLinked to original sources

bioRxiv2026

Xylulose 5-Phosphate/Phosphate Translocator Mediates Carbon Allocation for Anabolic Metabolism in the Chlamydomonas Chloroplast

Metabolite movement into chloroplasts is essential for sustaining chloroplast anabolic metabolism and cellular growth, and yet how the specific factors/transporters that enable import and support metabolism under heterotrophic conditions (in the dark in the presence of fixed carbon) remain poorly understood. Here, we identify CreTPT10, a chloroplast envelope-localized transporter in the unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas throughout) that, of the substrates tested, has the highest specificity for xylulose 5-phosphate (X5P). We also demonstrated that the loss of CreTPT10 caused a pronounced reduction in growth and respiratory activity in the dark, whereas no growth defects were observed in the tpt10 mutants when they were maintained in the light or under nutrient-limiting conditions. Furthermore, dark-grown tpt10 mutants exhibited markedly reduced levels of lipids, nucleotides, isoprenoids, and aromatic secondary metabolites, accompanied by coordinated repression of genes encoding enzymes associated with chloroplast-localized biosynthetic pathways. This metabolic suppression extended beyond the chloroplast, as genes associated with the mitochondrial respiratory chain and cell cycle progression were markedly downregulated in darkness. Together, these findings indicate that X5P import via CreTPT10 is critical for sustaining chloroplast anabolic metabolism and functionally coordinates chloroplast and mitochondrial energy metabolism to support heterotrophic growth.

plant biology↗

bioRxiv2026

Ovothiol A mediates singlet oxygen resistance and acclimation in Chlamydomonas

Light is essential for photosynthetic organisms, but excess light can generate toxic levels of reactive oxygen species (ROS). To neutralize these ROS, plants and algae produce a variety of antioxidants like carotenoids, tocopherols, and glutathione. However, the role of alternative ROS scavengers, such as ovothiols, has not been studied in the context of oxidative stress in photosynthetic organisms. Here, we report that many algal groups have the potential for the biosynthesis of ovothiols, a group of thiohistidines. We discovered that the model green microalga Chlamydomonas reinhardtii produces millimolar concentrations of ovothiol A, whose biosynthesis is mediated by the ovothiol synthase OVOA1. Using CRISPR-generated ovoa1 knockout mutants, we found that ovothiol production is essential for resistance and acclimation to singlet oxygen, a prominent ROS in photosynthetic organisms. Finally, we demonstrated that OVOA1 expression is activated by singlet oxygen and light signaling pathways in which we identified the major regulatory factors. Overall, our results show that ovothiol A is a major, previously overlooked antioxidant in Chlamydomonas. This work broadens our understanding of cellular mechanisms that combat the damaging effects of oxidative stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/702910v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cddb9corg.highwire.dtl.DTLVardef@10d0a43org.highwire.dtl.DTLVardef@11cc087org.highwire.dtl.DTLVardef@a40cc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Mitotic entry is controlled by the plant-specific phosphatase BSL1 and cyclin-dependent kinase B

Cell cycle regulation is well understood in opisthokonts (fungi and metazoans) but not in plants and Apicomplexa, as some cell cycle regulators are not conserved1-3. In opisthokonts, cell cycle progression requires dephosphorylation of cyclin-dependent kinase (CDK) by the CDC25 phosphatase4. Plants have no CDC25, and thus their mechanisms of cell cycle regulation remain elusive1,5,6. Here, we show that the BSL1 phosphatase dephosphorylates CDKB1 to promote mitotic entry in Chlamydomonas. Mutations of BSL1 or CDKB1 block mitotic entry after DNA replication. BSL1 shows dynamic localization through the cell cycle at the basal bodies, spindle poles, and cleavage furrow. CDKB1 is hyperphosphorylated at T14 and Y15 residues in the bsl1 mutant and in wild-type cells treated with DNA replication inhibitors. BSL1 binds to CDKB1 and dephosphorylates CDKB1 pT14/pY15 in vitro. Phospho-mimicking mutations of T14/Y15 inactivate CDKB1 function, whereas phospho-blocking mutations cause sensitivity to DNA replication inhibitors, which delay cytokinesis in wild-type cells more than cells expressing unphosphorylatable mutant CDKB1. These results indicate that CDKB1 T14/Y15 is phosphorylated to block mitotic entry before DNA replication is complete, and BSL1 dephosphorylates CDKB1 to promote mitosis. Our study demonstrates that BSL1, a phosphatase conserved in plants and Apicomplexa but absent in fungi and animals, is a CDKB1-activating mitosis-promoting factor that has evolved additional signaling functions in receptor kinase pathways in higher plants. One-Sentence SummaryBSL1 is a mitosis-promoting phosphatase that activates CDKB1 in plants.

