France

Land plants cover their epidermis with a specialised protective layer called the cuticle, a feature which is crucial for surviving the perils of a terrestrial environment, including desiccation, UV damage and pathogen attack, dangers which may worsen with our escalating climate crisis. Several cereal staples such as barley and wheat show further epidermal specialisation with a distinctive, glaucous wax bloom on reproductive stage tissues, a feature associated with improved drought tolerance. Understanding the mechanisms underpinning wax bloom development may help breed more climate resilient varieties. To date, studies on barley eceriferum mutants have identified several enzymes involved in cuticular wax biosynthesis. However, little is known about the upstream regulation of this process. My research addresses this knowledge gap by exploiting genetic resources to identify a key regulatory player in barley cuticle development. Here, I will discuss how I used high density genotyping, comparative gene expression and wax compositional analyses to identify a gene essential for wax bloom formation and the expression of cuticular metabolic genes in barley.

SEB Annual Conference 2022

Identification of a key regulator controlling cuticular wax in barley

technical paper

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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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

For marine species with complex life cycles, a critical question concerns the response of larval stages to warming. In species with feeding larvae, warming and food limitation may result in negative (additive or synergetic) effects on growth and survival. We experimentally evaluated the combined effects of temperature and food limitation on larval performance of the invasive crab Hemigrapsus sanguineus (local population of Helgoland, North Sea, German Bight). We then compared the performance of H. sanguineus with the native competitor (shore crab Carcinus maenas). Within the studied temperature range (15-24 °C) and under the conditions of food limitation (access to food for 6 hours a day), the primary driver of survival was temperature (low temperatures decreased survival especially at the more advanced stages). Food limitation decreased survival and growth at all temperatures but larvae metamorphosed to megalopa at all food limited conditions. The response of H. sanguineus differed from that of C. maenas where effects low temperatures have little impact on performance but high temperatures enhance the negative effects of food limitation. Hence, these two coexisting species appear to have different larval responses to warming and food limitation, with warming leading to a higher performance of the invasive relative to the native species.

Larval performance of the exotic Hemigrapsus sanguineus under increased temperature and food limitation

Two rounds of genome duplication (GD) in the ancestor of vertebrates, followed by additional GD during the evolution of ray-finned fishes (Actinopterygii), expanded certain gene families, including those encoding the hypoxia inducible transcription factor (HIF). The present study analyzed Actinopterygian genomes for duplicates of HIFα, the subunit that confers oxygen-dependent gene regulation. In contrast to tetrapod vertebrates that retain three HIFα genes from the ancestral vertebrate GD, four HIFα forms were found in the genomes of primitive Actinopterygians (spotted gar and Asian arowana). All four forms have been retained in zebrafish and related species (Otocephala) and salmonids and their sister taxa (northern pike) but one of them (HIF4α) was lost during the evolution of more derived fishes (Neoteleostei). In addition, the current analyses confirm that Otocephala retain duplicates of HIF1α and HIF2α from the teleost-specific GD, provide new evidence of salmonid-specific duplicates of HIF1α, HIF2α, and HIF3α, and reveal a broad distribution of a truncated form of HIF2α in salmonids and Neoteleostei. This study delivers a comprehensive view of HIFα evolution in the ray-finned fishes, highlights the need for a consistent nomenclature, and suggests avenues for future research on this critical transcription factor.

Genomic Analysis of Hypoxia Inducible Factor Alpha Reveals Missing Ohnologs and Evidence of Widespread Positive Selection in Ray-Finned Fishes

Sessile benthic species are submitted to fluctuating oxygen and temperature conditions that can negatively affect their energy and redox balance. Single effects of temperature and oxygen fluctuations have been extensively studied, but their combined effects are not yet well understood. We studied the bioenergetics and oxidative stress markers in the Pacific oyster Crassostrea gigas exposed for 10 days to daily immersion-emersion cycles under the control (15°C) (H), elevated (30°C) (HW), and fluctuating (15°C in water and 30°C in air) (HF) temperature. Oysters maintained under normoxia and 15°C were used as controls. Mortality increased in the oysters exposed to hypoxia and elevated temperature indicating acute thermal stress. Oysters co-exposed to fluctuating oxygen and temperature showed a decrease in the mitochondrial aerobic capacity but no onset of anaerobiosis shown by the lack of succinate accumulation. Glycogen content decreased in the digestive gland, whereas protein content increased in the gill of the HF group indicating higher protein deposition combined with higher energy demand during reoxygenation. In contrast, the HW group showed decreased protein levels reflecting heat-induced suppression of protein synthesis. Lipid peroxidation levels were elevated in the gills of the oysters from HW (but not H or HF group) indicating that antioxidant systems of oysters can cope with hypoxia-induced oxidative stress under the normal or fluctuating temperature but not during constant warming. Thus, long-term warming combined with intermittent hypoxia can lead to breakdown of physiological functions and higher summer mortality like already observed in the German Wadden Sea.

Effects of fluctuating oxygen and temperature regime on bioenergetics and oxidative stress of the pacific oyster Crassostrea gigas

Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients to prevent depletion during experiments. While this reduces the likelihood of cell starvation, it creates non-physiological culture conditions that have been shown to ‘re-wire’ cellular metabolism and broadly influence cell physiology. Recently, researchers have developed physiological media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, such physiologic media may be vulnerable to nutrient depletion over the course of an experiment  extending over several days. In this study we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax by several cancer cell lines (PC3, LNCaP, MCF-7 and SH-SY5Y) over 48 hours. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48h, cells were hypoglycemic (less than 2mM glucose). Most amino acids were similarly rapidly depleted to sub-physiological levels by 48h. In contrast, glucose and most amino acids remained within the physiological range at 24h. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of cellular starvation responses, amino acid biosynthesis, apoptotic signalling, and endoplasmic reticulum stress response. Alterations in mitochondrial respiration using Seahorse extracellular flux analysis were also observed. Taken together, these results exemplify the metabolic considerations for Plasmax use, highlighting that cell culture in Plasmax requires daily media exchange.

Rapid nutrient depletion by cancer cells cultured in the physiologic medium Plasmax  

Ocean acidification and warming caused by increased anthropogenic carbon dioxide emissions poses a tremendous challenge to marine calcifiers such as intertidal bivalves. The approach to studying acclimation potential or adaptation to climate change are often conducted under artificial conditions where food is provided ad libitum and do not consider tidal emersion. These limitations present problems for extrapolating results to natural ecosystems. For instance, food provisioning and regular emersion can enhance or limit acclimation capacities of marine organisms. Here we investigate the physiological response of young Pacific oyster Crassostrea gigas exposed for 3 months to ambient and future (+3°C, -0.3 pH unit) climate scenarios under two food regimes (ad libitum vs restricted) and two tidal levels (subtidal vs intertidal, emersion twice daily) under laboratory conditions. Oysters were sampled for biometric and physiological rate measurements (respiration and food intake), biochemistry (energetic reserves and enzyme assay) and histology to evaluate their reproductive status. At the end of the experiment, oysters were further challenged with a viral disease to investigate the physiological cost of acclimation to these conditions and their immune response. Preliminary analyses suggest that food level was the most influential factor, followed by climate scenario and tidal level. We indeed found that growth, food intake, reproduction and disease resistance of oysters were negatively affected by food restriction. Future conditions enhanced oyster physiological rates and sexual maturation, particularly at high food levels. Finally, tidal level had no major effect on these variables, suggesting physiological compensatory mechanisms during emersion. Our study suggests that the oyster is robust to future climatic conditions, even when feeding was restricted. A detailed analysis of the results will be presented at the conference.

