France

Programmed cell death (PCD) is a genetically managed biological process that results in the targeted sacrifice of unwanted or diseased cells. Plants use PCD for various purposes such as development, defence against pathogens, response to abiotic stress caused by UV radiation or heat shock. Several proteinases are involved in the PCD process; among them, cathepsin B (CathB) which plays an important role in pathogen defence, plant development, germination, senescence, microspore embryogenesis and abiotic stress response. A greater understanding of the molecular mechanisms underlying the processes downstream of CathB is the next important step for new discoveries in cell biology and applications in biotechnology. To achieve this goal, it is important to discover and test substrates cleaved by plant CathB. Up to 13 substrates for CathB were identified in animal cells, and we found orthologous genes in Arabidopsis thaliana for eight of them. Among these, we are testing some candidate substrates for CathB. To experimentally validate the cleavage site, we used leaf agro-infiltration to co-expressed substrate GFP-fusion and CathB in tobacco cells and carried out cleavage assays. in vivo and in vitro results will be presented. 
















SEB Annual Conference 2022

Characterising the proteolytic network of plant cathepsin B

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

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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Please register for the conference by clicking on this link:

https://www.sebiology.org/events/seb-annual-conference-2022/registration.html

The site will be open for registered participants closer to the event.

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 - Montpellier, France

poster

Polyploids are of common occurrence among the plants. Several studies have been conducted to understand the impact of polyploidy on the evolution of plant genome. Natural polyploids do occur in the wild, but it is difficult to decipher which changes are due to polyploidy as the plants accumulate variation over the history of their existence. It is important to study the impact of polyploidy away from natural variation. Therefore, we generated synthetic neoautopolyploids in two species of Vigna L. using colchicine-based cotton swab method. The polyploids were screened via flowcytometry and confirmed polyploids were taken to the field to study biological parameters. The two species were found to be behaving differently based on various parameters like photosynthetic efficiency, cell density, stomatal count, cell size and nucleus size. We will be studying inheritance pattern of these traits in subsequent generations. Our study provides a proof that polyploidy does not always have a positive scaling effect on plants at various biological parameters and the changes are species specific. We will be studying genomic changes in future and will correlate them with morphological changes observed.  

Keywords: Polyploids, WGD, Autopolyploid, Flowcytometry, morphometery

Whole Genome Duplication Show Species-Specific Morphological Changes In Neoautopolyploids

Rising temperatures pose a significant threat to the survival of ectothermic animals. Thermal physiological traits, including upper thermal limits, are proven useful traits to assess the vulnerability of ectotherms to changing temperatures. For instance, one may use upper thermal limits to estimate current and future thermal safety margins (i.e., the proximity of upper thermal limits to experienced temperatures), use this trait in coercion with other physiological traits in species distribution models, or investigate the plasticity, heritability and evolvability of these traits for buffering the impacts of changing temperatures. While datasets on thermal tolerance limits have been previously compiled, they often report single estimates for a given species, do not present measures of data dispersion, and are biased towards certain parts of the globe. In this project, we systematically searched the literature in seven languages and produced a dataset of over 3000 heat tolerance estimates for 616 amphibian species. I will first present how the dataset we compiled can be used to address various questions in ecophysiology, and its limitations. Indeed, despite its scope, this dataset also suffered from geographical biases and captured less than 10% of described amphibian species. Here, I will demonstrate how we can fill the missing parts of this puzzle using powerful imputation procedures and estimate the heat tolerance of most described amphibian species. I will then demonstrate how we used this comprehensive database to estimate the vulnerability of the worlds’ amphibians to current and projected temperatures. 

Finding the missing pieces of an ecological puzzle to assess the climate vulnerability of amphibians

Sea lamprey (Petromyzon marinus) invaded the Laurentian Great Lakes in the early 20th century and remain a threat to fisheries in the largest freshwater ecosystem of the world. Nowadays, population growth is restrained by a highly successful control program that mainly relies on the application of the lampricide 3-trifluoromethyl-4’-nitrophenol (TFM) to streams infested with larval sea lamprey. Recent work shows that the effectiveness of TFM is affected by temperature, with the toxicity to larval sea lamprey decreasing with increasing temperature. As such, we set out to unveil the thermal breadth of larval sea lamprey using intermittent-flow respirometry. To do so, we calculated thermal performance curves for this life stage using two approaches: First, we acclimated larvae to 7, 13, 18, 21, 25 and 28°C and tested their aerobic scope at the respective acclimation temperature. Second, we acclimated larvae to 6, 13 and 21°C and subsequently performed acute exposure tests (6, 10, 13, 15, 18, 21, 25, 28 and 31°C), with a temperature increase/decrease rate of approx. 0.1°C/min. Our results show a wide thermal tolerance, consistent with the plasticity required to become a successful invasive species. Further, sea lamprey larvae show a thermal optimum nearing 28°C, indicating that their tolerance to TFM will likely continue to increase as water temperatures in the Great Lakes continue to rise due to climate change.

