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Stomata are anatomical structures at the surface of leaves controlling gas exchanges between the plant and the atmosphere. The stomatal pore aperture is regulated by two specialized cells, the guard cells, having the capacity to reversibly change their volume and shape. The changes of the guard cells’ volume are under tight control of signaling cascades regulating the ionic transport processes across the plasma and the vacuolar membranes. In guard cells the vacuole can reversibly change its volume during the opening and closure of stomata. We have used a 3D reconstruction approach to visualize the dynamics changes subcellular compartments volumes in living guard cells. The method we developed allowed us to simultaneously quantify the volumetric modifications of the cytosol and the vacuole. Notably, we could follow modification of the subcellular organization in parallel with the ion transport dynamics at the plasma and vacuolar membranes. Our data show that the vacuole accounts for the whole guard cell volume changes. This suggests that the turgor changes of the guard cells are driven by the vacuole during stomata aperture. 

SEB Conference 2022

Subcellular volume changes in stomata opening: vacuolar vs cytosolic volume changes.

technical paper

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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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2022 found over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France.

The centriole is an evolutionary conserved organelle that coordinates fundamental biological processes including cell division, cellular signalling and cell motility. This organelle, of 500 nm long and 250 nm in diameter is composed of about 100 different proteins, some of which have been associated with human diseases. How these proteins are organized at the level of the centriole architecture and how associated mutations could be involved in pathologies is still poorly understood. I will present the latest work from my laboratory, which tackle these fundamental structural cell biology questions using cryo-tomography, cell biology and our newly developed ultrastructure expansion microscopy (U-ExM) method.

Expansion microscopy for Structural cell biology: from centriole architecture to human diseases

Determining how plasticity of developmental traits respond to environmental conditions is a challenge that must combine evolutionary sciences, ecology and developmental biology. Developmental plasticity is defined as the ability of an organism to adjust its development depending on environmental signals, thus producing alternative phenotypes precisely adjusted to the environment. Yet the mechanisms underlying developmental plasticity are not fully understood yet. Determining how plastic developmental changes that occur in response to environmental conditions are coordinated at the physiological, cellular, and molecular levels is a challenge that must combine ecology with developmental biology. The mechanisms that underlie the development of alternative phenotypes are still unclear for many systems and is one major goal of ecological developmental biology or Eco-Evo-Devo. We observed that clownfish (Amphiprion percula) young juveniles have a different rate of white bar formation depending on the sea anemone species, their obligate symbiotic partner, in which they are recruited: white bars develop more rapidly when fish are recruited in Stichodactyla gigantea than in Heteractis magnifica. Because the sister species A. ocellaris acquire their adult color pattern during metamorphosis under thyroid hormone (TH) control, we asked whether developmental plasticity in bar formation was associated with alteration in TH status as these are the main hormones triggering metamorphosis in vertebrates. We found that thyroid hormones regulate white bar formation and that a shift in hormone levels, associated with ecological differences, results in divergent color patterns in different sea anemone species in which the young fish is recruited. Taken together our results suggest that TH control the timing of adult color pattern formation and that shifts in gene expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development. We recently observed that the phenotypic change observed not only concerns pigmentation but also energy metabolism. The evolutionary implications of these observations will be discussed

Phenotypic plasticity in anemonefishes: An Eco/Evo/Devo analysis

As poikilotherms, fish exposed to environmental extremes temperatures experience a stressful metabolic challenge, which elicits physiological responses required to maintain cellular homeostasis. These responses include modified glucose or lipid metabolism, altered gene expression and alternative splicing, and endocrine and immune system activity. Additionally, heat seeking may not resolve the homeostatic needs of tropical poikilotherms under unpredictable extreme cold events, which occur frequently due to global climate changes. In spite of the physiological responses that occur in poikilothermic vertebrates, the prevailing notion is that their reduction in metabolic rate is passive. Here, we explored molecular hypothalamic and physiological responses to cold stress in the tropical cichlid, Nile tilapia (Oreochromis niloticus). When exposed to low temperatures, tilapia exhibited complex homeostatic responses, including increased plasma glucose and cortisol concomitant with reduced plasma lactate and metabolic rate. Hypothalamic transcriptome analysis revealed increased oxytocin expression. Blockage of oxytocin signaling using zebrafish CRISPR-knockout lines for oxytocin or its receptors, and pharmacological antagonist in tilapia, further affected temperature-dependent metabolic rate in two cold-exposed tropical fish species. This indicates that oxytocin, a known thermoregulator in homeotherms, actively regulates temperature-related homeostasis in fish. Overall, our findings show that the fish brain actively responds to cold temperature by regulating metabolic physiology. Moreover, we identify oxytocin signaling as an adaptive and evolutionarily conserved metabolic regulator of temperature-related homeostasis. 

