Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition

Approches quantitatives d’imagerie pour explorer les cellules photosynthétiques

Phytoplankton is the group of photosynthetic microorganisms (microalgae and cyanobacteria) living in suspension in marine and fresh waters. Through photosynthesis, phytoplankton produce large amounts of the oxygen essential for marine and terrestrial life. Marine microalgae are also promising organisms for biotechnological applications (human and animal food, biofuels). Because of their ecological and economic importance, the study of the phytoplankton responses to environmental challenged (including the ones induced by human activity and global warming) is a developing field of research. Phytoplankton activity is influenced by changes in the vertical stratification of the water column, which modulate light energy availability as well as nutrient supply to phytoplankton cells in a temperature-dependent manner. Based on light and nutrient availability, phytoplankton cells have evolved different lifestyles: autotrophy (photosynthetic activity), mixotrophy (simultaneous use of photosynthesis and respiration of exogenous carbon sources for growth) and photosymbiosis (symbiotic interactions with animal cells).In this thesis, I have studied the physiological responses of phytoplankton cells to environmental changes at the cellular and subcellular levels. To achieve this goal, I have developed a complete imaging workflow to perform quantitative morphometric analyses of entire algal cells, representatives of ecologically-successful and laboratory-model microalgal species. The protocol starts with FIB-SEM (Focused Ion Beam - Scanning Electron Microscopy) or SBF-SEM (Serial Block Facing – Scanning Electron Microscopy), to acquire high-resolution images. By implementing the 3D image analysis protocol, it is possible to obtained high-resolution whole cells models in three dimensions, suitable to perform quantitative analyses. Thanks to these tools, I have been able to image the adaptation of phytoplankton to various environmental conditions: i. changes in the size and morphology of plastids and mitochondria during light acclimation in diatoms, ii. changes in organelles interaction during nutrient acclimation in Nannochloropsis, iii. morphological changes occurring during photosymbiosis in Phaeocystis.Overall, this work reveals several scenarios of phytoplankton acclimation at both the cellular and subcellular levels. I have also validated the use of this protocol in plants in a study on chloroplast biogenesis during de-etiolation in Arabidopsis. of plastids.Le phytoplancton est le groupe de micro-organismes photosynthétiques (microalgues et cyanobactéries) vivant en suspension dans les eaux marines et douces. Grâce à la photosynthèse, le phytoplancton produit de grandes quantités d'oxygène indispensable à la vie marine et terrestre. Les microalgues marines sont également des organismes prometteurs pour les applications biotechnologiques (alimentation humaine et animale, biocarburants). En raison de leur importance écologique et économique, l'étude des réponses du phytoplancton aux défis environnementaux (y compris ceux induits par l'activité humaine et le réchauffement climatique) est un domaine de recherche en plein développement. L'activité du phytoplancton est influencée par les changements dans la stratification verticale de la colonne d'eau qui module, en fonction de la température, la disponibilité de l'énergie lumineuse ainsi que l'apport de nutriments aux cellules du phytoplancton. En raison de la disponibilité de la lumière et des nutriments, les cellules du phytoplancton ont évolué vers différents modes de vie : autotrophie (activité photosynthétique), mixotrophie (utilisation simultanée de la photosynthèse et de l’utilisation ? de sources de carbone extérieures pour la croissance) et photosymbiose (interactions symbiotiques avec des cellules animales).Dans cette thèse, j'ai étudié les réponses physiologiques des cellules du phytoplancton aux changements environnementaux au niveau cellulaire et sous-cellulaire. Pour atteindre cet objectif, j'ai mis au point un processus d'imagerie complet permettant d'effectuer des analyses morphométriques quantitatives de cellules entières d'algues représentatives à la fois d'espèces à succès écologique et de modèles de laboratoire. Le protocole commence avec l’acquisition de séries d’images hautes résolutions soit par FIB-SEM (Focused Ion Beam - Scanning Electron Microscopy) ou SBF-SEM (Serial Block Facing – Scanning Electron Microscopy). Le protocole d'analyse d'images 3D développé dans ce travail permet d'obtenir des modèles tridimensionnels à haute résolution de cellules entières permettant la réalisation d'analyses quantitatives. Grâce à ces outils, j'ai pu imager l'adaptation du phytoplancton à diverses conditions environnementales révélant ainsi : 1) le changement de taille et de morphologie des plastes et des mitochondries lors de l'acclimatation à la lumière dans les diatomées, 2) le changement dans l'interaction des organites chez Nannochloropsis lors de l'acclimatation aux nutriments, 3) les changements morphologiques qui surviennent lors de la photosymbiose dans l’algue Phaeocystis.Ces travaux révèlent plusieurs scénarios d'acclimatation du phytoplancton au niveau cellulaire et subcellulaire. J'ai également pu valider l'utilisation de ce protocole chez les plantes dans une étude sur la biogenèse des chloroplastes lors de la dé-étiolation des cotylédons d’Arabidopsis

