Frederik Sommer

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism

This work examines the mechanisms by which Chlamydomonas reinhardtii copes with nitrogen (N) limitation, finding transcriptomic and proteomic changes in multiple metabolic pathways and identifying an N-sparing mechanism that prioritizes respiratory metabolism and shifts the proteomic balance toward proteins with lower N contents, a result with implications for engineering of N-use efficiency. Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency.

Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii

Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully reversible cell cycle arrest after onset of stress. Our approach opens up new perspectives for plant systems biology and provides novel insights into plant stress acclimation.

Evidence for a Role of VIPP1 in the Structural Organization of the Photosynthetic Apparatus in Chlamydomonas

This work shows that suppressing the expression of the vesicle-inducing protein in plastids (VIPP1) in Chlamydomonas leads to aberrant structures at the origin of thylakoids and to structural defects particularly in photosystem II that render mutants sensitive to high light. The data indicate that VIPPs act in the biogenesis of thylakoid membrane core complexes, in particular the photosystems. The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b6f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the QA/QA− redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.

The CRR1 Nutritional Copper Sensor in Chlamydomonas Contains Two Distinct Metal-Responsive Domains

The binding of the SBP domain of CRR1 to copper response elements of target promoters in vitro is blocked by Cu(II) or Hg(II), which also deactivate transcription in vivo. Mutagenesis of the SBP domain implicates a conserved His residue in interaction with copper ions, suggesting a mechanism for copper sensing. A metallothionein-like domain at the CRR1 C terminus is implicated in zinc homeostasis. Copper response regulator 1 (CRR1), an SBP-domain transcription factor, is a global regulator of nutritional copper signaling in Chlamydomonas reinhardtii and activates genes necessary during periods of copper deficiency. We localized Chlamydomonas CRR1 to the nucleus in mustard (Sinapis alba) seedlings, a location consistent with its function as a transcription factor. The Zn binding SBP domain of CRR1 binds copper ions in vitro. Cu(I) can replace Zn(II), but the Cu(II) form is unstable. The DNA binding activity is inhibited in vitro by Cu(II) or Hg(II) ions, which also prevent activation of transcription in vivo, but not by Co(II) or Ni(II), which have no effect in vivo. Copper inhibition of DNA binding is reduced by mutation of a conserved His residue. These results implicate the SBP domain in copper sensing. Deletion of a C-terminal metallothionein-like Cys-rich domain impacted neither nutritional copper signaling nor the effect of mercuric supplementation, but rendered CRR1 insensitive to hypoxia and to nickel supplementation, which normally activate the copper deficiency regulon in wild-type cells. Strains carrying the crr1-ΔCys allele upregulate ZRT genes and hyperaccumulate Zn(II), suggesting that the effect of nickel ions may be revealing a role for the C-terminal domain of CRR1 in zinc homeostasis in Chlamydomonas.

FER1 and FER2 Encoding Two Ferritin Complexes in Chlamydomonas reinhardtii Chloroplasts Are Regulated by Iron

Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunit-specific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.

Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii

Systems analysis reveals that Chlamydomonas reinhardtii responds flexibly to an increase in light intensity. Rising metabolite levels and posttranslation regulation facilitate a rapid increase in the rate of carbon fixation and a slightly delayed increase in the rate of growth, while slower changes in protein abundance adjust allocation and relieve potential bottlenecks under the new conditions. We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

Conditional depletion of the chloroplast protease ClpP in the alga Chlamydomonas affects plastid protein homeostasis and leads to an autophagocytic and plastid unfolded protein-like response. It involves vacuolization of the cytoplasm and increased accumulation of small heat shock proteins, specific chaperones, proteases, and proteins implicated in thylakoid membrane maintenance and biogenesis. Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

Transcription Factor–Dependent Chromatin Remodeling at Heat Shock and Copper-Responsive Promoters in Chlamydomonas reinhardtii

Transcription factors mediating acclimation to abiotic stress in Chlamydomonas reinhardtii regulate the expression of their target genes via histone acetylation, histone methylation, nucleosome eviction, and polymerase loading/activation. At each target promoter, these means are employed quite individually to establish a characteristic chromatin state allowing for a fine-tuning of gene expression. How transcription factors affect chromatin structure to regulate gene expression in response to changes in environmental conditions is poorly understood in the green lineage. To shed light on this issue, we used chromatin immunoprecipitation and formaldehyde-assisted isolation of regulatory elements to investigate the chromatin structure at target genes of HSF1 and CRR1, key transcriptional regulators of the heat shock and copper starvation responses, respectively, in the unicellular green alga Chlamydomonas reinhardtii. Generally, we detected lower nucleosome occupancy, higher levels of histone H3/4 acetylation, and lower levels of histone H3 Lys 4 (H3K4) monomethylation at promoter regions of active genes compared with inactive promoters and transcribed and intergenic regions. Specifically, we find that activated HSF1 and CRR1 transcription factors mediate the acetylation of histones H3/4, nucleosome eviction, remodeling of the H3K4 mono- and dimethylation marks, and transcription initiation/elongation. By this, HSF1 and CRR1 quite individually remodel and activate target promoters that may be inactive and embedded into closed chromatin (HSP22F/CYC6) or weakly active and embedded into partially opened (CPX1) or completely opened chromatin (HSP70A/CRD1). We also observed HSF1-independent histone H3/4 deacetylation at the RBCS2 promoter after heat shock, suggesting interplay of specific and presumably more generally acting factors to adapt gene expression to the new requirements of a changing environment.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

Significance Eukaryotic algae, which play a fundamental role in global CO2 fixation, enhance the performance of the carbon-fixing enzyme Rubisco by placing it into an organelle called the pyrenoid. Despite the ubiquitous presence and biogeochemical importance of this organelle, how Rubisco assembles to form the pyrenoid remains a long-standing mystery. Our discovery of an abundant repeat protein that binds Rubisco in the pyrenoid represents a critical advance in our understanding of pyrenoid biogenesis. The repeat sequence of this protein suggests elegant models to explain the structural arrangement of Rubisco enzymes in the pyrenoid. Beyond advances in basic understanding, our findings open doors to the engineering of algal pyrenoids into crops to enhance yields. Biological carbon fixation is a key step in the global carbon cycle that regulates the atmospheres composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

Systems-Wide Analysis of Acclimation Responses to Long-Term Heat Stress and Recovery in the Photosynthetic Model Organism Chlamydomonas reinhardtii

Early acclimation responses of Chlamydomonas to long-term heat stress are directed toward restoration of protein homeostasis and membrane fluidity involving the redirection of photosynthetic electron flow from carbon fixation to saturated fatty acid synthesis, while late responses deal with the depletion of electron sinks. During recovery, cells aim at a rapid resumption of cell division/growth. We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42°C for 24 h and back to 25°C for ≥8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.