Oxytocin centrally regulates temperature-dependent metabolism in fish

Animals respond to environmental challenges with a suite of physiological responses – the stress response. As high temperatures become more frequent and intense globally, determining whether and how individuals vary in their stress response to heat is urgent. While there is an emerging interest in understanding avian stress response to heat, very often, only glucocorticoids are quantified. Here, we tested whether early-life conditions affect multiple traits of nestling stress physiology in response to heat. In the arid-adapted zebra finch, parents emit “heat-calls” when incubating under high temperatures, which adaptively alter offspring growth. We exposed nestlings to prenatal heat-calls or control-calls, and manipulated postnatal nest temperatures. At 13-day old, we collected blood samples in two experiments: under undisturbed “in-nest” conditions, or following a 2.5-hour heat-challenge. As biomarkers of acute, cellular and chronic stress, we measured, respectively, levels of corticosterone (CORT), constitutive heat-shock cognate 70 (HSC70) and stress-inducible heat-shock protein 90 (HSP90), and, the heterophil to lymphocyte (H/L) ratio. We found that none of the stress biomarkers co-varied in either experiment, likely due to distinct time-courses. After the heat-challenge, only the H/L ratio was higher than in the in-nest experiment. Furthermore, in the in-nest experiment, control-call nestlings had a marginally higher H/L ratio than heat-calls nestlings, and both HSPs increased with nest temperatures, in control-call individuals only. These results suggest a higher sensitivity of control-calls nestlings to summer nest temperatures. Overall, our study shows for the first time that a prenatal acoustic signal for heat affects physiological stress responses to postnatal thermal conditions. 

Prenatal acoustic signals and postnatal heat shape nestling stress physiology

Many pesticides are endocrine-active, even if the intended mechanism of action is not specified to directly interfere with outcomes such as development, reproduction, or sex determination. Pyrethroid pesticides, which are commonly used in both North America and the European Union, are based on toxins naturally produced by the chrysanthemum plant, but are chemically enhanced to increase persistence and toxicity to target organisms. These modifications make pyrethroids more likely to interact with endocrine targets such as the nuclear and membrane estrogen receptors, to modify downstream responses like the activity of steroidogenic enzymes, and to ultimately induce organismal responses indicative of endocrine disruption in fish (e.g. changes in gonad size, sex ratio). Previous work from our group confirmed these interactions for specific pyrethroid types, demonstrating that early life exposure can result in impacts that span multiple generations, and that increased temperature can exacerbate these effects. Recent findings indicate that other abiotic factors, such as salinity, can influence the direction and degree of endocrine-related responses, likely due to a combination of osmoregulatory modulation and differential chemical behavior that is dependent upon aqueous salinity. These observations are highly relevant in terms of predicted changes in temperature and salinity regimes over the coming decades, and our findings underscore the importance of understanding responses to endocrine-active compounds in the context of altered environmental conditions.

Endocrine disruption in the context of climate change: understanding responses in fish

On the upper surface of its leafs salvinia molesta has pillar shaped structures which are hydrophilic at the tip and hydrophobic at the shaft. When submerged under water, air is retained between the surface structure. Beside the storage of oxygen this feature enables a variety of further applications. The air layer could serve as barrier to prevent the settlement of biological organisms (antifouling) and reduce wall shear stress if water flows along the surface. These phenomena are from higher interest in case of ships or liquid transport in pipes. If an air retaining structure could be realised on such surfaces, a reduction of frictional drag, fuel consumption and CO2 production could follow. A requirement for this is the understanding of the physics and the influence of fundamental parameters such as structure geometry, size, contact angle of the structure material, pressure, or air saturation of the liquid. In this work the effect of different parameters is presented and discussed using numerical simulation, analytical approaches and experiments.

Air Retaining Surface Structure of Salvinia molesta - Physics, Applications and Challenges1

Muscles perform a number of roles, acting as motor, brakes and shock absorbers. Tendons also perform several roles, acting as transmission and/or elastic springs. But a distinct and perhaps largely overlooked role of isometric muscles and tendons is as functional tension bicycle spokes, becoming loaded in turn through simple geometry, and enabling horizontal translation during vertical weight support without high mechanical power demand. Here, quadrupedal mammal leg structure and function is considered in the context of enabling low mechanical demand at the level of the limb by maintain forces perpendicular to velocity, at the same time as providing low power supply by keeping muscles isometric when loaded, or unloaded when changing in length. These principles provide a simple account for the general structure of the scapula, serratus ventralis and triceps brachii of the forelimb, and the biceps femoris (curiously crossing across the femur), vastus, rectus femoris and tensor fasciae latae of the hindlimb. Different muscles experience tension at different stages in stance, each resulting in linkages where the links are isometric, and the limb supports predominantly vertical forces during horizontal translation.