Quantitative imaging methods to investigate photosynthetic cells

Le phytoplancton est le groupe de micro-organismes photosynthétiques (microalgues et cyanobactéries) vivant en suspension dans les eaux marines et douces. Grâce à la photosynthèse, le phytoplancton produit de grandes quantités d'oxygène indispensable à la vie marine et terrestre. Les microalgues marines sont également des organismes prometteurs pour les applications biotechnologiques (alimentation humaine et animale, biocarburants). En raison de leur importance écologique et économique, l'étude des réponses du phytoplancton aux défis environnementaux (y compris ceux induits par l'activité humaine et le réchauffement climatique) est un domaine de recherche en plein développement. L'activité du phytoplancton est influencée par les changements dans la stratification verticale de la colonne d'eau qui module, en fonction de la température, la disponibilité de l'énergie lumineuse ainsi que l'apport de nutriments aux cellules du phytoplancton. En raison de la disponibilité de la lumière et des nutriments, les cellules du phytoplancton ont évolué vers différents modes de vie : autotrophie (activité photosynthétique), mixotrophie (utilisation simultanée de la photosynthèse et de l’utilisation ? de sources de carbone extérieures pour la croissance) et photosymbiose (interactions symbiotiques avec des cellules animales).Dans cette thèse, j'ai étudié les réponses physiologiques des cellules du phytoplancton aux changements environnementaux au niveau cellulaire et sous-cellulaire. Pour atteindre cet objectif, j'ai mis au point un processus d'imagerie complet permettant d'effectuer des analyses morphométriques quantitatives de cellules entières d'algues représentatives à la fois d'espèces à succès écologique et de modèles de laboratoire. Le protocole commence avec l’acquisition de séries d’images hautes résolutions soit par FIB-SEM (Focused Ion Beam - Scanning Electron Microscopy) ou SBF-SEM (Serial Block Facing – Scanning Electron Microscopy). Le protocole d'analyse d'images 3D développé dans ce travail permet d'obtenir des modèles tridimensionnels à haute résolution de cellules entières permettant la réalisation d'analyses quantitatives. Grâce à ces outils, j'ai pu imager l'adaptation du phytoplancton à diverses conditions environnementales révélant ainsi : 1) le changement de taille et de morphologie des plastes et des mitochondries lors de l'acclimatation à la lumière dans les diatomées, 2) le changement dans l'interaction des organites chez Nannochloropsis lors de l'acclimatation aux nutriments, 3) les changements morphologiques qui surviennent lors de la photosymbiose dans l’algue Phaeocystis.Ces travaux révèlent plusieurs scénarios d'acclimatation du phytoplancton au niveau cellulaire et subcellulaire. J'ai également pu valider l'utilisation de ce protocole chez les plantes dans une étude sur la biogenèse des chloroplastes lors de la dé-étiolation des cotylédons d’Arabidopsis.Phytoplankton is the group of photosynthetic microorganisms (microalgae and cyanobacteria) living in suspension in marine and fresh waters. Through photosynthesis, phytoplankton produce large amounts of the oxygen essential for marine and terrestrial life. Marine microalgae are also promising organisms for biotechnological applications (human and animal food, biofuels). Because of their ecological and economic importance, the study of the phytoplankton responses to environmental challenged (including the ones induced by human activity and global warming) is a developing field of research. Phytoplankton activity is influenced by changes in the vertical stratification of the water column, which modulate light energy availability as well as nutrient supply to phytoplankton cells in a temperature-dependent manner. Based on light and nutrient availability, phytoplankton cells have evolved different lifestyles: autotrophy (photosynthetic activity), mixotrophy (simultaneous use of photosynthesis and respiration of exogenous carbon sources for growth) and photosymbiosis (symbiotic interactions with animal cells).In this thesis, I have studied the physiological responses of phytoplankton cells to environmental changes at the cellular and subcellular levels. To achieve this goal, I have developed a complete imaging workflow to perform quantitative morphometric analyses of entire algal cells, representatives of ecologically-successful and laboratory-model microalgal species. The protocol starts with FIB-SEM (Focused Ion Beam - Scanning Electron Microscopy) or SBF-SEM (Serial Block Facing – Scanning Electron Microscopy), to acquire high-resolution images. By implementing the 3D image analysis protocol, it is possible to obtained high-resolution whole cells models in three dimensions, suitable to perform quantitative analyses. Thanks to these tools, I have been able to image the adaptation of phytoplankton to various environmental conditions: i. changes in the size and morphology of plastids and mitochondria during light acclimation in diatoms, ii. changes in organelles interaction during nutrient acclimation in Nannochloropsis, iii. morphological changes occurring during photosymbiosis in Phaeocystis.Overall, this work reveals several scenarios of phytoplankton acclimation at both the cellular and subcellular levels. I have also validated the use of this protocol in plants in a study on chloroplast biogenesis during de-etiolation in Arabidopsis. of plastids

