Publications by authors named "Sabeeha S Merchant"

113 Publications

Single-cell visualization and quantification of trace metals in lysosome-related organelles.

Proc Natl Acad Sci U S A 2021 Apr;118(16)

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720;

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.
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http://dx.doi.org/10.1073/pnas.2026811118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072209PMC
April 2021

Co-Expression Networks in Chlamydomonas Reveal Significant Rhythmicity in Batch Cultures and Empower Gene Function Discovery.

Plant Cell 2021 Feb 2. Epub 2021 Feb 2.

Department of Chemistry and Biochemistry, University of California - Los Angeles, Los Angeles CA 90095, USA.

The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.
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http://dx.doi.org/10.1093/plcell/koab042DOI Listing
February 2021

Single-cell RNA sequencing of batch Chlamydomonas cultures reveals heterogeneity in their diurnal cycle phase.

Plant Cell 2021 Feb 2. Epub 2021 Feb 2.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA.

The photosynthetic unicellular alga Chlamydomonas (Chlamydomonas reinhardtii) is a versatile reference for algal biology because of its ease of culture in the laboratory. Genomic and systems biology approaches have previously described transcriptome responses to environmental changes using bulk data, thus representing the average behavior from pools of cells. Here, we apply single-cell RNA sequencing (scRNA-seq) to probe the heterogeneity of Chlamydomonas cell populations under three environments and in two genotypes differing by the presence of a cell wall. First, we determined that RNA can be extracted from single algal cells with or without a cell wall, offering the possibility to sample natural algal communities. Second, scRNA-seq successfully separated single cells into nonoverlapping cell clusters according to their growth conditions. Cells exposed to iron or nitrogen deficiency were easily distinguished despite a shared tendency to arrest photosynthesis and cell division to economize resources. Notably, these groups of cells not only recapitulated known patterns observed with bulk RNA-seq but also revealed their inherent heterogeneity. A substantial source of variation between cells originated from their endogenous diurnal phase, although cultures were grown in constant light. We exploited this result to show that circadian iron responses may be conserved from algae to land plants. We document experimentally that bulk RNA-seq data represent an average of typically hidden heterogeneity in the population.
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http://dx.doi.org/10.1093/plcell/koab025DOI Listing
February 2021

Widespread polycistronic gene expression in green algae.

Proc Natl Acad Sci U S A 2021 Feb;118(7)

UCLA DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: and Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between , , and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
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http://dx.doi.org/10.1073/pnas.2017714118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896298PMC
February 2021

An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas.

Nat Commun 2020 12 8;11(1):6269. Epub 2020 Dec 8.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476, Potsdam-Golm, Germany.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.
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http://dx.doi.org/10.1038/s41467-020-19983-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722844PMC
December 2020

From economy to luxury: Copper homeostasis in Chlamydomonas and other algae.

Biochim Biophys Acta Mol Cell Res 2020 11 13;1867(11):118822. Epub 2020 Aug 13.

Department of Biology, Brookhaven National Laboratory, Upton, NY, United States of America.

Plastocyanin and cytochrome c, abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(I). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR/SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem 1 accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the di‑iron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5' extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484467PMC
November 2020

The histone H3-H4 tetramer is a copper reductase enzyme.

Science 2020 07;369(6499):59-64

Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant H3-H4 tetramer is an oxidoreductase enzyme that binds Cu and catalyzes its reduction to Cu in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu ions in eukaryotes.
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http://dx.doi.org/10.1126/science.aba8740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842201PMC
July 2020

An atypical short-chain dehydrogenase-reductase functions in the relaxation of photoprotective qH in Arabidopsis.

Nat Plants 2020 02 13;6(2):154-166. Epub 2020 Feb 13.

Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.

