France

Microsphere super-resolution microscopy has recently received a lot of attention due to its simplicity and its capability to resolve features beyond the diffraction limit for living systems, in real time, without being invasive. Current systems which are able to image small biological structures are destructive, invasive (they use fluorescent dyes) or they provide reconstructed images (i.e. not a real time imaging of living organisms). The authors designed and built a bio-microscope tailored around the SMAL® (Super-Resolution Microsphere Amplifying Lens) objective lens which overcomes these drawbacks. The novelty of the setup is the ability to study cell’s structures and viruses which previously were not possible to be imaged using conventional white light microscopy. In this work, along various plant cells, label-free tobacco mosaic virus was employed for imaging in this work due to its stability, reliability, and its dimensions beyond the diffraction limit. The results were compared with images obtained using an X100/1.40 NA oil immersion objective lens fitted onto a white light microscope, in the same location. Microsphere assisted microscopy is a novel type of optical microscopy with great potential for high resolution imaging of in vivo biological structures and pathogens.

SEB Annual Conference 2022

Optical super-resolution imaging of tissues and tobacco mosaic virus (TMV) using microsphere technology

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

Improving the performance of the CO2-fixing enzyme Rubisco is among targets for increasing crop yields. Here, Earth system models (ESM) of canopy C3 and C4 photosynthesis were combined with species-specific Rubisco parameters to quantify the consequences of bioengineering foreign Rubiscos into C3 and C4 crops under field conditions. The sunlit/shaded model for canopy photosynthesis was used together with species-specific Rubisco kinetics parameters including maximum rate (Kcat), Michaelis-Menten constant for CO2 (Kc21%O2), specificity for CO2 to O2 (Sc/o), and associated heat activation (Ha) values. Canopy scale consequences of replacing native Rubiscos in wheat, maize and sugar beet with foreign enzymes from 27 species were modelled using data from Ameriflux and Fluxnet databases. Variation among the included Rubisco kinetics differentially affected modelled carbon uptake rates, and Rubiscos from several species of C4 grasses showed the greatest potential of over 50% carbon uptake improvement in wheat, and over 25% improvement in sugar beet and maize. 

Rubisco substitutions predicted to enhance crop performance through carbon uptake modelling

To cope with a sustained drop in temperature plants need to adjust their photosynthesis to better suit the prevailing conditions. Fully developed leaves with fixed morphology alter their protein content by upregulating limiting factors. This dynamic response is called dynamic acclimation and requires days to be completed. For acclimation to be completed energy and elements need to be invested in de novo protein synthesis. A limitation to soil nitrogen availability can hinder this response and affect plant survival after cold exposure. To test this hypothesis, we grew Arabidopsis thaliana plants in three different N soil availabilities and exposed them to cold for 7 days. Plants were grown in sand and fertilized with nutrient solution containing 2.5mM, 5mM and 15mM total N at 20oC day/ 18 oC night. After 8 weeks when the first fully developed leaves immerged plants were transferred at 5 oC for 7 days before acclimation was assessed. Our results indicate that N availability plays a pivot role in plant response to cold stress. Only plants grown at the highest N availability were able to upregulate their maximum photosynthetic capacity while plants grown at the lowest N availability were unable to cope with the temperature shift and as a result had lower photosynthetic capacity after cold exposure. Plants grown in 5mM total N were able to maintain the same photosynthetic capacity after cold exposure. 

Plants cold ressponse and how is affected by nitrogen availability

So called ‘drop-in’ Biofuels produced from plants are becoming increasingly important for the aviation and shipping industries, which urgently need to move away from fossil fuels in order to limit their impact on the climate. Algae farms provide a number of benefits over traditional biofuel crops, such as sugar cane, by alleviating competition for arable land. When exposed to high light, the green alga Chlamydomonas reinhardtii produces triacylglycerols (TAGs) which can be converted to drop-in biodiesel. Excess light results in the over-reduction of the photosynthetic electron transport chain, and it has been suggested that the production of TAGs is in response to this redox imbalance. A deeper understanding, therefore, of how Chlamydomonas cells address redox imbalance will help to inform future work aiming to maximise TAG production in these conditions. Our work has highlighted a potential role for the phenomenon known as light enhanced respiration (LER) in responding to redox stress induced by high light. We show that presence of acetate in the growth media may allow cells to better acclimate to high light by increasing the capacity of the cells for LER. Autotrophic cells (cells grown without acetate) appear to be more susceptible to photodamage than mixotrophic (grown with acetate) when grown in high light. Our results display the complexity of the high light response and provide preliminary evidence for the potential of the mitochondria as a tool for addressing redox imbalance. 

A role for mitochondria during high light stress in Chlamydomonas reinhardtii

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus anti-gravitropic offset (AGO) mechanisms. Here we report a new root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes a F-box and Tubby domain containing protein which is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wildtype. Transcript profiling revealed Hvegt1 roots deregulate cell wall-loosening enzymes and ROS cofactors. In situ analysis identified that HvEGT1 is highly expressed in inner root tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. Atomic Force Microscopy measurements revealed that elongating cell walls in Hvegt1 mutant roots are significantly less stiff than wildtype. Hence EGT1 appears to control root angle by regulating cell wall stiffness in elongating root tissues, counteracting the gravitropic machinery’s known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing a novel anti-gravitropic mechanism.

Root angle in cereals is controlled by a novel anti-gravitropic mechanism 

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. 
















Characterising the proteolytic network of plant cathepsin B

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.

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Optical super-resolution imaging of tissues and tobacco mosaic virus (TMV) using microsphere technology

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Rubisco substitutions predicted to enhance crop performance through carbon uptake modelling

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