3D‐reconstructions of zygospores in Zygnema vaginatum (Charophyta) reveal details of cell wall formation, suggesting adaptations to extreme habitats

International audienceThe streptophyte green algal class Zygnematophyceae is the immediate sister lineage to land plants. Their special form of sexual reproduction via conjugation might have played a key role during terrestrialization. Thus, studying Zygnematophyceae and conjugation is crucial for understanding the conquest of land. Moreover, sexual reproduction features are important for species determination. We present a phylogenetic analysis of a field‐sampled Zygnema strain and analyze its conjugation process and zygospore morphology, both at the micro‐ and nanoscale, including 3D‐reconstructions of the zygospore architecture. Vegetative filament size (26.18 ± 1.07 μm) and reproductive features allowed morphological determination of Zygnema vaginatum , which was combined with molecular analyses based on rbc L sequencing. Transmission electron microscopy (TEM) depicted a thin cell wall in young zygospores, while mature cells exhibited a tripartite wall, including a massive and sculptured mesospore. During development, cytological reorganizations were visualized by focused ion beam scanning electron microscopy (FIB‐SEM). Pyrenoids were reorganized, and the gyroid cubic central thylakoid membranes, as well as the surrounding starch granules, degraded (starch granule volume: 3.58 ± 2.35 μm 3 in young cells; 0.68 ± 0.74 μm 3 at an intermediate stage of zygospore maturation). Additionally, lipid droplets (LDs) changed drastically in shape and abundance during zygospore maturation (LD/cell volume: 11.77% in young cells; 8.79% in intermediate cells, 19.45% in old cells). In summary, we provide the first TEM images and 3D‐reconstructions of Zygnema zygospores, giving insights into the physiological processes involved in their maturation. These observations help to understand mechanisms that facilitated the transition from water to land in Zygnematophyceae

Two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis thaliana

Dynamics of chloroplast biogenesis Impact statement: Serial Block Face Scanning Electron Microscopy (SBF-SEM) associated with biomolecular analysis show that chloroplast differentiation proceeds by distinct 'Structure Establishment' and 'Chloroplast Proliferation' phases, each with differential protein and lipid regulation

A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in arabidopsis

Light triggers chloroplast differentiation whereby the etioplast transforms into a photosynthesizing chloroplast and the thylakoid rapidly emerges. However, the sequence of events during chloroplast differentiation remains poorly understood. Using Serial Block Face Scanning Electron Microscopy (SBF-SEM), we generated a series of chloroplast 3D reconstructions during differentiation, revealing chloroplast number and volume and the extent of envelope and thylakoid membrane surfaces. Furthermore, we used quantitative lipid and whole proteome data to complement the (ultra)structural data, providing a time-resolved, multi-dimensional description of chloroplast differentiation. This showed two distinct phases of chloroplast biogenesis: an initial photosynthesis-enabling ‘Structure Establishment Phase’ followed by a ‘Chloroplast Proliferation Phase’ during cell expansion. Moreover, these data detail thylakoid membrane expansion during de-etiolation at the seedling level and the relative contribution and differential regulation of proteins and lipids at each developmental stage. Altogether, we establish a roadmap for chloroplast differentiation, a critical process for plant photoautotrophic growth and survival

The Arabidopsis leaf quantitative atlas: a cellular and subcellular mapping through unified data integration

Quantitative analyses and models are required to connect a plant’s cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels

The Arabidopsis leaf quantitative atlas: a cellular and subcellular mapping through unified data integration

International audienceQuantitative analyses and models are required to connect a plant’s cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels

Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution

International audiencePhotoautotrophs’ environmental responses have been extensively studied at the organism and ecosystemlevel. However, less is known about their photosynthesis at the single-cell level. This information is neededto understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, ororgans. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology basedon the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturationpulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developingtissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marinephotosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link betweenlight penetration and photoprotection responses inside the different tissues that constitute a plant leafanatomy