Photosynthetic organisms experience wide fluctuations in light intensity and regulate light harvesting accordingly to prevent damage from excess energy. The antenna quenching component qH is a sustained form of energy dissipation that protects the photosynthetic apparatus under stress conditions. This photoprotective mechanism requires the plastid lipocalin LCNP and is prevented by SUPPRESSOR OF QUENCHING1 (SOQ1) under non-stress conditions. However, the molecular mechanism of qH relaxation has yet to be resolved. Here, we isolated and characterized RELAXATION OF QH1 (ROQH1), an atypical short-chain dehydrogenase-reductase that functions as a qH-relaxation factor in Arabidopsis. The ROQH1 gene belongs to the GreenCut2 inventory specific to photosynthetic organisms, and the ROQH1 protein localizes to the chloroplast stroma lamellae membrane. After a cold and high-light treatment, qH does not relax in roqh1 mutants and qH does not occur in leaves overexpressing ROQH1. When the soq1 and roqh1 mutations are combined, qH can neither be prevented nor relaxed and soq1 roqh1 displays constitutive qH and light-limited growth. We propose that LCNP and ROQH1 perform dosage-dependent, antagonistic functions to protect the photosynthetic apparatus and maintain light-harvesting efficiency in plants.
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http://dx.doi.org/10.1038/s41477-020-0591-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288749PMC
February 2020

Manganese co-localizes with calcium and phosphorus in acidocalcisomes and is mobilized in manganese-deficient conditions.

J Biol Chem 2019 11 16;294(46):17626-17641. Epub 2019 Sep 16.

Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.
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http://dx.doi.org/10.1074/jbc.RA119.009130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873200PMC
November 2019

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata.

BMC Genomics 2019 Jul 23;20(1):605. Epub 2019 Jul 23.

Department of Systematic and Evolutionary Botany, University of Zurich, Zurich, Switzerland.

Background: Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution.

Results: A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting.

Conclusions: The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.
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http://dx.doi.org/10.1186/s12864-019-5629-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652019PMC
July 2019

A Series of Fortunate Events: Introducing Chlamydomonas as a Reference Organism.

Plant Cell 2019 08 12;31(8):1682-1707. Epub 2019 Jun 12.

University of California, Los Angeles, Department of Chemistry and Biochemistry, Los Angeles, CA 90095.

The unicellular alga is a classical reference organism for studying photosynthesis, chloroplast biology, cell cycle control, and cilia structure and function. It is also an emerging model for studying sensory cilia, the production of high-value bioproducts, and in situ structural determination. Much of the early appeal of Chlamydomonas was rooted in its promise as a genetic system, but like other classic model organisms, this rise to prominence predated the discovery of the structure of DNA, whole-genome sequences, and molecular techniques for gene manipulation. The haploid genome of facilitates genetic analyses and offers many of the advantages of microbial systems applied to a photosynthetic organism. has contributed to our understanding of chloroplast-based photosynthesis and cilia biology. Despite pervasive transgene silencing, technological advances have allowed researchers to address outstanding lines of inquiry in algal research. The most thoroughly studied unicellular alga, , is the current standard for algal research, and although genome editing is still far from efficient and routine, it nevertheless serves as a template for other algae. We present a historical retrospective of the rise of to illuminate its past and present. We also present resources for current and future scientists who may wish to expand their studies to the realm of microalgae.
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http://dx.doi.org/10.1105/tpc.18.00952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713297PMC
August 2019

Comparative and Functional Algal Genomics.

Annu Rev Plant Biol 2019 04 1;70:605-638. Epub 2019 Mar 1.

Departments of Plant and Microbial Biology and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

Over 100 whole-genome sequences from algae are published or soon to be published. The rapidly increasing availability of these fundamental resources is changing how we understand one of the most diverse, complex, and understudied groups of photosynthetic eukaryotes. Genome sequences provide a window into the functional potential of individual algae, with phylogenomics and functional genomics as tools for contextualizing and transferring knowledge from reference organisms into less well-characterized systems. Remarkably, over half of the proteins encoded by algal genomes are of unknown function, highlighting the volume of functional capabilities yet to be discovered. In this review, we provide an overview of publicly available algal genomes, their associated protein inventories, and their quality, with a summary of the statuses of protein function understanding and predictions.
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http://dx.doi.org/10.1146/annurev-arplant-050718-095841DOI Listing
April 2019

Regulation of Oxygenic Photosynthesis during Trophic Transitions in the Green Alga .

