Stefan Schmollinger

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.

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.

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.

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cells entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.

A dedicated type II NADPH dehydrogenase performs the penultimate step in the biosynthesis of vitamin K 1 in synechocystis and arabidopsis

The biosynthesis of vitamin K1 requires an additional bona fide step that consists of the prerequisite reduction of the demethylnaphthoquinone ring prior to its transmethylation. Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process.

Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii

We found previously that the Chlamydomonas HSP70A promoter counteracts transcriptional silencing of downstream promoters in a transgene setting. To elucidate the underlying mechanisms, we analyzed chromatin state and transgene expression in transformants containing HSP70A-RBCS2-ble (AR-ble) constructs harboring deletions/mutations in the A promoter. We identified histone modifications at transgenic R promoters indicative for repressive chromatin, i.e. low levels of histone H3/4 acetylation and H3-lysine 4 trimethylation and high levels of H3-lysine 9 monomethylation. Transgenic A promoters also harbor lower levels of active chromatin marks than the native A promoter, but levels were higher than those at transgenic R promoters. Strikingly, in AR promoter fusions, the chromatin state at the A promoter was transferred to R. This effect required intact HSE4, HSE1/2 and TATA-box in the A promoter and was mediated by heat shock factor (HSF1). However, time-course analyses in strains inducibly depleted of HSF1 revealed that a transcriptionally competent chromatin state alone was not sufficient for activating the R promoter, but required constitutive HSF1 occupancy at transgenic A. We propose that HSF1 constitutively forms a scaffold at the transgenic A promoter, presumably containing mediator and TFIID, from which local chromatin remodeling and polymerase II recruitment to downstream promoters is realized.

Endoplasmic reticulum–mitochondria junction is required for iron homeostasis

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 (VPS13) 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.

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.

Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii.

We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.

A protocol for the identification of protein-protein interactions based on 15N metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants. Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.