Biology subjects

Gallaher, S. D.

Publications and source records attributed to Gallaher, S. D..

13 recordsLinked to original sources

bioRxiv2026

Two genomes in one mitochondrion: mitochondrial heteroplasmy and mitogenome dynamics in Chlamydomonas reinhardtii

The mitochondrial genome of the model green alga Chlamydomonas reinhardtii has long been described as a 15.8-kb linear molecule capped by inverted telomeres. By leveraging high-accuracy long-read sequencing to overcome limitations associated with conventional genome algorithm biases, we demonstrated that this classical paradigm was incomplete. Our analyses provided no evidence for canonical telomeric structures, instead revealed widespread structural heteroplasmy characterized by the coexistence of two distinct mitochondrial genome configurations. In addition to the historical 15.8-kb configuration (designated as config 2), we identified an expanded 19-kb configuration (config 1) containing 4,001-bp terminal inverted repeats that duplicate the cob and nad4 genes. Single-molecule long-read profiling captured these configurations as a dynamic network of multimeric mitochondrial DNA concatemers bridged with a conserved 17-bp homologous motif. Together, these structural features support a recombination-dependent replication (RDR) model that could enable mitochondrial DNA replication in the absence of conventional telomeric structures and reverse transcriptase-like (rtl) genes. We further identify widespread nuclear mitochondrial DNA segments (NUMTs), which complicate targeted amplification and quantification of these mitogenome configurations. Specifying NUMTs enabled configuration-specific digital PCR measurements across the CC-5325 lineage and additional field-isolated Chlamydomonas strains, revealing substantial variation in the relative abundance of the two mitogenome configurations. These findings revise a four-decade-old model of the C. reinhardtii mitogenome and provided a new framework for investigating mitochondrial genome architecture, structural heteroplasmy, and replication dynamics in photosynthetic eukaryotes.

plant biology↗

bioRxiv2025

Light on its feet: Acclimation to high and low diurnal light is flexible in Chlamydomonas reinhardtii

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods (Dupuis & Ojeda et al. 2025). Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 hours of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.

plant biology↗

bioRxiv2025

Mono-mix strategy enables comparative proteomics of a cross-kingdom microbial symbiosis

Cross-kingdom microbial symbioses, such as those between algae and bacteria, are key players in biogeochemical cycles. The molecular changes during initiation and establishment of symbiosis are of great interest, but quantitatively monitoring such changes can be challenging, particularly when the microorganisms differ greatly in size or are intimately associated. Here, we analyze output from data-dependent acquisition (DDA) LC-MS/MS proteomics experiments investigating the well-studied interaction between the alga Chlamydomonas reinhardtii and the heterotrophic bacterium Mesorhizobium japonicum. We found that detection of bacterial proteins decreased in coculture by 50% proteome-wide due to the abundance of algal proteins. As a result, standard differential expression analysis led to numerous false-positive reports of significantly downregulated proteins, where it was not possible to distinguish meaningful biological responses to symbiosis from artifacts of the reduced protein detection in coculture relative to monoculture. We show that data normalization alone does not eliminate the impact of altered detection on differential expression analysis of the cross-kingdom symbiosis. We assessed two additional strategies to overcome this methodological artifact inherent to DDA proteomics. In the first, we combined algal and bacterial monocultures at a relative abundance that mimicked the coculture, creating a "mono-mix" control to which the coculture could be compared. This approach enabled comparable detection of bacterial proteins in the coculture and the monoculture control. In the second strategy, we enhanced detection of lowly abundant bacterial proteins by using sample fractionation upstream of LC-MS/MS analysis. When these simple approaches were combined, they allowed for meaningful comparisons of nearly 10,000 algal proteins and over 4,000 bacterial proteins in response to symbiosis by DDA. They successfully recovered expected changes in the bacterial proteome in response to algal coculture, including upregulation of sugar-binding proteins and transporters. They also revealed novel proteomic responses to coculture that guide hypotheses about algal-bacterial interactions.

systems biology↗

bioRxiv2025

Allodiploid hybridization, loss-of-heterozygosity and aneuploidy in the green alga Auxenochlorella, an emerging model for discovery research and bioengineering