Effects of temperature acclimation on thermal performance for sea lamprey larvae

While group-living can increase foraging efficiency and feeding consistency, individual variation in behaviour can cause disparity in food intake among groupmates. For example, bolder individuals may secure more food if they are more exploratory or are willing to take risks to obtain food items. Temperature variation may further modulate phenotypic variation among groupmates and alter levels of feeding disparity within groups. 
We examined the effects of ambient temperature on group foraging in a social catfish, the bronze corydoras (Corydoras aeneus). Groups of 30 fish were placed within large mesocosms and were observed for feeding behaviour at both of 24°C or 28°C, with at least two weeks acclimation to each temperature. Feeding behaviour among individuals within groups was highly repeatably, with the same individuals being consistently first to feed when presented with food items, even when the same groups were tested across temperatures (R = 0.467). Temperature increased group activity and feeding rate, with groups finding food quicker at the warmer temperature, even though groups were more cohesive (individuals closer together) at the cooler temperature. Together, these data highlight individual variation within a group foraging context, whereby specific individuals are more successful at securing food than their groupmates. This will have implications for the relative costs and benefits of group membership for individuals.

Investigation into the effect of temperature on social foraging in Corydoras aeneus

Testing how evolutionary cold adaptation affects metabolism and performance of fish we compared the acute responses to warming of the stenothermal Antarctic eelpout Pachycara brachycephalum and of the eurythermal common eelpout Zoarces viviparus from the North Sea. We investigated whole animal oxygen consumption rate and ammonia excretion. As excretion of ammonia via the gills reflects whole animal metabolic patterns, especially nitrogen and amino acid metabolism, we also investigated the temperature dependent protein synthesis rate of gills in vivo and connected it to respiration and ammonia excretion. 
Both species were exposed to temperature changes within their species-specific thermal window:  P. brachycephalum -acclimated to 0°C- at a rate of 2°C day-1 until 10°C, and Z. viviparus - acclimated to 4°C - at a rate of 3°C day-1 until 22°C. Once temperature stabilized (within 12 hours), oxygen consumption rates were measured repeatedly and water samples were taken to determine the ammonia excretion of each fish individually. In a parallel experiment, we investigated the temperature dependent protein synthesis rate (Ks) of gills after intraperitoneal injections of 13C-labeled phenylalanine. The gill tissue was sampled 1.5 and 3 hours after the injection.
All measured parameters followed an exponential increase with warming for both species, however, with different Q10 values (e.g. Q10 protein synthesis rate P. brachycephalum 4.2 vs. 2.8 Z. viviparus). Protein synthesis rates at the same temperatures were similar in both species (P. brachycephalum vs. Z. viviparus: 4°C Ks= 2.5 vs. 2.6 % day-1; 10°C Ks= 4.5 vs. 4.3% day-1) indicating no cold compensation regarding protein synthesis in gills of the Antarctic eelpout. It will be discussed how these results are related to oxygen uptake and ammonia excretion.

The impact of acute warming on the respiratory system of fish – a comparison of eelpouts from Antarctica and temperate zones

Mitochondria are most known for their role in energy production, but they are also implicated in many other cellular functions (apoptosis, ROS production, cell proliferation, aging). In humans, the mitochondrial DNA was believed to encode only 37 genes involved in energy production and mitochondrial translation. However, recent studies have shown that within these genes could hide several small alternative genes (genes with translation initiation or termination sequences distinct from the reference gene sequences in which they are found) coding for micropeptides with a broad spectrum of functions that can be newly added to the large list of functions in which mitochondria are involved.

Following the discovery of the mitochondrial alternative gene mtaltnd4, we try to elucidate the functions of the gene and its peptide (MTALTND4) by studying: (1) the expression pattern of the peptide in different human tissues and blood plasma, (2) the involvement of the gene in the major mitochondrial functions by exposing cells to various treatments, (3) the interaction partners of MTALTND4 by mass spectrometry and (4) the impact of the peptide on cell transcriptome. 

We have found that mtaltnd4 may potentially produce a signaling protein in response to stress that would regulate certain cell metabolic processes and there is surely more to discover.











