Iron economy in Chlamydomonas reinhardtii.

While research on iron nutrition in plants has largely focused on iron-uptake pathways, photosynthetic microbes such as the unicellular green alga Chlamydomonas reinhardtii provide excellent experimental systems for understanding iron metabolism at the subcellular level. Several paradigms in iron homeostasis have been established in this alga, including photosystem remodeling in the chloroplast and preferential retention of some pathways and key iron-dependent proteins in response to suboptimal iron supply. This review presents our current understanding of iron homeostasis in Chlamydomonas, with specific attention on characterized responses to changes in iron supply, like iron-deficiency. An overview of frequently used methods for the investigation of iron-responsive gene expression, physiology and metabolism is also provided, including preparation of media, the effect of cell size, cell density and strain choice on quantitative measurements and methods for the determination of metal content and assessing the effect of iron supply on photosynthetic performance

Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii

With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment

Genome-wide analysis on Chlamydomonas reinhardtii reveals the impact of hydrogen peroxide on protein stress responses and overlap with other stress transcriptomes.

Reactive oxygen species (ROS) are produced by and have the potential to be damaging to all aerobic organisms. In photosynthetic organisms, they are an unavoidable byproduct of electron transfer in both the chloroplast and mitochondrion. Here, we employ the reference unicellular green alga Chlamydomonas reinhardtii to identify the effect of H2O2 on gene expression by monitoring the changes in the transcriptome in a time-course experiment. Comparison of transcriptomes from cells sampled immediately prior to the addition of H2O2 and 0.5 and 1 h subsequently revealed 1278 differentially abundant transcripts. Of those transcripts that increase in abundance, many encode proteins involved in ROS detoxification, protein degradation and stress responses, whereas among those that decrease are transcripts encoding proteins involved in photosynthesis and central carbon metabolism. In addition to these transcriptomic adjustments, we observe that addition of H2O2 is followed by an accumulation and oxidation of the total intracellular glutathione pool, and a decrease in photosynthetic O2 output. Additionally, we analyze our transcriptomes in the context of changes in transcript abundance in response to singlet O2 (O2*), and relate our H2O2 -induced transcripts to a diurnal transcriptome, where we demonstrate enrichments of H2O2 -induced transcripts early in the light phase, late in the light phase and 2 h prior to light. On this basis several genes that are highlighted in this work may be involved in previously undiscovered stress remediation pathways or acclimation responses

A Gateway platform for functional genomics in Haloferax volcanii

In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved in N4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions

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

Activation of Autophagy by Metals in Chlamydomonas reinhardtii

Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation of ATG8 that is independent of CRR1, a global regulator of copper signaling in Chlamydomonas. A similar effect on ATG8 was observed with copper and cobalt but not with cadmium or mercury ions. Transcriptome sequencing data revealed an increase in the abundance of the protein degradation machinery, including that responsible for autophagy, and a substantial overlap of that increased abundance with the hydrogen peroxide response in cells treated with nickel ions. Thus, our results indicate that metal stress triggers autophagy in Chlamydomonas and suggest that excess nickel may cause oxidative damage, which in turn activates degradative pathways, including autophagy, to clear impaired components and recover cellular homeostasisMinisterio de Economía y Competitividad BFU2012-35913Junta de Andalucía CVI-7336National Institutes of Health GM42143, R24 GM09247

Synergistic use of plant-prokaryote comparative genomics for functional annotations

<p>Abstract</p> <p>Background</p> <p>Identifying functions for all gene products in all sequenced organisms is a central challenge of the post-genomic era. However, at least 30-50% of the proteins encoded by any given genome are of unknown or vaguely known function, and a large number are wrongly annotated. Many of these ‘unknown’ proteins are common to prokaryotes and plants. We set out to predict and experimentally test the functions of such proteins. Our approach to functional prediction integrates comparative genomics based mainly on microbial genomes with functional genomic data from model microorganisms and post-genomic data from plants. This approach bridges the gap between automated homology-based annotations and the classical gene discovery efforts of experimentalists, and is more powerful than purely computational approaches to identifying gene-function associations.</p> <p>Results</p> <p>Among Arabidopsis genes, we focused on those (2,325 in total) that (i) are unique or belong to families with no more than three members, (ii) occur in prokaryotes, and (iii) have unknown or poorly known functions. Computer-assisted selection of promising targets for deeper analysis was based on homology-independent characteristics associated in the SEED database with the prokaryotic members of each family. In-depth comparative genomic analysis was performed for 360 top candidate families. From this pool, 78 families were connected to general areas of metabolism and, of these families, specific functional predictions were made for 41. Twenty-one predicted functions have been experimentally tested or are currently under investigation by our group in at least one prokaryotic organism (nine of them have been validated, four invalidated, and eight are in progress). Ten additional predictions have been independently validated by other groups. Discovering the function of very widespread but hitherto enigmatic proteins such as the YrdC or YgfZ families illustrates the power of our approach.</p> <p>Conclusions</p> <p>Our approach correctly predicted functions for 19 uncharacterized protein families from plants and prokaryotes; none of these functions had previously been correctly predicted by computational methods. The resulting annotations could be propagated with confidence to over six thousand homologous proteins encoded in over 900 bacterial, archaeal, and eukaryotic genomes currently available in public databases.</p

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of AtZNG1, AtMAP1A, AtMAP2A, and AtMAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted

Widespread polycistronic gene expression in green algae

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: Chlamydomonas reinhardtii and Chromochloris zofingiensis 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 C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage