Stefan Schmollinger

SUMMARY: An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg cell ⁻¹ of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis, and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 10 ⁶ molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 10 ⁶ complexes per cell. PSI averages 1.75 × 10 ⁶ complexes per cell and cytochrome b ₆ f averages 0.77 × 10 ⁶ complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artifacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Photosynthetic membranes undergo structural remodeling in response to environmental stress by altering fatty acid composition and desaturation levels. These changes, mediated by fatty acid desaturases (FADs), are essential for maintaining photosynthetic performance and adaptation. In this study, we demonstrate that copper-deficient Chlamydomonas reinhardtii cells upregulate the expression of the gene encoding stearoyl-ACP desaturase (SAD/FAB2). We propose that this four-fold induction reflects an increased physiological demand for its primary product, oleic acid (18:1∆9), and its subsequent downstream derivatives. The sad mutants exhibit a significant reduction in 18:1∆9 content compared to wild-type cells, which correlates with diminished growth rates. Although SAD abundance increases under Cu deficiency, loss of SAD strongly alters C18 fatty acid composition across Cu conditions, while the growth defect is most apparent under Cu-replete conditions. This suggests that SAD activity may be a limiting factor in copper-depleted environments, leading to slower growth and reduced 18:1∆9 levels in the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both of which are critical for photosynthetic function. The desaturation reaction catalyzed by SAD requires molecular oxygen and electrons supplied by ferredoxin (Fd). Using reciprocal IP-MS, we identified FDX5 as a Cu-deficiency specific SAD interacting protein. However, fdx5 mutants retained wild-type fatty acid profiles, indicating that FDX5 is not strictly required for SAD-dependent lipid desaturation and that another ferredoxin, likely FDX1, can compensate.

Chloroplast HSP70 is an essential component of the plastid proteostasis network, supporting protein folding, complex assembly and disassembly, and stress acclimation. Despite extensive genetic evidence for its essentiality, the cellular consequences of reduced chloroplast HSP70 activity remain poorly defined. Here, we investigated the function of the sole chloroplast HSP70 in Chlamydomonas reinhardtii , HSP70B, using an inducible artificial microRNA approach that reduced HSP70B abundance to below 30% of wild-type levels. HSP70B depletion resulted in cell division arrest and extensive proteome remodeling, characterized by strong upregulation of proteins involved in chloroplast protein quality control and membrane remodeling. Notably, this response was accompanied by increased abundance of protein quality control components in the endoplasmic reticulum, cytosol, and mitochondria, indicating pronounced proteostasis cross-talk between cellular compartments. In contrast, chloroplast and cytosolic ribosomes, photosynthetic and respiratory protein complexes, and central metabolic enzymes were broadly depleted, consistent with a collapse of cellular proteostasis. At the ultrastructural level, HSP70B-depleted cells exhibited lesions at thylakoid membrane conversion zones previously described in VIPP1-depleted cells. Accordingly, higher-order oligomeric forms of VIPP1 accumulated, and cells displayed extreme sensitivity to high-light stress. These findings confirm HSP70B as a key regulator of VIPP1 oligomer dynamics and highlight its central role in coordinating chloroplast membrane remodeling with cellular proteostasis in Chlamydomonas . One-sentence summary: Depletion of chloroplast HSP70B causes cell division arrest, proteostasis collapse, impaired VIPP1 oligomer dynamics with aberrant thylakoid structures, and increased light sensitivity.

SUMMARY: An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg / cell of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 10 ⁶ molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 10 ⁶ complexes per cell. PSI averages 1.75 × 10 ⁶ complexes per cell and cytochrome b ₆ f averages 0.77 × 10 ⁶ complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artefacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Plant-type ferredoxins (Fd) comprise small, soluble protein families that distribute electrons from photosystem I to various client proteins within the chloroplast stroma. In Chlamydomonas reinhardtii , the major, constitutively expressed FDX1/PetF supports Fd-NADP ⁺ reductase (FNR) in NADPH production. The highly similar FDX2 is present only when its preferred nitrogen (N) source ammonium is absent, supplying Fd-dependent nitrite reductase (NiR) for nitrate/nitrite assimilation. Surprisingly, despite accumulating to ∼10% of FDX1 abundance and preferential interaction with NiR, fdx2 mutants are asymptomatic when grown on nitrate, requiring to additionally deplete FDX1 for growth to be halted. A fdx1 knockout itself appears lethal, severe fdx1 knockdowns have reduced growth rates both in phototrophic and photoheterotrophic conditions, independent of the N source. Transcriptome analyses of fdx1 mutants revealed expression patterns similar to sulfur (S) deficient algae, and fdx1 strains have a reduced total cellular S content. S assimilation requires Fd-dependent sulfite reductase (SiR) activity, an enzyme distantly related to FDX2 client NiR. Expression defects are partially alleviated; growth and S content are less impacted with FDX2 expression. Our mutant analysis shows the two major Fds in Chlamydomonas focus on a specific subset of Fd-dependent metabolism, mostly supplying Fd-dependent enzymes involved in macronutrient assimilation (C/N/S).

Microalgae are promising sources to sustainably meet the global needs for energy and products. Algae grow under different trophic conditions, where nutritional status regulates biosynthetic pathways, energy production, and growth. The green alga Chromochloris zofingiensis has strong economic potential because it co‐produces biofuel precursors and the high‐value antioxidant astaxanthin while accumulating biomass when grown mixotrophically. As an emerging reference alga for photosynthesis, metabolism, and bioproduction, C. zofingiensis needs a defined, optimized medium to standardize experiments during fast growth for batch cultivation. Because the interplay of glucose treatment (+Glc) and mineral deficiency influences photosynthesis, growth, and the production of lipids and astaxanthin, we designed a replete nutrient medium tailored to the C. zofingiensis cellular ionome. We combined inductively coupled plasma mass spectrometry (ICP‐MS) and +Glc growth curves to determine a medium that is nutrient replete for at least 5 days of +Glc logarithmic growth. We found that there are high nutritional needs for phosphorus and sulfur during mixotrophy. Iron was the only element measured for which the cellular concentration correlated with exogenous concentration and was iteratively adjusted until the cellular ionome was consistent through the logarithmic growth phase. This Chromochloris‐Optimized Ratio of Elements (CORE) medium supports fast growth and high biomass and lipid accumulation without causing excess nutrient toxicity. This defined, nutrient‐replete standard is important for future C. zofingiensis investigations and can be adapted for other species to support high biomass for batch cultivation. The method used to develop CORE medium shows how ionomics informs replicable media design and may be applied in industrial settings to inform cost‐effective biofuel production.

Light capture and photosynthetic energy conversion depends on photosynthetic complexes that are embedded within lipid membranes. Components of these complexes are vulnerable to damage by reactive oxygen species, byproducts of photosynthesis that accumulate under environmental stress. Here we explore the basis for a lipid-based sensing mechanism allowing plants or algae to assess and respond to damage to the photosynthetic membranes. In Chlamydomonas reinhardtii, Low Carbon Inducible2 (LCI2) and Fatty Acid Desaturase4 (FAD4) are two proteins derived from the same locus by a differential splicing event, sharing an N-terminus encoded by the first two exons. FAD4 produces a 16-carbon, trans double bond-containing fatty acid found exclusively in phosphatidylglycerol of chloroplast membranes, while LCI2 recruits peroxidase activity to the membrane. The unique organization and transcriptional regulation of the LCI2/FAD4 locus represents a regulatory interface that allows cells to initiate the biosynthesis of a fatty acid unique to the photosynthetic membranes while also linking it to the production of an enzyme involved in the mitigation of reactive oxygen species.