The alternative gene mtaltnd4: uncovering the mitochondria’s hidden functions

The progression of climate warming will expose ectotherms to transient heatwave events and temperatures above their tolerance range at increased frequencies. It is therefore pivotal that we understand species’ physiological limits and the capacity for various controls to plastically alter these thresholds. Exercise training could have beneficial impacts on organismal heat tolerance through improvements in cardio-respiratory capacity, but this remains unexplored. Using juvenile Chinook salmon (Oncorhynchus tshawytscha), we tested the hypothesis that exercise training improves heat tolerance through enhancements in oxygen-carrying capacity. Fish were trained once daily at 60% of their maximum sustainable swim speed for 60 min. Tolerance to acute warming was assessed following three weeks of exercise training, measured as the critical thermal maximum (CTMax). CTMax measurements were coupled with examinations of the oxygen carrying capacity (haematocrit, haemoglobin concentration, relative ventricle size, and relative splenic mass) as critical components of the oxygen transport cascade in fish. Contrary to our hypothesis, we found that exercise training did not raise the CTMax of juvenile Chinook salmon with a mean CTMax increase of just 0.35°C compared to unexercised control fish. Training also failed to improve the oxygen carrying capacity of fish. Exercise training remains a novel strategy against acute warming that requires substantial fine-tuning before it can be applied to the management of commercial and wild fishes. 

Does exercise training improve heat tolerance in Chinook salmon (Oncorhynchus tshawytscha)?

Analogous to the situation in several lepidopteran and dipteran insects also the larval stages of some echinoderms and hemichordates (ambulacraria superphylum) evolved highly alkaline midguts up to pH 10.5. Despite its pivotal role in species´ sensitivity to changes in seawater pH, the underlying epithelial transport mechanisms are largely unknown. Using ion-selective microelectrodes we found that pluteus larva of the purple sea urchin not only have highly alkaline midgut fluids (pH ~9) but also a substantial reduced sodium concentration (150 mM) compared to the surrounding sea water (450 mM). We pharmacologically investigated the role of Na+/H+ exchangers in intracellular pH (pHi) regulation and midgut proton and sodium maintenance using the NHE inhibitor 5 (n ethyl n isopropyl)amiloride (EIPA). While life cell imaging demonstrated the importance of NHEs in pHi regulation of midgut epithelia cells, basolateral EIPA application decreased midgut pH whereas luminal application, via micro-injections, increased midgut [Na+], without affecting pH. Gene expression analyses identified putative NHE candidates for midgut pH and Na+ homeostasis. Based on this information an antibody was generated against the sea urchin Slc9a2, which showed that the protein is localized in luminal membranes of the midgut. Additionally the use of specific vivo morpholino knock down of spslc9a2 provoked an increase in midgut [Na+] without affecting pH underlining the role of this transporter in midgut Na+ maintenance. 
This work provides new insights of NHEs being involved in ion regulatory mechanisms, especially the maintenance of pH and [Na+] in larval midgut fluids and thereby show conserved features to insect and vertebrate digestive systems, which may contribute to the ability of sea urchin larvae to cope with changes in seawater pH.






Midgut Na+ and pH homeostasis is differentally regulated by Na+/H+ exchangers in the sea urchin larva

Otoliths are paired calcified structures and are used for hearing and keeping balance of teleost fishes. Otoliths are mainly composed by aragonite, one of the three calcium carbonate polymorphs. However, the otolith carbonate structures often transform from aragonite to another polymorph, vaterite and the mechanism for this transformation remains unclear. To examine the transformation mechanism, we established a new method for changing otolith carbonate polymorphs. Ten individuals of an inbred strain of Oryzias latipes were independently reared in aquaria with adding SrCl2・6H2O to tap water. The other set of individuals were reared in normal tap water as a control group. Sagittal otoliths were removed after 114¬¬–146 days of experimental period. To distinguish between aragonite and vaterite polymorphs, strontium (Sr) and magnesium (Mg) compositions of otoliths were analysed using an electron probe micro analyser. The individuals reared in Sr-rich water had otoliths of which the margins were replaced with vaterite, while control individuals had entirely aragonite otoliths. Our results clearly show that the addition of Sr to rearing water induces polymorphic transformation from aragonite to vaterite. Considering that the polymorphic transformation from inorganic calcite to inorganic aragonite is induced by adding Mg to water, thermodynamic mechanisms possibly affect the observed transformation. Our findings have a potential to explore the impact of having vaterite otolith on fishes as well as to improve understanding of vaterite-forming mechanisms.

A new method to induce polymorphic transformation from aragonite to vaterite in Oryzias otoliths

The adaptive significance of sequential hermaphroditism, typically found in tropical fish and marine invertebrates, has been successfully explained by the size-advantage principle (SAP) and notable exception has not been reported. Here we focus on a novel type of protandrous sequential hermaphroditism in an intertidal holothurian as a case not amenable to SAP. This species typically participates as a male in the first three spawning events, and then spawn eggs at the fourth, which is followed by the second sex change to release sperm again. By incorporating published empirical findings into evolutionary invasion analyses, we successfully specified the theoretical conditions that a population containing the reversible sex change is evolutionarily stable. Our scheme is a natural extension of the classical Fisher's principle of sex allocation, and is also capable of consistently explaining a strongly biased operational sex ratio found in sequential hermaphrodites.

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Characterising the proteolytic network of plant cathepsin B

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