Plant Cell 2019 03 20;31(3):579-601. Epub 2019 Feb 20.

Howard Hughes Medical Institute, Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102

Light and nutrients are critical regulators of photosynthesis and metabolism in plants and algae. Many algae have the metabolic flexibility to grow photoautotrophically, heterotrophically, or mixotrophically. Here, we describe reversible Glc-dependent repression/activation of oxygenic photosynthesis in the unicellular green alga We observed rapid and reversible changes in photosynthesis, in the photosynthetic apparatus, in thylakoid ultrastructure, and in energy stores including lipids and starch. Following Glc addition in the light, shuts off photosynthesis within days and accumulates large amounts of commercially relevant bioproducts, including triacylglycerols and the high-value nutraceutical ketocarotenoid astaxanthin, while increasing culture biomass. RNA sequencing reveals reversible changes in the transcriptome that form the basis of this metabolic regulation. Functional enrichment analyses show that Glc represses photosynthetic pathways while ketocarotenoid biosynthesis and heterotrophic carbon metabolism are upregulated. Because sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in both plants and animals, we have developed a simple algal model system to investigate conserved eukaryotic sugar responses as well as mechanisms of thylakoid breakdown and biogenesis in chloroplasts. Understanding regulation of photosynthesis and metabolism in algae could enable bioengineering to reroute metabolism toward beneficial bioproducts for energy, food, pharmaceuticals, and human health.
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http://dx.doi.org/10.1105/tpc.18.00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482638PMC
March 2019

Celebrates 30 Years of Publishing the Best Work in Plant Biology .

Plant Cell 2019 01 23;31(1). Epub 2019 Jan 23.

Director of PublicationsAmerican Society of Plant Biologists.

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http://dx.doi.org/10.1105/tpc.19.00040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391700PMC
January 2019

Multiomics resolution of molecular events during a day in the life of Chlamydomonas.

Proc Natl Acad Sci U S A 2019 02 18;116(6):2374-2383. Epub 2019 Jan 18.

Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

The unicellular green alga displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes , , and show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.
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http://dx.doi.org/10.1073/pnas.1815238116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369806PMC
February 2019

A Gelatin Microdroplet Platform for High-Throughput Sorting of Hyperproducing Single-Cell-Derived Microalgal Clones.

Small 2018 11 4;14(44):e1803315. Epub 2018 Oct 4.

Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, 90095, USA.

Microalgae are an attractive feedstock organism for sustainable production of biofuels, chemicals, and biomaterials, but the ability to rationally engineer microalgae to enhance production has been limited. To enable the evolution-based selection of new hyperproducing variants of microalgae, a method is developed that combines phase-transitioning monodisperse gelatin hydrogel droplets with commercial flow cytometric instruments for high-throughput screening and selection of clonal populations of cells with desirable properties, such as high lipid productivity per time traced over multiple cell cycles. It is found that gelatin microgels enable i) the growth and metabolite (e.g., chlorophyll and lipids) production of single microalgal cells within the compartments, ii) infusion of fluorescent reporter molecules into the hydrogel matrices following a sol-gel transition, iii) selection of high-producing clonal populations of cells using flow cytometry, and iv) cell recovery under mild conditions, enabling regrowth after sorting. This user-friendly method is easily integratable into directed cellular evolution pipelines for strain improvement and can be adopted for other applications that require high-throughput processing, e.g., cellular secretion phenotypes and intercellular interactions.
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http://dx.doi.org/10.1002/smll.201803315DOI Listing
November 2018

In situ architecture of the algal nuclear pore complex.

Nat Commun 2018 06 18;9(1):2361. Epub 2018 Jun 18.

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery.
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http://dx.doi.org/10.1038/s41467-018-04739-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006428PMC
June 2018

Using YFP as a Reporter of Gene Expression in the Green Alga Chlamydomonas reinhardtii.

Methods Mol Biol 2018 ;1755:135-148

Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA, USA.