plant biology↗

bioRxiv2024

Octadecanoids as emerging lipid mediators in cnidarian-dinoflagellate symbiosis

Oxylipin signaling has been suggested as a potential mechanism for the inter-partner recognition and homeostasis regulation of cnidarian-dinoflagellate symbiosis, which maintains the ecological viability of coral reefs. Here we assessed the effects of symbiosis and symbiont identity on a model cnidarian, the sea anemone Exaiptasia diaphana, using mass spectrometry to quantify octadecanoid oxylipins (i.e., 18-carbon-derived oxygenated fatty acids). A total of 84 octadecanoids were reported, and distinct stereospecificity was observed for the synthesis of R- and S-enantiomers for symbiont-free anemones and free-living cultured dinoflagellate symbionts, respectively. Symbiont-derived 13(S)-hydroxy-octadecatetraenoic acid (13(S)- HOTE) linked to a 13S-lipoxygnase was translocated to the host anemone with a 32-fold increase, suggesting it as a biomarker of symbiosis and as a potential agonist of host receptors that regulate inflammatory transcription. Only symbiosis with the native symbiont Breviolum minutum decreased the abundance of pro-inflammatory 9(R)-hydroxy-octadecadienoic acid (9(R)-HODE) in the host. In contrast, symbiosis with the non-native symbiont Durusdinium trenchii was marked by higher abundance of autoxidation-derived octadecanoids, corroborating previous evidence for cellular stress in this association. The putative octadecanoid signaling pathways reported here suggest foundational knowledge gaps that can support the bioengineering and selective breeding of more optimal host-symbiont pairings to enhance resilience and survival of coral reefs.

biochemistry↗

bioRxiv2024

Abundant and active community members respond to the diel cycle in hot spring phototrophic mats

Photosynthetic microbial mats in hot springs can provide insights into the diel behaviors of communities in extreme environments. In this habitat, photosynthesis dominates during the day, leading to super-oxic conditions, with a rapid transition to fermentation and anoxia at night. Multiple samples were collected from two springs over several years to generate metagenomic and metatranscriptomic datasets. Metagenome assembled genomes (MAGs) comprised 71 taxa (in 19 different phyla), of which twelve core taxa were present at high abundance in both springs. The eight most active taxa identified by metatranscriptomics were an oxygenic cyanobacterium (Synechococcus sp.), five anoxygenic phototrophs from three different phyla, and two understudied heterotrophs from phylum Armatimonadota. Surprisingly, in all eight taxa, a significant fraction of genes exhibited a diel expression pattern although peak timing varied considerably. The two abundant heterotrophs exhibit starkly different peak timing of expression, which we propose is shaped by their metabolic and genomic potential to use carbon sources that become differentially available during the diel cycle. Network analysis revealed pathway expression patterns that had not previously been linked to diel cycles, including ribosome biogenesis and chaperones. This provides a framework for analyzing metabolically coupled communities and the dominant role of the diel cycle.

microbiology↗

bioRxiv2024

Illuminating microbial mat assembly: Cyanobacteria and Chloroflexota cooperate to structure light-responsive biofilms