Auxenochlorella spp. are diploid oleaginous green algae whose streamlined genomes can be readily manipulated by homologous recombination, making them highly amenable to discovery research and bioengineering. Vegetatively diploid organisms experience specific evolutionary phenomena, including allodiploid hybridization, mitotic recombination, loss-of-heterozygosity and aneuploidy; however, studies of these forces have largely focused on yeasts. Here, we present a telomere-to-telomere phased diploid genome assembly of Auxenochlorella UTEX 250-A (haploid length 22 Mb) and introduce a genetic toolkit for site-specific manipulation of the nuclear genome in multiple strains, featuring several selectable markers, inducible promoters, and fluorescent reporters for protein localization. UTEX 250-A is an allodiploid hybrid of Auxenochlorella protothecoides and Auxenochlorella symbiontica, two species differentiated by extensive chromosomal rearrangements. UTEX 250-A haplotypes are a mosaic of each parental species following mitotic recombination, and two chromosomes are trisomic. Loss-of-heterozygosity events are pervasive across Auxenochlorella and can evolve rapidly in the laboratory. High-quality structural annotation yielded [~]7,500 genes per haplotype. Auxenochlorella have experienced gene family loss and reduction, including core photosynthesis genes, and exhibit periodic adenine and cytosine methylation at promoters and gene bodies, respectively. Approximately 10% of genes, especially those involved in DNA repair and sex, overlap antisense long noncoding RNAs, which may participate in a regulatory mechanism. We demonstrate the utility of Auxenochlorella for fundamental research by knockout of a chlorophyll biosynthesis enzyme, and confirm one trisomy by allele-specific transformation. These results demonstrate the generality of several evolutionary forces associated with vegetative diploidy and provide a foundation for use of Auxenochlorella as a reference organism. One-sentence summaryAuxenochlorella, green algae shaped by evolutionary forces acting on vegetative diploids, are amenable to discovery research and bioengineering via efficient site-specific homologous recombination

plant biology↗

bioRxiv2024

Fe starvation induces a second LHCI tetramer to photosystem I in green algae

Iron (Fe) availability limits photosynthesis at a global scale where Fe-rich photosystem (PS) I abundance is drastically reduced in Fe-poor environments. We used single-particle cryo-electron microscopy to reveal a unique Fe starvation-dependent arrangement of light-harvesting chlorophyll (LHC) proteins where Fe starvation-induced TIDI1 is found in an additional tetramer of LHC proteins associated with PSI in Dunaliella tertiolecta and Dunaliella salina. These cosmopolitan green algae are resilient to poor Fe nutrition. TIDI1 is a distinct LHC protein that co- occurs in diverse algae with flavodoxin (an Fe-independent replacement for the Fe-containing ferredoxin). The antenna expansion in eukaryotic algae we describe here is reminiscent of the iron-starvation induced (isiA-encoding) antenna ring in cyanobacteria, which typically co-occurs with isiB, encoding flavodoxin. Our work showcases the convergent strategies that evolved after the Great Oxidation Event to maintain PSI capacity.

plant biology↗

bioRxiv2024

Too dim, too bright, and just right: Systems analysis of the Chlamydomonas diurnal program upon acclimation to light stress and limitation

Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to inhibitory. To gain a comprehensive understanding of how diurnal regulatory circuits interface with sensing and response to various light intensities, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light. Although gene expression and photodamage are dynamic over the diurnal cycle, Chlamydomonas populations acclimated to low and high diurnal light exhibit constitutive phenotypes with respect to photosystem abundance, thylakoid architecture, and non-photochemical quenching that persist through the night. This suggests that cells "remember" or anticipate the daylight environment. The integrated data constitute an excellent resource for understanding gene regulatory mechanisms and photoprotection in eukaryotes under environmentally relevant conditions.

systems biology↗

bioRxiv2024

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae.

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistronic genes, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for polycistronic expression in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed 1) a preference for weak Kozak-like sequences for ORF 1 and 2) an underrepresentation of potential initiation codons before ORF 2, which are suitable conditions for leaky scanning to allow ORF 2 translation. We used mutational analysis in Auxenochlorella protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky ribosome scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames found across eukaryotic genomes and for transgene expression in synthetic biology applications. Significance StatementTextbook dogma states that nucleus-encoded genes are monocistronic, producing transcripts with a single translated open reading frame. However, highly conserved bicistronic loci are pervasive in green algae that are separated by several hundred million years of evolution, speaking to their ancestral origins and functions within the Chlorophyte lineage. A combination of bioinformatic analysis and in vivo gene manipulation supports leaky ribosomal scanning as the primary mechanism for translation of multiple ORFs from bicistronic transcripts. We have successfully tuned synthesis levels of two proteins encoded on one mRNA by modifying the ORF 1 Kozak-like sequence. These findings may have broad applications in synthetic biology.