The unicellular green alga Chlamydomonas reinhardtii is a valuable experimental system in plant biology for studying metal homeostasis. Analyzing transcriptional regulation with promoter-fusion constructs in C. reinhardtii is a powerful method for connecting metal-responsive regulation with cis-regulatory elements, but overcoming expression-level variability between transformants and optimizing experimental conditions can be laborious. Here, we provide detailed protocols for the high-throughput cultivation of C. reinhardtii and assaying Venus fluorescence as a reporter for promoter activity. We also describe procedural considerations for relating metal supply to transcriptional activity.
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http://dx.doi.org/10.1007/978-1-4939-7724-6_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448394PMC
January 2019

RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana.

Plant J 2018 04 12;94(1):146-156. Epub 2018 Mar 12.

Biophysics of Photosynthesis, VU University Amsterdam, Amsterdam, The Netherlands.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO ) and ribulose-1,5-bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD-like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.
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http://dx.doi.org/10.1111/tpj.13849DOI Listing
April 2018

Welcomes Assistant Features Editors.

Plant Cell 2018 01 19;30(1):1-2. Epub 2018 Jan 19.

Department of Chemistry and BiochemistryUniversity of California, Los AngelesEditor in Chief, The Plant Cell

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http://dx.doi.org/10.1105/tpc.18.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5810580PMC
January 2018

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.

Plant J 2018 02 7;93(3):545-565. Epub 2018 Jan 7.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.
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http://dx.doi.org/10.1111/tpj.13788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775909PMC
February 2018

Bilin-Dependent Photoacclimation in .

Plant Cell 2017 Nov 30;29(11):2711-2726. Epub 2017 Oct 30.

Department of Molecular and Cellular Biology, University of California, Davis, California 95616

In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant () of the green alga suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXα, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of , genetically complemented , and chemically rescued reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.
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http://dx.doi.org/10.1105/tpc.17.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728120PMC
November 2017

Journal Impact: Brave New World.

Plant Cell 2017 09 25;29(9):2071-2074. Epub 2017 Aug 25.

Department of Chemistry and Biochemistry University of California, Los Angeles Editor in Chief, The Plant Cell

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http://dx.doi.org/10.1105/tpc.17.00680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635977PMC
September 2017

LFO1 Is an IsdG Family Heme Oxygenase.

mSphere 2017 Jul-Aug;2(4). Epub 2017 Aug 16.

Department of Pathology, Microbiology, & Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Heme is essential for respiration across all domains of life. However, heme accumulation can lead to toxicity if cells are unable to either degrade or export heme or its toxic by-products. Under aerobic conditions, heme degradation is performed by heme oxygenases, enzymes which utilize oxygen to cleave the tetrapyrrole ring of heme. The HO-1 family of heme oxygenases has been identified in both bacterial and eukaryotic cells, whereas the IsdG family has thus far been described only in bacteria. We identified a hypothetical protein in the eukaryotic green alga , which encodes a protein containing an antibiotic biosynthesis monooxygenase (ABM) domain consistent with those associated with IsdG family members. This protein, which we have named LFO1, degrades heme, contains similarities in predicted secondary structures to IsdG family members, and retains the functionally conserved catalytic residues found in all IsdG family heme oxygenases. These data establish LFO1 as an IsdG family member and extend our knowledge of the distribution of IsdG family members beyond bacteria. To gain further insight into the distribution of the IsdG family, we used the LFO1 sequence to identify 866 IsdG family members, including representatives from all domains of life. These results indicate that the distribution of IsdG family heme oxygenases is more expansive than previously appreciated, underscoring the broad relevance of this enzyme family. This work establishes a protein in the freshwater alga as an IsdG family heme oxygenase. This protein, LFO1, exhibits predicted secondary structure and catalytic residues conserved in IsdG family members, in addition to a chloroplast localization sequence. Additionally, the catabolite that results from the degradation of heme by LFO1 is distinct from that of other heme degradation products. Using LFO1 as a seed, we performed phylogenetic analysis, revealing that the IsdG family is conserved in all domains of life. Additionally, contains two previously identified HO-1 family heme oxygenases, making the first organism shown to contain two families of heme oxygenases. These data indicate that may have unique mechanisms for regulating iron homeostasis within the chloroplast.
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http://dx.doi.org/10.1128/mSphere.00176-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557675PMC
August 2017

Regulating cellular trace metal economy in algae.