Microbial mats are stratified communities often dominated by unicellular and filamentous phototrophs within an exopolymer matrix. It is challenging to quantify the dynamic responses of community members in situ as they experience steep gradients and rapid fluctuations of light. To address this, we developed a binary consortium using two representative isolates from hot spring mats: the unicellular oxygenic phototrophic cyanobacterium Synechococcus OS-B (Syn OS-B) and the filamentous anoxygenic phototroph Chloroflexus MS-CIW-1 (Chfl MS-1). We quantified the motility of individual cells and entire colonies and demonstrated that Chfl MS-1 formed bundles of filaments that moved in all directions with no directional bias to light. Syn OS- B was slightly less motile but exhibited positive phototaxis. This binary consortium displayed cooperative behavior by moving further than either species alone and formed ordered arrays where both species aligned with the light source. No cooperative motility was observed when a non-motile pilB mutant of Syn OS-B was used instead of Syn OS-B. The binary consortium also produced more adherent biofilm than individual species, consistent with the close interspecies association revealed by electron microscopy. We propose that cyanobacteria and Chloroflexota cooperate in forming natural microbial mats, by colonizing new niches and building robust biofilms. SignificanceMicrobial mats are dense, layered communities with ancient origins and widespread occurrence, but how they assemble is not well understood. To investigate how microbial motility, physical interactions, and responses to light affect mat assembly, we developed a binary consortium from representative hot spring mat isolates. Individually, the Cyanobacteria and Chloroflexota isolates displayed significant differences in motility and biofilm formation. When combined, the consortium exhibited enhanced motility towards light and formed more robust biofilms. This model consortium approach complements in situ studies by directly testing the role of motility and physical cooperation in shaping microbial mats, and could inform biofilm applications in industrial settings.

microbiology↗

bioRxiv2024

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for [~]50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cells apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cells affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

plant biology↗

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↗

bioRxiv2023

Genome-wide distribution of 5-hydroxymethyluracil and chromatin accessibility in the Breviolum minutum genome

In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties were originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools. In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.

genomics↗

bioRxiv2022

Reversible restriction of electron flow across cytochrome b6f in dark acclimated cells limited for downstream electron sinks

Photosynthetic organisms frequently experience abiotic stresses that restrict their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers, photosystems I and II (PSI and PSII), resulting in a decline in primary productivity. This work describes a biological switch in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f complex when reductant and ATP generated by PET are in excess of the capacity of carbon metabolism to utilize these products; we specifically show a restriction at this switch when sta6 mutant cells, which cannot synthesize starch, are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction, which may be a form of photosynthetic control, causes diminished electron flow to PSI, which prevents PSI photodamage. When electron flow is blocked the plastid alternative oxidase (PTOX) may also become activated, functioning as an electron valve that dissipates some of the excitation energy absorbed by PSII thereby lessening PSII photoinhibition. Furthermore, illumination of the cells following the dark acclimation gradually diminishes the restriction at cytochrome b6f complex. Elucidating this photoprotective mechanism and its modulating factors may offer new insights into mechanisms associated with photosynthetic control and offer new directions for optimizing photosynthesis.

plant biology↗

bioRxiv2022

Chlamydomonas reinhardtii triose-phosphate/phosphate translocator3 (TPT3): A major chloroplast shunt for the export of fixed carbon and reductant

Modulation of export of photoassimilates from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study we identified chloroplast triose phosphate/phosphate translocators 2 and 3 (CreTPT2 and CreTPT3) in the green alga Chlamydomonas reinhardtii that exhibited similar substrate specificities but were differentially expressed over the diel cycle. We focused mostly on analyzing CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to the tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that impacted growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilate. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing the cells from experiencing oxidative stress and accumulating of reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the CreTPT transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

plant biology↗

bioRxiv2021

Photoprotection is regulated by light-independent CO2 availability

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

plant biology↗

bioRxiv2021

The chromatin organization of a chlorarachniophyte nucleomorph genome

Nucleomoprhs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features. In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions, and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain.

genomics↗

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↗

bioRxiv2020

Transcription-dependent domain-scale 3D genome organization in dinoflagellates

Dinoflagellate chromosomes represent a unique evolutionary experiment, as they exist in a permanently condensed, liquid crystalline state, are not packaged by histones, and contain genes organized into polycistronic arrays, with minimal transcriptional regulation. We analyze the 3D genome of Breviolum minutum, and find large topological domains without chromatin loops, demarcated by convergent gene array boundaries ("dinoTADs). Transcriptional inhibition degrades dinoTADs, implicating transcription-induced supercoiling as the primary topological force in dinoflagellates.

genomics↗

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↗