Elveflow (France)

The chapter explores the imperative role of genome editing techniques in enhancing tolerance against abiotic stress in crop plants, focusing on major stressors such as salt stress, drought, extreme temperatures, heavy metals, UV-B radiation, and mechanical stress. Abiotic stresses pose substantial challenges to global food security and necessitate sustainable and innovative solutions. Although conventional breeding has contributed significantly to addressing these challenges, the urgency for precise and effective gene editing tools is paramount. Recent breakthroughs in genome editing tools, notably CRISPR/Cas-based techniques, offer unprecedented opportunities to enhance crop traits efficiently. The chapter delves into the applications of genome editing tools to address specific stressors, illustrating the potential to modify single or multiple genes with high precision. Multiplex gene editing techniques enable simultaneous modifications, thereby conferring tolerance to numerous abiotic stresses. The chapter concludes by highlighting the immense potential of genome editing as a transformative tool to develop abiotic stress-tolerant crops, contributing significantly to sustainable agriculture and global food security.

The whitefly, a polyphagous insect pest feeding on nearly 1328 plant species, is a major threat to global cotton production and incurs up to 50% yield losses in cotton production in Pakistan. We investigated whether increased aspartate in phloem sap imparts whitefly toxicity and protects cotton plants from intense damage. The enzymatic step for aspartate production is carried through aspartate aminotransferase (AAT). In this study, we constitutively overexpressed the Oryza sativa cytoplasmic AAT (OsAAT2) under the CaMV35S promoter in Gossypium hirsutum cv. CIM-482. Real-time PCR analysis of the AAT transcripts revealed a 2.85- to 31.7-fold increase in mRNA levels between the different cotton lines. A substantial increase in the free-amino acid content of the major N-assimilation and transport amino acids (aspartate, glutamate, asparagine, and glutamine) was seen in the phloem sap of the transgenic cotton lines. The bioassay revealed that the two transgenic cotton lines with the highest free aspartate content in the phloem sap exhibited 97 and 94% mortality in the adult whitefly population and a 98 and 96% decline in subsequent nymph populations, respectively. There was also a significant change in the physiological behaviour of the transgenic cotton lines, with an increased net assimilation (A), gaseous exchange (Gs) and rate of transpiration (E). Improved morphological characteristics like plant height, total number of bolls and fiber yield were recorded in transgenic cotton lines. The AAT gene shows promise in mitigating whitefly infestations and enhancing the overall health and yield of cotton plants.

The ability of artificial microswimmers to respond to external stimuli and the mechanistical details of their origins belong to the most disputed challenges in interdisciplinary science. Therein, the creation of chemical gradients is technically challenging, because they quickly level out due to diffusion. Inspired by pivotal stopped flow experiments in chemical kinetics, we show that microfluidics gradient generation combined with a pressure feedback loop for precisely controlling the stop of the flows, can enable us to study mechanistical details of chemotaxis of artificial Janus micromotors, based on a catalytic reaction. We find that these copper Janus particles display a chemotactic motion along the concentration gradient in both, positive and negative direction and we demonstrate the mechanical reaction of the particles to unbalanced drag forces, explaining this behaviour.

Abstract Die Fähigkeit künstlicher Mikroschwimmer, auf äußere Reize zu reagieren und deren mechanistische Ursprünge, gehören zu den umstrittensten Fragen der interdisziplinären Wissenschaft. Die Erzeugung chemischer Gradienten ist dabei eine technische Herausforderung, da sie aufgrund von Diffusion schnell abflachen. Inspiriert von ‘Stop‐flow’ Experimenten aus der chemischen Kinetik zeigen wir, dass die Erzeugung eines mikrofluidischen Gradienten durch Kombination mit einer Druckrückkopplungsschleife zur präzisen Kontrolle des Stoppens erfolgen kann. Das ermöglicht es uns, die mechanistischen Details der Chemotaxis von künstlichen katalytischen Janus‐Mikromotoren zu untersuchen. Wir stellen fest, dass diese Kupfer‐Janus‐Partikel eine chemotaktische Bewegung entlang des Konzentrationsgradienten sowohl in positiver als auch in negativer Richtung zeigen, und wir demonstrieren die mechanische Reaktion der Partikel auf unausgewogene Widerstandskräfte, die dieses Verhalten erklären.

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand. In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient β-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for β-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

A new fabrication method of polymeric optical fiber tweezers with a multi-mode tip is presented. Preliminary results show higher robustness, improved ability for 2D trapping and differentiation of particles based on back-scattering analysis.