Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green algaChlamydomonas reinhardtiiunder highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35°C) and acute (40°C) high temperatures and subsequent recovery at 25°C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35°C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake and promoted growth. But 40°C disrupted cell division and growth. Both high temperatures induced photoprotection, while 40°C distorted thylakoid/pyrenoid ultrastructure, affected the carbon concentrating mechanism, and decreased photosynthetic efficiency. We demonstrated increased transcript/protein correlation during both heat treatments and hypothesize reduced post-transcriptional regulation during heat may help efficiently coordinate thermotolerance mechanisms. During recovery after both heat treatments, especially 40°C, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H₂O₂, during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod‐like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock‐out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane‐enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

While the composition and function of the major thylakoid membrane complexes are well understood, comparatively little is known about their biogenesis. The goal of this work was to shed more light on the role of auxiliary factors in the biogenesis of photosystem II (PSII). Here we have identified the homolog of LOW PSII ACCUMULATION 2 (LPA2) in Chlamydomonas. A Chlamydomonas reinhardtii lpa2 mutant grew slower in low light, was hypersensitive to high light, and exhibited aberrant structures in thylakoid membrane stacks. Chlorophyll fluorescence (F_v/F_m) was reduced by 38%. Synthesis and stability of newly made PSII core subunits D1, D2, CP43, and CP47 were not impaired. However, complexome profiling revealed that in the mutant CP43 was reduced to ~23% and D1, D2, and CP47 to ~30% of wild type levels. Levels of PSI and the cytochrome b₆f complex were unchanged, while levels of the ATP synthase were increased by ~29%. PSII supercomplexes, dimers, and monomers were reduced to ~7%, ~26%, and ~60% of wild type levels, while RC47 was increased ~6-fold and LHCII by ~27%. We propose that LPA2 catalyses a step during PSII assembly without which PSII monomers and further assemblies become unstable and prone to degradation. The LHCI antenna was more disconnected from PSI in the lpa2 mutant, presumably as an adaptive response to reduce excitation of PSI. From the co-migration profiles of 1734 membrane-associated proteins, we identified three novel putative PSII associated proteins with potential roles in regulating PSII complex dynamics, assembly, and chlorophyll breakdown.

Data visualization is integral for exploratory data analysis and data presentation. Consequently, every programming environment suitable for data analysis needs a feature-rich visualization library. In this paper, we present Plotly.NET - a fully featured charting library for the .NET ecosystem. Plotly.NET requires little ceremony to use, provides multiple API (application programming interface) layers for both low-code and maximum customizability, and can be used in many different visualization workflows - from interactive scripting and notebooks to user interface applications. Plotly.NET brings the charting capabilities of plotly.js to the .NET ecosystem, successfully combining the flexibility of the plotly grammar with the type-safety and expressiveness of .NET.

PSII assembly requires auxiliary factors, including Psb28. Although the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas (Chlamydomonas reinhardtii) psb28 null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers, and monomers, while overaccumulating early PSII assembly intermediates reaction center II (RCII), CP43mod, and D1mod. The mutant had less PSI and more cytochrome b6f complex, its thylakoids were organized mainly as monolayers, and it had a distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that THYLAKOID ENRICHED FRACTION 5 (TEF5), the homolog of Arabidopsis (Arabidopsis thaliana) PHOTOSYSTEM B PROTEIN 33/LIGHT HARVESTING-LIKE 8, comigrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant was severely impaired in PSII assembly and overaccumulated RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in RCII photoprotection or maturation. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria.