Crysten E. Blaby-Haas

Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage

Transcriptomes of iron-limited versus -deficient Chlamydomonas reinhardtii cells under photoheterotrophic versus photoautotrophic conditions are described. Increased transcript abundance was positively correlated with protein abundance, except for iron-containing proteins where negative correlation was found. Some responses, including upregulation of MDAR1 and CGLD27/At5g67370, are conserved in land plants. We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron.

The Chlamydomonas genome project: a decade on

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

The ins and outs of algal metal transport

Metal transporters are a central component in the interaction of algae with their environment. They represent the first line of defense to cellular perturbations in metal concentration, and by analyzing algal metal transporter repertoires, we gain insight into a fundamental aspect of algal biology. The ability of individual algae to thrive in environments with unique geochemistry, compared to non-algal species commonly used as reference organisms for metal homeostasis, provides an opportunity to broaden our understanding of biological metal requirements, preferences and trafficking. Chlamydomonas reinhardtii is the best developed reference organism for the study of algal biology, especially with respect to metal metabolism; however, the diversity of algal niches necessitates a comparative genomic analysis of all sequenced algal genomes. A comparison between known and putative proteins in animals, plants, fungi and algae using protein similarity networks has revealed the presence of novel metal metabolism components in Chlamydomonas including new iron and copper transporters. This analysis also supports the concept that, in terms of metal metabolism, algae from similar niches are more related to one another than to algae from the same phylogenetic clade. This article is part of a Special Issue entitled: Cell Biology of Metals.

Lysosome-related Organelles as Mediators of Metal Homeostasis

Metal ion assimilation is essential for all forms of life. However, organisms must properly control the availability of these nutrients within the cell to avoid inactivating proteins by mismetallation. To safeguard against an imbalance between supply and demand in eukaryotes, intracellular compartments contain metal transporters that load and unload metals. Although the vacuoles of Saccharomyces cerevisiae and Arabidopsis thaliana are well established locales for the storage of copper, zinc, iron, and manganese, related compartments are emerging as important mediators of metal homeostasis. Here we describe these compartments and review their metal transporter complement.

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.

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

Background: Yeast synthesizes coenzyme Q via a macromolecular protein complex. Results: The Q biosynthetic complex includes Coq8 and the uncharacterized protein YLR290C; the ylr290cΔ mutant exhibits impaired Q synthesis. Conclusion: YLR290C (Coq11) is a novel protein required for efficient yeast Q biosynthesis. Significance: Discovery and characterization of yeast Coq biosynthetic proteins leads to an improved understanding of coenzyme Q biosynthesis and regulation. Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.

Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

Significance The prevailing dogma is that access to copper (Cu+) is restricted to the extent that protein–protein interactions mediate the routing of Cu+ from transporters in the plasma membrane to target cuproenzymes or transporters within subcellular compartments. The soluble proteins that distribute Cu+ are called metallochaperones. Although the chloroplast requires Cu+, a chaperone that delivers this essential cofactor has remained a missing link in the model for plastid Cu+ delivery. Using a comparative genomic approach and validating by biochemical characterization, we have discovered a missing chaperone. Uniquely, the previously unidentified chaperone family has evolved from the transporter to which it delivers Cu+. We also uncover an interaction between the thylakoid-localized transporter and the Cu+ chaperone for stromal Cu/Zn superoxide dismutase, which highlights the complexity of Cu+ distribution networks. Metallochaperones traffic copper (Cu+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu+ chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu+ chaperone delivers Cu+ with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu+ import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu+-delivery mechanism and provides a unique snapshot into the evolution of a Cu+ distribution pathway. We also provide evidence for an interaction between PAA2, the Cu+-ATPase in thylakoids, and the Cu+-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu+ network that has evolved to fine-tune Cu+ distribution.

PHOTOSYSTEM II PROTEIN33, a protein conserved in the plastid lineage, is associated with the chloroplast thylakoid membrane and provides stability to photosystem II supercomplexes in arabidopsis

The study of a previously undescribed protein shows its involvement in the maintenance of the photosystem II light-harvesting supercomplex. Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Significance Fossil evidence shows that red algae (Rhodophyta) are one of the most ancient multicellular lineages. Their ecological, evolutionary, and commercial importance notwithstanding, few red algal nuclear genomes have been sequenced. Our analyses of the Porphyra umbilicalis genome provide insights into how this macrophyte thrives in the stressful intertidal zone and into the basis for its nutritional value as human food. Many of the novel traits (e.g., cytoskeletal organization, calcium signaling pathways) we find encoded in the Porphyra genome are extended to other red algal genomes, and our unexpected findings offer a potential explanation for why the red algae are constrained to small stature relative to other multicellular lineages. Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Iron sparing and recycling in a compartmentalized cell.

This review focuses on economizing, prioritizing and recycling iron in Chlamydomonas, a reference organism for discovering mechanisms of acclimation to poor iron nutrition in the plant lineage. The metabolic flexibility of Chlamydomonas offers a unique opportunity to distinguish the impact of iron nutrition on photosynthetic versus respiratory metabolism, and the contribution of subcellular compartments to iron storage and mobilization. Mechanisms of iron sparing include down regulation of protein abundance by transcript reduction or protein degradation. Two well-studied examples of hierarchical iron allocation are the maintenance of FeSOD in the plastid and heterotrophic metabolism in acetate-grown cells at the expense of photosynthetic metabolism. The latter implicates the existence of a pathway for inter-compartment iron recycling when access to iron becomes limiting.