plant biology↗

bioRxiv2024

Mutation of negative regulatory gene CEHC1 encoding an FBXO3 protein results in normoxic expression of HYDA genes in Chlamydomonas reinhardtii

Oxygen is known to prevent hydrogen production in Chlamydomonas, both by inhibiting the hydrogenase enzyme and by preventing the accumulation of HYDA-encoding transcripts. We developed a screen for mutants showing constitutive accumulation of HYDA1 transcripts in the presence of oxygen. A reporter gene required for ciliary motility, placed under the control of the HYDA1 promoter, conferred motility only in hypoxic conditions. By selecting for mutants able to swim even in the presence of oxygen we obtained strains that express the reporter gene constitutively. One mutant identified a gene encoding an F-box only protein 3 (FBXO3), known to participate in ubiquitylation and proteasomal degradation pathways in other eukaryotes. Transcriptome profiles revealed that the mutation, termed cehc1-1, leads to constitutive expression of HYDA1 and other genes regulated by hypoxia, and of many genes known to be targets of CRR1, a transcription factor in the nutritional copper signaling pathway. CRR1 was required for the constitutive expression of the HYDA1 reporter gene in cehc1-1 mutants. The CRR1 protein, which is normally degraded in Cu-supplemented cells, was stabilized in cehc1-1 cells, supporting the conclusion that CEHC1 acts to facilitate the degradation of CRR1. Our results reveal a novel negative regulator in the CRR1 pathway and possibly other pathways leading to complex metabolic changes associated with response to hypoxia.

plant biology↗

bioRxiv2024

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short- and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.

plant biology↗

bioRxiv2023

Iron rescues glucose-mediated photosynthesis repression during lipid accumulation in the green alga Chromochloris zofingiensis

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (-Fe). Cells grown in -Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.

plant biology↗

bioRxiv2023

Pumping Iron: A Multi-omics Analysis of Two Extremophilic Algae Reveals Mechanisms of Iron Economy

Marine algae are responsible for half of the worlds primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hyper-saline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multi-omics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a novel family of siderophore-iron uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, an LHCA-related protein found also in other chlorophytes. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms. Significance StatementDespite their small size, microalgae play a huge role in CO2 uptake via photosynthesis, and represent an important target for climate crisis mitigation efforts. Most photosynthesis proteins require iron as a co-factor so that insufficient iron often limits algal CO2 sequestration. With this in mind, we examined a genus of microalgae called Dunaliella that is exceptionally well-adapted to low iron environments. We produced complete genomes, transcriptomes, and proteomes for two species of Dunaliella that hail from radically different environments: one from coastal ocean waters and the other from salt flats. We identified dozens of genes and multiple, complementary strategies that both species utilize for iron-uptake and management that explain Dunaliellas remarkable ability to thrive on low iron.

systems biology↗

bioRxiv2022

JGI Plant Gene Atlas: An updateable transcriptome resource to improve structural annotations and functional gene descriptions across the plant kingdom

Gene functional descriptions, which are typically derived from sequence similarity to experimentally validated genes in a handful of model species, offer a crucial line of evidence when searching for candidate genes that underlie trait variation. Plant responses to environmental cues, including gene expression regulatory variation, represent important resources for understanding gene function and crucial targets for plant improvement through gene editing and other biotechnologies. However, even after years of effort and numerous large-scale functional characterization studies, biological roles of large proportions of protein coding genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, a public and updateable data resource consisting of transcript abundance assays from 2,090 samples derived from 604 tissues or conditions across 18 diverse species. We integrated across these diverse conditions and genotypes by analyzing expression profiles, building gene clusters that exhibited tissue/condition specific expression, and testing for transcriptional modulation in response to environmental queues. For example, we discovered extensive phylogenetically constrained and condition-specific expression profiles across many gene families and genes without any functional annotation. Such conserved expression patterns and other tightly co-expressed gene clusters let us assign expression derived functional descriptions to 64,620 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome-next.jgi.doe.gov), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

plant biology↗

bioRxiv2022

The Chlamydomonas Genome Project, version 6: reference assemblies for mating type plus and minus strains reveal extensive structural mutation in the laboratory

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of RECQ3 helicase, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results across laboratories and over time. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference.

genomics↗