Physiological and immune response of oysters to climate change as a function of diet and tidal regime

The genus Primula L. (Primulaceae) represents approximately 430 species, distributed all over the Northern Hemisphere in temperate to alpine regions. It is an important floristic element of the Himalayan flora and is considered an ecological indicator of the high-altitude vegetation. Many Primula species show two morphs (pin and thrum) based on style length. The stigma of pin plants is positioned at the entrance of the flower, exhibiting a long style, while in thrum flowers, the style is much shorter, leaving the stigma around halfway up the floral tube. Among these species, Primula denticulata exhibits a large extent of occurrence in a broad altitudinal range (2096-3700 m). A study was undertaken to understand the frequency, morphological and phenological variations between two morphs of the species along an altitudinal gradient in the Western Himalayas. We recorded the numbers of pin and thrum flower morphs at three different elevations (2337m, 3000m, and 3700m) during 2021 and 2022. Our results indicate variation in morph frequency at middle elevation i.e., 3000m, while at lower and higher elevations, both the morphs have an equal frequency. A delay was observed in the onset of phenological events with increasing elevation. We observed more numbers of floral visitors at lower and middle elevations than at higher elevations. The stigmatic surface of pin and thrum morphs were found to be different under Scanning Electron Microscope (SEM). Along with these morphological and phenological observations, genetic diversity analysis of P. denticulata is underway with 290 samples collected from different populations along the altitudinal gradient and 100 primer pairs designed from Simple Sequence Repeats (SSRs). As a result of this study, the correlation of morphological and phenological studies with genetic variation existing along the altitudinal gradient can be observed. The study will show the impact of changing environmental conditions on the diversity of the plant along the altitudinal gradient.
Keywords: Primula denticulata, Morph frequency, Altitudinal gradient, Morphological variations, Phenological variations, Changing environmental conditions








Effect of morphological, phenological variations and morph frequencies on population genetic diversity along an altitudinal gradient in heterostylous Primula denticulata

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.

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

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

Hydrogen peroxide (H2O2) is widely used as a signalling molecule. It is critical to measure H2O2 with a high spatial and temporal resolution to better understand its role as a messenger. The most frequent approach to detect H2O2 has been using chemical probes, which have the disadvantage of limited specificity and cannot report any dynamics or spatial information. Genetically encoded probes have made significant advances to overcome these limitations. Probes of the HyPer family consist of a circularly permuted yellow fluorescent protein (cpYFP) core and a sensing domain from a bacterial H2O2-activated transcription factor (OxyR). These, however, have a pronounced pH sensitivity. Other H2O2 sensors, such as roGFP2-Orp1, have shown to be pH-insensitive but lack the responsiveness to detect low nanomolar H2O2 changes, possibly due to the strong reducing effect of the glutathione redox potential (EGSH). A new ultrasensitive HyPer7 fuses the OxyR of N. meningitidis to an optimized cpYFP largely pH-independent. Here, we have generated and characterized stable Arabidopsis thaliana plants expressing a cytosolic version of HyPer7 and prove that the reduction of this sensor is mostly EGSH-independent and highly responsive to externally imposed oxidation via H2O2 feeding. Furthermore, HyPer7 could sense stress-induced H2O2 in live-cell plants exposed to herbicides, such as methyl viologen or the flagellin-induced apoplastic ROS burst, much better than the previously mentioned H2O2 probes. HyPer7 sensitivity also allowed us to see that the laser light used for excitation is sufficient to induce H2O2 release from chloroplasts in amounts only detected by this ultrasensitive sensor.

The latest HyPe(r) in plant H2O2 biosensing

Membrane trafficking and organelle biogenesis play important roles in plant growth and development, as well as responses to external signals. The plant endomembrane system contains several functionally distinct membrane-enclosed organelles, including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) or early endosomes (EE), prevacuolar compartment (PVC) or multivesicular body (MVB) and vacuole. One of our major research programs has been focused on illustrating the underlying mechanisms of vacuolar trafficking and vacuole biogenesis and function in plant cells. One major approach has been the whole-cell electron tomography (ET) analysis with nanometer resolution of vacuole formation and distribution in different cell types of developmental stages, including root cells, stomatal lineage cells and developing pollens in Arabidopsis. Here I will present our recent work and models of vacuole biogenesis and function in plants from the ET analysis. Supported by grants from the Research Grants Council of Hong Kong and CUHK. 

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