Legs as linkages: thinking of isometric muscles and tendons as bicycle spokes

Cereal grains are among the most important sources of energy for humans and domestic animals, owing to large and nutritious endosperm tissues. We study nuclear organization and chromatin in seeds of cereals using cultivated barley (Hordeum vulgare subsp. vulgare) - a diploid temperate zone model cereal used for both food and feed. Barley has a large genome (2n = 2x = 14, 1C = 5.1 Gbp) with interphase chromosomes organized in Rabl configuration with centromeres and telomeres clustering at opposite nuclear poles. Though considered to be typical for large cereal genomes, observations of Rabl configuration are based mostly on nuclei from meristematic tissues. We explored interphase chromosome organization in different tissues of developing barley grains and will show that Rabl chromosome organization is not a general rule in barley and it diminishes with the increasing nuclear DNA content and seed age. Furthermore, we performed transcriptome profiling of different barley grain tissues that allowed determining roles of different biological processes and also chromatin regulatory pathways during this critical stage of development. In summary, all these facets of our barley chromatin research create a fruitful environment for future functional analyses of nuclear organization and epigenetic regulation of gene expression in this agriculturally important species.

The dynamics of chromatin changes during barley seed development

Reactive oxygen species (ROS) are by-products of oxidative metabolism, and while low levels contribute to cell signalling, high levels can be toxic. In mammals, ischaemia-reperfusion leads to production of toxic levels of ROS, which cause oxidative tissue damage. In contrast, anoxia-tolerant species, such as the freshwater turtle, do not sustain oxidative tissue damage after anoxia and reoxygenation. How anoxia-tolerant animals are able to prevent ROS dysregulation and oxidative damage with anoxia is unclear. We investigated ROS production and antioxidant capacity in the turtle heart upon anoxia-reoxygenation. Using the in vivo mass-spectrometric H2O2-probe MitoB, we found that there is no excessive production of ROS in the turtle heart after whole animal anoxia-reoxygenation. We hypothesized that this was due to the fact that turtles avoid large accumulation of succinate in the heart and preserve energy levels. This would prevent reverse electron transfer and extensive ROS production from complex I after anoxia. However, addition of exogenous succinate and manipulation of membrane potential with oligomycin did not induce excessive ROS production in turtle heart strips in vitro. This suggests that other factors, such as S-nitrosation of complex I, supercomplex stability or high antioxidant capacity may also be involved in preventing ROS after anoxia in the turtle heart. Together, this study shows that turtles are able to avoid toxic levels of ROS in the heart and oxidative tissue damage after anoxia and reoxygenation. 

Low in vivo production of reactive oxygen species after anoxia and reoxygenation prevents oxidative damage in the turtle heart

Surviving in low oxygen environments requires complex and integrated physiological responses, many of which center on economizing energy budgets. At the cellular level, significant reductions in protein synthesis and even translational arrest (in anoxia) can save as much as 90% of routine ATP demand. This suggests strategic investment in the protein complement of essential tissues, like the heart, that must maintain some level of function for the animal to survive through and recover from a hypoxic bout. The use of quantitative proteomics in animal models of hypoxia tolerance has unveiled new ideas about how the cardiac proteome is influenced by hypoxia exposure, as well as the ontogenic constraints on this aspect of cardiac plasticity. For example, developmental hypoxia exposure of American alligators (Alligator mississippiensis) induces persistent shifts in the cardiac proteome that, paradoxically, reflect an increased capacity for translation, proteolysis, and metabolic capacity. Concomitant with these changes was an equitable down-regulation of seemingly random cardiac proteins, which may offset the cellular energy budget. In Western painted turtles (Chrysemys picta bellii), the cold acclimation required to prepare tissues for chronic anoxia during winter dormancy induced general down-regulation of electron transport system proteins in adult but not hatchling turtles. Interestingly, both studies revealed 
‘age’ as a key explanatory variable in the cardiac proteome. This suggests a robust developmental program in the ectotherm heart that is resilient even to potent environmental stressors like oxygen and temperature, and warrants further investigations into the developmental thresholds of hypoxia tolerance.






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