Carbon and zinc (Zn) metabolism are intrinsically connected in phototrophs, as crucial components involved in CO ₂ assimilation, like carbonic anhydrases, are highly abundant Zn proteins. Utilizing these and other proteins, the eukaryotic green algae Chlamydomonas reinhardtii can maintain phototrophic growth in low CO ₂ environments by inducing a carbon concentrating mechanism (CCM). In this work we show that Chlamydomonas dynamically increases its Zn content to accommodate the higher intracellular Zn demand in low CO ₂ environments. This increase requires the presence of Cia5, a major regulator of the CCM in Chlamydomonas. How Cia5 regulates expression of thousands of low CO ₂ -inducible genes remains enigmatic, its transcript and protein abundance is unchanged in different CO ₂ environments, even in the presence of an additional reduced carbon source, acetate. We show here that the Cia5 protein is not present in Zn-limitation, despite CIA5 transcription being unchanged. We used a CRISPR knock-in approach to express Cia5-HA from its endogenous locus and used two independent Cia5-HA expressing strains for affinity purification and identified a protein belonging to a conserved family of metal binding GTPases, ZNG3, as a constitutive interaction partner. Like Cia5, ZNG3 is constitutively expressed, co-expressed with Cia5 along the diurnal cycle and is Cia5-dependently induced in low CO ₂ environments. Surprisingly, zng3 mutants do not phenocopy cia5 mutants and grow well in low CO ₂ conditions. Instead, zng3 mutants are unable to grow like wildtype if excess carbon is available in the form of high CO ₂ or acetate. Transcriptomics of wildtype and zng3 mutants grown with different carbon sources revealed that transcriptional induction of the majority of genes involved in the CCM is maintained in low CO ₂ grown zng3 mutants, while the degree of induction in a subset of LCI genes is reduced ( HLA3 , CAH4 and CAH5 ). Genes encoding proteins involved in plastid quality control were induced in zng3 mutants grown on acetate and high CO ₂ , as well as other, related metallochaperones. We hypothesize that Zn trafficking towards the plastid is mis regulated in zng3 mutants resulting in protein mis-metalation and unfolding. Taken together, we propose that ZNG3 and Cia5 coordinate Zn and CO ₂ metabolism, affecting intracellular Zn trafficking and modulate the CO ₂ response.

Summary: Microalgae are promising sources to sustainably meet the global needs for energy and products. Algae grow under different trophic conditions, where nutritional status regulates biosynthetic pathways, energy production, and growth. The green alga Chromochloris zofingiensis has strong economic potential because it co-produces biofuel precursors and the high-value antioxidant astaxanthin while accumulating biomass when grown mixotrophically. As an emerging reference alga for photosynthesis, metabolism, and bioproduction, C. zofingiensis needs a defined, optimized medium to standardize experiments during fast growth. Because the interplay of glucose consumption (+Glc) and mineral deficiency influences photosynthesis, growth, and the production of lipids and astaxanthin, we designed a replete nutrient medium tailored to the C. zofingiensis cellular ionome. We combined inductively coupled plasma mass spectrometry (ICP-MS) and +Glc growth curves to determine a medium that is nutrient replete for at least 5 days of +Glc logarithmic growth. We found that there are high nutritional needs for phosphorus and sulfur during mixotrophy. Iron was the only element measured for which the cellular concentration correlated with exogenous concentration and was iteratively adjusted until the internal ionome was consistent through the logarithmic growth phase. This C hromochloris - O ptimized R atio of E lements (CORE) medium supports fast growth and high biomass without causing excess nutrient toxicity. This defined, nutrient-replete standard is important for future C. zofingiensis investigations and can be adapted for other species to support high biomass. The method used to develop CORE medium shows how ionomics informs replicable media design and may be applied in industrial settings to inform cost-effective biofuel production. Significance Statement: Studying how carbon sources and mineral nutrients interplay to regulate algal metabolism can be exploited to discover and control pathways in photosynthesis and biofuel production. Here we design a medium from the cellular ionome of Chromochloris zofingiensis , a powerful algal model for photosynthesis, metabolism, and bioproducts, to provide a defined, replete standard for mixotrophic and heterotrophic growth of green algae. These media design principles show how accounting for increased nutritional demands based on carbon substrate can ensure experimental replicability when probing diverse algal metabolisms.

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.