Curr Opin Plant Biol 2017 10 30;39:88-96. Epub 2017 Jun 30.

Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, USA; Institute for Genomics and Proteomics, University of California, Los Angeles, 611 Charles E. Young Drive East, Los Angeles, USA.

As indispensable protein cofactors, Fe, Mn, Cu and Zn are at the center of multifaceted acclimation mechanisms that have evolved to ensure extracellular supply meets intracellular demand. Starting with selective transport at the plasma membrane and ending in protein metalation, metal homeostasis in algae involves regulated trafficking of metal ions across membranes, intracellular compartmentalization by proteins and organelles, and metal-sparing/recycling mechanisms to optimize metal-use efficiency. Overlaid on these processes are additional circuits that respond to the metabolic state as well as to the prior metal status of the cell. In this review, we focus on recent progress made toward understanding the pathways by which the single-celled, green alga Chlamydomonas reinhardtii controls its cellular trace metal economy. We also compare these mechanisms to characterized and putative processes in other algal lineages. Photosynthetic microbes continue to provide insight into cellular regulation and handling of Cu, Fe, Zn and Mn as a function of the nutritional supply and cellular demand for metal cofactors. New experimental tools such as RNA-Seq and subcellular metal imaging are bringing us closer to a molecular understanding of acclimation to supply dynamics in algae and beyond.
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http://dx.doi.org/10.1016/j.pbi.2017.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595633PMC
October 2017

Endoplasmic reticulum-mitochondria junction is required for iron homeostasis.

J Biol Chem 2017 08 21;292(32):13197-13204. Epub 2017 Jun 21.

From the Department of Biological Chemistry,

The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 () gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.
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http://dx.doi.org/10.1074/jbc.M117.784249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555183PMC
August 2017

A bioactive peptide amidating enzyme is required for ciliogenesis.

Elife 2017 05 17;6. Epub 2017 May 17.

Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, United States.

The pathways controlling cilium biogenesis in different cell types have not been fully elucidated. We recently identified peptidylglycine α-amidating monooxygenase (PAM), an enzyme required for generating amidated bioactive signaling peptides, in and mammalian cilia. Here, we show that PAM is required for the normal assembly of motile and primary cilia in , planaria and mice. PAM knockdown lines failed to assemble cilia beyond the transition zone, had abnormal Golgi architecture and altered levels of cilia assembly components. Decreased PAM gene expression reduced motile ciliary density on the ventral surface of planaria and resulted in the appearance of cytosolic axonemes lacking a ciliary membrane. The architecture of primary cilia on neuroepithelial cells in mouse embryos was also aberrant. Our data suggest that PAM activity and alterations in post-Golgi trafficking contribute to the observed ciliogenesis defects and provide an unanticipated, highly conserved link between PAM, amidation and ciliary assembly.
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http://dx.doi.org/10.7554/eLife.25728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461114PMC
May 2017

Chromosome-level genome assembly and transcriptome of the green alga illuminates astaxanthin production.

Proc Natl Acad Sci U S A 2017 05 8;114(21):E4296-E4305. Epub 2017 May 8.

Howard Hughes Medical Institute, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102;

Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. There is interest in the unicellular green alga , because it produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. To advance understanding of its biology and facilitate commercial development, we present a chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions. The assembly, derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealed a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. The genome has uniform gene density over chromosomes, low repetitive sequence content (∼6%), and a high fraction of protein-coding sequence (∼39%) with relatively long coding exons and few coding introns. Functional annotation of gene models identified orthologous families for the majority (∼73%) of genes. Synteny analysis uncovered localized but scrambled blocks of genes in putative orthologous relationships with other green algae. Two genes encoding beta-ketolase (), the key enzyme synthesizing astaxanthin, were found in the genome, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants showed that is required for the production of astaxanthin. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production.
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http://dx.doi.org/10.1073/pnas.1619928114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448231PMC
May 2017